WO2024143863A1 - Method for culturing dendritic cells having enhanced immunogenicity mediated by adjacent immune cells activated by interleukin-33, and method for activating cytotoxic t cells using dendritic cells - Google Patents

Abstract

The present invention relates to a method for culturing dendritic cells. When the dendritic cell culturing method according to the present invention and dendritic cells prepared by the dendritic cell culturing method are used, dendritic cells having enhanced immunogenicity can be prepared using a culture medium derived from peripheral immune cells affected by interleukin-33, and cytotoxic T cells can be activated by using the dendritic cells.

Description

A method of culturing dendritic cells to improve immunogenicity mediated by adjacent immune cells activated with interleukin-33, and a method of activating cytotoxic T cells using the dendritic cells.

This invention was made under project number 2017R1A5A1014560 under the support of the Ministry of Science and ICT of the Republic of Korea. The research management agency for the project is the National Research Foundation of Korea, the research project name is “Group Research Support”, and the research project name is “Non-lymphoid organ immunity research.” Center”, the host institution is Sungkyunkwan University, and the research period is 2022.03.01-2023.02.28. am.

This patent application claims priority to Korean Patent Application No. 10-2022-0191212 filed with the Korean Intellectual Property Office on December 30, 2022, the disclosure of which is incorporated herein by reference.

The present invention includes the steps of (a) culturing a cell population containing basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; and (c) isolating dendritic cells from the resulting product.

Dendritic cells are representative antigen-presenting cells that can activate cytotoxic T cells by presenting antigens to them, and the development of cell therapies for tumor treatment is actively in progress.

Because the number of dendritic cells in the body is insufficient, in vitro cultured dendritic cells are used to treat tumors. Most existing in vitro cultured dendritic cells have used GM-CSF (Granulocyte-macrophage colony-stimulating factor), and recently, Flt3L (FMS-like tyrosine kinase 3 ligand) has been used to produce cells more similar to in vivo dendritic cells. It is being produced. Although genetic modification or adjuvants are being used to increase the immunogenicity of these GM/FL-BMDCs, unexpected side effects such as off-target effects or excessive inflammation caused by inflammatory cytokines are occurring. This exists. In addition, the methods presented above only focus on increasing the expression of specific costimulatory molecules and changing cytokines that can stimulate T cells by affecting dendritic cells at the time of completion of differentiation. As such, most in vitro cultured dendritic cell treatments are aimed at enhancing immunogenicity in the state of complete differentiation of dendritic cells, but there has been little research on ways to form new dendritic cell subpopulations and increase immunogenicity by controlling the intermediate stages of differentiation. The situation remains at the laboratory level.

Interleukin-33 (IL-33) is a nuclear cytokine belonging to the interleukin 1 family. Additionally, IL-33 is secreted by damaged or necrotic cells as a warning and is well known to be associated with type 2 immunity. The role of IL-33 in tumors is controversial. In the tumor environment, the IL-33/ST2 (Suppressor of Tumorigenicity 2) axis has been reported to promote T reg expansion and alter the immune system in a tumor-promoting direction. Meanwhile, it has been shown that IL-33 mediates anti-tumor immunity, including efficient induction of anti-tumor responses and antigen-specific T cells when IL-33 is used as an adjuvant. However, the detailed immunological mechanism of anti-tumor immunity induced by adjuvant IL-33 has not yet been elucidated.

Numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.

The present inventors have made extensive research efforts to develop an effective method for producing dendritic cells with the aim of increasing the immunogenicity of dendritic cells cultured in vitro. As a result, the anti-tumor immunity of dendritic cells obtained by culturing a cell population containing basophils with a culture medium containing interleukin-33 (IL-33) and treating the supernatant of the culture medium was compared to that of the existing culture medium. The present invention was completed by confirming that it was significantly higher than that of dendritic cell therapeutic agents.

Therefore, the object of the present invention is the following steps: (a) culturing a cell population containing basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; and (c) isolating dendritic cells from the resulting product.

Another object of the present invention is the following steps: (a) culturing a cell population containing basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; and (c) separating dendritic cells from the resulting product.

Another object of the present invention is the following steps: (a) culturing a cell population containing basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; (c) producing dendritic cells using a dendritic cell culture method comprising the step of isolating dendritic cells from the resulting product; and (d) co-culturing the dendritic cells and cytotoxic T cells.

Another object of the present invention is the following steps: (a) culturing a cell population containing basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; and (c) isolating the dendritic cells from the resulting product. The present invention provides a pharmaceutical composition for treating cancer containing dendritic cells prepared by a dendritic cell culturing method.

Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.

According to one aspect of the present invention, the present invention includes the steps of (a) culturing a cell population including basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; and (c) separating dendritic cells from the resulting product.

The present inventors have made extensive research efforts to develop an effective method for producing dendritic cells with the aim of increasing the immunogenicity of dendritic cells cultured in vitro. As a result, the anti-tumor immunity of dendritic cells obtained by culturing a cell population containing basophils with a culture medium containing interleukin-33 (IL-33) and treating the supernatant of the culture medium was compared to that of the existing culture medium. The present invention was completed by confirming that it was significantly higher than that of dendritic cell therapeutic agents.

The “cell population” may be CD11c ⁻ . CD11c, also known as integrin αX, is a cell adhesion molecule. CD11c is known to be expressed on monocytes, macrophages, granulocytes, and myeloid dendritic cells, and binds to CD18 to participate in cell adhesion.

In one embodiment of the present invention, the cell population containing the basophils may be Flt3L cultured bone marrow-derived cells.

In the present invention, the term "Flt3L" refers to Fms-related tyrosine kinase 3 ligand, an abbreviation for FMS-like tyrosine kinase 3 ligand, which induces the proliferation of hematopoietic progenitor cells in addition to the formation of new blood vessels, thereby promoting hematopoiesis. It refers to a cytokine that functions as a growth factor that activates progenitor cells and increases the number of immune cells. The Flt3L may be of human origin and may be a protein having the amino acid sequence of GenBank: AAA19825.1. These bone marrow cells cultured with Flt3L may be Flt3L cultured bone marrow-derived cells, which may be cells containing basophils.

As used herein, the term “dendritic cells (DC)” refers to the most core professional antigen-presenting cells of the immune system that can induce both innate immune response and adaptive immune response. As an antigen-presenting cell (APC), it can activate naive and memory immune responses that have never encountered an antigen. In its immature state, DC acts like a sentinel, constantly patrolling in search of antigens. Generally, when an APC takes up an antigen, the exogenous antigen is presented primarily through MHC-II (major histocompatibility complex class II) (CD4 ⁺ T cell activation) and the endogenous antigen is presented through MHC-I (CD8 ⁺ T cell activation) ). However, DCs have a special ability to cross present exogenous antigens not only through MHC-II but also through MHC-I. Therefore, DCs can activate CD4 ⁺ and CD8 ⁺ T cells more effectively.

