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WO2001026679A2 - Cellules et molecules impliquees dans l'immunoregulation - Google Patents

Cellules et molecules impliquees dans l'immunoregulation Download PDF

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WO2001026679A2
WO2001026679A2 PCT/CA2000/001172 CA0001172W WO0126679A2 WO 2001026679 A2 WO2001026679 A2 WO 2001026679A2 CA 0001172 W CA0001172 W CA 0001172W WO 0126679 A2 WO0126679 A2 WO 0126679A2
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cells
regulatory
cell
mice
osteopontin
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WO2001026679A3 (fr
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Li Zhang
Zhu Xu Zhang
Liming Yang
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    • C12N2501/60—Transcription factors

Definitions

  • the present invention relates to methods and compositions for modulating an immune response.
  • the invention includes novel cells and molecules that are useful in suppressing immune responses in an antigen-specific manner.
  • autoimmune disease allograft rejection and graft versus host disease
  • autoimmune diseases including multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, refer to a large group of chronic diseases affecting over 80 million people worldwide. Most of autoimmune diseases are progressive, and leading to malfunction of affected tissue and organs, and in severe cases, the death of the patients. Transplantation is the current therapeutic modality for patients with end-stage organ failure. Joint efforts of clinicians and immunologists have now made it possible for 80-95% of patients to live with a functional allograft for one year post-transplantation.
  • the present inventors have isolated novel regulatory T cells that have many important roles in immune regulation including inducing antigen specific tolerance, preventing allo- and xeno-graft rejection, and graft versus host disease.
  • the phenotype of the novel regulatory T cells is CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l ⁇
  • the cells do not express IL-2, IL-4, IL-10 and IL-13 but do express IFN- ⁇ , TNF- ⁇ and TGF- ⁇ mRNA after activation.
  • Treatment of the regulatory T cells with IL-10, cyclosporin A, anti-IFN- ⁇ , anti-TCR antibodies or regulatory T cell specific monoclonal antibodies abrogates suppression by the cells.
  • the present invention provides an isolated regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l-. More specifically, the regulatory T cells have the phenotype CD3 + ⁇ TCR + CD4-CD8-CDlla + CD18 + CD25 + CD28"CD44"NK1.1". The regulatory T cells also express Ly-6A and osteopontin.
  • the present invention also includes methods for activating or expanding the regulatory T cells both in vitro and in vivo.
  • the present invention also includes the use of the novel regulatory cells to suppress an immune response. Accordingly, the present invention provides a method of suppressing an immune response comprising administering an effective amount of a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28 " NKl.l- to an animal in need of such treatment. In one embodiment, the present invention provides a method of inducing immune tolerance in a recipient animal comprising administering an effective amount of a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l- to an animal in need of such treatment. In a particular embodiment, the novel regulatory cells may be used to prevent graft rejection.
  • the present invention provides a method of preventing or treating graft versus host disease comprising administering an effective amount of a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l- to an animal in need of such treatment.
  • the present invention provides a method of preventing or treating an autoimmune disease comprising administering an effective amount of a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l- to an animal in need of such treatment.
  • the present invention also includes antibodies to the novel regulatory cells of the invention and the use of the antibodies in stimulating or inhibiting the regulatory T cells. As such, the antibodies are useful in therapies to suppress or enhance an immune response.
  • the present inventors have also found that the proteins Ly-6A and osteopontin are highly expressed on the above described regulatory T cells but not on non-regulatory cells. Further, they have also shown that blocking Ly-6A or osteopontin abolishes suppression by these cells.
  • the present invention provides a method of suppressing an immune response comprising administering an effective amount of an Ly-6A protein, a nucleic acid sequence encoding an Ly-6A protein, osteopontin or a nucleic acid sequence encoding osteopontin to an animal in need of such treatment.
  • the present invention provides a method of inducing immune tolerance in a recipient animal comprising administering an effective amount of an Ly-6A protein, a nucleic acid sequence encoding an Ly-6A protein, osteopontin or a nucleic acid sequence encoding osteopontin to the recipient animal.
  • the Ly-6A or osteopontin may be used to prevent graft rejection.
  • the present invention provides a method of preventing or treating graft versus host disease comprising administering an effective amount of an Ly-6A protein, a nucleic acid sequence encoding an Ly-6A protein, osteopontin or a nucleic acid sequence encoding osteopontin to an animal.
  • the present invention provides a method of preventing or treating an autoimmune disease comprising administering an effective amount of an Ly-6A protein, a nucleic acid sequence encoding an Ly-6A protein, osteopontin or a nucleic acid sequence encoding osteopontin to an animal having, suspected of having, or susceptible to having an autoimmune disease.
  • the invention also includes pharmaceutical compositions containing the novel regulatory cells, antibodies to the novel cells, Ly-6A proteins, nucleic acids encoding Ly-6A protein, osteopontin or a nucleic acid sequence encoding osteopontin for use in inducing tolerance in transplantation or autoimmune disease.
  • the present invention provides a method of preventing immune suppression comprising administering an effective amount of an agent that inhib its a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l " ; Ly-6A or osteopontin to an animal in need thereof.
  • the agent is an antibody that binds the regulatory cells or an antibody that binds Ly-6A or osteopontin or an antisense oligonucleotide that inhibits the expression of Ly-6A or osteopontin.
  • the invention also includes pharmaceutical compositions containing the above inhibitors for use in inducing or augmenting an immune response.
  • Figure 1(a) is a schematic illustration of the 2C T cell.
  • Figure 1(b) is a schematic illustration of breeding (2Cxdm2)Fl and (B6xBALB/c) F1 -transgene negative (Tg ⁇ ) animals.
  • Figure 1(c) is a schematic illustration of the experimental model.
  • FIG. 2 (a) Spleen cells from DST-treated tolerant mice specifically enhance L d+ skin allograft survival when transferred to na ⁇ ve syngeneic mice. (2Cxdm2) F1 mice were given DST from
  • All of the mice were given skin grafts from both (B6xBALB/c) F1 and C3H.
  • all DST-treated mice accepted (B6xBALB/c) F1 grafts but rejected C3H grafts (tolerant), whereas non-DST-treated mice rejected both grafts (non- tolerant).
  • 1B2 + CD4 + (grey) or 1B2 + DN (black) cells from tolerant mice 120 days after skin grafting were added into the MLR cultures as putative regulatory cells at various ratios as indicated.
  • Cells were cultured in ⁇ MEM supplemented with 10% FCS, 30 U/ml of rIL-2 and rIL-4. Proliferation was measured by [ 3 H]-TdR incorporation. The cultures to which no putative regulatory cells were added were used as controls. The results represent 3 independent experiments each with 5 replicates.
  • Figure 3 Dose dependent inhibition of syngeneic CD8 + T cells in vitro by DN T cells.
  • 1B2 + DN clones generated from tolerant (TN02, ⁇ and TN12, ⁇ ) and naive (CN04, X) mice were added to the MLR cultures as putative regulatory cells, and a 1B2 + CD8 + T cell clone (C02, ⁇ ) was used as a control.
  • Cell proliferation was measured by [ 3 H]-TdR incorporation. The data are expressed as percent inhibition of proliferation compared to the controls to which no putative regulatory T cells were added. The experiments were repeated 4 times and the results represent the 5 other DN clones and 2 other 1B2 + CD8 + T cell clones (not shown).
  • CD8 + and DN T cells were purified from spleens of (B6xdm2) F1 ( ⁇ ) and MRL/+ (•, H-2 K ) mice.
  • (B6xdm2) T cells were stimulated with irradiated C3H splenocytes and MRL/+ T cells were stimulated with irradiated (B6xBALB/c) F1 splenocytes.
  • CD8 + T cells were used as responders (2,000 /well), and varying numbers of syngeneic DN T cells were added to the corresponding MLR cultures as putative regulatory cells. Cell proliferation was measured by [ 3 H]-TdR incorporation. The data are expressed as percent inhibition of proliferation compared to the controls to which no DN T cells were added. The data represent 2 experiments each with 5 replicate cultures.
  • FIG. 4 (a) Regulatory T cells express a unique combination of cell surface markers. Regulatory and non-regulatory T cell clones (i-vi) as well as fresh spleen cells from tolerant mice 120 days after skin grafting (vii-ix) were stained with mAbs specific for the ⁇ TCR (1B2), ⁇ TCR, CD4, CD8, CD25, CD28, CD30, CD44, CD45, CD62L, CD69, CDlla, CD18, and NK1.1. The relative levels of expression of 1B2 and CD8, CD25, CD28, CD30, and CD44 on DN regulatory T cells (grey) and 1B2 + CD8 + non-regulatory T cells (white) are shown. The negative control is shown in Black (iii). Data represent 8 regulatory, 3 non-regulatory T cell clones and spleen T cells from 5 tolerant animals. The observed pattern of expression remained the same before and after stimulation.
  • DN T cells induce suppression by killing activated CD8 + T cells through the Fas/FasL pathway.
  • Regulatory and non-regulatory T cell clones mediate cytotoxicity through different pathways.
  • 1B2 + CD8 + or 1B2 + DN T cells were stimulated independently by L d+ spleen cells and used as effector cells.
  • Fas Fc fusion protein blocks DN T cell-mediated cytotoxicity.
  • DN T cells are able to lyse activated CD8 + cells from normal, but not Fas-mutant Ipr mice.
  • Primary CD8 + T cells were purified from the spleen of MRL/+ ( ⁇ ), MRL/lpr (D), (B6xdm2) F1 (A), and B6/lpr ( ⁇ ) mice.
  • Primary ⁇ TCR+CD3 + DN T cells were purified from both (B6xdm2) F1 (dash lines) and MRL/Zpr mice (solid lines).
