Kay E. Gurley

Induction of long-term specific tolerance to allografts in rats by therapy with an anti-CD3-like monoclonal antibody

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. In vivo, G4.18 was not toxic even in high doses, and this was thought to be due to the inability of this mAb to activate T cells in vitro because the rat lacks Fc receptors for mouse IgGS. Therapy with G4.18 resulted in transient modulation of TCR/CD3 on T cells and depletion of these cells from blood. G4.18 had no depleting effects by lymph node or spleen cells but caused marked, transient thymic involution. Therapy with G4.18 also induced indefinite survival (&100 days) of PVG (RTF) heart grafts but not skin grafts in DA (RTFα) hosts. These hosts with long-surviving cardiac transplants, when grafted from PVG skin, accepted these grafts but rejected third-party skin in first-set. Thus G4.18 was shown to induce long-term specific tolerance to an organ allograft.

Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. V. Dependence of CD4+ suppressor cells on the presence of alloantigen and cytokines, including interleukin 2.

CD+ cells from CsA-treated DA rats with long-surviving PVG heart allografts specifically suppress the capacity of naive CD4+ cells to restore allograft rejection in irradiated DA rats, but have normal donor-specific alloreactivity in MLC. CD4+ suppressor cells from CsA-treated DA rats cultured for 3 days against either PVG or DA spleen cells lost the capacity to transfer suppression into irradiated DA rats grafted with PVG hearts and regained the ability to mediate rejection. However, these cells retained suppressor function when stimulated with donor-specific alloantigen in media supplemented with 20% Con A supernatant. CD4+ cells from CsA-treated rats cultured against either third-party stimulator cells or syngeneic cells expressing anti-PVG idiotype in media supplemented with Con A supernatant failed to maintain suppressor cell function. CD4+ cells from CsA-treated rats cultured in media supplemented with Con A supernatant alone also failed to maintain suppressor function. Suppressor cell function in culture was not maintained by rIL-2. mAb to the IL-2 receptor a chain (CD25) prevented the maintenance of suppressor cell function in media supplemented with Con A supernatant. Con A supernatant is rich in IFN-γ, but addition of an anti-IFN-γ mAb to the culture did not affect the maintenance of suppressor cells. These studies demonstrate that the CD4+ suppressor cell from CsA-treated rats with long-surviving grafts is short-lived; its survival is dependent upon contact with specific alloanti-gens and cytokines, one of which is IL-2. In the absence of cytokines and/or specific alloantigen, the CD4+ cells regain the capacity to initiate graft rejection in irradiated rats, suggesting that within the CD4+ subpopulation there is a fragile balance between cells with the capacity to suppress and effect rejection.

Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. VI. In vitro alloreactivity of T cell subsets from rats with long-surviving allografts.

DA rats treated with a short course of cyclosporine develop specific unresponsiveness to RT1-incompatible PVG donor heart allografts. CD4+ cells, not CD8+ cells, transfer unresponsiveness to irradiated rats. However, host-derived CD8+ cells are important in reestablishing unresponsiveness. In this study, unfractionated lymphoid cells and W3/25+ (CD4+) cells from CsA-treated rats with long-surviving PVG allografts demonstrated normal alloreactivity to PVG alloantigen in the mixed lymphocyte culture and failed to suppress the proliferative response of naive W3/25+ cells to donor-specific alloantigen. MRC OX8+ (CD8+) cells did not proliferate. Sera from CsA-treated rats had no effect on the MLC reactivity of cells from CsA-treated rats, suggesting that blocking or antiidiotypic antibodies did not diminish alloreactivity. IL-2 production by W3/25+ cells from CsA-treated rats was similar to that by W3/25+ cells from naive rats. Specific cytotoxic T cells to PVG were generated in MLC, and the frequency of precursor cytotoxic lymphocytes in CsA-treated rats was similar to that in naive DA rats. In an in vitro assay testing response to idiotype, neither W3/25+ or MRC OX8+ cells from unresponsive rats proliferated. As CD4+ cells from CsA-treated rats lose their capacity to adoptively transfer specific unresponsiveness unless maintained in a cytokine-rich supernatant, all in vitro assays were performed with and without added cytokines, but no change in reactivity consistent with suppression was observed in any assay. CD4+ suppressor cells had no effect on conventional in vitro assays of alloreactivity, preventing the detection of the unresponsiveness in vitro.

Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. II. Sequential changes in alloreactivity of T cell subsets.

A 10-day course of cyclosporine treatment inhibits the capacity of DA rats to reject PVG heart grafts and leads to the development of specific unresponsiveness and indefinite graft survival, which is mediated by a W3/25+ (CD4+) suppressor cell. In this study the sequential changes in the alloreactivity of the CD4+ and CD8+ subsets of CsA-treated DA rats were examined. During the induction phase, 8 and 20 days posttransplant, W3/25+ cells retained normal alloreactivity in that they adoptively restored PVG heart graft rejection in irradiated DA rats. By day 50 they had lost their capacity to restore rejection of PVG grafts but still retained their capacity to effect third party W/F graft rejection. W3/25+ cells from control grafted rats adoptively restored PVG graft rejection even at 75 days posttransplant, suggesting that the loss of alloreactivity of W3/25+ cells in CsA-treated rats was due to the prevention of rejection by CsA, and not a consequence of sensitization to alloantigen. MRCOX8+ cells from CsA-treated rats showed some evidence of sensitization at days 8 and 20 but lost this by day 50. These studies showed that during the induction phase, normal alloreactivity of W3/25+ cells is retained and sensitization of MRCOX8+ cells occurs. Specific loss of reactivity and suppressor potential of W3/25+ cells developed later, when specific unresponsiveness to second donor strain grafts developed in these hosts.

Induction of tolerance to heart allografts in rats using posttransplant total lymphoid irradiation and anti-T cell antibodies

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.

Bone marrow transplantation following total lymphoid irradiation. I. Correlation with field size and suppressor cell induction.

Total lymphoid irradiation (TLI) induces a unique state of immunosuppression. Although permanent bone marrow chimerism has been obtained in rodents prepared by TLI, uniform marrow engraftment has been more difficult to obtain in larger mammals. Accordingly, studies were performed to assess the immunologic perturbations induced by TLI in inbred LEW rats, and to explore the effect of altering field size of irradiation on the induction of suppressor cells and the success of allogeneic bone marrow transplantation. Additional abdominal shielding to protect a single kidney (right) from irradiation during TLI presented successful of bone marrow engraftment (WF leads to LEW, N = 5) but chimerism was uniformly obtained (N = 3) using the full irradiation field (P less than .05) Lymphopenia and a relative monocytosis were noted in all rats subjected to TLI. Although TLI using the full irradiation field eliminated alloreactivity of nylon-wool-purified spleen cells, significant, if reduced, alloreactivity was noted in rats subjected to TLI using smaller irradiation fields. Irradiated (1500 rads) nylon-wool-purified splenic T cells of rats subjected to TLI using the full field effected significantly greater suppression (P less than .001) of a normal mixed lymphocyte culture than did cells from rats subjected to TLI with right kidney shields in place (relative response reduced to 15.2 +/- 5.7% versus 64.3 +/- 11.7%). Success of bone marrow engraftment in rats prepared by TLI was correlated, therefore, with the induction of a profound lymphopenia, elimination of alloreactivity, and the development of a potent splenic suppressor system.