After acquiring an antigen and undergoing a maturation process, DC moves to lymphoid organs and presents the antigen to naive T cells. T cell activation requires not only antigen presentation by APC but also stimulation by costimulatory molecules (CD80, CD86, CD40, etc.) and pro-inflammatory cytokines expressed on the APC surface. Through these signals, fully mature DC induces CD4 ⁺ T cells to differentiate into Th1 (T helper 1) cells and also activates CD8 ⁺ T cells (cytolytic T lymphocytes). However, in the absence of stimulation by APC costimulatory molecules and pro-inflammatory cytokines or when stimulated by immunosuppressive cytokines, CD4 ⁺ T cells differentiate into Th2 cells or regulatory T cells (Treg).

Dendritic cells can be broadly divided into dendritic cells derived from blood monocytes and dendritic cells present in lymphoid organs and bone marrow. Monocyte-derived dendritic cells are also called DC1, and when blood monocytes are stimulated with LPS or TNF-α, they differentiate into dendritic cells. Monocyte-derived dendritic cells secrete IL-12, a major stimulatory factor for T cells. When dendritic cells present in lymphoid organs and bone marrow react to antigens, they activate large amounts of IFN-α and IFN-β, activating natural killer cells.

In one embodiment of the present invention, the cell population containing the basophils is peripheral blood mononuclear cell (PBMC).

In the present invention, the term “peripheral blood mononuclear cells” refers to cells with spherical nuclei present in the blood. These peripheral blood mononuclear cells include immune cells such as B lymphocytes, T lymphocytes, monocytes, macrophages, dendritic cells, and natural killer cells (NK cells).

In one embodiment of the present invention, the peripheral blood mononuclear cells are cells from which T lymphocytes, B lymphocytes, monocytes, natural killer cells, or a combination thereof have been removed. More specifically, the peripheral blood mononuclear cells may be basophils.

In one embodiment of the present invention, the dendritic cells are monocyte-derived dendritic cells or bone marrow-derived dendritic cells.

In one embodiment of the present invention, the dendritic cells induce the expression of at least one of MHC-I, MHC-II, IL-12, and co-stimulatory molecule CD86.

The costimulatory molecule is a molecule required for activation of naive lymphocytes. Two signaling processes are required to activate a naive T or B lymphocyte into an effector lymphocyte. First, an appropriate foreign antigen must be delivered, and second, signal transduction by co-stimulatory molecules must occur. These two signals must be transmitted simultaneously to activate naïve lymphocytes into effector lymphocytes, and if even one signal is lacking, activation of lymphocytes does not occur. CD86 is a costimulatory molecule required for priming and activation of naive and memory T cells.

In one embodiment of the present invention, the method of culturing dendritic cells includes adding at least one of GM-CSF (Granulocyte-macrophage colony-stimulating factor), IL-13, IL-9, and IL-5 to the supernatant of (b). One more thing is included.

In one embodiment of the present invention, the method of culturing dendritic cells further includes administering a dendritic cell maturation-inducing factor to the result of step (c).

The dendritic cell maturation-inducing factor may include one or more of IL-1β, IL-6, TNF-α, IFN-γ, PGE2, Picibanil, and Poly I:C (polyinosinic-polycytidylic acid).

In one embodiment of the present invention, the dendritic cell maturation inducing factor is poly I:C.

According to another aspect of the present invention, the present invention includes the steps of (a) culturing a cell population containing basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; and (c) isolating the dendritic cells from the resulting product.

The “dendritic cell” of the present invention includes the “method for cultivating dendritic cells,” which is another aspect of the present invention described above, and redundant content is used. In order to avoid excessive complexity of the description in the present specification, redundant description is omitted. do.

According to another aspect of the present invention, the present invention includes the steps of: (a) culturing a cell population containing basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; (c) producing dendritic cells using a dendritic cell culture method comprising the step of isolating dendritic cells from the resulting product; and (d) co-culturing the dendritic cells and cytotoxic T cells.

The “method for increasing the activity of cytotoxic T cells” of the present invention includes the “method for cultivating dendritic cells,” which is another aspect of the present invention described above, so overlapping content is cited and the excessive complexity of the description in the present specification is used. To avoid duplicate entries, omit them.

The “cytotoxic T cells” play an important role in cellular immune responses centered on lymphocytes, which play an important role in immunity against cancer. Immune responses are initiated or regulated by stimulation by dendritic cells.

In one embodiment of the present invention, the dendritic cells and cytotoxic T cells can be co-cultured for 1 to 10 days, specifically for 4 days.

In one embodiment of the present invention, the ratio of dendritic cells and cytotoxic T cells in step (b) may be 1:5 to 1:50, and specifically 1:20.

In the present invention, the activated T cells can evade the defense mechanisms of cancer cells, treat cancer or neoplastic conditions, or prevent recurrence, progression, or metastasis of cancer.

According to another aspect of the present invention, (a) culturing a cell population including basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; and (c) isolating the dendritic cells from the resulting product. It provides a pharmaceutical composition for treating cancer containing dendritic cells prepared by a method of culturing dendritic cells.

As used herein, the term “treatment” means reducing, suppressing, alleviating or eradicating a disease state.

The "pharmaceutical composition for treating cancer containing dendritic cells" of the present invention includes the "dendritic cells produced by the dendritic cell culture method", which is another aspect of the present invention described above, and the overlapping content is cited. , to avoid excessive complexity of the description in this specification, redundant description is omitted.

The pharmaceutical composition of the present invention may additionally contain pharmaceutically acceptable excipients, carriers, or diluents.

“Pharmaceutically acceptable” means that the preparation is sterile and free of pyrogens. Suitable pharmaceutical carriers are well known in the pharmaceutical arts. The carrier(s) must be “acceptable” in the sense that it does not adversely affect the efficacy of the active ingredient of the present invention and is not harmful to its recipient. Typically, the carrier will be sterile, pyrogen-free water or saline; However, other acceptable carriers may be used. Accordingly, “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” are intended simply to act as a carrier, i.e., not to have biological activity of their own. includes the compound(s) used to form part of the preparation, which is not included in the preparation. Pharmaceutically acceptable carriers or excipients are generally safe and non-toxic. Pharmaceutically acceptable carriers and/or excipients as used herein include all of one or more carriers and/or excipients.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work.

Excipients can be one or more carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition to enable, for example, lyophilization. Examples of polymers include starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates, polyethylene. Glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinylacetate of different degrees of hydrolysis, and polyvinylpyrrolidone of all different molecular weights, these are for example , are added to the composition to control viscosity, to achieve adhesion, or to protect the lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, phosphoric acids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids of all different acyl chain lengths and saturations, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin; They are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid build-up or favorable pigment properties.

Diluent is intended to mean an aqueous or non-aqueous solution intended to dilute the peptide in pharmaceutical preparations. The diluent may be one or more saline, water, polyethylene glycol, propylene glycol, ethanol, or oil (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

Diluents can also function as buffers. The term “buffer” is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilizing pH. Examples of buffers include Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartarate. , AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES. am.

In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences ( ^19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, for example, intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intrathecal administration, intracerebral administration, intrasternal administration, topical administration, intranasal administration, intrapulmonary administration. and intrarectal administration, etc., but is not limited thereto.

The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art. Alternatively, it can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.

According to another aspect of the present invention, the present invention provides a method of treating cancer, comprising administering to a subject a composition containing dendritic cells prepared by a culture method comprising the following steps: do.

