WO2020215013A1 - Tolerogenic dendritic cells and uses thereof - Google Patents

Abstract

A tolerogenic dendritic cell is genetically or pharmacologically modified to alter their cellular metabolism by suppressing expression of Kelch-like ECH-associated protein (Keap1) or iNOS or by inducing Nrf2 and/or suppressing NfkB signaling.

Description

TOLEROGENIC DENDRITIC CELLS AND USES THEREOF

RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application

No. 62/835,323, filed April 17, 2019, the subject matter of which are incorporated herein by reference in their entirety.

BACKGROUND

[0002] Aplastic anemia is a life-threatening rare disease that occurs when one’s own immune system damages blood-making bone marrow cells, which gradually stop producing red and white blood cells and platelets. In the United States, it is estimated that there are between 500-1000 new cases annually, with unique challenges facing patients diagnosed later in life. Patients must receive frequent blood transfusions, take multiple immunosuppressive agents to suppress the autoimmune response that damages the marrow, take other drugs to prevent infections, and limit contact with the outside world to avoid infection and even minor injury. Over the long term, most patients eventually die of infections or of complications of their therapy. Thus, there is a desperate need for safer, more effective and less costly therapies for this disease, particularly for elderly patients for whom survival rates are unacceptably low.

[0003] While treatment paradigms have improved, the overall approach in the United States (US) has been similar for many decades. For young patients with a HLA matched sibling donor, Hematopoietic Stem Cell Transplantation (HSCT) is the preferred approach, whereas Immunosuppressive Therapy (1ST) comprised of Horse Antithymocyte Globulin (ATG) and Cyclosporine (CSA) is utilized in those that lack one. When 1ST fails to keep the disease in check (in as many as 30 to 40 percent of patients) Eltrombopag (Promacta) is used but works only in about 30 percent of patients and usually leads to only a partial, not a complete, response.

[0004] Upon failure of 1ST, these patients may undergo unrelated donor HSCT if their health status allows. The outlook is bleak for those patients for whom HSCT is not an option, as long-term (10-year) survival with immunosuppression ranging from 40% to 50%. For this group of patients on chronic 1ST, there is an added risk of somatic mutations resulting in evolution of myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) and the frequent recurrence of aplastic anemia. Using next-generation sequencing in a study of 439 patients with aplastic anemia, genetic abnormalities were detected in 50% of patients after 6 months of 1ST initiation, and one third of these patients had acquired somatic mutations in myeloid cancer candidate genes.

SUMMARY

[0005] Embodiments described herein relate to tolerogenic dendritic cells (TolDCs), methods of generating a population of TolDCs, the use of TolDCs in increasing immune tolerance, and the use of the TolDCs in treating inflammatory and immune disorders in a subject in need thereof.

[0006] It was found that TolDCs can be generated from dendritic cells through their metabolic reprograming by targeted activation of the nuclear factor (erythroid-derived 2)- like-2 factor (Nrf2), indicucible nitric oxide synthase (iNOS), and several other key regulators of dendritic cell metabolosim, such as genetic or pharmacologic manipulation of Tgf-b, smad7, and other targets of nfkB signaling. Targeted activation of Nrf2 is a novel approach to metabolic reprogramming of dendritic cells (DCs), which acquire a stable, immune suppressive or‘tolerizing’ phenotype. Unlike mature DCs (mDCs), the dominant anti-inflammatory signature of TolDCs is characterized by reduced expression of cell surface stimulatory ligands, decreased secretion of immunosuppressive cytokines and a distinct cellular metabolic profile that regulates T cell polarization.

[0007] Under quiescent conditions, Nrf2 is anchored in the cytoplasm through binding to Kelch-like ECH-associated protein (Keapl), which, in turn, facilitates the ubiquitination and proteolysis of Nrf2. It was found that disruption of Nrf2 binding to Keapl in DCs, a key mechanism for the repressive effects of Keapl on Nrf2, can activate Nrf2 and confer a tolerogenic phenotype to DCs.

[0008] In some embodiments, TolDCs described herein can include DCs that are administered an Nrf2 activator. The Nrf2 activator can include a triterpenoid administered at an amount effective to generate the population of tolerogenic dendritic cells. In some embodiments, the triterpenoid is at least one of CDDO-IM, CDDO-2P-IM, or CDDO-3P-IM.

[0009] In other embodiments, TolDCs described herein can include DCs that are genetically modified to inhibit, suppress, and/or disrupt expression of Keapl. The inhibition, suppression, and/or disruption of Keapl can include a deletion of at least a portion of Keapl gene to inhibit expression or function of Keapl including Keapl binding to Nrf2. [00010] In some embodiments, the Keapl expression in the DCs can be disrupted by gene editing. The gene editing be performed using at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and at least one guide RNA (gRNA) having a spacer sequence complementary to a target sequence in a Keapl DNA. The CRIS PR-associated endonuclease can be selected from a wild-type Cas9, a human-optimized Cas9, a nickase mutant Cas9, SpCas9(K855a), SpCas9(K810A/K1003 A/rl060A), or SpCas9(K848A/K1003A/R1060A).

[00011] The DCs used to generate the TolDCs can be obtained by isolating monocytes from the subject and culturing the monocytes with GM-CSF and IL-4 to generate immature dendritic cells. The monocytes can be isolated from bone marrow or peripheral blood of the subject.

[00012] In some embodiments the TolDCs generated by adminstration of an Nrf2 activator or by disruption of Keapl expression or binding to Nrf2 of the DCs can have a phenotype characterized by the production of multiple immune suppressive cytokines, including at least one of IL-4, IL-10 or TGF-b, high levels of HemeOxygenase- 1 (HOI) and low levels of inducible nitric oxide synthase (iNOS) (/._<?., HO-l^Hl,iNOS^low expression) with decreased NO production, promoting expansion of regulatory (suppressor) T cells, suppression of T cell activation and suppression of production of at least one of TNFa, IFN- g, or IL-12 in human mixed lymphocyte reaction (MLR) assays, exhibition of a shift from glycolytic metabolism to oxidative phosphorylation (OXPHOS) during ex vivo TolDC expansion, and/or activation of Nrf2 target gene expression.

[00013] In other embodiments, the TolDCs generated by adminstration of an Nrf2 activator or by disruption of Keapl expression or binding to Nrf2 of the DCs can have a phenotype characterized by the production of IL-10 and TGF-b and suppression of TNFa and IL-12 in human mixed lymphocyte reaction (MLR) assays.

[00014] In still other embodiments, the TolDCs generated by adminstration of an Nrf2 activator or by disruption of Keapl expression or binding to Nrf2 of the DCs can have a phenotype characterized by the production of IL-4, IL-10 and TGF-b and suppression of TNFa, IFN-g, and IL-12 in human mixed lymphocyte reaction (MLR) assays.

[00015] Other embodiments described herein relate to methods increasing immune tolerance in a subject in need thereof. The method can include administering to the subject a therapeu tic ally effective amount of TolDCs generated by administration of an Nrf2 activator or genetically modified such that the expression of Keapl in the TolDCs is inhibited, suppressed, and/or disrupted.

[00016] In some embodiments, the subject to which the TolDCs are administered has an inflammatory condition, an allergy, or an autoimmune disorder. In other embodiments, the subject has received a tissue or organ transplant.

[00017] In other embodiments, the TolDCs are administered to a subject following a hematopoetic cell transplant with bone marrow, hematopoetic stem cells, or umbilical cord blood.

[00018] In other embodiments, the subject has aplastic anemia, myelodysplasia, myelofibrosis, thrombocytopenia due to other bone marrow diseases, drug induced thrombocytopenia, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenia, or thrombocytopenia following viral infections, neutropenia due to other bone marrow diseases, drug induced neutropenia, autoimmune neutropenia, idiopathic neutropenia, or neutropenia following viral infections, drug induced cytopenias, immune cytopenias, cytopenias following viral infections, or cytopenias.

[00019] In still other embodiments, the subject has aplastic anemia, myelodysplasia, myelofibrosis, anemia due to other disorder of bone marrow, drug induced anemia, immune mediated anemias, anemia of chronic disease, anemia following viral infections, or anemia of unknown cause.

[00020] In some embodiments, the tolerogenic dendritic cells are administered to the subject following chemotherapy administration, radiation therapy, or immunosuppressive therapy.

[00021] Still other embodiments relate to a method of treating an inflammatory or immune condition in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of TolDCs that are generated by administration of an Nrf2 activator or genetically modified such that the expression of Keapl in the TolDCs is inhibited, suppressed, and/or disrupted.

[00022] In some embodiments, the inflammatory or immune condition comprises at least one of achlorhydra autoimmune active chronic hepatitis, acute disseminated

encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison’s disease,

agammaglobulinemia, alopecia areata, Alzheimer’s disease, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-gbm/tbm nephritis, antiphospholipid syndrome, antisynthetase syndrome, aplastic anemia, arthritis, atopic allergy, atopic dermatitis, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenia purpura, autoimmune uveitis, balo disease/balo concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaff’s encephalitis, blau syndrome, bullous pemphigoid, Castleman's disease, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, coeliac disease, Cogan syndrome, cold agglutinin disease, cranial arteritis, crest syndrome, Crohns disease, Cushing's syndrome, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, Dressler's syndrome, discoid lupus erythematosus, eczema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressive, fibromyalgia, fibromyositis, fibrosing aveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-barre syndrome (gbs), Hashimoto’s encephalitis, Hashimoto's thyroiditis, henoch-schonlein purpura, hidradenitis suppurativa, Hughes syndrome, inflammatory bowel disease (IBD), idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, iga nephropathy, inflammatory demyelinating polyneuopathy, interstitial cystitis, irritable bowel syndrome (ibs), Kawasaki's disease, lichen planus, Lou Gehrig’s disease, lupoid hepatitis, lupus erythematosus, meniere's disease, microscopic polyangiitis, mixed connective tissue disease, morphea, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neuromyotonia, occular cicatricial pemphigoid, opsoclonus myoclonus syndrome, ord thyroiditis, Parkinson’s disease, pars planitis, pemphigus, pemphigus vulgaris, pernicious anaemia, polymyalgia rheumatic, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, raynaud phenomenon, relapsing polychondritis, Reiter’s syndrome, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome, spondyloarthropathy, sticky blood syndrome, still's disease, stiff person syndrome, sydenham chorea, sweet syndrome, takayasu’s arteritis, temporal arteritis, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson’s syndrome, Wiskott-Aldrich syndrome as well as hypersensitivity reactions of the skin, atherosclerosis, ischemia- reperfusion injury, myocardial infarction, and restenosis.

[00023] Still other embodiments relate to a method of generating TolDCs. The method can include isolating bone marrow from the subject. The isolated bone marrow is then cultured with GM-CSF and IL-4 to generate a population of immature dendritic cells. The method further includes activating Nrf2 signaling and/or suppressing NfkB signaling and/or disrupting Keapl expression in the immature dendritic cells to generate the population of tolerogenic dendritic cells.

[00024] In some embodiments, Keap 1 expression can be disrupted by deleting at least a portion of Keapl gene to inhibit expression or function of Keapl in immature dendritic cells. For example, Keapl expression in the dendritic cells can be disrupted by administering to the dendritic cells at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRIS PR) -associated endonuclease, and at least one guide RNA (gRNA) having a spacer sequence complementary to a target sequence in a Keap DNA.

[00025] In other embodiments, Nrf2 signaling can be activated and/or NfkB signaling can be suppressed by administering to the immature dendritic cells an amount of triterpenoid effective to generate the population of tolerogenic dendritic cells. In some embodiments, the triterpenoid is at least one of CDDO-IM, CDDO-2P-IM, or CDDO-3P-IM.

[00026] In still other embodiments, the triterpenoid can be administered to the immature dendritic cells in combination with GM-CSF and/or LPS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0001] Figs. l(A-B) illustrates image and plots showing BMDC generation and characterization by CD1 lc. (A) BMDCs were expanded from hematopoietic progenitors isolated from C57BL/6 mice. The observation of cluster formation through the microscope has demonstrated during the period of differentiation (all images- 50x) (B) BMDCs were harvested on day 7 and analyzed by flow cytometry for CD1 lc expression. Graphs depict the percentage of the expanded CD1 lc+ cell population.

[0002] Fig. 2 illustrates plots showing DC cell surface ligand expression is unaltered by CDDO-DFPA. Cells were pre-treated in the presence or absence of CDDO-DFPA (200 nM) for 1 hour prior to stimulation with LPS (100 ng/ml) for 24 hrs. Cell surface expression of CD80, CD86, MHC II, and PD-L1 was analyzed by flow cytometry.

[0003] Figs. 3(A-F) illustrate graphs showing CDDO-DFPA altered the genetic and protein phenotype of immunogenic DCs. BMDCs were pre-treated in the presence or absence of CDDO-DFPA (50-400 nM) for 1 hour prior to addition of LPS (100 ng/ml), and either harvested for RNA extraction (4 hrs.) or allowed to condition culture medium for 24 hrs. prior to collection for cytokine analyses. The levels of IFN-y (A), IL-12 (B), EDN-1 (C), TNFoc (D), IL-6 (E), and IL-23 (F) were measured by qRT-PCR and ELISA. The results are expressed as mean ± S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t-test.

[0004] Figs. 4(A-D) illustrate graphs and an immunoblot showing CDDO-DFPA induced TolDCs phenotype confirmed by gene and protein expression. BMDCs were pre treated in the presence or absence of CDDO-DFPA (10-400 nM) for 1 hour prior to addition of LPS (100 ng/ml), and cells were harvested for RNA extraction after 24 hrs. The levels of IL-4 (A), IL-10 (B), and TGF-b (C) were measured by qRT-PCR. (D) Cell protein lysate (12 hrs.) were collected for analyses and levels of HO-1, and b-actin expression were analyzed by Western blotting. The results are expressed as mean ± S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t-test.

[0005] Fig. 5 illustrates plots showing CDDO-DFPA exposed DCs suppress T cell proliferation. DCs were pre-treated with CDDO-DFPA (100-400 nM) for 1 hour only, then washed and co-cultured with CFSE stained T cells at a 1:10 ratio. Splenic T cells and DCs were isolated from C57BL/6 OTII transgenic mice and C57BL/6 mice, respectively. CDDO- DFPA pretreated DCs were co-cultured with CFSE stained T cells with (w/) or without (w/o) OVA addition during incubation. T cell proliferation was determined by flow cytometry at day 2. Graphs depict the percentage of dividing T cells relative to numbers T cell division. The data is a representation of 3 independent experiments. [0006] Figs. 6(A-F) illustrate plots and graphs showing the characterization of mitochondrial function of Nrf2^+/+ and Nrf2^_/ DCs. Nrf2^+/+ or Nrf2^_/ BMDCs were pretreated in the presence or absence of CDDO-DFPA (400 nM) for 1 hour before exposure to LPS (10 ng/ml) for 24 hours. (A) Schematic representation of real-time mitochondrial respiration. OCR analysis included 4 consecutive stages, starting from basal respiration and after the addition of oligomycin (mitochondrial complex V inhibition), FCCP (maximal respiration induction), and rotenone/antimycin A (ETC inhibition). (B) Representative kinetic study of mitochondrial OCR (pmol/min) in Nrf2^+/+ DCs (light blue), Nrf2^+/+ DCs+LPS (blue), and Nrf2^+/+ DCs+LPS+CDDO-DFPA (purple) with sequential addition of oligomycin, FCCP, and rotenone/antimycin A. (C) OCR quantification of basal respiration, ATP production, maximal respiration, and spare capacity of Nrf2^+/+ DCs (light blue), Nrf2^+/+ DCs+LPS (blue), and Nrf2^+/+ DCs+LPS+CDDO-DFPA (purple). (D) Representative kinetic study of mitochondrial OCR (pmol/min) in Nrf2 ^/_ DCs (red), Nrf2^_/ DCs+LPS (yellow), and Nrf2^_/

DCs+LPS+CDDO-DFPA (brown) with sequential addition of oligomycin, FCCP, and rotenone/antimycin A. (E) OCR quantification of basal respiration, ATP production, maximal respiration, and spare capacity of Nrf2 ^/_ DCs (red), Nrf2^_/ DCs+LPS (yellow), and Nrf2^_/ DCs+LPS+CDDO-DFPA (brown). (F) OCR quantification of basal respiration, ATP production, maximal respiration, and spare capacity of Nrf2+/+ (light blue) and Nrf2-/- DCs (red). The results are expressed as mean + S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t-test.

[0007] Figs. 7(A-F) illustrate plots and graphs showing the Characterization of the glycolytic function of Nrf2^+/+ and Nrf2^_/ DCs. Nrf2^+/+ or Nrf2^_/ BMDCs were pretreated in the presence or absence of CDDO-DFPA (400 nM) for 1 hour before exposure to LPS (10 ng/ml) for 24 hours. (A) Schematic representation of a real-time glycolysis. The ECAR assay consisted 4 stages, starting from basal (glucose-free) followed by the addition of glucose (glycolysis induction), oligomycin (maximal glycolysis induction), and 2-DG

(glycolysis inhibition). (B) Representative kinetic study of glycolytic ECAR (mpH/min) in Nrf2^+/+ DCs (light blue), Nrf2^+/+ DCs+LPS (blue), and Nrf2^+/+ DCs+LPS+CDDO-DFPA (purple) with sequential addition of glucose, oligomycin, and 2-DG. (C) ECAR quantification of basal, glycolysis, glycolytic capacity, and glycolytic reserve of Nrf2^+/+ DCs (light blue), Nrf2^+/+ DCs+LPS (blue), and Nrf2^+/+ DCs+LPS+CDDO-DFPA (purple). (D) Representative kinetic study of glycolytic ECAR (mpH/min) in Nrf2 ^/_ DCs (red), Nrf2^_/ DCs+LPS (yellow), and Nrf2^_/ DCs+LPS+CDDO-DFPA (brown) with sequential addition of glucose, oligomycin, and 2-DG. (C) ECAR quantification of basal, glycolysis, glycolytic capacity, and glycolytic reserve of Nrf2 ^/_ DCs (red), Nrf2^_/ DCs+LPS (yellow), and Nrf2^_/

DCs+LPS+CDDO-DFPA (brown). The results are expressed as mean + S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t-test.

[0008] Figs. 8(A-D) illustrate immunoblots and graphs showing Nrf2 activation in DCs reduced LPS-derived iNOS expression and NO production. Nrf2^+/+ or Nrf2 ^/_ BMDCs were pretreated in the presence or absence of CDDO-DFPA (100 and 200 nM for Western blotting and 50, 100, and 200 nM for qRT-PCR) for 1 hour before exposure to LPS (10 ng/ml) for 6 or 24 hours. (A) Total cellular lysates were analyzed for iNOS, HO-1, Nrf2, and b-actin expression by Western blotting. Experiments were repeated a minimum of three times. (B & C) Cells were harvested for RNA extraction. The levels of iNOS (B) and Hmox-1 (C) were measured by qRT-PCR. (D) Conditioned medium was collected for NO production by analysis of nitrite levels. The results are expressed as mean + S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t- test.

[0009] Figs. 9(A-K) illustrate plots and images showing TolDC therapy ameliorated the symptoms of AA mice. TolDCs were administered by i.v. on day 0, 3, and 5 following injection of LN cells to the recipient mice. (A) Survival curve for AA mice. Kaplan-Meier survival curve followed by the Mantel-Cox log-rank test within 30 days (n = 7-8 mice in each group). *P < 0.05. Representative data of complete blood counts, including WBC (B), Hb (C), HCT (D), and PLT (E) at indicated time points after injection of LN cells. All data are presented as the mean + S.E.M. *P < 0.05. Multiple t-tests with Holm-Sidak analysis. Mice irradiated only (IR only, green), LN cells injected mice (AA, blue), AA mice with TolDC administration (AA+TolDCs, red). (F) Sternums were collected on day 14 for H&E and IHC examination. Representative sections of Normal, IR only, AA, and AA+TolDCs mice stained with H&E to assess BM cellularity and hemorrhage. Infiltration of T cells was assessed by CD3 antibody. iNOS, Nrf2, and HO-1 expression were also analyzed by IHC. A pathologist blinded to subject identity scored sections taken from each animal for H&E (G), T cell (H) infiltration, iNOS (I), Nrf2 (J), HO-1 (K) expression. Scale bars = 50 pm. Quantification data were presented as the mean ± S.E.M. (n = 3 mice in Normal and IR only group n = 8 mice in AA and AA+TolDCs group). ***P < 0.001, Unpaired student t-test.

[00010] Figs. 10(A-N) illustrate plots showing TolDC therapy regulated the cell proliferation and differentiation in spleen and BM of AA mice. TolDCs were administered by i.v. on day 0, 3, and 5 after injection of LN cells to the recipient mice. Mice were euthanized on day 10 or day 14 for harvesting splenocytes and BM cells, respectively. The proliferation of CD4 and CD8 T cells in the spleen (A) and BM (D) were measured by flow cytometry. Cells were stimulated with PMA/ionomycin/Golgistop for 4 hours and subjected to flow cytometry to determine the differentiation of Thl7 and Treg subsets among CD4+ T cells based on their expression of IL-17 and CD25/Foxp3, respectively (B, C, and E). The population of HSCs was measured as lin Vc-Kit⁺ cells in BM (F). Quantification of data in spleen (G) and BM (H) was presented as the mean (n = 2-5 mice in each group). *P < 0.05, **P < 0.01, One-way ANOVA with the Bonferroni corrections.

[00011] Figs. 1 l(A-G) illustrate images and plots showing BM biopsies from AA patients exhibited the milieu for immunogenic DCs. (A) Biopsies from AA patients and healthy donors were collected for H&E and IHC examination. Representative sections of AA patient and healthy donor were stained with H&E to assess BM cellularity and hemorrhage. Infiltration of T cells and DCs was assessed by CD3 and CDl lc antibodies, respectively. iNOS, Nrf2, and HO-1 expression were also analyzed by IHC. A pathologist blinded to subject identity scored sections taken from each sample for H&E (B), T cell (C) and DC (D) infiltration, iNOS (E), Nrf2 (F), and HO-1 (G) expression. Scale bars = 50 pm. Quantification data were presented as the mean ± S.E.M. (n = 4 patients in each group). ***P < 0.001, unpaired student t-test.

[00012] Fig. 12 illustrates images showing the pro -inflammatory immune

microenvironment in BM of patients with SAA. The bone marrow (BM) of SAA patients (top row) exhibits high iNOS, low Nrf2 and low HO-1 relative to the BM of normal patients (bottom row).

[00013] Fig. 13 illustrates a schematic multifunctional Triterpenoid activators of Nrf2. Small molecules in the triterpenoid family activate Nrf2 via reaction with Keapl, protecting Nrf2 from degradation. They also regulate NFkB signaling through direct binding to TkB.

[00014] Fig. 14 illustrates a schematic showing the experimental design. CByB6Fl mice will be exposed to a sublethal dose (5Gy) of total body irradiation by a 137 cesium g source irradiator 6 hours before intravenous (i.v.) injection of 5 x 10⁶ LN cells isolated from

C57BL/6 mice. Treatment groups receive (i.v.) 5 x 10⁶ TolDCs on day 0, 3, 5, 9, 12, and 18. Mice in both control and treatment groups are bled from the tail tip at different time points to measure blood counts using a Hemavet 950 analyzer (Drew Scientific). In some

experiments, mice will be euthanized to collect splenocytes and BM cells for analyses at day 10 and 14, respectively. Sternums will be collected on day 14 for hematoxylin and eosin (H&E) and IHC examination in each experiment.

[00015] Figs. 15(A-D) illustrate a schematic and graph showing molecular and phenotypic characterization of Keapl-KO dendritic cells. (A) The immature BMDC were harvested at day7 and (2xl0e⁵ cells) were transfected with CRISPR Keapl RNP by Neon Electroporator with the condition of 1500V/30 ms/ 1 pulse. Next day, the transfected DCs were treated with 200 nM of CDDO-DFPA(DFPA), CDDO-IM(IM), or CDDO-3P-IM(3P) 1 hour prior to EPS (lOOng/ml) treatment. The treated cells were harvested for further molecular and phenotypic analyses including ICE, Flow cytometry, and ELISA assays. (B) the ICE analysis have shown that CRISPR Keapl.75 RNP induced 85% indel mutations (ICE score) and estimated to have 79% of Keapl-KO (KO score). (C) The phenotype of Keapl- KO DCs( CD1 lc, CD86, and MHCII) was characterized by flow cytometry and Flowjo analysis. The classical DC markers(% of parent) were not significantly changed by induction of Keapl-KO. (D)The secretion of pro -inflammatory cytokine (TNFa) and the anti inflammatory cytokine (IL-10) by Keapl-KO DC were compared to that of control DCs treated with CDDO and LPS as indicated.

[00016] Figs. 16(A-C) illustrate a schematic, graph, and table showing an optimized protocol for tolerogenic dendritic cell production. (A)The schematic diagram shows the optimized method for dendritic cell differentiation and production of CLM18.3 (B)The immature DCs were harvested on day 8 and treated again in Celloram’s induction cocktail for another day (C) The optimized protocol yielded iDC, mDC, and TolDC(CLM18.3) on day 9 as indicated. The cell number seeded at day 0 was set to 100%.

[00017] Figs. 17(A-D) illustrate graphs and plots showing purity, activity, and cytokines of CLM18.3 (A) CLM18.3 showed > 80% CDl lc+ and slight reduction of CD80, CD86, and MHCII. (B) CLM18.3 expressed low TNF-a, IL-12, but high TGF-b. (QCLM18.3 suppressed OVA-peptide specific T cell proliferation compared to control DC. As a proof- of-principle study, CRISPR-ko of Keapl also suppressed T cell proliferation (D) The Keapl- ko DC secrets low TNF-cr, IL-12, but high TGF- ? and IL-10.

[00018] Figs. 18(A-E) illustrate plots showing CLM-18.3 TolDCs exhibit unique transcriptome signatures. (A) Antigen Processing and Presentation Pathways and (B) Allograft Rejection Pathways. CLM18.3(18.3); mature DC (mDC) (C) MHC-I gene expression, (D) MHC-II gene expression, (E) the gene expression significantly related to tolerogenic dendritic cell function

[00019] Figs. 19(A-E) illustrate a schematic, graphs, and plots showing CLM18.3 significantly improved clinical scores and survival in mouse GvHD model (A) The experimental plan of GVHD model (B) CD4 and CD8 T cell subset analysis in each treatment group. (C) The level of TGF- ? in serum at day 14 and day 30 (D)The clinical score of each group measured by 5 clinical criteria (weight, posture, activity, Fur, and skin) (E)The Kaplan-Meier survival curve showed the survival benefit of CLM18.3 treated group.

[00020] Figs. 20(A-E) illustrate a schematic, graphs, and plots showing CLM18.3 significantly prolonged the survival in mouse aplastic anemia model (A) The experimental plan of aplastic anemia model (B)The Kaplan-Meier survival curve showed the survival benefit of CLM18.3 treated group in contrast to aplastic anemia group(red). (C) The level of TGF- ? and IL-10 in serum at day 17 (D) Blood counting showed the reconstitution of blood in each group (White blood cell, Hematocrit, platelet)(E) CD4 and CD8 T cell subset analysis in each treatment group(Left) and the preservation of Lin cKit⁺ hematopoietic stem cell population in bone marrow (right).

DETAILED DESCRIPTION

[00021] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

Commonly understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Edition, Springer- Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present application.

[00022] The term "dendritic cell" refers a special antigen-presenting cell presenting various antigen samples along with MHC Class I complex or MHC Class II complex to a T cell by absorbing an antigen in cells. The dendritic cells may be divided into the immature dendritic cells and mature dendritic cells according to the expression level of the surface phenotype or maturity. The expression profile of surface markers of the dendritic cells may be easy through a flow cytometry assay that is known in the art.

[00023] The term "immature dendritic cells," refers to dendritic cells, in which the cells are found at the initial mature state of the dendritic cells, CD 14 that is a surface phenotype of a mononuclear cell is not expressed, and CD40, CD80, and CD86 that are a co-stimulatory molecule are expressed in a low level.

[00024] The term, "mature dendritic cells," means the cells, in which the immature dendritic cells are matured, and then the mature dendritic cells are formed. The mature dendritic cells have ability capable of inducing an immune reaction by increasing the expressions of MHC class II, CD40, CD80, and CD86, releasing a pro-inflammatory cytokine, and then activating a naive T cell.

[00025] The term“Tolerogenic dendritic cells" or "TolDCs" refers to dendritic cells capable of suppressing immune responses or generating tolerogenic immune responses, such as polyclonal or antigen- specific regulatory T-cells and/or B-cells or suppressive T cell- mediated immune responses. Tolerogenic DCs can be characterized by specific tolerogenic immune response induction ex vivo and/or in vivo.

[00026] Embodiments described herein relate to tolerogenic dendritic cells (TolDCs), methods of generating a population of TolDCs, the use of TolDCs in increasing immune tolerance, and to the use of the TolDCs in treating inflammatory and immune disorders in a subject in need thereof.

[00027] It was found that TolDCs can be generated from dendritic cells through targeted activation of the nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2). Targeted activation of Nrf2 is a novel approach to metabolic reprogramming of dendritic cells (DCs), which acquire a stable, immune suppressive orTolerizing’ phenotype. Unlike mature DCs (mDCs), the dominant anti-inflammatory signature of TolDCs is characterized by reduced expression of cell surface stimulatory ligands, decreased secretion of immunosuppressive cytokines and a distinct cellular metabolic profile that regulates T cell polarization.

[00028] Under quiescent conditions, Nrf2 is anchored in the cytoplasm through binding to Kelch-like ECH-associated protein (Keapl), which, in turn, facilitates the ubiquitination and proteolysis of Nrf2. It was found that disruption of Nrf2 binding to Keapl in DCs, a key mechanism for the repressive effects of Keapl on Nrf2, can activate Nrf2 and confer a tolerogenic phenotype to DCs.

[00029] Nrf2 activation of the DCs can be increased and/or promoted in several ways including: direct activation of Nrf2 (e.g., by using small molecules); inhibition of genes that express Keapl (e.g., by blocking the expression or activity of the genes and/or proteins); activation of genes and/or proteins that inhibit one or more of, the activity and function of Keapl (e.g., by increasing the expression or activity of the genes and/or proteins);

introduction of genes and/or proteins that negatively regulate the binding of Nrf2 to Keapl (e.g., by using recombinant gene expression vectors, recombinant viral vectors or

recombinant polypeptides); gene replacement with, for instance, a hypomorphic mutant of Keapl (e.g., by homologous recombination, overexpression using recombinant gene expression or viral vectors, or mutagenesis), or genetic or pharmacologic manipulation of Tgf-b, smad7, and other targets of nfkB signaling.

[00030] In some embodiments, TolDCs described herein can include DCs that are administered an Nrf2 activator.

[00031] As used herein, the term "Nrf2 Activator" means an agent that after

administration results in a stimulated and/or increased nuclear translocation of Nrf2 protein and causes the subsequent increases in expression of one or more ARE-regulated genes by acting directly on Nrf2, Keapl, and or the Nrf2-Keapl complex.

