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The mechanisms by which CD4T cells, especially CD4CD25T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved.

Specific transplant tolerance is mediated by CD4T cells that die unless supported by T-cell derived cytokines and donor antigen. This study examined the role of Th1 and Th2 cytokines in the maintenance of tolerance.

DA rats treated with cyclosporine for 2 weeks after being grafted with an RT1 -incompatible PVG heart graft did not reject the graft and developed a state of specific unresponsiveness to graft antigens. The cellular mechanisms maintaining this state of unresponsiveness were studied by testing the capacity of lymphocytes from these animals to effect or inhibit graft rejection in irradiated grafted hosts. Whole lymph node and spleen cell populations, and the T cell subpopulation separated from the latter, failed to restore the rejection of PVG hearts in irradiated DA recipients but restored third-party Wistar-Furth (W/F) rejection. Both whole spleen cells and the splenic T cell subpopulation had the capacity to suppress the ability of normal DA lymphocytes to cause graft rejection. Suppression was not dependent upon a state of chimerism in grafted cyclosporine-treated animals, and was not associated with any measurable alterations in the proportion of cytotoxic/suppressor T cells in lymphoid tissues. These studies show that the state of specific unresponsiveness that follows the treatment of heart grafted rats with cyclosporine is dependent, in part, upon active suppression that is induced or mediated by T lymphocytes. Many features of the immune reactivity of cyclosporine-treated grafted rats support the hypothesis that the mechanism of specific suppression in these animals is akin to that of enhancement, rather than to that of transplantation tolerance induced in neonatal rats.

A quantitative adoptive transfer assay was used to investigate the role of the spleen in cell-mediated rejection of directly vascularized heart grafts. In this assay, the cell-mediated rejection response can be examined directly by testing the capacity of inocula of T cells to effect rejection of DA heart grafts in PVG rats whose own lymphocytes have been destroyed by whole body irradiation. The capacity of a variety of inocula, including lymph node cells (LNCs), spleen cells, and T cells from lymph node and spleen, to restore rejection were compared in groups of splenectomized and nonsplenectomized hosts. In both groups all inocula restored rejection toward normal. Only in experiments testing inocula equivalent to a small fraction of the naive peripheral lymphocyte pool was rejection delayed in the splenectomized hosts, and this was only a delay of a few days. These results showed that in the absence of the spleen, the primary rejection responses can be generated. In addition, it was demonstrated that the normal spleen contains only a small fraction of the T cell pool with the capacity to effect rejection. Memory T cells were also shown to mediate rejection in splenectomized hosts.

T cell receptor Vβ gene usage in renal allograft-derived T cell lines was investigated using a semiquantitative, polymerase chain reaction (PCR)-based technique. A panel of Vβ-specific primers was carefully designed to have uniform hybridization characterstics, enhancing comparability, and high specificity based on 3' mismatches between primers and nontarget V regions. PCR data were in concordance with previous phenotypic, functional, and Southern blot analyses, but the PCR technique proved to be more sensitive and comprehensive. For example, the EH3 cell line was confirmed to be polyclonal, with a number of different Vβ genes mediating anti-DR8-linked reactivity. Sequence analysis showed the most prevalent signals obtained by PCR, those for Vβ7 and Vβ20, probably corresponded to the only two rearrangements detected by Southern. The J2 cell line was shown to be polyclonal in early culture, when it was mostly CD8+and B35-reactive. Late cultures and a subclone were CD4+, possessed DR3-linked reactivity, and evidenced only one PCR signal: Vβ6. Similarly, the MH3 cell line was shown to be polyclonal early in culture, when it was reactive toward A11 and A29. In late cultures, when Southern analysis indicated clonality, only anti-A11 reactivity was maintained, and the predominant PCR signal was for Vβ17. Other alloreactivities were also attributed to specific Vβ. The Kng cell line, alloreactive to A28, yielded two strong PCR signals; Vβ2 and Vβ9. The Mijo line, DR1-reactive, gave a predominant signal for Vβ12. Thus in spite of variable in vitro selection occurring in some

