Mahzuz Karim

CD25+CD4+ Regulatory T Cells Prevent Graft Rejection: CTLA-4- and IL-10-Dependent Immunoregulation of Alloresponses

Specific and selective immunological unresponsiveness to donor alloantigens can be induced in vivo. We have shown previously that CD25+CD4+ T cells from mice exhibiting long-term operational tolerance to donor alloantigens can regulate rejection of allogeneic skin grafts mediated by CD45RBhighCD4+ T cells. In this study, we wished to determine whether donor-specific regulatory cells can be generated during the induction phase of unresponsiveness, i.e., before transplantation. We provide evidence that pretreatment with anti-CD4 Ab plus a donor-specific transfusion generates donor-specific regulatory CD25+CD4+ T cells that can suppress rejection of skin grafts mediated by naive CD45RBhighCD4+ T cells. Regulatory cells were contained only in the CD25+ fraction, as equivalent numbers of CD25−CD4+ T cells were unable to regulate rejection. This pretreatment strategy led to increased expression of CD122 by the CD25+CD4+ T cells. Blockade of both the IL-10 and CTLA-4 pathways abrogated immunoregulation mediated by CD25+ T cells, suggesting that IL-10 and CTLA-4 are required for the functional activity of this population of immunoregulatory T cells. In clinical transplantation, the generation of regulatory T cells that could provide dynamic control of rejection responses is a possible route to permanent graft survival without the need for long-term immunosuppression.

IFN-γ production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo

The significance of cytokine production by CD4+ regulatory T (T reg) cells after antigen exposure in vivo and its impact on their regulatory activity remains unclear. Pretreatment with donor alloantigen under the cover of anti-CD4 therapy generates alloantigen reactive T reg cells that can prevent rejection of donor-specific skin grafts that are mediated by naive CD45RBhighCD4+ T cells. To examine the kinetics and importance of cytokine gene transcription by such alloantigen-reactive T reg cells, pretreated mice were rechallenged with donor alloantigen in vivo. CD25+CD4+ T cells, but not CD25−CD4+ T cells, showed a fivefold increase in IFN-γ mRNA expression within 24 h of reencountering alloantigen in vivo. This expression kinetic was highly antigen-specific and was of functional significance. Neutralizing IFN-γ at the time of cotransfer of alloantigen reactive T reg cells, together with CD45RBhighCD4+ effector T cells into Rag −/− skin graft recipients, resulted in skin graft necrosis in all recipients; the generation and function of alloantigen-reactive T reg cells was impaired dramatically in IFN-γ–deficient mice. These data support a unique role for IFN-γ in the functional activity of alloantigen-reactive T reg cells during the development of operational tolerance to donor alloantigens in vivo.

Alloantigen-Induced CD25+CD4+ Regulatory T Cells Can Develop In Vivo from CD25−CD4+ Precursors in a Thymus-Independent Process

The capacity of naturally occurring autoreactive CD25+CD4+ regulatory T cells (Treg) to control immune responses both in vivo and in vitro is now well established. It has been demonstrated that these cells undergo positive selection within the thymus and appear to enter the periphery as committed CD25+CD4+ Treg. We have shown previously that CD25+CD4+ Treg with the capacity to prevent skin allograft rejection can be generated by pretreatment with donor alloantigen under the cover of anti-CD4 therapy. Here we demonstrate that this process does not require an intact thymus. Furthermore, generation of these Treg is not dependent on the expansion of CD25+CD4+ thymic emigrants, because depletion of CD25+ cells before pretreatment does not prevent Treg development, and Treg can be generated from CD25−CD4+ precursors. Taken together, these results clearly demonstrate that CD25+CD4+ Treg can be generated in the periphery from CD25−CD4+ precursors in a pathway distinct to that by which naturally occurring autoreactive CD25+CD4+ Treg develop. These observations may have important implications for the design of protocols, both experimental and clinical, for the induction of tolerance to autoantigens or alloantigens in adults with limited thymic function.

Regulatory T cells in transplantation.

