We have previously demonstrated in thymectomized and T-cell-depleted mice that porcine thymus grafts can replace the host thymus to support normal mouse thymopoiesis and repopulate the peripheral T cell repertoire (7
). Importantly, these murine T cells developing in porcine thymus grafts show specific tolerance to the donor, even by the stringent test of skin grafting (7
). This approach has recently been extended to allogeneic and xenogeneic (pig to baboon) large animal models (3
), suggesting that it has considerable potential to promote the success of xenotransplantation.
Positive selection in porcine thymic xenografts in immunocompetent mice is mediated entirely by the porcine MHC of the thymic epithelium, but both porcine donor antigens and recipient MHC antigens contribute to negative selection (26
). Nevertheless, the murine T cells that repopulate the periphery can recognize antigens presented by host MHC molecules and can clear opportunistic infections (9
). These data suggest that recipient mouse-restricted immune reactivity reflects cross-reactivity on murine MHC of a broad repertoire of T cells selected in the porcine thymus grafts.
More recently, we extended these findings to the pig/human combination using immunodeficient mice and demonstrated that normal human thymopoiesis could take place in xenogeneic porcine thymus grafts (10
). These results were replicated here, with normal proportions of all human thymocyte subsets in the swine thymus graft. The slight reduction in percentages of DP and SP thymocytes and increase in “double negative” cells in the xenogeneic grafts () probably reflects the presence of porcine thymocytes that often persist long-term in the porcine grafts, as we have previously reported (10
). We also confirmed here that the human T cells developing in porcine thymus grafts are tolerant to the xenogeneic source animal in MLR assays. Remarkably, this unresponsiveness is specific for the MHC (the SLA) of the xenograft donor, as these human T cells showed normal responses to SLA-mismatched porcine stimulators, as well as to allogeneic human stimulators. In our previous study, the failure to overcome tolerance to the donor porcine SLA with exogenous IL-2 suggested that the tolerance was achieved by intrathymic deletion rather than by an anergy mechanism (10
). Similar TCR Vβ usage was observed in human thymocytes developing in porcine versus human thymic grafts, suggesting that a polyclonal repertoire was generated in the xenogeneic thymus graft.
We have now addressed the polyclonality of the human T cell repertoire developing in a xenogeneic thymus in a more direct fashion, using spectratype analysis. Our studies reveal similar, polyclonal, normal TCR repertoires among single positive human thymocytes generated in xenogeneic porcine and autologous human thymus grafts. Given that all positive selection of murine T cells in porcine thymus grafts in immunocompetent mice is mediated by the porcine donor MHC, whereas negative selection is mediated by both mouse and pig MHC (26
), it is reasonable to assume that this diverse human TCR repertoire results from positive selection by porcine thymic epithelium and negative selection on human, porcine and murine elements in the grafts, which we have previously shown to be present (10
). The ability of a porcine thymic epithelium to positively select human TCR may reflect in part the extensive homology between SLA and HLA. SLA class Ia (30
) and class Ib genes (31
) show homology to human class Ia and Ib molecules, respectively. Class II genes also showed strong homology between SLA and HLA (32
). However, we favor another explanation, given that murine T cells positively selected by porcine thymic epithelium also show evidence for polyclonality (see above) (29
), despite the lower species homology of pig and mouse MHC molecules. TCR structure seems to have evolved to have reactivity to MHC (34
) and this evolution likely occurred before mammalian speciation, resulting in conservation of inherent TCR MHC reactivity between species. This reactivity may ensure the selection of a polyclonal repertoire in the xenogeneic thymic environment.
While the failure to amplify Vβ10 from single positive cells in porcine thymus grafts might suggest that this family of TCR could be deleted by a porcine endogenous superantigen, we have previously succeeded in detecting Vβ10+
human thymocytes in porcine thymus grafts (10
), arguing against this possibility. Nevertheless, these previous studies involved whole thymocyte cell suspensions rather than single positive thymocytes, and it is possible that deletion due to a porcine endogenous superantigen occurs at the late double positive and/or single positive stage.
The double grafting experiments performed here may be relevant to the question of whether or not recipient thymectomy will be needed to achieve xenogeneic tolerance through the thymus transplantation approach. In immunocompetent, T cell-depleted mice, recipient thymectomy was found to be essential for the successful engraftment and function of porcine thymus xenografts (37
). Recipient thymectomy was also found to be essential for xenogeneic thymus engraftment in a pig to baboon model (13
). In a separate ongoing study, we have observed that untreated humanized mice are capable of rejecting porcine thymic tissue grafted after human immune reconstitution has occurred (K. Habiro and Y. Yang, personal communication). However, the data presented here show that two of three animals receiving porcine thymus grafts simultaneously with human thymus tissue and human HSCs accepted the pig thymus long-term, despite the lack of xenogeneic MLR tolerance observed among thymocytes in the human graft. These studies raise the possibility that post-thymic mechanisms, with or without a role for presentation of soluble porcine antigen in the human thymus, may lead to tolerance of human thymocytes after they leave the human thymus graft. Not all self-reactive thymocytes are normally deleted (38
), and regulatory T cells play an important role in suppressing autoreactivity of these cells (41
). Likewise, regulatory cells developing in porcine thymus grafts may suppress the ability of non-tolerant human T cells developing in human thymus grafts to reject porcine thymic grafts. Indeed, thymic epithelium has been shown to be capable of inducing dominant tolerance to its antigens in an allogeneic model (42
). It is also possible that the thymus is an immunoprotected site and that non-tolerant T cells generated in the human thymus grafts simply “ignored” the porcine thymus grafts, which were healed in by the time human T cells had developed in the human grafts. However, previous thymic grafting studies in allogeneic mouse models showed that a second thymus graft could tolerize T cells developing in a first thymus graft to minor antigens but not to MHC alloantigens (43
). Since we did not analyze tolerance of peripheral T cells in these mice, further studies will be needed to address their tolerance.
The studies presented here demonstrate that the addition of human CD34 cells i.v. can enhance human T cell reconstitution from a porcine graft. Improved human immune reconstitution, including T cells, B cells, dendritic cells, the ability to reject xenografts and the ability to mount specific class-switched antibody responses have recently been demonstrated in HU/HU mice receiving i.v. injections of fetal CD34+ cells from the same human donor (23
). The presence of human APC in the periphery seems to enhance the survival and function of T cells generated in a human thymus graft, consistent with studies showing a need for self MHC/peptide in the periphery (47
). Our demonstration of a similar phenomenon in mice receiving porcine thymic xenografts with human FL and CD34 cells, in which the thymic epithelium and human APC in the periphery are completely MHC-mismatched, is somewhat surprising. We hypothesize that because human T cells interact more efficiently with human than mouse APCs in the periphery and because the human TCR repertoire developing in a porcine thymus is diverse, that cross-reactivity with human MHC of TCR selected on porcine MHC allows human APC to provide MHC/peptide signals that support the survival and function of the human T cells. Our observations suggest an excellent model for dissecting the impact of disparity between thymic epithelium (pig) and APC in the periphery (human) on human T cell function. For example, several studies in rodents suggest that self-tolerance may be incomplete, with an increased tendency to develop autoimmunity, when T cells develop in a xenogeneic thymus graft (28
) The improved understanding of immune function obtained from such studies in humanized mice should help to bring the xenogeneic thymic transplantation approach closer to clinical application for the induction of tolerance.