Methods of inducing t-cell tolerance to tissue or organ transplant

 

(57) Abstract:

The invention relates to medicine and relates to methods of inducing T-cell tolerance to tissue or organ transplant. The inventive methods include the introduction of the subject 1) allogeneic or xenogeneic cells, which expresses donor antigens and which has a ligand on a cell surface which interacts with a receptor on the surface of the recipient cell which mediates contact-dependent helper-effector function and 2) receptor antagonist that inhibits the interaction of the ligand with the receptor. The advantage of the invention consists in the induction of tolerance to donor antigens and creates the opportunity for the successful transplantation of donor tissue or organ without immunopositive rejection of the donor graft. 3 C. and 35 C.p. f-crystals, 2 tab., 7 Il.

To induce antigen-specific T-cell activation and clonal expansion of the surface of resting T-lymphocytes must be delivered two signals provided by antigen presenting cells (APC) (Jenkins M. and Schwartz, R. (1987) J. Exp.Med. 165, 302-319; Mueller D. L. et al. (1990) J. Immunol. 144, 3701-3709; Williams I. R. and Unnanue E. R. (1990) J. Immunol. 145, 85-93). The first signal is avania foreign Utegenova peptide, submitted in connection with the major histocompatibility complex (HCGS). The second signal, called costimulate, induces T-cell proliferation and the appearance of functionality (Schwartz, R. H. (1990) Science 248, 1349-1356). Costimulate is neither antigen-specific nor limited HCGS and, I believe, is ensured by one or more separate molecules cell surface downregulation of agriculture (M. K. Jenkins et al. (1988) J. Immunol. 140, 3324-3330; Linsley, P. S. et al. (1991) J. Exp. Med. 173, 721-730; Gimmi, C. D. et al. (1991) Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Yong J. W. et al. (1992) J. Clin. Invest. 90, 229-237; Koulova L. et al. (1991) J. Exp. Med. 173, 759-762; H. Reiser 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, 774-780; Dustin M. I. et al. (1989) J. Exp. Med. 169, 503; R. J. Armitage et al. (1992) Nature 357, 80-82; Y. Liu et al. (1992) J. Exp. Med. 175, 437-445). One co-stimulatory cascade included in T-cell activation, includes a molecule CD28 on the surface of T-cells. This molecule can receive co-stimulatory signal delivered by the ligand on B cells or other APK. The ligands for CD28 include family members, B7, B-limfozitah activation antigens, such as B7-1 and/or B7-2 (Freedman, A. S. et al. (1987) J. Immunol. 137, 3260-3267; Freeman, G. J. et al. (1989) J. Immunol. 143, 2714-2722; Freeman, G. J. et al. (1991) J. Exp. Med. 174, 625-631; Freeman, G. J. et al. (1993) Science 262, 909-911; Azuma, M. et al. (1993) Nature 366, 76-79; Freeman, G. J. et al. (Aktivirovannyh T-cells, although the role of CTLA4 in costimulation unclear.

Shipping to T-cell antigen-specific co-stimulatory signal by the signal leads to the activity of T-cells, which may include both the proliferation of T-cells and secretion of cytokines. In contrast, believe that shipping to T-cell antigen-specific signal in the absence of co-stimulatory signal induces T-cell immunological status of neuvecelle or anergy inducyruya thus, antigen-specific tolerance in T-cell.

The interaction between T-cells and B-cells plays a Central role in immune responses. Induction of humoral immunity to thymus-dependent antigens require "assistance" provided by T-cells-helper (hereinafter - Th-cells). Although some assistance is provided by B-lymphocytes, is transmitted soluble molecules secreted by Th-cells (for example, lymphokines, such as IL-4 and IL-5), activation of B-cells requires contact-dependent interaction between B-cells and Th cells. Cm. Hirohata et al. J. Immunol. , 140: 3736-3744 (1988); Barlett et al., J. Immunol., 143: 1745-1754 (1989). This indicates that the activation of B-cells involves binding interaction of molecules on the cell surface of B-cells and Th cells. Sledovatel is independent of the interaction between molecules on B-cells and T-cells also supports the observation that that isolated plasma membrane of activated T-cells can provide helper functions required for the activation of B-cells. Cm. Brian, Proc. Natl. Acad. Sci. USA, 85: 564-568 (1988); Hodgkin et al., J. Immunol., 145: 2025-2034 (1990); Noelle et al., J. Immunol., 146: 1118-1124 (1991).

Molecule CD40 identified on the surfaces of immature and Mature B-lymphocytes and a cross-linking antibody induces proliferation of B-cells. Cm. Valle et al., Eur. J. Immunol., 19: 1463-1467 (1989); Gordon et al., J. Immunol., 140: 1425-1430 (1988); Gruber et al., J. Immunol., 142: 4144-4152 (1989). Molecules CD40 cloned and characterized. Cm. Stamenkovic et al. , EMBO J. 8: 1403-1410 (1989). The ligand for CD40 - gp39 (also known as CD40-ligand or CD4OL) molecular cloned and characterized. Cm. Armitage et al., Nature, 357: 80-82 (1992); Lederman et al. , J. Exp. Med., 175: 1091-1101 (1992); Hollenbaugh et al., EMBO J. 11: 4313-4319 (1992). Protein gp39 antigen is expressed on activated, but not resting, CD4+- Th-cells. Cm. Spriggs et al., J. Exp. Med., 176: 1543-1550 (1992); Lane et al., Eur. J. Immunol., 22: 2573-2578 (1992); Roy et al., J. Immunol. , 151: 1-14 (1993). Cells transfetsirovannyh genome gp39, and expressing the protein Dr on its surface, can induce B-cell proliferation and together with other stimulatory signals can induce the production of antibodies. Cm. Armitage et al., Nature, 357: 80-82 (1992); Hollenbaugh et al., EMBO J. 11: 4313-4319 (1992).

awesime helper-effector functions of T-cells, important to induce immune responses that 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 the activation reactions of B-cells to antigen. The present invention is based, at least in part, on the discovery that molecules on the cell surface, which mediates contact-dependent helper-effector functions of T-cells also play a critical role in the response of T-cells to alloantigens. In particular, we discovered that under certain conditions, the interference in the interaction Dr with ligand for allogenic cell, which represents alloantigen T-cell can induce tolerance in T-cell. Preferably, allogenic cell, which represents alloantigen T-cell, requires interaction between the ligand gp39, T-cell, to be able to provide the signals required for activation of T-cells. Inhibition of the interaction of the ligand gp39 in allogeneic cell with gp39, T-cell prevents the activation of T-cells and induces rather alloantigen-specific tolerance in T-cells. The induction of T-cell tolerance to alloantigens, as described in the present invention, can be used as primariamente suitable to induce T-cell tolerance to donor tissue or organ in the recipient tissue or organ. The above methods include the introduction of the transplant recipient 1) allogenic or xenogenic cells that Express at least one donor antigen and which have a ligand on a cell surface which interacts with a receptor on the surface of recipient T-cells, which mediates contact-dependent helper-effector functions; 2) antagonist molecules on the surface of recipient T-cells, which mediates contact-dependent helper-effector functions. Antagonist inhibits the interaction between a molecule on T-cells and its ligand on the allogeneic or xenogeneic cell.