[00032] Nrf2 Activators may comprise a Michael addition acceptor, one or more fumaric acid esters, i.e., fumaric acid mono- and/or diesters which may be selected from the group of monoalkyl hydrogen fumarate and dialkyl fumarate, such as monomethyl hydrogen fumarate, dimethyl fumarate, monoethyl hydrogen fumarate, and diethyl fumarate, ethacrynic acid, bardoxolone methyl (methyl 2-cyano-3,12-dioxooleana-l,9(l l)dien-28-oate), isothiocyanate such as sulforaphane, l,2-dithiole-3-thione such as oltipraz, 3,5-di-tert-butyl-4- hydroxytoluene, 3-hydroxycoumarin, 2-cyano-3,12-dioxoolean-l,9-dien-28-oic acid and its methyl (CDDO-Me, bardoxolone methyl) and imidazolide (CDDO-Im) derivatives, pyridyl analogues thereof, such as l-[2-Cyano-3,12-dioxooleana-l,9(l l)-dien-28-oyl]-4(-pyridin-2- yl)- 1 H-imidazole (CDDO-2P-Im), 1 -[2-Cyano-3 , 12-dioxooleana- 1,9(11 )-dien-28-oyl] -4(- pyridin-3-yl)-lH-imidazole (CDDO-3P-Im), triterpenoid 2-cyano-3,12-dioxooleana-l,9- dien-28-oic acid-difluoro-propyl-amide, (CDDO-DFPA, RTA-408) or a pharmacologically active derivative or analog of the aforementioned agents.

[00033] Nrf2 activators may be classified based on their chemical structures: Diphenols, Michael reaction acceptors, isothiocyanates, thiocarbamates, trivalent arsenicals, 1,2-dithiole- 3-thiones, hydroperoxides, vicinal dimercaptans, heavy metals, polyenes, and triterpenoids.

In general, Nrf2 Activators are chemically reactive in that they may be electrophiles, substrates for glutathione transferases, and/or can modify sulfhydryl groups by alkylation, oxidation, or reduction.

[00034] In another embodiment, Nrf2 Activators are compounds that bond covalently to Keapl protein, such as by a sulfur atom of an amino acid residue of Keapl.

[00035] In another embodiment, the Nrf 2 Activators are bardoxolone methyl and dialkyl fumarate such as dimethyl fumarate and diethyl fumarate.

[00036] In another embodiment, Nrf2 activators are selected from: Chalcone derivatives such as 2-trifluoromethyl-2'-methoxychalcone, auranofin, ebselen, 1,2-naphthoquinone, cynnamic aldehyde, caffeic acid and its esters, curcumin, reservatrol, artesunate, tert- butylhydroquinone, and -quinone, (tBHQ, tBQ), vitamins Kl, K2 and K3, menadione, fumaric acid esters, i.e. fumaric acid mono- and/or diester which may be selected from the group of monoalkyl hydrogen fumarate and dialkyl fumarate, such as monomethyl hydrogen fumarate, dimethyl fumarate (DMF), monoethyl hydrogen fumarate, and diethyl fumarate, 2- cyclopentenones, ethacrynic acid and its alkyl esters, bardoxolone methyl (methyl 2-cyano- 3,12-dioxooleana-l,9(l l)dien-28-oate) (CDDO-Me, RTA 402), ethyl 2-cyano-3, 12- dioxooleana- 1,9(1 l)dien-28-oate, 2-cyano-3, 12-dioxooleana- 1,9(1 l)dien-28-oic acid

(CDDO), l[2-Cyano-3,12-dioxooleana-l,9(l l)-dien-28-oyl]imidazole (CDDO-Im), (2- cyano-N-methyl-3,12-dioxooleana-l,9(l l)-dien-28 amide (CDDO-methyl amide, CDDO- MA), l-[2-Cyano-3,12-dioxooleana-l,9(l l)-dien-28-oyl]-4(-pyridin-2-yl)-lH-imidazole (CDDO-2P-Im) , 1 - [2-Cy ano-3 , 12-dioxooleana- 1,9(11 )-dien-28 -oyl] -4(-pyridin-3 -yl)- 1 Id- imidazole (CDDO-3P-Im), isothiocyanate such as sulforaphane, l,2-dithiole-3-thione such as oltipraz, 3,5-di-tert-butyl-4-hydroxytoluene, 3-hydroxycoumarin, 4-hydroxynonenal, 4- oxononenal, malondialdehyde, (E)-2-hexenal, capsaicin, allicin, allylisothiocyanate, 6- methylthiohexyl isothiocyanate, 7-methylthioheptyl isothiocyanate, sulforaphane, 8- methylthiooctyl isothiocyanate, corticosteroids, such as dexamethasone, 8-iso prostaglandin A2, alkyl pyruvate, such as methyl and ethyl pyruvate, diethyl or dimethyl oxaloproprionate, 2-acetamidoacrylate, methyl or ethyl-2-acetamidoacrylate, hypoestoxide, parthenolide, eriodictyol, 4-hydroxy-2-nonenal, 4-oxo-2nonenal, geranial, zemmbone, aurone,

isoliquiritigenin, xanthohumol, [10]-Shogaol, eugenol, l'-acetoxychavicol acetate, allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, 4-(methylthio)-3-butenyl isothiocyanate and 6-methylsulfinylhexyl isothiocyanate, ferulic acid and its esters, such as ferulic acid ethyl ester, and ferulic acid methyl ester, sofalcone, 4-methyl daphnetin, imperatorin, auraptene, poncimarin, bis[2-hydroxybenzylidene]acetones, alicylcurcuminoid, 4-bromo flavone, b-naphthoflavone, sappanone A, aurones and its corresponding indole derivatives such as benzylidene-indolin-2-ones, perillaldehyde, quercetin, fisetin, koparin, genistein, tanshinone HA, BHA, BHT, PMX-290, AL-1, avicin D, gedunin, fisetin, andrographolide, and tricyclic bis(cyano enone) TBE-31 [(+/-)-(4bS,8aR,10aS)-10a-ethynyl- 4-b,8,8-trimethyl-3,7-dioxo-3,4-b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile], as well as other triterpenoids disclosed in U.S. Pat. Nos. 6 6,326,507, 6,552,075, 6,974,801,

7,288,568, 7,863,327, 7,915,402, 7,943,778, 8,034,955, 8,071,632, 8,124,656, 8,124,799, 8,129,429, 10,501,420, US 2009/0060873, US 2009/0048204, WO 2008/136838, WO

2009/023232, and WO 2009/146216 all of which are incorporated by reference in their entirety.

[00037] In another embodiment, Nrf2 activators are selected from: carnosic acid, 2-naphthoquinone, cynnamic aldehyde, caffeic acid and its esters, curcumin, reservatrol, artesunate, tert-butylhydroquinone, vitamins Kl, K2 and K3, fumaric acid esters, i. e. , fumaric acid mono- and/or diester which is preferably selected from the group of monoalkyl hydrogen fumarate and dialkyl fumarate, such as monomethyl hydrogen fumarate, dimethyl fumarate, monoethyl hydrogen fumarate, and diethyl fumarate, isothiocyanate such as sulforaphane, l,2-dithiole-3-thione such as oltipraz, 3,5-di-tert-butyl-4-hydroxytoluene, 3- hydroxycoumarin, 4-hydroxynonenal, 4-oxononenal, malondialdehyde, (E)-2-hexenal, capsaicin, allicin, allylisothiocyanate, 6-methylthiohexyl isothiocyanate, 7-methylthioheptyl isothiocyanate, sulforaphane, 8-methylthiooctyl isothiocyanate, 8-iso prostaglandin A2, alkyl pyruvate, such as methyl and ethyl pyruvate, diethyl or dimethyl oxaloproprionate, 2- acetamidoacrylate, methyl or ethyl-2-acetamidoacrylate, hypoestoxide, parthenolide, eriodictyol, 4-Hydroxy-2-nonenal, 4-oxo-2nonenal, geranial, zerumbone, aurone, isoliquiritigenin, xanthohumol, [10]-Shogaol, eugenol, l'-acetoxychavicol acetate, allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, 4-(Methylthio)-3-butenyl isothiocyanate and 6-methylsulfinylhexyl isothiocyanate and the respective quinone or hydroquinone forms of the aforementioned quinone and hydroquinone derivatives.

[00038] In another embodiment, Nrf2 Activators may be Michael reaction acceptors such as dimethylfumarate, monomethyl hydrogen fumarate isothiocyanates and 1,2-dithiole- 3-thiones. In another embodiment, Nrf2 Activators are selected from monomethyl hydrogen fumarate, dimethyl fumarate, oltipraz, 1,2-naphthoquinone, tert-butylhydroquinone, methyl or ethyl pyruvate, 3,5-di-tert-butyl-4-hydroxytoluene, diethyl and dimethyl oxaloproprionate, hypoestoxide, parthenolide, eriodictyol, 4-Hydroxy-2-nonenal, 4-oxo-2nonenal, geranial, zerumbone, aurone, isoliquiritigenin, xanthohumol, [10]-Shogaol, eugenol, l'- acetoxychavicol acetate, allyl isothiocyanate, benzyl isothiocyanate, phenethyl

isothiocyanate, 4-(Methylthio)-3-butenyl isothiocyanate and 6-Methylsulfinylhexyl isothiocyanate.

[00039] In other embodiments, a TolDCs described herein can include DCs that are genetically modified to inhibit, suppress, and/or disrupt expression of Keapl. The inhibition, suppression, and/or disruption of Keapl can include a deletion of at least a portion of Keapl gene to inhibit expression or function of Keapl including Keapl binding to Nrf2.

[00040] In some embodiments, the Keapl expression in the DCs can be disrupted by gene editing. Gene editing means for inhibiting, disrupting, and/or suppressing Keapl expression can include RNA-guided CRISPR technology. In a CRISPR system, CRISPR clusters encode spacers, which are sequences complementary to target sequences

("protospacers") in a viral nucleic acid, or in another nucleic acid to be targeted. CRISPR clusters are transcribed and processed into mature CRISPR RNAs (crRNAs). CRISPR clusters also encode CRISPR associated (Cas) proteins, which include DNA endonucleases. The crRNA binds to target DNA sequence, whereupon the Cas endonuclease cleaves the target DNA at or adjacent to the target sequence.

[00041] One useful CRISPR system includes the CRISPR associated endonuclease Cas9. Cas9 is guided by a mature crRNA that contains about 20-30 base pairs (bp) of spacer and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease Ill-aided processing of pre-crRNA. The crRNA TracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the target sequence on the target DNA. Cas9 recognizes a trinucleotide (NGG) photospacer adjacent motif (PAM) to decide the cut site (the 3^rd nucleotide from PAM). The crRNA and tracrRNA can be expressed separately or engineered into an artificial chimeric small guide RNA (sgRNA) via a synthetic stem loop (AGAAAU) to mimic the natural crRNA/tracrRNA duplex. Such sgRNAs, can be synthesized or in vitro transcribed for direct RNA transfection, or they can be expressed in situ, e.g., from U6 or Hl-promoted RNA expression vectors. The term "guide RNA" (gRNA) will be used to denote either a crRNA TracrRNA duplex or an sgRNA. It will be understood the term "gRNA complementary to" a target sequence indicates a gRNA whose spacer sequence is complementary to the target sequence.

[00042] Other CRISPR systems that can be used include CRISPR/Cpfl, which is a DNA-editing technology analogous to the CRISPR/Cas9 system, characterized in 2015 by Feng Zhang's group from the Broad Institute and MIT. Cpfl is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpfl genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpfl is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. Cpfl is further described below.

[00043] In one embodiment, the CRISPR-associated endonuclease is a Cas9 nuclease. The Cas9 nuclease can be a sequence from Staphylococcus aureus. The Cas9 nuclease can also have a nucleotide sequence identical to the wild type Streptococcus pyogenes sequence. In some embodiments, the CRISPR-associated endonuclease can be a sequence from other species, for example, other Streptococcus species, such as Thermophiles; Psuedomonas aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microogranisms. Alternatively, the wild type Streptococcus pyogenes Cas9 sequence can be modified. For example, the nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, i.e., "humanized." A humanized Cas9 nuclease sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1 GL669193757; KM099232.1 GL669193761; or KM099233.1 GL669193765. Alternatively, the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as PX330 or PX260 from Addgene (Cambridge, Mass.). In some embodiments, the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GL669193757; KM099232.1 GL669193761; or KM099233.1 GL669193765 or Cas9 amino acid sequence of PX330 or PX260 (Addgene, Cambridge, Mass.).

[00044] The Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas9 by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations).

One or more of the substitution mutations can be a substitution (e.g., a conservative amino acid substitution). For example, a biologically active variant of a Cas9 polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a wild type Cas9 polypeptide. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

[00045] The amino acid residues in the Cas9 amino acid sequence can be non-naturally occurring amino acid residues. Naturally occurring amino acid residues include those naturally encoded by the genetic code as well as non-standard amino acids (e.g., amino acids having the D-configuration instead of the L-configuration). The present peptides can also include amino acid residues that are modified versions of standard residues (e.g., pyrrolysine can be used in place of lysine and selenocysteine can be used in place of cysteine). Non- naturally occurring amino acid residues are those that have not been found in nature, but that conform to the basic formula of an amino acid and can be incorporated into a peptide. These include D-alloisoleucine(2R,3S)-2amino-3-methylpentanoic acid and L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid. For other examples, one can consult textbooks or the worldwide web (a site is currently maintained by the California Institute of Technology and displays structures of non-natural amino acids that have been successfully incorporated into functional proteins).

[00046] The Cas9 nuclease sequence can be a mutated sequence. For example, the Cas9 nuclease can be mutated in the conserved HNH and RuvC domains, which are involved in strand specific cleavage. For example, an aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single- stranded breaks, and the subsequent preferential repair through HDR22 can potentially decrease the frequency of unwanted InDel mutations from off-target double- stranded breaks.

[00047] In addition to the wild type and variant Cas9 endonucleases previously described, the methods described herein can also encompass CRISPR systems including "enhanced-specificity" S. pyogenes Cas9 variants (eSpCas9), which dramatically reduce off- target cleavage. These variants are engineered with alanine substitutions to neutralize positively charged sites in a groove that interacts with the non-target strand of DNA. This modification reduces interaction of Cas9 with the non-target strand, thereby encouraging re hybridization between target and non-target strands. The effect of this modification is a requirement for more stringent Watson-Crick pairing between the gRNA and the target DNA strand, which limits off-target cleavage.

[00048] Especially preferred are three variants found to have the best cleavage efficiency and fewest off-target effects: SpCas9(K855a), SpCas9(K810A/K1003A/rl060A) (a.k.a. eSpCas9 1.0), and SpCas9(K848A/K1003A/R1060A) (a.k.a. eSPCas9 1.1). Techniques for cloning and inducing cellular expression of these enhanced-specificity variants are well known. It will be appreciated that other Cas9 variants are known and the method described herein are not limited to the Cas9 variants described herein.

[00049] In some embodiments, gene editing compositions can include a CRISPR- associated endonuclease polypeptide encoded by any of the nucleic acid sequences described above. Polypeptides can be generated by a variety of methods including, for example, recombinant techniques or chemical synthesis. Once generated, polypeptides can be isolated and purified to any desired extent by means well known in the art. For example, one can use lyophilization following, for example, reversed phase (preferably) or normal phase HPLC, or size exclusion or partition chromatography on polysaccharide gel media such as Sephadex G- 25. The composition of the final polypeptide may be confirmed by amino acid analysis after degradation of the peptide by standard means, by amino acid sequencing, or by FAB -MS techniques.

[00050] In some embodiments, an engineered CRISPR system includes Cas9 and one or more gRNAs complementary to a Keapl sequence. [00051] The inhibition, disruption, and/or suppression of Keapl in the DCs and TolDCs can also be performed using siRNA, miRNAs (micro-RNAs), shRNAs (short hairpin RNAs), or RNAis (RNA interference) that target critical RNAs (mRNA) that translate (non-coding or coding) proteins involved with the formation or expresssion of Keapl. The siRNA, miRNAs, shRNAs, or RNAi can be included in the expression vectors described herein along with the gene editing compositions. These RNA interference approaches are there to suppress the expression of Keapl.

[00052] shRNAs or siRNAs can be used to produce short double stranded RNA molecules which are processed by Dicer and single stranded RNA base-pairs with a target mRNA. Argonaute proteins then assist with mRNA degradation or translation inhibition.

This results in post transcriptional down-regulation of gene expression but does not change the genetic code.

[00053] shRNA is double stranded RNA created from a DNA construct encoding a sequence of single stranded RNA and its complement that are separated by a stuffer fragment that allows the RNA molecule to fold back on itself to create a hairpin loop. shRNA can come in two different designs of a simple stem-loop and a microRNA adapted shRNA. A simple stem-loop shRNA has a 50-70 nucleotide transcript that forms a stem-loop structure consisting of a 19 to 29 bp region of double stranded RNA (the stem) bridged by a region of predominantly single-stranded RNA (the loop) and a dinucleotide 3' overhang. A microRNA adapted shRNA is greater than 250 nucleotides and more closely resembles native pri- microRNA molecules and consists of a shRNA stem structure which may include

microRNA-like mismatches, bridged by a loop and flanked by 5' and 3' endogenous microRNA sequences.

[00054] Use of shRNA in RNAi instead of siRNA can be preferred as it has a low rate of degradation and turnover. siRNA can have variable transfection efficiencies that limits siRNA-mediated RNAi to only those cells capable of transfection. After the vector has integrated into the host genome, shRNA is transcribed in the nucleus by polymerase II or polymerase III. Also, shRNA can be delivered into mammalian cells through infection with viral vectors unlike siRNA.

[00055] In some embodiments, an effective amount of Nrf2 actiator and/or a gene composition directed against Keapl can be administered to DCs obtained from a subject being treated, i.e., autologous DCs and/or from another subject, i.e., allogenic DCs. The DCs can be isolated from a subject ( e.g ., bone marrow) or generated from precursor DCs, in bone marrow or peripheral blood. Techniques known to one skilled in the art may be used to obtain/generate DCs from bone marrow and/or peripheral blood mononuclear cells. Cells isolated from the bone marrow or blood, including hematopoietic progenitor cells and monocytes, of a patient may be cultured in the presence of factors, such as the combination of GM-CSF and IL-4, IL-13, IL-15 and IFN-a, or Flt3L, to differentiate into immature DCs after a period of, e.g., 4 to 5 days. In some embodiments, isolated bone marrow cells can be cultured in the presence of GM-CSF and IL-4 to generate a population of immature DCs.

[00056] Techniques known to one skilled in the art can be used to assess/confirm the presence of immature DCs. For example, the presence of dendritic cells can be

assessed/confirmed by detecting the expression of DC surface markers using techniques, such as FACS.

[00057] An effective amount of a composition including an agent that activates Nrf2 (Nrf2 activator) and/or isolated nucleic acid encoding a CRISPR-associated endonuclease with at least one isolated nucleic acid encoding at least gRNA including a spacer sequence complementary to a target sequence in a Keapl DNA can be administered to the immature DCs. The Nrf2 activator can include an amount of a triterpenoid effective to generate the population of tolerogenic dendritic cells. In some embodiments, the triterpenoid is at least one of CDDO-IM, CDDO-2P-IM, or CDDO-3P-IM.

[00058] In still other embodiments, at least one or more of lipopolysaccharides (LPS), rapamycin, corticosteroids, IL-10, vitamin D3, dexamethasone, BAY 11-7085, and, optionally, GM-CSF, can be administered in combination with the triterpenoid to induce DC maturation along with DC tolerance. For example, LPS and GM-CSF can be administered in combination with the triterpenoid to induce DC maturation along with DC tolerance.

[00059] The CRISPR-associated endonuclease and the at least one gRNA can be expressed in the DC of the patient which can include, but not limited to human KEAP1 gRNA for chrl9: 10500014 (+); chrl9:10499916(-) ; chrl9:10499891(-) ; chrl9: 10499865 (+) ; chrl9:10499821(-). The CRISPR-associated endonuclease can be any of those gene editors described above. The siRNA, miRNAs, shRNAs, or RNAi can also be included in the composition. The target sequence in the Keapl genome can then be cleaved disrupting the Keapl genome. Disrupting the Keapl can suppress Keapl expression, and promote Nrf2 activation generating the TolDCs described herein. [00060] In some embodiments, the TolDCs generated by administration of an agent that activates Nrf2 and/or by disruption of Keapl expression or binding to Nrf2 of the DCs can have a phenotype characterized by the production of multiple immune suppressive cytokines, including IL-4, IL-10 and TGF-b, high levels of HemeOxygenase-1 (HOI) and low levels of inducible nitric oxide synthase (iNOS) (/._<?., HO-l^Hl,iNOS^low expression) with decreased NO production, promoting expansion of regulatory (suppressor) T cells, suppression of T cell activation and production of TNFa, IFN-g, and IL-12 in human mixed lymphocyte reaction (MLR) assays, exhibition of a shift from glycolytic metabolism to oxidative phosphorylation (OXPHOS) during ex vivo TolDC expansion, and/or activation of Nrf2 target gene expression.

[00061] In some embodiments an agent that activates Nrf2 can be used in combination with with gene editing to disrupt Keapl expression or binding to Nrf2 of the DCs. Nrf2 activators and Keapl expression inhibitors may have complementary mechanisms of action. Administration of each agent alone may result in generation of TolDCs, but, because each agent may cause this generation of TolDCs through separate mechanisms, one agent may increase the baseline sensitivity of the system to the other agent. Thus, agents with complimentary mechanisms of action may act such that the therapeutically effective dose of either agent or both may be reduced relative to mono-therapy doses. Further, the combined therapeutically effective dose of both agents may be less than an additive substitution of one agent for the other. Put another way, the therapeutic effect when the Keapl inhibitor and the Nrf2 Activator are used together may be more than additive, i.e., greater than the sum of the effects that result from using each agent alone.

[00062] In an embodiment, the combined use of a Keapl expression inhibitor and an Nrf2 activator may eliminate, reduced incidence, or reduce severity of adverse effect(s) associated with use of the the Nrf2 activator as a mono-therapy. In another embodiment, the combined use of Keapl expression inhibitor and an Nrf2 Activator may reduce the dose of one or both of the agents employed in the combination treatment, and, the side effect(s) that may be observed in mono-therapy with the agents may be avoided or reduced. For example, dimethyl fumarate may potentially cause reduction in white cell count, flushing, redness, itching, skin rash, nausea, vomiting, diarrhea, stomach or abdominal pain, indigestion, and/or dyspepsia when administered in therapeutically effective amounts. [00063] The TolDCs described herein can be used in methods of treating various immune conditions and disorders. For example, compositions comprising TolDCs can be used in conjunction with tissue or organ transplantation for improving graft tolerance, prolonging survival of a transplanted tissue or organ, and treating graft- versus-host disease.

[00064] In addition, TolDCs described herein can be used for decreasing inflammation such as caused by an autoimmune disease, allergic response, neurodegenerative disease, a cardiovascular disease, damaged tissue, or a wound. Inflammatory conditions and autoimmune diseases that may be treated with TolDCs by the methods described herein can include, but are not limited to multiple sclerosis (MS), rheumatoid arthritis (RA), post- traumatic arthritis, reactive arthritis, psoriasis, pemphigus vulgaris, Sjogren's disease, autoimmune thyroid disease (AITD), Hashimoto's thyroiditis, myasthenia gravis, diabetes mellitus type 1, stomatitis, lupus erythematosus, acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic dermatitis, autoimmune aplastic anemia, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hemolytic anemia, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Balo disease/Balo concentric sclerosis, Behcet's disease, Berger's disease, Bickerstaffs

encephalitis, Blau syndrome, Bullous pemphigoid, Castleman's disease, celiac disease,

Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's Syndrome, cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diffuse cutaneous systemic sclerosis, Dressler's syndrome, eczema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressiva, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy, Henoch-Schonlein purpura, gestational pemphigoid, hidradenitis suppurativa, Hughes-Stovin syndrome,

hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, IgA nephropathy, inclusion body myositis, chronic inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), lupoid hepatitis, Majeed syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease, microscopic colitis, myositis, narcolepsy,

neuromyelitis optica, neuromyotonia, occular cicatricial pemphigoid, opsoclonus myoclonus syndrome, Ord's thyroiditis, palindromic rheumatism, PANDAS, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonage-Turner syndrome, Pars planitis, pemphigus vulgaris, pernicious anaemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, relapsing polychondritis, Reiter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, sarcoidosis, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, serum sickness, Sjogren's syndrome, spondyloarthropathy, Still's disease, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, thrombocytopenia, Tolosa- Hunt syndrome, transverse myelitis, undifferentiated connective tissue disease,

undifferentiated spondyloarthropathy, urticarial vasculitis, vasculitis, vitiligo, Wegener's granulomatosis, autoimmune cardiomyopathy, ischemic heart disease, atherosclerosis, cancer, fibrosis, inflammatory bowel disease, inflammatory myopathy, giant cell arteritis (GCA), asthma, allergy, Parkinson's disease, schizophrenia, and Alzheimer's disease.

[00065] In other embodiments, the TolDCs described herein can be administered to a subject to mitigate bone marrow graft rejection, to enhance bone marrow graft engraftment, to enhance engraftment of a hematopoietic stem cell graft, or an umbilical cord blood stem cell graft, to enhance engraftment of a hematopoietic stem cell graft, or an umbilical cord stem cell graft, and/or to decrease the number of units of umbilical cord blood required for transplantation into the subject. The administration can be, for example, following treatment of the subject or the marrow of the subject with radiation therapy, chemotherapy, or immunosuppressive therapy.

[00066] In other embodiments, the TolDCs described herein can be administered to a recipient of a bone marrow transplant, of a hematopoietic stem cell transplant, or of an umbilical cord blood stem cell transplant, in order to decrease the administration of other treatments or growth factors.

[00067] In still other embodiments , the TolDCs described herein can be administered to a subject to enhance recovery following bone marrow transplantation, following umbilical cord blood transplantation, following transplantation with hematopoietic stem cells, following conventional chemotherapy, following radiation treatment, and in individuals with anemias from diseases that include but are not limited to aplastic anemia, myelodysplasia, myelofibrosis, anemia from other bone marrow diseases, drug induced anemia, immune mediated anemias, anemia of chronic disease, idiopathic anemia, and following infections with viruses that include, but are not limited to, HIV, CMV, and parvovirus.

[00068] In other embodiments, the subject has aplastic anemia, myelodysplasia, myelofibrosis, thrombocytopenia due to other bone marrow diseases, drug induced thrombocytopenia, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenia, or thrombocytopenia following viral infections, neutropenia due to other bone marrow diseases, drug induced neutropenia, autoimmune neutropenia, idiopathic neutropenia, or neutropenia following viral infections, drug induced cytopenias, immune cytopenias, cytopenias following viral infections, or cytopenias.

[00069] In still other embodiments, the subject has aplastic anemia, myelodysplasia, myelofibrosis, anemia due to other disorder of bone marrow, drug induced anemia, immune mediated anemias, anemia of chronic disease, anemia following viral infections, or anemia of unknown cause.

[00070] In some embodiments, the TolDCs described herein, a composition(s) comprising such stable TolDCs, or combination therapies are administered to a subject suffering from or diagnosed with an autoimmune disease, graft rejection or graft-versus-host disease. In other embodiments, TolDCs described herein, a composition(s) comprising such stable TolDCs, or combination therapies are administered to a subject predisposed or susceptible to developing an autoimmune disease, graft rejection or graft-versus-host disease. [00071] In some embodiments, TolDCs described herein, a composition(s) comprising such TolDCs, or combination therapies are administered to a mammal. In certain

embodiments, TolDCs described herein, a composition(s) comprising such TolDCs, or combination therapies are administered to a mammal which is 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 10 to 15 years old, 15 to 20 years old, 20 to 25 years old, 25 to 30 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old.

[00072] The TolDCs described herein or a composition(s) comprising such stable TolDCs can be administered via any route known in the art. TolDCs described herein or a composition(s) comprising such TolDCs can be administered by, for example, infusion or bolus injection, and may be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known and can be used to deliver TolDCs described herein or a composition(s) comprising such TolDCs.

[00073] Methods of administration include but, are not limited to, parenteral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or intracerebral. In a specific embodiment, TolDCs described herein or a composition(s) comprising such TolDCs are/is intravenously, intradermally or subcutaneously administered to the patient. In another specific embodiment, TolDCs described herein or a composition(s) comprising such TolDCs are/is administered to the patient by direct intranodal delivery. The mode of administration is left to the discretion of the practitioner.

[00074] In specific embodiments, it may be desirable to administer TolDCs described herein or a composition(s) comprising such TolDCs locally. In specific embodiments, TolDCs described herein or a composition(s) comprising such TolDCs are/is administrated at the site of the autoimmune disease, graft rejection or graft- versus-host disease by local infusion. For example, in the case of rheumatoid arthritis, TolDCs described herein or a composition(s) comprising such stable TolDCs can be administrated directly intra-articularly.

[00075] The amount TolDCs described herein, or the amount of a composition comprising TolDCs, that will be effective in the treatment of an autoimmune disease, graft rejection or graft-versus-host disease can be determined by standard clinical techniques. In vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend, e.g., on the route of administration, the type of symptoms, and the seriousness of the symptoms, and should be decided according to the judgment of the practitioner and each patient's or subject's circumstances.

[00076] Doses of TolDCs for administration to a subject by any route of administration can be at least 100, 200, 300, 400, 500, 700, 1,000, 5,000, 10,000, 25,000, 50,000, or 100,000 cells. In specific embodiments, the number of TolDCs is at least 100, 200, 300, 400, 500 cells. In other embodiments, the number of TolDCs is at least 300, 400, 500, 700, 1,000 cells. In yet other specific embodiments, the number of TolDCs is at least 700, 1,000, 5,000, 10,000 cells. In some embodiments, the number of TolDCs at least 5,000, 10,000, 25,000, 50,000, or 100,000 cells. In yet another embodiment, the number of TolDCs is at least 50,000, or 100,000 cells. In other embodiments, the number of TolDCs is at least 1 xlO⁶, 5 x 10⁶, 1 x 10⁷, 5 x 10⁷, 1 x 10⁸, 5 x 10⁸ or more cells. In specific embodiments, the number of stable semi-mature tolDCs is between 1 x 10² to 1 x 10⁴, 5 x 10⁴ to 5 x 10⁶, 1 x 10⁵ to 1 x 10⁷, 1 x 10⁵ to 5 x 10⁸, 1 x 10⁶ to 1 x 10⁸, or 1 x 10⁶ to 1 x 10⁷, or 1 x 10⁴ to 1 x 10⁵ cells.

[00077] In certain embodiments, a subject is administered TolDCs described herein or a composition thereof in an amount effective to inhibit or reduce symptoms associated with the autoimmune disease, graft rejection or graft-versus-host disease by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In certain embodiments to treat, a subject is administered TolDCs described herein or a composition thereof in an amount effective to inhibit or reduce symptoms associated with the autoimmune disease, graft rejection or graft-versus-host disease by at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, or 2- to 5-fold, 2- to 10-fold, 5- to 10-fold, or 5- to 20-fold relative to a negative control as determined using an assay described herein or other known to one of skill in the art.

[00078] In certain embodiments to, a subject is administered TolDCs described herein or a composition thereof in an amount effective to decrease an autoimmune response or graft rejection by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In some embodiments, a subject is administered TolDCs described herein or a composition thereof in an amount effective to decrease an autoimmune response or graft rejection by at least 1.5-fold, 2-fold, 2.5-fold, 3- fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, or 2 to 5-fold, 2 to 10-fold, 5 to 10-fold, or 5 to 20-fold relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[00079] In certain embodiments, a dose of TolDCs described herein or a composition thereof is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks or once a month, or less. In other embodiments, two, three or four doses of TolDCs described herein or composition thereof is administered to a subject every day, every couple of days, every third day, once a week or once every two weeks. In some embodiments, a dose(s) of TolDCs described herein or a composition thereof is administered for 2 days, 3 days, 5 days, 7 days,

14 days, 21 days, 28 days or 31 days. In certain embodiments, a dose of TolDCs described herein or a composition thereof is administered for 0.5 month, 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.