  • CD8 + and DN T cells from (B6xdm2) F1 and B6/lpr mice were stimulated by irradiated (20 Gy) C3H (H-2 K ) splenocytes, and CD8 + and DN T cells from MRL/+ and MRL/ lpr mice were stimulated by irradiated (B6xBALB/c) F1 cells.
  • Activated CD3 + DN T cells were collected and used as effector cells at E:T ratios as indicated.
  • Fresh irradiated (B6xBALB/c) F1 splenocytes were added during the cytotoxicity assay. Specific lysis of target cells was determined as described in the methods. The data are expressed as mean percent killing of 3 replicate cultures from 2 independent experiments.
  • DN T cells do not induce bystander killing in activated CD8 + T cells that express a different TCR specificity.
  • Spleen cells from mice expressing ⁇ TCR-transgene specific for the LCMV-gp were stimulated in vitro with peptide p33 (peptide p33-41, a gift from Dr. P. Ohashi).
  • Female T3.70 + CD8 + anti-male HY transgenic T cells (anti-HY) were stimulated with irradiated male B6 spleen cells.
  • C3H (H-2 K ) T cells were stimulated with irradiated SJL (H-2 S ) spleen cells.
  • 1B2 + CD8 + T cells were stimulated by irradiated (B6xBALB/c) F1 spleen cells.
  • Activated CD8 + T cells from the above cultures were collected, labeled with lO ⁇ Ci/ml of [ 3 H]-TdR at 37°C overnight, and used as targets.
  • 1B2 + DN T cells were stimulated independently by (B6xBALB/c) F1 spleen cells, then co-cultured with various target cells at 37°C for 18 hrs at varying E:T ratios as indicated. Fresh, appropriate, irradiated spleen cells were added during cytotoxicity assays.
  • TCR -L d interaction is critical for DN T cell-mediated suppression.
  • the DN cells were either left untreated (solid line) or preincubated with anti-L d mAb (20 ⁇ g/ml, dashed line) for 1 hour before addition to the cultures.
  • Labeled CD8 + T cells were either left untreated ( ⁇ ), or preincubated with 1B2 (A) or irrelevant isotype matched control (T) mAb (lOO ⁇ g/ml) for 1 hour and then washed prior to being used as targets.
  • the effector and target cells were co-cultured for 18 hours in the presence of rIL-2 and rIL-4 (30U/ml) and specific lysis of target cells was determined using the JAM assay. The results represent 5 replicate cultures.
  • Figure 7 is a schematic diagram showing 1B2 + DN T cell suppression of
  • Ly-6A knockout DN T cells fail to kill activated syngeneic CD8 + T cells.
  • DN and CD8 + T cells were purified from the spleen of normal B6 (open bars) and B6-Ly-6A knockout (closed bars) mice (both are H-2b), and stimulated by irradiated splenocytes from BALB/c mice (H-2d) separately.
  • Activated CD8 + T cells from B6 or B6-Ly-6A knockout mice were labelled with lO ⁇ Ci/ml of [ 3 H]-TdR and used as targets.
  • % Specific killing (S-E)/S x 100, where E (experimental) is cpm of retained DNA in the presence of DN effector cells, and S (spontaneous) is cpm of retained DNA in the absence of DN T cells.
  • Figure 9 (a) Expansion of donor-derived DN T cells and elimination of anti-host CD8 + T cells following infusion of anti-L d spleen cells. (B6xC.B-17) F1 scid mice
  • mice were lethally irradiated (8.5 Gy) and reconstituted with 4xl0 7 splenocytes either from B6 (semi-allogeneic, left panel) or (2Cxdm2) F1 (L d mismatched, right panel) mice.
  • Liver histology is shown at 100 days after infusion of allogeneic cells.
  • the left panel shows infiltrating mononuclear cells, proliferation of bioduct, and abnormal portal and venous structures, a typical lesion of chronic graft verses host disease in the liver.
  • the right panel shows that the liver histology is normal.
  • the hepatocytes, liver cell cords, portal and venous structures are all normal. There is no evidence of graft versus host disease.
  • Figure 10 (a) Specific recognition of cell surface molecules on DN regulatory T cells by the monoclonal antibodies (mAbs).
  • the DN regulatory T cell clone TN12.2 and CD8 + non-regulatory T cell clone (C02) were stained with the mAb P3C2/G1, followed by an anti-rat secondary PE labelled mAb. Cells were analyzed using a flow cytometer. The data shown is representative of 25 mAbs that the inventors have generated so far.
  • mAb generated against DN regulatory T cell clones can also specifically bind to primary activated DN regulatory T cells.
  • DN regulatory T cells and CD8 + T cells purified from the spleen of B6 mice were activated in vitro and stained with mAb P3C2/G1, and analyzed as described in (a).
  • mAbs are able to reverse DN regulatory T cell-mediated suppression of 1B2 + CD8 + T cells. Suppression assays were set up in 96 well plates as described in Figure 2(b). The DN T cell clone TN12.2 was used as suppressor cells, and 1B2 + CD8 + T cells from the spleen of (2Cxdm2) F1 mice were used as responders. In addition, lOO ⁇ l of supernatant from P3C2/A2 or control P3G3/D1 mAb hybridomas was added to each well. As controls, supernatant was added to DN T cells clones in the absence of 1B2 + CD8 + responder cells.
  • FIG. 11 (a) In vitro generated DN regulatory T cell lines are resistant to TCR cross-linking induced apoptosis. 1B2 + DN T cell clones (TN02, TN12 and CN04), 1B2 + CD8 + T cell clone (T01) and primary activated 1B2 + CD8 + T cells (2Cxdm2) F1 were cross-linked with plate-bond 1B2 mAb (65 ⁇ g/ml). At various time points after TCR-cross-linking, the percentage of dead cells in each culture was determined by trypan blue staining.
  • IL-10 increases susceptibility of DN T cells to apoptosis.
  • TN12 DN T cells were cross-linked in the absence (open bars) or presence of IL-10 (100 ng/ml, black bars). Percentage cell death was determined as described in (a).
  • Figure 12 IL-10, CsA, anti-TCR and anti-IFN- ⁇ antibodies abrogate DN regulatory T cell mediated suppression.
  • Figure 13 Expression and function of Osteopontin (OPN) in DN regulatory T cells.
  • DN regulatory T cells (CN04) were stained by either purified mouse anti-rat OPN mAb MPIIIB10 (DSHB, The University of Iowa) followed by FITC-labelled anti-mouse monoclonal antibody or secondary monoclonal antibody alone as a control. Data were analysed using a Flow cytometer.
  • Figure 14 (a) Pretransplant DST and short-term CD4 depletion leads to permanent acceptance ofxenogeneic vascularized cardiac grafts.
  • Bar graft shows the % of 1B2 + CD8 + and 1B2 + DN T cells in DST treated accepted and non-DST treated rejecting skin allografts pooled from 5 mice at 7 days after transplantation.
  • FIG 16 Increase of IL-4 and IFN- ⁇ in accepted skin allografts.
  • (2Cxdm2) F1 mice were treated as in Figure 15. After 7 days the skin from accepted (B6xBALB/c) F1 grafts and rejecting C3H grafts were harvested, and stained for the anti-L d transgenic TCR (1B2), CD4, CD8 and various cytokines as indicated. The cells were analyzed by using a flow cytometer. The markers indicate the percentage of cells producing each cytokines. Data shown here are the pooled results from 5 mice. DETAILED DESCRIPTION OF THE INVENTION I. Novel Regulatory T cells The present inventors have isolated novel regulatory T cells that have important functions in immune regulation.
  • novel regulatory cells are distinguished from previously described regulatory cells as they possess a unique phenotype and express a unique array of cytokines.
  • the novel cells are CD3 + ⁇ TCR + CD4 " CD8 " CDlla + CD18 + CD25 + CD28-CD44-NK1.1-. These cell surface markers distinguish the cells from any previously described T cell subset such as helper, cytotoxic or memory T cells.
  • the novel regulatory cells are also distinguished from CD4 " CD8 " T cells and regulatory/suppressor T cells described by others.
  • novel regulatory cells do not express IL-2, IL-4, IL-10 and IL-13 but do express IFN- ⁇ , TNF- ⁇ and TGF- ⁇ mRNA after activation which distinguish them from Thl, Th2 or Th3/Trl cells. Further , the inventors have shown that the regulatory T cells of the invention also express Ly-6A and osteopontin which has not been previously described on other regulatory T cells.
  • the present invention provides an isolated immune regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4"CD8- CD44-CD28"NK1.1-.
  • the regulatory cells having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l" are sometimes referred to herein as "the novel regulatory cells” or "the regulatory T cells".
  • the term "a regulatory T cell” includes one or more of the regulatory T cells of the invention.
  • the inventors have also shown that suppression by the novel regulatory T cells requires cell contact and is not mediated through a soluble factor released by the cells.
  • the novel regulatory T cells also require signals through both the T cell receptor (TCR) and Fas /Fas ligand to mediate suppression.
  • TCR T cell receptor
  • the suppressive properties of the novel regulatory T cells can be abolished by IL-10, cyclosporin A (CsA), anti-regulatory T cell antibodies, anti-IFN- ⁇ and anti-TCR antibodies ( Figure 12b). Accordingly, IL-10, CsA, anti-regulatory T cell antibodies, anti-IFN- ⁇ antibodies and anti-TCR antibodies can be used to inhibit the function of the regulatory T cells.
  • the present invention also includes the generation of the novel regulatory T cells in vitro.
  • the cells may be isolated from normal animals, for example by isolating lymphocytes, labelling T cells and sorting for cells containing the desired phenotype using a FACS sorter.
  • the inventors have demonstrated that the novel regulatory T cells generally require IL-2 and IL-4 to proliferate and to suppress.