In a preferred embodiment of the invention, the receptor on the surface of T-cells, which mediates contact-dependent helper-effector functions, is a gp39. In this embodiment of the invention, the antagonist is a molecule that inhibits the interaction of gp39 on T-cell ligand gp39 in the allogeneic or xenogeneic cell. Especially preferred gp39 antagonist is an antibody against gp39. In another embodiment of the present invention, the gp39 antagonist is a soluble form of the ligand gp39, such as soluble is modnye cells, such as B-cells. Alternatively, allogeneic or xenogeneic cells represent a small resting B-cells. Allogenic or xenogenic cells and the antagonist is typically administered to the recipient prior to transplantation in the recipient tissue or organ. For example, lymphoid cells (such as B-cells) from a donor tissue or organ is administered to the recipient together with the antagonist before transplantation to the recipient tissue or organ.

The methods of the present invention can be applied, for example, to induce T-cell tolerance to transplanted tissues or organs, such as liver, kidney, heart, lung, skin, muscle, nervous tissue, stomach and intestine. In one of the embodiments of the present invention transplantirovannam fabric contains pancreatic islets. Accordingly, the invention relates to a method of treating diabetes comprising the administration to a patient in need of such treatment, 1), allogeneic or xenogeneic cells, which Express a donor antigens; 2) receptor antagonist on the surface of T-cells of the recipient, which mediates contact-dependent helper-effector functions, such as the gp39 antagonist (e.g., antibody against gp39); and 3) donor pancreatic islets.

Krylatov pancreatic islets transplanted mice with "chemical" diabetes, which was previously introduced one antibody against gp39 or previously introduced some nefrackzionirovannam or fractionated allogeneic spleen cells.

Fig. 2A and Fig. 2B are graphical depictions of term viability of the transplant of pancreatic islets transplanted to mice with "chemical" diabetes who previously received a single dose of fractionated allogenic spleen cells together with treatment with antibody against gp39 (MR1) or within 2 weeks (Fig. 2A), or within 7 weeks (Fig. 2B), when this term viability determined to reduce the concentration of glucose in plasma. Each curve represents the results obtained for a separate mouse. Where there's no shading icons apply to recipients who have islet allografts were spontaneously stopped working. Shaded icons are mice, in which the grafts were functioning at the end of the experiment.

In Fig. 3A, 3B and 3C depict the profiles of flow cytometry staining of activated within 6 hours of human peripheral blood lymphocytes with one of CD40g (Fig. 3A), mAb (monoclinic crystal. antibody) 4D9-8 (Fig. aktivirovannyh within 6 hours of human peripheral blood lymphocytes grown in the presence of ciclosporin And painted one of the mAb 4D9-8 (Fig. 4A), mAb 4D9-9 (Fig. 4B) or CD40Ig (Fig. 4B).

In Fig. 5A and 5B depict the profiles of flow cytometry staining of activated within 6 hours of human peripheral blood lymphocytes CD40Ig in the presence of its mAb 4D9-8 (Fig. 5A) or its mAb 4D9-9 (Fig. 5B).

In Fig. 6 are graphs of inhibition of proliferation of human B-cells soluble gp39 and IL-4, when cells are grown in the presence of a monoclonal antibody 4D9-8 AND 4D9-9, 24-31, 24-43, 89-76 or 89-79 to human gp39.

In Fig. 7 graphically depicts the inhibition reaction ALLO-specific mixed culture of lymphocytes, when cells are grown in the presence of a monoclonal antibody 24-31 or 89-79 against human gp39.

Detailed description of the invention

The present invention relates to a method of inducing in vivo T-cell tolerance to donor tissue graft or organ transplant recipient. The above methods include the introduction of the transplant recipient 1) allogeneic or xenogeneic cells, which Express a donor antigens and which have a ligand on the cell surface that mutual is factorie function; 2) antagonist receptor on the surface of T-cells, which inhibits the interaction between ligand and receptor. As installed here, the term "recipient" refers to the subject, which should take tissue or organ transplanted tissue or organ or transplanted tissue or organ. As installed here, "allogeneic" cells from different individuals of the same species as the recipient, and they Express "alloantigen", which differ from antigens expressed by cells of the recipient. "Xenogenic" cells from species other than the recipient, and they Express "xenoantigen", which differ from antigens expressed by cells of the recipient. As installed here, the term "donor antigens" refers to antigens expressed donor tissue or donor organ that is transplanted to the recipient. Donor antigens can be alloantigens or xenoantigens, depending on the source of the graft. Allogenic or xenogenic cells entered the recipient as part of the scheme of inducing tolerance, Express donor antigens, i.e., Express some or all of the same antigens present in the donor tissue or organ, which transplant ntata, but you can get them from one or more sources, with donor common antigenic determinants.

In addition to allogeneic and xenogeneic cells to the recipient as part of the scheme of inducing tolerance introduce antagonist molecules on T-cells, which mediates contact-dependent helper-effector functions. As defined here, the molecule or receptor, which mediates contact-dependent helper-effector functions, is a molecule or receptor, which is expressed on Th-cell and interacts with the ligand on the effector cell (e.g., B-cell), and the interaction of the molecule with its ligand is necessary for the generation of reactions of effector cells (i.e., the activation of B-cells). Now discovered that, in addition to involvement in the response of effector cells, such molecule or receptor involved in the response of T cells to the antigen. Preferably the molecule on the T cell which mediates contact-dependent helper-effector function is a gp39. Accordingly, in preferred embodiments of the invention the methods of the invention include the introduction of the recipient of a transplant of allogeneic or xenogeneic cells and gp39 antagonist. Activation of redah and gp39 on allogenic or xenogenic cells. When the inhibition of the interaction of gp39 antagonist T cells of the recipient are not activated donor antigens expressed allogenic and xenogenic cells, but rather in them is called tolerance to donor antigens. Induction of tolerance to donor antigens in the recipient creates an opportunity, therefore, for the successful transplantation of donor tissue or organ without immunopositive rejection of the donor graft.

