MXPA96005051A - Methods to induce tolerance to t cells for a tissue grafting uórg - Google Patents

Abstract

The present invention relates to methods for inducing tolerance to T cells for a tissue graft or organ in a transplant recipient. The methods involve administering to a patient: 1) an allogenic or xenogeneic cell which expresses the antigens of the donor and which has a ligand on the surface of the cell, which interacts with a receptor on a surface of a recipient T cell, which mediates the helper effector function dependent on contact and 2) receptor antagonist which inhibits the interaction of the ligand with the receptor. In a preferred embodiment, the allogeneic or xenogeneic cell is a B cell, preferably a B cell at rest and the molecule on the surface of the T cell which mediates the contact-dependent helper effector function is gp39. A preferred gp39 antagonist is an anti-gp39 antibody. The allogeneic or xenogeneic cell and the gp39 antagonist are typically administered to a recipient of the transplant prior to transplantation of the organ tissue. The methods of the invention can be used to induce tolerance to T cells for transplants such as liver, kidney, heart, lung, skin, muscle, neuronal tissue, stomach and intestine. Also disclosed is a method for treating diabetes that comprises administering to a patient alogeneic or xenogeneic cells expressing the antigens of the donor, a gp39 antagonist and pancreatic islets.

Description

MBTQDOS TO INDUCE THE TOLLERY TO THE CBLULAfl T PABA my TWTOTQ PB T1JXPQ P QRQAyQ ANTgCBDBNTBS DK THE INVENTION To induce the activation of antigen-specific T cells and clonal expansion, two signals provided by the cells presenting the antigen (APC) should be delivered to the surface of resting T lymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp. Med. 165, 302-319; Mueller, DL, et al. (1990) J. I mol, 144, 3701-3709; Williams, IR and Unanue, ER (1990) J. Immunol 145, 85-93). The first signal, which confers specificity for the immune response, is mediated by means of the T cell receptor (TCR) after the antigenic peptide recognition presented in the context of the main hisocompatibility complex (MHC). The second signal, called co-stimulation, induces the T cells to proliferate and become functional (Schwartz, R. H. (1990) Science 248, 1349-1356). The co-stimulation is neither antigen-specific nor restricted by MHC and is believed to be provided by one or more cell surface molecules expressed by the APC (Jenkins, MK, et al (1988) J. Immunol.140, 3324-3330; Linsley, PS, et al. (1991) J. Exp. Med. 173, 721-730; Gimmi, CD et al., (1991) Proc. Natl.

RBP: 23418 Acad. Sci. USA. 88, 6575-6579; Young, J. W., et al. (1992) J. Clin Invest. 90, 229-237; Kouloval, L., et al. (1991) J. Exp. Med. 173, 759-762; Reiser, H. et al. (1992) Proc. Natl. Acad. Sci. USA 89, 271-275; van-Seventer, G.A. et al. (1990) J. Immunol. 144, 4579-4586; LaSalle, J. M. et al., (1991) J. Immunol. 147; * 774-80; Dustin, M. I., et al, (1989) J. Exp. Med. 169, 503; Armitage, R. J., et al. (1992) Nature 357, 80-82; Liu, Y., et al. (1992) J. Exp. Med. 175, 437-445). A co-stimulatory route in a tailspin in the activation of T cells involves the CD28 molecule on the surface of T cells. This molecule can receive a costimulatory signal supplied by a ligand in B cells or other APCs. Ligands for CD 28 include members of the B7 family of B cell activation antigens, such as B7 / 1 and / or B7-2 (Freedman, AS et al (1987) J. Immunol., 137, 3260-3267; Freeman, GJ et al. (1989) J. Immunol., 143, 2714-2722; Freeman, GJ, et al. (1991) J. Exp. Med. 174, 625-631; Freeman, GJ, et al. (1993) Science 262, 909-911; Azuma, M. et al. (1993) Nature 366, 76-79; Freeman, GJ et al. (1993) J. Exp. Med. 178, 2185-2192). B7-1 and B7-2 are also ligands for other molecules, CTLA4, presented on the surface of activated T cells, although the role of CTLA4 in co-stimulation is unclear. The delivery to a T cell of a specific signal of the antigen with a costimulatory signal, leads to the activation of the T cell, which can include both the proliferation of T cells and cytokine secretion. In contrast, the delivery to a T cell of a specific antigen signal in the absence of a costimulatory signal is thought to induce a state of irresponsibility or anergy in the T cells, whereby the specific tolerance for the antigen is induced. in the T cell. < --- The interrelations between T cells and laß B cells play a central role in immune responses. The induction of humoral immunity for thymus-dependent antigens requires "help" provided by cooperating T cells (hereinafter Th). Although some help provided for B lymphocytes is mediated by soluble molecules released by Th cells (eg, lymphocytes such as HIN-4 and HIN-5), activation of B cells also requires a contact-dependent interaction between C cells and Th cells. Hirohata et al. ., J. Immunol., 140: 3736-3744 (1988); Bartlett et al., J. Immunol, 143: 1745-1754 (1989). This indicates that activation of B cells involves a mandatory interaction between cell surface molecules on B cells and Th cells. The molecule or molecules in the T cell, therefore, mediates the effector functions cooperators dependent on the contact of the T cell.

The contact-dependent interaction between the molecules on B cells and T cells is further supported by the observation that plasma membranes isolated from activated T cells can provide cooperating functions necessary for the activation of B cells, Proc., Natl . Acad. Sci. ITTS, 85: 564-568 (1988); Hodgkin et al., J. Immunol., 145: 2025-2034 (1990); Noelle et al., J. Immunol, 146: 1118-1124 (1991). "" • One molecule, CD40, has been identified on the The surface of immature and mature B lymphocytes, which, when cross-linked by antibodies, induces the proliferation of B cells. Valle et al., Eur. J. Immunuol., 19: 1463-1467 (1989). CD40 has been molecularly cloned and characterized. Stamenkovic et al., EMBO J., 8: 1403-1410 (1989). A ligand for CD40, gp39 (also called CD40 or CD40L ligand) has also been molecularly cloned and characterized. Armitage et al., Nature, 357-: 80-82 (1192); Lederman et al., J. Exp. Med., 175-1091-1101 (1992); Hollenbaugh et al., EMBO J., 11: 4313-4319 (1992). The protein gp39 is expressed on activated cells, but not at rest, CD4 + Th. Spriggs et al., J. Exp. Med., 176: 1543-1550 (1992); Lane et al., Eur J. Immnuol., 22: 2573-2578 (1992); Roy et al., J. Immunol, 151: 1-14 (1993). Cells transfected with the gp 39 gene and expressing the protein gp39 on its surface can activate the proliferation of B cells and together with other stipulating signals, can induce the production of antibodies. Ar itage et al., Nature, 357: 80-82 (1992); Hollenbaugh et al., EMBO J., 11: 4313-4319 (1992).

^ BRRVK DESCRIPTION D «THE INVENTION The molecules of the cell surfaces, the V "- which mediate the effector functions. dependent on the contact of T cells, are important to induce immune responses which require the help of T cells. For example, the interaction of gp39 on t cells with Cd40 on B cells plays a central role in activating the responses of B cells to a antigen. The present invention is based, at least in part, on the discovery that surface molecules of The cell, which mediates the cooperative effector functions dependent on the contact of the T cells, also plays a critical role in the response of the cells.