(a) culturing a cell population containing basophils with a culture medium containing IL-33 and obtaining a supernatant;

(b) culturing the supernatant by treating dendritic cells or their progenitor cells; and

(c) separating dendritic cells from the resulting product.

In the present invention, the term “subject” refers to a subject in need of prevention, improvement or treatment of cancer. Specifically, the subject may be a subject diagnosed with cancer, but is not limited thereto.

The features and advantages of the present invention are summarized as follows:

(a) The present invention includes the steps of culturing a cell population including basophils with a culture medium containing IL-33 and obtaining a supernatant; Processing the supernatant to dendritic cells or their progenitor cells and culturing them; and isolating dendritic cells from the resulting product.

(b) The present invention includes the steps of culturing a cell population including basophils with a culture medium containing IL-33 and obtaining a supernatant; Processing the supernatant to dendritic cells or their progenitor cells and culturing them; And it provides dendritic cells prepared by a dendritic cell culture method comprising the step of isolating dendritic cells from the resultant.

(c) The present invention includes the steps of culturing a cell population including basophils with a culture medium containing IL-33 and obtaining a supernatant; Processing the supernatant to dendritic cells or their progenitor cells and culturing them; Preparing dendritic cells by a dendritic cell culture method comprising the step of isolating dendritic cells from the resulting product; and co-culturing the dendritic cells and cytotoxic T cells.

(d) The present invention includes the steps of culturing a cell population including basophils with a culture medium containing IL-33 and obtaining a supernatant; Processing the supernatant to dendritic cells or their progenitor cells and culturing them; and a pharmaceutical composition for treating cancer containing dendritic cells prepared by a dendritic cell culturing method comprising the step of isolating dendritic cells from the resulting product.

When using the dendritic cell culture method according to the present invention, culturing a cell population including basophils with a culture medium containing IL-33 and obtaining a supernatant; Dendritic cells with improved immunogenicity compared to existing dendritic cell treatments can be cultured, including the step of treating and culturing the supernatant into dendritic cells or their progenitor cells.

Figure 1A shows that during the generation of Flt3-BMDCs (FL-DCs), 5 ng/ml IL-33 was added at different time points (day 0, 3, 5, 7, or 9), cells were harvested on day 10, and CD103+ This is the result of examining the expression of CD103 by evaluating cDC1 (n=3 mice per group).

Figure 1b shows that FL-DCs generated on day 5 with IL-33 treatment (FL-33-DC) or untreated (FL-DC) were harvested on day 9, pulsed with OVA protein for 18 h, and then incubated for 4 days. These are the results of co-culturing with CTV-labeled OT-I T cells for one day and evaluating the proliferation of IFNγ ⁺ effector T cells (n=3 mice per group).

Figure 2a shows the results of administering GM-CSF or IL-33 on day 5 during FL-DCs generation, collecting cells on day 10, and evaluating CD103 ⁺ cDC1s (control group (FL-DCs), GM-CSF treated group) (FL -GM-DCs) and IL-33 treated group (FL-33-DCs)) (n=4 mice per group).

Figure 2b shows the results of each group of FL-DCs, FL-GM-DCs, and FL-33-DCs pulsed with OVA protein for 18 hours, cDC1s isolated, and co-cultured with CTV-labeled OT-I T cells for 4 days. This is the result of evaluating the proliferation of IFNγ ⁺ effector T cells (n=4 mice per group).

Figure 2c shows the results of analyzing the expression of MHC molecules and costimulatory molecules in cDC1 of each group of FL-DCs, FL-GM-DCs, and FL-33-DCs (n=3 mice per group).

Figure 2d shows the results of measuring IL-12 in the culture medium after cDC1 was isolated from each group of FL-DCs, FL-GM-DCs, and FL-33-DCs and treated with LPS for 18 hours (n=3 mice per group).

Figure 2e shows the results of measuring the antigen uptake ability within cDC1 of each group of FL-DCs, FL-GM-DCs, and FL-33-DCs using DQ-OVA (n=3 mice per group).

Figure 3a shows the results of culturing BM cells from WT and ST2 ^-/- mice in the presence of Flt3L, adding IL-33 on the 5th day of culture, collecting cells on the 10th day, and evaluating CD103 ⁺ cDC1 (group per mouse n=3)

Figure 3b shows the results of evaluating ST2 expression levels in DC precursors of bone marrow (BM) and spleen cDCs, along with ILCs of BM as positive controls (n=3 mice per group).

Figure 3c shows the process of mixing WT (CD45.1 ⁺ ) and ST2 ^-/- (CD45.2 ⁺ ) BM cells at a 1:1 ratio and culturing them in the presence of Flt3L, and adding IL-33 on the 5th day of culture. Cells were collected on day 10 and CD103 ⁺ cDC1 was evaluated in CD45.1 ⁺ and CD45.2 ⁺ cells (n=3 mice per group).

Figure 3d shows the process of placing WT Flt3L-BMDCs and ST2 ^-/- Flt3L-BMDCs into the upper and lower chambers of a transwell, respectively, on the 5th day of culture and culturing them for an additional 5 days in the presence of IL-33, and CD103 ⁺ cDC1s in the lower chamber. This is the result of evaluating (n=3 mice per group).

Figure 4A is a heatmap of gene expression changes between CD103 ⁺ cDC1 of FL-33-DCs and FL-GM-DCs.

Figure 4b shows the Gene Set Enrichment Analysis (GSEA) results of FL-33-cDC1s vs. FL-GM-cDC1s.

Figure 4C shows the results of gene ontology analysis of cellular component genes upregulated in FL-33-cDC1s vs. FL-GM-cDC1s (FC > 2 and p < 0.2).

Figure 4D is a heatmap of the top 10 genes in the “Membrane” annotation.

Figure 4e shows the results confirming FCGR3 expression in cDC1s of FL-DCs, FL-GM-DCs, and FL-33-DCs (n=3 mice per group).

Figure 5a shows that in order to identify bystander cells that are key to FCGR3+CD103+cDC1 differentiation by IL-33, day 5 WT FL-DC cells were divided into CD11c ^- and CD11c ⁺ cells, and ST2-KO FL-DC were used in transwells. This is the result of co-culture in the presence of IL-33 (n=3 mice per group).

Figure 5b is a schematic diagram of constructing FL-33-DCs from ST2-KO BM cells in the presence of CD11c ⁻ subfraction from day 5 WT FL-DCs.

Figure 5c shows that on day 5 of WT FL-DC culture, CD11c ^- cells were divided into FcεRIα ^- and FcεRIα ⁺ , FcεRIα ^- cells were divided into CD127 ⁺ ILCs and CD127 ^- non-ILCs, and FcεRIα ⁺ cells were divided into CD49 ⁺ basophils and CD49b ^- parts. (Top) Results of FCGR3 ⁺ CD103 ⁺ cDC1s analysis after additional co-culture with ST2 ^-/- FL-DC (day 5) in the presence of IL-33 (bottom).

Figure 5d shows the results of analysis of cytokine-related transcripts after IL-33 treatment of CD11c ^- cells on day 5 of FL-DC.

Figure 5e shows the results of treatment with neutralizing antibodies for each cytokine and analysis of FCGR3 ⁺ CD103 ⁺ cDC1s when producing FL-33-DC.