There has recently been an explosion of renewed interest in regulatory T cells, particularly those within the CD4(+)CD25(+) population. It is becoming increasingly apparent that these cells exist not only as naturally occurring cells that may contribute to the maintenance of self-tolerance, but they also have the potential to prevent rejection of allografts in experimental models. Such cells have now been identified in humans as well as in rodents.

Pretransplant blood transfusion without additional immunotherapy generates CD25+CD4+ regulatory T cells: a potential explanation for the blood-transfusion effect.

Background. Preoperative blood transfusion has had a significant historic impact on graft outcome in clinical kidney transplantation, and the effect has been widely replicated in many experimental transplant models. Although the mechanisms underlying the blood-transfusion effect are poorly understood, one possibility is that preexposure to alloantigen results in the induction of regulatory cells with the capacity to control the effector arm of the immune response. Methods. Recent studies in autoimmune models have shown that T cells with regulatory function can be isolated from unmanipulated animals on the basis of CD25 expression, and we have recently shown that pretreatment of recipient mice with donor alloantigen combined with anti-CD4 antibody therapy generates CD25+CD4+ T cells that can prevent graft rejection. We therefore used this sensitive adoptive transfer mouse model to ask whether blood transfusion in the absence of any other treatment can also lead to the generation of alloreactive CD25+CD4+ regulatory T cells. Results. Although a single donor-specific transfusion (DST) fails to induce dominant regulation, we demonstrate that pretreatment with multiple DSTs generates CD25+CD4+ T cells that are as effective as those that result from blood transfusion under anti-CD4 antibody cover. More importantly, our results show that these cells also develop following multiple transfusions of unrelated (random) blood. Conclusion. These results provide a basis for understanding the blood-transfusion effect in transplantation and, by providing a link between naturally occurring regulatory cells and those induced by alloantigen, may shed new light on the fundamental basis of the effect itself.

Exhaustive differentiation of alloreactive CD8+ T cells: critical for determination of graft acceptance or rejection.

Background. The precise role that CD8+ T cells play in the rejection and acceptance of different types of allograft is unclear and has been shown to vary between donor–recipient combinations. Methods. The response of adoptively transferred CD8+ T cells reactive to the donor alloantigen H2Kb was examined after transplantation of H2Kb+ liver, kidney, and heart grafts in mice. Results. After transfer of 6×106 alloreactive CD8+ T cells to T-cell depleted syngeneic mice spontaneous long-term acceptance of liver grafts was observed, whereas kidney and heart grafts were acutely rejected. Within 5 days of liver transplantation, we found that the entire H2Kb-reactive T-cell pool was stimulated to proliferate and differentiate into memory or effector cells that were detectable within lymphoid tissues as well as the liver graft itself. However, despite the generation of effector or memory T cells, liver allografts were accepted, which correlated with the exhaustion or deletion of such cells. In contrast, although activation and proliferation of H2Kb-reactive CD8+ T cells was observed after transplantation of heart or kidney grafts, unactivated, H2Kb-reactive CD8+ T cells were still present in the spleen even long term. Interestingly, differences in the effector function of liver and kidney graft infiltrating donor-reactive CD8+ T cells were not detected after adoptive transfer into immunodeficient mice, despite a reduction in Th1-type cytokines within liver grafts. Conclusions. The rapid and extensive initial activation and differentiation of donor-reactive CD8+ T cells that occurs after liver transplantation leads to clonal exhaustion or deletion of the alloreactive CD8+ T-cell repertoire resulting in spontaneous tolerance induction.

CD25+CD4+ regulatory T cells develop in mice not only during spontaneous acceptance of liver allografts but also after acute allograft rejection.

Background. Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25+CD4+ regulatory T cells. Methods. CD25+ and CD25−CD4+ T cells, isolated from CBA. Ca (H2k) recipients of C57BL/10 (B10; H2b) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1−/− mice to investigate their influence on skin allograft rejection mediated by CD45RBhighCD4+ effector T Cells. Results. Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25+CD4+ T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25−CD4+ T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25+CD4+ T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. Conclusions. Our data provide evidence that the presence of CD25+CD4+ regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.