T cells to alloantigens. In particular, it has been discovered that under appropriate conditions, interference with an interaction of gp39 with a ligand in an allogenic cell, which is presenting alloantigens to the T cell, can induce tolerance in the T cell. Preferably, the allogenetic cell which presents alloantigens to the T cells requires an interaction between a gp39 ligand on the cell and gp39 on the T cell that is going to be able to provide signals necessary for the activation of T-cells. By inhibiting the interaction of the gp39 ligand in the allogeneic cell with gp 39 on the T cell, T-cell activation is prevented and it also induces tolerance of the alloantigen-specific T cells. The induction of tolerance of T cells to alloantigens as described herein. It can be used as a preparative regimen for tissue or organ transplantation. Accordingly, the methods of the invention are particularly useful for inducing tolerance to T cells to a tissue or donor organ in a tissue or organ receptor. The methods involve administering to a recipient of the transplant: 1) an allogenic or xenogenetic cell which expresses at least one antigen of the donor and which has a ligand on a surface of the cell, which interacts with a receptor on the surface of a receiving T cell, which mediates the cooperative effector effectors dependent on contact; 2) an antagonist of the molecule on the surface of the recipient T cell, which mediates cooperative effector functions dependent on contact. The antagonist inhibits an interaction between the molecule on the T cell and its ligand on the allogenic or xenogenetic cell. In a preferred embodiment, the receptor on the surface of a recipient T cell, which mediates the helper effector functions of the contact is gp39. In this embodiment, the antagonist is a molecule which inhibits the interaction of gp39 in a T cell with a gp39 ligand on an allogenic or xenogenetic cell. A particularly preferred gp39 antagonist is an an-gp39 antibody. In another embodiment, the gp39 antagonist is a soluble form of a gp39 ligand, for example soluble CD40. The allogeneic or xenogenetic cell, which is administered to the receptor, is preferably a lymphoid cell, for example a B cell. Alternatively, the allogenic or xenogenetic cell is a small resting B cell. The allogeneic or xenogenetic cell and the antagonist (e.g., the gp39 antibody) is typically administered to a recipient individual prior to transplantation of the tissue or organ into the patient. For example, the lymphoid cells (e.g., B cells) of the tissue or organ donor are administered to the recipient, together with the antagonist, prior to transplantation of the tissue or organ into the recipient. The methods of the present invention can be used, for example, to induce tolerance to T cells for the transplanted tissue or organ such as liver, kidney, heart, lung, skin, muscles, neuronal tissue, stomach and intestines. In one embodiment, the transplanted tissue comprises pancreatic islets. Accordingly, the invention provides a method for treating diabetes comprising administering to the patient in need of treatment 1) allogenic or xenogenetic cell which express antigens of the donor; 2) an antagonist of a receptor on the surface of the receptor T cells, which mediate the effector functions. cooperative contact-dependent, such as gp39 antagonists (eg, a gp39 antibody) and 3) pancreatic donor islets.

BRJY1 PBggRIP IQN pg LOg PIgyjQg 15 Figure 1 is a graphic representation of the allografts of the transplanted pancreatic islet in chemically diabetic mice, pretreated with the anti-gp39 antibody alone or pretreated with allogenic splenic cells without fractionated or fractionated, alone. Laß Figures 2A and 2bv are graphical representations of the survival of transplanted pancreatic islet allografts, as measured by a decrease in plasma glucose concentration, in diabetic mice chemically pretreated with a single dose of fractionated alogenic splenic cells, together with an anti-prag39 antibody (MRI) treatment is already given for 2 weeks (panel A) or 7 weeks (panel B). Each curve represents data from an individual mouse. The empty symbols identify receptors in which the allograft of the island spontaneously failed. The full symbols indicate mice whose islet grafts were functional at the end of the experiment. Figures 3A B and C are cytometric flow profiles representing the staining of activated human peripheral blood lymphocytes for 6 hours either with CD40Ig (panel A), mAb 4D9-8 (panel B) or mAb 4D-9-9 (panel B) or CD40Ig (panel C). Figures 4A, B and C are flow cytometric profiles representing the staining of human peripheral blood lymphocytes, activated for 6 hours cultured in the presence of cyclosporin A stained with either mAb 4D8 (panel a), mAb 4D9-9 (panel B ) or CD40Ig (panel C). Figures 5A B are flow cytometric profiles representing the staining of human peripheral blood lymphocytes, activated for 6 hours with CD40Ig in the presence of unlabeled 4D9-8 mAb (panel A) or unlabeled 4D9-9 mAb (panel B). Figure 6 is a graphical representation of the inhibition of human B cell β proliferation induced by soluble gp39 and LI-4 when cells are cultured in the presence of mAbs 4D9-8, 4D9-9, 24-31, 24-13 , 89.-76 or 89-79, anti-human gp39. Figure 7 is a graphical representation of the inhibition of an alloespecific mixed lymphocyte response, when the cells are cultured in the presence of mAb 24-31 or 89-79, antihuman gp39.

DETAILED DESCRIPTION DR THE INVENTION This invention features methods for inducing tolerance of T cells in vivo to a donor tissue or an organ transplant in a transplant recipient. The methods involve administering to the recipient 1) an allogeneic or xenogenetic cell which expresses the donor antigens and the. which have a ligand on a cell surface, which interacts with a receptor on the surface of a receptor T cell, which mediates the contact-dependent helper effector function and 2) a receptor antagonist on the surface of the T cell , which inhibits the interaction of the ligand and the receptor. As used herein, the term "recipient" refers to an individual in whom a tissue or organ graft is to be transplanted, is being transplanted or has been transplanted. As defined herein, an "allogenic" cell is obtained from a different individual of the same species as the receptor and expresses "alloantigens" which differ from the antigens expressed by the recipient cells. A "xenogenetic" cell is obtained from a species different from that of the receptor and expresses "xenoantigens," which differ from the antigens expressed by the recipient cells. * As used herein, the term "donor antigens" refers to to the antigens expressed by the tissue graft or donor organ that is to be transplanted into the recipient. Donor antigens can be alloantigens or xenoantigens, depending on the source of the graft. The allogeneic or xenogenetic cell administered to the recipient as part of the tolerization regimen expresses antigens of the donor, that is, expresses some or all of the same antigens present in the tissue or donor organ to be transplanted. The allogenic or xenogenetic cell is preferably obtained from the tissue or organ graft donor, but can be obtained from one or more sources having antigenic determinants common with the donor. In addition to the allogeneic or xenogenetic cell, a molecule antagonist on the T cells, which mediate the contact-dependent helper effector functions, is administered to the recipient as part of the tolerization regimen. As defined herein, a molecule or receptor which mediates the contact-dependent helper effector functions, is one which is expressed on a Th cell and interacts with a ligand on an effector cell (e.g., a B cell), wherein the interaction of the molecule with its ligand is necessary for the generation of an effector cell response (for example, activation of the B cell). In addition to being involved in effector cell responses, it has now been found that such a molecule or receptor is involved in the response of the T cell to the antigen. Preferably, the molecule on a T cell which mediates the functions cooperating effectors dependent on the contact is gp39. Accordingly, in preferred embodiments, the methods of the invention involve administering to a recipient of the transplant an allogeneic or xenogenetic cell and a gp39 antagonist. Activation of T cells receptors by the allogeneic or xenogenetic cell implies an interaction between gp39 on the recipient T cells and a * "* ligand gp39 on the allogenic or xenogenetic cell.

By inhibiting this interaction with a gp39 antagonist, the T cells of the receptor are not activated by the antigens donors expressed by the allogeneic or xenogenetic cell, but on the contrary they are tolerated for the donor antigens. The induction of tolerance to the donor antigens in the recipient, thus allows the successful transplantation of the tissue or donor organ without rejection mediated by the immune system of the donor graft.

Various aspects of the invention are described in greater detail in the following subsections.

I. Sp39 Antagonists 5 In accordance with the methods of the invention, a gp39 antagonist is administered to a receptor to interfere with the interaction of gp39 on the recipient T cells with a gp39 ligand on an allogenic or xenogeneic O cell, such as a B cell administered to the recipient. A gp39 antagonist is defined as a molecule which interferes with this interaction. The gp39 antagonist can be an antibody directed against gp39 (eg, a monoclonal antibody against gp39), a fragment or derivative of an antibody directed against gp39 (e.g., FAb or F (ab) '2 fragments, chimeric or ^ humanized antibodies) soluble forms of a gp39 ligand (for example, soluble CD40), soluble forms of a fusion protein of a gp39 ligand (for example, soluble CD40Ig) or pharmaceutical agents which disintegrate or interfere with the gp39-CD40 interaction.