[00080] The dosages of prophylactic or therapeutic agents which have been or are currently used for the treatment of autoimmune diseases, graft rejection, or graft-versus-host disease can be determined using references available to a clinician such as, e.g., the

Physicians' Desk Reference (68th ed. 2014).

[00081] The above-described administration schedules are provided for illustrative purposes only and should not be considered limiting.

Example 1

[00082] In this example we provide a detailed protocol with step by step method to isolate immature dendritic cells (iDCs) from hematopoietic progenitors of mice and analyze the efficacy of any interest of agents in converting these iDCs into TolDCs by their functional and phenotypic characterization in vitro and in vivo. This is an elaborate method to characterize the TolDCs by their surface ligands, cytokine profile, and immunosuppressive functions in vitro. PROTOCOL

Preparation of bone marrow-derived dendritic cells (BMDCs)

[00083] 1. Use appropriate method to euthanize C57BL/6 mice. Isolate and clean tibia-fibula and femur bones with 70% ethanol.

[00084] 2. Trim both ends of tibias and femurs. Use 3 ml PBS in 3 ml syringe with a 23G needle to flush the contents of marrow from one end of bones to a conical tube containing 9 ml PBS. Repeat this step 3 times for each end of bones.

[00085] 3. Centrifuge the cell suspension at 300 x g for 5 mins.

[00086] 4. Remove the supernatant and re-suspend the cell pellet with 1 ml ACK lysing buffer for 5 mins.

[00087] 5. Add 9 ml PBS to dilute ACK lysing buffer and centrifuge at 300 x g for 5 mins.

[00088] 6. Remove the supernatant and resuspend with 10 ml culture medium

(RPMI-1640 plus L-glutamine, 10% FBS, 1% non-essential amino acid (100X), 10 mM HEPES, 50 nM b-mercaptoethanol, and 5% penicillin/streptomycin). Note: Endotoxin level has to be less than 0.1 EU/ml in FBS.

[00089] 7. Pass the cell suspension through a 40 mhi cell strainer.

[00090] 8. Adjust the cell number to 1 x 10⁶ cells/ml with 15 ng/ml granulocyte- macrophage colony- stimulating factor (GM-CSF) and 10 ng/ml IL-4.

[00091] 9. Plate 3 ml of 1 x 10⁶ cells/ml in each well of 6-well plate and incubate the cells at 37°C, 5% CO2 and 95% humidity in the CO2 incubator.

[00092] 10. On day 3, remove all 3 ml culture medium from each well, add 2 ml fresh PBS in each well, and then gently swirl the plate to ensure removing all the non adherent cells.

[00093] 11. Replace 3 ml fresh culture medium with 15 ng/ml GM-CSF and 10 ng/ml IL-4 in each well.

[00094] 12. On day 5, directly add another 3 ml fresh culture medium with 15 ng/ml GM-CSF and 10 ng/ml IL-4 in each well. The total volume in each well is now 6 ml.

[00095] 13. On day 7, to harvest BMDCs, gently pipette the culture medium in each well to dislodge the loosely-adherent BMDCs into suspension. The adherent macrophages are still attached to the plate. [00096] 14. Centrifuge the cell suspension at 300 x g for 5 mins and resuspend with fresh culture medium for further experiments. BMDCs can be identified with fluorescence-labeled CD 11c antibody by flow cytometry.

Characterize TolDC gene and protein profile

[00097] 1. Plate 2 ml of 1 x 10⁶ BMDCs/ml per well in 6 well-plate with culture medium in the presence or absence of 100-400 nM CDDO-DFPA for incubating 1 hr. Other agents for induction of TolDCs can be applied at this step, such as IL-10, vitamin D3, dexamethasone or BAY 11-7085.

[00098] 2. Add 10 or 100 ng/ml of LPS for incubating 4-24 hrs. (incubation time is different due to mRNA or protein measurement).

[00099] 3. Harvest the cell suspension and centrifuge at 300 x g for 5 mins to collect the cells and supernatant, respectively.

[000100] 4. TolDCs can be directly analyzed for the cell surface ligands by flow cytometry, such as stimulatory ligands: CD40, CD80, CD86, MHC-II, OX40L, ICOSL, or inhibitory ligands: PD-L1, PD-L2, ILT3, ILT4.

[000101] 5. The RNA extraction and supernatant from TolDCs can be analyzed for the cytokine profile at gene and protein level by quantitative real-time PCR (qRT-PCR) and ELISA, respectively. For example, inflammatory cytokines: TNF-oc, IFNy, EDN-1, IL-6, IL- 12, and IL-23, or anti-inflammatory cytokines: IL-4, IL-10, IL-15, TGF-b, and HO-1.

Evaluate the function of TolDCs in vitro and in vivo

T-cell syngeneic proliferation assay

[000102] 1. To obtain splenic CD4+ T cells, use the appropriate method to euthanize OT-II TCR transgenic mice and isolate the spleen in PBS.

[000103] 2. Use the back of push-stick of 3 ml syringe to mince the spleen by passing a 40 mhi cell strainer.

[000104] 3. Collect the cell suspension and centrifuge at 300 x g for 5 mins.

[000105] 4. Resuspend the cell pellet in 400 mΐ of MACS buffer (PBS, 0.5% BSA, and 2 mM EDTA).

[000106] 5. Add 100 mΐ of CD4+ T Cell Biotin-Antibody Cocktail (Miltenyi Biotec

Inc.) at 4°C for 5 mins. [000107] 6. Add 300 mΐ of MACS buffer and 200 mΐ of Anti-Biotin MicroBeads

(Miltenyi Biotec Inc.) at 4°C for 10 mins.

[000108] 7. Place LS Column and Pre-Separation Filter (Miltenyi Biotec Inc.) together in the magnetic field and rinse it with 3 ml of MACS buffer.

[000109] 8. Add 9 ml of MACS buffer in the cells and centrifuge at 300 x g for 5 mins.

[000110] 9. Resuspend the cell pellet in 3 ml of MACS buffer and apply onto the

LS Column. Collect flow-through containing CD4+ T cells.

[000111] 10. Wash column with another 3 ml of MACS buffer and also collect the flow-through.

[000112] 11. To obtain splenic Pan DCs, use the appropriate method to euthanize

C57BL/6 mice and isolate the spleen in 2 ml of collagenase D solution (2 mg/ml collagenase D dissolved in HBSS containing calcium, magnesium).

[000113] 12. Inject 1 ml of collagenase D solution to the spleen two times by a 1 ml syringe and a 25G needle. Cut the spleen into small pieces with small scissors.

[000114] 13. Shake and incubate at room temperature for 25 mins.

[000115] 14. Add 500 mΐ of 0.5 M EDTA at room temperature for 5 mins.

[000116] 15. Resuspend the cell pellet in 350 mΐ of MACS buffer, 50 mΐ of FcR

Blocking Reagent (Miltenyi Biotec Inc.), and 100 mΐ of Pan Dendritic Cell Biotin- Antibody Cocktail (Miltenyi Biotec Inc.) at 4°C for 10 mins.

[000117] 16. Wash the cells by adding 9 ml of MACS buffer and centrifuge at 300 x g for 5 mins.

[000118] 17. Resuspend the cell pellet in 800 mΐ of MACS buffer and add 200 mΐ of

Anti-Biotin MicroBeads at 4°C for 10 mins.

[000119] 18. Wash column with another 3 ml of MACS buffer two times and also collect the flow-through.

[000120] 19. Treat the DCs in the presence or absence of 100-400 nM CDDO-

DFPA at 37°C for 1 hr. and also label the CD4+ T cells with 1 mM CFSE at 37°C for 15 mins.

[000121] 20. Culture IOOmI of 2 x 10⁴DCs and IOOmI of 2 x 10⁵ T cells (1:10 ratio) in a 96-well plate with 100 ng/ml of ovalbumin (OVA) peptide 323-329. [000122] 21. After 2 days, measure the CFSE intensity of T cells by flow cytometry.

REPRESENTATIVE RESULTS

The differentiation and selection of BMDCs

[000123] Bone marrow progenitor cells were cultured in complete RPMI medium in the presence of GM-CSF and IL-4 to differentiate into iDCs for 7 days (Fig. 1A). On day 1, cells were in small size and showed spherical morphology. Washing with PBS before the replacement of fresh medium on Day 3 helped cells forming clusters and increased CD1 lc+ cell population. On day 4, BMDCs were enlarged in size and initiated the cluster formation. Adhered macrophages were also converted and observed at the bottom of the plate with an elongated shape. On day 5 large size of clusters of BMDCs are formed. On day 6, a large number of semi-adherent and floating BMDCs were also observed. BMDCs were harvested on day 7 and analyzed by flow cytometry for CD1 lc expression as a specific marker of murine DCs. As shown in a representative flow cytometry plot in Fig. IB, around 83.6% of BMDCs expressing CD1 lc were obtained by this method.

Induction and genetic characterization of TolDCs

[000124] Some of the TolDC-induced agents, such as vitamin D3 and dexamethasone, have shown the down-regulated expression of DC surface ligands, including MHC II and costimulatory molecules, CD40, CD80, and CD86. However, in LPS or CD40L-induced maturation of DCs, calcineurin inhibitors cyclosporin A and FK506 showed no effect on CD83, CD80, CD86, and MHC II expression. As evident by flow cytometry analysis, our TolDC-induced agent, CDDO-DFPA also didn’t reveal any significant effect on LPS-induced surface ligand expression of DCs, including MHC II, CD80, CD86, and PD-L1. (Fig. 2).

[000125] In addition, a comparison of BMDCs exposed to LPS with or without CDDO- DFPA by qRT-PCR and ELISA showed that CDDO-DFPA treatment significantly reduced the BMDC expression of pro-inflammatory cytokine genes such as IFN-g, IL-12, EDN1, TNFa, IL-6, and IL-23 induced by LPS activation (Figure 3A-3F). Both IFN-g and IL-12 are necessary for Thl cell differentiation. The latter two (IL-6, and IL-23) are necessary for Thl7 cell differentiation. BMDCs treated with CDDO-DFPA also showed increased expression of anti-inflammatory cytokine genes such as IL-4, IL-10, TGF-b and HO-1 (Figs. 4A-4D). IL-4 promotes the differentiation of CD4 T cells toward the Th2 phenotype, whereas IL-10 and TGF-b are known to exert anti-inflammatory activity and suppress autoimmunity through mechanisms that include the induction of Treg. It is noteworthy that the distinctive IL- 12-;IL-10+ cytokine production profile, the inhibition of EDN-1, and induction of HO-1 expression induced by CDDO-DFPA, are all known to authenticate DCs tolerogenic function.

Cellular and functional characterization of TolDCs In vitro

[000126] DCs promote T cell proliferation through their engagement of costimulatory ligands and through the elaboration of cytokines and other soluble mediators. Our data thus far suggest that CDDO-DFPA has the capacity to modulate the T cell response by altering gene expression and function of DCs. Therefore, using in vitro model of syngeneic stimulation, we examined how CDDO-DFPA modified DC-mediated T cell proliferation. Isolated DCs were pretreated with CDDO-DFPA and washed prior to co-culture with CFSE stained T cells with OVA peptide. We found that TolDCs, induced by CDDO-DFPA significantly suppressed the T cell proliferation (Fig. 5).

[000127] This Example describes an efficient protocol to generate iDCs and differentiate them into TolDCs. We analyzed the TolDC surface ligands by flow cytometry and then the cytokine profile was measured by qRT-PCR and ELISA. Later, the immunoregulatory function of TolDCs was confirmed by ensuring their capacity to reduce T cell proliferation.

In the end, the functional utility of these TolDC were evaluated by testing them into preclinical murine model of MS, EAE.

[000128] The iDCs were generated and differentiated from bone marrow precursors of mice with the combination of GM-CSF and IL-4. Other protocols have used forms -like tyrosine kinase 3 ligands (Flt3L) in the culture medium to generate iDCs. Although, the use of Flt3L increases the yield of iDCs, these iDCs usually take 2 more days (9 days) to harvest, compared to GM-CSF/IL-4 addition (7 days). More importantly, iDC generated from Flt3L induces the differentiation of both cDCs and pDCs. But GM-CSF/IL-4 induce the differentiation of iDC more toward to cDCs only. It has been shown that iDCs generated from these two methods produce morphologically different cells, which represent different surface marker, and cytokine profile upon their activation. Furthermore, their migration ability, and antigen- specific T cell responses also vary. Since, GM-CSF/IL-4 induced BMDCs are superior at T cell stimulation and the production of inflammatory mediators following LPS treatment, we found it more suitable in our experiments.

[000129] In this protocol, CDDO-DFPA (synthetic triterpenoid) and LPS were added as a DC tolerance and maturation inducer, respectively. We found that after harvesting iDCs from BMDC generation method, pretreatment with CDDO-DFPA induced the tolerogenic DC phenotype. Unlike CDDO-DFPA, which was added in the culture after iDC harvest (day 7), vitamin D3 has also been shown and could be added in the culture during the iDC differentiation (day 2, 4, 6). It has been described that adding vitamin D3 results in diverse mechanisms both during and after iDC differentiation. Adding CDDO-DFPA during the iDC differentiation showed no difference in the purity of CD11C+ DCs, but dose-dependently lowered the yield of iDCs on day 7 (data not shown). Our data suggest that the concentration of CDDO-DFPA we used may be toxic to bone marrow progenitor cells, even though iDC showed normal viability with the concentration of CDDO-DFPA. On the other hand, iDCs can also be matured by other inducers than LPS, such as CD40L, TNF-a, and IFN-g.

However; DC maturation by LPS through Toll-like receptors 4(TLR4) leads the activation of several transcription factors, including nuclear factor-kB (NF-KB), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal- regulated protein kinase (ERK1/2). The maturation of DCs by CD40L and TNF-a through CD40 and TNF receptors, respectively induces the NF-KB pathway. Whereas, IFN-g stimulates a different pathway including the activation of Janus kinase (JNK), tyrosine kinase (TYK), and signal transducer and activator of transcription proteins (STATs). But the downstream effects are also complemented by NF-KB pathway. In addition, the gene expression profile of CD40L/TNF-a-based DC maturation suggests that these DCs polarize T cells toward to Th2 cell response. Furthermore, it has also been confirmed that DC maturation by IFN-g is strongly biased towards a Thl cell response. Therefore, other DC maturation inducers for any specific T cell subsets differentiation are not considered in this protocol.

Example 2

[000130] In this Example, we investigate the role of Nrf2 in TolDCs by its genetic (Nrf2 ^/_ ) or pharmacological (CDDO-DFPA) manipulation in these cells. Here, we show that Nrf2 regulates DC tolerance by modulating their cytokine profile and cellular metabolism. CDDO-DFPA-induced Nrf2 activation, resulted in a significant anti-inflammatory transcriptome response, enhanced HO-1 expression, suppressed NO production, and a metabolic shift from glycolysis to OXPHOS in DCs. Our data correlates with

immunohistochemical (IHC) analyses of BM biopsies of severe aplastic anemia (AA) patients, in which the invariably high iNOS and low Nrf2 expression is in striking contrast to normal BM donors. Finally, administration of CDDO-DFPA-induced TolDCs in a murine BMF model of AA increased animal survival rate, BM cellularity, and hematopoiesis. In these mice, TolDC therapy not only regulated the proliferation of DCs, T cells, and HSCs but also retained the Thl7/Treg balance. These results reveal the Nrf2-dependent mechanisms of TolDC induction and highlight their therapeutic utility in the treatment of A A and other autoimmune diseases.

Methods

Patient information

[000131] BM biopsies were obtained from patients diagnosed with severe AA according to the International AA Study Group criteria. Inherited BMF syndromes and paroxysmal nocturnal hemoglobinuria were ruled out. These biopsies were obtained under an IRB- approved protocol and used for IHC staining.

Animals

[000132] C57BL/6 and BALB/c mice were inbred and then crossbred (C57BL/6 x

BALB/c) to generate FI (CByB6Fl) mice. Colonies of OT-II T cell receptor (TCR) transgenic and Nrf2^_/ mice colonies were maintained for use in the in vitro and in vivo assays described in this report. All studies were performed in compliance with procedures approved by the Case Western Reserve University School of Medicine’s Institutional Animal Care and Use Committee.

BM-derived dendritic cells (BMDCs) preparation

[000133] Cells were isolated from BM of C57BL/6 or Nrf2^_/ mice and were differentiated into BMDCs. In brief, isolated BM cells were cultured for 7 days in RPMI-1640 plus L- glutamine medium containing 10% FBS, 50 nM b-mercaptoethanol, and 5%

penicillin/streptomycin with GM-CSF (15 ng/ml) and IL-4 (10 ng/ml). At the end of this culture period, cells were harvested and CDl lc expression (80-85% of the expanded cell population) was confirmed by FACS analysis.

Quantitative real-time PCR (qRT-PCR)

[000134] Total RNA was isolated from cells using RNAqueous®-Micro Total RNA Isolation Kit (ThermoFisher Scientific Inc.) and subjected to cDNA synthesis using

Superscript® III CellsDirect™ cDNA Synthesis Kit (ThermoFisher Scientific Inc.). qRT- PCR of individual genes was performed on CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Inc.) using the primers and probes (Applied Biosystems Inc.): TNFa (Mm00443258), IFN-g (Mm01168134_ml), IL-12a (Mm00434165), IL-4

(Mm99999154_ml), IL-10 (Mm01288386_ml), TGF-bI (Mm01178820_ml), iNOS (Mm00440502_ml), and Hmox-1 (Mm00516005_ml). The results were generated and normalized to GAPDH (Mm99999915).

T-cell proliferation assay

[000135] Splenic CD4⁺ T cells were isolated from OT-II TCR transgenic mice using a CD4⁺ T Cell Isolation Kit (Miltenyi Biotec Inc.) and labeled with CFSE as described previously. Splenic DCs were isolated from C57BL/6 mice using a Pan Dendritic Cell Isolation Kit (Miltenyi Biotec Inc.). Both DCs and T cells were co-cultured at 1:10 ratio in presence or absence of 100 ng/ml of ovalbumin (OVA) peptide 323-329 (InvivoGen Inc.). T cell proliferation was measured by analyzing CFSE intensity by flow cytometry after 72 hours.

Metabolism assays

[000136] The OXPHOS and glycolysis level of DCs were analyzed through assessment of mitochondrial oxygen consumption rate (OCR, pmol/min) and extracellular acidification rate (ECAR, mpH/min), respectively by an XFp extracellular flux analyzer (Agilent

Technologies). For OCR studies, 6 x 10⁴ BMDCs/well were treated for 1 hour with or without CDDO-DFPA followed by LPS (10 ng/ml) for 24 hours in poly-D-lysine coated XFp Cell Culture Miniplates. Cells were centrifuged and washed with assay medium (XF Base Medium, 10 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine, pH 7.4) and incubated in a non-C0₂ 37°C incubator for 45 mins before analysis. A complete OCR study was performed in 4 consecutive stages: basal respiration, mitochondrial complex V inhibition (1 mM oligomycin), maximal respiration induction (0.5 mM carbonyl cyanide-4- (trifluoromethoxy)phenylhydrazone [FCCP]), and electron transportation chain (ETC) inhibition (0.5 mM rotenone/antimycin A). For ECAR assays, cells were washed in assay medium (XF Base Medium, 2 mM L-glutamine, pH 7.4). A complete ECAR assay consisted of 4 stages: basal, glycolysis induction (10 mM glucose), maximal glycolysis induction (1 mM oligomycin), and glycolysis inhibition (50 mM 2-deoxy-D-glucose [2-DG]). The quantified results for OXPHOS and glycolysis were generated by Report Generator (Wave software, version 2.3).

Western blot analysis and NO production measurement

[000137] Total cell lysates were prepared with RIPA buffer containing protease inhibitors. Western blot analysis was performed using iNOS, HO-1, Nrf2, and b-actin (control) antibodies (Santa Cruz Biotechnology) as previously described. NO production was measured as nitrite from DC culture supernatants using the colorimetric NO assay kit (ThermoFisher Scientific Inc), according to the manufacturer’s protocol.

A A murine model and TolDC treatment

[000138] Inguinal, brachial, axillary, and mesenteric lymph nodes (LNs) were extracted from C57BL/6 mice and filtered through a 40-pm nylon mesh to obtain a single-cell suspension. About 5 x 10⁶ of these LN cells were then infused by intravenous (i.v.) injection in 8-10 week-old CByB6Fl mice exposed to a sublethal dose (5 gray [Gy]) total body irradiation by a ¹³⁷cesium g source irradiator (J.L. Shepherd) 6 hours before injection, as previously described. The treatment group received (i.v.) 5 x 10⁶ TolDCs (CByB6Fl BMDCs treated with 400 nM CDDO-DFPA for 1 hour followed by 10 ng/ml LPS treatment for 24 hours) on day 0, 3, and 5. Both control and treatment groups of mice were bled from the tip of the tail at different time points to measure blood counts using a Hemavet 950 analyzer (Drew Scientific). In some experiments, mice were euthanized to collect splenocytes from spleen (day 10) and BM cells from tibia and femur (day 14). Sternums were collected on day 14 for hematoxylin and eosin (H&E) and IHC examination in each experiment.

Histology and IHC

[000139] Human BM biopsies or murine sternum sections were prepared and stained for H&E or immunostained using antibodies against CD3, CDl lc, iNOS, Nrf2, or HO-1 to study pathology and immune infiltration as previously described earlier. Images were acquired using a confocal microscope and combined using LSM 510 imaging software (Carl Zeiss, Inc.). Each H&E stained section was assigned a histological score for cellularity from 1 to 4 by an observer blinded to sample identity, where 1 represents normal 2- mild cellular loss, 3- moderate cellular loss, and 4- severe cytopenia. Similarly, IHC score ranged from 0 to 4 by an observer blinded to sample identity, where 0 represents negative immunostaining and 4 represents the highest number of positively immunostained cells. Because of the loss of cellularity in AA samples, the IHC was normalized to positively stained cells/100 cells.

Flow cytometry and intracellular cytokine staining

[000140] Splenocytes and BM cells were harvested from mice following AA induction and were stimulated with 50 ng/ml of PMA, 1 pg/ml of ionomycin, and 10 mg/ml of

GolgiStop (BD Bioscience). After 4 hours, cells were fixed, permeabilized (BD

bio science/eBio science Inc.), and immunostained for flow cytometry analysis. Fluorescein- conjugated monoclonal antibodies for mouse CD4, CD8, and CD25 were from BD

Biosciences, whereas IL-17, c-Kit, lineage cocktail (lin), and Foxp3 antibodies were from eBioscience. Stained cells were analyzed by a BD FACSCalibur flow cytometer (BD

Biosciences) and data were analyzed by a FlowJo software Version 10.0.7 (TreeStar).

Results

Induction of the TolDC phenotype is Nrf2-dependent

[000141] To evaluate the role of Nrf2 in regulating the DC phenotype, we adopted the loss and gain of function approach to test the modulation of the cytokine transcriptome by utilizing either Nrf2^_/ or CDDO-DFPA-treated Nrf2^+/+ DCs, respectively. When exposed to LPS, Nrf2^_/ DCs attained a more mature phenotype, in comparison to Nrf2^+/+ DCs, as evident by increased gene expression of the inflammatory cytokines IFN-g and IL-12, but not TNFoc. CDDO-DFPA treatment alone didn’t alter this cytokine response in DCs. However, when LPS-treated DCs were exposed to CDDO-DFPA, we observed suppressed TNFoc, IFN-g, and IF- 12 gene expression in Nrf2^+/+ but not Nrf2^_/ DCs.

[000142] Although EPS treatment significantly suppressed expression of the anti inflammatory cytokine IF-4 in both Nrf2^+/+ and Nrf2 ^A DCs, EPS-induced TGF-bI suppression was hampered in Nrf2^_/ DCs (Figure- IE). Both of these cytokines were restored towards normalcy with increasing doses of CDDO-DFPA treatment in Nrf2^+/+ but not in Nrf2 ^/_ DCs. Since IL-10 production by LPS has been reported to be triggered by an Nrf2 independent pathway in DCs, we tested these claims by treating Nrf2^+/+ and Nrf2^_/ iDCs with CDDO-DFPA alone or in combination with LPS. We observed increased IL-10 expression in CDDO-DFPA-treated Nrf2^+/+ but not in Nrf2^_/ DCs, suggesting an Nrf2-dependent action of CDDO-DFPA. LPS-induced IL-10 expression masked the differences among the CDDO- DFPA-treated and -untreated groups, therefore we could not evaluate the role of Nrf2 in the induction of IL-10 during DC maturation (data not shown).

[000143] Since DCs regulate T cell proliferation through the modulation of cytokines and other soluble mediators, we examined the involvement of Nrf2 in DC-mediated T cell proliferation. When CFSE-stained OT-II T cells were cultured with or without OVA peptide, Nrf2^_/ DCs were able to induce higher T cell proliferation compared to Nrf2^+/+ DCs in the presence of OVA peptide. Treatment with CDDO-DFPA suppressed this DC-induced T cell proliferation at a higher rate in Nrf2^+/+ DCs cultures (62% at 200nM and 81% at 400nM CDDO-DFPA) compared to Nrf2 ^/_DCs cultures (43% at 200nM and 51% at 400nM CDDO- DFPA). These results establish an Nrf2-dependent TolDC phenotype triggered by DC exposure to CDDO-DFPA.

Nrf2-mediated dichotomous metabolic reprogramming defines DC differentiation

[000144] There is a rapidly growing interest in the role of metabolic changes in the modulation of phenotype and function of DCs. Although OXPHOS and fatty acid oxidation are two major energy sources of iDCs and TolDCs, the LPS-stimulated maturation triggers a shift towards a glycolytic metabolic state in these cells, with glucose as the preferred carbon source. This dichotomous metabolic reprogramming results in the differential cellular function of mDCs and TolDCs. In order to study the functional role of Nrf2 in this process, we examined the modulations of OXPHOS and glycolytic metabolic levels in Nrf2^+/+ and Nrf2^_/ DCs in either the presence or absence of LPS stimulation, with or without prior exposure to the synthetic triterpenoid, CDDO-DFPA. The OCR and ECAR rate of mitochondrial respiration of DCs was measured in real time as depicted in Fig. 6A and 7A.

[000145] Both Nrf2^+/+ and Nrf2^_/ DCs displayed characteristic changes of OCR in response to addition of oligomycin (for inhibition of the mitochondrial ATP-synthase), FCCP (for uncoupling of OXPHOS from ATP synthesis), and Rot/AA (for inhibition of the ETC) (Fig. 6B and 6D). Although Nrf2^+/+ DCs displayed higher levels of basal and maximal OCR following FCCP addition when compared to Nrf2^_/ DCs, they both remained unresponsive to FCCP upon LPS stimulation. CDDO-DFPA treatment partially restored the OCR level after FCCP addition in Nrf2^+/+ but not in Nrf2^_/ DCs. Quantitative analysis confirmed that after LPS treatment, Nrf2 activation significantly increased basal respiration, ATP production, maximal respiration, and spare capacity in Nrf2^+/+ DCs (Fig. 6C), whereas Nrf2^_/ DCs showed no effect (Fig. 2E).

[000146] We observed no measurable difference in ECAR between Nrf2^+/+ and Nrf2^_/ DCs following addition of glucose (for fueling glycolysis), oligomycin (for inhibition of the mitochondrial ATP-synthase), and 2-DG (for competitive inhibition of glucose) (Fig. 7B and 7D). LPS treatment triggered oligomycin-induced maximal ECAR levels in both Nrf2^+/+ and Nrf2^_/ DCs. CDDO-DFPA treatment diminished this response in Nrf2^+/+ DCs. However, in contrast, it increased ECAR levels in Nrf2^_/ DCs. Our quantitative analysis revealed that CDDO-DFPA treatment in Nrf2^+/+ DCs significantly reduced the ECAR basal level, decreased glycolysis, and restored glycolytic reserve without altering glycolytic capacity in the LPS-treated group (Fig. 7C). Nrf2 ^/_ DCs remained unresponsive to CDDO-DFPA treatment in these parameters (Fig. 7E). This data indicates that the lower energy production from glycolysis in Nrf2^+/+ DCs (treated with LPS and CDDO-DFPA) is not due to impairment of glycolytic capacity, but rather is a result of compensated energy production by OXPHOS. These results are consistent with our previous observation in Fig. 6.

[000147] In totality these data indicate that Nrf2^_/ DCs possess a more mature phenotype at a baseline. Moreover, DCs completely shut down the use of OXPHOS as an energy source during maturation and Nrf2 activation promotes a metabolic signature in TolDCs that maintains OXPHOS as the energy source. Nrf2 activation induces a TolDC phenotype which maintains the same level of glycolytic capacity as seen in mDCs, but uses less glycolysis and more OXPHOS as the energy source.

Nrf2 regulates DC metabolism through iNOS and HO-1 expression

[000148] We next examined the molecular mechanisms responsible for the Nrf2-driven metabolic reprograming of DCs and the higher OXPHOS observed in TolDCs. The functional inhibition of iNOS -derived NO has been shown to be responsible for LPS -induced OXPHOS impairment. Therefore, we analyzed the mRNA and protein expression of iNOS and secreted nitrites to measure NO production in Nrf2^+/+ and Nrf2^_/ DCs treated with or without CDDO-DFPA in either the presence or absence of LPS. As expected, Nrf2^+/+ DCs showed increased Nrf2 protein expression in response to CDDO-DFPA treatment (Fig. 8A). We also observed increased mRNA and protein expression of iNOS in response to LPS treatment in both Nrf2^+/+ and Nrf2^_/ DCs. However, exposure to CDDO-DFPA diminished this response only in Nrf2^+/+ DCs in a dose dependent manner (Figs. 8A and 8B), suggesting an Nrf2-dependent action of CDDO-DFPA. It has been reported that HO-1, an Nrf2- dependent antioxidant, reduces the activity and expression of iNOS. Therefore, we next analyzed the expression of HO-1 in our experiments. We found increased Hmox-1 mRNA and HO-1 protein expression in response to CDDO-DFPA treatment in Nrf2^+/+but not in Nrf2^_/ DCs (Figs. 8A and 8B). Since HO-1 has been shown to reduce NO production, either directly through scavenging or indirectly through the inhibition of iNOS, we next examined the nitrite levels in culture supernatants of DCs stimulated with or without LPS in the presence or absence of CDDO-DFPA. Our data demonstrate that LPS-induced NO production was significantly decreased upon CDDO-DFPA treatment in a dose dependent manner, but only in Nrf2^+/+ and not in Nrf2^_/ DCs (Fig. 8D). These results suggest that activation of Nrf2 in DCs is responsible for inducing HO-1 and diminishing iNOS and NO expression to further restore the OXPHOS metabolic signature of TolDCs.

TolDCs exhibit capacity to suppress clinical pathology and symptoms in mice with AA

[000149] We next investigated the mechanism of immunoregulatory function and the therapeutic utility of TolDCs in the previously described AA murine model. In this model, the survival rate improved from 17 to 28 days in AA mice receiving TolDCs compared to the untreated control group (Fig. 9A). As expected, from day 8 to 17, we observed a significant decline in white blood cells (WBCs), hemoglobin (Hb), hematocrit (HCT), and platelets (PLTs) of AA mice when compared to the only irradiation (IR) exposed group (Fig. 9B-9E). The TolDC treatment didn’t alter either WBCs or PLTs counts (Fig. 9B and 9E), however; Hb and HCT values were significantly improved in the treated group (Figs. 9C and 9D). Histological analysis of BM collected from TolDCs-treated AA mice showed significantly improved BM cellularity and reduced areas of hemorrhage compared to untreated AA mice (Figs. 9F and 9G). Our IHC analyses also revealed a significant decline in T cell infiltration (Figs. 9F and 9H) and iNOS expression (Figs. 9F and 91) in the BM of TolDCs-treated AA mice when compared to untreated AA mice. TolDCs- treated AA mice also exhibit significantly increased Nrf2 (Figs. 9F and 9J) and HO-1 (Figs. 9F and 9K) protein expression compared to untreated AA mice. These results reveal the potential therapeutic utility of TolDCs in the treatment of AA.