Various aspects of the present invention are described in more detail in the following sections.

I. the gp39 Antagonist

In accordance with the methods of the invention, the recipient enter the gp39 antagonist, to prevent the interaction of gp39 on T cells of the recipient with ligand gp39 on allogenic and xenogenic cells, such as B-cells, is introduced to the recipient. Agonist gp39 is defined as a molecule that interferes with this interaction. Agonist gp39 may be an antibody directed against gp39 (e.g., monoclonal antibody against gp39), fragment or derivative of the antibody directed against gp39 (e.g., Fab fragments or F(ab')2, chimeric antibodies or humanized antibodies) soluble form of the ligand gp39 (najczesciej means, which destroy or inhibit the interaction of gp39 - CD40.

A. Antibodies

Mammal (e.g., mouse, hamster, or rabbit) can be immunized with immunogenic form of gp39 protein or protein fragment (e.g., a peptide fragment), which reveals the formation of antibodies in a mammal. Cell that expresses gp39 on its surface, can also be used as an immunogen. Alternative immunogen include purified protein gp39 or protein fragments. Cleaning gp39 from expressing gp39 cells can be accomplished by standard methods of purification. In addition, the cell master can be expressed cDNA gp39 (Armitage et al., Nature, 357: 80-82 (1992); Lederman et al., J. Exp. Med., 175: 1091-1101 (1992); Hollenbaugh et al., EMBO J. 11: 4313-4319 (1992)), for example, in the lines of bacterial cells or mammalian cells, and gp39 purified from cell cultures by standard methods. Alternatively, using known methods (for example, F-moc or T-boc chemical synthesis) based on the amino acid sequence gp39 (described in Armitage et al., Nature, 357: 80-82 (1992); Lederman et al., J. Exp. Med., 175: 1091-1101 (1992); Hollenbaugh et al. , EMBO J., 11: 4313-4319 (1992)) can be synthesized peptides gp39. Methods of providing the immunogenicity of protein include conjugation with carrier the adjuvant. The development of immunization can be monitored by detection of antibody titers in serum or plasma. To assess the levels of antibodies can be used in standard ELISA or other methods of immunoassay with the immunogen as antigen.

After immunization can be obtained antisera and, if desirable, of sera can be distinguished polyclonal antibodies. To obtain monoclonal antibodies producing antibodies cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard methods of fusion of somatic cells, these cells immortalized and formed cell hybridoma. Such methods are well known in the art. For example, the hybridoma technique originally developed by Kohler and Milstein (Nature (1975) 256: 495-497), as well as developed other techniques such as the hybridoma method with human B-cells (Kozbar et al., Immunol. Today (1983) 4:72), the EBV-hybridoma method for producing human monoclonal antibodies (Cole et al., Monoclonal Antibodies in Cancer Therapy (1985) (Allen R. Bliss, Inc., pages 77-96), and screening of libraries of chimeric antibodies (Huse et al., Science (1989) 246: 1275). The hybrid cells to generate antibodies specifically reactive to the protein or peptide, mogus the term "antibody" is supposed to denote its fragments, which are specifically reactive against gp39 protein, or peptide, or fusion protein gp39. Antibodies can be fragmented using known techniques and the fragments sceneroot for use in the same way described above for whole antibodies. For example, fragments F(ab')2can be generated by treatment of the antibody with pepsin. The resulting fragment F(ab')2can be processed to restore the disulfide bonds with the formation of fragments, Fab'. It is also assumed that the antibodies in the present invention include bespecifically and chimeric molecules having part of anti-gp39.

When antibodies produced in the subjects, non-human, are used to treat people, they are recognized with varying degrees of success as foreign, and the patient may be caused by the immune reaction. One approach for minimizing or eliminating this problem, which should be preferred in General immunosuppression, is the production of derivatives of chimeric antibodies, i.e., antibody molecules that combine variable region of the animal, non-human, and human constant region. Chimeric molecules Academy of Sciences of the ski constant region. There are a lot of approaches to producing chimeric antibodies, and they can be used to generate chimeric antibody containing the variable region of immunoglobulin that recognizes gp39. 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. 4 816 557; Boss et al. , U.S. patent No. 4 816 397; Tanaguchi et al., publication of the European patent EP 1 711 496; publication of European patent 0 173 494, United Kingdom patent GB 2 177 096B. It is expected that such chimeric antibodies are humans less immunogenic than the corresponding nehelenia antibodies.

For the purposes of treatment of the human monoclonal or chimeric antibodies specifically reactive with gp39 protein or peptide can then be humanized by producing chimeras of human variable regions, in which parts of the variable regions, especially the conservative spanning region antigen-binding domain, are of human origin and only the hypervariable sites have superhuman origin. Such modified immunoglobulin molecule can be obtained by any of several methods known in the art (see, for example. Teng et al., Proc. Natl. Acad. Sci. U. S. A., 80: 7308-7312 (1983); Kozbor et al., Immunology Today, 4: EP 0 239 400. Humanized antibodies can be produced commercially, for example Scotgen Limited, 2 Holly Road, Twickencham, Middlesex, UK.

Another method of generating specific antibodies, or fragments of antibodies reactive against the protein or peptide gp39, is skanirovaniya expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria, the protein or peptide gp39. For example, complete Fab fragments, VH-region and FV-field 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). Screening such libraries with, for example gp39 peptide, can identify the fragments of immunoglobulins, reactive with gp39. Alternatively, for the production of antibodies or their fragments can be used SCID-hu mouse (available from Genpharm).

The methodology for production of monoclonal antibodies directed against gp39, including human gp39 and murine gp39, and monoclonal antibodies suitable for use in the methods of the present invention, is described in detail in example 2.

Monoclonal antibodies against human gp39 of the present invention ). Preferred antibodies are monoclonal antibodies 3E4, 2H5, 2H8, 4D9-8 AND 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. Hybridoma 89-76 and 24-31 producing antibodies 89-76 and 24-31, respectively, deposited under the terms Budapeshtskogo Treaty in the American type culture collection, Parklawn Drive, Rockville, Maryland, September 2, 1994. Hybridoma 89-76 assigned inventory number ATCC HB117-13, and hybridoma 24-31 assigned inventory number ATCC HB11712. Antibody 24-31 and 89-76 are isotype IgG1.

In another embodiment of the invention for use in the methods of the present invention mAb against human gp39 binds an epitope recognized by a monoclonal antibody selected from the group consisting of 3E4, 2H5, 2H8, 4D9-8 AND 4D9-9, 24-31, 89-76 and 89-79. Preferably mAb against human gp39 binds the epitope recognized by the monoclonal antibody 24-31 or a monoclonal antibody 89-76. The ability of the mAb to bind an epitope that is specified in any of the above antibodies can be determined by standard tests of cross-competition. For example, the antibody that binds the same epitopes that are recognized by mAb 24-31, will compete with Kvasiny mAb 24-31 epitope, will not compete with the binding of labeled 24-31 with activated T-cells.