Figure 5f shows the results of sorting CD11c ^- cells on day 5 of FL-DC and analyzing each cytokine in the culture medium after treatment with IL-33 (n=4 mice per group).

Figure 5g shows FL-DC, FL-GM-DC, and FL-33-DC generated in the presence or absence of a cytokine neutralizing antibody mixture during the production of FL-33-DC, after pulsing with OVA protein for 18 hours, cDC1. This is the result of evaluating proliferating IFN-γ ⁺ effector CD8 ⁺ T cells after isolation and co-culture with CTV-labeled OT-I T cells for 4 days (n=3 mice per group).

Figure 6a shows the results of evaluating tumor growth in EG.7 TB mice administered or not administered FL-DC, FL-GM-DC, and FL-33-DC vaccines (n=4 mice per group).

Figure 6b shows the results of observing effector CD8 ⁺ T cells in the spleens of mice administered or not administered the FL-DC, FL-GM-DC, and FL-33-DC vaccines (n=4 mice per group).

Figure 6c shows the results of measuring CTL activity in the TIL of mice administered or not administered the FL-DC, FL-GM-DC, and FL-33-DC vaccines (n=4 mice per group).

Figure 6d shows a representative lung metastatic tumor image (left) and the number of tumor nodules measured in mice intravenously injected with B16F10 tumor cells administered or not with FL-DC, FL-GM-DC, and FL-33-DC vaccines. Results (n=3 mice per group).

Figure 6e shows the results of measuring IFNγ ⁺ effector T cells in the lungs of mice administered or not administered the FL-DC, FL-GM-DC, and FL-33-DC vaccines (n=3 mice per group).

Figure 6f shows the results of measuring CTL activity in the lungs of mice administered or not administered FL-DC, FL-GM-DC, and FL-33-DC vaccines (n=3 mice per group).

Figure 7a shows a schematic diagram of the production of human monocyte-derived DC (hMo-DC) in the presence of culture supernatant of Lin ^- human PBMCs treated with IL-33 and the results of confirming IL1RL1, ST2 mRNA, at each step.

Figure 7b shows the surface phenotype of hMo-Dc produced by the method shown in Figure 7a (n=4 per group).

Figure 7c shows the results of measuring IL-12 in the culture medium after treating hM0-Dc produced by the method shown in Figure 7a with poly I:C for 18 hours (n=4 per group).

Figure 7d shows the results of measuring the proliferation of IFNγ ⁺ effector T cells in CD8 + T cells after co-culture with hMo-Dc produced by the method shown in Figure 7a (n=4 per group).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental method

1. Mouse Preparation

Wild-type C57BL/6 mice were purchased from DBL (Eumseong, Korea). ST2 ^-/- (C.129P2-Il1rl1 ^tm1Anjm ) mice were purchased from Dr. It was a gift from Andrew N. McKenzie (Institute of Molecular Biology, Medical Research Council, Cambridge, UK). OT-I [C57BL/6-Tg (TcraTcrb)1100Mjb/J] mice were purchased from Jackson Laboratory. All mice were inbred on a C57BL/6 background and maintained in a specific pathogen-free facility at Sungkyunkwan University in accordance with institutional/university animal care and use guidelines.

2. Preparation of bone marrow-derived dendritic cells (FL-BMDCs or FL-DCs) using Flt3L

Mice were sacrificed using CO ₂ . The tibia and femur of mice were collected and washed with PBS using a 3ml syringe to isolate BM cells. Red blood cells were lysed with ACK lysis buffer for 5 minutes and washed twice. BM cells were grown in complete RPMI medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, and supplemented with 100 ng/ml recombinant human Flt3L, 10 mM HEPES, and 55 μM β-mercaptoethanol. Suspension culture was performed at 2 x 10 ⁶ /ml. Cells were plated at 1 ml/well in a 12-well plate and incubated at 37°C, 5% CO ₂ for 10 days. Dendritic cells prepared in this way were named FL-DCs. Recombinant mouse IL-33 (mIL-33) or recombinant mouse GM-CSF (mGM-CSF) was administered at 5 ng/ml on the 5th day of FL-DC preparation and cultured for an additional 5 days, and the prepared dendritic cells were They were named FL-33-DCs or FL-GM-DCs, respectively. On the last day, cells were used for subsequent experiments.

3. Production of mouse tumor model

E·G7 (OVA-expressing EL4; lymphoma) and TC-1 (hybridoma) cells were purchased from ATCC, and B16F10-OVA (OVA-expressing B16F10; C57BL/6 mouse-derived cutaneous melanoma) cells were provided by a professor at Sungkyunkwan University. It was provided by Lim Yong-taek. OVA stands for ovalbumin. These cell lines were cultured in RPMI medium containing FBS and 100 U/ml penicillin/streptomycin. 5 x 10 ⁵ B16F10-OVA cells were inoculated subcutaneously on the right flank of the mouse. The size of the tumor was monitored every 2 days starting from day 8 after tumor injection, and the size of the tumor was calculated as V = (short axis) ² x (long axis) x 1/2.

4. Cell isolation from tissue

Spleens or tumor-draining lymph nodes from TF or TB mice were grained in RPMI medium, passed through a 70-μm cell strainer, and centrifuged. The supernatant was discarded, red blood cells were lysed with ACK lysis buffer, and cells were washed twice with PBS. Tumor tissues from TB mice were dissected, minced in RPMI medium at 4°C, and then physically grained. The tissue was then filtered through a 70-μm cell strainer. To isolate tumor-infiltrating lymphocytes (TILs), 40%/80% Percoll density gradient centrifugation was performed at 325xg for 23 minutes. The interfacial layer was collected and washed twice with PBS. Cells were used for subsequent experiments. For sorting, suspended cells were filtered once more in round-bottom polystyrene tubes with 40-μm cell strainer caps (Falcon).

5. CTL (cytotoxic T lymphocyte) analysis

CD8 ⁺ T cells isolated from TILs of cytokine-administered or DC-inoculated TB mice were cocultured with Cell Trace Far Red (CTF)-labeled target cells (EG.7 cells) at different ratios for 4 h. After propidium iodide staining, CTL activity was assessed by measuring the proportion of PI ⁺ cells in CTF-labeled cells by flow cytometry.