TolDC treatment regulates both T cell and HSC homeostasis in AA mice

[000150] Effector T cells, especially Thl7 cells, actively contribute to the destruction of HSCs in the BM during AA. In order to test the efficacy of TolDC treatment in modulating this immune response, we next analyzed the T cell repertoire and HSCs in AA mice with or without TolDC treatment. The marked expansion of both CD4⁺ and CD8⁺ T cells observed in the spleens (Figs. 10A, 10G, and 10H) and BM (Figs. 10D, 10K, and 10L) of AA mice at day 10 and 14, respectively, was significantly reduced by TolDC treatment. The dichotomy in the generation of Thl7 that induce autoimmunity and Tregs that inhibit autoimmune tissue injury has been previously reported. Moreover, a higher Thl7/Treg ratio has also been found in AA patients. Therefore, we next analyzed the abundance of populations of both Thl7 (CD4⁺IL-17⁺) and Tregs (CD4⁺CD25⁺Foxp3⁺) by cell surface phenotypes and their characteristic intracellular markers in AA mice treated with or without TolDCs. We observed a significant decrease in the Thl7 cell population in spleens (Figs. 10B and 101) and BM (Figs. 10E and 10M) as well as increased Tregs in the spleen (Figs. IOC and 10J) of TolDCs- treated mice when compared to untreated AA mice. Finally, we analyzed the HSC population in BM of these mice by using lin and c-kit⁺ as cell surface markers. As expected, AA mice had lower numbers of HSCs when compared to normal mice, but TolDC treatment restored these HSC populations and showed significantly higher lin and c-kit⁺ cells compared to the untreated group (Figs. 10F and 10N). These results provide the first scientific evidence of the therapeutic utility of TolDCs converted ex vivo by CDDO-DFPA exposure, and specifically their capacity to ameliorate disease severity in an AA murine model by regulating the T cell homeostasis.

Evidence for suppressed Nrf2 signaling in the hypoplastic BM of AA patients associated with an immunogenic DC milieu

[000151] AA is characterized by a marked expansion of T cells and immunogenic DCs in blood and BM. In order to investigate the significance of Nrf2 signaling in BM

microenvironment in humans with AA and the relationship to immunogenic DCs, we compared the expression of iNOS, Nrf2, HOI, CD3, and CD1 lc by IHC in BM biopsies of 4 severe AA patients and 4 healthy donors. Our histological analysis (by H&E) revealed a significantly reduced BM cellularity in AA patients compared with their healthy counterparts (Figs. 11A and 1 IB). Our IHC analysis showed a significantly higher number of infiltrating T cells (anti-CD3) (Figs. 11A and 11C) and DCs (anti-CDl lc) (Figs. 11A and 11D) in severe AA patients compared to healthy donors. In addition, it has been clearly demonstrated that DCs shift their immature form to an active one, which promotes the over-function of T cells and hematopoiesis failure in AA patients. The abundance of signals inducing mDCs, and not TolDCs, in the BM microenvironment become the key elements to cause the destruction of HSCs in AA. Therefore, we next analyzed the expression of iNOS, Nrf2, and HO-1, which critically regulate the metabolic pathway of TolDCs and mDCs. The images and quantified analyses showed that AA patients have a significantly higher level of iNOS and reduction of Nrf2 and HO-1 expression when compared with healthy donors (Figs. 11A and 11E-11G). These results demonstrate the clinical significance of Nrf2 signaling in influencing the BM microenvironment of AA patients in which the infiltration of immunogenic DCs accompanies or correlates with hematopoietic tissue destruction.

Example 3

[000152] This example describes methods to reproducibly generate TolDCs through targeted activation of the nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) by repression of Kelchlike ECH-associated protein 1 (Keapl). Underquiescent conditions, Nrf2 is anchored in the cytoplasm through binding to Keapl, which, in turn, facilitates the ubiquitination and proteolysis of Nrf2, a key mechanism for the repressive effects of Keapl on Nrf2. In previous studies, we have shown an absolute requirement for Nrf2 for induction of the TolDC phenotype by small molecules in the triterpenoid family. Here we show that disruption of Keapl expression will repress glycolytic metabolism and confer a tolerogenic phenotype to DCs.

[000153] Keapl gene deletion in mouse DCs promotes a shift to OXPHOS and consequently confers a tolerogenic phenotype to mouse DCs. Therapeutic efficacy of Keapl ^/_ TolDCs can be assessed through adoptive transfer experiments in the established model of sever aplastic anemia (SAA), as defined by assessment of normal hematopoiesis, serum inflammatory cytokine profiles and survival. [000154] In aplastic anemia (AA), progressive changes in the bone marrow (BM) microenvironment underlie an immune dysfunction that contributes directly to disease progression and therapy resistance. This biology has been effectively demonstrated in validated, immune mediated models of this disease. We can employ the well-characterized CByB6Fl model of AA in which lymph node (LN) cells extracted from C57BL/6 are adoptively transferred into recipient CByB6Fl mice, generated by a cross of C57BL/6 and BALB/c mice, which are inbred and then crossbred (C57BL/6 x BALB/c) to generate the FI (CByB6Fl) mice. Recipient mice invariably develop impaired hematopoietic function and die within four weeks. This model recapitulates all aspects of human A A and it has been utilized to evaluate novel therapeutic strategies for AA. While other preclinical models of AA have been described, this model permits rapid evaluation of novel agents, either alone and in combination with approved therapies.

Linking Nrf2 activation to the metabolic phenotype characteristic of TolDCs

[000155] Efforts to define the mechanisms underlying the“tolerogenic” potential of DCs have revealed a signature metabolic shift such that TolDCs favor oxidative phosphorylation and fatty acid oxidation. We previously showed the utility of small molecule activators of Nrf2 in the metabolic reprogramming of TolDCs and to demonstrate the therapeutic potential of this population when expanded ex vivo.

[000156] An absolute requirement for Nrf2 activity for induction of the TolDC phenotype was demonstrated by Celloram in studies utilizing Nrf2^_/ DCs. Specifically, exposure of DCs to selected pharmacologic activators of Nrf2 ex vivo resulted in a TolDC phenotype as evidenced by induction of IL-4, IL-10, and TGF-b and suppression of TNFa, IFN-g, and IL- 12 levels in Nrf2^+/+ DCs but not in Nrf2^_/ DCs. Indeed, these small molecule activators of Nrf2 induced patterns of oxidative phosphorylation (OXPHOS) and glycolysis that are characteristic of TolDCs, but they failed to do so in Nrf2^_/ DCs. We have also shown a significantly enhanced HO-1 and reduced iNOS/NO production in Nrf2^+/+ relative to Nrf2^_/ DCs, suggesting Nrf2-dependent TolDC induction is linked to suppression of the inhibitory effect of NO on OXPHOS.

Relevance of Nrf2 activity to the pathogenesis of S AA in humans

[000157] TolDCs generated by small molecule Nrf2 activators improves hematopoiesis and enhances survival in this established murine model of AA, and the response to the TolDCs is associated with a significant reduction in Thl7 cells and an increase in Treg cells. The clinical relevance of these observations was demonstrated through immunohistochemical (IHC) analyses of bone marrow biopsies from patients with S AA, which show an increased in T cells, elevated iNOS expression and decreased Nrf2 and HO-1 expression compared to normal subjects (Fig. 12). Importantly, an imbalance of Thl7 and Treg cell populations in patients with SAA has been linked to disease pathogenesis and progression by several investigators, providing a rationale to explore the unique potential for CLM-18 to restore the Thl7/Treg balance in SAA.

The application of TolDC for induction of tolerance in SAA

[000158] The capacity of DCs to induce antigen- specific tolerance has been previously described. In studies of organ allograft rejection, donor-derived DCs prolong graft acceptance, which may be more durable when combined with co-stimulatory blockade. In some cases, graft tolerance induced by donor DCs has been reported to involve delivery of donor antigen to recipient antigen presenting cells. Donor-derived DCs induce specific tolerance when given simultaneously with the organ graft. The capacity of ex vivo expanded TolDCs to treat autoimmune disease has also been explored in preclinical models and is now the focus of ongoing clinical trials. However, there has been no published report of any effort to examine the potential of TolDCs as a therapy for SAA. As described in Examples 1 and 2, we developed a unique method for ex vivo expansion of highly functional TolDCs achieved through the activation of Nrf2. The effort described in this example is the first to explore their therapeutic potential for SAA.

Disruption of Keapl gene expression in DCs is an improved strategy for Nrf2 activation in TolDCs

[000159] We have shown the utility of TolDCs expanded ex vivo through the use of small molecule activators of Nrf2. There are two important considerations that are highly relevant to the advancement of this strategy toward clinical application. The first consideration is the potential for patient exposure to the Nrf2 activating agent if carried with the cell product.

This is considered highly unlikely given the small amount (nM concentrations) required for the ex vivo induction of the TolDC phenotype from peripheral blood monocytes. However, a demonstration of the absence of the compound or drug in the cell product may be required by the FDA. The second consideration is the multi-functional nature of the most potent and effective activators of Nrf2 in our system, all of which are small molecules in the triterpenoid family. Most members of the triterpenoid family have the capacity to interact with other proteins, including key regulators of NFkB signaling (Fig. 13). Targeted disruption of Keapl gene expression is an alternative approach to specifically activate Nrf2 in TolDCs. This unique strategy may have two advantages. First, it may provide a more effective, robust and durable activation of Nrf2, obviating the influence of a pharmacologic Nrf2 activator on other signaling pathways in DCs. Second, it will eliminate considerations of patient exposure to small molecule activators of Nrf2 that may have potential to persist in the cell product.

Therefore, we proposed disrupting Keapl gene in both human and mouse TolDCs through use of the CRISPR/Cas9 syste. We can show Keapl ⁷ TolDC offers protection in the preclinical model of SAA, benchmarking against the previously established CLM-18, and induced using small molecule activators of Nrf2.

[000160] Several strategies have been developed to disrupt the aberrant immune response in patients with SAA. However, an approach based on the administration of ex vivo expanded, autologous tolerogenic DCs has not been explored. We show the potential of this approach, utilizing an established preclinical model of SAA to demonstrate efficacy of their TolDC product, CLM-18. These studies defined Nrf2 as a principal intracellular mediator of the metabolic shift required for induction of the tolerogenic DC phenotype. In this Example, we propose to optimize CLM-18 through use of the CRISPR/Cas9 system to specifically disrupt Keapl gene expression and thereby silence the principal negative regulator of Nrf2 activity. Major advantages of this approach are the potential to expand autologous TolDCs ex vivo without the use of any pharmacologic activators of Nrf2 in the culture system, and the ability to obviate any off-target effects of such agents. We developed CRISPR/Cas9 methods for use in targeting Keapl gene expression during the induction of TolDCs, demonstrating the ease of use during TolDC induction ex vivo. The readout will be clear, including:

evaluation of Keapl mutational spectrum by TIDE analysis, estimating the frequencies of insertions and deletions (Indels) in a pool of dendritic cells transfected with Cas9-RNP, and protein expression, metabolic profile, cytokine and chemokine gene expression profile, assessment of Nrf2 activity based on transcriptome analyses of Nrf2 target gene expression, as well as DC expression of HO-1 and iNOS.

[000161] We can perform in vitro experiments designed to show knock out (KO) of Keapl gene expression in human monocytes (using CRISPR/Cas9) promotes a shift from glycolytic metabolism to oxidative phosphorylation (OXPHOS) during the ex vivo generation of human DCs. We predict the latter will convey a more stable, highly functional TolDC phenotype. Analyses of DC metabolism will be performed in collaboration with the academic partner. Analyses of TolDC phenotype and function can be performed and benchmarked against human DCs for ex vivo expansion in the presence of small molecule activators of Nrf2.

Efficient ex vivo expansion of autologous human TolDC by disruption of Keapl gene expression

[000162] We can demonstrate Keapl-/- (Keapl ^KO) TolDC exhibit the key characteristics that have been defined for TolDCs generate by small molecule activators of Nrf2, principally: 1) HO-l^Hl, iNOS^low DCs; 2) production of IL-4, IL-10, and TGF-b; 3) suppression of T cell activation and production of TNF-a, IFN-g, and IF- 12 in human mixed lymphocyte reaction (MFR) assays; 4) A predominant shift from glycolytic metabolism to oxidative

phosphorylation (OXPHOS) during ex vivo TolDC expansion. Additionally, transcriptome analyses demonstrating activation of Nrf2 target gene expression in Keapl ^KO TolDCs will confirm activation of Nrf2 activity as a consequence of Keapl gene deletion.

Details of the CRISPR Methods and TolDC expansion procedures

[000163] We developed methods for CRISPRRNP delivery to murine bone marrow derived dendritic cells (BMDC) by NEON electroporation. For genetic modification of DC, electroporation-meditated CRISPR- KO methods offer greater efficiency than lipid-based methods. A plasmid DNA-free ribonucleoprotein (RNP) CRISPR system, consisting of Cas9 protein and guide RNA, has been selected to increase the chance of a safe gene modification. The Cas9 RNP is a functional complex which works immediately after it enters the cell as subsequent transcription and translation are not required. Moreover, the complex is rapidly degraded afterwards from the cell, minimizing the chance for off-target cleavage events when compared to plasmid DNA-based systems.

[000164] During development and optimization of this procedure for application in DCs, ROSA26 RNP was used as a positive control and SIRPa RNP as a DC surface marker which can be readily measured by flow cytometry. Each RNP was delivered to BMDC by the NEON Electroporator (Invitrogen) as indicated. Differentiation of BMDC was induced by culturing bone marrow cells for 7 days in the differentiation medium with GM-CSF (20 ng/ml) and IL-4 (15 ng/ml) and the day 7 BMDC (2xl0⁵/well) were used for CRISPR/Cas9 RNP delivery. NEON Electroporation of DCs involved 1500V/30 ms/1 pulse, followed by an additional two days of culture in vitro. Genomic DNAs from the BMDCs were harvested for CRISPR-PCR which specifically amplified the sequences around CRISPR-targeted site. The PCR products, which may include mutated sequences triggered by CRISPR/Cas9, were denatured and renatured for hetero-dimer DNA complex formation which then cleaved by T7 endonuclease I. The cleaved DNA was separated in 2% agarose gel and the approximate percent of insertional and deletional mutations (Indel%) was calculated for each condition. We observed approximately 55% Indel for ROSA26 RNP and 65% Indel for SIRPa RNP regardless of the Cas9/gRNA concentration. Efficacy is demonstrated by DC surface expression of SIRPa, determined by flow cytometry, with over 50% reduction in median fluorescent intensity (MFI) compared to that of ROSA26 RNP control cells (not shown). Histogram analysis clearly shows SIRPa RNP increased the SIRPa negative cell population as compared to ROSA26 control, confirming utility of the approach for targeting Keapl during ex vivo expansion of TolDC. Generation and characterization of human Keapl ^KO TolDC from peripheral blood monocytes (PBMs).

[000165] The protocol for generation of DCs from human PBMs is a 7-day procedure. As above, day 7 DCs generated from PBMs can be subjected to Keapl gene deletion through NEON electroporation of CRISPR-RNP sequences, and activated (after two days) prior to evaluation of the TolDC phenotype. The Keapl KO TolDCs can be compared to negative controls (receiving ROSA26 RNP) and to the positive control TolDCs generated from small molecule activators of Nrf2 based on DC exposure to small molecule activators of Nrf2 in culture. Phenotypic characterization will include flow cytometric analysis for the level change of surface expression of CD1 lc, CD80, CD86, and MHCII and other markers of mature DC, as well as assessment of the key features described in the milestone section (above). Finally, the ability of each TolDC product to suppress APC-mediated T cell activation will be assessed by the academic partner using human T cell isolates and monocyte-derived mature DCs from multiple donors in a classical mixed lymphocyte reaction (MLR). Readouts include assessment of T cell surface markers associated with activation, cytokine release ( e.g IFNy) and/or proliferation of T cells are all standard readouts enabling quantitative assessment of KeaplKO TolDC function in the MLR assay, relative the previously characterized CLM-18 product. [000166] Alternatively, the combination of Keapl gene deletion with small molecule activators of Nrf2 can be used as an approach that enhances the expansion of a more potent TolDC product. We are prepared to consider this alternative and/or change the TolDC culture conditions to yield a product with the greatest capacity to induce immune tolerance.

[000167] We can test whether Keapl gene deletion in mouse DCs promotes a shift to OXPHOS and consequently confers a tolerogenic phenotype to mouse DCs. Therapeutic efficacy of KeaplKO TolDCs can be assessed through adoptive transfer experiments in the established model of SAA, as defined by assessment of normal hematopoiesis, serum inflammatory cytokine profiles and survival. The production of murine KeaplKO TolDC using the established BMDC culture system will be used, and analyses of their in vivo efficacy will be as described above (Fig. 14). Administration of KeaplKO TolDC beginning day 1 and concomitant with the administration of lymph node cells of C57BL/6 mice permits assessment of the capacity of the cell product to delay progression of SAA and to ameliorate disease severity in a model that has a well-defined, highly reproducible and rapid time to progression. All BMDC derived cell products will undergo testing that includes viability, composition and function to ensure a product with consistent purity and potency. Mice in each group will be monitored daily for response to treatment and for development of complications related to the disease so that all mice may be euthanized when ill and captured for analyses of tissues so as not to lose any data points.

[000168] We anticipate that efficacy of KeaplKO TolDC in the CByB6Fl model of SAA will be as least equivalent to that observed for TolDC induced via exposure to an activator of Nrf2. However, we may find that the clinical response achieved may also depend on the number of KeaplKO TolDCs administered. An observation such as this will provide an opportunity to define the effects of increasing KeaplKO TolDC cell dose on the progression and severity of SAA. In addition, while we have established the system for CRISPR/Cas9 mediated deletion of Keapl in BMDC, we are prepared to compare this approach to an alternative strategy in which BMDC may be generated directly from mice with a tamoxifen- inducible CMVCre-Keaplfl/fl in which Cre-mediated deletion of Keapl is induced by treatment with tamoxifen (1 mg mouse 1 dayl; ip injection). Deletion of Keapl would be determined as previously described, and activation of Nrf2 would be confirmed by measuring expression of its downstream target NADPH quinone oxidoreductase 1 (Nqol) by quantitative PCR (TaqMan, Applied Biosystems), as for all TolDCs generated via Keapl gene deletion.

Example 4

[000169] Differentiation of BMDC was induced by culturing bone marrow cells for 7 days in the differentiation medium with GM-CSF (20 ng/ml) and IL-4 (15 ng/ml) and the day 7 BMDC (2xl0⁵/well) were used for CRISPR/Cas9 RNP delivery. Each control and crKeapl RNP was delivered to BMDC by the NEON Electroporator (Invitrogen) as indicated

(Fig. 15A). NEON Electroporation of DCs involved 1500V/30 ms/1 pulse, followed by an additional two days of culture in vitro. Genomic DNAs from the BMDCs were harvested for CRISPR-PCR which specifically amplified the sequences around CRISPR-targeted site. The CRISPR-PCR products, which may include mutated sequences triggered by CRISPR/Cas9, were sequenced by Sanger sequencing method. The approximate percent of insertional and deletional mutations (Indel%) was calculated using ICE analysis provided by Synthego, where ICE score indicates indel% and KO score presents the potential percentage of Keapl - KO in the mixture. We observed approximately 85% ICE score and 79% KO score for crKeapl.75 RNP (Fig. 15B). There was no significant change of DC surface markers in Keapl-KO DC cells as compared to those of control cells (Fig. 15C). The secreted cytokines, however, were apparently altered in the Keapl-KO DC, where pro-inflammatory cytokine TNFcr was reduced approximately over 60% of control (Fig. 15D). These results support the rationale that unleashing Nrf2 by knock-out of Keapl would reprogram immature dendritic cells toward tolerogenic phenotype, confirming utility of the approach for targeting Keapl during ex vivo expansion of TolDC.

Example 5

[000170] This example describes methods to reproducibly generate TolDCs through targeted activation of the nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) of immature dendrwith l-[2-Cyano-3,12-dioxooleana-l,9(l l)-dien-28-oyl]-4(-pyridin-3-yl)-lH- imidazole (CDDO-3P-Im).

[000171] Figs. 16(A-C) illustrate a schematic, graph, and table showing an optimized protocol for tolerogenic dendritic cell production. (A)The schematic diagram shows the optimized method for dendritic cell differentiation and production of CLM18.3 (B)The immature DCs were harvested on day 8 and treated again in Celloram’s induction cocktail for another day (C) The optimized protocol yielded iDC, mDC, and TolDC(CLM18.3) on day 9 as indicated. The cell number seeded at day 0 was set to 100%.

[000172] Figs. 17(A-D) illustrate graphs and plots showing purity, activity, and cytokines of CLM18.3 (A) CLM18.3 showed > 80% CDl lc+ and slight reduction of CD80, CD86, and MHCII. (B) CLM18.3 expressed low TNF-a, IL-12, but high TGF-jff. (C)CLM18.3 suppressed OVA-peptide specific T cell proliferation compared to control DC. As a proof-of- principle study, CRISPR-ko of Keapl also suppressed T cell proliferation (D) The Keapl-ko DC secrets low TNF-cr, IL-12, but high TGF- ? and IL-10.

[000173] Figs. 18(A-E) illustrate plots showing CLM-18.3 TolDCs exhibit unique transcriptome signatures. (A) Antigen Processing and Presentation Pathways and (B) Allograft Rejection Pathways. CLM18.3(18.3); mature DC (mDC) (C) MHC-I gene expression, (D) MHC-II gene expression, (E) the gene expression significantly related to tolerogenic dendritic cell function

[000174] Figs. 19(A-E) illustrate a schematic, graphs, and plots showing CLM18.3 significantly improved clinical scores and survival in mouse GvHD model (A) The experimental plan of GVHD model (B) CD4 and CD8 T cell subset analysis in each treatment group. (C) The level of TGF- ? in serum at day 14 and day 30 (D)The clinical score of each group measured by 5 clinical criteria (weight, posture, activity, Fur, and skin) (E)The Kaplan-Meier survival curve showed the survival benefit of CLM18.3 treated group.

[000175] Figs. 20(A-E) illustrate a schematic, graphs, and plots showing CLM18.3 significantly prolonged the survival in mouse aplastic anemia model (A) The experimental plan of aplastic anemia model (B)The Kaplan-Meier survival curve showed the survival benefit of CLM18.3 treated group in contrast to aplastic anemia group(red). (C) The level of TGF- ? and IL-10 in serum at day 17 (D) Blood counting showed the reconstitution of blood in each group (White blood cell, Hematocrit, platelet)(E) CD4 and CD8 T cell subset analysis in each treatment group(Left) and the preservation of Lin cKit⁺ hematopoietic stem cell population in bone marrow (right).

[000176] While this application has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the application encompassed by the appended claims. All patents, publications and references cited in the foregoing specification are herein incorporated by reference in their entirety.

1

TOLEROGENIC DENDRITIC CELLS AND USES THEREOF RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application

No._-62/835,323, filed April 17, 2019, the subject matter of which are incorporated herein by reference in their entirety.

BACKGROUND

[0002] Aplastic anemia is a life-threatening rare disease that occurs when one’s own immune system damages blood-making bone marrow cells, which gradually stop producing red and white blood cells and platelets. In the United States, it is estimated that there are between 500-1000 new cases annually, with unique challenges facing patients diagnosed later in life. Patients must receive frequent blood transfusions, take multiple immunosuppressive agents to suppress the autoimmune response that damages the marrow, take other drugs to prevent infections, and limit contact with the outside world to avoid infection and even minor injury. Over the long term, most patients eventually die of infections or of complications of their therapy. Thus, there is a desperate need for safer, more effective and less costly therapies for this disease, particularly for elderly patients for whom survival rates are unacceptably low.

[0003] While treatment paradigms have improved, the overall approach in the United States (US) has been similar for many decades. For young patients with a HLA matched sibling donor, Hematopoietic Stem Cell Transplantation (HSCT) is the preferred approach, whereas Immunosuppressive Therapy (1ST) comprised of Horse Antithymocyte Globulin (ATG) and Cyclosporine (CSA) is utilized in those that lack one. When 1ST fails to keep the disease in check (in as many as 30 to 40 percent of patients) Eltrombopag (Promacta) is used but works only in about 30 percent of patients and usually leads to only a partial, not a complete, response.

[0004] Upon failure of 1ST, these patients may undergo unrelated donor HSCT if their health status allows. The outlook is bleak for those patients for whom HSCT is not an option, as long-term (10-year) survival with immunosuppression ranging from 40% to 50%. For this group of patients on chronic 1ST, there is an added risk of somatic mutations resulting in evolution of myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) and the frequent recurrence of aplastic anemia. Using next-generation sequencing in a study of 439 patients with aplastic anemia, genetic abnormalities were detected in 50% of patients after 6 2 months of 1ST initiation, and one third of these patients had acquired somatic mutations in myeloid cancer candidate genes.

SUMMARY

[0005] Embodiments described herein relate to tolerogenic dendritic cells (TolDCs), methods of generating a population of TolDCs, the use of TolDCs in increasing immune tolerance, and the use of the TolDCs in treating inflammatory and immune disorders in a subject in need thereof.

[0006] It was found that TolDCs can be generated from dendritic cells through their metabolic reprograming by targeted activation of the nuclear factor (erythroid-derived 2)- like-2 factor (Nrf2), indicucible nitric oxide synthase (iNOS), and several other key regulators of dendritic cell metabolosim, such as genetic or pharmacologic manipulation of Tgf-b, smad7, and other targets of nfkB signaling. Targeted activation of Nrf2 is a novel approach to metabolic reprogramming of dendritic cells (DCs), which acquire a stable, immune suppressive or‘tolerizing’ phenotype. Unlike mature DCs (mDCs), the dominant anti-inflammatory signature of TolDCs is characterized by reduced expression of cell surface stimulatory ligands, decreased secretion of immunosuppressive cytokines and a distinct cellular metabolic profile that regulates T cell polarization.

[0007] Under quiescent conditions, Nrf2 is anchored in the cytoplasm through binding to Kelch-like ECH-associated protein (Keapl), which, in turn, facilitates the ubiquitination and proteolysis of Nrf2. It was found that disruption of Nrf2 binding to Keapl in DCs, a key mechanism for the repressive effects of Keapl on Nrf2, can activate Nrf2 and confer a tolerogenic phenotype to DCs.

[0008] In some embodiments, TolDCs described herein can include DCs that are administered an Nrf2 activator. The Nrf2 activator can include a triterpenoid administered at an amount effective to generate the population of tolerogenic dendritic cells. In some embodiments, the triterpenoid is at least one of CDDO-IM, CDDO-2P-IM, or CDDO-3P-IM.

[0009] In other embodiments, TolDCs described herein can include DCs that are genetically modified to inhibit, suppress, and/or disrupt expression of Keapl. The inhibition, suppression, and/or disruption of Keapl can include a deletion of at least a portion of Keapl gene to inhibit expression or function of Keapl including Keapl binding to Nrf2. -3-

[00010] In some embodiments, the Keapl expression in the DCs can be disrupted by gene editing. The gene editing be performed using at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and at least one guide RNA (gRNA) having a spacer sequence complementary to a target sequence in a Keapl DNA. The CRIS PR-associated endonuclease can be selected from a wild-type Cas9, a human-optimized Cas9, a nickase mutant Cas9, SpCas9(K855a), SpCas9(K810A/K1003 A/rl060A), or SpCas9(K848A/K1003A/R1060A).

[00011] The DCs used to generate the TolDCs can be obtained by isolating monocytes from the subject and culturing the monocytes with GM-CSF and IL-4 to generate immature dendritic cells. The monocytes can be isolated from bone marrow or peripheral blood of the subject.

[00012] In some embodiments the TolDCs generated by adminstration of an Nrf2 activator or by disruption of Keapl expression or binding to Nrf2 of the DCs can have a phenotype characterized by the production of multiple immune suppressive cytokines, including at least one of IL-4, IL-10 or TGF-b, high levels of HemeOxygenase- 1 (HOI) and low levels of inducible nitric oxide synthase (iNOS) (/._<?., HO-l^Hl,iNOS^low expression) with decreased NO production, promoting expansion of regulatory (suppressor) T cells, suppression of T cell activation and suppression of production of at least one of TNFa, IFN- g, or IL-12 in human mixed lymphocyte reaction (MLR) assays, exhibition of a shift from glycolytic metabolism to oxidative phosphorylation (OXPHOS) during ex vivo TolDC expansion, and/or activation of Nrf2 target gene expression.

[00013] In other embodiments, the TolDCs generated by adminstration of an Nrf2 activator or by disruption of Keapl expression or binding to Nrf2 of the DCs can have a phenotype characterized by the production of IL-10 and TGF-b and suppression of TNFa and IL-12 in human mixed lymphocyte reaction (MLR) assays.

[00014] In still other embodiments, the TolDCs generated by adminstration of an Nrf2 activator or by disruption of Keapl expression or binding to Nrf2 of the DCs can have a phenotype characterized by the production of IL-4, IL-10 and TGF-b and suppression of TNFa, IFN-g, and IL-12 in human mixed lymphocyte reaction (MLR) assays.

[00015] Other embodiments described herein relate to methods increasing immune tolerance in a subject in need thereof. The method can include administering to the subject a -4- therapeu tic ally effective amount of TolDCs generated by administration of an Nrf2 activator or genetically modified such that the expression of Keapl in the TolDCs is inhibited, suppressed, and/or disrupted.

[00016] In some embodiments, the subject to which the TolDCs are administered has an inflammatory condition, an allergy, or an autoimmune disorder. In other embodiments, the subject has received a tissue or organ transplant.

[00017] In other embodiments, the TolDCs are administered to a subject following a hematopoetic cell transplant with bone marrow, hematopoetic stem cells, or umbilical cord blood.

[00018] In other embodiments, the subject has aplastic anemia, myelodysplasia, myelofibrosis, thrombocytopenia due to other bone marrow diseases, drug induced thrombocytopenia, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenia, or thrombocytopenia following viral infections, neutropenia due to other bone marrow diseases, drug induced neutropenia, autoimmune neutropenia, idiopathic neutropenia, or neutropenia following viral infections, drug induced cytopenias, immune cytopenias, cytopenias following viral infections, or cytopenias.

[00019] In still other embodiments, the subject has aplastic anemia, myelodysplasia, myelofibrosis, anemia due to other disorder of bone marrow, drug induced anemia, immune mediated anemias, anemia of chronic disease, anemia following viral infections, or anemia of unknown cause.

[00020] In some embodiments, the tolerogenic dendritic cells are administered to the subject following chemotherapy administration, radiation therapy, or immunosuppressive therapy.

[00021] Still other embodiments relate to a method of treating an inflammatory or immune condition in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of TolDCs that are generated by administration of an Nrf2 activator or genetically modified such that the expression of Keapl in the TolDCs is inhibited, suppressed, and/or disrupted.