B. Soluble ligands for gp39

Other antagonists gp39 that you can enter to induce tolerance in T-cells include soluble forms of the ligand gp39. Monovalent soluble ligand gp39, such as soluble CD40 may contact gp39, inhibiting thus, the interaction of gp39 with CD40 on B-cells. The term "soluble" indicates that the ligand is not constantly associated with the cellular membrane. Soluble ligand gp39 may be obtained by chemical synthesis or preferably, by recombinant DNA, for example, by the expression of only the extracellular domain of the ligand (in the absence of the transmembrane and cytoplasmic domains). Preferred soluble ligand gp39 is soluble CD40. Alternatively, soluble ligand gp39 may be in the form of a fused protein. This protein contains at least a portion of the ligand, gp39, is attached to the second molecule. For example, CD40 can be expressed in the form of a fused protein with immunoglobulin (i.e., protein CD40Ig). In one of the embodiments of the invention is produced by a protein containing the amino acid residues uncle the CH2 and CH3 heavy chain of the immunoglobulin, i.e. Cyl, with the formation of the fused protein CD40Ig (see, e.g., LinsLey et al., (1991) J. Exp. Med. 1783: 721-730; Capon et al., (1989) Nature 337: 525-531; and Capon, U.S. patent 5 116 964). Protein can be obtained by chemical synthesis or preferably, by recombinant DNA-based cDNA CD40 (Stamenkovic et al., EMBO J., 8: 1403-1410 (1989)).

II. Cells for the induction of antigen-specific tolerance

The present invention is based, at least in part, on the discovery that the performance of alloantigens T cells allogeneic cells in the presence of a gp39 antagonist results in T-cell tolerance to alloantigens. Cells that are able to induce tolerance by this mechanism include cells that present antigen and activate T cells by interacting with gp39 (i.e., for delivering corresponding signals to T-cell activation T-cells requires the interaction between gp39, T-cell and ligand gp39 in the cell presenting the antigen). Inhibition of the interaction of the ligand on the allogeneic or xenogeneic cell with gp39 on the recipient T-cells prevents T-cell activation of ALLO - or xenoantigens and, rather, induces T-cell tolerance to antigens. Preventing the activation of T cells across which the molecules of the B7 family on B-cell), and thus the cell delivers to T-cell only antigenic signal in the absence of co-stimulatory signal inducyruya, thus, tolerance.

Accordingly, the methods of the present invention recipient injected allogeneic or xenogeneic cells. Allogeneic or xenogeneic cell is able to present antigen to T cells of the recipient and represents, for example, B-lymphocyte - cell "professionally" representing the antigen (e.g., monocyte, dendritic cell, a cell of Langerhans), or other cell which presents antigen to immune cells (e.g., keratinocyte, endothelial cell, astrocytomas, fibroblast, oligodendrocyte). Moreover, preferably, allogeneic or xenogeneic cell had a reduced ability to stimulate co-stimulatory signal in recipient T-cells. For example, allogenic or xenogenic cell may lose expression or to Express only a low level of co-stimulatory molecules such as B7 protein family (e.g., B7-1 and B7-2). Expression of co-stimulatory molecules on the potential of allogenic or xenogenic cells used in the methods of the present invention, can be estimated by standard methods ="ptx2">

Preferred allogenic or xenogenic cells to induce T-cell tolerance are lymphoid cells, such as lymphocytes in peripheral blood or splenic cells. Preferred lymphoid cells to induce T-cell tolerance are B-cells. B-cells can be isolated in pure form from a mixed population of cells (e.g., of other types of cells in the peripheral blood or spleen) standard methods of cell division. For example, adherent cells can be removed by cultivation of splenic cells in plastic cups and retrieval of population non-stick cells. T-cells can be removed from a mixed population of cells by treatment with antibody against T-cells (e.g., anti-Thy1.1 and/or anti-Thy1.2) and complement. In one of the embodiments of the invention as antigen-presenting cells are resting lymphoid cells, preferably resting B-cells. Resting lymphoid cells, such as resting B-cells, can be identified by methods known in the art, for example, based on their small size and density. Resting lymphoid cells can be selected, for example, flow elutriation population is small, resting lymphoid cells, purified from cells that can activate T-cell responses can be obtained by collecting fractions (fractions) at 14-19 ml/min, preferably at 19 ml/min (at 3200 rpm). On the other hand, small resting lymphocytes (for example, B-cells) can be isolated by centrifugation in a discontinuous density gradient, for example when using gradient ficoll or percoll, and after centrifugation can be obtained layer containing small resting lymphocytes. Small resting B-cells can also be distinguished from activated B-cells by standard test methods (e.g., immunofluorescence), expression of costimulatory molecules such as B7-1 and/or B7-2, on the surface of activated B-cells.

Allogenic or xenogenic cells entered the recipient, there are, at least in part, as representing the donor recipient antigens to T cells. Thus, the cells Express antigens that are also expressed donor tissue or organ. Usually this can be done through the use of allogeneic or xenogeneic cells obtained from donor tissue or organ transplant. For example, the weave and used in the methods of the present invention. Alternatively, allogeneic or xenogeneic cells can be obtained from a source other than the donor tissue or organ, if such cells have antigenic determinants in common with the tissue or organ donor. For example, can be used allogenic or xenogenic cells, which Express a (mostly or all) of the same antigens of the major histocompatibility complex that donor tissue or organ. Thus, can be used allogeneic or xengine cells from a source that is haplotype GCGS compatible with the donor tissue or organ (E.g., the closest relative of the donor graft).

III. Introduction cells and gp39 antagonist

T-cell tolerance to an organ or tissue transplant can be induced by injecting the recipient of a transplant of a gp39 antagonist in combination with allogeneic or xenogeneic cells, which Express a donor antigens and interact with recipient T-cells through gp39. In a preferred embodiment of the invention, allogeneic or xenogeneic cell and the gp39 antagonist is administered to the recipient together or at the same time. On the other hand, antag is t an antibody with a long half-life. In a preferred embodiment of the invention the antagonist and allogenic or xenogenic cells administered to the recipient prior to transplantation in the recipient organ or tissue (i.e., the recipient shall obtain the antagonist and cells). For example, the introduction of allogeneic and xenogeneic cells and antagonist can be made for several days (for example, five to eight) days before the transplant tissue or organ.