'A. Antibodies A mammal (a mouse, a hamster or a rabbit) can be immunized with an immunogenic form of the 5 gp39 protein or fragment of the protein (e.g., a peptide fragment) which produces an antibody response in the mammal. A cell which expresses gp39 on its surface, can also be used as the immunogen. _._ ,. Alternative immunogens include the purified gp39 iO protein or fragments of the protein. The gp39 can be purified from a cell expressing gp39 by standard purification techniques. Additionally, the cDNA of gp39 (Armitage et al., Nature, 357: 80-82 (1992); Lederman et al., Exp. Med. 175: 1091-1101 (1992); Hollenbaugh; et al., EMBO J., 11: 4913-4319 (1992)) can be expressed in a host cell, for example a cell line of / "• - mammalian bacterium and gp39 protein purified from cell cultures by standard techniques.Alternatively, gp39 peptides can be synthesized based on the amino acid sequence of gp39 (described in Armitage,., Nature 357: 80-82; Lederman et al., J. Exp. Med., 175: 1091-1101 (1992); Hollenbaugh et al., EMBO J., 11-4313: 4319 (1992)) using known techniques, (e.g., chemical synthesis F-moc or T-boc). The techniques to confer immunogenicity in a protein includes conjugation with carriers or other techniques well known in the art. For example, the protein can be administered in the presence of the adjuvant. The progress of immunization can be verified by detection of antibody titers in the plasma or serum. The standard ELISA immunoassay or other immunoassay can be used with the immunogen as an antigen to assess antibody levels. After immunization, the accessories can be obtained and if desired the monoclonal antibodies are isolated from the serum. To produce monoclonal antibodies, cells that produce antibodies (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures by immortalizing these cells and producing hybridoma cells. Such techniques are well known in the art.

For example, the hybridoma technique originally developed by Kohler and Milstein (Nature (1975) 254: 495-497) as well as other techniques such as the human B-cell hybridoma technique (Kozbar et al., Immunol. Today (1983) 4:72), the EBV hybridoma technique for producing human monoclonal antibodies (Colé et al., Monoclonal Antibodies in Cancer Therapy (1985) (Alien R. Bliss, Inc. pages 77-96), and screening for combinatorial antibody libraries. (Huse et al., Science (1989) 246: 1275) Hybridoma cells can be screened in unchemically for the production of antibodies, specifically reactive with the protein or peptide and isolated monoclonal antibodies. present, it is intended to include its fragments, which are specifically reactive a gp39 protein or its peptide or the gp39 fusion protein.Antibodies can be fragmented using standard techniques. and the fragments selected for utility in the same manner as described above for whole antibodies. For example, F (ab) 2 fragments can be generated by treating the antibody with pepsin. The resulting F (ab) 2 fragment can be treated to reduce the disulfide bridges to produce the FAB 'fragments. The antibody in the present invention is further intended to include biospecific and chimeric molecules that have an anti-gp39 moiety. When antibodies produced in non-human individuals are used therapeutically in humans, they are recognized to varying degrees as being extraneous to an immune response that can be generated in the patient. One approach to minimize or eliminate this problem, which is preferably of general immunosuppression, is to produce chimeric antibody derivatives, ie, antibody molecules that combine an animal, non-human variable region and a human constant region. Chimeric antibody molecules can include, for example, the antigen binding domain of an antibody of a mouse, rat or other species, with human constant regions. A variety of chimeric antibodies to prepare iso chimeric antibodies have been described and "can be used to prepare chimeric antibodies containing the variable region of immunoglobulin which is reconnected to gp39 See, for example, Morrison et al., Proc.Natl. Acad. Sci. USA 81-6851 (1985), Takeda et al., Nature 314: 452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397: Tanaguchiet al., European Patent Publication Ep 171496, European Patent Publication 0173494, UK Patent GB 2177096B It is expected that such chimeric antibodies will be less immunogenic in a human individual than the corresponding non-chimeric antibody. human therapeutic, monoclonal or chimeric antibodies specifically reactive with a gp39 protein or peptide, can be further humanized by the production of human variable region chimeras, in which parts of the variable regions, especially the conserved structure regions of the antigen binding domain, are of human origin and only the hypervariable regions are of non-human origin. Such altered immunoglobulin molecules can be made by any of several techniques known in the art (eg, Teng et al., Proc. Natl. Acad. Sci. USA 80: 7308-7312 (1983); Kozbor et al., Immunology Today , 4: 7279 (1983), Olsson et al., Meth Enzymol., 92-3-16 (1982).), And are preferably made in accordance with the teachings of PCT Publication WO92 / 06193 or EP 0239400. Humanized antibodies can be commercially produced, for example, by Scotgen Limited, 2, Holly Road, Twickenham, Middlesex, Great Britain. Another method of generating specific antibodies, or antibody fragments, reagents against a gp39 protein or peptide is to screen expression libraries encoding the immunoglobulin genes or their portions, expressed in bacteria with a gp39 protein or peptide, by For example, the complete FAB fragments, VH regions and FV regions can be expressed in bacteria using phage display libraries. See, for example Ward et al., Nature 341: 544-546: (1989); Huseet al., Science, 246; 1275-1281 (1981); and McCafferty et al., Nature, 348: 552-554 (1990). Screening of such libraries for example with a gp39 peptide can identify immunoglobulin fragments reactive with gp39. Alternatively, the mouse SCID-hu (available from Genpharm) can be used to produce antibodies, or their fragments.

, .. * - Methodologies for producing monoclonal antibodies directed against gp39, including human gp39 and mouse gp39 and any of the monoclonal antibodies suitable for use in the methods of the invention, are describe in greater detail in Example 2. The anti-human gp39 monoclonal antibodies of the invention are preferred for use in inducing tolerance of antigen-specific T cells. Preferred antibodies include 3E4 monoclonal antibodies, 2H5, 2H8, 4D9-8, 4D9-9, 24-31, 24-43, 89-76 and 89-79, described in Example 2. Particularly preferred antibodies are monoclonal antibodies 89-76 and 24-31. Hybridomas 89-76 and 24-31, producing antibodies 89-76 and 24-31, respectively, were deposited under the stipulations of the Budapest Treaty at the American Type Culture Collection, Parklawn Drive, Rockville, Md. On September 2, 1994. Hybridoma 89-76 was assigned ATCC Accession Number HB11713 and hybridoma 24-31 was assigned ATCC Accession Number Hb 11712. Antibodies 24-31 and 89-76 are of the IgGI isotype. In another embodiment, the antihuman gp39 mAb for use in the methods of the invention binds an epitope recognized by a monoclonal antibody selected from a group consisting of 3E4, 2H5, 2H8, 4D9-8, 4D9-9, 24-31, 24- 25 43, 89-76 and 89-79. More preferably, the anti-human gp39 mAb is hybridized to an epitope recognized by monoclonal antibody 24-31 or monoclonal antibody 89-76. The ability of a mAb to bind an epitope recognized by any of the antibodies mentioned in the above, can be determined by standard cross-competition assays. For example, an antibody that binds to the same epitope recognized by a mAb 24-31, will compete for the labeled 24-31 linkage to the activated T cells, whereas an antibody that binds to a different epitope than that recognized by the mAb 24-31 will not compete for the binding of 24-31 labeled for activated T cells.