[00022] In some embodiments, the inflammatory or immune condition comprises at least one of achlorhydra autoimmune active chronic hepatitis, acute disseminated

encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison’s disease,

agammaglobulinemia, alopecia areata, Alzheimer’s disease, amyotrophic lateral sclerosis, 5 ankylosing spondylitis, anti-gbm/tbm nephritis, antiphospholipid syndrome, antisynthetase syndrome, aplastic anemia, arthritis, atopic allergy, atopic dermatitis, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenia purpura, autoimmune uveitis, balo disease/balo concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaff’s encephalitis, blau syndrome, bullous pemphigoid, Castleman's disease, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, coeliac disease, Cogan syndrome, cold agglutinin disease, cranial arteritis, crest syndrome, Crohns disease, Cushing's syndrome, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, Dressler's syndrome, discoid lupus erythematosus, eczema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressive, fibromyalgia, fibromyositis, fibrosing aveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-barre syndrome (gbs), Hashimoto’s encephalitis, Hashimoto's thyroiditis, henoch-schonlein purpura, hidradenitis suppurativa, Hughes syndrome, inflammatory bowel disease (IBD), idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, iga nephropathy, inflammatory demyelinating polyneuopathy, interstitial cystitis, irritable bowel syndrome (ibs), Kawasaki's disease, lichen planus, Lou Gehrig’s disease, lupoid hepatitis, lupus erythematosus, meniere's disease, microscopic polyangiitis, mixed connective tissue disease, morphea, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neuromyotonia, occular cicatricial pemphigoid, opsoclonus myoclonus syndrome, ord thyroiditis, Parkinson’s disease, pars planitis, pemphigus, pemphigus vulgaris, pernicious anaemia, polymyalgia rheumatic, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, raynaud phenomenon, relapsing polychondritis, Reiter’s syndrome, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome, 6 spondyloarthropathy, sticky blood syndrome, still's disease, stiff person syndrome, sydenham chorea, sweet syndrome, takayasu’s arteritis, temporal arteritis, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson’s syndrome, Wiskott-Aldrich syndrome as well as hypersensitivity reactions of the skin, atherosclerosis, ischemia- reperfusion injury, myocardial infarction, and restenosis.

[00023] Still other embodiments relate to a method of generating TolDCs. The method can include isolating bone marrow from the subject. The isolated bone marrow is then cultured with GM-CSF and IL-4 to generate a population of immature dendritic cells. The method further includes activating Nrf2 signaling and/or suppressing NfkB signaling and/or disrupting Keapl expression in the immature dendritic cells to generate the population of tolerogenic dendritic cells.

[00024] In some embodiments, Keap 1 expression can be disrupted by deleting at least a portion of Keapl gene to inhibit expression or function of Keapl in immature dendritic cells. For example, Keapl expression in the dendritic cells can be disrupted by administering to the dendritic cells at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRIS PR) -associated endonuclease, and at least one guide RNA (gRNA) having a spacer sequence complementary to a target sequence in a Keap DNA.

[00025] In other embodiments, Nrf2 signaling can be activated and/or NfkB signaling can be suppressed by administering to the immature dendritic cells an amount of triterpenoid effective to generate the population of tolerogenic dendritic cells. In some embodiments, the triterpenoid is at least one of CDDO-IM, CDDO-2P-IM, or CDDO-3P-IM.

[00026] In still other embodiments, the triterpenoid can be administered to the immature dendritic cells in combination with GM-CSF and/or LPS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0001] Figs. l(A-B) illustrates image and plots showing BMDC generation and characterization by CD1 lc. (A) BMDCs were expanded from hematopoietic progenitors isolated from C57BL/6 mice. The observation of cluster formation through the microscope has demonstrated during the period of differentiation (all images- 50x) (B) BMDCs were -7- harvested on day 7 and analyzed by flow cytometry for CD1 lc expression. Graphs depict the percentage of the expanded CD1 lc+ cell population.

[0002] Fig. 2 illustrates plots showing DC cell surface ligand expression is unaltered by CDDO-DFPA. Cells were pre-treated in the presence or absence of CDDO-DFPA (200 nM) for 1 hour prior to stimulation with LPS (100 ng/ml) for 24 hrs. Cell surface expression of CD80, CD86, MHC II, and PD-L1 was analyzed by flow cytometry.

[0003] Figs. 3(A-F) illustrate graphs showing CDDO-DFPA altered the genetic and protein phenotype of immunogenic DCs. BMDCs were pre-treated in the presence or absence of CDDO-DFPA (50-400 nM) for 1 hour prior to addition of LPS (100 ng/ml), and either harvested for RNA extraction (4 hrs.) or allowed to condition culture medium for 24 hrs. prior to collection for cytokine analyses. The levels of IFN-y (A), IL-12 (B), EDN-1 (C), TNFoc (D), IL-6 (E), and IL-23 (F) were measured by qRT-PCR and ELISA. The results are expressed as mean ± S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t-test.

[0004] Figs. 4(A-D) illustrate graphs and an immunoblot showing CDDO-DFPA induced TolDCs phenotype confirmed by gene and protein expression. BMDCs were pre treated in the presence or absence of CDDO-DFPA (10-400 nM) for 1 hour prior to addition of LPS (100 ng/ml), and cells were harvested for RNA extraction after 24 hrs. The levels of IL-4 (A), IL-10 (B), and TGF-b (C) were measured by qRT-PCR. (D) Cell protein lysate (12 hrs.) were collected for analyses and levels of HO-1, and b-actin expression were analyzed by Western blotting. The results are expressed as mean ± S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t-test.

[0005] Fig. 5 illustrates plots showing CDDO-DFPA exposed DCs suppress T cell proliferation. DCs were pre-treated with CDDO-DFPA (100-400 nM) for 1 hour only, then washed and co-cultured with CFSE stained T cells at a 1:10 ratio. Splenic T cells and DCs were isolated from C57BL/6 OTII transgenic mice and C57BL/6 mice, respectively. CDDO- DFPA pretreated DCs were co-cultured with CFSE stained T cells with (w/) or without (w/o) OVA addition during incubation. T cell proliferation was determined by flow cytometry at day 2. Graphs depict the percentage of dividing T cells relative to numbers T cell division. The data is a representation of 3 independent experiments. 8

[0006] Figs. 6(A-F) illustrate plots and graphs showing the characterization of mitochondrial function of Nrf2^+/+ and Nrf2^_/ DCs. Nrf2^+/+ or Nrf2^_/ BMDCs were pretreated in the presence or absence of CDDO-DFPA (400 nM) for 1 hour before exposure to LPS (10 ng/ml) for 24 hours. (A) Schematic representation of real-time mitochondrial respiration. OCR analysis included 4 consecutive stages, starting from basal respiration and after the addition of oligomycin (mitochondrial complex V inhibition), FCCP (maximal respiration induction), and rotenone/antimycin A (ETC inhibition). (B) Representative kinetic study of mitochondrial OCR (pmol/min) in Nrf2^+/+ DCs (light blue), Nrf2^+/+ DCs+LPS (blue), and Nrf2^+/+ DCs+LPS+CDDO-DFPA (purple) with sequential addition of oligomycin, FCCP, and rotenone/antimycin A. (C) OCR quantification of basal respiration, ATP production, maximal respiration, and spare capacity of Nrf2^+/+ DCs (light blue), Nrf2^+/+ DCs+LPS (blue), and Nrf2^+/+ DCs+LPS+CDDO-DFPA (purple). (D) Representative kinetic study of mitochondrial OCR (pmol/min) in Nrf2 ^/_ DCs (red), Nrf2^_/ DCs+LPS (yellow), and Nrf2^_/

DCs+LPS+CDDO-DFPA (brown) with sequential addition of oligomycin, FCCP, and rotenone/antimycin A. (E) OCR quantification of basal respiration, ATP production, maximal respiration, and spare capacity of Nrf2 ^/_ DCs (red), Nrf2^_/ DCs+LPS (yellow), and Nrf2^_/ DCs+LPS+CDDO-DFPA (brown). (F) OCR quantification of basal respiration, ATP production, maximal respiration, and spare capacity of Nrf2+/+ (light blue) and Nrf2-/- DCs (red). The results are expressed as mean + S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t-test.

[0007] Figs. 7(A-F) illustrate plots and graphs showing the Characterization of the glycolytic function of Nrf2^+/+ and Nrf2^_/ DCs. Nrf2^+/+ or Nrf2^_/ BMDCs were pretreated in the presence or absence of CDDO-DFPA (400 nM) for 1 hour before exposure to LPS (10 ng/ml) for 24 hours. (A) Schematic representation of a real-time glycolysis. The ECAR assay consisted 4 stages, starting from basal (glucose-free) followed by the addition of glucose (glycolysis induction), oligomycin (maximal glycolysis induction), and 2-DG

(glycolysis inhibition). (B) Representative kinetic study of glycolytic ECAR (mpH/min) in Nrf2^+/+ DCs (light blue), Nrf2^+/+ DCs+LPS (blue), and Nrf2^+/+ DCs+LPS+CDDO-DFPA (purple) with sequential addition of glucose, oligomycin, and 2-DG. (C) ECAR quantification of basal, glycolysis, glycolytic capacity, and glycolytic reserve of Nrf2^+/+ DCs (light blue), Nrf2^+/+ DCs+LPS (blue), and Nrf2^+/+ DCs+LPS+CDDO-DFPA (purple). (D) Representative kinetic study of glycolytic ECAR (mpH/min) in Nrf2 ^/_ DCs (red), Nrf2^_/ DCs+LPS (yellow), -9- and Nrf2^_/ DCs+LPS+CDDO-DFPA (brown) with sequential addition of glucose, oligomycin, and 2-DG. (C) ECAR quantification of basal, glycolysis, glycolytic capacity, and glycolytic reserve of Nrf2 ^/_ DCs (red), Nrf2^_/ DCs+LPS (yellow), and Nrf2^_/

DCs+LPS+CDDO-DFPA (brown). The results are expressed as mean + S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t-test.

[0008] Figs. 8(A-D) illustrate immunoblots and graphs showing Nrf2 activation in DCs reduced LPS-derived iNOS expression and NO production. Nrf2^+/+ or Nrf2 ^/_ BMDCs were pretreated in the presence or absence of CDDO-DFPA (100 and 200 nM for Western blotting and 50, 100, and 200 nM for qRT-PCR) for 1 hour before exposure to LPS (10 ng/ml) for 6 or 24 hours. (A) Total cellular lysates were analyzed for iNOS, HO-1, Nrf2, and b-actin expression by Western blotting. Experiments were repeated a minimum of three times. (B & C) Cells were harvested for RNA extraction. The levels of iNOS (B) and Hmox-1 (C) were measured by qRT-PCR. (D) Conditioned medium was collected for NO production by analysis of nitrite levels. The results are expressed as mean + S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-treated groups. Unpaired student t- test.

[0009] Figs. 9(A-K) illustrate plots and images showing TolDC therapy ameliorated the symptoms of AA mice. TolDCs were administered by i.v. on day 0, 3, and 5 following injection of LN cells to the recipient mice. (A) Survival curve for AA mice. Kaplan-Meier survival curve followed by the Mantel-Cox log-rank test within 30 days (n = 7-8 mice in each group). *P < 0.05. Representative data of complete blood counts, including WBC (B), Hb (C), HCT (D), and PLT (E) at indicated time points after injection of LN cells. All data are presented as the mean + S.E.M. *P < 0.05. Multiple t-tests with Holm-Sidak analysis. Mice irradiated only (IR only, green), LN cells injected mice (AA, blue), AA mice with TolDC administration (AA+TolDCs, red). (F) Sternums were collected on day 14 for H&E and IHC examination. Representative sections of Normal, IR only, AA, and AA+TolDCs mice stained with H&E to assess BM cellularity and hemorrhage. Infiltration of T cells was assessed by CD3 antibody. iNOS, Nrf2, and HO-1 expression were also analyzed by IHC. A pathologist blinded to subject identity scored sections taken from each animal for H&E (G), T cell (H) infiltration, iNOS (I), Nrf2 (J), HO-1 (K) expression. Scale bars = 50 pm. Quantification data 10 were presented as the mean ± S.E.M. (n = 3 mice in Normal and IR only group n = 8 mice in AA and AA+TolDCs group). ***P < 0.001, Unpaired student t-test.

[00010] Figs. 10(A-N) illustrate plots showing TolDC therapy regulated the cell proliferation and differentiation in spleen and BM of AA mice. TolDCs were administered by i.v. on day 0, 3, and 5 after injection of LN cells to the recipient mice. Mice were euthanized on day 10 or day 14 for harvesting splenocytes and BM cells, respectively. The proliferation of CD4 and CD8 T cells in the spleen (A) and BM (D) were measured by flow cytometry. Cells were stimulated with PMA/ionomycin/Golgistop for 4 hours and subjected to flow cytometry to determine the differentiation of Thl7 and Treg subsets among CD4+ T cells based on their expression of IL-17 and CD25/Foxp3, respectively (B, C, and E). The population of HSCs was measured as lin Vc-Kit⁺ cells in BM (F). Quantification of data in spleen (G) and BM (H) was presented as the mean (n = 2-5 mice in each group). *P < 0.05, **P < 0.01, One-way ANOVA with the Bonferroni corrections.

[00011] Figs. 1 l(A-G) illustrate images and plots showing BM biopsies from AA patients exhibited the milieu for immunogenic DCs. (A) Biopsies from AA patients and healthy donors were collected for H&E and IHC examination. Representative sections of AA patient and healthy donor were stained with H&E to assess BM cellularity and hemorrhage. Infiltration of T cells and DCs was assessed by CD3 and CDl lc antibodies, respectively. iNOS, Nrf2, and HO-1 expression were also analyzed by IHC. A pathologist blinded to subject identity scored sections taken from each sample for H&E (B), T cell (C) and DC (D) infiltration, iNOS (E), Nrf2 (F), and HO-1 (G) expression. Scale bars = 50 pm. Quantification data were presented as the mean ± S.E.M. (n = 4 patients in each group). ***P < 0.001, unpaired student t-test.

[00012] Fig. 12 illustrates images showing the pro -inflammatory immune

microenvironment in BM of patients with SAA. The bone marrow (BM) of SAA patients (top row) exhibits high iNOS, low Nrf2 and low HO-1 relative to the BM of normal patients (bottom row).

[00013] Fig. 13 illustrates a schematic multifunctional Triterpenoid activators of Nrf2. Small molecules in the triterpenoid family activate Nrf2 via reaction with Keapl, protecting Nrf2 from degradation. They also regulate NFkB signaling through direct binding to TkB.

[00014] Fig. 14 illustrates a schematic showing the experimental design. CByB6Fl mice will be exposed to a sublethal dose (5Gy) of total body irradiation by a 137 cesium g source 11 irradiator 6 hours before intravenous (i.v.) injection of 5 x 10⁶ LN cells isolated from

C57BL/6 mice. Treatment groups receive (i.v.) 5 x 10⁶ TolDCs on day 0, 3, 5, 9, 12, and 18. Mice in both control and treatment groups are bled from the tail tip at different time points to measure blood counts using a Hemavet 950 analyzer (Drew Scientific). In some

experiments, mice will be euthanized to collect splenocytes and BM cells for analyses at day 10 and 14, respectively. Sternums will be collected on day 14 for hematoxylin and eosin (H&E) and IHC examination in each experiment.

[00015] Figs. 15(A-D) illustrate a schematic and graph showing molecular and phenotypic characterization of Keapl-KO dendritic cells. (A) The immature BMDC were harvested at day7 and (2xl0e⁵ cells) were transfected with CRISPR Keapl RNP by Neon Electroporator with the condition of 1500V/30 ms/ 1 pulse. Next day, the transfected DCs were treated with 200 nM of CDDO-DFPA(DFPA), CDDO-IM(IM), or CDDO-3P-IM(3P) 1 hour prior to EPS (lOOng/ml) treatment. The treated cells were harvested for further molecular and phenotypic analyses including ICE, Flow cytometry, and ELISA assays. (B) the ICE analysis have shown that CRISPR Keapl.75 RNP induced 85% indel mutations (ICE score) and estimated to have 79% of Keapl-KO (KO score). (C) The phenotype of Keapl- KO DCs( CD1 lc, CD86, and MHCII) was characterized by flow cytometry and Flowjo analysis. The classical DC markers(% of parent) were not significantly changed by induction of Keapl-KO. (D)The secretion of pro -inflammatory cytokine (TNFa) and the anti inflammatory cytokine (IL-10) by Keapl-KO DC were compared to that of control DCs treated with CDDO and LPS as indicated.

[00016] Figs. 16(A-C) illustrate a schematic, graph, and table showing an optimized protocol for tolerogenic dendritic cell production. (A)The schematic diagram shows the optimized method for dendritic cell differentiation and production of CLM18.3 (B)The immature DCs were harvested on day 8 and treated again in Celloram’s induction cocktail for another day (C) The optimized protocol yielded iDC, mDC, and TolDC(CLM18.3) on day 9 as indicated. The cell number seeded at day 0 was set to 100%.

[00017] Figs. 17(A-D) illustrate graphs and plots showing purity, activity, and cytokines of CLM18.3 (A) CLM18.3 showed > 80% CDl lc+ and slight reduction of CD80, CD86, and MHCII. (B) CLM18.3 expressed low TNF-a, IL-12, but high TGF-b. (QCLM18.3 suppressed OVA-peptide specific T cell proliferation compared to control DC._ As a proof- 12 of-principle study, CRISPR-ko of Keapl also suppressed T cell proliferation (D) The Keapl- ko DC secrets low TNF-cr, IL-12, but high TGF- ? and IL-10.

[00018] Figs. 18(A-E) illustrate plots showing CLM-18.3 TolDCs exhibit unique transcriptome signatures. (A) Antigen Processing and Presentation Pathways and (B) Allograft Rejection Pathways. CLM18.3(18.3); mature DC (mDC) (C) MHC-I gene expression, (D) MHC-II gene expression, (E) the gene expression significantly related to tolerogenic dendritic cell function

[00019] Figs. 19(A-E) illustrate a schematic, graphs, and plots showing CLM18.3 significantly improved clinical scores and survival in mouse GvHD model (A) The experimental plan of GVHD model (B) CD4 and CD8 T cell subset analysis in each treatment group. (C) The level of TGF- ? in serum at day 14 and day 30 (D)The clinical score of each group measured by 5 clinical criteria (weight, posture, activity, Fur, and skin) (E)The Kaplan-Meier survival curve showed the survival benefit of CLM18.3 treated group.

[00020] Figs. 20(A-E) illustrate a schematic, graphs, and plots showing CLM18.3 significantly prolonged the survival in mouse aplastic anemia model (A) The experimental plan of aplastic anemia model (B)The Kaplan-Meier survival curve showed the survival benefit of CLM18.3 treated group in contrast to aplastic anemia group(red). (C) The level of TGF- ? and IL-10 in serum at day 17 (D) Blood counting showed the reconstitution of blood in each group (White blood cell, Hematocrit, platelet)(E) CD4 and CD8 T cell subset analysis in each treatment group(Left) and the preservation of Lin cKit⁺ hematopoietic stem cell population in bone marrow (right).

DETAILED DESCRIPTION

[00021] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

Commonly understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Edition, Springer- Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994. The -13- definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present application.

[00022] The term "dendritic cell" refers a special antigen-presenting cell presenting various antigen samples along with MHC Class I complex or MHC Class II complex to a T cell by absorbing an antigen in cells. The dendritic cells may be divided into the immature dendritic cells and mature dendritic cells according to the expression level of the surface phenotype or maturity. The expression profile of surface markers of the dendritic cells may be easy through a flow cytometry assay that is known in the art.

[00023] The term "immature dendritic cells," refers to dendritic cells, in which the cells are found at the initial mature state of the dendritic cells, CD 14 that is a surface phenotype of a mononuclear cell is not expressed, and CD40, CD80, and CD86 that are a co-stimulatory molecule are expressed in a low level.

[00024] The term, "mature dendritic cells," means the cells, in which the immature dendritic cells are matured, and then the mature dendritic cells are formed. The mature dendritic cells have ability capable of inducing an immune reaction by increasing the expressions of MHC class II, CD40, CD80, and CD86, releasing a pro-inflammatory cytokine, and then activating a naive T cell.

[00025] The term“Tolerogenic dendritic cells" or "TolDCs" refers to dendritic cells capable of suppressing immune responses or generating tolerogenic immune responses, such as polyclonal or antigen- specific regulatory T-cells and/or B-cells or suppressive T cell- mediated immune responses. Tolerogenic DCs can be characterized by specific tolerogenic immune response induction ex vivo and/or in vivo.

[00026] Embodiments described herein relate to tolerogenic dendritic cells (TolDCs), methods of generating a population of TolDCs, the use of TolDCs in increasing immune tolerance, and to the use of the TolDCs in treating inflammatory and immune disorders in a subject in need thereof.

[00027] It was found that TolDCs can be generated from dendritic cells through targeted activation of the nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2). Targeted activation of Nrf2 is a novel approach to metabolic reprogramming of dendritic cells (DCs), which acquire a stable, immune suppressive orTolerizing’ phenotype. Unlike mature DCs (mDCs), the dominant anti-inflammatory signature of TolDCs is characterized by reduced expression -14- of cell surface stimulatory ligands, decreased secretion of immunosuppressive cytokines and a distinct cellular metabolic profile that regulates T cell polarization.

[00028] Under quiescent conditions, Nrf2 is anchored in the cytoplasm through binding to Kelch-like ECH-associated protein (Keapl), which, in turn, facilitates the ubiquitination and proteolysis of Nrf2. It was found that disruption of Nrf2 binding to Keapl in DCs, a key mechanism for the repressive effects of Keapl on Nrf2, can activate Nrf2 and confer a tolerogenic phenotype to DCs.

[00029] Nrf2 activation of the DCs can be increased and/or promoted in several ways including: direct activation of Nrf2 (e.g., by using small molecules); inhibition of genes that express Keapl (e.g., by blocking the expression or activity of the genes and/or proteins); activation of genes and/or proteins that inhibit one or more of, the activity and function of Keapl (e.g., by increasing the expression or activity of the genes and/or proteins);

introduction of genes and/or proteins that negatively regulate the binding of Nrf2 to Keapl (e.g., by using recombinant gene expression vectors, recombinant viral vectors or

recombinant polypeptides); gene replacement with, for instance, a hypomorphic mutant of Keapl (e.g., by homologous recombination, overexpression using recombinant gene expression or viral vectors, or mutagenesis), or genetic or pharmacologic manipulation of Tgf-b, smad7, and other targets of nfkB signaling.

[00030] In some embodiments, TolDCs described herein can include DCs that are administered an Nrf2 activator.

[00031] As used herein, the term "Nrf2 Activator" means an agent that after

administration results in a stimulated and/or increased nuclear translocation of Nrf2 protein and causes the subsequent increases in expression of one or more ARE-regulated genes by acting directly on Nrf2, Keapl, and or the Nrf2-Keapl complex.

[00032] Nrf2 Activators may comprise a Michael addition acceptor, one or more fumaric acid esters, i.e., fumaric acid mono- and/or diesters which may be selected from the group of monoalkyl hydrogen fumarate and dialkyl fumarate, such as monomethyl hydrogen fumarate, dimethyl fumarate, monoethyl hydrogen fumarate, and diethyl fumarate, ethacrynic acid, bardoxolone methyl (methyl 2-cyano-3,12-dioxooleana-l,9(l l)dien-28-oate), isothiocyanate such as sulforaphane, l,2-dithiole-3-thione such as oltipraz, 3,5-di-tert-butyl-4- hydroxytoluene, 3-hydroxycoumarin, 2-cyano-3,12-dioxoolean-l,9-dien-28-oic acid and its methyl (CDDO-Me, bardoxolone methyl) and imidazolide (CDDO-Im) derivatives, pyridyl -15- analogues thereof, such as l-[2-Cyano-3,12-dioxooleana-l,9(l l)-dien-28-oyl]-4(-pyridin-2- yl)- 1 H-imidazole (CDDO-2P-Im), 1 -[2-Cyano-3 , 12-dioxooleana- 1,9(11 )-dien-28-oyl] -4(- pyridin-3-yl)-lH-imidazole (CDDO-3P-Im), triterpenoid 2-cyano-3,12-dioxooleana-l,9- dien-28-oic acid-difluoro-propyl-amide, (CDDO-DFPA, RTA-408) or a pharmacologically active derivative or analog of the aforementioned agents.

[00033] Nrf2 activators may be classified based on their chemical structures: Diphenols, Michael reaction acceptors, isothiocyanates, thiocarbamates, trivalent arsenicals, 1,2-dithiole- 3-thiones, hydroperoxides, vicinal dimercaptans, heavy metals, polyenes, and triterpenoids.

In general, Nrf2 Activators are chemically reactive in that they may be electrophiles, substrates for glutathione transferases, and/or can modify sulfhydryl groups by alkylation, oxidation, or reduction.

[00034] In another embodiment, Nrf2 Activators are compounds that bond covalently to Keapl protein, such as by a sulfur atom of an amino acid residue of Keapl.

[00035] In another embodiment, the Nrf 2 Activators are bardoxolone methyl and dialkyl fumarate such as dimethyl fumarate and diethyl fumarate.

[00036] In another embodiment, Nrf2 activators are selected from: Chalcone derivatives such as 2-trifluoromethyl-2'-methoxychalcone, auranofin, ebselen, 1,2-naphthoquinone, cynnamic aldehyde, caffeic acid and its esters, curcumin, reservatrol, artesunate, tert- butylhydroquinone, and -quinone, (tBHQ, tBQ), vitamins Kl, K2 and K3, menadione, fumaric acid esters, i.e. fumaric acid mono- and/or diester which may be selected from the group of monoalkyl hydrogen fumarate and dialkyl fumarate, such as monomethyl hydrogen fumarate, dimethyl fumarate (DMF), monoethyl hydrogen fumarate, and diethyl fumarate, 2- cyclopentenones, ethacrynic acid and its alkyl esters, bardoxolone methyl (methyl 2-cyano- 3,12-dioxooleana-l,9(l l)dien-28-oate) (CDDO-Me, RTA 402), ethyl 2-cyano-3, 12- dioxooleana- 1,9(1 l)dien-28-oate, 2-cyano-3, 12-dioxooleana- 1,9(1 l)dien-28-oic acid

(CDDO), l[2-Cyano-3,12-dioxooleana-l,9(l l)-dien-28-oyl]imidazole (CDDO-Im), (2- cyano-N-methyl-3,12-dioxooleana-l,9(l l)-dien-28 amide (CDDO-methyl amide, CDDO- MA), l-[2-Cyano-3,12-dioxooleana-l,9(l l)-dien-28-oyl]-4(-pyridin-2-yl)-lH-imidazole (CDDO-2P-Im) , 1 - [2-Cy ano-3 , 12-dioxooleana- 1,9(11 )-dien-28 -oyl] -4(-pyridin-3 -yl)- 1 Id- imidazole (CDDO-3P-Im), isothiocyanate such as sulforaphane, l,2-dithiole-3-thione such as oltipraz, 3,5-di-tert-butyl-4-hydroxytoluene, 3-hydroxycoumarin, 4-hydroxynonenal, 4- oxononenal, malondialdehyde, (E)-2-hexenal, capsaicin, allicin, allylisothiocyanate, 6- -16- methylthiohexyl isothiocyanate, 7-methylthioheptyl isothiocyanate, sulforaphane, 8- methylthiooctyl isothiocyanate, corticosteroids, such as dexamethasone, 8-iso prostaglandin A2, alkyl pyruvate, such as methyl and ethyl pyruvate, diethyl or dimethyl oxaloproprionate, 2-acetamidoacrylate, methyl or ethyl-2-acetamidoacrylate, hypoestoxide, parthenolide, eriodictyol, 4-hydroxy-2-nonenal, 4-oxo-2nonenal, geranial, zemmbone, aurone,

isoliquiritigenin, xanthohumol, [10]-Shogaol, eugenol, l'-acetoxychavicol acetate, allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, 4-(methylthio)-3-butenyl isothiocyanate and 6-methylsulfinylhexyl isothiocyanate, ferulic acid and its esters, such as ferulic acid ethyl ester, and ferulic acid methyl ester, sofalcone, 4-methyl daphnetin, imperatorin, auraptene, poncimarin, bis[2-hydroxybenzylidene]acetones, alicylcurcuminoid, 4-bromo flavone, b-naphthoflavone, sappanone A, aurones and its corresponding indole derivatives such as benzylidene-indolin-2-ones, perillaldehyde, quercetin, fisetin, koparin, genistein, tanshinone HA, BHA, BHT, PMX-290, AL-1, avicin D, gedunin, fisetin, andrographolide, and tricyclic bis(cyano enone) TBE-31 [(+/-)-(4bS,8aR,10aS)-10a-ethynyl- 4-b,8,8-trimethyl-3,7-dioxo-3,4-b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile], as well as other triterpenoids disclosed in U.S. Pat. Nos. 6 6,326,507, 6,552,075, 6,974,801,

7,288,568, 7,863,327, 7,915,402, 7,943,778, 8,034,955, 8,071,632, 8,124,656, 8,124,799, 8,129,429, 10,501,420, US 2009/0060873, US 2009/0048204, WO 2008/136838, WO

2009/023232, and WO 2009/146216 all of which are incorporated by reference in their entirety.

another embodiment, Nrf2 activators are selected from: carnosic acid,

[00037] 2-naphthoquinone, cynnamic aldehyde, caffeic acid and its esters, curcumin, reservatrol, artesunate, tert-butylhydroquinone, vitamins Kl, K2 and K3, fumaric acid esters, i.e., fumaric acid mono- and/or diester which is preferably selected from the group of monoalkyl hydrogen fumarate and dialkyl fumarate, such as monomethyl hydrogen fumarate, dimethyl fumarate, monoethyl hydrogen fumarate, and diethyl fumarate, isothiocyanate such as sulforaphane, l,2-dithiole-3-thione such as oltipraz, 3,5-di-tert-butyl-4-hydroxytoluene, 3- hydroxycoumarin, 4-hydroxynonenal, 4-oxononenal, malondialdehyde, (E)-2-hexenal, capsaicin, allicin, allylisothiocyanate, 6-methylthiohexyl isothiocyanate, 7-methylthioheptyl isothiocyanate, sulforaphane, 8-methylthiooctyl isothiocyanate, 8-iso prostaglandin A2, alkyl pyruvate, such as methyl and ethyl pyruvate, diethyl or dimethyl oxaloproprionate, 2- acetamidoacrylate, methyl or ethyl-2-acetamidoacrylate, hypoestoxide, parthenolide, -17- eriodictyol, 4-Hydroxy-2-nonenal, 4-oxo-2nonenal, geranial, zerumbone, aurone,

isoliquiritigenin, xanthohumol, [10]-Shogaol, eugenol, l'-acetoxychavicol acetate, allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, 4-(Methylthio)-3-butenyl isothiocyanate and 6-methylsulfinylhexyl isothiocyanate and the respective quinone or hydroquinone forms of the aforementioned quinone and hydroquinone derivatives.

[00038] In another embodiment, Nrf2 Activators may be Michael reaction acceptors such as dimethylfumarate, monomethyl hydrogen fumarate isothiocyanates and 1,2-dithiole- 3-thiones. In another embodiment, Nrf2 Activators are selected from monomethyl hydrogen fumarate, dimethyl fumarate, oltipraz, 1,2-naphthoquinone, tert-butylhydroquinone, methyl or ethyl pyruvate, 3,5-di-tert-butyl-4-hydroxytoluene, diethyl and dimethyl oxaloproprionate, hypoestoxide, parthenolide, eriodictyol, 4-Hydroxy-2-nonenal, 4-oxo-2nonenal, geranial, zerumbone, aurone, isoliquiritigenin, xanthohumol, [10]-Shogaol, eugenol, l'- acetoxychavicol acetate, allyl isothiocyanate, benzyl isothiocyanate, phenethyl

isothiocyanate, 4-(Methylthio)-3-butenyl isothiocyanate and 6-Methylsulfinylhexyl isothiocyanate.