Found that the introduction of a single dose of allogeneic cells (in combination with the antagonist) is sufficient for the induction of T cell tolerance to donor tissue or organ (see example 1). The number of input cells may vary depending on the type of cells used, the type of tissue or organ transplant, weight of the recipient, the General condition of the recipient and other variables known to the specialist. An appropriate number of cells for use in the method of the present invention may be determined by the expert in this field of technology in the usual ways (for example, as described in example 1). Cells are introduced in the form and way, which are suitable for the induction of T-cell tolerance in the recipient. The cells can be introduced in a physiologically acceptable solution, such as for the military.

Antagonist according to the invention is administered to a subject in a biologically compatible form suitable for pharmaceutical injection in vivo to induce T-cell tolerance. "Biologically compatible form suitable for administration in vivo" means the form of the antagonist, in which any toxic effects are determined by therapeutic effects of the compounds. The term "subject" is supposed to refer to living organisms, which can be caused by an immune reaction, for example, mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species. The gp39 antagonist can be in any pharmacological form, optionally, with a pharmaceutically acceptable carrier. The introduction of a therapeutically active amount of an antagonist is defined as the introduction of a quantity, effective dose, and the time necessary to achieve the desired result (for example, T-cell tolerance). For example, a therapeutically active amount of a gp39 antagonist may vary according to factors such as stage of disease, age, sex and weight of individuals and the ability of the antagonist to cause the desired response in the individual. Schema dosage can be adjusted to provide the op who may be proportionally reduced, in accordance with the needs of therapeutic situation. As described in example 1, for the treatment of antibody against gp39 scheme effective treatment may include the establishment of the introduction of antibodies before transplantation of a tissue or organ (e.g., five to eight days before transplantation) with subsequent repeated introduction of antibodies (e.g., every second day) for several weeks (e.g., two to seven weeks) after transplantation.

The active compound (e.g., antagonist, such as an antibody) can be entered in the usual way, such as by injection (subcutaneous, intravenous, etc), oral administration, inhalation, transdermal application, or rectal administration. Depending on the method of administration, the active compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions that may inactivate the compound. The preferred method of administration is intravenous injection.

To enter the gp39 antagonist way other than parenteral administration, it may be necessary to apply the antagonist of the coating material or to introduce this material together with the antagonist, which will prevent its inactivation. For example and inhibitors or in an appropriate carrier, such as liposomes. Pharmaceutically acceptable diluents are saline and aqueous buffer solutions. Enzyme inhibitors are inhibitors of pancreatic trypsin, diisopropylphenol (DEP) and the drug. Liposomes include emulsions water in oil in water", as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

The active compound may also be injected parenterally, or IPR. Can also be obtained dispersions in glycerol, liquid polyethylene glycols, mixtures and oils. Under normal conditions of storage and use, these preparations may contain a preservative to prevent microbial growth.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where all dissolved in water) or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate use. In all cases, the composition must be sterile and must be fluid to the extent that it is easy to use syringe. It must be stable under conditions of manufacture and storage and must be protected from contamination by microorganisms, so the Yu, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol, and so forth), and suitable mixtures. Proper fluidity can be maintained, for example, using a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it is preferable to include in the composition isotonic agents such as sugars, polyols, such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable compositions can be invoked by incorporating in the composition an agent that slows down the absorption, for example, aluminum monostearate or gelatin.

Sterile injectable compositions can be obtained by incorporating the active compound (e.g., gp39 antagonist) in the required amount in an appropriate solvent in combination with one or more ingredients listed above, in accordance with the requirements, followed the spruce, which contains the basic dispersion medium and the required other ingredients from those mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of obtaining are vacuum drying and drying by freezing, which give a powder of the active ingredient (e.g., antagonist) plus any additional desired ingredient from their solution, pre-sterilized by filtration.

When the active connection is suitably protected, as described above, the protein can be entered orally, for example, with an inert diluent or an assimilable edible carrier. Used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and retarders, etc., the Use of such media and agents with pharmaceutically active substances is well known in the art. The use of any conventional media or agent in pharmaceutical compositions considered in all cases, except for their incompatibility with the active connection. The composition may also include additional active connected ease of administration and uniformity of dosage. Under the unit dosage form refers to physically separate units, suitable for the treatment of mammals as a standard doses; each unit contains a predetermined quantity of active compound calculated to obtain the desired therapeutic effect, in combination with the required pharmaceutical carrier. The definition of a unit dosage form according to the invention is dictated by and directly dependent on (a) the special properties of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technology of the preparation of compositions such an active compound for the treatment of sensitive individuals.

After or together with the scheme described here the evocation of tolerance, the donor tissue or organ transplant recipient graft in the usual way.

IV. The application of the methods of the invention

The methods of the present invention can be used in many different situations during the transplantation of tissue and organs. The above methods can be used to induce T-cell tolerance in transplant recipient tissue or organ, such as liver, kidney, heart, lung, skin, muscle, nervous tissue, galshoyani, which entails the transplant tissue or organ (e.g. liver transplantation in the treatment of hypercholesterolemia, transplantation of muscle cells in the treatment of muscular dystrophy, transplantation of neural tissue in the treatment of disease

Huntington or Parkinson's disease, and so on). In a preferred embodiment of the invention transplantirovannam fabric contains pancreatic islets. Accordingly, the invention concerns a method for the treatment of diabetes by transplantation of cells of pancreatic islets. The said method comprises the administration to a subject in need of such treatment, 1), allogeneic or xenogeneic cells, which Express a donor antigens, 2) antagonist molecules expressed on T cells of the recipient, which mediates contact-dependent helper-effector function, such as a gp39 antagonist (e.g., antibodies against gp39), and 3) donor cells of pancreatic islets. Preferably, allogenic or xenogenic cells and the antagonist is administered to the recipient before introducing him pancreatic islets.

The present invention also further illustrated by the following examples, which should not be construed as limiting. All references, patents and op>Example 1. Induction of tolerance to allografts pancreatic islets through the introduction of a recipient of allogeneic cells and anti-39

It is now established that allotransplantation depends on the total immunosupresive that nonspecific cuts immunoelectron functions. However, immunosuppressive pharmaceutical drugs can cause significant side effects. In addition, this approach reflects allotransplantation islets of Langerhans for diabetes (see, for example, Robertson R. P, (1992) N. Engl. J. Med. 327, 1861). Therapy with antibodies directed against T-cells, may allow for successful allotransplantation of islets rodents, but this approach gives too the same results for total immunosuppression (Carpenter C. B. (1990) N. Engl. Med. 322, 1224; Roark J. H. et al. (1992) Transplantation 54, 1098; Kahan, B. D. (1992) Curr. Opin. Immunol. 4, 553). In this example, the tolerance to the islet grafts induced a recipient of a transplant by manipulating the presentation of alloantigen T cells to prevent their activation. The term cell viability allografts in mice C57BL/6 (H-2bwith "chemical" diabetes check, using the methodology described below.