In DA rats grafted with PVG hearts a short course of cyclosporine induces a state of specific unresponsiveness. In animals with grafts surviving >75 days, the W3/25+(CD4+) subset loses its capacity to mediate rejection of PVG hut not third-party heart grafts when transferred into irradiated DA hosts. In this study we examined whether there was an associated change in the capacity of peripheral lymphoid T cell subsets from unresponsive animals to induce graft versus host (GVH) reactivity. First we demonstrated that there is synergy between naive CD4+and CD8+cells in the popliteal lymph node PLN assay, but that alone, only CD4+and not CD8+cells proliferate. Unfractionated and CD4+cells from unresponsive animals produced similar PLN enlargement in both donor-specific (DAxPVG)F1hosts and third-party (W/FxDA)F1, hosts. This enlargement was comparable to that produced cells from naive and specifically sensitized hosts. MBC OX8+cells from both unresponsive and naive hosts did not produce PLN enlargement unless large numbers were injected; small numbers of sensitized MRC OX8+cells produced specific PLN enlargement. CD4+cells from CsA-treated DA did not respond to DA anti-PVG idiotypein an in vivo assay adapted from the host versus graft (HVG) PLN assay. As the PLN assay does not test cells capacity to effect tissue damage, cells from CsA-treated DA rats were tested in a lethal GVHD assay. These cells had the same capacity to induce lethal GVH in irradiated (DAxPVG)F1and (DAxW/F)F1hosts. The normal response of cells from unresponsive animals in both proliferative and effector GVH assays slows that cells with the potential to respond to PVG alloantigen and mediate tissue damage are present in unresponsive animals but are prevented from mediating rejection, possibly due to the relatively weak. immune stimulus of an organ graft.

Monoclonal antibodies to CDS have been shown to activate T cells in vivo and in vitro but have also been shown to render T cells anergic in vitro. In this study G4.18, a mouse IgG3 mAb, was produced that appeared to recognize CDS by its binding to all peripheral T cells, including a population not recognized by mAb to TCR-α/β that was presumed to be TCR-γ/δ cells. It precipitated molecules in the 24–26 kd region consistent with the CD3 complex as well as molecules ∼45 and ∼49 kd that corresponded to TCR α and β chains and a 92-kd complex. Incubating T cells for 24 hr with saturating concentrations of G4.18 caused modulation of the TCR complex. In vitro, it activated T cells but only if prebound to plastic. In solution it inhibited MLC and CML, but not PHA or Con A activation.

This study examined whether posttransplant anti-T cell monoclonal or polyclonal antibody therapy could provide a window of treatment to allow posttransplant total lymphoid irradiation (TLI) to induce tolerance. These experiments were conducted in a high responder strain combination of an ACI cardiac allograft into a Lewis rat. In this situation, treatment with antibody or posttransplant TLI alone is insufficient to induce tolerance, while similar treatments alone have been shown to induce tolerance in low responder strains. The affects of three anti-T cell therapies were compared: anti-CD4 mAb therapy, anti-CD3 mAb, and rabbit antithymocyte globulin (RATG). None of these antibody therapies alone prolonged graft survival indefinitely. Combining anti-CD4 therapy with posttransplant TLI markedly delayed rejection but failed to induce long-term graft survival. Tolerance could be induced by a combination of anti-pan T cell antibody (anti-CD3) and TLI, and, all grafts survived beyond 100 days. RATG failed to prevent graft rejection when used alone or in combination with TLI. However, posttransplant therapy with a combination of RATG, TLI, and single-donor blood transfusion resulted in graft survival beyond 100 days. Recipients bearing long-term donor grafts rejected third-party (PVG) grafts within 2 weeks. Low density donor bone marrow cells used instead of a blood transfusion did not facilitate tolerance. The results indicate that monoclonal or polyclonal anti-pan T cell antibodies, TLI, and a donor blood cell infusion function synergistically in facilitating tolerance to allografts in the posttransplant period.

The functional specificity and T cell receptor (TCR) Vβ gene expression of three class II HLA-DR1-reactive human T cell populations were examined. WP2, a renal allograft-derived, long-term CD4T cell line, was specifically cytotoxic for DR1, one of the mismatched antigens present on the allograft. Initial studies of WP2 using six TCR Vβ-specific mAb revealed a predominance of T cells expressing a member of the Vβ8 gene family. A smaller, yet significant, number of cells expressed TCR using Vβ5.1. Semiquantitative Vβ-specific polymerase chain reaction (PCR) analyses of RNA derived from this T cell line confirmed the presence of Vβ8 and Vβ5.1 messages. The PCR signal for Vβ8 was the strongest, followed by those for Vβ4 and Vβ5. An earlier WP2 culture had a very similar PCR profile, with a dominant signal for Vβ8, although signals for Vβ4 and Vβ5 were considerably lower. Previous PCR analyses of eight other renal allograft-derived, long-term T cell lines, grown under identical in vitro conditions, revealed no other example of predominant usage of Vβ8. We established two replicate long-term, anti-DR1 mixed lymphocyte reactions using PBL from two unrelated normal donors as responder and stimulator. The MLR were given alloantigen every 10 days, and RNA was obtained from the cultured cells immediately prior to each stimulation. PCR analyses of RNA taken at 10-day intervals over a total of 60–70 days indicated that, although the MLR were initially quite heterogeneous with regard to Vβ message expression, by the end of the fourth or fifth Ag cycle the predominant PCR signals observed in both MLR were for Vβ8. These results suggest that T cells using Vβ8 gene-encoded segments as part of their TCR may have a selective advantage in responses to DR1.