[00039] In other embodiments, a TolDCs described herein can include DCs that are genetically modified to inhibit, suppress, and/or disrupt expression of Keapl. The inhibition, suppression, and/or disruption of Keapl can include a deletion of at least a portion of Keapl gene to inhibit expression or function of Keapl including Keapl binding to Nrf2.

[00040] In some embodiments, the Keapl expression in the DCs can be disrupted by gene editing. Gene editing means for inhibiting, disrupting, and/or suppressing Keapl expression can include RNA-guided CRISPR technology. In a CRISPR system, CRISPR clusters encode spacers, which are sequences complementary to target sequences

("protospacers") in a viral nucleic acid, or in another nucleic acid to be targeted. CRISPR clusters are transcribed and processed into mature CRISPR RNAs (crRNAs). CRISPR clusters also encode CRISPR associated (Cas) proteins, which include DNA endonucleases. The crRNA binds to target DNA sequence, whereupon the Cas endonuclease cleaves the target DNA at or adjacent to the target sequence.

[00041] One useful CRISPR system includes the CRISPR associated endonuclease Cas9. Cas9 is guided by a mature crRNA that contains about 20-30 base pairs (bp) of spacer and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease Ill-aided processing of pre-crRNA. The crRNA TracrRNA duplex directs Cas9 to target DNA via -18- complementary base pairing between the spacer on the crRNA and the target sequence on the target DNA. Cas9 recognizes a trinucleotide (NGG) photospacer adjacent motif (PAM) to decide the cut site (the 3^rd nucleotide from PAM). The crRNA and tracrRNA can be expressed separately or engineered into an artificial chimeric small guide RNA (sgRNA) via a synthetic stem loop (AGAAAU) to mimic the natural crRNA/tracrRNA duplex. Such sgRNAs, can be synthesized or in vitro transcribed for direct RNA transfection, or they can be expressed in situ, e.g., from U6 or Hl-promoted RNA expression vectors. The term "guide RNA" (gRNA) will be used to denote either a crRNA TracrRNA duplex or an sgRNA. It will be understood the term "gRNA complementary to" a target sequence indicates a gRNA whose spacer sequence is complementary to the target sequence.

[00042] Other CRISPR systems that can be used include CRISPR/Cpfl, which is a DNA-editing technology analogous to the CRISPR/Cas9 system, characterized in 2015 by Feng Zhang's group from the Broad Institute and MIT. Cpfl is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpfl genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpfl is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. Cpfl is further described below.

[00043] In one embodiment, the CRISPR-associated endonuclease is a Cas9 nuclease. The Cas9 nuclease can be a sequence from Staphylococcus aureus. The Cas9 nuclease can also have a nucleotide sequence identical to the wild type Streptococcus pyogenes sequence. In some embodiments, the CRISPR-associated endonuclease can be a sequence from other species, for example, other Streptococcus species, such as Thermophiles; Psuedomonas aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microogranisms. Alternatively, the wild type Streptococcus pyogenes Cas9 sequence can be modified. For example, the nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, i.e., "humanized." A humanized Cas9 nuclease sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1 GL669193757; KM099232.1 GL669193761; or KM099233.1 GL669193765. Alternatively, the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as PX330 or PX260 from Addgene (Cambridge, Mass.). In some embodiments, the Cas9 -19- endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GL669193757; KM099232.1 GL669193761; or KM099233.1 GL669193765 or Cas9 amino acid sequence of PX330 or PX260 (Addgene, Cambridge, Mass.).

[00044] The Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas9 by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations).

One or more of the substitution mutations can be a substitution (e.g., a conservative amino acid substitution). For example, a biologically active variant of a Cas9 polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a wild type Cas9 polypeptide. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

[00045] The amino acid residues in the Cas9 amino acid sequence can be non-naturally occurring amino acid residues. Naturally occurring amino acid residues include those naturally encoded by the genetic code as well as non-standard amino acids (e.g., amino acids having the D-configuration instead of the L-configuration). The present peptides can also include amino acid residues that are modified versions of standard residues (e.g., pyrrolysine can be used in place of lysine and selenocysteine can be used in place of cysteine). Non- naturally occurring amino acid residues are those that have not been found in nature, but that conform to the basic formula of an amino acid and can be incorporated into a peptide. These include D-alloisoleucine(2R,3S)-2amino-3-methylpentanoic acid and L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid. For other examples, one can consult textbooks or the worldwide web (a site is currently maintained by the California Institute of Technology and displays structures of non-natural amino acids that have been successfully incorporated into functional proteins).

[00046] The Cas9 nuclease sequence can be a mutated sequence. For example, the Cas9 nuclease can be mutated in the conserved HNH and RuvC domains, which are involved in strand specific cleavage. For example, an aspartate-to-alanine (D10A) mutation in the RuvC 20 catalytic domain allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single- stranded breaks, and the subsequent preferential repair through HDR22 can potentially decrease the frequency of unwanted InDel mutations from off-target double- stranded breaks.

[00047] In addition to the wild type and variant Cas9 endonucleases previously described, the methods described herein can also encompass CRISPR systems including "enhanced-specificity" S. pyogenes Cas9 variants (eSpCas9), which dramatically reduce off- target cleavage. These variants are engineered with alanine substitutions to neutralize positively charged sites in a groove that interacts with the non-target strand of DNA. This modification reduces interaction of Cas9 with the non-target strand, thereby encouraging re hybridization between target and non-target strands. The effect of this modification is a requirement for more stringent Watson-Crick pairing between the gRNA and the target DNA strand, which limits off-target cleavage.

[00048] Especially preferred are three variants found to have the best cleavage efficiency and fewest off-target effects: SpCas9(K855a), SpCas9(K810A/K1003A/rl060A) (a.k.a. eSpCas9 1.0), and SpCas9(K848A/K1003A/R1060A) (a.k.a. eSPCas9 1.1). Techniques for cloning and inducing cellular expression of these enhanced-specificity variants are well known. It will be appreciated that other Cas9 variants are known and the method described herein are not limited to the Cas9 variants described herein.

[00049] In some embodiments, gene editing compositions can include a CRISPR- associated endonuclease polypeptide encoded by any of the nucleic acid sequences described above. Polypeptides can be generated by a variety of methods including, for example, recombinant techniques or chemical synthesis. Once generated, polypeptides can be isolated and purified to any desired extent by means well known in the art. For example, one can use lyophilization following, for example, reversed phase (preferably) or normal phase HPLC, or size exclusion or partition chromatography on polysaccharide gel media such as Sephadex G- 25. The composition of the final polypeptide may be confirmed by amino acid analysis after degradation of the peptide by standard means, by amino acid sequencing, or by FAB -MS techniques.

[00050] In some embodiments, an engineered CRISPR system includes Cas9 and one or more gRNAs complementary to a Keapl sequence. 21

[00051] The inhibition, disruption, and/or suppression of Keapl in the DCs and TolDCs can also be performed using siRNA, miRNAs (micro-RNAs), shRNAs (short hairpin RNAs), or RNAis (RNA interference) that target critical RNAs (mRNA) that translate (non-coding or coding) proteins involved with the formation or expresssion of Keapl. The siRNA, miRNAs, shRNAs, or RNAi can be included in the expression vectors described herein along with the gene editing compositions. These RNA interference approaches are there to suppress the expression of Keapl.

[00052] shRNAs or siRNAs can be used to produce short double stranded RNA molecules which are processed by Dicer and single stranded RNA base-pairs with a target mRNA. Argonaute proteins then assist with mRNA degradation or translation inhibition.

This results in post transcriptional down-regulation of gene expression but does not change the genetic code.

[00053] shRNA is double stranded RNA created from a DNA construct encoding a sequence of single stranded RNA and its complement that are separated by a stuffer fragment that allows the RNA molecule to fold back on itself to create a hairpin loop. shRNA can come in two different designs of a simple stem-loop and a microRNA adapted shRNA. A simple stem-loop shRNA has a 50-70 nucleotide transcript that forms a stem-loop structure consisting of a 19 to 29 bp region of double stranded RNA (the stem) bridged by a region of predominantly single-stranded RNA (the loop) and a dinucleotide 3' overhang. A microRNA adapted shRNA is greater than 250 nucleotides and more closely resembles native pri- microRNA molecules and consists of a shRNA stem structure which may include

microRNA-like mismatches, bridged by a loop and flanked by 5' and 3' endogenous microRNA sequences.

[00054] Use of shRNA in RNAi instead of siRNA can be preferred as it has a low rate of degradation and turnover. siRNA can have variable transfection efficiencies that limits siRNA-mediated RNAi to only those cells capable of transfection. After the vector has integrated into the host genome, shRNA is transcribed in the nucleus by polymerase II or polymerase III. Also, shRNA can be delivered into mammalian cells through infection with viral vectors unlike siRNA.

[00055] In some embodiments, an effective amount of Nrf2 actiator and/or a gene composition directed against Keapl can be administered to DCs obtained from a subject being treated, i.e., autologous DCs and/or from another subject, i.e., allogenic DCs. The DCs 22 can be isolated from a subject ( e.g ., bone marrow) or generated from precursor DCs, in bone marrow or peripheral blood. Techniques known to one skilled in the art may be used to obtain/generate DCs from bone marrow and/or peripheral blood mononuclear cells. Cells isolated from the bone marrow or blood, including hematopoietic progenitor cells and monocytes, of a patient may be cultured in the presence of factors, such as the combination of GM-CSF and IL-4, IL-13, IL-15 and IFN-a, or Flt3L, to differentiate into immature DCs after a period of, e.g., 4 to 5 days. In some embodiments, isolated bone marrow cells can be cultured in the presence of GM-CSF and IL-4 to generate a population of immature DCs.

[00056] Techniques known to one skilled in the art can be used to assess/confirm the presence of immature DCs. For example, the presence of dendritic cells can be

assessed/confirmed by detecting the expression of DC surface markers using techniques, such as FACS.

[00057] An effective amount of a composition including an agent that activates Nrf2 (Nrf2 activator) and/or isolated nucleic acid encoding a CRISPR-associated endonuclease with at least one isolated nucleic acid encoding at least gRNA including a spacer sequence complementary to a target sequence in a Keapl DNA can be administered to the immature DCs. The Nrf2 activator can include an amount of a triterpenoid effective to generate the population of tolerogenic dendritic cells. In some embodiments, the triterpenoid is at least one of CDDO-IM, CDDO-2P-IM, or CDDO-3P-IM.

[00058] In still other embodiments, at least one or more of lipopolysaccharides (LPS), rapamycin, corticosteroids, IL-10, vitamin D3, dexamethasone, BAY 11-7085, and, optionally, GM-CSF, can be administered in combination with the triterpenoid to induce DC maturation along with DC tolerance. For example, LPS and GM-CSF can be administered in combination with the triterpenoid to induce DC maturation along with DC tolerance.

[00059] The CRISPR-associated endonuclease and the at least one gRNA can be expressed in the DC of the patient which can include, but not limited to human KEAP1 gRNA for chrl9: 10500014 (+); chrl9:10499916(-) ; chrl9:10499891(-) ; chrl9: 10499865 (+) ; chrl9:10499821(-). The CRISPR-associated endonuclease can be any of those gene editors described above. The siRNA, miRNAs, shRNAs, or RNAi can also be included in the composition. The target sequence in the Keapl genome can then be cleaved disrupting the Keapl genome. Disrupting the Keapl can suppress Keapl expression, and promote Nrf2 activation generating the TolDCs described herein. -23-

[00060] In some embodiments, the TolDCs generated by administration of an agent that activates Nrf2 and/or by disruption of Keapl expression or binding to Nrf2 of the DCs can have a phenotype characterized by the production of multiple immune suppressive cytokines, including IL-4, IL-10 and TGF-b, high levels of HemeOxygenase-1 (HOI) and low levels of inducible nitric oxide synthase (iNOS) (/._<?., HO-l^Hl,iNOS^low expression) with decreased NO production, promoting expansion of regulatory (suppressor) T cells, suppression of T cell activation and production of TNFa, IFN-g, and IL-12 in human mixed lymphocyte reaction (MLR) assays, exhibition of a shift from glycolytic metabolism to oxidative phosphorylation (OXPHOS) during ex vivo TolDC expansion, and/or activation of Nrf2 target gene expression.

[00061] In some embodiments an agent that activates Nrf2 can be used in combination with with gene editing to disrupt Keapl expression or binding to Nrf2 of the DCs. Nrf2 activators and Keapl expression inhibitors may have complementary mechanisms of action. Administration of each agent alone may result in generation of TolDCs, but, because each agent may cause this generation of TolDCs through separate mechanisms, one agent may increase the baseline sensitivity of the system to the other agent. Thus, agents with complimentary mechanisms of action may act such that the therapeutically effective dose of either agent or both may be reduced relative to mono-therapy doses. Further, the combined therapeutically effective dose of both agents may be less than an additive substitution of one agent for the other. Put another way, the therapeutic effect when the Keapl inhibitor and the Nrf2 Activator are used together may be more than additive, i.e., greater than the sum of the effects that result from using each agent alone.

[00062] In an embodiment, the combined use of a Keapl expression inhibitor and an Nrf2 activator may eliminate, reduced incidence, or reduce severity of adverse effect(s) associated with use of the the Nrf2 activator as a mono-therapy. In another embodiment, the combined use of Keapl expression inhibitor and an Nrf2 Activator may reduce the dose of one or both of the agents employed in the combination treatment, and, the side effect(s) that may be observed in mono-therapy with the agents may be avoided or reduced. For example, dimethyl fumarate may potentially cause reduction in white cell count, flushing, redness, itching, skin rash, nausea, vomiting, diarrhea, stomach or abdominal pain, indigestion, and/or dyspepsia when administered in therapeutically effective amounts. -24-

[00063] The TolDCs described herein can be used in methods of treating various immune conditions and disorders. For example, compositions comprising TolDCs can be used in conjunction with tissue or organ transplantation for improving graft tolerance, prolonging survival of a transplanted tissue or organ, and treating graft- versus-host disease.

[00064] In addition, TolDCs described herein can be used for decreasing inflammation such as caused by an autoimmune disease, allergic response, neurodegenerative disease, a cardiovascular disease, damaged tissue, or a wound. Inflammatory conditions and autoimmune diseases that may be treated with TolDCs by the methods described herein can include, but are not limited to multiple sclerosis (MS), rheumatoid arthritis (RA), post- traumatic arthritis, reactive arthritis, psoriasis, pemphigus vulgaris, Sjogren's disease, autoimmune thyroid disease (AITD), Hashimoto's thyroiditis, myasthenia gravis, diabetes mellitus type 1, stomatitis, lupus erythematosus, acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic dermatitis, autoimmune aplastic anemia, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hemolytic anemia, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Balo disease/Balo concentric sclerosis, Behcet's disease, Berger's disease, Bickerstaffs

encephalitis, Blau syndrome, Bullous pemphigoid, Castleman's disease, celiac disease,

Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's Syndrome, cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diffuse cutaneous systemic sclerosis, Dressler's syndrome, eczema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressiva, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid, glomerulonephritis, Goodpasture's syndrome, Graves' disease, -25-

Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy, Henoch-Schonlein purpura, gestational pemphigoid, hidradenitis suppurativa, Hughes-Stovin syndrome,

hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, IgA nephropathy, inclusion body myositis, chronic inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), lupoid hepatitis, Majeed syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease, microscopic colitis, myositis, narcolepsy,

neuromyelitis optica, neuromyotonia, occular cicatricial pemphigoid, opsoclonus myoclonus syndrome, Ord's thyroiditis, palindromic rheumatism, PANDAS, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonage-Turner syndrome, Pars planitis, pemphigus vulgaris, pernicious anaemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, relapsing polychondritis, Reiter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, sarcoidosis, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, serum sickness, Sjogren's syndrome, spondyloarthropathy, Still's disease, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, thrombocytopenia, Tolosa- Hunt syndrome, transverse myelitis, undifferentiated connective tissue disease,

undifferentiated spondyloarthropathy, urticarial vasculitis, vasculitis, vitiligo, Wegener's granulomatosis, autoimmune cardiomyopathy, ischemic heart disease, atherosclerosis, cancer, fibrosis, inflammatory bowel disease, inflammatory myopathy, giant cell arteritis (GCA), asthma, allergy, Parkinson's disease, schizophrenia, and Alzheimer's disease.

[00065] In other embodiments, the TolDCs described herein can be administered to a subject to mitigate bone marrow graft rejection, to enhance bone marrow graft engraftment, to enhance engraftment of a hematopoietic stem cell graft, or an umbilical cord blood stem cell graft, to enhance engraftment of a hematopoietic stem cell graft, or an umbilical cord stem cell graft, and/or to decrease the number of units of umbilical cord blood required for 26 transplantation into the subject. The administration can be, for example, following treatment of the subject or the marrow of the subject with radiation therapy, chemotherapy, or immunosuppressive therapy.

[00066] In other embodiments, the TolDCs described herein can be administered to a recipient of a bone marrow transplant, of a hematopoietic stem cell transplant, or of an umbilical cord blood stem cell transplant, in order to decrease the administration of other treatments or growth factors.

[00067] In still other embodiments , the TolDCs described herein can be administered to a subject to enhance recovery following bone marrow transplantation, following umbilical cord blood transplantation, following transplantation with hematopoietic stem cells, following conventional chemotherapy, following radiation treatment, and in individuals with anemias from diseases that include but are not limited to aplastic anemia, myelodysplasia, myelofibrosis, anemia from other bone marrow diseases, drug induced anemia, immune mediated anemias, anemia of chronic disease, idiopathic anemia, and following infections with viruses that include, but are not limited to, HIV, CMV, and parvovirus.

[00068] In other embodiments, the subject has aplastic anemia, myelodysplasia, myelofibrosis, thrombocytopenia due to other bone marrow diseases, drug induced thrombocytopenia, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenia, or thrombocytopenia following viral infections, neutropenia due to other bone marrow diseases, drug induced neutropenia, autoimmune neutropenia, idiopathic neutropenia, or neutropenia following viral infections, drug induced cytopenias, immune cytopenias, cytopenias following viral infections, or cytopenias.

[00069] In still other embodiments, the subject has aplastic anemia, myelodysplasia, myelofibrosis, anemia due to other disorder of bone marrow, drug induced anemia, immune mediated anemias, anemia of chronic disease, anemia following viral infections, or anemia of unknown cause.

[00070] In some embodiments, the TolDCs described herein, a composition(s) comprising such stable TolDCs, or combination therapies are administered to a subject suffering from or diagnosed with an autoimmune disease, graft rejection or graft-versus-host disease. In other embodiments, TolDCs described herein, a composition(s) comprising such stable TolDCs, or combination therapies are administered to a subject predisposed or susceptible to developing an autoimmune disease, graft rejection or graft-versus-host disease. -27-

[00071] In some embodiments, TolDCs described herein, a composition(s) comprising such TolDCs, or combination therapies are administered to a mammal. In certain

embodiments, TolDCs described herein, a composition(s) comprising such TolDCs, or combination therapies are administered to a mammal which is 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 10 to 15 years old, 15 to 20 years old, 20 to 25 years old, 25 to 30 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old.

[00072] The TolDCs described herein or a composition(s) comprising such stable TolDCs can be administered via any route known in the art. TolDCs described herein or a composition(s) comprising such TolDCs can be administered by, for example, infusion or bolus injection, and may be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known and can be used to deliver TolDCs described herein or a composition(s) comprising such TolDCs.

[00073] Methods of administration include but, are not limited to, parenteral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous or intracerebral. In a specific embodiment, TolDCs described herein or a composition(s) comprising such TolDCs are/is intravenously, intradermally or subcutaneously administered to the patient. In another specific embodiment, TolDCs described herein or a composition(s) comprising such TolDCs are/is administered to the patient by direct intranodal delivery. The mode of administration is left to the discretion of the practitioner.

[00074] In specific embodiments, it may be desirable to administer TolDCs described herein or a composition(s) comprising such TolDCs locally. In specific embodiments, TolDCs described herein or a composition(s) comprising such TolDCs are/is administrated at the site of the autoimmune disease, graft rejection or graft- versus-host disease by local infusion. For example, in the case of rheumatoid arthritis, TolDCs described herein or a composition(s) comprising such stable TolDCs can be administrated directly intra-articularly.

[00075] The amount TolDCs described herein, or the amount of a composition comprising TolDCs, that will be effective in the treatment of an autoimmune disease, graft rejection or graft-versus-host disease can be determined by standard clinical techniques. In vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. -28-

The precise dose to be employed will also depend, e.g., on the route of administration, the type of symptoms, and the seriousness of the symptoms, and should be decided according to the judgment of the practitioner and each patient's or subject's circumstances.

[00076] Doses of TolDCs for administration to a subject by any route of administration can be at least 100, 200, 300, 400, 500, 700, 1,000, 5,000, 10,000, 25,000, 50,000, or 100,000 cells. In specific embodiments, the number of TolDCs is at least 100, 200, 300, 400, 500 cells. In other embodiments, the number of TolDCs is at least 300, 400, 500, 700, 1,000 cells. In yet other specific embodiments, the number of TolDCs is at least 700, 1,000, 5,000, 10,000 cells. In some embodiments, the number of TolDCs at least 5,000, 10,000, 25,000, 50,000, or 100,000 cells. In yet another embodiment, the number of TolDCs is at least 50,000, or 100,000 cells. In other embodiments, the number of TolDCs is at least 1 xlO⁶, 5 x 10⁶, 1 x 10⁷, 5 x 10⁷, 1 x 10⁸, 5 x 10⁸ or more cells. In specific embodiments, the number of stable semi-mature tolDCs is between 1 x 10² to 1 x 10⁴, 5 x 10⁴ to 5 x 10⁶, 1 x 10⁵ to 1 x 10⁷, 1 x 10⁵ to 5 x 10⁸, 1 x 10⁶ to 1 x 10⁸, or 1 x 10⁶ to 1 x 10⁷, or 1 x 10⁴ to 1 x 10⁵ cells.

[00077] In certain embodiments, a subject is administered TolDCs described herein or a composition thereof in an amount effective to inhibit or reduce symptoms associated with the autoimmune disease, graft rejection or graft-versus-host disease by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In certain embodiments to treat, a subject is administered TolDCs described herein or a composition thereof in an amount effective to inhibit or reduce symptoms associated with the autoimmune disease, graft rejection or graft-versus-host disease by at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, or 2- to 5-fold, 2- to 10-fold, 5- to 10-fold, or 5- to 20-fold relative to a negative control as determined using an assay described herein or other known to one of skill in the art.

[00078] In certain embodiments to, a subject is administered TolDCs described herein or a composition thereof in an amount effective to decrease an autoimmune response or graft rejection by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to -29-

80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In some embodiments, a subject is administered TolDCs described herein or a composition thereof in an amount effective to decrease an autoimmune response or graft rejection by at least 1.5-fold, 2-fold, 2.5-fold, 3- fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, or 2 to 5-fold, 2 to 10-fold, 5 to 10-fold, or 5 to 20-fold relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[00079] In certain embodiments, a dose of TolDCs described herein or a composition thereof is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks or once a month, or less. In other embodiments, two, three or four doses of TolDCs described herein or composition thereof is administered to a subject every day, every couple of days, every third day, once a week or once every two weeks. In some embodiments, a dose(s) of TolDCs described herein or a composition thereof is administered for 2 days, 3 days, 5 days, 7 days,

14 days, 21 days, 28 days or 31 days. In certain embodiments, a dose of TolDCs described herein or a composition thereof is administered for 0.5 month, 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.

[00080] The dosages of prophylactic or therapeutic agents which have been or are currently used for the treatment of autoimmune diseases, graft rejection, or graft-versus-host disease can be determined using references available to a clinician such as, e.g., the

Physicians' Desk Reference (68th ed. 2014).

[00081] The above-described administration schedules are provided for illustrative purposes only and should not be considered limiting.

Example 1

[00082] In this example we provide a detailed protocol with step by step method to isolate immature dendritic cells (iDCs) from hematopoietic progenitors of mice and analyze the efficacy of any interest of agents in converting these iDCs into TolDCs by their functional and phenotypic characterization in vitro and in vivo. This is an elaborate method to characterize the TolDCs by their surface ligands, cytokine profile, and immunosuppressive functions in vitro. -30-

PROTOCOL

Preparation of bone marrow-derived dendritic cells (BMDCs)

[00083] 1. Use appropriate method to euthanize C57BL/6 mice._ Isolate and clean tibia-fibula and femur bones with 70% ethanol.

[00084] 2. Trim both ends of tibias and femurs. Use 3 ml PBS in 3 ml syringe with a 23G needle to flush the contents of marrow from one end of bones to a conical tube containing 9 ml PBS. Repeat this step 3 times for each end of bones.

[00085] 3. Centrifuge the cell suspension at 300 x g for 5 mins.

[00086] 4. Remove the supernatant and re-suspend the cell pellet with 1 ml ACK lysing buffer for 5 mins.

[00087] 5. Add 9 ml PBS to dilute ACK lysing buffer and centrifuge at 300 x g for 5 mins.

[00088] 6. Remove the supernatant and resuspend with 10 ml culture medium

(RPMI-1640 plus L-glutamine, 10% FBS, 1% non-essential amino acid (100X), 10 mM HEPES, 50 nM b-mercaptoethanol, and 5% penicillin/streptomycin). Note: Endotoxin level has to be less than 0.1 EU/ml in FBS.

[00089] 7. Pass the cell suspension through a 40 mhi cell strainer.

[00090] 8. Adjust the cell number to 1 x 10⁶ cells/ml with 15 ng/ml granulocyte- macrophage colony- stimulating factor (GM-CSF) and 10 ng/ml IL-4.

[00091] 9. Plate 3 ml of 1 x 10⁶ cells/ml in each well of 6-well plate and incubate the cells at 37°C, 5% CO2 and 95% humidity in the CO2 incubator.

[00092] 10. On day 3, remove all 3 ml culture medium from each well, add 2 ml fresh PBS in each well, and then gently swirl the plate to ensure removing all the non adherent cells.

[00093] 11. Replace 3 ml fresh culture medium with 15 ng/ml GM-CSF and 10 ng/ml IL-4 in each well.

[00094] 12. On day 5, directly add another 3 ml fresh culture medium with 15 ng/ml GM-CSF and 10 ng/ml IL-4 in each well. The total volume in each well is now 6 ml.

[00095] 13. On day 7, to harvest BMDCs, gently pipette the culture medium in each well to dislodge the loosely-adherent BMDCs into suspension. The adherent macrophages are still attached to the plate. -31-

[00096] 14. Centrifuge the cell suspension at 300 x g for 5 mins and resuspend with fresh culture medium for further experiments. BMDCs can be identified with fluorescence-labeled CD 11c antibody by flow cytometry.

Characterize TolDC gene and protein profile

[00097] 1. Plate 2 ml of 1 x 10⁶ BMDCs/ml per well in 6 well-plate with culture medium in the presence or absence of 100-400 nM CDDO-DFPA for incubating 1 hr. Other agents for induction of TolDCs can be applied at this step, such as IL-10, vitamin D3, dexamethasone or BAY 11-7085.

[00098] 2. Add 10 or 100 ng/ml of LPS for incubating 4-24 hrs. (incubation time is different due to mRNA or protein measurement).

[00099] 3. Harvest the cell suspension and centrifuge at 300 x g for 5 mins to collect the cells and supernatant, respectively.

[000100] 4. TolDCs can be directly analyzed for the cell surface ligands by flow cytometry, such as stimulatory ligands: CD40, CD80, CD86, MHC-II, OX40L, ICOSL, or inhibitory ligands: PD-L1, PD-L2, ILT3, ILT4.

[000101] 5. The RNA extraction and supernatant from TolDCs can be analyzed for the cytokine profile at gene and protein level by quantitative real-time PCR (qRT-PCR) and ELISA, respectively. For example, inflammatory cytokines: TNF-oc, IFNy, EDN-1, IL-6, IL- 12, and IL-23, or anti-inflammatory cytokines: IL-4, IL-10, IL-15, TGF-b, and HO-1.

Evaluate the function of TolDCs in vitro and in vivo

T-cell syngeneic proliferation assay

[000102] 1. To obtain splenic CD4+ T cells, use the appropriate method to euthanize OT-II TCR transgenic mice and isolate the spleen in PBS.

[000103] 2. Use the back of push-stick of 3 ml syringe to mince the spleen by passing a 40 mhi cell strainer.

[000104] 3. Collect the cell suspension and centrifuge at 300 x g for 5 mins.

[000105] 4. Resuspend the cell pellet in 400 mΐ of MACS buffer (PBS, 0.5% BSA, and 2 mM EDTA).

[000106] 5. Add 100 mΐ of CD4+ T Cell Biotin-Antibody Cocktail (Miltenyi Biotec

Inc.) at 4°C for 5 mins. -32-

[000107] 6. Add 300 mΐ of MACS buffer and 200 mΐ of Anti-Biotin MicroBeads

(Miltenyi Biotec Inc.) at 4°C for 10 mins.

[000108] 7. Place LS Column and Pre-Separation Filter (Miltenyi Biotec Inc.) together in the magnetic field and rinse it with 3 ml of MACS buffer.

[000109] 8. Add 9 ml of MACS buffer in the cells and centrifuge at 300 x g for 5 mins.

[000110] 9. Resuspend the cell pellet in 3 ml of MACS buffer and apply onto the

LS Column. Collect flow-through containing CD4+ T cells.

[000111] 10. Wash column with another 3 ml of MACS buffer and also collect the flow-through.

[000112] 11. To obtain splenic Pan DCs, use the appropriate method to euthanize

C57BL/6 mice and isolate the spleen in 2 ml of collagenase D solution (2 mg/ml collagenase D dissolved in HBSS containing calcium, magnesium).

[000113] 12. Inject 1 ml of collagenase D solution to the spleen two times by a 1 ml syringe and a 25G needle. Cut the spleen into small pieces with small scissors.

[000114] 13. Shake and incubate at room temperature for 25 mins.

[000115] 14. Add 500 mΐ of 0.5 M EDTA at room temperature for 5 mins.

[000116] 15. Resuspend the cell pellet in 350 mΐ of MACS buffer, 50 mΐ of FcR

Blocking Reagent (Miltenyi Biotec Inc.), and 100 mΐ of Pan Dendritic Cell Biotin- Antibody Cocktail (Miltenyi Biotec Inc.) at 4°C for 10 mins.

[000117] 16. Wash the cells by adding 9 ml of MACS buffer and centrifuge at 300 x g for 5 mins.

[000118] 17. Resuspend the cell pellet in 800 mΐ of MACS buffer and add 200 mΐ of

Anti-Biotin MicroBeads at 4°C for 10 mins.

[000119] 18. Wash column with another 3 ml of MACS buffer two times and also collect the flow-through.

[000120] 19. Treat the DCs in the presence or absence of 100-400 nM CDDO-

DFPA at 37°C for 1 hr. and also label the CD4+ T cells with 1 mM CFSE at 37°C for 15 mins.

[000121] 20. Culture IOOmI of 2 x 10⁴DCs and IOOmI of 2 x 10⁵ T cells (1:10 ratio) in a 96-well plate with 100 ng/ml of ovalbumin (OVA) peptide 323-329. -33-

[000122] 21. After 2 days, measure the CFSE intensity of T cells by flow cytometry.

