Harnessing regulatory T cells for clinical use in transplantation: the end of the beginning.

Owing to the adverse effects of immunosuppression and an inability to prevent chronic rejection, there is a pressing need for alternative strategies to control alloimmunity. In three decades, regulatory T cells (Tregs) have evolved from a hypothetical mediator of adoptively transferred tolerance to a well-defined population that can be expanded ex vivo and returned safely to patients in clinical trials. Herein, we review the historical developments that have permitted these advances and the current status of clinical trials examining Tregs as a cellular therapy in transplantation. We conclude by discussing the critical unanswered questions that face this field in the coming years.

Alloantigen-induced specific immunological unresponsiveness.

When the immune system encounters alloantigen it can respond in any one of a number of different ways. The choice that is made will take into account factors such as where, when and how the contact with the alloantigen takes place, as well as the environmental conditions that prevail at the time the alloantigen is encountered. Alloantigen administration before transplantation either alone or in combination with therapeutic agents that modulate the functional activity of the responding leucocytes can be a powerful way of inducing specific unresponsiveness to alloantigens in vivo. The molecular mechanisms that influence the way the outcome of the immune response to alloantigen develops, either activation or unresponsiveness to the triggering antigen, hold the key to our ability to manipulate the immune system effectively by exposing it to donor antigen for therapeutic purposes. This review will focus on alloantigen-induced immunological unresponsiveness and how insights into the mechanisms of unresponsiveness have driven the development of novel tolerance-induction strategies that show promise for translation into the clinic in the future.

IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo.

We present evidence that donor-reactive CD4(+) T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4(+) T cells contained within the CD45RB(high) fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RB(low) CD4(+) and CD25(+)CD4(+) populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RB(high) CD4(+) T cells. Analysis of the mechanism of immunoregulation transferred by CD45RB(low) CD4(+) T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RB(low) CD4(+) T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RB(high) CD4(+) T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RB(low) CD4(+) T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.

Immunoregulation by CD4 T cells in the induction of specific immunological unresponsiveness to alloantigens in vivo: evidence for a reduction in the frequency of alloantigen-specific cytotoxic T cells in vitro.

Donor-specific unresponsiveness to allogeneic cardiac allografts in mice can be induced by the combined pretreatment with donor alloantigen and anti-CD4 antibody (anti-CD4+DST). We have investigated whether the induction of unresponsiveness in this model is due to the presence of T cells that regulate immune responsiveness towards the allograft. First, we analysed the functional characteristics of splenocytes from pretreated mice at the time of transplantation. A significant reduction in the frequency of donor specific cytotoxic precursor was found only after the anti-CD4+DST treatment. Next, we designed an in vitro assay to identify the phenotype of the splenocyte population responsible. CD4+ and CD4- fractions were purified from mice treated with anti-CD4+DST or anti-CD4 alone (controls) by cell sorting. Interestingly, only the addition of CD4+ cells from anti-CD4+DST treated mice resulted in a selective reduction and a bimodal distribution in the donor specific CTLp response, indicating the presence of a regulatory population. CD4+ cells from controls did not have this effect. These in vitro findings were substantiated by adoptive transfer experiments in vivo. These data demonstrate that CD4+ cells with the ability to regulate immune responsiveness to a cardiac allograft are present at the time of transplantation following pretreatment with donor alloantigen in combination with anti-CD4.

Evidence that the continued presence of the organ graft and not peripheral donor microchimerism is essential for maintenance of tolerance to alloantigen in vivo in anti-CD4 treated recipients.

The source of donor alloantigen required to maintain tolerance in vivo was evaluated in anti-Cd4 monoclonal antibody (mAb) treated mice. Treatment with a depleting anti-Cd4 mAb at the time of transplantation (day -1,0) induces tolerance to C57BL/10 (H2b) vascularized cardiac allografts in C3H.He (H2k) mice. The presence of the vascularized allograft was found to be essential for the induction of tolerance in this experimental model; it is the only source of donor alloantigen during the induction phase of unresponsiveness (0-50 days). In the maintenance phase (>50 days) donor alloantigen is potentially available from two sources, the organ graft itself or donor cells that have migrated out of the graft and are resident in the periphery (donor microchimerism). We show that the vascularized cardiac allograft is essential for the maintenance of tolerance to donor alloantigen in vivo. When the primary heart graft remained in situ, tolerance to donor alloantigens, as assessed by the survival of a second heart graft, was maintained indefinitely (>250 days) (MST of second C57 heart grafts >100 days). However, when the primary heart graft was removed 50 days after transplantation, a time point when tolerance to donor alloantigens was demonstrable in vivo, tolerance was lost 200 days later (MST of second C57 heart grafts 31 days). No evidence of donor microchimerism in the recipient was obtained using allele specific polymerase chain reaction (pcr) analysis for donor class I antigen. Persistence of donor alloantigen in the form of the vascularized organ graft is therefore required for both the induction and maintenance of tolerance to alloantigen in vivo in this experimental model.