B. Lisandos Solubles for OP39 Other gp 39 antagonists which can be administered to induce tolerance to T cells, / • "-. Include soluble forms of a gp39 ligand.A soluble monovalent ligand of gp39, such as soluble CD40, can bind to gp39, thereby inhibiting the interaction of gp39 with CD40 in B cells. The term "soluble" indicates that the ligand is not permanently associated with a cell membrane. A soluble GP39 ligand can be prepared by chemical synthesis or preferably by recombinant DNA techniques, for example by expression only of the extracellular domain (absent the transmembrane and cytoplasmic ligand domains). A preferred soluble gp39 ligand is soluble CD40. Alternatively, a soluble GP39 ligand may be in the form of a fusion protein. Such a fusion protein comprises at least a portion of gp39 ligand bound to a second molecule. For example, CD40 * can be expressed as a fusion protein with immunoglobulin (ie, a CD40Ig fusion protein). In one embodiment, a fusion protein is produced which comprises amino acid residues of a portion of the extracellular domain of CD40 bound to the amino acid residues of a sequence corresponding to the hinge, CH2 and CH3 regions of an immunoglobulin heavy chain, for example CI , to form a CD40Ig fusion protein (e.g., Linsley et al (1991) J. Exp. Med. 1783: 721-730; Capon et al., (1989) Nature 337-525-531; and Capon US 5,116,964). The fusion protein can be produced by chemical synthesis, or preferably by recombinant DNA techniques based on CD40 cDNA (Stamenkovic et al., EMBO J. 8: 1403-1410 (1989).

II. Cells for the Induction of Specific Tolerance to the Angle The present invention is based, at least in part, on the discovery that the presentation of alloantigens to T cells by allogeneic cells in the presence of a gp39 antagonist results in tolerance of T cells to the alonatins. The cells which are capable of inducing tolerance by this mechanism include those which present the antigen and T cells activated by the interaction with gp39 (i.e., an interaction between gp39 in the T cells and a gp39 ligand in the cell presenting the antigen, it is necessary to - "*" supply the appropriate signals for the activation of T cells to T cells). Inhibition of the ligand interaction in the allogeneic or xenogenetic cell with gp39- in the recipient T cells prevents the activation of the T cells by allo- or genoantigens, and, in addition, induces tolerance to the T cells for the antigens. Interference with the Activation of T cells by means of gp39 can prevent the induction of costimulatory molecules in the allogeneic or xenogenetic "" 'cell (eg family molecules).

B7 in a B cell), such that the cell provides only one antigenic signal to the T cell in the absence of a costimulatory signal, inducing tolerance in this way. Accordingly, in the methods of the invention, a allogeneic or xenogenetic cell is administered to a recipient individual. The allogeneic or xenogenetic cell is capable of presenting the antigen to the T cells of the recipient and for example, it is a B lymphocyte, a cell presenting the "professional" antigen (eg, a dendritic cell, monocyte, Langerhan cell) or another cell which presents the antigen to the immune cells (for example, a keratinocyte, endothelial cell, astrocyte, fibroblast, oligondrocyte). Furthermore, it is preferable that the allogeneic or xenogenetic cell has a reduced ability to stimulate or co-stimulate in the recipient T cells (for example, the allogeneic or xenogenetic cell may lack The expression of o express only low levels of costimulatory molecules such as the B7 family of proteins (for example B7-1 and B7-2). The expression of costimulatory molecules in cells. allogeneic or xenogenetic potentials that are going to be used in the method of The invention can be evaluated by standard techniques, for example by flow cytometry using f-directed antibodies against costimulatory molecules. The preferred allogeneic or xenogenetic cells to induce tolerance of T cells are lymphoid cells, by For example, lymphocytes of peripheral cells or of expénic cells. The preferred lymphoid β cells to induce tolerance of T cells are B cells. B cells can be purified from a mixed population of cells (for example, other types of cells in peripheral blood or the spleen) by separation techniques. of standard cells. For example, adherent cells can be removed by cultivating non-adherent cells on plastic discs and recovering the population of non-adherent cells: T cells can be removed from a mixed population of 5 cells by treatment with an anti-T cell antibody (e.g. . * anti-Thyl.ly/ or anti-Thyl.2) and the add-on. In one embodiment, resting lymphoid cells, preferably B cells at rest, are used as the cells that present the antigen. The lymphoid cells at rest, such as resting B cells can be isolated by techniques known in the art, for example based on their small size and density. The resting lymphoid cells can be isolated, for example, by counterflow centrifugation elution as described in Example 1. Using the counter-flow centrifugal elution, a population of resting, small, depleted lymphoid cells of cells which can activate the T cell response β can be obtained by harvesting a fraction or fractions at 14-19 ml / min, preferably 19 ml / min to 3,200 pro). Alternatively, the small lymphocyte (for example cell B) can be isolated by centrifugation in a discontinuous density gradient, for example using a Ficoll or Percoll gradient and a layer containing resting lymphocytes, small can be obtained after centrifugation. The small, resting B cells can also be distinguished from activated B cells by assay for the expression of costimulatory molecules such as B7-1 and / or B7-2 on the surface of activated B cells by standard techniques (e.g., immunofluorescence ). 5 Allogeneic or xenogeneic cells administered * for the receptor function, at least in part, present donor antigens to the recipient T cells. In this way, the express cells the - .. r- antigens which are also expressed by the tissue or donor organ. Typically, this can be done using allogeneic or xenogeneic cells obtained from the tissue or organ graft donor. For example, peripheral lymphoid cells, B cells or clinical cells of the tissue or organ donor can. be isolated or used in the methods of. the invention. Alternatively, alogeneic or xenogeneic cells can be obtained from a source '* "apart from the tissue or organ donor while the cells have antigenic determinants in common with the tissue or donor organ, eg, the allogeneic cells or xenogeneic which express (most or all) of the same major histocompatibility complex as the donor tissue or organ can be used. In this way, the allogeneic or xenogeneic cells can be used from a source, which is MCH haplotype coupled with the t * donor of the tissue or organ (for example, a close one in relation to the donor of the graft).

III: Administration of Cells and Antagonists of ctp3ft The "tolerance of the cells to an organ or tissue graft can be induced according to the invention, by the administration to the transplant recipient of a f ^ -gp39 antagonist together with a cell, allogeneic or xenogeneic which expresses donor antigens and interacts with the recipient T cells by means of gp39. In a preferred embodiment, the allogeneic or xenogeneic cell and the antagonist gp39 are administered to the container simultaneously or contemporaneously. Alternatively, the gp39 antagonist can be administered before the allogeneic or xenogeneic cells are administered, for example, when the antagonist is an antibody with a prolonged half-life. In a preferred embodiment, the antagonist and the allogeneic or xenogeneic cells are administered to the recipient before of the organ or tissue transplantation in the recipient (ie, the recipient is pre-treated with the antagonist and the cells). For example, the administration of allogeneic or xenogeneic cells and the antagonist may be carried out several days (e.g., five to eight days) before the tissue or organ transplantation.

** Administration of a single dose of allogeneic cells (in combination with the antagonist) has been found to be sufficient for the induction of tolerance to T cells for a tissue or donor organ 5 (see Example 1) . The number of cells administered may vary depending on the type of cell used, the type of tissue or organ graft, the weight of the recipient, the general condition of the recipient and other variables known to a skilled technician. of cells for used in the method of the invention can be determined by one skilled in the art by conventional methods (e.g., as described in Example 1). The cells are administered in a form and in a way which is suitable for the induction of tolerance of T cells in the receptor. The cells can be administered in a physiologically acceptable solution, such as buffered saline, or a similar vehicle. The cells are preferably administered intravenously. An antagonist of the invention is administered in an individual in a biologically compatible form, suitable for in vivo pharmaceutical administration to induce tolerance to T cells. By "biologically compatible form for administration in vivo" is meant a form of the antagonist to be administered in which Any of the toxic effects are considered by the therapeutic effects of the compound. The term "individual" is intended to include living organisms in which an immune response can be produced, for example mammals. Examples of individuals include humans, dogs, cats, mice, rats and their transgenic species. An antagonist of% gp39 can be administered in any pharmacological form optionally with a pharmaceutically acceptable carrier. The administration of a therapeutically active amount of the antagonist is defined as an effective amount, at doses and for periods of time necessary to achieve the desired result (e.g., tolerance to T cells. For example, a therapeutically active amount of a gp39 antagonist may vary according to factors such as disease status, age, sex and weight of the individual and the ability of the antagonist to produce a desired response in the individual.Dose regimens can be adjusted to provide the optical therapeutic response.For example, several divided doses can be administered daily or the dosiß can be reduced proportionally as indicated by the exigencies of the therapeutic situation As described in Example 1, for treatment with an anti-gp39 antibody, an effective treatment regimen may include initiation of antibody administration before tissue transplantation or organ (for example, five to eight days before the transplant), followed by readministration No antibody (for example, every day) for several weeks (e.g. two to seven weeks) after transplantation. The active compound (for example, an antagonist such as an antibody) can be administered in a convenient manner t * al as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application or rectal application. Depending on the route of , r administration, the active compound can be. coated in a material to protect the compound from the action of enzymes, acids and other natural conditions, which can inactivate the compound. A preferred route of administration is by intravenous injection. To administer a different gp39 antagonist of parenteral administration, it may be necessary to coat the antagonist with or co-administer the antagonist with a * material to avoid inactivation. For example, an antagonist can be administered to an individual in an appropriate carrier or diluent, co-administered with inhibitors of the enzyme or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Inhibitors of the enzyme include inhibitors of pancreatic trypsin, diisopropyl fluorophosphate (DEP) and trasilol.