  • the inventors have further developed methods to activate and expand antigen-specific regulatory T cells in vitro by stimulating the novel regulatory T cells with allogeneic lymphocytes in the presence of IL-2 and IL-4. Accordingly, IL-2 and IL-4 can be used to increase the number and improve the function of the novel regulatory cells.
  • the present invention provides a method of expanding a population of regulatory T cells having the phenotype CD3 + ⁇ TCR + CD4-CD8"CD44- CD28 " NK1.1 " in vitro comprising:
  • the sample may be any sample that contains the regulatory T cells or precursors of the regulatory T cells including, but not limited to, blood, bone marrow, lymphoid tissue, epithelia, thymus, liver, spleen, cancerous tissues, lymph node tissue, infected tissue, fetal tissue and fractions or enriched portions thereof.
  • the sample is preferably blood including peripheral blood or fractions thereof, including buffy coat cells, mononuclear cells and low density mononuclear cells (LDMNC).
  • the regulatory T cells may be obtained from a sample of blood using techniques known in the art such as density gradient centrifugation.
  • the sample or fraction thereof Prior to stimulating the sample or fraction thereof (such as LDMNC) with antigen, the sample or fraction thereof may be depleted of other cell types such as B cells, NK cells and CD4 + or CD8 + T cells.
  • the sample may be depleted of certain cell types using techniques known in the art.
  • the cells of a particular phenotype may be depleted by culturing the starting sample or fraction thereof with an antibody cocktail containing antibodies specific for markers on the cells to be depleted.
  • the antibodies in the cocktail are tetrameric antibody complexes as described in United States Patent No. 4,868,109 to Lansdorp.
  • the antigen can be any antigen depending on the desired antigen specificity of the regulatory T cells.
  • the antigen may be a donor specific antigen or cells containing a donor specific antigen when the cells are used to prevent graft rejection, an autoantigen when the cells are used to treat autoimmune disease and an allergen when the cells are used to treat an allergy.
  • the antigens can be used in any form including purified peptides, soluble proteins, plasmid expressing cDNA encoding specific antigens, cell lines expressing specific antigens (EBV transformed cell lines, dendritic cells, fibroblasts transfected with specific antigens such as foreign MHC molecules), molecules that cause autoimmune diseases and allergy.
  • the cells are cultured with the antigen "under conditions suitable for the expansion of the regulatory T cells” which means in an appropriate culture medium and for a suitable period of time to allow for the expansion of the regulatory T cells.
  • the appropriate culture medium is any media that supports the expansion of the regulatory T cells and it preferably contains IL-2 and IL-4.
  • expansion it is meant that the number of the regulatory T cells after step (c) in the above method is higher than the number of regulatory T cells in the initial sample.
  • IL-10 can convert the apoptosis-resistant regulatory T cells to apoptosis sensitive phenotype.
  • certain antibodies to the novel regulatory cells may also induce apoptosis in the cells. Accordingly, IL-10 and/or antibodies that bind the novel regulatory cells can be used to reduce the number of the regulatory T cells and can be used in therapies where immune suppression is not desired.
  • the inventors have generated monoclonal antibodies (mAbs) by immunization of animals with regulatory T cells of the invention.
  • the inventors have so far generated 25 mAbs that can specifically bind to the surface of the regulatory T cells.
  • Some of the mAbs can convert apoptosis-resistant regulatory T cells into apoptosis-sensitive phenotype.
  • Some mAbs can enhance growth of regulatory T cells and some can abolish the suppressive function of regulatory T cells.
  • the present invention provides an antibody that can bind to a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l ⁇
  • the invention includes use of antibodies that bind to the regulatory cells for isolation and purification of regulatory T cells.
  • the present invention also includes the use of the antibodies for up- or down-regulation of survival and function of regulatory T cells.
  • the present invention also includes use of the antibodies for identification of proteins that are crucial for the function and survival of regulatory T cells.
  • the invention also includes the use of the antibodies and their therapeutic modifications in prevention and treatment of diseases such as graft rejection, autoimmune diseases, allergy and AIDS which are discussed in greater detail below.
  • the invention also incudes modified forms of the antibodies of the invention. Modifications of monoclonal antibodies include the generation of recombinant mAbs fused with human immunoglobulin Fc portion and the conjugation of the mAbs with enzymes, isotopes, etc.
  • the present inventors have isolated several genes that are expressed in the novel T regulatory cells of the invention but are not expressed in non-regulatory cells.
  • the inventors have shown that treatment of the novel regulatory T cells with IL-10 switches the cells from a regulatory to a non-regulatory phenotype.
  • IL-10 may regulate the expression of certain molecules necessary for suppression by the novel regulatory cells. Consequently, using PCR-selected cDNA subtraction, the inventors have identified genes that are differentially expressed in regulatory cells but not non-regulatory cells.
  • the inventors have generated cDNA libraries from both regulatory and non-regulatory T cells.
  • Ly-6A is a glycosyl phosphatidylinositol (GPI)-anchored cell surface molecule expressed on most peripheral lymphocytes, thymocytes and other cells.
  • GPI glycosyl phosphatidylinositol
  • Ly-6A protein includes the full-length Ly-6A protein as well as fragments or portions of the protein. Preferred fragments or portions of the protein are those that are sufficient to suppress an immune response.
  • the Ly-6A protein also includes fragments that can be used to prepare antibodies.
  • the Ly-6A protein may be prepared as a soluble fusion protein.
  • the fusion protein may contain the extracellular domain of Ly-6A linked to an immunoglobulin (Ig) Fc Region.
  • the Ly-6A fusion protein may be prepared using techniques known in the art. Generally, a DNA sequence encoding the extracellular domain of Ly-6A is linked to a DNA sequence encoding the Fc of the Ig and expressed in an appropriate expression system where the Ly-6A - Fclg fusion protein is produced.
  • the Ly-6A protein may be obtained from known sources or prepared using recombinant DNA techniques. The protein may have any of the known published sequences for Ly-6A.
  • the protein may also be modified to contain amino acid substitutions, insertions and/or deletions that do not alter the immunosuppressive properties of the protein.
  • conserveed amino acid substitutions involve replacing one or more amino acids of the Ly-6A amino acid sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting analog should be functionally equivalent to the Ly-6A protein.
  • Non-conserved substitutions involve replacing one or more amino acids of the Ly-6A amino acid sequence with one or more amino acids which possess dissimilar charge, size, and /or hydrophobicity characteristics.
  • the Ly-6A protein may be modified to make it more therapeutically effective or suitable.
  • the Ly-6A protein may be cyclized as cyclization allows a peptide to assume a more favourable conformation.
  • Cyclization of the Ly-6A peptides may be achieved using techniques known in the art.
  • disulphide bonds may be formed between two appropriately spaced components having free sulfhydryl groups.
  • the bonds may be formed between side chains of amino acids, non-amino acid components or a combination of the two.
  • the Ly-6A protein or peptides of the present invention may be converted into pharmaceutical salts by reacting with inorganic acids including hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids including formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulphonic acid, and tolunesulphonic acids.
  • inorganic acids including hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid, etc.
  • organic acids including formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulphonic acid, and tolune
  • osteopontin may be used to enhance immune tolerance or suppression and inhibiting osteopontin may be used to enhance an immune response.
  • osteopontin protein includes the full-length osteopontin protein as well as fragments or portions of the protein. Preferred fragments or portions of the protein are those that are sufficient to suppress an immune response.
  • the osteopontin protein also includes fragments that can be used to prepare antibodies.
  • the osteopontin protein may be prepared as a soluble fusion protein.
  • the fusion protein may contain the extracellular domain of osteopontin linked to an immunoglobulin (Ig) Fc Region.
  • the osteopontin fusion may be prepared using techniques known in the art.
  • a DNA sequence encoding the extracellular domain of osteopontin is linked to a DNA sequence encoding the Fc of the Ig and expressed in an appropriate expression system where the osteopontin - Fclg fusion protein is produced.
  • the osteopontin protein may be obtained from known sources or prepared using recombinant DNA techniques.
  • the protein may have any of the known published sequences for osteopontin.
  • the protein may also be modified to contain amino acid substitutions, insertions and/ or deletions that do not alter the immunosuppressive properties of the protein. conserveed amino acid substitutions involve replacing one or more amino acids of the osteopontin amino acid sequence with amino acids of similar charge, size, and /or hydrophobicity characteristics.
  • Non-conserved substitutions involve replacing one or more amino acids of the osteopontin amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics.
  • the osteopontin protein may be modified to make it more therapeutically effective or suitable.
  • the osteopontin protein may be cyclized as cyclization allows a peptide to assume a more favourable conformation.
  • Cyclization of the osteopontin pep tides may be achieved using techniques known in the art.
  • disulphide bonds may be formed between two appropriately spaced components having free sulfhydryl groups.
  • the bonds may be formed between side chains of amino acids, non-amino acid components or a combination of the two.
  • the osteopontin protein or peptides of the present invention may be converted into pharmaceutical salts by reacting with inorganic acids including hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids including formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulphonic acid, and tolunesulphonic acids.
  • inorganic acids including hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid, etc.
  • organic acids including formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulphonic acid, and tol
  • the present invention includes the use of (a) the novel regulatory cells; (b) agents that stimulate the regulatory cells such as antibodies to the novel regulatory cells; (c) an Ly-6A protein or nucleic acid encoding an Ly-6A protein; and (d) an osteopontin protein or a nucleic acid encoding an osteopontin protein to suppress an immune response or to prepare a medicament to suppress an immune response.
  • the present invention provides a method of suppressing an immune response comprising administering an effective amount of a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28"NKl.l- to an animal in need of such treatment.
  • administering includes administering regulatory T cells that have been expanded or cultured in vitro and expanding or stimulating regulatory T cells that are present in the animal directly in vivo.
