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T cell clones are proving to be useful for analysis of various immunological phenomena. Although some clones grow well in IL 2-containing CM alone, the combination of antigen, "filler" cells, and CM improves cloning efficiency and may favor the retention of stable clones expressing a "normal" phenotype. The antigen recognition repertoire of T cells appears to be much larger than was predicted using conventional approaches. CTLs can react with at least 23 different determinants on the H-2Kb molecule of the MHC. CTLs demonstrate MHC-restricted recognition of viral and haptenic determinants, but the restriction is not strict for all cloned cells, and a variety of reactivity patterns are found. Antigen-responsive cloned T cells appear to recognize antigens in association with alloantigens encoded by the I-A region of the MHC. However, the relevant structures formed in hybrid animals include all possible combinations of peptides including those formed by trans-complementation of I-A and I-E region gene products. Culture supernatants from antigen-responsive cloned T cells contain a variety of biological activities including IL 2, B cell-stimulating factor, colony-stimulating factor, migration inhibition factor, interferon, and factors that modulate expression of Ia antigens on macrophages and influence production of complement components. These factors may be important in mediating interactions between T lymphocytes and other cells. Cloned T cells have proved to be useful for studying the importance of particular phenotypic characteristics associated with given immunological functions. Although expression of Lyt-2 generally correlates with cytolytic activity and Lyt-1 with helper activity, important exceptions have been found with T cell clones. Cloned T cells promise to be useful for further characterization of the molecular basis of immune responses.

Cloned populations of T cells have been used to derive clone-specific monoclonal antibodies. These antibodies influence the specific immunological functions of the cloned T cells, suggesting that these antibodies react with the T cell receptor for antigen. Immunoprecipitates have been obtained with some of these antibodies, and the characteristics of the polypeptides from different cells are surprising similar. At present, little is known about this molecular complex other than that the two polypeptides are disulfide-linked and have apparent molecular weights of about 45,000 daltons and 43,000 daltons. This molecular complex, at least in human T cells, is associated with, but not covalently linked with, another molecular complex (Leu4/T3). However, modern techniques of cellular and molecular immunology should provide information very soon about the actual role of these molecules in T cell recognition of antigen. Indeed, cDNA clones that may encode genes for one of the peptide chains have been obtained.

Previous studies using flow cytofluorometry demonstrated that passively acquired alloantigens encoded by the I-A and H-2K/D regions of the MHC could be identified on the surface of murine cloned T lymphocytes. The present studies were designed to further characterize the nature of that bound alloantigenic material on cloned lines of alloreactive amplifier T cells (Th). Immune precipitation and polyacrylamide gel electrophoresis were used to substantiate that allogeneic I-A and H-2K/D antigens were present on he surface of these cloned Th. Using proteolytic digestion with papain, bound allogeneic I-A and H-2K/D was removed from the surface of a cloned Th designated L2. Allogeneic I-A or surface of a cloned Th designated L2. Allogeneic I-A or H-2K/D antigens were not reexpressed if papain digested cells were incubated overnight in culture medium without addition of fresh alloantigen. Further, cloned cells did not release detectable amounts of interleukin 2 (IL-2) in response to Con A stimulation immediately following enzymatic digestion. After overnight culture, papain-digested cells released amounts of IL-2 similar to untreated controls. The proliferative response of these cells measured by 3H-thymidine incorporation, however, was significantly less than that observed using undigested controls, unless alloantigen was provided in the form of fresh irradiated stimulating cells. The addition of partially purified preparations of monoclonal anti Ia antibodies to cultures of cloned Th resulted in marked reduction in incorporation of 3HTdR. These findings are consistent with the hypothesis that modulation of the Th proliferative response may occur at least partially through binding of allogeneic material to the cell surface.

We have investigated the relative roles of the Lyt-2+ and L3T4+ T lymphocyte subsets in rejection of class-I-MHC-antigen-disparate skin and tumor allografts. To deplete T cells in vivo, rat anti-Lyt-2 or anti-L3T4 monoclonal antibodies (mAb) were administered to adult-thymectomized (ATX) recipient mice prior to transplantation. BALB/c (H-2d) recipient mice rejected the Ia- Sarcoma I (Sa1) (H-2a) tissue culture-derived tumor after depletion of the L3T4+ T cell subset in vivo. In contrast, depletion of the Lyt-2+ T cell subset permitted lethal tumor growth in all recipient mice. To determine the role of particular T cell subsets in rejection of Ld class-I-MHC-antigen-disparate allografts, BALB/c skin was transplanted to BALB/c-H-2dm2 recipient mice. Skin grafts were rejected by control mice with a mean survival time (MST) of 14.5 days. The MST of skin grafts for mice treated with anti-L3T4 mAb was 16.6 days. In contrast, administration of anti-Lyt-2 mAb alone (MST = greater than 47 days) or together with anti-L3T4 mAb (MST = greater than 50 days) caused prolonged or indefinite graft survival in all recipient mice. Depletion of specific T cell subsets was confirmed by flow cytometric analysis and by analysis of T cell function in vitro. These results suggest that Lyt-2+ T lymphocytes are essential for rejection of class-I-MHC-disparate allografts; indirect presentation of alloantigen to L3T4+ T cells may not be necessary for rejection.

Enhancement of LBN F1 renal allograft survival in Lewis (L) rats is achieved by injecting the recipient i.v. with donor antigen (LBN F1 spleen cells) 1 day before transplantation and antidonor antibody (L anti-BN alloantiserum) at the time of transplantation. Treatment with this combination of antigen and antibody also induces the recipient to make L anti-(L anti-BN) anti-idiotypic antibody that reaches peak titers within 10 days. The degree of graft enhancement achieved was increased greatly by delaying transplantation until the peak of the anti-idiotypic antibody response 10 days after treatment with antigen and antibody. Two in vitro assays for cellular immunity (51Cr release and microcytotoxicity) failed to demonstrate antidonor activity in spleen cells from recipients for which transplantation had been delayed 10 days. The close correlation of enhancement, absence of cellular immunity in vitro, and the kinetics of the anti-idiotypic antibody response suggest that anti-idiotypic antibody may prevent either sensitization and generation of effector T lymphocytes or the destructive potential of sensitized cells.

Lewis rats receiving an LBN kidney allograft demonstrate no signs of rejection if they are pretreated with donor spleen cells and antiserum reactive with the donor alloantigen. We examined the cellular reactivity of long-term kidney allograft survivors. Normal proliferative and cytolytic responses were obtained with spleen cells from long-term survivors, in marked contrast to the diminished responses of cells from neonatally tolerant rats or the heightened cytolytic response of cells from rats that had rejected a renal allograft. Serum from long-term renal allograft survivors as well as serum obtained from rats at the time of transplantation did not suppress proliferative or cytolytic responses of normal cells. The results of this study suggest that long-term renal allograft survivors possess the precursors of those cells which are responsible for proliferative and cytolytic responses in mixed leukocyte cultures, but that they have not been sensitized to their renal allograft.