Induction deposition (140 mg/kg). Permanent diabetes is confirmed by the demonstration of glucose concentration in plasma, 400 mg/ml, in three cases in one week.

Fractionation of allogeneic splenic cells

Donor allogeneic cells for pretreatment of recipients of transplants receive from hybrid animals (C57BALB/c) (H-2b/d)F1to prevent graft versus host. To highlight small limfocitna cell suspensions of splenic cells from 8-week-old female mice (C57BALB/c)F1purified from erythrocytes and then fractionary in size by lutricia, as described in Tony H. P. et al. (1985) J. Exp. Med. 161, 223; and E. Gosselin J et al. (1988) J. Immunol. 140, 1408. Briefly, small lymphocytes secrete flowing through elutriating centrifugation, using, for example, a centrifuge model J-6B (Beckman Instruments, Palo Alto CA). Approximately 1-5 108cells in 8 ml of culture medium or balanced salt solution with 1.5% fetal bovine serum treated with desoksiribonukleaza placed in outracing the camera at the original speed counter-current of 13.5 ml/min, and rotate at 4oC with constant speed 3200 rpm Fraction of small cells with a very small admixture of large cells of buirette. In the experiments described here, the fraction of small cells harvested at 19 ml/min (at 3200 rpm). This fraction is completely cleared, according to the data of the auxiliary cellular resistance to radiation (3000 rad), in the analysis of T-cell lines specific for either rabbit IgG and H2d(CDC35), or for alloreactive H2b(D10.G4). Small cells and nefrackzionirovannam cells washed twice in serum-free medium before injection into the tail vein of recipients of allografts. Receive approximately 40-100 106nefrackzionirovannam splenic cells (C57BALB/c)F1(H-2b/dor 40-100 106elutriating small splenic cells (C57BALB/c)F1(H-2b/d).

Pre-treatment of recipients of transplants

Recipients of grafts pre-enter or nefrackzionirovannam allogeneic splenic cells (C57BALB/c)F1(H-2b/d), or elutriating "faction, 19" splenic cells of small diameter, which are cleared from the activity of the APC (selected as described above) or monoclonal antibody against gp39 (MR1, see example 2, experiment 3); or a combination of allogeneic cells and antibodies against gp39. Cell fractions 19 checked by two different intervallum not impose any allogeneic cells, neither antibody. Allogeneic cells are administered to transplant recipients by injection into the tail vein for five to eight days before the transplantation of islet transplant. The antibody MR1 administered at the dose of 250 μg/mouse twice a week, starting 7 days before transplantation of islets and continuing within 2-7 weeks or until adverse outcome of transplantation. The first injection of antibodies is usually given on the same day that the first injection of allogeneic splenic cells.

Transplantation of islet transplant

Allogeneic islets BALB/c(H-2d) allocate a modified way collagenase digestion (Gottlieb, P. A., et al. (1990) Diabets 39, 643). The Islands at a dose of 30 islets on g body mass are implanted in subrenal capsule of recipient mice (C57B1/6J(H-2b) immediately after the separation. Term viability of the graft is determined by maintaining the concentration of plasma glucose level of 200 mg/ml

Results

In the first series of experiments, recipients of islet grafts previously introduced either alone allogeneic splenic cells, or one antibody against gp39. As can be seen in Fig. 1, in the absence of previously entered splenic cells, all islet tra is sposobnosti islets has also been observed in animals, which was introduced only nefrackzionirovannam splenic cells containing the normal activity of the APC (63 days; the interval 4-12 days; N=7) or low dose (40-44 106cells) fractions 19, purified from agriculture, splenic cells (73 days; the interval of 3-14 days, N=16). In contrast, injection of a higher dose fractions 19, purified from agriculture, small splenocytes (75-88 106cells) prolongs the viability of allografts (1910 days; the interval 7-40 day; N=16). This effect term viability of the graft statistically quite significant (Fto 3.58=17,3, p < 0,001, when compared with groups not receiving anything treated nefrackzionirovannam splenic cells or a smaller dose of splenic cells fraction 19), but was not permanent. Long but limited period of viability of allogeneic islets recipients with diabetes who received small cell fraction 19, purified from agriculture, leads to the conclusion that some of these cells could not support term viability of the graft. Additional groups of recipients of grafts introduced 77-88 106cell fractions 20. This faction also, the majority of which are small lymphocytes, but their population differs from the population fractions 19 to those charenee = 8,5 day; the interval 6-12). Another group of recipients of grafts were injected only monoclonal antibody against gp39 - MR1. Fig. 1 shows that islet allografts fail within 15 days from 7/11 mice that received only anti-gp39 mAb. The remaining four mice had functioning grafts at the end of the experiment at 48 hours. The results show that the introduction of a recipient of one of gp39 antibodies against MR1 can extend the viability of islet transplant (mean = 20-19 day; interval 9-uncertainty., N=5). The degree of prolongation statistically similar extent, achieved with the use of higher doses of some of the splenic cells of fraction 19, and was significantly higher than the level reached in the other three groups (p < 0,05).

A series of experiments, described above, shows that a single administration of high doses of fraction 19 of the splenic cells, purified from APC or anti-gp39 mAb may increase the pot life of the transplant of pancreatic islets when compared with the absence of such introduction. However, any processing only one medication is not effective at inducing the recipient's long-term tolerance to islet transplant. Found that the combination of the agent. The results are shown in Fig. 2, each curve represents data for a single mouse. Where there's no shading icons are recipients whose transplants collapse spontaneously. Shaded icons are mice, in which islet grafts were functioning at the end of the experiment. Fig. 2 shows that an indefinite term viability is achieved in all animals treated for 7 weeks with anti-gp39 mAb, and one-time injection of fraction 19 - splenic cells, purified from AIC (N= 6). To change this scheme by reducing the duration of the introduction of anti-gp39 weakens, but does not cancel, the favorable effect on the pot life of the transplant. An indefinite term viability of the graft is achieved in 6/8 recipients, when anti-gp39 mAb is administered only during the 2 weeks in combination with fraction 19 of the splenic cells (Fig. 2A). An indefinite term viability of the grafts was also observed in recipients treated with anti-gp39 within 2-7 weeks in combination with one injection nefrackzionirovannam allogeneic splenic cells.