  • the regulatory T cells are prepared in vitro as described above and injected into the animal.
  • the regulatory T cells are directly induced or stimulated in vivo, for example using IL-2 and IL-4 or antibodies to the cells and /or by stimulating the cells with antigen or one MHC Class I locus mismatched allogeneic lymphocytes.
  • the present invention provides a method of suppressing an immune response comprising administering an effective amount of an agent that stimulates a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44 CD28'NKl.l- to an animal in need of such treatment.
  • the agent is an antibody that stimulates the regulatory cells.
  • the present invention provides a method of suppressing an immune response comprising administering an effective amount of an Ly-6A protein or a nucleic acid sequence encoding an Ly-6A protein to an animal in need of such treatment.
  • the present invention provides a method of suppressing an immune response comprising administering an effective amount of an osteopontin protein or a nucleic acid sequence encoding an osteopontin protein to an animal in need of such treatment.
  • an "effective amount" of the active agent i.e., regulatory T cells, antibodies, osteopontin or Ly-6A protein or nucleic acid of the present invention
  • the effective amount of the active agent may vary according to factors such as the disease state, age, sex, and weight of the animal. Dosage procedures may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • animal as used herein includes all members of the animal kingdom including humans.
  • the methods of suppressing an immune response using the regulatory T cells or agents that stimulate the regulatory T cells of the invention have wide ranging applications.
  • the regulatory T cells are antigen specific and therefore they can be used to specifically suppress an immune response to any antigen including the antigens present on pathogens (such as viruses, bacteria, fungi, etc.) and grafts as well as the antigens implicated in autoimmune disease and allergies. Generating an antigen specific immune response is much more desirable than the non-specific immune suppressants that are known in the art.
  • the present invention provides a method of inducing specific immune tolerance in a recipient animal comprising administering an effective amount of the regulatory T cells, antibodies to the regulatory cells, Ly-6A protein or a nucleic acid sequence encoding an Ly-6A protein or osteopontin or a nucleic acid sequence encoding osteopontin to the recipient animal.
  • inducing immune tolerance means rendering the immune system unresponsive to a particular antigen without inducing a prolonged generalized immune deficiency.
  • antigen means a substance that is capable of inducing an immune response.
  • immune tolerance means rendering the immune system unresponsive to an auto-antigen that the host is recognizing as foreign, thus causing an autoimmune response.
  • immune tolerance means rendering the immune system unresponsive to an allergen that generally causes an immune response in the host.
  • immune tolerance means rendering the immune system unresponsive to the antigens on the transplant.
  • An alloantigen refers to an antigen found only in some members of a species, such as blood group antigens.
  • a xenoantigen refers to an antigen that is present in members of one species but not members of another.
  • an allograft is a graft between members of the same species and a xenograft is a graft between members of a different species.
  • the recipient can be any member of the animal kingdom including rodents, pigs, cats, dogs, ruminants, non-human primates and preferably humans.
  • the method of the invention is used to prevent rejection of a transplanted graft.
  • the graft may be organs, tissues, cells or the like.
  • the organ, tissue or cells to be transplanted can be from the same species as the recipient (allograft) or can be from another species (xenograft).
  • the graft can be any tissue, organ or cell including heart, liver, kidney, lung, pancreas, pancreatic islets, brain tissue, cornea, bone, intestine, skin and hematopoietic cells.
  • the present inventors have demonstrated that injection of the regulatory T cells into animals can significantly enhance skin allograft survival in a dose-dependant and antigen-specific manner. Accordingly, in one embodiment, the present invention provides a method of preventing graft rejection in a recipient animal comprising administering an effective amount of a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l" to an animal in need of such treatment. The present inventors have further identified the methods to increase the number and function of the novel regulatory T cells within allografts by infusion of one MHC class I locus mismatched allogeneic lymphocytes, which in turn leads to permanent acceptance of the allografts.
  • graft survival could be enhanced by increasing local IL-2 and IL-4 levels by either injection of rIL-2 and rIL-4 or increase IL-2/4 producing cells locally in conjunction with local injection of regulatory T cells.
  • the method can be used to enhance the survival of a cardiac xenograft.
  • the inventors have demonstrated that pre-transplant DST in conjunction with several injections of mAb to selectively deplete CD4 + T cells leads to a long term survival of rat cardiac xenografts in mice ( Figure 14a). Because the recipients are non-thymectomized, new anti-donor T cells generated from the thymus must have been inactivated by regulatory T cells in the recipient in order to maintain the xenografts. CD4 + T cells have been speculated to play a role in allograft tolerance.
  • CD4 + T cells are physically depleted in this model, it is unlikely that CD4 + T cells are responsible for the xenograft survival. Furthermore, depletion of CD4 + T cells leads to an increase of the proportion of the regulatory T cells in the recipients.
  • the inventors have demonstrated that the number of the novel regulatory T cells in accepted xenografts is significantly increased in DST and anti-CD4 treated animals ( Figure 14b), and that the regulatory T cells can specifically kill anti-donor T cells (Example 1). Therefore it is plausible that the xenograft tolerance seen after treatment with DST and anti-CD4 depleting mAb is due to enhancement of the number and function of donor-specific DN regulatory T cells.
  • donor-specific regulatory T cells can be generated in the following way. Before organ transplantation the regulatory T cells can be purified from blood or bone marrow of the recipient. The purified regulatory T cells can be stimulated with donor antigens in various forms including irradiated donor lymphocytes or recipient cells (e.g., dendritic cells or fibroblasts) that have been genetically manipulated to express one donor MHC antigen. Suitable concentrations of IL-2 and IL-4 will be added to the culture. The donor-specific regulatory T cells generated in such way can be injected back into the recipients before transplantation with or without co-injection of rIL-2 and rIL-4. Because these regulatory T cells can specifically kill the recipient CD8 + T cells that are activated by the same donor as the inventors have demonstrated, the regulatory T cells should be able to protect the graft from being attacked by anti-donor cytotoxic T cells.
  • donor antigens in various forms including irradiated donor lymphocytes or recipient cells (e.g., dendritic cells or fibro
  • the method of the invention may be also used to prevent graft versus host disease wherein the immune cells in the transplant mount an immune attack on the recipient's organs and tissues. This can occur when the tissue to be transplanted contains immune cells such as when bone marrow or lymphoid tissue is transplanted when treating leukemias, aplastic anemias and enzyme or immune deficiencies, for example.
  • the present invention provides a method of preventing or inhibiting graft versus host disease in a recipient animal comprising administering an effective amount of the regulatory T cells, antibodies to the regulatory T cells, an Ly-6A protein or a nucleic acid sequence encoding an Ly-6A protein or osteopontin or a nucleic acid sequence encoding osteopontin to the recipient animal.
  • regulatory T cells can be purified from blood or bone marrow of graft donor and expanded in vitro.
  • the activated regulatory T cells can be injected back into the recipient before bone marrow transplantation from the same donor.
  • the donor cytotoxic T cells that are activated by host alloantigens will be killed by the donor-derived regulatory T cells therefore to prevent GVHD.
  • Donor cells that are activated by third party antigens, including viruses and bacteria, will not be killed and thus can fight off infections.
  • the inventors have postulated novel mechanisms by which the regulatory T cells prevent GVHD.
  • bone marrow transplantation mature donor T cells CD4 + and CD8 +
  • CD4 + and CD8 + will recognize alloantigens expressed on the host, be activated and express high level of Fas.
  • These activated donor T cells will destroy target cells and tissues that express the host alloantigens and cause GVHD.
  • the inventors have demonstrated that the novel regulatory T cells constitutively express a high level of Fas ligand.
  • APC antigen-presenting cells
  • the regulatory T cells can "steal" host alloantigens from the surface of APC through the anti-host TCR, and turn themselves into killer cells.
  • the regulatory T cells express the "stolen" host alloantigens on their surface, they can attract the activated anti-host cytotoxic T cells. Once the anti-host cytotoxic T cells recognized the alloantigens on the regulatory T cells, the latter will send death signals through Fas ligand to the former. Unlike the anti-host cytotoxic T cells, which kill target cells through perforin-mediated pathway, the killing mediated by the regulatory T cells requires direct cell-cell contact and depends on Fas-Fas ligand interaction. Most host tissues, although they express MHC class I molecules, do not express Fas, and will not be destroyed directly by the regulatory T cells. Therefore the regulatory T cell themselves do not cause GVHD.
  • the method of the present invention may also be used to treat or prevent autoimmune disease.
  • the immune system of the host fails to recognize a particular antigen as "self” and an immune reaction is mounted against the host's tissues expressing the antigen.
  • the immune system is tolerant to its own host's tissues and autoimmunity can be thought of as a breakdown of tolerance in the immune system.
  • the present invention provides a method of preventing or treating an autoimmune disease comprising administering an effective amount of the regulatory T cells, antibodies to the regulatory T cells, Ly-6A protein or a nucleic acid sequence encoding an Ly-6A protein or osteopontin or a nucleic acid sequence encoding osteopontin to an animal having, suspected of having, or susceptible to having an autoimmune disease.
  • the invention also includes the use of the regulatory T cells, antibodies to the regulatory T cells, Ly-6A protein or a nucleic acid sequence encoding an Ly-6A protein or osteopontin or a nucleic acid sequence encoding osteopontin to prevent or treat an autoimmune disease or to prepare a medicament to prevent or treat an autoimmune disease.