The liposomes include water-in-oil emulsions in water as well as conventional liposomes (Strejan et al., (1984) J. Nexir or Immunol 7:27). The active compound can also be administered parenterally or intraperitoneally. The depressions can also be prepared in glycerol, liquid polyethylene glycols and their mixtures and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where they are soluble in water) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent that it is easy to apply with a syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The Porter may be a solvent or a dispersion medium, containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of such a coating as lecithin, for the maintenance of the required particle size in the case of dispersion and for the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be approximated by the inclusion in the composition of an agent which retards absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (eg, a gp39 antagonist) in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated therein. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which produces a powder of the active ingredient (eg, antagonist) plus any additional desired ingredient of a previously filtered solution. sterile of it. When the active compound is protected suitably, as described above, the protein can be administered orally, for example, with an inert diluent or an edible assimilable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvent, medium or Dispersion, coatings, antibacterial agents and antifungals, isotonic retardation and absorption agents and the like The use of such media and agents for pharmaceutically active substances is well known in the art. means or As conventional agents are incompatible with the active compound, the use thereof in the therapeutic compositions is contemplated The supplemental active compounds can also be incorporated into the compositions. parenterals in the form of dose unit for the case of administration and uniformity of the dose. The dosage unit form as used herein, refers to physically discrete units, suitable as unit doses for the mammalian individuals that are to be treaties; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for dosage forms of the invention will be dictated by, and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved and (b) the limitations inherent in the technique of forming compounds such as an active compound for the treatment of sensitivity in individuals. Subsequent to, or in conjunction with the tolerization regimen described herein, a tissue or donor organ is transplanted into a transplant recipient by conventional techniques.

IV. Uses of the Methods of the Invention The methods of the invention are applicable to a wide variety of tissue and organ transplant situations. The methods can be used to induce tolerance to T cells in a recipient of a tissue or organ graft such as pancreatic islets, liver, kidney, heart, lung, skin, muscle, neuronal tissue, stomach and intestines. In this way, the methods of the invention can be applied in treatment of diseases or conditions which are related to the transplantation of tissue or organ (for example, liver transplantation to treat hypercholesterolemia, muscle cell transplantation to treat dystrophy muscle, neuronal tissue transplantation to treat Huntington's disease or Parkinson's disease, etc.). In a preferred embodiment, the transplanted tissue comprises pancreatic islets. Accordingly, the invention encompasses a method for the treatment of diabetes by transplantation of pancreatic islet cells. The method comprises: administering to an individual in need of treatment l) alogeneic or xenogeneic cells which express the antigens of the donor, 2) an antagonist of a molecule expressed on the recipient T cells which mediate the contact-dependent effector function of the contact , such as a gp39 antagonist (eg, antibody, anti-gp39) and 3) cells of the donor pancreatic islet. Preferably, allogeneic or xenogeneic cells and antagonists are administered to the recipient prior to administration of the pancreatic islets. The invention is further illustrated by the following example, which should not be considered as limiting. The contents of all references, patents and published patent applications cited throughout this application, are hereby incorporated by reference.

EXAMPLE 1: Induction of Tolerance to Pancreatic Islßtaß Allografts by Treatment of the Receptor with Alogeneic and Anti-39 Cells Contemporary allograft studies »* depends on generalized immunosuppression that does not specifically cut off immune effector functions. However, immunosuppressive pharmaceutical agents can cause - • / significant side effects. Besides, the The alotransplantation of the Langerhans Cay for the treatment of diabetes has proved to be refractory to this approach (see, for example, Robertson, R.P. (1992), N. Engel J. Med 327, 1861). Laa therapies with antibodies directed against T cells can allow allograft successful islets in rodents, but this approach also results uniformly in generalized immunosuppression (Carpenter, CB (1990) N. Engl, J. Med 322, 1224; Roark, JH et al. (1992) Tranaplantation 54, 1098; Kahan , BD (1992) Curr Opin. I Munol, 4, 553). In this example, tolerance to islet allografts is induced in a transplant recipient by manipulation of the alloantigen presentation to the T cells to prevent its activation. The survival of islet allografts in C57BL / 6 (H-26) diabetic mice was chemically examined using the following methodology: Induction of Diabetes Male C57B1 / 6I (H-26) mice become diabetic by intravenous administration of streptozotocin (140 mg / kg). Permanent diabetes was confirmed by the demonstration of a plasma glucose concentration of > .400 mg / dl on three occasions over a period of one week. -v 0 Fractionation of Alogeneic Splenic Cells Donor allogeneic cells for the treatment of graft recipients were obtained from hybrid animals (C57 x BALB / c9 (H-2b / d) F1 to avoid graft disease against the host.) To isolate small lymphocyte cells, cell suspensions Spleens from female mice F1 (C57 x BALB / c) eight weeks old are depleted of erythrocytes and then fractionated by elutriation size as described in Tony, HP et al (1985) J. Exp. Med. 161 , 223, and Gosselin EJ et al. (1988) J. I unol, 140, 14JD8 In summary, small lymphocytes were isolated by counterflow centrifugal e? Uence, for example, using a model J-6B centrifuge (Beckman Instruments , Palo Alto CA.) Approximately 1-5 x 108 cells in 8 ml of 5 culture medium or salt solution balanced with 1.5% fetal bovine serum, treated with deoxyribonuclease, loaded into the elutriation chamber with a speed of Initial counter-current flow of 13.5 ml / minute and centrifuged at 4 ° C at a constant speed of 3,200 rpm. A fraction of small cells with very few large contaminating cells are eluted at 14-19 ml / minute, although the exact flow rate may depend on the medium in which the cells are suspended. In the experiments described herein, the fraction of small cells was collected at 19 ml / minute (at 3,200 rpm). This fraction was completely depleted of the radiation-resistant accessory cell fraction (3000 rads) when tested with specific T-cell lines for either rabbit IgG and H2 < (CDC35) or alloreactive for H2D (D10.G4). Small cells and unfractionated cells are washed twice in serum-free medium before injection into the tail vein in the allograft recipients. Approximately 40-100 x 106 (57 x BALB / c) F ^^ (H-2 ° / d) splenic cells unfractionated or 40-100 x 106 (C57 x BALB / c) Fj ^ (H-2 ° d ) of small decanted cells were used. retratflmientp of the Rseptoreg of the Inierto Graft recipients were pretreated with either allogeneic splenic cells (C57 x BALB / c) F1 (H-2D / d) unfractionated, small diameter "fraction 19" splenic cells decanted that had been devoid of APC activity (isolated as described in the foregoing), an anti-gp39 monoclonal antibody (MR1, see Example 2, Experiment 3), or a combination of allogeneic cells and the a »nti-gp39 antibody. The cells of fraction 19 were tested at two different dose ranges, a low dose of 40-44 x 10 6 cells or a high dose of 77-88 x 10 6 cells. The control animals did not receive allogeneic cellsß or treatment with the antibody. The allogeneic cells were administered to the graft recipients by injection into the tail vein from five to eight days before transplanting the islet allograft. Treatment with the MR1 antibody was at a dose of 250 g / mouse twice weekly, beginning 7 days after transplanting the islet and continuing for 2-7 weeks or until the graft is rejected. The first injection of the antibody was typically given on the same day as the first injection of allogeneic splenic cells.