REPRESENTATIVE RESULTS÷

The differentiation and selection of BMDCs

[000123] Bone marrow progenitor cells were cultured in complete RPMI medium in the presence of GM-CSF and IL-4 to differentiate into iDCs for 7 days (Fig. 1A). On day 1, cells were in small size and showed spherical morphology. Washing with PBS before the replacement of fresh medium on Day 3 helped cells forming clusters and increased CD1 lc+ cell population. On day 4, BMDCs were enlarged in size and initiated the cluster formation. Adhered macrophages were also converted and observed at the bottom of the plate with an elongated shape. On day 5 large size of clusters of BMDCs are formed. On day 6, a large number of semi-adherent and floating BMDCs were also observed. BMDCs were harvested on day 7 and analyzed by flow cytometry for CD1 lc expression as a specific marker of murine DCs. As shown in a representative flow cytometry plot in Fig. IB, around 83.6% of BMDCs expressing CD1 lc were obtained by this method.

Induction and genetic characterization of TolDCs

[000124] Some of the TolDC-induced agents, such as vitamin D3 and dexamethasone, have shown the down-regulated expression of DC surface ligands, including MHC II and costimulatory molecules, CD40, CD80, and CD86. However, in LPS or CD40L-induced maturation of DCs, calcineurin inhibitors cyclosporin A and FK506 showed no effect on CD83, CD80, CD86, and MHC II expression. As evident by flow cytometry analysis, our TolDC-induced agent, CDDO-DFPA also didn’t reveal any significant effect on LPS-induced surface ligand expression of DCs, including MHC II, CD80, CD86, and PD-L1. (Fig. 2).

[000125] In addition, a comparison of BMDCs exposed to LPS with or without CDDO- DFPA by qRT-PCR and ELISA showed that CDDO-DFPA treatment significantly reduced the BMDC expression of pro-inflammatory cytokine genes such as IFN-g, IL-12, EDN1, TNFa, IL-6, and IL-23 induced by LPS activation (Figure 3A-3F). Both IFN-g and IL-12 are necessary for Thl cell differentiation. The latter two (IL-6, and IL-23) are necessary for Thl7 cell differentiation. BMDCs treated with CDDO-DFPA also showed increased expression of anti-inflammatory cytokine genes such as IL-4, IL-10, TGF-b and HO-1 (Figs. we 4A-4D). -34-

IL-4 promotes the differentiation of CD4 T cells toward the Th2 phenotype, whereas IL-10 and TGF-b are known to exert anti-inflammatory activity and suppress autoimmunity through mechanisms that include the induction of Treg. It is noteworthy that the distinctive IL- 12-;IL-10+ cytokine production profile, the inhibition of EDN-1, and induction of HO-1 expression induced by CDDO-DFPA, are all known to authenticate DCs tolerogenic function.

Cellular and functional characterization of TolDCs In vitro

[000126] DCs promote T cell proliferation through their engagement of costimulatory ligands and through the elaboration of cytokines and other soluble mediators. Our data thus far suggest that CDDO-DFPA has the capacity to modulate the T cell response by altering gene expression and function of DCs. Therefore, using in vitro model of syngeneic stimulation, we examined how CDDO-DFPA modified DC-mediated T cell proliferation. Isolated DCs were pretreated with CDDO-DFPA and washed prior to co-culture with CFSE stained T cells with OVA peptide. We found that TolDCs, induced by CDDO-DFPA significantly suppressed the T cell proliferation (Fig. 5).

[000127] This Example describes an efficient protocol to generate iDCs and differentiate them into TolDCs. We analyzed the TolDC surface ligands by flow cytometry and then the cytokine profile was measured by qRT-PCR and ELISA. Later, the immunoregulatory function of TolDCs was confirmed by ensuring their capacity to reduce T cell proliferation.

In the end, the functional utility of these TolDC were evaluated by testing them into preclinical murine model of MS, EAE.

[000128] The iDCs were generated and differentiated from bone marrow precursors of mice with the combination of GM-CSF and IL-4. Other protocols have used forms -like tyrosine kinase 3 ligands (Flt3L) in the culture medium to generate iDCs. Although, the use of Flt3L increases the yield of iDCs, these iDCs usually take 2 more days (9 days) to harvest, compared to GM-CSF/IL-4 addition (7 days). More importantly, iDC generated from Flt3L induces the differentiation of both cDCs and pDCs. But GM-CSF/IL-4 induce the differentiation of iDC more toward to cDCs only. It has been shown that iDCs generated from these two methods produce morphologically different cells, which represent different surface marker, and cytokine profile upon their activation. Furthermore, their migration ability, and antigen- specific T cell responses also vary. Since, GM-CSF/IL-4 induced -35-

BMDCs are superior at T cell stimulation and the production of inflammatory mediators following LPS treatment, we found it more suitable in our experiments.

[000129] In this protocol, CDDO-DFPA (synthetic triterpenoid) and LPS were added as a DC tolerance and maturation inducer, respectively. We found that after harvesting iDCs from BMDC generation method, pretreatment with CDDO-DFPA induced the tolerogenic DC phenotype. Unlike CDDO-DFPA, which was added in the culture after iDC harvest (day 7), vitamin D3 has also been shown and could be added in the culture during the iDC differentiation (day 2, 4, 6). It has been described that adding vitamin D3 results in diverse mechanisms both during and after iDC differentiation. Adding CDDO-DFPA during the iDC differentiation showed no difference in the purity of CD11C+ DCs, but dose-dependently lowered the yield of iDCs on day 7 (data not shown). Our data suggest that the concentration of CDDO-DFPA we used may be toxic to bone marrow progenitor cells, even though iDC showed normal viability with the concentration of CDDO-DFPA. -On the other hand, iDCs can also be matured by other inducers than LPS, such as CD40L, TNF-a, and IFN-g.

However; DC maturation by LPS through Toll-like receptors 4(TLR4) leads the activation of several transcription factors, including nuclear factor-kB (NF-KB), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal- regulated protein kinase (ERK1/2). The maturation of DCs by CD40L and TNF-a through CD40 and TNF receptors, respectively induces the NF-KB pathway. Whereas, IFN-g stimulates a different pathway including the activation of Janus kinase (JNK), tyrosine kinase (TYK), and signal transducer and activator of transcription proteins (STATs). But the downstream effects are also complemented by NF-KB pathway. In addition, the gene expression profile of CD40L/TNF-a-based DC maturation suggests that these DCs polarize T cells toward to Th2 cell response. Furthermore, it has also been confirmed that DC maturation by IFN-g is strongly biased towards a Thl cell response. Therefore, other DC maturation inducers for any specific T cell subsets differentiation are not considered in this protocol.

Example 2

[000130] In this Example, we investigate the role of Nrf2 in TolDCs by its genetic (Nrf2 ^/_ ) or pharmacological (CDDO-DFPA) manipulation in these cells. _ Here, we show that Nrf2 regulates DC tolerance by modulating their cytokine profile and cellular metabolism. -36-

CDDO-DFPA-induced Nrf2 activation, resulted in a significant anti-inflammatory transcriptome response, enhanced HO-1 expression, suppressed NO production, and a metabolic shift from glycolysis to OXPHOS in DCs. Our data correlates with

immunohistochemical (IHC) analyses of BM biopsies of severe aplastic anemia (AA) patients, in which the invariably high iNOS and low Nrf2 expression is in striking contrast to normal BM donors. Finally, administration of CDDO-DFPA-induced TolDCs in a murine BMF model of AA increased animal survival rate, BM cellularity, and hematopoiesis. In these mice, TolDC therapy not only regulated the proliferation of DCs, T cells, and HSCs but also retained the Thl7/Treg balance. These results reveal the Nrf2-dependent mechanisms of TolDC induction and highlight their therapeutic utility in the treatment of A A and other autoimmune diseases.

Methods

Patient information

[000131] BM biopsies were obtained from patients diagnosed with severe AA according to the International AA Study Group criteria. Inherited BMF syndromes and paroxysmal nocturnal hemoglobinuria were ruled out. These biopsies were obtained under an IRB- approved protocol and used for IHC staining.

Animals

[000132] C57BL/6 and BALB/c mice were inbred and then crossbred (C57BL/6 x

BALB/c) to generate FI (CByB6Fl) mice. Colonies of OT-II T cell receptor (TCR) transgenic and Nrf2^_/ mice colonies were maintained for use in the in vitro and in vivo assays described in this report. All studies were performed in compliance with procedures approved by the Case Western Reserve University School of Medicine’s Institutional Animal Care and Use Committee.

BM-derived dendritic cells (BMDCs) preparation

[000133] Cells were isolated from BM of C57BL/6 or Nrf2^_/ mice and were differentiated into BMDCs. In brief, isolated BM cells were cultured for 7 days in RPMI-1640 plus L- glutamine medium containing 10% FBS, 50 nM b-mercaptoethanol, and 5%

penicillin/streptomycin with GM-CSF (15 ng/ml) and IL-4 (10 ng/ml). At the end of this -37- culture period, cells were harvested and CDl lc expression (80-85% of the expanded cell population) was confirmed by FACS analysis.

Quantitative real-time PCR (qRT-PCR)

[000134] Total RNA was isolated from cells using RNAqueous®-Micro Total RNA Isolation Kit (ThermoFisher Scientific Inc.) and subjected to cDNA synthesis using

Superscript® III CellsDirect™ cDNA Synthesis Kit (ThermoFisher Scientific Inc.). qRT- PCR of individual genes was performed on CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Inc.) using the primers and probes (Applied Biosystems Inc.): TNFa (Mm00443258), IFN-g (Mm01168134_ml), IL-12a (Mm00434165), IL-4

(Mm99999154_ml), IL-10 (Mm01288386_ml), TGF-bI (Mm01178820_ml), iNOS (Mm00440502_ml), and Hmox-1 (Mm00516005_ml). The results were generated and normalized to GAPDH (Mm99999915).

T-cell proliferation assay

[000135] Splenic CD4⁺ T cells were isolated from OT-II TCR transgenic mice using a CD4⁺ T Cell Isolation Kit (Miltenyi Biotec Inc.) and labeled with CFSE as described previously. Splenic DCs were isolated from C57BL/6 mice using a Pan Dendritic Cell Isolation Kit (Miltenyi Biotec Inc.). Both DCs and T cells were co-cultured at 1:10 ratio in presence or absence of 100 ng/ml of ovalbumin (OVA) peptide 323-329 (InvivoGen Inc.). T cell proliferation was measured by analyzing CFSE intensity by flow cytometry after 72 hours.

Metabolism assays

[000136] The OXPHOS and glycolysis level of DCs were analyzed through assessment of mitochondrial oxygen consumption rate (OCR, pmol/min) and extracellular acidification rate (ECAR, mpH/min), respectively by an XFp extracellular flux analyzer (Agilent

Technologies). For OCR studies, 6 x 10⁴ BMDCs/well were treated for 1 hour with or without CDDO-DFPA followed by LPS (10 ng/ml) for 24 hours in poly-D-lysine coated XFp Cell Culture Miniplates. Cells were centrifuged and washed with assay medium (XF Base Medium, 10 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine, pH 7.4) and incubated in a non-C0₂ 37°C incubator for 45 mins before analysis. A complete OCR study was performed in 4 consecutive stages: basal respiration, mitochondrial complex V inhibition -38-

(1 mM oligomycin), maximal respiration induction (0.5 mM carbonyl cyanide-4- (trifluoromethoxy)phenylhydrazone [FCCP]), and electron transportation chain (ETC) inhibition (0.5 mM rotenone/antimycin A). For ECAR assays, cells were washed in assay medium (XF Base Medium, 2 mM L-glutamine, pH 7.4). A complete ECAR assay consisted of 4 stages: basal, glycolysis induction (10 mM glucose), maximal glycolysis induction (1 mM oligomycin), and glycolysis inhibition (50 mM 2-deoxy-D-glucose [2-DG]). The quantified results for OXPHOS and glycolysis were generated by Report Generator (Wave software, version 2.3).

Western blot analysis and NO production measurement

[000137] Total cell lysates were prepared with RIPA buffer containing protease inhibitors. Western blot analysis was performed using iNOS, HO-1, Nrf2, and b-actin (control) antibodies (Santa Cruz Biotechnology) as previously described. NO production was measured as nitrite from DC culture supernatants using the colorimetric NO assay kit (ThermoFisher Scientific Inc), according to the manufacturer’s protocol.

A A murine model and TolDC treatment

[000138] Inguinal, brachial, axillary, and mesenteric lymph nodes (LNs) were extracted from C57BL/6 mice and filtered through a 40-pm nylon mesh to obtain a single-cell suspension. About 5 x 10⁶ of these LN cells were then infused by intravenous (i.v.) injection in 8-10 week-old CByB6Fl mice exposed to a sublethal dose (5 gray [Gy]) total body irradiation by a ¹³⁷cesium g source irradiator (J.L. Shepherd) 6 hours before injection, as previously described. The treatment group received (i.v.) 5 x 10⁶ TolDCs (CByB6Fl BMDCs treated with 400 nM CDDO-DFPA for 1 hour followed by 10 ng/ml LPS treatment for 24 hours) on day 0, 3, and 5. Both control and treatment groups of mice were bled from the tip of the tail at different time points to measure blood counts using a Hemavet 950 analyzer (Drew Scientific). In some experiments, mice were euthanized to collect splenocytes from spleen (day 10) and BM cells from tibia and femur (day 14). Sternums were collected on day 14 for hematoxylin and eosin (H&E) and IHC examination in each experiment.

Histology and IHC

[000139] Human BM biopsies or murine sternum sections were prepared and stained for H&E or immunostained using antibodies against CD3, CDl lc, iNOS, Nrf2, or HO-1 to study -39- pathology and immune infiltration as previously described earlier. Images were acquired using a confocal microscope and combined using LSM 510 imaging software (Carl Zeiss, Inc.). Each H&E stained section was assigned a histological score for cellularity from 1 to 4 by an observer blinded to sample identity, where 1 represents normal 2- mild cellular loss, 3- moderate cellular loss, and 4- severe cytopenia. Similarly, IHC score ranged from 0 to 4 by an observer blinded to sample identity, where 0 represents negative immunostaining and 4 represents the highest number of positively immunostained cells. Because of the loss of cellularity in AA samples, the IHC was normalized to positively stained cells/100 cells.

Flow cytometry and intracellular cytokine staining

[000140] Splenocytes and BM cells were harvested from mice following AA induction and were stimulated with 50 ng/ml of PMA, 1 pg/ml of ionomycin, and 10 mg/ml of

GolgiStop (BD Bioscience). After 4 hours, cells were fixed, permeabilized (BD

bio science/eBio science Inc.), and immunostained for flow cytometry analysis. Fluorescein- conjugated monoclonal antibodies for mouse CD4, CD8, and CD25 were from BD

Biosciences, whereas IL-17, c-Kit, lineage cocktail (lin), and Foxp3 antibodies were from eBioscience. Stained cells were analyzed by a BD FACSCalibur flow cytometer (BD

Biosciences) and data were analyzed by a FlowJo software Version 10.0.7 (TreeStar).

Results

Induction of the TolDC phenotype is Nrf2-dependent

[000141] To evaluate the role of Nrf2 in regulating the DC phenotype, we adopted the loss and gain of function approach to test the modulation of the cytokine transcriptome by utilizing either Nrf2^_/ or CDDO-DFPA-treated Nrf2^+/+ DCs, respectively. When exposed to LPS, Nrf2^_/ DCs attained a more mature phenotype, in comparison to Nrf2^+/+ DCs, as evident by increased gene expression of the inflammatory cytokines IFN-g and IL-12, but not TNFoc. CDDO-DFPA treatment alone didn’t alter this cytokine response in DCs. However, when LPS-treated DCs were exposed to CDDO-DFPA, we observed suppressed TNFoc, IFN-g, and IF- 12 gene expression in Nrf2^+/+ but not Nrf2^_/ DCs.

[000142] Although EPS treatment significantly suppressed expression of the anti inflammatory cytokine IF-4 in both Nrf2^+/+ and Nrf2 ^A DCs, EPS-induced TGF-bI suppression was hampered in Nrf2^_/ DCs (Figure- IE). Both of these cytokines were restored -40- towards normalcy with increasing doses of CDDO-DFPA treatment in Nrf2^+/+ but not in Nrf2 ^/_ DCs. Since IL-10 production by LPS has been reported to be triggered by an Nrf2 independent pathway in DCs, we tested these claims by treating Nrf2^+/+ and Nrf2^_/ iDCs with CDDO-DFPA alone or in combination with LPS. We observed increased IL-10 expression in CDDO-DFPA-treated Nrf2^+/+ but not in Nrf2^_/ DCs, suggesting an Nrf2-dependent action of CDDO-DFPA. LPS-induced IL-10 expression masked the differences among the CDDO- DFPA-treated and -untreated groups, therefore we could not evaluate the role of Nrf2 in the induction of IL-10 during DC maturation (data not shown).

[000143] Since DCs regulate T cell proliferation through the modulation of cytokines and other soluble mediators, we examined the involvement of Nrf2 in DC-mediated T cell proliferation. When CFSE-stained OT-II T cells were cultured with or without OVA peptide, Nrf2^_/ DCs were able to induce higher T cell proliferation compared to Nrf2^+/+ DCs in the presence of OVA peptide. Treatment with CDDO-DFPA suppressed this DC-induced T cell proliferation at a higher rate in Nrf2^+/+ DCs cultures (62% at 200nM and 81% at 400nM CDDO-DFPA) compared to Nrf2 ^/_DCs cultures (43% at 200nM and 51% at 400nM CDDO- DFPA). These results establish an Nrf2-dependent TolDC phenotype triggered by DC exposure to CDDO-DFPA.

Nrf2-mediated dichotomous metabolic reprogramming defines DC differentiation

[000144] There is a rapidly growing interest in the role of metabolic changes in the modulation of phenotype and function of DCs. Although OXPHOS and fatty acid oxidation are two major energy sources of iDCs and TolDCs, the LPS-stimulated maturation triggers a shift towards a glycolytic metabolic state in these cells, with glucose as the preferred carbon source. This dichotomous metabolic reprogramming results in the differential cellular function of mDCs and TolDCs. In order to study the functional role of Nrf2 in this process, we examined the modulations of OXPHOS and glycolytic metabolic levels in Nrf2^+/+ and Nrf2^_/ DCs in either the presence or absence of LPS stimulation, with or without prior exposure to the synthetic triterpenoid, CDDO-DFPA. The OCR and ECAR rate of mitochondrial respiration of DCs was measured in real time as depicted in Fig. 6A and 7A.

[000145] Both Nrf2^+/+ and Nrf2^_/ DCs displayed characteristic changes of OCR in response to addition of oligomycin (for inhibition of the mitochondrial ATP-synthase), FCCP (for uncoupling of OXPHOS from ATP synthesis), and Rot/AA (for inhibition of the ETC) -41-

(Fig. 6B and 6D). Although Nrf2^+/+ DCs displayed higher levels of basal and maximal OCR following FCCP addition when compared to Nrf2^_/ DCs, they both remained unresponsive to FCCP upon LPS stimulation. CDDO-DFPA treatment partially restored the OCR level after FCCP addition in Nrf2^+/+ but not in Nrf2^_/ DCs. Quantitative analysis confirmed that after LPS treatment, Nrf2 activation significantly increased basal respiration, ATP production, maximal respiration, and spare capacity in Nrf2^+/+ DCs (Fig. 6C), whereas Nrf2^_/ DCs showed no effect (Fig. 2E).

[000146] We observed no measurable difference in ECAR between Nrf2^+/+ and Nrf2^_/ DCs following addition of glucose (for fueling glycolysis), oligomycin (for inhibition of the mitochondrial ATP-synthase), and 2-DG (for competitive inhibition of glucose) (Fig. 7B and 7D). LPS treatment triggered oligomycin-induced maximal ECAR levels in both Nrf2^+/+ and Nrf2^_/ DCs. CDDO-DFPA treatment diminished this response in Nrf2^+/+ DCs. However, in contrast, it increased ECAR levels in Nrf2^_/ DCs. Our quantitative analysis revealed that CDDO-DFPA treatment in Nrf2^+/+ DCs significantly reduced the ECAR basal level, decreased glycolysis, and restored glycolytic reserve without altering glycolytic capacity in the LPS-treated group (Fig. 7C). Nrf2 ^/_ DCs remained unresponsive to CDDO-DFPA treatment in these parameters (Fig. 7E). This data indicates that the lower energy production from glycolysis in Nrf2^+/+ DCs (treated with LPS and CDDO-DFPA) is not due to impairment of glycolytic capacity, but rather is a result of compensated energy production by OXPHOS. These results are consistent with our previous observation in Fig. 6.

[000147] In totality these data indicate that Nrf2^_/ DCs possess a more mature phenotype at a baseline. Moreover, DCs completely shut down the use of OXPHOS as an energy source during maturation and Nrf2 activation promotes a metabolic signature in TolDCs that maintains OXPHOS as the energy source. Nrf2 activation induces a TolDC phenotype which maintains the same level of glycolytic capacity as seen in mDCs, but uses less glycolysis and more OXPHOS as the energy source.

Nrf2 regulates DC metabolism through iNOS and HO-1 expression

[000148] We next examined the molecular mechanisms responsible for the Nrf2-driven metabolic reprograming of DCs and the higher OXPHOS observed in TolDCs. The functional inhibition of iNOS -derived NO has been shown to be responsible for LPS -induced OXPHOS impairment. Therefore, we analyzed the mRNA and protein expression of iNOS -42- and secreted nitrites to measure NO production in Nrf2^+/+ and Nrf2^_/ DCs treated with or without CDDO-DFPA in either the presence or absence of LPS. As expected, Nrf2^+/+ DCs showed increased Nrf2 protein expression in response to CDDO-DFPA treatment (Fig. 8A). We also observed increased mRNA and protein expression of iNOS in response to LPS treatment in both Nrf2^+/+ and Nrf2^_/ DCs. However, exposure to CDDO-DFPA diminished this response only in Nrf2^+/+ DCs in a dose dependent manner (Figs. 8A and 8B), suggesting an Nrf2-dependent action of CDDO-DFPA. It has been reported that HO-1, an Nrf2- dependent antioxidant, reduces the activity and expression of iNOS. Therefore, we next analyzed the expression of HO-1 in our experiments. We found increased Hmox-1 mRNA and HO-1 protein expression in response to CDDO-DFPA treatment in Nrf2^+/+but not in Nrf2^_/ DCs (Figs. 8A and 8B). Since HO-1 has been shown to reduce NO production, either directly through scavenging or indirectly through the inhibition of iNOS, we next examined the nitrite levels in culture supernatants of DCs stimulated with or without LPS in the presence or absence of CDDO-DFPA. Our data demonstrate that LPS-induced NO production was significantly decreased upon CDDO-DFPA treatment in a dose dependent manner, but only in Nrf2^+/+ and not in Nrf2^_/ DCs (Fig. 8D). These results suggest that activation of Nrf2 in DCs is responsible for inducing HO-1 and diminishing iNOS and NO expression to further restore the OXPHOS metabolic signature of TolDCs.

TolDCs exhibit capacity to suppress clinical pathology and symptoms in mice with AA

[000149] We next investigated the mechanism of immunoregulatory function and the therapeutic utility of TolDCs in the previously described AA murine model. In this model, the survival rate improved from 17 to 28 days in AA mice receiving TolDCs compared to the untreated control group (Fig. 9A). As expected, from day 8 to 17, we observed a significant decline in white blood cells (WBCs), hemoglobin (Hb), hematocrit (HCT), and platelets (PLTs) of AA mice when compared to the only irradiation (IR) exposed group (Fig. 9B-9E). The TolDC treatment didn’t alter either WBCs or PLTs counts (Fig. 9B and 9E), however; Hb and HCT values were significantly improved in the treated group (Figs. 9C and 9D). Histological analysis of BM collected from TolDCs-treated AA mice showed significantly improved BM cellularity and reduced areas of hemorrhage compared to untreated AA mice (Figs. 9F and 9G). Our IHC analyses also revealed a significant decline in T cell infiltration (Figs. 9F and 9H) and iNOS expression (Figs. 9F and 91) in the BM of TolDCs-treated AA -43- mice when compared to untreated AA mice. TolDCs- treated AA mice also exhibit significantly increased Nrf2 (Figs. 9F and 9J) and HO-1 (Figs. 9F and 9K) protein expression compared to untreated AA mice. These results reveal the potential therapeutic utility of TolDCs in the treatment of AA.

TolDC treatment regulates both T cell and HSC homeostasis in AA mice

[000150] Effector T cells, especially Thl7 cells, actively contribute to the destruction of HSCs in the BM during AA. In order to test the efficacy of TolDC treatment in modulating this immune response, we next analyzed the T cell repertoire and HSCs in AA mice with or without TolDC treatment. The marked expansion of both CD4⁺ and CD8⁺ T cells observed in the spleens (Figs. 10A, 10G, and 10H) and BM (Figs. 10D, 10K, and 10L) of AA mice at day 10 and 14, respectively, was significantly reduced by TolDC treatment. The dichotomy in the generation of Thl7 that induce autoimmunity and Tregs that inhibit autoimmune tissue injury has been previously reported. Moreover, a higher Thl7/Treg ratio has also been found in AA patients. Therefore, we next analyzed the abundance of populations of both Thl7 (CD4⁺IL-17⁺) and Tregs (CD4⁺CD25⁺Foxp3⁺) by cell surface phenotypes and their characteristic intracellular markers in AA mice treated with or without TolDCs. We observed a significant decrease in the Thl7 cell population in spleens (Figs. 10B and 101) and BM (Figs. 10E and 10M) as well as increased Tregs in the spleen (Figs. IOC and 10J) of TolDCs- treated mice when compared to untreated AA mice. Finally, we analyzed the HSC population in BM of these mice by using lin and c-kit⁺ as cell surface markers. As expected, AA mice had lower numbers of HSCs when compared to normal mice, but TolDC treatment restored these HSC populations and showed significantly higher lin and c-kit⁺ cells compared to the untreated group (Figs. 10F and 10N). These results provide the first scientific evidence of the therapeutic utility of TolDCs converted ex vivo by CDDO-DFPA exposure, and specifically their capacity to ameliorate disease severity in an AA murine model by regulating the T cell homeostasis.

Evidence for suppressed Nrf2 signaling in the hypoplastic BM of AA patients associated with an immunogenic DC milieu

[000151] AA is characterized by a marked expansion of T cells and immunogenic DCs in blood and BM. In order to investigate the significance of Nrf2 signaling in BM

microenvironment in humans with AA and the relationship to immunogenic DCs, we -44- compared the expression of iNOS, Nrf2, HOI, CD3, and CD1 lc by IHC in BM biopsies of 4 severe AA patients and 4 healthy donors. Our histological analysis (by H&E) revealed a significantly reduced BM cellularity in AA patients compared with their healthy counterparts (Figs. 11A and 1 IB). Our IHC analysis showed a significantly higher number of infiltrating T cells (anti-CD3) (Figs. 11A and 11C) and DCs (anti-CDl lc) (Figs. 11A and 11D) in severe AA patients compared to healthy donors. In addition, it has been clearly demonstrated that DCs shift their immature form to an active one, which promotes the over-function of T cells and hematopoiesis failure in AA patients. The abundance of signals inducing mDCs, and not TolDCs, in the BM microenvironment become the key elements to cause the destruction of HSCs in AA. Therefore, we next analyzed the expression of iNOS, Nrf2, and HO-1, which critically regulate the metabolic pathway of TolDCs and mDCs. The images and quantified analyses showed that AA patients have a significantly higher level of iNOS and reduction of Nrf2 and HO-1 expression when compared with healthy donors (Figs. 11A and 11E-11G). These results demonstrate the clinical significance of Nrf2 signaling in influencing the BM microenvironment of AA patients in which the infiltration of immunogenic DCs accompanies or correlates with hematopoietic tissue destruction.

Example 3

[000152] This example describes methods to reproducibly generate TolDCs through targeted activation of the nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) by repression of Kelchlike ECH-associated protein 1 (Keapl). Underquiescent conditions, Nrf2 is anchored in the cytoplasm through binding to Keapl, which, in turn, facilitates the ubiquitination and proteolysis of Nrf2, a key mechanism for the repressive effects of Keapl on Nrf2. In previous studies, we have shown an absolute requirement for Nrf2 for induction of the TolDC phenotype by small molecules in the triterpenoid family. Here we show that disruption of Keapl expression will repress glycolytic metabolism and confer a tolerogenic phenotype to DCs.

[000153] Keapl gene deletion in mouse DCs promotes a shift to OXPHOS and consequently confers a tolerogenic phenotype to mouse DCs. Therapeutic efficacy of Keapl ^/_ TolDCs can be assessed through adoptive transfer experiments in the established model of sever aplastic anemia (SAA), as defined by assessment of normal hematopoiesis, serum inflammatory cytokine profiles and survival. -45-

[000154] In aplastic anemia (AA), progressive changes in the bone marrow (BM) microenvironment underlie an immune dysfunction that contributes directly to disease progression and therapy resistance. This biology has been effectively demonstrated in validated, immune mediated models of this disease. We can employ the well-characterized CByB6Fl model of AA in which lymph node (LN) cells extracted from C57BL/6 are adoptively transferred into recipient CByB6Fl mice, generated by a cross of C57BL/6 and BALB/c mice, which are inbred and then crossbred (C57BL/6 x BALB/c) to generate the FI (CByB6Fl) mice. Recipient mice invariably develop impaired hematopoietic function and die within four weeks. This model recapitulates all aspects of human A A and it has been utilized to evaluate novel therapeutic strategies for AA. While other preclinical models of AA have been described, this model permits rapid evaluation of novel agents, either alone and in combination with approved therapies.

Linking Nrf2 activation to the metabolic phenotype characteristic of TolDCs

[000155] Efforts to define the mechanisms underlying the“tolerogenic” potential of DCs have revealed a signature metabolic shift such that TolDCs favor oxidative phosphorylation and fatty acid oxidation. We previously showed the utility of small molecule activators of Nrf2 in the metabolic reprogramming of TolDCs and to demonstrate the therapeutic potential of this population when expanded ex vivo.

[000156] An absolute requirement for Nrf2 activity for induction of the TolDC phenotype was demonstrated by Celloram in studies utilizing Nrf2^_/ DCs. Specifically, exposure of DCs to selected pharmacologic activators of Nrf2 ex vivo resulted in a TolDC phenotype as evidenced by induction of IL-4, IL-10, and TGF-b and suppression of TNFa, IFN-g, and IL- 12 levels in Nrf2^+/+ DCs but not in Nrf2^_/ DCs. Indeed, these small molecule activators of Nrf2 induced patterns of oxidative phosphorylation (OXPHOS) and glycolysis that are characteristic of TolDCs, but they failed to do so in Nrf2^_/ DCs. We have also shown a significantly enhanced HO-1 and reduced iNOS/NO production in Nrf2^+/+ relative to Nrf2^_/ DCs, suggesting Nrf2-dependent TolDC induction is linked to suppression of the inhibitory effect of NO on OXPHOS.

Relevance of Nrf2 activity to the pathogenesis of S AA in humans

[000157] TolDCs generated by small molecule Nrf2 activators improves hematopoiesis and enhances survival in this established murine model of AA, and the response to the -46-

TolDCs is associated with a significant reduction in Thl7 cells and an increase in Treg cells. The clinical relevance of these observations was demonstrated through immunohistochemical (IHC) analyses of bone marrow biopsies from patients with S AA, which show an increased in T cells, elevated iNOS expression and decreased Nrf2 and HO-1 expression compared to normal subjects (Fig. 12). Importantly, an imbalance of Thl7 and Treg cell populations in patients with SAA has been linked to disease pathogenesis and progression by several investigators, providing a rationale to explore the unique potential for CLM-18 to restore the Thl7/Treg balance in SAA.