  • Autoimmune diseases that may be treated or prevented according to the present invention include, but are not limited to, type 1 insulin-dependent diabetes mellitus, multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, adult respiratory distress syndrome, dermatitis, meningitis, thrombotic thrombocytopenic purpura, Sj ⁇ gren's syndrome, encephalitis, uveitic, leukocyte adhesion deficiency, rheumatic fever, Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis, primary biniary cirrhosis, pemphigus, pemphigoid, necrotizing vasculitis, myasthenia gravis, polymyositis, sarcoidosis, granulomatosis, vasculitis, pernicious anemia, CNS inflammatory disorder, antigen-antibody complex mediated diseases, autoimmune haemolytic anemia,
  • the regulatory T cells can be purified from a patient and stimulated with antigens that are known to be involved in the induction/progression of autoimmune diseases, such as collagen in arthritis, myelin basic protein in multiple sclerosis, etc.
  • the antigen-specific regulatory T cells generated in vitro will be injected back to the patient.
  • the regulatory T cells will be able to specifically kill activated CD8 + and/or CD4 + T cells that cause autoimmune diseases.
  • the method of the present invention may also be used to treat or prevent an allergic reaction.
  • an allergic reaction the immune system mounts an attack against a generally harmless, innocuous antigen or allergen.
  • Allergies that may be prevented or treated using the methods of the invention include, but are not limited to, hay fever, asthma, atopic eczema as well as allergies to poison oak and ivy, house dust mites, bee pollen, nuts, shellfish, penicillin and numerous others.
  • the present invention provides a method of preventing or treating an allergy comprising administering an effective amount of the regulatory T cells, antibodies, Ly-6A protein or a nucleic acid sequence encoding an Ly-6A protein or osteopontin or a nucleic acid sequence encoding osteopontin to an animal having or suspected of having an allergy.
  • the invention also includes the use of the regulatory T cells, antibodies to the regulatory T cells, Ly-6A protein or a nucleic acid sequence encoding an Ly-6A protein or osteopontin or a nucleic acid sequence encoding osteopontin to prevent or treat an allergy or to prepare a medicament to prevent or treat an allergy.
  • antigen-specific regulatory T cells will be purified from patients and stimulated in vitro with corresponding allergens, and inject back to the patients.
  • the present inventors have also shown that the regulatory T cells activated in vitro by a specific antigen can specifically kill the CD8 + T cells that are activated by the same antigen but not those activated by different antigen ( Figures 5-7).
  • the regulatory T cells can be used to specifically suppress an immune response caused by CD8 + or cytotoxic T cells.
  • the present invention provides a method of suppressing a cytotoxic T cell response to an antigen comprising administering an effective amount of a regulatory T cell having the phenotype CD3 + ⁇ TCR + CD4-CD8-CD44-CD28-NKl.l- and having specificity for the antigen, to an animal in need thereof.
  • the regulatory cells can be expanded in vitro with the antigen causing the CT1 response as described above.
  • the regulatory T cells can also be stimulated in vivo for example by using antibodies that activate the regulatory cells.
  • the present invention also includes methods of preventing immune suppression (or enhancing an immune response) by administering an agent that inhibits the regulatory T cells, Ly-6A or osteopontin.
  • an agent that inhibits the regulatory T cells, Ly-6A or osteopontin that inhibits the regulatory T cells, Ly-6A or osteopontin.
  • the present invention provides a method of preventing immune suppression comprising administering an effective amount of an agent that inhibits the activation and /or the function of the regulatory T cells to an animal in need thereof.
  • Agents that inhibit the regulatory T cells include CsA, IL-10, anti-IFN ⁇ and anti-TCR antibodies in addition to antibodies to the regulatory cells.
  • the present invention provides a method of preventing immune suppression comprising administering an effective amount of an agent that inhibits Ly-6A to an animal in need thereof.
  • the present invention provides a method of preventing immune suppression comprising administering an effective amount of an agent that inhibits osteopontin to an animal in need thereof.
  • the present invention provides a method of preventing immune suppression comprising administering an effective amount of an agent that binds to the regulatory T cells, Ly-6A or osteopontin to an animal in need thereof.
  • the agent that binds the regulatory T cells, Ly-6A or osteopontin is an antibody.
  • Antibodies to the regulatory T cells are described above. In particular, the inventors have shown that some antibodies to the regulatory T cells can induce apoptosis and are therefore useful in suppressing the regulatory T cells.
  • Antibodies to the regulatory T cells, Ly-6A or osteopontin may be prepared using techniques known in the art such as those described by Kohler and Milstein, Nature 256, 495 (1975) and in U.S. Patent Nos. RE 32,011; 4,902,614; 4,543,439; and 4,411,993, which are incorporated herein by reference. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference). Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab') 2 ) and recombinantly produced binding partners.
  • antibody producing cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
  • myeloma cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
  • Such techniques are well known in the art, (e.g., the hybridoma technique originally developed by Kohler and Milstein) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies in Cancer Therapy (1985) Allen R.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the regulatory T cells, Ly-6A or osteopontin and the monoclonal antibodies can be isolated. Therefore, the invention also contemplates hybridoma cells secreting monoclonal antibodies with specificity for regulatory T cells, Ly-6A or osteopontin of the invention.
  • Chimeric antibody derivatives i.e., antibody molecules that combine a non- human animal variable region and a human constant region are also contemplated within the scope of the invention.
  • Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions.
  • Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes a protein of the invention (See, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al, Nature 314, 452 (1985), Cabilly et al, U.S. Patent No.
  • Monoclonal or chimeric antibodies specifically reactive with regulatory T cells, Ly-6A or osteopontin of the invention as described herein can be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin.
  • Such irnmunoglobulin molecules may be made by techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al, Immunology Today, 4, 7279 (1983); Olsson et al, Meth. Enzymol., 92, 3-16 (1982)), and PCT Publication WO92/06193 or EP 0239400).
  • Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain).
  • Specific antibodies, or antibody fragments, reactive against Ly-6A or osteopontin may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with Ly-6A or osteopontin.
  • complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al., Nature 341, 544- 546: (1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).
  • the agent that inhibits the regulatory T cells is an agent that interferes with the Fas-FasL interaction between the regulatory T cells and their target.
  • the agent may be a soluble Fas fusion protein (such as Fas-Fc) which binds to the FasL on the regulatory T cells and inhibits their function.
  • the agent that inhibits Ly-6A or osteopontin is an antisense oligonucleotide that inhibits the expression of Ly-6A or osteopontin.
  • Antisense oligonucleotides that are complimentary to a nucleic acid sequence from an Ly-6A gene or an osteopontin gene can be used in the methods of the present invention to inhibit Ly-6A or osteopontin.
  • the present inventors have prepared antisense oligonucleotides to Ly-6A which are described in Example 2.
  • the present invention provides a method of preventing immune suppression comprising administering an effective amount of an antisense oligonucleotide that is complimentary to a nucleic acid sequence from an Ly-6A gene to an animal in need thereof.
  • the present invention also provides, a method of preventing immune suppression comprising administering an effective amount of an antisense oligonucleotide that is complimentary to a nucleic acid sequence from an osteopontin gene to an animal in need thereof.
  • antisense oligonucleotide as used herein means a nucleotide sequence that is complimentary to its target.
  • oligonucleotide refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages.
  • the term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases.
  • the term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide.
  • the antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines,
  • antisense oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.
  • phosphorothioate bonds link all the nucleotides.
  • the antisense oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents.
  • An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254, 1497). PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro.
  • PNA peptide nucleic acid
  • oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506).
  • Oligonucleotides may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide.
  • Antisense oligonucleotides may also have sugar mimetics.
  • the antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the antisense nucleic acid molecules of the invention or a fragment thereof may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides.
  • the antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced, (c) Compositions
  • the invention also includes pharmaceutical compositions containing the regulatory T cells, the antibodies to the T cells, Ly-6A or osteopontin proteins or nucleic acids for use in inducing immune suppression as well as pharmaceutical compositions containing inhibitors of these for use in preventing immune suppression.
  • compositions can be for intralesional, intravenous, topical, rectal, parenteral, local, inhalant or subcutaneous, intradermal, intramuscular, intrathecal, transperitoneal, oral, and intracerebral use.
  • the composition can be in liquid, solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets, solutions or suspensions.
  • compositions of the invention can be intended for administration to humans or animals. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration.
  • compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • the pharmaceutical compositions may additionally contain other agents such as immunosuppressive drugs or antibodies to enhance immune tolerance or immunostimulatory agents to enhance the immune response through the novel antigen specific regulatory T cells.
  • the pharmaceutical composition for use in inducing immune tolerance comprises an effective amount of the regulatory T cells, an antibody against the regulatory T cells, an Ly-6A protein or osteopontin in admixture with a pharmaceutically acceptable diluent or carrier.
  • the Ly-6A protein or osteopontin is preferably prepared as an immunoadhesion molecule in soluble form which can be administered to the patient.
  • the composition preferably contains Ly-6A proteins or osteopontin in soluble form which may be injected intravenously or perfused directly at the site of the transplantation.
  • the pharmaceutical composition for use in inducing immune tolerance comprises an effective amount of a nucleic acid molecule encoding an Ly-6A protein or osteopontin in admixture with a pharmaceutically acceptable diluent or carrier.
  • the nucleic acid molecules of the invention encoding an Ly-6A protein or osteopontin may be used in gene therapy to induce immune tolerance.
  • Recombinant molecules comprising a nucleic acid sequence encoding an Ly-6A protein or osteopontin, or fragment thereof, may be directly introduced into cells or tissues in vivo using delivery vehicles such as retroviral vectors, adenoviral vectors and DNA e virus vectors. They may also be introduced into cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. Recombinant molecules may also be delivered in the form of an aerosol or by lavage.
  • the nucleic acid molecules of the invention may also be applied extracellularly such as by direct injection into cells.
  • the nucleic acid molecules encoding Ly-6A or osteopontin are preferably prepared as a fusion with a nucleic acid molecule encoding an immunoglobulin (Ig) Fc region.