6. Flow cytometry or cell sorting (mouse)

Cells were analyzed on a FACSCanto II (BD Bioscience) and sorted on a FACSAria Fusion (BD Bioscience). Data analysis was performed using FlowJo software version 10 (TreeStar). All cells were suspended in FACS flow buffer (BD Bioscience) and stained with appropriate antibodies and fixable viability dye eFlour506 for 15 minutes at 4°C in the dark. The cells were then washed twice and used in subsequent experiments. For cDC1 analysis and sorting, single cells were cultured with anti-CD103-FITC or APC, anti-FCGR3-PE, anti-XCR1-PE or APC, B220-PerCPcy5.5 (for FL-DCs), anti-CD11c-PEcy7, anti- -Stained with MHC-II-APCcy7 and anti-CD11b-Pacific blue. In vivo cDC1s in organs including the spleen are total cDCs, and MHC-II⁺CD11c^high After gating with XCR1⁺was gated. In vitro cDC1s derived from FL-DC type as total cDCs, B220^-MHC-II⁺CD11c⁺Gated with and then additionally XCR1⁺was gated. Anti-CD86-FITC, anti-CD80-PE, anti-FCGR3-PE, anti-CD40-PerCPcy5.5 and anti-MHC-I-Pacific Blue were additionally stained to probe surface molecules. CD8⁺ For T cell surface staining, single cells were stained with anti-CD62L-FITC, anti-CD8-PEcy7, anti-CD44-APC and anti-CD3-APCcy7, and CD3⁺ CD8⁺CD44 on T cells^highCD62L^- The cells were considered activated T cells.CD8⁺ For intracellular staining of T cells, single cells were stimulated for 4 h with cell activation cocktail (Biolegend), followed by anti-IL-9-PE, anti-CD8-PEcy7, anti-IFN-γ-APC, and anti- CD3-APCcy7 was stained according to the protocol of the Fixation/Permeablization Kit (BD biosciences). BM single cells were incubated with anti-CD3/B220/Ly6G/CD11b (Lin)-FITC, anti-CD135-PE, anti-CD11c-PerCPcy5.5, anti-CD115-PEcy7, anti-CD115-PEcy7, to stain ST2 expression in DC precursors. -ST2-APC, anti-MHC-II-APCcy7 and anti-CD117-Pacific blue were stained. Afterwards, ST2 expression was analyzed in the next gate. MDP: Lin^-MHC-II^-CD11c^-CD135⁺CD115⁺CD117⁺; CDP: Lin^-MHC-II^-CD11c^-CD135⁺CD115⁺CD117^-; Pre-cDC: Lin^-MHC-II^-CD11c⁺CD135⁺ _.Naive BM single cells or single cells from several-day-old FL-DCs were incubated with anti-Lin-FITC, anti-CD127-PE, and anti-Lin-FITC to stain the type 2 innate lymphoid cells (ILC2s) population. Stained with -CD25-PerCPcy5.5 and anti-ST2-APC. ST2 expression is dependent on Lin in BM cells^-CD127⁺ Total ILCs were analyzed and the ILC2 population was Lin^-CD127⁺CD25⁺ST2⁺was analyzed. CD11c^- For basophil/ILC analysis and sorting in cells, whole spleen cells or single cells from day 5 FL-DC were incubated with anti-FcεRIα-FITC, anti-CD49b-PE, anti-CD3/B220/CD11b-PerCPcy5.5, anti- Stained with -CD127-PEcy7 and anti-CD11c-eF450. CD11c in spleen cells^-FcεRIα⁺CD49b⁺ The cells, basophils, were isolated or depleted. Total CD11c in FL-DCs at day 5^- Cells were isolated or cells were subdivided into the following gates. LC; FcεRIα^-CD3/B220/CD11b^-CD127⁺; non-ILC: FcεRIα^-CD127^-; CD49b^- part time job; FcεRIα⁺CD49b^-; basophils; FcεRIα⁺CD49b⁺. Cells were analyzed by the above gate, and sorted cells were used in subsequent experiments.

7. DC/T cell co-culture

The spleens of OT-I mice were grained in RPMI medium, passed through a 70 μm cell strainer, and centrifuged. The supernatant was discarded, and red blood cells were lysed with ACK lysis buffer. CD8 ⁺ T cells were purified using the Mouse CD8 ⁺ T Cell Isolation Kit II (Miltenyi Biotec) and labeled with CTV for 15 minutes at 37°C. In vivo cDC1s derived from the spleen were pulsed with OVA protein for 18 h. Afterwards, DCs were co-cultured with 2.5 x 10 ⁵ CD8 ⁺ T cells at a ratio of 1:20 (DC:T) for 4 days. FL-DCs generated in vitro were pulsed with OVA for 18 hours before cell collection. After isolating cDC1s or total cDCs, 2.5 x 10 ⁵ CD8 ⁺ T cells were co-cultured at a ratio of 1:5 (DC:T) for 4 days. Cell proliferation and cytokine expression of DC viability of CTV ⁻ cells or CD8 ⁺ T cells were assessed by flow cytometry.

8. Antigen uptake/processing assay

2 x 10 ⁵ XCR1 ⁺ cells were isolated from FL-DCs, FL-GM-DCs and FL-33-DCs. 1 μg/ml of DQ-OVA containing RPMI was administered and cultured at 37°C for 3 hours. Afterwards, cold RPMI was added, cells were washed twice with PBS, and DQ-OVA expression was analyzed.

9. WT and ST2-KO mixed cell culture

BM cells were collected from CD45.1 ⁺ WT and CD45.2 ⁺ ST2-KO mice. Mixed 1:1 and cultured in the absence or presence of mIL-33. WT or ST2-KO derived cells were distinguished by staining with anti-CD45.1-Pacific blue and anti-CD45.2-PerCPcy5.5.

10. RNA isolation

CD103 ⁺ cDC1s from FL-33-DCs or FL-GM-DCs were sorted into 5 ml round bottom polystyrene tubes using a BD FACSAria Fusion with a 70 μm nozzle. On day 5 of FL-DC culture, WT or ST2-KO CD11c- cells were sorted using BD FACSAria Fusion, treated with IL-33 for 6 h, and then collected. Cells were lysed with 500 μl of Trizol (Life Technologies). RNA purification was performed using the PicoPure RNA Isolation Kit (Applied Biosystems) according to the manufacturer's protocol. RNA quality was assessed with an Agilent 2100 bioanalyzer using an RNA 6000 Nano Chip (Agilent Technologies, The Netherlands), and RNA quantification was performed using an ND 2000 Spectrophotometer (Thermo Inc., USA) from ebiogen Inc (Seoul, Korea). .

11. RNA sequencing and data analysis

RNA libraries were constructed using the QuantSeq 3' mRNA Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer's instructions. The library was amplified and purified to add the complete adapter sequence required for cluster generation. Afterwards, high-throughput single-end 75-bp sequencing was performed with NextSeq 500 (Illumina, Inc., USA). QuantSeq 3' mRNA-Seq aligned reads were performed using Bowtie2. The alignment file was used to assemble the transcriptome, estimate its abundance, and detect differential expression of genes. Differentially expressed genes were determined based on counts of unique and multiple alignments using coverage in Bedtools. RC (read count) data was processed based on the quantile normalization method using EdgeR. This sequencing procedure from purified RNA was performed at ebiogen Inc (Seoul, Korea). Gene ontology analysis of genes upregulated in FL-33-DCs was based on a search performed in DAVID (http://david.abcc.ncifcrf.gov/). GSEA (Gene Set Enrichment Analysis) was performed using GSEA v4.0.3 software. Gene sets for analysis were obtained from the Gene Set Knowledgebase (/ge-lab.org/gs/), a curated functional genomics database for murine transcriptomics. To visualize the GSEA results, an enrichment plot was used. Enrichment score and FDR (false discovery rate) values were applied after gene set permutations were analyzed 1000 times.

12. Transwell culture

On day 5 of FL-DC generation, ST2-KO cells were plated in 24-well Transwell lower chambers. WT cells were plated in the upper chamber and 5 ng/ml mIL-33 was administered for an additional 5 days. Afterwards, cells in the lower chamber were analyzed.