The application of TolDC for induction of tolerance in SAA

[000158] The capacity of DCs to induce antigen- specific tolerance has been previously described. In studies of organ allograft rejection, donor-derived DCs prolong graft acceptance, which may be more durable when combined with co-stimulatory blockade. In some cases, graft tolerance induced by donor DCs has been reported to involve delivery of donor antigen to recipient antigen presenting cells. Donor-derived DCs induce specific tolerance when given simultaneously with the organ graft. The capacity of ex vivo expanded TolDCs to treat autoimmune disease has also been explored in preclinical models and is now the focus of ongoing clinical trials. However, there has been no published report of any effort to examine the potential of TolDCs as a therapy for SAA. As described in Examples 1 and 2, we developed a unique method for ex vivo expansion of highly functional TolDCs achieved through the activation of Nrf2. The effort described in this example is the first to explore their therapeutic potential for SAA.

Disruption of Keapl gene expression in DCs is an improved strategy for Nrf2 activation in TolDCs

[000159] We have shown the utility of TolDCs expanded ex vivo through the use of small molecule activators of Nrf2. There are two important considerations that are highly relevant to the advancement of this strategy toward clinical application. The first consideration is the potential for patient exposure to the Nrf2 activating agent if carried with the cell product.

This is considered highly unlikely given the small amount (nM concentrations) required for the ex vivo induction of the TolDC phenotype from peripheral blood monocytes. However, a demonstration of the absence of the compound or drug in the cell product may be required by the FDA. The second consideration is the multi-functional nature of the most potent and -47- effective activators of Nrf2 in our system, all of which are small molecules in the triterpenoid family. Most members of the triterpenoid family have the capacity to interact with other proteins, including key regulators of NFkB signaling (Fig. 13). Targeted disruption of Keapl gene expression is an alternative approach to specifically activate Nrf2 in TolDCs. This unique strategy may have two advantages. First, it may provide a more effective, robust and durable activation of Nrf2, obviating the influence of a pharmacologic Nrf2 activator on other signaling pathways in DCs. Second, it will eliminate considerations of patient exposure to small molecule activators of Nrf2 that may have potential to persist in the cell product.

Therefore, we proposed disrupting Keapl gene in both human and mouse TolDCs through use of the CRISPR/Cas9 syste. We can show Keapl ⁷ TolDC offers protection in the preclinical model of SAA, benchmarking against the previously established CLM-18, and induced using small molecule activators of Nrf2.

[000160] Several strategies have been developed to disrupt the aberrant immune response in patients with SAA. However, an approach based on the administration of ex vivo expanded, autologous tolerogenic DCs has not been explored. We show the potential of this approach, utilizing an established preclinical model of SAA to demonstrate efficacy of their TolDC product, CLM-18. These studies defined Nrf2 as a principal intracellular mediator of the metabolic shift required for induction of the tolerogenic DC phenotype. In this Example, we propose to optimize CLM-18 through use of the CRISPR/Cas9 system to specifically disrupt Keapl gene expression and thereby silence the principal negative regulator of Nrf2 activity. Major advantages of this approach are the potential to expand autologous TolDCs ex vivo without the use of any pharmacologic activators of Nrf2 in the culture system, and the ability to obviate any off-target effects of such agents. We developed CRISPR/Cas9 methods for use in targeting Keapl gene expression during the induction of TolDCs, demonstrating the ease of use during TolDC induction ex vivo. The readout will be clear, including:

evaluation of Keapl mutational spectrum by TIDE analysis, estimating the frequencies of insertions and deletions (Indels) in a pool of dendritic cells transfected with Cas9-RNP, and protein expression, metabolic profile, cytokine and chemokine gene expression profile, assessment of Nrf2 activity based on transcriptome analyses of Nrf2 target gene expression, as well as DC expression of HO-1 and iNOS.

[000161] We can perform in vitro experiments designed to show knock out (KO) of Keapl gene expression in human monocytes (using CRISPR/Cas9) promotes a shift from -48- glycolytic metabolism to oxidative phosphorylation (OXPHOS) during the ex vivo generation of human DCs. We predict the latter will convey a more stable, highly functional TolDC phenotype. Analyses of DC metabolism will be performed in collaboration with the academic partner. Analyses of TolDC phenotype and function can be performed and benchmarked against human DCs for ex vivo expansion in the presence of small molecule activators of Nrf2.

Efficient ex vivo expansion of autologous human TolDC by disruption of Keapl gene expression

[000162] We can demonstrate Keapl-/- (Keapl ^KO) TolDC exhibit the key characteristics that have been defined for TolDCs generate by small molecule activators of Nrf2, principally: 1) HO-l^Hl, iNOS^low DCs; 2) production of IL-4, IL-10, and TGF-b; 3) suppression of T cell activation and production of TNF-a, IFN-g, and IF- 12 in human mixed lymphocyte reaction (MFR) assays; 4) A predominant shift from glycolytic metabolism to oxidative

phosphorylation (OXPHOS) during ex vivo TolDC expansion. Additionally, transcriptome analyses demonstrating activation of Nrf2 target gene expression in Keapl ^KO TolDCs will confirm activation of Nrf2 activity as a consequence of Keapl gene deletion.

Details of the CRISPR Methods and TolDC expansion procedures

[000163] We developed methods for CRISPRRNP delivery to murine bone marrow derived dendritic cells (BMDC) by NEON electroporation. For genetic modification of DC, electroporation-meditated CRISPR- KO methods offer greater efficiency than lipid-based methods. A plasmid DNA-free ribonucleoprotein (RNP) CRISPR system, consisting of Cas9 protein and guide RNA, has been selected to increase the chance of a safe gene modification. The Cas9 RNP is a functional complex which works immediately after it enters the cell as subsequent transcription and translation are not required. Moreover, the complex is rapidly degraded afterwards from the cell, minimizing the chance for off-target cleavage events when compared to plasmid DNA-based systems.

[000164] During development and optimization of this procedure for application in DCs, ROSA26 RNP was used as a positive control and SIRPa RNP as a DC surface marker which can be readily measured by flow cytometry. Each RNP was delivered to BMDC by the NEON Electroporator (Invitrogen) as indicated. Differentiation of BMDC was induced by culturing bone marrow cells for 7 days in the differentiation medium with GM-CSF (20 -49- ng/ml) and IL-4 (15 ng/ml) and the day 7 BMDC (2xl0⁵/well) were used for CRISPR/Cas9 RNP delivery. NEON Electroporation of DCs involved 1500V/30 ms/1 pulse, followed by an additional two days of culture in vitro. Genomic DNAs from the BMDCs were harvested for CRISPR-PCR which specifically amplified the sequences around CRISPR-targeted site. The PCR products, which may include mutated sequences triggered by CRISPR/Cas9, were denatured and renatured for hetero-dimer DNA complex formation which then cleaved by T7 endonuclease I. The cleaved DNA was separated in 2% agarose gel and the approximate percent of insertional and deletional mutations (Indel%) was calculated for each condition. We observed approximately 55% Indel for ROSA26 RNP and 65% Indel for SIRPa RNP regardless of the Cas9/gRNA concentration. Efficacy is demonstrated by DC surface expression of SIRPa, determined by flow cytometry, with over 50% reduction in median fluorescent intensity (MFI) compared to that of ROSA26 RNP control cells (not shown). Histogram analysis clearly shows SIRPa RNP increased the SIRPa negative cell population as compared to ROSA26 control, confirming utility of the approach for targeting Keapl during ex vivo expansion of TolDC. Generation and characterization of human Keapl ^KO TolDC from peripheral blood monocytes (PBMs).

[000165] The protocol for generation of DCs from human PBMs is a 7-day procedure. As above, day 7 DCs generated from PBMs can be subjected to Keapl gene deletion through NEON electroporation of CRISPR-RNP sequences, and activated (after two days) prior to evaluation of the TolDC phenotype. The Keapl KO TolDCs can be compared to negative controls (receiving ROSA26 RNP) and to the positive control TolDCs generated from small molecule activators of Nrf2 based on DC exposure to small molecule activators of Nrf2 in culture. Phenotypic characterization will include flow cytometric analysis for the level change of surface expression of CD1 lc, CD80, CD86, and MHCII and other markers of mature DC, as well as assessment of the key features described in the milestone section (above). Finally, the ability of each TolDC product to suppress APC-mediated T cell activation will be assessed by the academic partner using human T cell isolates and monocyte-derived mature DCs from multiple donors in a classical mixed lymphocyte reaction (MLR). Readouts include assessment of T cell surface markers associated with activation, cytokine release ( e.g IFNy) and/or proliferation of T cells are all standard readouts enabling quantitative assessment of KeaplKO TolDC function in the MLR assay, relative the previously characterized CLM-18 product. -50-

[000166] Alternatively, the combination of Keapl gene deletion with small molecule activators of Nrf2 can be used as an approach that enhances the expansion of a more potent TolDC product. We are prepared to consider this alternative and/or change the TolDC culture conditions to yield a product with the greatest capacity to induce immune tolerance.

[000167] We can test whether Keapl gene deletion in mouse DCs promotes a shift to OXPHOS and consequently confers a tolerogenic phenotype to mouse DCs. Therapeutic efficacy of KeaplKO TolDCs can be assessed through adoptive transfer experiments in the established model of SAA, as defined by assessment of normal hematopoiesis, serum inflammatory cytokine profiles and survival. The production of murine KeaplKO TolDC using the established BMDC culture system will be used, and analyses of their in vivo efficacy will be as described above (Fig. 14). Administration of KeaplKO TolDC beginning day 1 and concomitant with the administration of lymph node cells of C57BL/6 mice permits assessment of the capacity of the cell product to delay progression of SAA and to ameliorate disease severity in a model that has a well-defined, highly reproducible and rapid time to progression. All BMDC derived cell products will undergo testing that includes viability, composition and function to ensure a product with consistent purity and potency. Mice in each group will be monitored daily for response to treatment and for development of complications related to the disease so that all mice may be euthanized when ill and captured for analyses of tissues so as not to lose any data points.

[000168] We anticipate that efficacy of KeaplKO TolDC in the CByB6Fl model of SAA will be as least equivalent to that observed for TolDC induced via exposure to an activator of Nrf2. However, we may find that the clinical response achieved may also depend on the number of KeaplKO TolDCs administered. An observation such as this will provide an opportunity to define the effects of increasing KeaplKO TolDC cell dose on the progression and severity of SAA. In addition, while we have established the system for CRISPR/Cas9 mediated deletion of Keapl in BMDC, we are prepared to compare this approach to an alternative strategy in which BMDC may be generated directly from mice with a tamoxifen- inducible CMVCre-Keaplfl/fl in which Cre-mediated deletion of Keapl is induced by treatment with tamoxifen (1 mg mouse 1 dayl; ip injection). Deletion of Keapl would be determined as previously described, and activation of Nrf2 would be confirmed by measuring expression of its downstream target NADPH quinone oxidoreductase 1 (Nqol) by -51- quantitative PCR (TaqMan, Applied Biosystems), as for all TolDCs generated via Keapl gene deletion.

Example 4

[000169] Differentiation of BMDC was induced by culturing bone marrow cells for 7 days in the differentiation medium with GM-CSF (20 ng/ml) and IL-4 (15 ng/ml) and the day 7 BMDC (2xl0⁵/well) were used for CRISPR/Cas9 RNP delivery. Each control and crKeapl RNP was delivered to BMDC by the NEON Electroporator (Invitrogen) as indicated

(Fig._-15A). NEON Electroporation of DCs involved 1500V/30 ms/1 pulse, followed by an additional two days of culture in vitro. Genomic DNAs from the BMDCs were harvested for CRISPR-PCR which specifically amplified the sequences around CRISPR-targeted site. The CRISPR-PCR products, which may include mutated sequences triggered by CRISPR/Cas9, were sequenced by Sanger sequencing method. The approximate percent of insertional and deletional mutations (Indel%) was calculated using ICE analysis provided by Synthego, where ICE score indicates indel% and KO score presents the potential percentage of Keapl - KO in the mixture. We observed approximately 85% ICE score and 79% KO score for crKeapl.75 RNP (Fig. 15B). There was no significant change of DC surface markers in Keapl-KO DC cells as compared to those of control cells (Fig. 15C). The secreted cytokines, however, were apparently altered in the Keapl-KO DC, where pro-inflammatory cytokine TNFcr was reduced approximately over 60% of control (Fig. 15D). These results support the rationale that unleashing Nrf2 by knock-out of Keapl would reprogram immature dendritic cells toward tolerogenic phenotype, confirming utility of the approach for targeting Keapl during ex vivo expansion of TolDC.

Example 5

[000170] This example describes methods to reproducibly generate TolDCs through targeted activation of the nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) of immature dendrwith l-[2-Cyano-3,12-dioxooleana-l,9(l l)-dien-28-oyl]-4(-pyridin-3-yl)-lH- imidazole (CDDO-3P-Im).

[000171] Figs. 16(A-C) illustrate a schematic, graph, and table showing an optimized protocol for tolerogenic dendritic cell production. (A)The schematic diagram shows the optimized method for dendritic cell differentiation and production of CLM18.3 (B)The immature DCs were harvested on day 8 and treated again in Celloram’s induction cocktail for -52- another day (C) The optimized protocol yielded iDC, mDC, and TolDC(CLM18.3) on day 9 as indicated. The cell number seeded at day 0 was set to 100%.

[000172] Figs. 17(A-D) illustrate graphs and plots showing purity, activity, and cytokines of CLM18.3 (A) CLM18.3 showed > 80% CDl lc+ and slight reduction of CD80, CD86, and MHCII. (B) CLM18.3 expressed low TNF-a, IL-12, but high TGF-jff. (C)CLM18.3 suppressed OVA-peptide specific T cell proliferation compared to control DC. As a proof-of- principle study, CRISPR-ko of Keapl also suppressed T cell proliferation (D) The Keapl-ko DC secrets low TNF-cr, IL-12, but high TGF- ? and IL-10.

[000173] Figs. 18(A-E) illustrate plots showing CLM-18.3 TolDCs exhibit unique transcriptome signatures. (A) Antigen Processing and Presentation Pathways and (B) Allograft Rejection Pathways. CLM18.3(18.3); mature DC (mDC) (C) MHC-I gene expression, (D) MHC-II gene expression, (E) the gene expression significantly related to tolerogenic dendritic cell function

[000174] Figs. 19(A-E) illustrate a schematic, graphs, and plots showing CLM18.3 significantly improved clinical scores and survival in mouse GvHD model (A) The experimental plan of GVHD model (B) CD4 and CD8 T cell subset analysis in each treatment group. (C) The level of TGF- ? in serum at day 14 and day 30 (D)The clinical score of each group measured by 5 clinical criteria (weight, posture, activity, Fur, and skin) (E)The Kaplan-Meier survival curve showed the survival benefit of CLM18.3 treated group.

[000175] Figs. 20(A-E) illustrate a schematic, graphs, and plots showing CLM18.3 significantly prolonged the survival in mouse aplastic anemia model (A) The experimental plan of aplastic anemia model (B)The Kaplan-Meier survival curve showed the survival benefit of CLM18.3 treated group in contrast to aplastic anemia group(red). (C) The level of TGF- ? and IL-10 in serum at day 17 (D) Blood counting showed the reconstitution of blood in each group (White blood cell, Hematocrit, platelet)(E) CD4 and CD8 T cell subset analysis in each treatment group(Left) and the preservation of Lin cKit⁺ hematopoietic stem cell population in bone marrow (right).

[0001761 While this application has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the application encompassed by the appended claims. All patents, publications and 53 references cited in the foregoing specification are herein incorporated by reference in their entirety.

Claims

-54- Having described the invention, the following is claimed:

1. A tolerogenic dendritic cell genetically or pharmacologically modified to modulate cell metabolism and suppress expression of Kelch-like ECH-associated protein (Keapl) and iNos or activate Nrf2 signaling and/or suppress NfkB signaling.

2. The tolerogenic dendritic cell of claim 1, comprising one or more of the following characteristics:

(a) HO-l^Hl, iNOS^low expression;

(b) production of at least one of IL-4, IL-10, and TGF-b;

(c) suppression of T cell activation and suppression of production of at least one of TNFoc, IFN-g, and IF- 12 in human mixed lymphocyte reaction (MFR) assays;

(d) a predominant shift from glycolytic metabolism to oxidative phosphorylation (OXPHOS) during ex vivo TolDC expansion; or

(e) activation of Nrf2 target gene expression.

3. The tolerogenic dendritic cell of claim 2, comprising at least the following characteristics: production of IF- 10 and TGF-b and suppression of TNFoc and IF- 12 in human mixed lymphocyte reaction (MFR) assays.

4. The tolerogenic dendritic cell of claims 1 or 2, comprising at least the following characteristics: production of IF-4, IF- 10, and TGF-b and suppression of TNFoc, IFN-g, and IF- 12 in human mixed lymphocyte reaction (MFR) assays.

5. The tolerogenic dendritic cell of any of claims 1 to 4, comprising a deletion of at least a portion of Keapl gene to inhibit expression or function of Keapl.

6. A method of generating a population of tolerogenic dendritic cells, the method comprising:

activating Nrf2 signaling and/or suppressing NfkB signaling and/or disrupting Keapl expression in immature dendritic cells. -55-

7. The method of claim 6, the tolerogenic dendritic cells including one or more of the following characteristics:

(a) HO-l^Hl, iNOS^low expression;

(b) production of at least one of IL-4, IL-10, and TGF-b;

(c) suppression of T cell activation and suppression of production of at least one of TNFoc, IFN-g, and IL-12 in human mixed lymphocyte reaction (MLR) assays;

(d) a predominant shift from glycolytic metabolism to oxidative phosphorylation (OXPHOS) during ex vivo TolDC expansion; or

(e) activation of Nrf2 target gene expression.

8. The method of claims 6 or 7, the tolerogenic dendritic cells comprising at least the following characteristics: production of IL-10 and TGF-b and suppression of TNFoc and IL-12 in human mixed lymphocyte reaction (MLR) assays.

9. The method of any of claims 6 to 8, the tolerogenic dendritic cells comprising at least the following characteristics: production of IL-4, IL-10, and TGF-b and suppression of TNFoc, IFN-g, and IL-12 in human mixed lymphocyte reaction (MLR) assays.

10. The method of any of claim 6 to 9, comprising deleting at least a portion of Keapl gene to inhibit expression or function of Keapl in immature dendritic cells.

11. The method of claim 10, wherein Keapl expression in the immature dendritic cells is disrupted by gene editing.

12. The method of claim 11, wherein Keapl expression in the dendritic cells is disrupted by administering to the dendritic cells at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and at least one guide RNA (gRNA) having a spacer sequence complementary to a target sequence in a Keapl DNA. -56-

13. The method according to claim 12, wherein the CRISPR-associated endonuclease is selected from a wild-type Cas9, a human-optimized Cas9, a nickase mutant Cas9, SpCas9(K855a), SpCas9(K810A/K1003A/rl060A), or

SpCas9(K848A/K1003A/R1060A).

14. The method of any of claims 6 to 13, further comprising isolating monocytes from the subject and culturing the monocytes with GM-CSF and IL-4 to generate immature dendritic cells.

15. The method of any of claims 6 to 14, further comprising activating Nrf2 signaling and/or suppressing NfkB signaling by administering to the immature dendritic cells an amount of triterpenoid effective to generate the population of tolerogenic dendritic cells.

16. The method of claim 15, wherein the triterpenoid is at least one of CDDO-IM, CDDO-2P-IM, or CDDO-3P-IM.

17. The method of claim 15 or 16, wherein triterpenoid is administered to the immature dendritic cell in combination with GM-CSF and/or LPS.

18. A method of increasing immune tolerance in a subject in need thereof, the method comprising:

administering to the subject a therapeutically effective amount of tolerogenic dendritic cells of any of claim 1 to 5

19. The method of claim 18, wherein the subject has an inflammatory condition, an allergy, or an autoimmune disorder.

20. The method of claim 18, wherein the subject has received a tissue or organ transplant. -57-

21. The method of claim 18, wherein the subject has myelodysplasia, myelofibrosis, thrombocytopenia due to other bone marrow diseases, drug induced thrombocytopenia, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenia, thrombocytopenia following viral infections, neutropenia due to other bone marrow diseases, drug induced neutropenia, autoimmune neutropenia, idiopathic neutropenia, neutropenia following viral infections, cytopenias, immune cytopenias, cytopenias following viral infections, or cytopenias.

22. The method of claim 18, wherein the subject has aplastic anemia, myelodysplasia, myelofibrosis, anemia due to other disorder of bone marrow, drug induced anemia, immune mediated anemias, anemia of chronic disease, anemia following viral infections, or anemia of unknown cause.

23. The method of claim 18, wherein the tolerogenic dendritic cells are administered to the subject following chemotherapy administration, radiation therapy, or immunosuppressive therapy.

24. The method of claim 18, wherein the tolerogenic dendritic cells are administered to the subject following a hematopoetic cell transplant with bone marrow, hematopoetic stem cells, or umbilical cord blood.

25. A method of treating an inflammatory or immune condition in a subject in need thereof, the method comprising:

Administering to the subject a therapeutically effective amount of a tolerogenic dendritic cell of any of claim 1 to 5.

26. The method of claim 25, wherein the inflammatory or immune condition comprises at least one of achlorhydra autoimmune active chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison’s disease, agammaglobulinemia, alopecia areata, Alzheimer’s disease, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-gbm/tbm nephritis, antiphospholipid syndrome, antisynthetase syndrome, aplastic anemia, arthritis, atopic allergy, atopic dermatitis, autoimmune 58 cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenia purpura, autoimmune uveitis, balo disease/balo concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaff’s encephalitis, blau syndrome, bullous pemphigoid, Castleman's disease, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, coeliac disease, Cogan syndrome, cold agglutinin disease, cranial arteritis, crest syndrome, Crohns disease, Cushing's syndrome, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, Dressler's syndrome, discoid lupus erythematosus, eczema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressive, fibromyalgia, fibromyositis, fibrosing aveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-barre syndrome (gbs), Hashimoto’s encephalitis, Hashimoto's thyroiditis, henoch-schonlein purpura, hidradenitis suppurativa, Hughes syndrome, inflammatory bowel disease (IBD), idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, iga nephropathy, inflammatory demyelinating polyneuopathy, interstitial cystitis, irritable bowel syndrome (ibs), Kawasaki's disease, lichen planus, Lou Gehrig’s disease, lupoid hepatitis, lupus erythematosus, meniere's disease, microscopic polyangiitis, mixed connective tissue disease, morphea, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neuromyotonia, occular cicatricial pemphigoid, opsoclonus myoclonus syndrome, ord thyroiditis, Parkinson’s disease, pars planitis, pemphigus, pemphigus vulgaris, pernicious anaemia, polymyalgia rheumatic, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, raynaud phenomenon, relapsing polychondritis, Reiter’s syndrome, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome,

spondyloarthropathy, sticky blood syndrome, still's disease, stiff person syndrome, sydenham chorea, sweet syndrome, takayasu’s arteritis, temporal arteritis, transverse myelitis, ulcerative -59- colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson’s syndrome, Wiskott-Aldrich syndrome as well as hypersensitivity reactions of the skin, atherosclerosis, ischemia- reperfusion injury, myocardial infarction, and restenosis.

27. A method of treating acute rejection of transplanted organs in a subject in need thereof, the method comprising:

administering to the subject a therapeutically effective amount of a tolerogenic dendritic cell of any of claim 1 to 5.

28. A method of treating aplastic anemia in a subject in need thereof, the method comprising:

administering to the subject a therapeutically effective amount of a tolerogenic dendritic cell of any of claim 1 to 5.

-54-

[000176] -

Having described the invention, the following is claimed:

1. A tolerogenic dendritic cell genetically or pharmacologically modified to modulate cell metabolism and suppress expression of Kelch-like ECH-associated protein (Keapl) and iNos or activate Nrf2 signaling and/or suppress NfkB signaling.

2. The tolero genictolero gnic dendritic cell of claim 1, comprising one or more of the following characteristics:

(a) HO-l^Hl, iNOS^low expression;

(b) production of at least one of IL-4, IL-10, and TGF-b;

(c) suppression of T cell activation and suppression of production of at least one of TNFoc, IFN-g, and IF- 12 in human mixed lymphocyte reaction (MFR) assays;

(d) a predominant shift from glycolytic metabolism to oxidative phosphorylation (OXPHOS) during ex vivo TolDC expansion; or

(e) activation of Nrf2 target gene expression.

3. The tolerogenic dendritic cell of claim 2, comprising at least the following characteristics: production of IF- 10 and TGF-b and suppression of TNFoc and IF- 12 in human mixed lymphocyte reaction (MFR) assays.

4. The tolerogenic dendritic cell of claims 1 or 2, comprising at least the following characteristics: production of IF-4, IF- 10, and TGF-b and suppression of TNFoc, IFN-g, and IF- 12 in human mixed lymphocyte reaction (MFR) assays.

5. The tolerogenic dendritic cell of any of claims 1 to 4, comprising a deletion of at least a portion of Keapl gene to inhibit expression or function of Keapl.

6. A method of generating a population of tolerogenic dendritic cells, the method comprising: -55- activating Nrf2 signaling and/or suppressing NfkB signaling and/or disrupting Keapl expression in immature dendritic cells.

7. The method of claim 6, the tolerogenic dendritic cells including one or more of the following characteristics:

(a) HO-l^Hl, iNOS^low expression;

(b) production of at least one of IL-4, IL-10, and TGF-b;

(c) suppression of T cell activation and suppression of production of at least one of TNFoc, IFN-g, and IL-12 in human mixed lymphocyte reaction (MLR) assays;

(d) a predominant shift from glycolytic metabolism to oxidative phosphorylation (OXPHOS) during ex vivo TolDC expansion; or

(e) activation of Nrf2 target gene expression.

8. The method of claims 6 or 7, the tolerogenic dendritic cells comprising at least the following characteristics: production of IL-10 and TGF-b and suppression of TNFoc and IL-12 in human mixed lymphocyte reaction (MLR) assays.

9. The method of any of claims 6 to 8, the tolerogenic dendritic cells comprising at least the following characteristics: production of IL-4, IL-10, and TGF-b and suppression of TNFoc, IFN-g, and IL-12 in human mixed lymphocyte reaction (MLR) assays.

10. The method of any of claim 6 to 9, comprising deleting at least a portion of Keapl gene to inhibit expression or function of Keapl in immature dendritic cells.

11. The method of claim 10, wherein Keapl expression in the immature dendritic cells is disrupted by gene editing.

12. The method of claim 11, wherein Keapl expression in the dendritic cells is disrupted by administering to the dendritic cells at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and at least one guide RNA (gRNA) having a spacer sequence complementary to a target sequence in a Keapl DNA. -56-

13. The method according to claim 12, wherein the CRISPR-associated endonuclease is selected from a wild-type Cas9, a human-optimized Cas9, a nickase mutant Cas9, SpCas9(K855a), SpCas9(K810A/K1003A/rl060A), or

SpCas9(K848A/K1003A/R1060A).

14. The method of any of claims 6 to 13, further comprising isolating monocytes from the subject and culturing the monocytes with GM-CSF and IL-4 to generate immature dendritic cells.

15. The method of any of claims 6 to 14, further comprising activating Nrf2 signaling and/or suppressing NfkB signaling by administering to the immature dendritic cells an amount of triterpenoid effective to generate the population of tolerogenic dendritic cells.

16. The method of claim 15, wherein the triterpenoid is at least one of CDDO-IM, CDDO-2P-IM, or CDDO-3P-IM.

17.- The method of claim 15 or 16, wherein triterpenoid is administered to the immature dendritic cell in combination with GM-CSF and/or LPS.

18. A method of increasing immune tolerance in a subject in need thereof, the method comprising:

administering to the subject a therapeutically effective amount of tolerogenic dendritic cells of any of claim 1 to 5

19. The method of claim 18, wherein the subject has an inflammatory condition, an allergy, or an autoimmune disorder.

20. The method of claim 18, wherein the subject has received a tissue or organ transplant. -57-

21.- The method of claim 18, wherein the subject has myelodysplasia, myelofibrosis, thrombocytopenia due to other bone marrow diseases, drug induced thrombocytopenia, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenia, thrombocytopenia following viral infections, neutropenia due to other bone marrow diseases, drug induced neutropenia, autoimmune neutropenia, idiopathic neutropenia, neutropenia following viral infections, cytopenias, immune cytopenias, cytopenias following viral infections, or cytopenias.

-22.- The method of claim 18, wherein the subject has aplastic anemia, myelodysplasia, myelofibrosis, anemia due to other disorder of bone marrow, drug induced anemia, immune mediated anemias, anemia of chronic disease, anemia following viral infections, or anemia of unknown cause.

23.- The method of claim 18, wherein the tolerogenic dendritic cells are administered to the subject following chemotherapy administration, radiation therapy, or immunosuppressive therapy.

24. The method of claim 18, wherein the tolerogenic dendritic cells are administered to the subject following a hematopoetic cell transplant with bone marrow, hematopoetic stem cells, or umbilical cord blood.

25. A method of treating an inflammatory or immune condition in a subject in need thereof, the method comprising:

Administering to the subject a therapeutically effective amount of a tolerogenic dendritic cell of any of claim 1 to 5.

26. The method of claim 25, wherein the inflammatory or immune condition comprises at least one of achlorhydra autoimmune active chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison’s disease, agammaglobulinemia, alopecia areata, Alzheimer’s disease, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-gbm/tbm nephritis, antiphospholipid syndrome, antisynthetase syndrome, aplastic anemia, arthritis, atopic allergy, atopic dermatitis, autoimmune 58 cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenia purpura, autoimmune uveitis, balo disease/balo concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaff’s encephalitis, blau syndrome, bullous pemphigoid, Castleman's disease, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, coeliac disease, Cogan syndrome, cold agglutinin disease, cranial arteritis, crest syndrome, Crohns disease, Cushing's syndrome, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, Dressler's syndrome, discoid lupus erythematosus, eczema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressive, fibromyalgia, fibromyositis, fibrosing aveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-barre syndrome (gbs), Hashimoto’s encephalitis, Hashimoto's thyroiditis, henoch-schonlein purpura, hidradenitis suppurativa, Hughes syndrome, inflammatory bowel disease (IBD), idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, iga nephropathy, inflammatory demyelinating polyneuopathy, interstitial cystitis, irritable bowel syndrome (ibs), Kawasaki's disease, lichen planus, Lou Gehrig’s disease, lupoid hepatitis, lupus erythematosus, meniere's disease, microscopic polyangiitis, mixed connective tissue disease, morphea, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neuromyotonia, occular cicatricial pemphigoid, opsoclonus myoclonus syndrome, ord thyroiditis, Parkinson’s disease, pars planitis, pemphigus, pemphigus vulgaris, pernicious anaemia, polymyalgia rheumatic, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, raynaud phenomenon, relapsing polychondritis, Reiter’s syndrome, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome,

spondyloarthropathy, sticky blood syndrome, still's disease, stiff person syndrome, sydenham chorea, sweet syndrome, takayasu’s arteritis, temporal arteritis, transverse myelitis, ulcerative -59- colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson’s syndrome, Wiskott-Aldrich syndrome as well as hypersensitivity reactions of the skin, atherosclerosis, ischemia- reperfusion injury, myocardial infarction, and restenosis.

27. A method of treating acute rejection of transplanted organs in a subject in need thereof, the method comprising:

administering to the subject a therapeutically effective amount of a tolerogenic dendritic cell of any of claim 1 to 5.

28. A method of treating aplastic anemia in a subject in need thereof, the method comprising:

administering to the subject a therapeutically effective amount of a tolerogenic dendritic cell of any of claim 1 to 5.