  • Ig immunoglobulin
  • the Ly-6A or osteopontin protein will be expressed in vivo as a soluble fusion protein.
  • the pharmaceutical composition for use in preventing immune suppression or enhancing an immune response comprises an effective amount of an agent that inhibits the regulatory T cells, Ly-6A, or osteopontin in admixture with a pharmaceutically acceptable diluent or carrier.
  • an agent that inhibits the regulatory T cells, Ly-6A, or osteopontin in admixture with a pharmaceutically acceptable diluent or carrier.
  • Such compositions may be administered as a vaccine either alone or in combination with other active agents or antigens.
  • the Ly-6A inhibitors may act like an adjuvant by potentiating the immune response to the antigen in the vaccine.
  • the pharmaceutical composition for use in preventing immune suppression comprises an effective amount of an antibody to the regulatory cells or Ly-6A or osteopontin in admixture with a pharmaceutically acceptable diluent or carrier.
  • the antibodies may be delivered intravenously.
  • the pharmaceutical composition for use in preventing immune suppression comprises an effective amount of an antisense oligonucleotide nucleic acid complimentary to a nucleic acid sequence from an Ly-6A gene or osteopontin in admixture with a pharmaceutically acceptable diluent or carrier.
  • the oligonucleotide molecules may be administered as described above for the compositions containing Ly-6A or osteopontin nucleic acid sequences.
  • DST pretransplantation DST can enhance allograft survival (10,11).
  • the suggested mechanisms whereby the introduction of donor cells leads to tolerance include clonal deletion, clonal anergy, suppression by regulatory cells, regulation of cell surface molecules or cytokines expression, and promotion of microchimerism (21,35-37). Although informative, these studies are not definitive.
  • the major obstacle to understanding the mechanism of donor specific transfusion (DST) -induced tolerance is the lack of a specific cell marker(s) for detecting donor-specific antigen-reactive cells in vivo. This limitation precludes tracing the cellular and molecular events occurring within patients or normal (i.e. non-transgenic) animals after encountering donor antigen. In order to obtain a system in which a T cell response to a defined allogeneic
  • mice carry functionally re-arranged TCR ⁇ - (one copy) and ⁇ -chain (eight copies) transgenes derived from a cytotoxic T cell clone 2C, which is alloreactive for class I MHC Ag L d .
  • the specificity for L d requires the transgenic ⁇ - and ⁇ -chains as well as the CD8 co-receptor (38,39).
  • the 2C clonotypic TCR (both - and ⁇ -chains) is recognized by the mAb 1B2 (40).
  • mice on C57BL/6 (B6) background were bred with dm2 mice (a BALB/c L d loss mutant, H-2D d+ , K d+ , I-E d+ , I-A d+ , L d" ).
  • the (2Cxdm2) F1 mice (H-2 b / d , L d ", either 1B2 + or 1B2 " ) were used as recipients.
  • (B6xBALB/c) F1 mice (BYJ F1 , H-2 b / d , L d+ ) mismatched only for L d with the (2Cxdm2) F1 mice were used as lymphocyte and graft donors. As a result, the only immune response in this system is derived from recipient cells reacting to L d Ag expressed on the donor.
  • the (2Cxdm2) F1 mice were intravenously injected with viable L d+ (B6xBALB/c) F1 lymphocytes followed by transplantation of skin grafts obtained from both (B6xBALB/c) F 1
  • the regulatory T cells reported by others are CD4 + (13,42,45).
  • 1B2 + CD8 + , 1B2 + CD4 + and 1B2 + CD4'CD8- (double negative, DN) T cells were purified from the spleen of tolerant mice, and tested for their regulatory function in vitro .
  • a dose-dependent inhibition of proliferation of na ⁇ ve anti-L d T cells was observed only in cultures to which 1B2 + DN cells from tolerant mice were added.
  • ⁇ T cells in the periphery of mice or humans express the CD4 or CD8 co-receptor. About 1-5% of peripheral T cells are double negative (DN) (50). Although extensive studies have been done on CD4 + or CD8 + T cells, little is known about the function and homeostasis of DN T cells. This paucity of information is largely due to the relatively small number of DN T cells in the periphery, which make such studies more difficult. In order to characterize the DN regulatory T cells and understand the mechanisms of suppression, the inventors generated panels of 1B2 + DN and 1B2 + CD8 + T cell clones from the spleen of tolerant mice.
  • the transgenic TCR can be detected by the clonotypic monoclonal antibody (mAb) 1B2. To determine which subset of T cells from tolerant
  • (2Cxdm2) F1 mice was responsible for the inhibition of the anti-L d response mediated by na ⁇ ve T cells, 1B2 + CD8 + , 1B2 + CD4 + and 1B2 + CD4-CD8 " (double negative, DN) T cells were purified from the spleen of tolerant mice 120 days after skin grafting, and tested for their regulatory function in vitro .
  • a dose-dependent inhibition of proliferation of na ⁇ ve anti-L d T cells in mixed lymphocyte reactions (MLR) was observed only in cultures to which 1B2 + DN T cells from tolerant mice were added (Figure 2b). No inhibition was seen when 1B2 + CD8 + or 1B2 + CD4 + T cells from tolerant mice were added to the MLR.
  • DN T cells obtained from both transgenic and normal mice can inhibit allogeneic immune responses mediated by T cells of the same TCR specificity.
  • mice also have regulatory function.
  • ⁇ TCR + CD3 + DN T cells were purified from normal (B6xdm2) F1 and (MRL/+, H-2 k ) mice, and their ability to suppress syngeneic
  • CD8 + cells was examined. As seen in 1B2 + DN T cell clones, a dose-dependent inhibition of proliferation of syngeneic CD8 + T cells by activated CD3 + DN T cells was also observed when the same culture conditions (i.e., alloantigen plus IL-2 and IL-4) were used (Figure 3d). This finding suggests that peripheral DN T cells from both transgenic and normal mice are able to function as regulatory T cells, obviating concerns that the DN regulatory T cells observed in the TCR transgenic animals are a non-physiologically relevant oddity.
  • the DN regulatory T cell clones can be generated from both tolerant and na ⁇ ve animals ( Figure 3a), suggesting that the DN regulatory T cell precursors may exist in the spleen of normal mice.
  • the role of DST may be to promote the activation/function of DN T cells.
  • DN regulatory T cells express a unique set of cell surface markers.
  • Regulatory clones obtained from both tolerant and na ⁇ ve animals express equivalent levels of ⁇ TCR, CDlla, CD18, CD25, CD69, CD45, CD62L, Ly-6A, and CTLA-4 when compared with non-regulatory 1B2 + CD8 + T cell clones or primary activated 1B2 + CD8 + T cells. They are negative for TCR and NK1.1. Interestingly, unlike 1B2 + CD8 + T cells or clones, none of the DN regulatory T cell clones express CD28 or CD44 at any time point after activation.
  • peripheral DN T cells also differ from bone marrow derived DN natural suppressor T cells which express NK1.1 (51-54) and DN T cell clones described by others (55). These findings may explain why earlier attempts to identify a single unique marker on CD4 + or CD8 + suppressor T cells failed.
  • RT-PCR Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) revealed that both regulatory and non-regulatory T cell clones express equivalent levels of IFN- ⁇ , TGF- ⁇ , and TNF- ⁇ transcripts, and none of them express IL-2, IL-4, or IL-13 mRNA at any time after activation.
  • IL-10 was only expressed in primary activated 1B2 + CD8 + T cells and 1B2 + CD8 + clones, but not in any DN regulatory T cell clones ( Figure 4b).
  • the regulatory T cells were incubated with varying doses of Fas-Fc fusion protein prior to and during the cytotoxicity assays.
  • the ability of DN regulatory T cells to kill activated 1B2 + CD8 + T cells was blocked in a dose-dependant manner by Fas-Fc fusion protein. This finding indicates that blocking FasL on the DN T cell abolishes DN T cell-mediated cytotoxicity.
  • DN T cell suppression is antigen specific, which is consistent with the in vitro finding that activated Fas + L d+ T cells are killed by anti-L d DN T cells.
  • the finding that syngeneic L d " 1B2 + CD8 + T cells were also killed suggests the possibility that DN T cells may mediate a non-specific bystander killing through Fas/FasL interactions, as seen in some CD4 + and CD8 + T cells.
  • 1B2 + DN T cells were stimulated by L d+ cells and used as effector cells.
  • L d Fas + CD8 + T cells with different antigen specificities including a) 1B2 + CD8 + T cells (anti-L d ); b) female anti-male HY TCR transgenic T cells (anti-HY); c) anti-lymphocytic choriomeningitis virus glycoprotein TCR transgenic T cells (anti-LCMV-gp); d) C3H anti-SJL non-transgenic T cells (anti-H-2S) were used as targets. Although the target cells are all L d" , and express a similar level of Fas following activation, only those 1B2 + CD8 + T cells that carry the same TCR specificity as the DN T cells were killed.
  • 1B2 + DN and 1B2 + CD8 + cells were independently incubated with irradiated L d+ APC, and their ability to pick-up and express L d was monitored at different time points by triple staining of cultured cells with 1B2, anti-CD8, and anti-L d mAbs.
  • expression of L d on both 1B2 + DN and 1B2 + CD8 + T cells was observed within minutes after incubation with L d+ spleen cells. By 12 hours, there was no detectable L d on the surface of 1B2 + CD8 + T cells.
  • the antigen-specific regulatory T cells express a unique combination of cell surface markers ( ⁇ TCR + CD4"CD8-CDlla + CD18 + CD25 + CD28"CD44-NKl.l-Ly-6A + ) and array of cytokines (express IFN- ⁇ , TGF- ⁇ , and TNF- ⁇ , but not IL-2, IL-4, IL-10 and IL-13), which make them distinguishable from any previously reported T cell subsets.