13. Cytokine measurement

¹ x 10 ⁶ Afterwards, the culture supernatant was collected. Cytokine levels were measured using ELISA MAX™ Deluxe Set Mouse IL-12(p70) (Biolegend). 2 x ¹⁰⁴ CD127 ^- non-ILCs or CD127 ⁺ ILCs of 5-day-old FcεRIα ^- cells and CD49b ⁺ basophils or CD49b ^- sections of FcεRIα + cells were incubated ^with FL-DCs in the presence or absence of IL-33. Cultured for 36 hours in a 96-well plate. Afterwards, the culture supernatant was collected. Cytokine levels were measured using mouse GM-CSF, IL-5, and IL-9 ELISA MAX Deluxe (Biolegend) and mouse IL-13 Qunatikine ELISA (R&D Systems).

14. Co-culture of ST2-KO cells with WT immune cell fractions

CD11c ⁻ and CD11c ⁺ cells were isolated at day 5 from WT spleen cells or FL-DCs using FACSAria Fusion (BD Biosciences). Cells (1 × 10 ⁶ ) were cocultured with ST2-KO spleen cells for 2 days or at day 5 with ST2-KO FL-DCs for an additional 5 days. On day 5 of FL-DC culture, CD11c- cells were divided into FcεRIα ^- CD127 ^- non-ILCs, FcεRIα ^- CD127 ⁺ ILCs, FcεRIα ⁺ CD49b ^- cells, and FcεRIα ⁺ CD49b ⁺ basophils. Non-ILCs (9 x 10 ⁵ cells) or other cell fractions (2 x 10 ⁴ cells) were cocultured with ST2-KO FL-DCs on day 5 for an additional 5 days. WT splenic basophils were isolated from CD11c ⁻ cells or depleted from CD11c ⁻ cells by FACS sorting. Basophil-containing or depleted CD11c ^- cells (1 x 10 ⁶ ) or FACS-sorted basophils (2 x 10 ⁴ cells) were co-cultured with ST2-KO spleen cells for 2 days. Co-cultures were performed in the absence or presence of 5 ng/ml IL-33.

15. Cytokine blocking in FL-33-DCs

5 ng/ml of rmIL-33 was mixed with anti-GM-CSF (10 μg/ml), anti-IL-13 (2.5 μg/ml), anti-IL-9 (10 μg/ml), or anti-IL-9 (10 μg/ml). On day 5, FL-DCs were treated with 5 (10 μg/ml) for an additional 5 days. Afterwards, cells were collected and used in subsequent experiments.

16. DC-based tumor immunotherapy

The tumor immunotherapy model used C57BL/6 WT mice with some modifications. 5 x 10 ⁵ EG.7 cells were inoculated subcutaneously into mice. On day 9, FL-DCs, FL-GM-DCs, and FL-33-DCs were pulsed with OVA protein for 18 hours. ^Afterwards , ^live Tumor size was measured every 2 days starting on day 8 after tumor cell injection. Immune cell analysis was performed on day 18. For the lung metastasis model, 1.5 x 10 ⁵ B16F10-OVA cells were injected intravenously into mice. ^{Live ​} Injected. At day 18 after tumor cell injection, lungs were collected for nodule count and immune cell analysis. To investigate immune cell analysis in the lungs, lungs were minced and dissociated in digestion buffer (86 μg/ml collagenase IV from Clostridium histolyticum in RPMI) for 45 min at 37°C. After filtration through a 70-μm cell strainer, residual red blood cells were lysed using ACK lysis buffer. Cells were washed twice and used in subsequent experiments.

17. Isolation of peripheral blood mononuclear cells (PBMCs) from human blood

Blood from healthy volunteers was collected in heparin-coated vacutainers (BD Bioscience). Peripheral blood monomuclear cells (PBMCs) were isolated using Ficoll-Paque (1.077 g/ml, GE Healthcare). Briefly, blood was added to Ficoll and centrifuged at 2000 rpm for 20 minutes. The interfacial layer was collected and residual red blood cells were lysed using ACK lysis buffer. Cells were washed twice and used in subsequent experiments.

18. Cell isolation from human PBMCs

Cells were analyzed on a FACSCanto II and sorted on a FACSAria Fusion. Single cells of human PBMCs were stained with anti-CD3/CD16/CD19/CD56/CD66b-PerCPcy5.5, anti-CD14-PEcy7 and anti-CD8-APCcy7. Cells were separated by the following gates; Lin ^- : CD3/CD16/CD19/CD56/CD66b ^- CD14 ^- ; Classical monocytes: CD3/CD16/CD19/CD56/CD66b ^- CD14 ⁺ ; CD8 ⁺ T cells; CD3 ⁺ CD8 ⁺ . Lin ⁻ cells and monocytes were used to generate Mo-DC. CD8 ⁺ T cells were stained with CTV, and CTV-labeled CD8 ⁺ T cells were used in allogeneic CD8 ⁺ T/Mo-DC co-culture.

19. Generation of human Mo-DCs

Lin ^- cells were cultured for 3 days at ⁵ . Afterwards, the culture supernatant was collected. The previously known monocyte culture method was used with slight modifications. Monocytes were ^grown at 5 Suspended. After 3 days, the medium was replaced with fresh medium with all additives added, and the collected supernatant was dosed at 50%. On day 7, cells were collected and used for subsequent experiments. Anti-HLA-A,B,C(HLA-I)-FITC, anti-HLA-DP,DQ,DR(HLA-II)-FITC, anti-CD40-FITC, anti-CD83-FITC, anti-CD80- PE, anti-CD86-PE and anti-CD11c-APC were used for surface phenotyping of hMo-DCs. For IL-12 measurement, 30 μg/ml poly I:C was administered on day 6, left for 24 hours, and culture supernatant was collected. IL-12 levels were measured using the LEGEND MAX™ Human IL-12(p70) ELISA kit (Biolegend). For allogeneic CD8 ⁺ T cell response, 5 x 10 ⁴ Mo-DCs were co-cultured with 1.5 x 10 ⁵ CTV-labeled allogeneic CD8 ⁺ T cells for 4 days. Cells were stimulated with activation cocktail before analysis. Afterwards, cells were stained and analyzed with anti-CD3-PerCPcy5.5, anti-IFN-γ-PEcy7, and anti-CD8-APCcy7 according to the protocol of the Fixation/Permeablization kit (BD biosciences).

20. Statistics

Statistical significance was determined using Prism 9.1 software (GraphPad). The significance of differences between two or multiple groups was assessed using unpaired t-test with Welch's correction, one-way ANOVA with Dunnett T3 post-test, or two-way ANOVA with Tukey post-test. All data are mean ± SD.

Experiment result

1. CD103 in Flt3L-BMDC treated with IL-33 ⁺ It increases the cDC1s population and strongly induces cytotoxic T cell immunity.

