Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. IV. Examination of T cell subsets in graft-versus-host assays.

In DA rats grafted with PVG hearts a short course of cyclosporine induces a state of specific unresponsiveness. In animals with grafts surviving greater than 75 days, the W3/25+ (CD4+) subset loses its capacity to mediate rejection of PVG but 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)F1 hosts and third-party (W/FxDA)F1 hosts. This enlargement was comparable to that produced cells from naive and specifically sensitized hosts. MRC 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 idiotype in 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)F1 and (DAxW/F)F1 hosts. The normal response of cells from unresponsive animals in both proliferative and effector GVH assays shows 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.

Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. III. Further characterization of the CD4+ suppressor cell and its mechanisms of action.

The cellular basis of the specific unresponsiveness that develops in DA rats treated with cyclosporine (CSA) for 10 d after grafting a PVG heart was examined using an adoptive transfer assay. CD4+ cells from rats with long survival grafts specifically lack the capacity to restore PVG heart graft rejection, and can also inhibit the capacity of naive T cells to restore rejection, while in the first few weeks post-transplant, both CD4+ and CD8+ T cells from CSA-treated hosts have the capacity to effect PVG graft rejection. In this study, we demonstrated the CD4+ suppressor cells also had the capacity to inhibit restoration of rejection by CD4+ cells from CSA-treated DA rats recently transplanted with PVG hearts, and from rats sensitized to third party, but not from those specifically sensitized to PVG. They also inhibited the capacity of both naive CD8+ and sensitized CD8+ cells to effect rejection. These results showed that the CD4+ suppressor cell was capable of overriding the capacity to effect rejection of the CD4+ cell and activated CD8+ cells that were present in the CSA-treated host shortly after transplantation. The failure of naive CD8+ cells to augment suppression and the capacity of CD4+ suppressor cells to transfer unresponsiveness to irradiated hosts in which regeneration of CD8+ cells was abolished by thymectomy suggested that it was the CD4+ cell alone that mediated suppression. However, the failure of CD4+ suppressor cells to reinduce unresponsiveness in irradiated hosts whose CD8+ cells had been depleted by therapy with the mAb MRC Ox8 showed that a radioresistant CD8+ cell was required to reestablish the state of specific unresponsiveness. The induction of CD4+ suppressor cells in thymectomized hosts suggested that these cells were derived from long-lived CD4+ lymphocytes. However, their sensitivity to cyclophosphamide and their loss of suppressor function both after removal of the graft and after 3 d in culture demonstrated that the suppressor cell itself had a short lifespan. The CD4+ suppressor was shown to be MRC Ox22+ (CD45R+), MRC Ox17+ (MHC class II), and MRC Ox39+ (CD25, IL-2-R). These studies demonstrated the CD4+ suppressive cell identified in rats with specific unresponsiveness induced by CSA therapy had many features of the suppressor inducer cell identified in in vitro studies of the alloimmune response.(ABSTRACT TRUNCATED AT 400 WORDS)

T cell subsets mediating lethal graft versus host disease: demonstration that synergy between CD4+ and CD8+ T cells is the predominant mechanism in low responder rat strains.

T cell subsets from rat strains that have been characterized as high and low responders to alloantigen were examined for their capacity to mediate lethal graft versus host disease (GVHD) across strain combinations incompatible for class I, class II, and non-MHC antigens. Inocula of 5 X 10(7) lymph node and spleen cells (LC) from low responder DA (RT1a) and high responder W/F (RT1u) strains caused lethal GVHD in (W/F X DA)F1 hybrids given 6 Gy whole body irradiation. W/F CD4+ (W3/25+) cells (2 X 10(7], equal to the number in 5 X 10(7) LC mediated lethal GVHD but 10(8) DA CD4+ cells were required to cause lethal GVHD. CD8+ (MRC OX8+) cells (5 X 10(7] from W/F rats alone caused lethal GVHD but those from DA rats could not. Mixtures of CD4+ and CD8+ DA T cells, equivalent to the number in 5 X 10(7) LC, did mediate lethal GVHD, demonstrating that synergy between the subsets was the predominant mechanism with DA cells. These results suggest that differences in alloreactivity between the strains tested may be due to alternate requirements for the alloactivation of T cell subsets; the high responder subsets being self-sufficient and the low responder subsets being dependent upon each other.