  • the regulatory T cells can be obtained by either pre- transplantation DST or in vitro stimulation of na ⁇ ve cells with alloantigen in the presence of exogenous IL-2/IL-4, it suggests that the regulatory T cell precursors may pre-exist in normal individuals. Once the proper conditions are provided, the regulatory T cells will activate, proliferate, and specifically inhibit CD8 + T cells activated by the same alloantigen, and thus prevent rejection of specific allografts.
  • the role of DST in the induction of donor-specific tolerance may be to promote the activation and expansion of the regulatory T cells in vivo by providing antigen stimulation and IL-2 /IL-4.
  • the following results support this notion: 1) Both antigen and exogenous IL-2 and IL-4 are required for the regulatory T cells to survive and proliferate in vitro. 2) The level of IL-4 in the sera of DST recipients increased significantly. 3) The number of regulatory T cells in the accepted L d+ skin allografts and cardiac xenographts of DST-treated tolerant animals was significantly increased ( Figure 15 and 14, respectively).
  • regulatory T cells Down-regulation of specific immune responses by regulatory T cells in vitro and in vivo has been observed in numerous cases. However, the mechanism by which regulatory T cells mediate antigen-specific suppression remains unclear. Various mechanisms such as competition with antigen-specific T cells for APC or growth factors, and the production of suppressive cytokines have been proposed. The inventors demonstrated, in their model, that regulatory T cell mediated suppression requires direct contact with activated CD8 + T cells, suggesting that suppression is not simply due to secretion of inhibitory cytokines or soluble factors. Since regulatory T cell mediated suppression can not be abolished by the addition of an excessive number of APC or IL-2 /IL-4, it indicates that suppression is neither due to competition for the surface area on APC nor growth factors with CD8 + cells.
  • the inventors demonstrate that regulatory T cells are able to kill activated CD8 + T cells through the Fas/FasL pathway. This killing can be inhibited by Fas-Fc fusion protein, and the ability of regulatory T cells to kill syngeneic CD8 + T cells from Fas-mutant Ipr mice was significantly impaired. Together, these results demonstrate that Fas/FasL interactions are involved, at least partially, in regulatory T cell-mediated suppression.
  • regulatory T cells are able to kill both activated L d+ allogeneic and anti-L d syngeneic T cells, but not other CD8 + T cells activated by 3rd party antigens suggests the involvement of specific antigen recognition during suppression.
  • the regulatory T cells lost their ability to kill activated 1B2 + CD8 + T cells when pre-incubated with anti-L d mAb.
  • Fas + syngeneic CD8 + T cells recognize the alloantigen expressed on the surface of regulatory T cells, the latter will send a death signal through their FasL to the CD8 + T cells.
  • Activated T cells that express Fas but cannot interact specifically with regulatory T cells through antigen-TCR binding will not be affected.
  • CD8 + T cells that express L d can specifically interact with the TCR of regulatory T cells and are therefore also killed.
  • the mechanism of suppression utilized by regulatory T cells in this model ( Figure 7) is clearly different from any previously proposed models including veto cells.
  • mice C57BL/6 (B6, H-2 b ), (B6xBALB/c) F1 (H-2 b / d ), BALB/c-H-2-dm2 (dm2, a BALB/c L d loss mutant), C3H (H-2 k ), SJL (H-2 S ), B6/lpr (H-2 b ), MRL/+ (H-2 k ) and MRL/ Ipr (H-2 k ) mice were purchased from Jackson Laboratories (Bar Harbor, ME).
  • Anti-HY TCR transgenic mice were obtained from Dr. H.S. Teh and anti-LCMV-gp transgenic mice is a gift of Dr. P. Ohashi.
  • a breeding stock of 2C transgenic mice was kindly provided by Dr. D. Y. Loh.
  • a large fraction of T cells in the periphery of the 2C mice express a transgenic TCR reactive against L d class I MHC. These T cells can be detected by a clonotypic mAb 1B2 and are predominantly CD8 + .
  • the specificity for L d requires both transgenic ⁇ - and ⁇ -chains.
  • 2C transgenic mice were first back-crossed onto B6 mice for more than 10 generations to obtain the transgene on B6 background and then bred with dm2 mice.
  • mice were used for in vivo and in vitro studies. All mice were maintained in the specific pathogen free animal colony at the Ontario Cancer Institute. DST, skin grafting and adoptive transfer of lymphocytes. (2Cxdm2) F1 mice were either infused with viable spleen cells collected from (B6xBALB/c) F1 mice (DST-treated) or left non-injected (control).
  • the 1B2 + /CD3 + CD8 + , 1B2 + /CD3 + CD4 + and 1B2 + /CD3 + CD8"CD4" T cells were sorted by using a cell sorter (Coulter Epics V, Hialeah, FL) and the purity and viability of the cells after sorting were more than 95%.
  • a cell sorter Coulter Epics V, Hialeah, FL
  • Spleen cells were collected from naive or tolerant (B62Cxdm2) F1 mice 120 days after acceptance of (B6xBALB/c) F1 skin allografts. These cells were used to generate T cell clones using standard cloning and subcloning procedures.
  • 5xl0 4 cells were cultured irradiated L d+ cells in an ⁇ -Minimum Essential Media ( ⁇ -MEM) supplemented with FCS, rIL-2 and rIL-4. The cells were incubated at 37°C with 5% C0 2 .
  • the T cell clones were stimulated in the above manner every 3-4 days, and used as suppressor cells.
  • Spleen cells from both na ⁇ ve and tolerant mice were collected, triple stained with 1B2-Red 670, CD8-FITC or CD4/CD8-FITC (both from PharMingen) and one of the following PE-conjugated antibodies (anti-CD28 or anti-CD44, both from PharMingen). Data were acquired and analyzed on an EPICS XL-MCL flow cytometer (COULTER CORPORATION, Miami, FL).
  • MLR Na ⁇ ve splenic CD8 + T cells (1000 cells/well) were co-cultured in 96-well plates with irradiated (20 Gy) sex-matched splenocytes (3xl0 5 cells/well) from (B6xBALB/c) F1 mice in ⁇ -MEM supplemented with FCS and rIL-2 and rIL-4 as sources of growth factor.
  • Various numbers of purified putative suppressor T cells were added to MLR. After a 3-day incubation, ICi of [ 3 H]-TdR was added to each well. Eighteen hours later, cells were harvested and counted in a beta counter. Cultures to which no putative suppressor cells were added were used as controls.
  • DN T cells were stimulated by irradiated allogeneic splenocytes for 2-3 days in the presence of IL-2/IL-4. Viable cells were harvested and seeded into 96-well microtiter plates as effector cells. Target cells were stimulated with appropriate antigens in vitro for 2-3 days. Activated CD8 + T cells were collected, labeled with O.lmCi/ml of 51 Cr for 1 hr or lO ⁇ Ci/ml of [ 3 H]-TdR at 37°C overnight and used as targets.
  • RT-PCR analysis 1B2 + CD8 + or 1B2 + DN T cells were activated by either irradiated L d + spleen cells or plate-bound 1B2 antibody. Before activation, and 4, 10, 20, and 90 hrs after activation, viable cells were collected for RNA extraction using TriZol reagent (GIBCO, BRL). cDNA was prepared from RNA with 0.5mg/ml pd(N)6 Random Hexamer Primer (Pharmacia) and 300 units of murine MLV reverse transcriptase (GIBCO BRL). 2 ⁇ l of the cDNA mixture was used in a PCR reaction with 10 pmol of forward and reverse primers and 2.5U of Taq DNA polymerase (Gibcol BRL).
  • 1B2 + DN T cells and clones from tolerant mice can specifically inhibit anti-L d responses in vitro and in vivo. Since agents such as IL-10 and CsA abrogate the suppressive function of the DN T cells, it provides an excellent model to study the differentially expressed molecules in regulatory and non-regulatory T cells. Identification and characterization of the molecules responsible for suppression will not only provide insight into mechanism(s) leading to Ag-specific suppression, but also may lead to the development of novel therapeutic modalities.
  • IL-10 switches DN T cell clone (TNI 2) from a regulatory to a non-regulatory phenotype ( Figure 12a). This finding suggests that IL-10 may regulate the expression of certain molecules crucial for DN T cell-mediated suppression.
  • PCR-Selected cDNA subtraction technique 59
  • the inventors have, in a forward reaction, identified genes that are differentially expressed in regulatory and non-regulatory T cells.
  • Ly-6A Figure 8a
  • Ly-6A (Scal-1 or TAP) is a glycosyl phosphatidylinositol (GPI)-anchored cell surface molecule expressed on most peripheral lymphocytes, thymocytes, and a variety of other cells (60-64). Ly-6A is involved in cell adhesion and T cell activation (65-68). The consequence of Ly-6A stimulation, however, may either enhance or inhibit T cell activity (65,69-72). The mechanisms underlying the differential roles of Ly-6A on T cell activity is not known and the ligand for Ly-6A remains to be identified.
  • GPI glycosyl phosphatidylinositol
  • DN regulatory T cells require direct cell-cell contact with CD8 + T cells ( Figure 4c). Furthermore, these DN T cells do not express detectable levels of CD4, CD8, CD28, or CTLA-4, indicating that the classical co-stimulatory molecules are not regulating DN T cell function. Interestingly, after incubation with rIL-10, which converts the regulatory T cell phenotype into a non-regulatory one, the expression of Ly-6A is significantly reduced (Figure 8a).
  • Ly-6A-Fc fusion protein for the following purposes: to study the potential of the fusion protein in modulating the DN regulatory T cell mediated suppression, and to identify and isolate the counterligand(s).