The expression of CD103 was examined while adding IL-33 at different time points during the generation of Flt3-BMDCs (FL-DCs). The CD103 ⁺ cDC1 population increased significantly when IL-33 was added for up to 5 days and then decreased (Figure 1A). These results support the hypothesis that the CD103 ⁺ cDC1s population develops de novo from DC precursors in the presence of IL-33, rather than from established splenic cDCs. FL-DCs treated with IL-33 (FL-33-DC) primed CD8 ⁺ T cells more strongly than FL-DCs not treated with IL-33 (Figure 1b). This means that upon treatment with IL-33 in the intermediate stage of dendritic cell differentiation, immunogenic CD103 ⁺ cDC1s can be newly induced, thereby strongly inducing cytotoxic T cell immunity.

2. IL-33 mediated CD103 ⁺ cDC1s induce GM-CSF-mediated CD103 ⁺ It induces cytotoxic T cell immunity more strongly than cDC1s.

As GM-CSF is also known to induce the production of CD103 ⁺ DC1s, as illustrated in Figure 1a, IL-33 and GM-CSF were added on day 5 during Flt3L-BMDC culture, respectively, to produce FL-33-DCs and FL- GM-DCs were prepared. As shown in Figure 2A, CD103 ⁺ cDC1s were the major population in both FL-33-DCs and FL-GM-DCs. As shown in Figure 2b, when co-cultured with OT-1 T cells, FL-33-DCs were confirmed to be more effective than FL-GM-DCs in priming antigen-specific T cells. These results suggest that adjuvant IL-33-induced CD103 ⁺ cDC1s, a major population of cDC1s, are better than GM-CSF-induced CD103 ⁺ cDC1s in inducing antitumor immunity of IFN-γ ⁺ CD8 ⁺ T cells in mice. This suggests that the effect is excellent. Analysis of surface phenotype (MHC-1 and CD40) (Figure 2c), IL-12 expression (Figure 2d) and antigen uptake capacity (Figure 2e) showed that CD103 ⁺ cDC1s from FL-33-DCs were significantly different from those of FL-GM-DCs or It was confirmed that CD103- control FL-DCs showed significantly higher immunogenicity than CD103 ⁺ cDC1s.

These results indicate that CD103 ⁺ cDC1s induced by IL-33 are different from CD103 ⁺ cDC1s induced by GM-CSF and have a better effect of inducing T cell priming than CD103 ⁺ cDC1s induced by GM-CSF.

3. Immunogenic CD103 ⁺ cDC1s development is ST2 primed with IL-33 ⁺ It depends on exogenous factors secreted by immune cells.

ST2 is well known as a receptor for IL-33. As shown in Figure 3a, when IL-33 was treated during the production of WT and ST2-KO mouse FL-DCs, CD103 ⁺ cDC1 was developed normally in the WT group, but was not induced in the ST2-KO group. ST2 expression was examined in DC precursors and purified cDCs. As shown in Figure 3b, it was confirmed that not only BM cDC precursors but also splenic cDCs did not express ST2, unlike BM ILCs used as a positive control. These data imply that IL-33-induced CD103 ⁺ cDC1 development relies on exogenous factors from other ST2 ⁺ immune cells rather than the IL-33/ST2 intrinsic signaling pathway in cDCs.

As shown in Figure 3c, when WT and ST2-KO mouse BM cells were co-cultured and FL-DC production was performed in the presence of IL-33, a population of CD103 ⁺ cDC1s was efficiently induced even in ST2-KO mouse BM cells. It was confirmed that this was the case. These results imply that CD103 ⁺ cDC1 development in the spleens of IL-33-treated mice and during generation of FL-33-DCs is dependent on exogenous factors derived from ST2 ⁺ bystander immune cells. As shown in Figure 3D, when ST2-KO BM cells were cultured in a transwell system, CD103 ⁺ cDC1s were induced in the presence of WT cells and IL-33. Additionally, the CD103 ⁺ cDC1 population was efficiently induced in ST2-KO FL-DCs generated with culture supernatants of WT FL-33-DCs.

These data imply that soluble factors secreted by ST2 ⁺ bystander immune cells are involved in the development of CD103 ⁺ cDC1 induced by IL-33.

4. Highly immunogenic CD103 induced by IL-33 ⁺ cDC1s exhibit GM-CSF-induced gene expression patterns, particularly expression of FCGR3, CD103 ⁺ Distinguished from cDC1s.

To evaluate genotypic differences between IL-33-induced CD103 ⁺ cDC1s and GM-CSF-induced CD103 ⁺ cDC1s, RNA sequencing of CD103 ⁺ cDC1s isolated from FL-33-DCs and FL-GM-DCs, respectively, was performed. carried out.

As shown in Figure 4a, IL-33-induced CD103 ⁺ cDC1s were confirmed to be distinct from GM-CSF-derived CD103 ⁺ cDC1s in 1076 differentially expressed genes (|log ₂ fold change| ≥ 1, p -value < 0.2). As shown in Figure 4b, in Gene Set Enrichment Analysis (GSEA), the gene set of primary immunodeficiency was negatively enriched in IL-33-induced CD103 ⁺ cDC1s compared to GM-CSF-induced CD103 ⁺ cDC1s. , gene sets of proteasome and glycolysis/gluconeogenesis, which are associated with activation or cross-priming of DCs, were confirmed to be positively enriched. These data support our finding that IL-33-induced CD103 ⁺ cDC1s are more immunogenic than GM-CSF-induced CD103 ⁺ cDC1s. As shown in Figure 4C , to identify specific surface marker(s) for IL-33-induced CD103 ⁺ cDC1s, we analyzed the top enrichment terms for cellular components in the gene ontology enrichment of the 524 upregulated genes. , DAVID Bioinformatics Resources analysis confirmed that there was a high frequency of “Membrane” annotations related to the cell surface. As shown in Figure 4D, among the top 10 up-regulated genes investigated in the “Membrane” annotation, Fcgr3 was selected as a candidate for IL-33-induced cDC1 marker. Because, in DAVID Bioinformatics Resources, FCGR3 phagocytosis, recognition, positive regulation of tumor necrosis factor production and MHC class I ((https:/david.ncifcrf.gov/annotationReport.jsp?annot=86,91 This is because it has immunological characteristics such as antigen processing/presentation through ,92,78,27,35,43,52,50,73,89&currentList=0).). As shown in Figure 4e, FCGR3 was also expressed on the surface of FL-33-DCs produced in vitro, but was not detected on FL-DCs or FL-GM-DCs. Therefore, IL-33-induced CD103 ⁺ cDC1s are clearly distinct from GM-CSF-induced CD103 ⁺ cDC1s in their gene expression profile, suggesting that FCGR3 can be used as a distinguishable marker for IL-33-induced immunogenic CD103 ⁺ cDC1s. was confirmed.

5. ST2 primed with IL-33 ⁺ Basophils are FCGR3 ⁺ CD103 ⁺ It plays an important role in the development and immunogenicity of cDC1s.