To confirm graft function and absence of insulin secretion remaining native ostrovka unilateral nephrectomy results in a recurrence of hyperglycemia (>300 mg/ml) within 3 days.

Islet allografts and natural pancreas were studied histologically in all animals or when the transplant was destroyed, or at the end of the experiment. Histological sections of islet allografts in kidney recipients fractionated allogeneic small lymphocytes and long (7 weeks) introduction mAb MR1 shows abundant intact islets visible below the renal capsule devoid of mononuclear infiltration and contain well-granulated insulin - and glucagon-positive cells. In contrast, histological sections of islet allografts in kidney recipients who received one anti-gp39 mAb, show characteristic intense inflammation with mononuclear cells and destruction associated islet cells. In all pancreatic glands owners morphology of islets with a single streptozotocin diabetes.

Example 2. The production and study of antibodies against gp39

Experience 1. Antibodies directed against human gp39

For the induction of antigen-specific T-cell tolerance in humans, preferably an antibody directed against human gp39. For producing murine monaslim fused protein gp39 gp39-CD8 in complete Freund's adjuvant (CFA). Then, after 6 weeks, mice injected with soluble gp39-CD8 in incomplete Freund's adjuvant (IFA). Soluble gp39-CD8 give in soluble form through 4 weeks after the second immunization. Then, 2 weeks later, mice revaccinated activated human peripheral blood lymphocytes, and after 2 weeks they finally revaccinated soluble gp39-CD8. Splenocytes were fused with the partner cells merging NS-1 to 4 days after the last immunization the immunization protocols.

Clones producing antibodies against human gp39, allocate on the basis of the process of multiple screening. First, clones sceneroot method of fixation on bacteriological cups using gp39-CD8. Then the positive clones sceneroot against control fused protein CD8 - CD72-CD8. Clones that were identified as positive in the analysis of fixation on bacteriological cups are removed. The remaining clones then sceneroot with resting and activated within 6 hours human peripheral blood lymphocytes (PBL) using flow cytometrical analysis. Positive are hybridoma, coloring activated, but not resting PBL. Finally, the remaining clones tested for their ability blocks is in sceneroot first against gp39-CD8 and CD72-CD8 in the analysis of fixation on bacteriological cups. Discovered that of these clones 30 are fixed on the cups gp39, but not CD8. These clones then sceneroot for detection of gp39 on activated human PBL. Approximately 15 clones are molecule on activated PBL, but not resting cells. Further specificity is confirmed by determining the ability of the clones to block the binding of CD40Ig this analysis. Such clones have become 3E4, 2H5 and 2H8. Such clones are preferred for use in the ways described here. Clones that are positive on activated, but not resting PBL, also sceneroot on the reactivity with a clone of activated rat T-cells POMC8. Clone 2H8 expresses the cross-reactivity with this line of rat T-cells.

Experience 2. Antibodies directed against human gp39

The immunization procedure similar to the procedure described in example 1 is used to produce additional antibodies directed against human gp39. One mouse BALB/c mice subjected to immunization soluble gp39-CD8 in CFA, and later, 4 weeks later, she injected activated within 6 hours human peripheral blood lymphocytes. Subsequently, the mouse revaccinated soluble gp39-CD8 for 4 days prior to fusion of the splenocytes with the cytometry staining of activated within 6 hours of PBL. Selected clones coloring activated, but not resting, human PBL. For further analysis, select the 6 clones - 4D9-8 AND 4D9-9, 24-31, 24-43, 89-76 and 89-79.

The specificity of the selected antibodies is confirmed by several tests. First, analysis by flow cytometry shows that all 6 mAb paint activated, but not resting, T cells from peripheral blood (see, as a typical example, Fig. 3B and 3C, showing staining of activated T-cells 4D9-8 and 4D9-9, respectively). The expression of molecules recognized by each of the six antibodies detected within 4 hours after activation, is the maximum 6-8 hours after activation and is not detected 24 hours after activation. All six mAb recognizes a molecule expressed on activated CD3+PBL mainly phenotype CD4+but part of CD8+T-cells also Express molecule. The expression of molecules recognized these six antibodies inhibited by the presence in the culture medium of cyclosporine And, as the expression of gp39 (see, for example, Fig. 4A and 4B, showing staining of T-cells treated with cyclosporine, 4D9-8 and 4D9-9, respectively). The kinetics and distribution of expression of molecules recognized by these national antibodies block the staining gp39 CD40Ig (see, for example, in Fig. 5A and 5B, which shows the inhibition of staining gp39 CD40Ig in the presence 4D9-8 and 4D9-9, respectively). When analysed by ELISA method all six mAb recognize gp39-CD8, soluble merged form molecules gp39. In addition, all six mAb form immunoprecipitate molecule of approximately 36 KD of the labeled35S-methionine activated human PBL. Immunoprecipitation molecule is identical to the molecule, precipitiously fused protein of human CD40Ig.

Functional activity of six selected monoclonal antibody 4D9-8 AND 4D9-9, 24-31, 24-43, 89-76 and 89-79) check as follows. First determine the ability of the mAb to inhibit the proliferation of purified human B-cells grown with IL-4 and soluble gp39. Purified human T cells grown with gp39 and IL-4 in the presence or absence of purified monoclonal antibodies or CD40Ig at doses from 0 to 12.5 µg/ml Proliferation of B-cells determined 3 days after introduction into the culture of thymidine. The results are given in Fig. 6) show that all six mAb can inhibit the proliferation of B-cells induced gp39 and IL-4. The most effective in the inhibition induced proliferation of B-cells, are mAb 89-76 and 24-31.

Then the consistent anti-CD3 activated T-cells and IL-2. Purified IgD+human B-cells by positive selection with cell sorting device with excitation fluorescence (FACS), and then cultured with anti-CD3 activated human T cells (treated with mitomycin C) and IL-2 for 6 days in the presence or absence of purified monoclonal antibodies against gp39 at a dosage of from 0 to 10 µg/ml For 6 days by the method of ELISA test production of IgM, IgG and IgA. The results are given below in table. 1) show that all six antibodies can inhibit T-cell-dependent B-cell differentiation, measured by the production of IgM, IgG and IgA.

To check the effect of mAb against gp39, T-cell response, monoclonal antibodies include in the standard reaction of the mixed culture of lymphocytes (SCR). Cultivate 300000 human peripheral blood lymphocytes (responders = P) with 100000 irradiated allogeneic peripheral blood lymphocytes (stimulants = C) in the presence or in the absence of mAb against gp39 (10 µg/ml). Culture mark pulse 3H-thymidine for 4, 5 and 6 days and 18 hours later collect cells grown in culture. All six mAb against human gp39 inhibit allospecific reaction when measured by SCR (see, as a typical example, Fig.La positive control using protein CTLA4-immunoglobulin, and mAb against CD28).