Mechanisms maintaining antibody-induced enhancement of allografts. II. Mediation of specific suppression by short lived CD4+ T cells.

In DA rats grafted with PVG hearts, the injection of 1 ml of Wistar-Furth x DA)F1 anti-PVG serum on the day of grafting prevents rejection and induces a state of specific unresponsiveness. An adoptive transfer assay was used to test the capacity of T cell subsets, taken from rats given enhancing serum, to either restore rejection or to transfer unresponsiveness to syngeneic hosts irradiated with 9 Gy and grafted with donor (PVG) or third party (Wistar-Furth) hearts. W3/25+ (CD4+) cells from these animals retained some capacity to restore rejection until 50 days posttransplant, after which they invariably failed to restore PVG graft rejection but retained the capacity to effect Wistar-Furth rejection. At this time CD4+ cells were also capable of inhibiting naive but not specifically sensitized CD4+ cells capacity to restore PVG graft rejection in irradiated hosts. The development of CD4+ suppressor cells was concurrent with the appearance of clinically evident unresponsiveness in the host. MRC Ox8+ (CD8+) cells from enhanced rats when mixed with naive CD4+ cells delayed rejection in adoptive recipients but did not reestablish unresponsiveness. Paradoxically, the CD4+ cells that transfer unresponsiveness to the adoptive host proliferate such as normal cells in MLC to both donor and third party alloantigen. Unfractionated cells, CD4+ or CD8+ cells did not proliferate to relevant idiotype in vitro. The CD4+ cells after 3 days in culture, with either alloantigen or idiotype-bearing stimulator cells, lost their capacity to suppress in the adoptive transfer assay. The maintenance of specific unresponsiveness was thus shown to be due to a CD4+ suppressor T cell whose function was lost in culture, and therefore could not be detected in MLC or idiotype assays.

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.

Graft-versus-host resistance induced by tolerant cell populations. Evidence against clonal deletion as a mechanism of transplantation tolerance.

Characterization of the effect of immunization of F1 hybrid hosts with low doses of parental cells has shown that the F1 hybrid response to the receptor for the unshared MHC antigen on the immunizing cell induces specific resistance to a GVH challenge from cells of the same parental strain. We have shown that cells from parental rats tolerant to the unshared MHC antigens are capable of inducing GVH resistance in F1 hybrids. Unlike cells from normal parental rats that induce GVH resistance only when given in low immunizing doses of 10(6) cells, 10(6)-10(8) cells from tolerant donors effectively immunize F1 hybrids. This effect does not appear to be the result of passive transfer of suppressor cells from the tolerant donor. An alternative explanation is that tolerant populations contain cells that express the receptor for the tolerated alloantigen. The finding that normal parental populations that have been deleted of receptor-bearing cells by passage through semiallogeneic intermediate hosts do not induce GVH resistance, whereas tolerant cell populations do, confirms that clonal deletion does not adequately account for the functional characteristics of the tolerant cells. Attempts to delete putative receptor-bearing cells from the tolerant population however produced equivocal results.

The regulation of autoantibody production in Heymann's nephritis by T lymphocyte subsets.

The role of T lymphocyte subsets in the regulation of the autoantibody response to renal tubular antigen and the development of Heymann's nephritis (HN) in different rat strains was investigated in the present study. WF rats were highly susceptible to HN induction, PVG rats were moderately susceptible, whereas DA and BN were resistant. The induction of an autoantibody response to renal tubular antigen and HN in PVG rats was T cell-dependent as they were abrogated by neonatal thymectomy and T cell depletion after adult thymectomy, whole body irradiation and bone marrow reconstitution. Reconstitution of neonatal thymectomized and adult thymectomized whole body irradiated and bone marrow reconstitution of PVG rats with different T lymphocyte subsets revealed that the induction of an autoantibody response to renal tubular antigen in these animals required help from a long lived, W3/25+ T cell subset and was down regulated by a MRC OX8+ T cell subset. Adoptive transfer experiments demonstrated that the suppressor T cells involved were short lived and were probably generated by the interaction between a W3/25+ inducer and a MRC OX8+ precursor subpopulation. The suppressor precursor T cells were recently derived from the thymus as the suppressor T cell response was abrogated by adult thymectomy, an effect which could only be fully restored by thymic grafting or injection of adult thymocytes but not by the injection of normal spleen or lymph node cells. As adult thymectomy had less effect on the autoantibody response and HN development in the highly susceptible WF rats, the difference in disease susceptibility between the WF and PVG rats may be the result of different suppressor T cell recruitment from the adult thymus. Since adult thymectomy did not affect disease development in the DA and BN rats, the recently thymus-derived suppressor T cells did not contribute to innate disease resistance in these rat strains.