  • the cDNA encoding the extracellular region of Ly-6A is amplified by PCR, and inserted into a plasmid expressing mouse Fc ⁇ 2a obtained from Dr. T Strom (73) to create a fusion gene of Ly-6A and Fc ⁇ 2a.
  • the fusion gene will then be cut from the original plasmid and cloned into the eukaryotic expression vector pBK/CMV (Stratagene). Upon sequence confirmation, the construct will be transfected into CHO cells by electroporation. Transfected cells will be selected with G418, and subcloned. High producing clones will be selected by screening culture supernatants in ELISA using Ly-6A specific mAb as the capture antibody, and Alkaline Phosphatase-coupled anti-Fc ⁇ 2a as the detection antibody. Western blot analysis will be used to confirm the size and specificity of the Ly-6A-Fc using anti-Ly-6A and anti-Fc ⁇ 2a mAbs (both from PharMingen).
  • Ly-6A-Fc protein The ability of the Ly-6A-Fc protein to modulate the DN regulatory T cell mediated suppression will first be determined in vitro. Anti-L d MLRs will be set-up to which DN regulatory T cell clones will be added at various ratios. Varying doses of Ly-6A-Fc protein will be either preincubated with the DN regulatory T cells, the stimulators or added directly into the cultures at various time points. Proliferation and the number of CD8 + and DN regulatory T cells in the culture will be determined as described above. If the addition of Ly-6A-Fc to the culture alters the DN-mediated Ag-specific suppression in vitro, its role in allograft rejection will also be determined in vivo.
  • mice (B6 x dm2) F1 mice will be divided into 4 groups (10 mice per group). One group of mice will be injected with DN regulatory T cells, one with Ly-6A-Fc, one injected with both DN regulatory T cells and Ly-6A-Fc protein, and an additional control group will be treated with saline. All mice will be transplanted with cardiac allografts from both
  • Transplantation of allogeneic lymphocytes could be a potent therapeutic modality for the treatment of various diseases if graft versus host disease (GVHD) could be prevented .
  • GVHD graft versus host disease
  • DST pre-transplant donor-specific transfusion
  • the inventors have recently analysed the mechanism underlying DST-induced donor-specific tolerance, and demonstrated that a novel subset of regulatory T cells play an important role in the induction of antigen-specific tolerance (Example 1).
  • the regulatory cells can specifically lyse target cells that express the same TCR, but not 3rd party controls (Figure 5).
  • Preincubation of the regulatory T cells with either anti-TCR mAb (Figure 6c and 12b) or Fas-Fc fusion protein ( Figure 5b) abolish DN regulatory T cell mediated cytotoxicity.
  • mice were sacrificed for pathohistologic evaluation of GVHD between 100 and 150 days after infusion of allogeneic lymphocytes.
  • Tissue samples from each of the four major sites of GVHD involvement hepatic parenchyma, biliary system, small intestine and skin
  • lymphocytic infiltration and GVHD No evidence for acute or chronic GVHD was observed.
  • No difference in the histology of liver, skin or small intestine could be observed between normal and (2Cxdm2) F1 -injected (B6xC.B-17) F1 scid mice.
  • the spleen and lymph nodes were harvested from (B6xC.B-17) F1 scid recipients, and the number of CD8 + , CD4 + , CD3 + CD8' and NK1.1 + cells were stained with appropriate mAbs and analyzed by using a flow cytometer. A vigorous expansion followed by a massive apoptotic cell death of 1B2 + CD8 + cells was observed within the first few days after the infusion of (2Cxdm2) F1 cells. At 3 weeks after injection, the majority of 1B2 + CD8 + T cells was deleted from the periphery (Figure 9a).
  • mice Eight weeks after the first infusion, the recipient mice were given a second injection of na ⁇ ve splenocytes from both male (2Cxdm2) F1 and female anti-HY TCR transgenic mice.
  • Some male na ⁇ ve (B6xBALB/c) F1 mice were injected with the same number of (2Cxdm2) F1 and anti-HY cells as controls.
  • the spleen and lymph nodes were harvested, cells were stained with mAbs specific for 1B2, T3.70, and CD8, and analyzed using flow cytometer. Both the percentage and total number of anti-L d and anti-HY CD8 + T cells in the spleen and lymph nodes of mice that were given only one injection were similar to those that received two injections.
  • CD8 + donor T cells in GVHD By contrast, little is done to reveal the function of regulatory T cells in this context. As the majority of T cells in (2Cxdm2) F1 -reconstituted mice are regulatory T cells, it is possible that these donor-derived regulatory T cells are responsible for inhibition of newly infused na ⁇ ve anti-host T cells. To test this hypothesis, 1B2 + DN T cells were purified from (2Cxdm2) F1 reconstituted mice and used as effector cells. Their ability to kill activated anti-host T cells were examined in vitro. As seen in Example 1, the cytotoxicity was seen only when 1B2 + CD8 + T cells were used as target cells. No killing was seen when a 3rd party anti-HY T cells were used as targets. These findings indicate that regulatory T cells may prevent GVHD by killing activated anti-host T cells.
  • Pretransplant DST stimulates donor-specific regulatory T cells in recipients and results in permanent acceptance of allo- and xeno-grafts.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigens
  • transplantation in clinic bone marrow and organ (such as kidney) transplantation, completely match for 6 HLA loci is sought.
  • HLA are highly polymorphic, it is difficult to find a donor with all the 6 HLA loci matched with the recipient.
  • graft rejection still takes place due to the incompatibility of minor histocompatibility antigens between donor and recipient.
  • the inventors have demonstrated in mice, that DST of one MHC molecule mismatched lymphocytes before transplantation leads to permanent acceptance of skin allografts of the lymphocyte donor origin.
  • the inventors have demonstrated that the underlying mechanism of DST-induced tolerance is that recipient DN regulatory T cells are activated and expanded after infusion of one class I molecule mismatched lymphocytes. As one class I mismatched donors are still difficult to find, the inventors can transfect recipient cells with one HLA antigen and use these cells to induce specific tolerance to that particular HLA alloantigen.
  • regulatory T cell clones were incubated in the presence or absence of TCR cross-linking. At various time points after incubation, cells were collected and stained with Annexin V a marker for early apoptosis. The number of apoptotic regulatory T cells in each culture was determined FACS. As shown in Figure 10c, some of the mAbs can convert our apoptosis resistant regulatory T cell clones into apoptosis sensitive phenotype regardless of cross-linking of TCR. This data indicate that some of the mAbs can bind to surface molecules expressed on apoptosis-resistant cells and induce death of these cells.
  • IL-10 may regulate the expression of certain molecules crucial for regulatory T cell-mediated suppression.
  • the inventors have identified genes that are differentially expressed in regulatory and non-regulatory T cells.
  • the inventors have now determined that osteopontin is also highly expressed on the regulatory cells.
  • Northern blot analysis has confirmed a higher level of expression of Eta-1 in the regulatory TN12 cells compared to C02 non-regulatory T cells and IL-10-treated TN12 cells ( Figure 13a).

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Abstract

La présente invention concerne des cellules et des molécules impliquées dans l'immunorégualtion. Ces cellules sont des lymphocytes T régulateurs dotés du phénotype CD3+αβTCR?+CD4-CD8-CD44-CD28-NK1.1-¿. Ces lymphocytes régulateurs expriment des niveaux élevés de Ly-6A et d'ostéopontine alors que des cellules non régulatrices ne le font pas. Une inhibition de Ly-6A ou d'ostéopontine inhibe les propriétés suppressives des cellules.
PCT/CA2000/001172 1999-10-08 2000-10-06 Cellules et molecules impliquees dans l'immunoregulation Ceased WO2001026679A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003066072A3 (fr) * 2002-02-07 2004-06-17 Univ Paris Curie Thérapie cellulaire à l'aide de lymphocytes t immorégulateurs
WO2004050706A3 (fr) * 2002-12-03 2004-09-16 Medical Res Council Lymphocytes t regulateurs
EP1455841A4 (fr) * 2001-11-21 2004-12-08 Univ Leland Stanford Junior Compositions et methodes associees a l'osteopontine
WO2015006519A1 (fr) * 2013-07-10 2015-01-15 The United States Of America, As Represented By The Secretary, Departement Of Health &Human Services Induction de lymphocytes t régulateurs spécifiques d'antigènes, médiée par des cellules apoptotiques, pour le traitement de maladies auto-immunes chez l'homme et les animaux

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2124858A1 (fr) * 1991-11-05 1993-05-13 Samuel Strober Cellules suppressives et cellules meres
JPH0733680A (ja) * 1993-07-16 1995-02-03 Kirin Brewery Co Ltd 免疫抑制剤
WO1998056405A1 (fr) * 1997-06-10 1998-12-17 Whitehead Institute For Biomedical Research Procede utilisant l'osteopontine pour moduler une reponse immunitaire

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1455841A4 (fr) * 2001-11-21 2004-12-08 Univ Leland Stanford Junior Compositions et methodes associees a l'osteopontine
US7282490B2 (en) 2001-11-21 2007-10-16 The Board Of Trustees Of The Leland Stanford Junior University Osteopontin-related compositions and methods
WO2003066072A3 (fr) * 2002-02-07 2004-06-17 Univ Paris Curie Thérapie cellulaire à l'aide de lymphocytes t immorégulateurs
WO2004050706A3 (fr) * 2002-12-03 2004-09-16 Medical Res Council Lymphocytes t regulateurs
WO2015006519A1 (fr) * 2013-07-10 2015-01-15 The United States Of America, As Represented By The Secretary, Departement Of Health &Human Services Induction de lymphocytes t régulateurs spécifiques d'antigènes, médiée par des cellules apoptotiques, pour le traitement de maladies auto-immunes chez l'homme et les animaux

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