Tranßolantß from Aloinierto de Isletaa The BALB / c allogeneic islets (H-2d) were isolated by a modified collagenase digestion method (Gottlieb, P.A., et al. (1990) Diabetes 39, 643). The islands at a dose of 30 islets / g of body weight were implanted in the subrrenal capsule of the C57B1 / 61 (H-2b) recipient mice immediately after isolation. Graft survival is defined as a maintenance of a plasma glucose concentration of < 200 mg / dl.

Results and In a first series of experiments, the islet allograft receptors are pretreated with either alogeneic splenic cells alone or anti-gp39 antibody alone. As shown in Figure 1, in the absence of splenic cell pretreatment, all islet allografts were rejected at 13 days after transplant (9 ± 2 d, range 5-13 d; N »23). Little survival of the islet was also observed in animals treated only with unfractionated splenic cells containing normal APC activity (6 ± 3 d, range 4-12 d, N = »7) or low doses (40-44 x 106 cells) of splenic cells depleted of Fraction 19 APC (7 ± 3 d, range 3-14 d, N »16). In contrast, the injection of a larger dose of exhausted small β splenocytes from Fraction 19 APC (75-88 x 106 cells) prolonged allograft survival (19 ± 10 d, range 7-40 d, N = 16). This effect on the duration of survival of the graft was statistically significant (^ .sβ "17-3 'p <0.001 when compared with groups treated with nothing, with complete splenic transfusions, or the lowest dose of splenic cells fraction 19) but was not permanent. Prolonged but finite survival of allogeneic islets in small cell diabetic receptors Fraction 19, "depleted in APC suggested that these cells alone can not sustain allograft survival. An additional group of graft recipients were treated with 77-88 x 106 cells fraction 20. This fraction was also formed mostly of small lymphocytes but differs from the population of fraction 19 in that it contains the measurable APC fraction. The receptors of this cell (N »6) rejected all their grafts quickly (mean» 8.5 d, range 6-12). Another group of graft recipients were treated with only one anti-gp39 monoclonal antibody, MRl. Figure 1 illustrates that islet allografts failed at 15 days in 7/11 mice treated only with the anti-gp39 monoclonal antibody. The remaining four mice had functional grafts at the end of the experiment on day 48. The results show that. administration of the anti-gp39 MRl antibody receptor can only prolong islet allograft survival (mean 20 = 19 d, indefinite range: N »5). The degree of prolongation was statistically similar to that achieved using a higher dose of splenic cells Fraction 19 and significantly longer than that achieved in the other three groups (p <0.05). The series of experiments described above, indicated that high doses of splenic cells depleted in APC Fraction 19 or treatment with the anti-gp39mAb msnoclonal antibody alone, can increase the survival of the pancreatic islet allograft, compared to those without treatment . However, neither treatment was effective in inducing the treatment. long-term tolerance for islet allografts in recipients. In the next series of experiments, the treatment of allogeneic splenic cells was combined with the anti-gp-39 treatment of the receptor. The combined administration of allogenic and anti-gp39 splenic cells was found to be more effective than any reagent alone. The results are shown in Figure 2, in which each curve represents the data of an individual mouse. The empty symbols identify receptors in which the allograft of the island failed spontaneously. The filled symbols indicate the mice whose islet grafts were functional at the end of the experiment. Figure 2 (panel B), shows that indefinite graft survival is achieved in all animals treated for 7 weeks with the anti-gp39 monoclonal antibody and a single injection of splenic cells depleted in APC Fraction 19 (N-69). duration of the reduction of anti-gp39 treatment weakened, but did not eliminate the favorable effect on graft survival Indefinite graft survival was achieved in 6/8 recipients when the anti-gp39 monoclonal antibody was administered for only 2 weeks in combination with splenic cells Fraction 19 (Figure 2, panel A) The survival of the infinite graft was also observed in recipients treated with anti-gp39 for 2 or 7 weeks in combination with an injection of unfractionated allogeneic splenic cells. of islet graft and the absence of insulin secretion by residual native islets, not destroyed by trafficking With streptozotocin, the kidneys that carry the subrrenal implants were removed. In all cases, unilateral nephrectomy resulted in recurrence of hyperglycemia (>300 mg / dl) after 3 days. Islet allografts and native pancreas were studied histologically in all animals, either when the graft failed or at the end of the experiment. The histological sections of the islet allografts in the kidneys of the recipients of the fractionated allogeneic small lymphocytes and the treatment with the continuous monoclonal antibody MRl mAb (7 weeks) presented abundant intact islets visible below the renal capsule, which were devoid of mononuclear infiltration and had well-granulated insulin and positive glucagon cells. In contrast, the histological sections of the islet allografts in the kidneys of the recipients treated with the anti-gp39 monoclonal antibody only showed intense characteristics of mononuclear cell inflammation and the destruction of islet cells. In all host pancreas, the morphology of the islet was uniformly consistent with -diabetes by streptozotocin.

EXAMPLE Production and Characterization of Antibodies at > 39 Experiment 1 - Antibodies directed against human gp39 For the induction of antigen-specific β-cell β tolerance in a human subject, it is preferable to administer an antibody directed against human gp39. The following methodology was used to produce mouse anti-human gp39 monoclonal antibodies. Balb / c mice were immunized with a soluble gp39 fusion protein, gp39-CD8 in complete Freund's Adjuvant (CFA). The mice were subsequently stimulated six weeks later with a soluble gp39-CD8 in incomplete Freund's adjuvant (IFA). The soluble gp39-CD8 is given in soluble form 4 weeks after secondary immunization. The mice are then boosted with human peripheral blood lymphocytes, activated two weeks later, followed by a final boost with soluble gp39-CD8 after an additional 2 weeks. The splenocytes were fused ct > n the fusion partner in NS-1 on day 4 after the final immunization as in the standard protocols. Clones that produce human anti-gp-39 antibodies are selected based on a multiple screening process. The clones are initially screened by a plaque binding assay using gp39-CD8. The positive clones are then selected against a control CD8 fusion protein, CD72-CD8. Clones which are classified as positive in the CD8-CD72 plate-binding assay are deleted. The remaining clones are subsequently screened at rest and active human peripheral blood lymphocytes for 6 hours (PBL) by flow cytometry analysis. Hybridomas stain activated but not at rest, PBL are considered positive. Finally, the remaining clones are tested for their ability to block the binding of CD40Ig to the drive plate at gp39. Approximately 300 clones are initially screened against gp39-CD8 and CD72-CD8 in plaque binding assays. Of those clones, 30 were found to detect the gp39 attached to the plate and not CD8. These clones are next selected for the detection of gp39 in human PBL.

Approximately 15 clones detected a molecule in activated PBL, but not in resting cells. The specificity was further confirmed by determining the ability of the clones to block the detection of CD40Ig of gp39 bound to the plate. 3 out of 10 clones tested blocked the binding of CD40Ig in this assay. These clones were 3E4, 2H5 and 2H8. Such clones are preferred for use in the methods described herein. The clones which proved to be positive in activated PBL but not in resting PBL were also screened for reactivity with an activated rat T cell clone, POMC8. Clone 2H8 expressed cross-reactivity with this rat T-cell line.