To identify, recognize whether all six monoclonal antibodies to specific epitopes on the molecule of human gp39, carry out experiments with cross-blocking. First activated human PBL block each of the six mAb (25 μg/ml). Cells are washed and then painted 10 μg/ml biotinylated antibody, followed by reaction with featuretraditional Avidya (PE-Av). Cell staining PE-Av analyze FACS. The results are shown below in table. 2.

All antibodies block the binding of CD40Ig with activated human PBL. However, the data in the table. 2, clearly shows the inefficiency of certain antibodies in blocking the binding of other antibodies to activated human PBL, which leads to the assumption that they recognize separate epitopes on molecules of human gp39.

Hybridoma 89-76 and 24-31 producing antibodies 89-76 and 24-31, respectively, deposited according to the Budapest Treaty in the American type culture collection, Parklawn Drive, Rockville, Maryland, September 2, 1994. Hybridoma 89-76 assigned inventory number ATCC HB11713 and hybridoma 24-31 assigned inventory number ATCC HB11712.

Experience 3. Antibodies directed against mising the e antibody against murine gp39 - MR1. For producing monoclonal antibody MR1 used the way described below, and can be used to generate other antibodies directed to gp39.

Hamsters subjected to immunization intraperitonal 5-106activated Th1 cells (d1.6) with weekly intervals for six weeks. When serum titers against murine Th1 exceeds 1:10000, will be merged cells with polyethylene glycol using immune Hamachi splenocytes and NS-1. Supernatant from cells containing the growing hybridoma, sceneroot flow cytometry on resting and activated Th1. One particular hybridoma, which produces Mab that selectively recognizes activated Th, then test and subcloning to get MR1. MR1 get in ascites and purified ion-exchange HPLC. Hybridoma MR1 deposited with the American type culture collection and assigned inventory number HB11048.

Equivalents

Specialists in this field of technology will see or be able to carry out using the usual experimental methods, many equivalents of the specific embodiments of the present invention described herein. Such equivalents are considered as included in the volume below the fo is it as references.

1. Method of inducing T-cell tolerance of a tissue or organ in a recipient to a donor tissue or organ, including the introduction referred to the recipient (a) allogenic or xenogenic cells, which expresses at least one donor antigen and which has a ligand on the cell surface that interact with a receptor on the surface of recipient T-cells, which mediates contact-dependent helper-effector function, and b) an antagonist of a receptor on the surface of T-cells, which inhibits the interaction of the ligand with the receptor.

2. The method according to p. 1, characterized in that the receptor on the surface of recipient T-cells, which mediates contactability helper-effector function, is a gp 39.

3. The method according to p. 2, wherein the receptor antagonist is a monoclonal antibody against gp 39.

4. The method according to p. 3, characterized in that the antibody against gp 39 is an antibody against gp 39 people.

5. The method according to p. 3, wherein the monoclonal antibody is an MRI.

6. The method according to p. 3, wherein the monoclonal antibody is a chimeric monoclonalantibody monoclonal antibody.

8. The method according to p. 1, wherein the allogeneic or xenogeneic cell is a lymphoid resting In the cage.

9. The method according to p. 1, wherein the allogeneic or xenogeneic cell and receptor antagonist is administered to the recipient prior to transplantation of a tissue or organ.

10. The method according to p. 1, wherein the tissue or organ includes pancreatic islets.

11. The method according to p. 1, wherein the tissue or organ selected from the group comprising liver, kidney, heart, lung, skin, muscle, nervous tissue, stomach and intestine.

12. Method of inducing T-cell tolerance of a tissue or organ in a recipient to a donor tissue or organ, including the introduction referred to the recipient (a) allogenic or xenogenic cells, which expresses at least one donor antigen, and b) antagonist gp 39.

13. The method according to p. 12, wherein the antagonist gp 39 is a monoclonal antibody against gp 39.

14. The method according to p. 13, characterized in that the antibody against gp 39 is an antibody against gp 39 people.

15. The method according to p. 13, wherein the monoclonal antibody is the monoclonal antibody.

17. The method according to p. 13, wherein the monoclonal antibody is humanitariannet monoclonal antibody.

18. The method according to p. 12, wherein the antagonist gp 39 is a soluble form of the ligand gp 39, representing a protein SD.

19. The method according to p. 12, wherein the allogeneic or xenogeneic cell is a lymphoid resting In the cage.

20. The method according to p. 12, wherein the allogeneic or xenogeneic cell and receptor antagonist is administered to the recipient prior to transplantation of a tissue or organ.

21. The method according to p. 12, wherein the tissue or organ includes pancreatic islets.

22. The method according to p. 12, wherein the tissue or organ selected from the group comprising liver, kidney, heart, lung, skin, muscle, nervous tissue, stomach or intestine.

23. Method of inducing T-cell tolerance to donor tissue or organ in the recipient tissue or organ under item 1, characterized in that in the treatment of diabetes additionally injected donor cells of pancreatic islets.

24. The method according to p. 23, characterized in that the antibody against gp 39 is a monoclonal antib gp 39 people.

26. The method according to p. 24, wherein the monoclonal antibody is an MRI.

27. The method according to p. 24, wherein the monoclonal antibody is a chimeric monoclonal antibody.

28. The method according to p. 24, wherein the monoclonal antibody is humanitariannet monoclonal antibody.

29. The method according to p. 23, wherein the antagonist gp 39 is a soluble form of the ligand gp 39, representing protein SD.

30. The method according to p. 23, wherein the allogeneic or xenogeneic cell is a lymphoid resting In the cage.

31. The method according to p. 23, wherein the allogeneic or xenogeneic cell and receptor antagonist is administered to the recipient prior to transplantation of the cells of pancreatic islets.

32. Method of inducing T-cell tolerance of a tissue or organ in a recipient to a donor tissue or organ, including the introduction of said recipient and donor allogeneic cells, and b) antibodies against gp 39, with donor allogeneic cell and antibody against gp 39 is administered to the recipient prior to transplantation of a tissue or organ.

33. The method according to p. 32, from the .32, characterized in that the antibody against gp 39 is an antibody against gp 39 people.

35. The method according to p.,33 characterized in that the monoclonal antibody is an MRI.

36. The method according to p. 34, wherein the monoclonal antibody is a chimeric monoclonal antibody.

37. The method according to p. 34, wherein the monoclonal antibody is humanitariannet monoclonal antibody.

38. The method according to p. 35, wherein the allogeneic donor cell is a lymphoid resting In the cage.

 

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