Using FCGR3 as a phenotypic marker for highly immunogenic CD103 ⁺ cDC1s, bystander immune cells and exogenous soluble factors required for the development of IL-33-derived highly immunogenic FCGR3 ⁺ CD103 ⁺ cDC1s were investigated. As shown in Figure 5A, the FCGR3 ⁺ CD103 ⁺ cDC1s population was produced effectively in ST2-KO FL-33-DC cultures only when the CD11c ⁻ fraction of WT FL-DC cultures was placed in the upper chamber of the Transwell culture at day 5. It has been done. These data imply that the IL-33-derived exogenous factor required for the development of FCGR3 ⁺ CD103 ⁺ cDC1s originates from the CD11c ⁻ cell fraction. To identify bystander immune cells in the CD11c ⁻ fraction, FL-33-DCs were generated from ST2-KO BM cells in the presence of CD11c ⁻ subfraction from day 5 WT FL-DCs, as shown in Figure 5B. As shown in the upper left part of Figure 5c, CD11c ^- cells were divided into FcεRIα ^- and FcεRIα ⁺ , FcεRIα ^- cells were divided into CD127 ⁺ ILCs and CD127 ^- non-ILCs, and FcεRIα ⁺ cells were divided into CD49 ⁺ basophils and CD49b ^- parts. . As shown in the upper right part of Figure 5C, among the CD11c ⁻ subfractions collected on day 5 of WT FL-DC culture, FcεRIα ⁺ CD49b ⁺ basophils showed the strongest induction of FCGR3 ⁺ CD103 ⁺ cDC1 in the presence of IL-33. In addition, FcεRIα ^- CD127 ⁺ ILC2 and FcεRIα ⁺ CD49 ^- mast cell fractions were also confirmed to be partially involved in the production of FCGR3 ⁺ CD103 ⁺ cDC1s in ST2-KO FL-33-DC culture. To search for extrinsic factors, transcriptome analysis was performed, and IL-33-related cytokine genes such as Csf2, Il13, Il9, and Il5 were significantly increased in IL-33-primed WT CD11c ⁻ cells, but not in ST2-KO CD11c ⁻ cells. This was confirmed not to be the case in cells (Figure 5d). Additionally, as shown in Figure 5E, among the CD11c ⁻ cell fraction, basophils were identified as the most important in the expression of these cytokines, especially IL-13 expression. As shown in Figure 5f, when IL-33-related cytokines were blocked, FCGR3 ⁺ CD103 ⁺ cDC1 development in FL-33-DC cultures was mainly due to blockade of GM-CSF and IL-13 and IL-9 and IL-5. It was confirmed that it was significantly inhibited by partial blocking. As shown in Figure 5g, the T cell priming capacity of FL-33-DCs was confirmed to be completely abolished by blocking these cytokines during FL-33-DCs production.

Taken together, exogenous lytic factors (including GM-CSF, IL-13, IL-9, and IL-5) secreted by IL-33-influenced ST2 ⁺ basophils are critical for the development of immunogenic FCGR3 ⁺ CD103 ⁺ cDC1s. It can be seen that it plays a role.

6. FL-33-DCs showed a more potent effect than untreated FL-DCs or FL-GM-DCs in DC-based tumor immunotherapy.

As shown in Figure 6a, it was confirmed that the FL-33-DC vaccine inhibited tumor growth in TB mice more strongly than other DC vaccine candidates in DC-based tumor immunotherapy. Effector CD8 ⁺ T cells (Figure 6b) and antigen-specific (Ag-specific) CTL responses (Figure 6c) were significantly enhanced in the spleen of FL-33-DCs-inoculated mice compared to other BMDC-inoculated mice. As shown in Figure 6d, it was confirmed that the number of tumor nodules in the mice inoculated with FL-33-DCs was significantly reduced in the metastatic tumor model compared to the other DCs inoculated groups. As shown in Figures 6e and 6f, it was confirmed that the IFN-γ ⁺ effector CD8 ⁺ T cells and CTL responses in the lung were significantly improved in FL-33-DCs-inoculated mice compared to other DCs-inoculated mice. These data imply that the FL-33-DC vaccine is more effective in inducing antitumor immunity and tumor immunotherapy than standard FL-DCs.

7. Human monocyte-derived DCs (hMo-DCs) displayed an enhanced immunogenic phenotype when cultures were treated with supernatants from IL-33-primed PBMC cultures.

As shown in Figure 7A, to determine whether IL-33-mediated enhancement of DC immunogenicity could be used for human DC vaccine preparation, IL-33-treated Lin ^- PBMC (CD3/CD14/CD16/ The supernatant of the CD19/CD56/CD66b-depleted) culture was applied. (The reason for this application is that neither monocytes nor Mo-DCs expressed ST2 in all culture steps, and only Lin ⁻ cells expressed ST2; Figure 7a). As shown in Figure 7b, it was confirmed that the IL-33-primed Lin ^- cell supernatant significantly increased the expression of MHC I, MHC II, and the co-stimulatory molecule CD86. Additionally, as shown in Figure 7c, hMo-DCs treated with the supernatant of IL-33 primed PBMCs were confirmed to secrete more IL-12 than the untreated control group. As shown in Figure 7D, hMo-DCs treated with IL-33 primed PBMC supernatant were found to prime and activate allogeneic CD8 ⁺ T cells more strongly than those treated with control supernatant. These data imply that IL-33-induced exogenous factors secreted by ST2 ⁺ bystander immune cells such as basophils increase hMo-DCs immunogenicity.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention.

Claims (15)

A method of culturing dendritic cells comprising the following steps. (a) culturing a cell population containing basophils with a culture medium containing IL-33 and obtaining a supernatant; (b) culturing the supernatant by treating dendritic cells or their progenitor cells; and (c) separating dendritic cells from the resulting product. The method of culturing dendritic cells according to claim 1, wherein the cell population including basophils is peripheral blood mononuclear cells (PBMC). The method of claim 2, wherein the peripheral blood mononuclear cells are dendritic cells from which T lymphocytes, B lymphocytes, monocytes, natural killer cells (NK cells), or combinations thereof have been removed. Culture method. The method of claim 1, wherein the dendritic cells are monocyte-derived dendritic cells or bone marrow-derived dendritic cells. The method of culturing dendritic cells according to claim 1, wherein the dendritic cells induce expression of at least one of MHC-I, MHC-II, IL-12, and co-stimulatory molecule CD86. The method of culturing dendritic cells according to claim 1, wherein the supernatant of (b) further includes at least one of GM-CSF, IL-13, IL-9, and IL-5. The method of culturing dendritic cells according to claim 1, further comprising administering a dendritic cell maturation inducing factor to the result of step (c). The method of culturing dendritic cells according to claim 7, wherein the dendritic cell maturation inducing factor is poly I:C. Dendritic cells produced by the culture method of any one of claims 1 to 8. A method of increasing the activity of cytotoxic T cells comprising the following steps: (a) preparing dendritic cells by the culture method of any one of claims 1 to 8; and (b) co-culturing the dendritic cells and cytotoxic T cells. The method of claim 10, wherein the co-culture is performed for 1 to 10 days. Method for increasing the activity of cytotoxic T cells. The method of claim 10, wherein in step (b), the ratio of dendritic cells to cytotoxic T cells is 1:5 to 1:50. A pharmaceutical composition for treating cancer, comprising dendritic cells prepared by the culture method of any one of claims 1 to 8. The pharmaceutical composition for treating cancer according to claim 13, wherein the dendritic cells induce the expression of at least one of MHC-I, MHC-II, IL-12, and co-stimulatory molecule CD86. A method of treating cancer, comprising administering to a subject a composition containing dendritic cells prepared by the culture method of any one of claims 1 to 8.

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