Experiment 2 - Antibodies directed against human QP39 An immunization procedure similar to that described in experiment 1 was used to produce additional antibodies directed against human gp39. A Balb / c mouse was immunized with CFA soluble gp39-CD8, followed by stimulation with human peripheral blood lymphocytes activated for 6 hours, 4 weeks later. The mouse was subsequently reinforced with soluble gp39-CD8, 4 days before fusion of the splenocytes with the fusion partner in NS-1 by standard protocols. Hybridoma cloning was screened by flow cytometry staining of the activated human PBL for 6 hours. Staining of activated clones but not of resting human PBLs were selected. Six clones, 4D9-8, 4D9-9, 24-31, 24-43, 89-76 and 89-79, were selected for further analysis. The specificity of the selected antibodies was confirmed by several tests. First, the analysis. flow cytometric, showed that all six monoclonal antibodies are stained, activated, but not peripheral blood T cells at rest (see Figure 3B and 3C for a representative example, which represents the staining of T cells activated with 4D9-8 and 4D9-9, respectively). The expression of the molecule recognized by each of the six antibodies is detectable at 4 hours of activation, is maximum between 6-8 hours after activation and is not detectable for 24 hours after activation. All six monoclonal antibodies recognized a molecule expressed on the activated CD3 + PBL, predominantly of the CD4 + phenotype but a portion of the CD8 + T cells also expressed the molecule. The expression of the molecule recognized by the six monoclonal antibodies is inhibited by the presence of cyclosporin A in the culture medium, since it is the expression of gp39 (see Figure 4A and 4B for a representative example, which represents staining of cells T treated with ciclosporin with 4D9-8 and '4D9-9, respectively). The kinetics and distribution of the expression of the molecule recognized by these mAbs are identical to that of gp39, as detected by the fusion protein of human CD40Ig. In addition, all six Inonoclonal antibodies block the staining of gp39 by CD40Ig (see Figures 5A and 5B for a representative example, which represents the inhibition of gp39 staining by CD40Ig in the presence of 4D9r8 and 4D9-9, respectively). In an ELISA, all six monoclonal antibodies recognized gp39CD8, a soluble fusion form of the gp39 molecule. In addition, all six monoclonal antibodies immunoprecipitate a molecule of approximately 36 kd units of the activated human PBLs, labeled with 35S-methionine. The immunoprecipitated molecule is identical to that precipitated by the human CD40Ig fusion protein. The functional activity of the six selected monoclonal antibodies (4D9-8, 4D9-9, 24-32, 24-43, 89-76 and 89-79) were tested as follows. First, the ability of monoclonal antibodies to inhibit the proliferation of purified, human D cells cultured with IL-4 and soluble gp39 was measured. The purified human D cells were cultured with gp39 and IL-4 in the presence or absence of purified monoclonal antibodies or CD40Ig at doses between 0 and 12.5 μg / ml. The proliferation of B cells was determined after 3 days in culture by incorporation of thymidine. The results (shown in Figure 6) of showed that all six monoclonal antibodies can inhibit B cell proliferation induced by gp39 and IL-4. Monoclonal antibodies 89-76 and 24-31 were more effective in inhibiting the proliferation of induced B cells. Next, the ability of the monoclonal antibodies to inhibit the differentiation of B cells, as measured by Ig-induced T cell production by anti-CD3 and IL-2 was examined. Purified human Ig + cellsβ were prepared by positive selection with FACS and then cultured with human T cells activated with anti-CD3 (treated with mitomycin C) and IL-2 for 6 days in the presence or absence of the anti-gp monoclonal antibodies. 39 purified as doses between 0 and 10 μg / ml. The production of IgM, IgG and IgA was evaluated by ELISA on day 6. The results (shown in the following Table 1) showed that all six antibodies can inhibit B-cell differentiation dependent on the T cell as measured by the production of IgM, IgG and IGA.

Table 1 Emdiu S? Éh no m 17400 6710 4471 4DM ai 4J13 2130 2S19 LO 4394 2551 1519 10.0 10U 319 396 4D9: 0.1 3594 919 1731 14 2659 1233 1606 10-0 374 441 266 24 * 31 i 3 * 3 911 344 14 1217 314 165! L0 1120 396 23 24-43 0.1 4227 4132 432 14 3193 2130 192 lao 7021 1232 1001 19-76 0.1 37 © 1069 344 14 2110 352 171 104 f t 551 19 «* 7 and 976! 1924 3021 14 2314 460 156 104 m 135 434 To examine the effect of anti-gp39 monoclonal antibodies on the β-cellβ response, the monoclonal antibody was included in standard mixed lymphocyte (MLR) reactions. 300,000 human peripheral blood lymphocytes (responders »R) were cultured with 100,000 peripheral blood lymphocytes allogeneic, irradiated (stimulators »C) in the presence or absence of the anti-gp39 monoclonal antibody (10.μg / ml). Cultures were labeled with thymidine-3H on day 4, 5 or 6 and harvested 18 hours later. All six anti-human gp39 monoclonal antibodies inhibited the aloespecific * responses as measured by MLR (see Figure 7 for a representative example representing the inhibition of the aloespecific responses when R and S are incubated in the presence of 24-31 or 89 -76; a CTLA4-immunoglobulin fusion protein and an anti-CD28 monoclonal antibody were used as positive controls). To determine if the six monoclonal antibodies recognized distinct apitopes in the human gp39 molecule, cross blocking experiments were carried out. The activated human PDL were first blocked with each of the six monoclonal antibodies (25 μg / ml unconjugated antibodies). The β cells were washed and then 10 μg / nl of antibody conjugated with biotin were stained, followed by the avidin reaction conjugated with phytoerythrin (PE-Av). Staining of the cells with PE-Av was analyzed by FACS the results are shown in the following in Table 2.

Table 2 Blocking Ap < fcwrift < Jt? FtHrftp? H 409-1 4DM 24-31 24-43 S9-76 «9-79 none +++ +++ + 4 ++ + * ++ ++++ * + ** 4094 ND - +++ * + * + * +++ ++ * 4D9 9 ++ * ND ++ * ++ * + ++ 4 ++ • »• 24-3 + ND ++ 4 * + ++ 24-43 + * * +++ ND ++ + • 99-76 * + +++ + * + ND + * + 09-79 + + * +++ +++ +++ ND intensity of staining and «i pore«? t_-it poctt ^ (++ ** «MI 200¡ ++ *"> 12S ** «M? 506 +> MI> 2-apo there is stain left over the bottom) .ND "not determined.

All antibodies blocked the binding of CD40Ig to the activated human PBL. However, the data shown in Table 2 clearly demonstrate the failure of some antibodies to block the binding of other antibodies to activated human PDL, suggesting that they recognize distinct apitopes on human gp39 molecules. Hybridomas 89-76 and 24-31, which produce antibodies 89-76 and 24-31, respectively, were deposited under the stipulations of the Budapest Treaty at the American Type Culture Collection Parkland Drive, Rockville, Md. September 1994. Hybridomas 89-76 were assigned ATCC Accession Number HB11713 and hybridoma 24-31 was assigned ATCC Accession Number HB11712.

Experiment 3 - Antibodies directed against mouse gp39 In one embodiment of the invention, the gp39 antagonist is a mouse anti-gp39 monoclonal antibody, MRl. The following method was used to produce the monoclonal antibody MRl and can be used to generate other antibodies directed towards gp39. The Hamsters were immunized intraperitoneally with 5-106 activated Thl cells (di.6) at weekly intervals for 6 weeks. When the serum titer against murine T1 was greater than about 1: 10,000, the cell fusions are performed with polyethylene glycol using splenocytes from immune hamster and NS-1. The supernatant of the wells containing the growing hybridomas was screened by flow cytometry in T1 at rest and activated. A particular hybridoma which produces an antibody that selectively recognized activated T ^ was also tested and subcloned to derive MRl. MRl was produced in ascites and purified by ion exchange CLAP. A MRl hybridoma had been deposited in the American Type Culture Collection and assigned Accession Number HB11048.