Postirradiation recovery of lymphoid cells in the rat.

Whole-body irradiation has been extensively used to ablate immune responsiveness in rodent recipients in adoptive allograft assays. This study was undertaken to determine the relative radioresistance and the tempo of regeneration, following whole-body irradiation, of cells involved in the allograft response. Six distinct cell populations have been identified in the lymphoid tissues of rats subjected to sublethal whole-body irradiation. The relative representation of these subpopulations was significantly different from that in nonirradiated controls. NK cells, macrophages, and plasma cells, which are present in very low numbers in cell suspensions prepared from normal lymphoid tissues, made up a significant proportion of the residual/regenerating population in the tissues of rats recovering from whole-body irradiation. More significantly perhaps, the mature T cell populations showed a significant increase in the T cytotoxic/suppressor to T helper cell ratio. These observations support the suggestion that a number of the cell types within the mixed cell population observed in the rejecting indicator grafts of irradiated recipients in adoptive allograft assays are host derived. The finding that the T cytotoxic/suppressor population is apparently more radioresistant than the T helper population supports a conclusion that graft rejection in irradiated recipients, restored with pure populations of T helper cells, may not be directly mediated by the injected cells but may be the result of collaboration between these and host-derived cytotoxic cell populations.

The mechanism of prolonged graft survival following removal of the regional lymph node.

The immune responsiveness of rats in which the rejection of RT-1-incompatible neonatal heart grafts had been prevented by removal of the draining lymph node was examined. It was found that within three days of node removal animals developed a state of specific unresponsiveness characterized by the failure to reject secondary grafts made into the contralateral leg with intact draining nodes. Studies of recipients with long-surviving grafts revealed that their serum contained significant levels of cytotoxic alloantibody although their cells behaved like those from naive donors in mixed lymphocyte interactions, graft-versus-host responses, and adoptive allograft assays. There was no evidence for the presence of sensitized T cells or suppressor T cells in the tissues of recipients with long-surviving grafts. The findings support the suggestion that prolonged graft survival following node removal is due to enhancement actively induced by the passage of graft antigen into the circulation at a time when the induction of the cell-mediated response has been aborted by removal of the node regional to the graft.

Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. Mediation of specific suppression by T helper/inducer cells.

DA rats grafted with major histocompatibility complex-incompatible PVG heart grafts and treated with cyclosporine (CY) for 10 d do not reject their grafts, and develop a state of specific unresponsiveness toward PVG allografts. Cells from these animals tested in an adoptive transfer assay were incapable of restoring PVG graft rejection, and capable of specifically inhibiting the capacity of adoptively transferred normal lymph node cells (LNC) to do so. They effected third party Wistar/Furth (W/F) graft rejection, however. Adoptive transfer assays with purified subpopulations of the lymphocytes that mediated this effect showed that W3/25+ T cells of the helper/inducer subclass, when injected alone, failed to restore rejection, and were also able, when injected with normal LNC or the W/25+ cells separated from them, to prevent these cells from effecting rejection. MRC OX8+ T cells of the cytotoxic/suppressor subclass, B cells, and serum from rats with long-surviving grafts all failed to inhibit the allograft responsiveness of normal LNC, and thus were not identified as mediators of the state of specific unresponsiveness. These results show that the specific unresponsiveness that develops in rats with long-surviving grafts, and which, in part at least, is responsible for prolonged graft survival, is due to an alteration in the alloreactivity of the helper/inducer subclass of T cells. These cells not only lack the capacity to initiate a rejection response against the alloantigens of the graft, but also have the ability to inhibit the capacity of normal W3/25+ cells to do so.