EQUIVALENTS Those skilled in the art will recognize or be able to achieve using no more than routine experimentation, many equivalents for the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. The contents of all references and patent applications published citations throughout this application are incorporated herein by reference. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers. Having described the invention as above, the contents of the following are claimed as property:

Claims (50)

1. CLAIMS l. A composition suitable for inducing tolerance to T cells for a tissue or donor organ in a tissue or organ receptor, characterized in that it comprises the following components: (a) an allogeneic or xenogeneic cell, which expresses at least one donor antigen and which has a ligand on a surface of the cell, which interacts with a receptor on a surface of a recipient T cell, which mediates the contact-dependent cooperating effector functions and (b) a receptor antagonist on the surface of the T cell, which inhibits the interaction of the ligand with the receptor.
2. 2. The composition according to claim 1, characterized in that the receptor on the surface of the receptor T cell, which mediates the contact-dependent effector function is gp39.
3. 3. The composition according to claim 2, characterized in that the antagonist is an anti-gp39 antibody. - "V
4. 4. The composition according to claim 3, characterized in that the gp39 antibody is a monoclonal antibody.
5. 5. The composition according to claim 3, characterized in that the anti-gp39 antibody is a human anti-gp39 antibody.
6. 6. The composition according to claim 4, characterized in that the monoclonal antibody is MRl.
7. 7. The composition according to claim 4, characterized in that the antibody 15 monoclonal is a chimeric monoclonal antibody.
8. 8. The composition according to claim 4, characterized in that the monoclonal antibody is a humanized momoclonal antibody.
9. 9. The composition according to claim 1, characterized in that the allogeneic or xenogeneic cell is a lymphoid cell.
10. 10. The composition according to claim 9, characterized in that the lymphoid cell is a B cell.
11. 11. The composition according to claim 10, characterized in that the B cell is a B cell at rest.
12. 12. The composition according to claim 1, characterized in that the allogeneic or xenogeneic cell and the antagonists are administered to the recipient before transplantation of the tissue or organ.
13. 13. The composition according to claim 1, characterized in that the tissue or organ comprises pancreatic islets.
14. 14. The composition according to claim 1, characterized in that the tissue or organ is selected from the group consisting of liver, kidney, heart, lung, skin, muscle, neuronal tissue, stomach and intestine.
15. 15. The composition for inducing tolerance to T cells for a tissue or donor organ in a y-tissue or organ receptor, characterized in that it comprises administering to the recipient a) a allogeneic and xenogeneic cell which expresses at least one donor antigen; and 5 b) a gp39 antagonist.
16. 16. The composition according to claim 15, characterized in that the gp39 antagonist is an anti-gp39 antibody.
17. 17. The composition according to claim 16, characterized in that the anti-gp39 antibody is a monoclonal antibody.
18. 18. The composition according to claim 16, characterized in that the anti-gp39 antibody is a human anti-gp39 antibody.
19. 19. The composition according to claim 17, characterized in that the monoclonal antibody is MRl.
20. 20. The composition according to claim 17, characterized in that the antibody 25 monoclonal eß a chimeric monoclonal antibody.
21. The composition according to claim 17, characterized in that the monoclonal antibody is a humanized monoclonal antibody.
22. 22. The composition according to claim 15, characterized in that the gp39 antagonist is a soluble form of a gp39 ligand.
23. 23. The composition according to claim 22, characterized in that the soluble form of the gp39 ligand is a CD40 fusion protein.
24. 24. The composition according to claim 15, characterized in that the allogeneic cell 15 or xenogeneic is a lymphoid cell.
25. 25. The composition according to claim 24, characterized in that the lymphoid cell is a B cell.
26. The composition according to claim 25, characterized in that the B cell is a B cell at rest.
27. 27. The composition according to claim 15, characterized in that the allogeneic or xenogeneic cell and the antagonist are administered to the recipient before the transplantation of the tissue or organ.
28. 28. * The composition according to claim 15, characterized in that the tissue or organ comprises pancreatic islets.
29. 29. The composition according to claim 15, characterized in that the tissue or organ is selected from the group consisting of liver, kidney, heart, lung, skin, muscle, neuronal tissue, stomach and intestine.
30. 30. The composition for treating diabetes, characterized in that it comprises administering to an individual in need of treatment: a) an allogenic or xenogeneic cell, which expresses at least one donor antigen; b) a gp39 antagonist; and c) donor pancreatic islet cells.
31. 31. The composition of claim 30, characterized in that the anti-gp39 antibody is a monoclonal antibody. ^ -.
32. 32. The composition according to claim 30, characterized in that the anti-gp39 antibody is a human anti-gp39 antibody.
33. 33. The composition according to claim 31, characterized in that the monoclonal antibody is MRl. • r,
34. 34. The composition according to claim 31, characterized in that the monoclonal antibody is a chimeric monoclonal antibody.
35. 35. The composition according to claim 31, characterized in that the antibody 15 monoclonal is a humanized monoclonal antibody.
36. 36. The composition according to claim 30, characterized in that the gp39 antagonist is a soluble form of a gp39 ligand.
37. 37. The composition according to claim 36, characterized in that the soluble form of a gp39 ligand is a CD40 fusion protein.
38. 38. The composition according to claim 30, characterized in that the allogeneic or xenogeneic cell is a lymphoid cell.
39. 39. The composition according to claim 38, characterized in that the lymphoid cell is a B cell.
40. 40. The composition according to claim 39, characterized in that the B cell is a resting B cell.
41. 41. The composition according to claim 30, characterized in that the allogeneic or xenogeneic cell and the antagonist are administered to the recipient before transplantation of pancreatic islet cells.
42. 42. A composition for inducing tolerance to T cells for a tissue or donor organ in a tissue or organ receptor, characterized in that it comprises administering to the recipient a) a donor allogeneic cell; and b) an anti-gp39 antibody, wherein the donor allogeneic cell and the anti-gp 39 antibody are administered to the recipient prior to transplantation of the tissue or organ.
43. 43. The composition according to claim 42, characterized in that the anti-gp39 antibody is a monoclonal antibody.
44. 44. The composition according to claim 42, characterized in that the anti-gp39 antibody is a human anti-gp39 antibody.
45. 45. The composition according to claim 43, characterized in that the monoclonal antibody is MRl.
46. 46. The composition according to claim 44, characterized in that the monoclonal antibody is a chimeric monoclonal antibody.
47. 47. The composition according to claim 44, characterized in that the monoclonal antibody is a humanized monoclonal antibody.
48. 48. The composition according to claim 42, characterized in that the allogeneic donor cell is a lymphoid cell.
49. 49. The composition according to «* claim 48, characterized in that the lymphoid cell is a B cell.
50. 50. The composition according to claim 49, characterized in that the B cell is a B cell at rest. SUMMARY DB THE INVENTION Methods for inducing tolerance to T cells for a tissue or organ graft in a transplant recipient are described. The methods involve administering to a patient: 1) an allogeneic or xenogeneic cell which expresses the antigens of the donor and which has a ligand on the surface of the cell, which interacts with a receptor on the surface of a T cell .. receptor, which mediates the cooperative effector function dependent on the contact; and 2) receptor antagonist, which inhibits > the interaction of the ligand with the receptor. In a preferred embodiment, the allogeneic or xenogeneic cell is a B cell, preferably a B cell at rest and the molecule on the surface of the T cell which mediates the contact-dependent helper effector function is gp39. A preferred gp39 antagonist is an anti-gp39 antibody. The allogeneic or xenogeneic cell and the gp39 antagonist are typically administered to a recipient of the transplant prior to transplantation of the tissue or organ. The methods of the invention can be used to induce tolerance to T cells for transplants such as liver, kidney, heart, lung, skin, muscle, neuronal tissue, stomach and intestine. A method for treating diabetic is also described which comprises administering to a patient allogeneic or xenogeneic cells expressing the antigens of the donor, a gp39 antagonist and pancreatic islets.