Cytokines and peripheral tolerance to alloantigen.

The induction of peripheral tolerance to alloantigen is accompanied in many cases by a decrease in the production of cytokines such as IL-2 and IFN gamma, yet a sustained production of cytokines such as IL-10 and IL-4. Whether or not this altered pattern of cytokine production in tolerant animals is causally related to the induction and/or maintenance of the tolerant state has yet to be fully determined, although experiments blocking selectively the action of IL-2 with CD25 antibodies suggest that manipulation of cytokine production may at least be a route to tolerance. Alternative methods for directly influencing the cytokine balance are sought and recent experiments on the CD28/CTLA-4-B7 interaction suggest a possible approach.

Lymphocyte changes associated with prolongation of cardiac allograft survival in adult mice using anti-CD4 monoclonal antibody.

This study investigated the effect of anti-CD4 MoAb treatment on lymphocyte phenotype and function and correlated these changes with the prolongation of cardiac allograft survival in adult mice. Indefinite survival of heterotopic cardiac allografts was obtained in several fully allogeneic strain combinations when two doses of the anti-CD4 MoAb, YTS 191.1, were given at the time of transplantation. A dose response analysis in the C57BL/10 to C3H/He strain combination showed that very low doses of YTS 191.1 (25 micrograms/dose) were able to induce prolonged allograft survival when administered perioperatively. At the time of transplantation the immunosuppression induced by administration of the anti-CD4 MoAb is not antigen-specific, as heart grafts from different donor strains, mismatched for both major and minor histocompatibility antigens, showed prolonged survival in treated recipients. Immunocompetence was restored by 6 weeks after MoAb treatment, as recipients regained the ability to reject a cardiac allograft transplanted at this time point. However, while recovery of immunocompetence could be demonstrated in vivo, leucocytes isolated from the peripheral lymphoid organs of treated mice continued to be hyporesponsive in mixed leucocyte culture (MLC). Phenotypic analysis of the peripheral lymphoid tissues showed that C3H/He recipients treated with 25 micrograms/dose of YTS 191.1 had a marked, but not complete, elimination of the CD4+ subset at the time of transplantation, which was gradually restored to 50% of normal by 6 weeks after treatment. Thus, complete elimination of the CD4+ subset was not required to achieve indefinite allograft survival, and immunocompetence, as assessed in vivo, returned even when the CD4+ subset was present at half the normal level. Low doses of anti-CD4 MoAb (25 micrograms) had no effect on the expression of the CD4 molecule by thymocytes, and yet thymocytes were hyporesponsive to alloantigen in vitro. At higher doses of YTS 191.1, immature CD4+8+ thymocytes were selectively depleted. These results suggest that anti-CD4 MoAb therapy may modulate the intrathymic T cell selection process. These studies provide further insight into the mechanism of action of low dose, depleting anti-CD4 MoAb therapy in allograft rejection, and form a basis from which rational modifications to therapeutic protocols in transplantation models can be made.

The assessment of transplantation tolerance induced by anti-CD4 monoclonal antibody in the murine model.

Transplantation tolerance can be induced in the adult mouse by the selective manipulation of the CD4+ T cell subset. C3H/He recipients with prolonged survival (> 120 days) of C57BL/10 cardiac allografts induced by treatment, at the time of transplantation, with the anti-CD4 mAb, YTS 191.1, were skin grafted simultaneously with donor-specific and third-party (BALB/c) skin. The development of donor-specific tolerance was proved by the specific prolongation of C57BL/10 skin graft survival, while third-party grafts were rejected. Further investigation of recipients with long-term surviving primary heart allografts showed that donor-specific tolerance was associated with organ-specific differences. Secondary cardiac allografts were universally accepted, even at 42 days after the primary heart transplant, while prolonged survival of donor-specific skin grafts was not obtained until more than 120 days after primary cardiac transplantation. Analysis of leukocyte reactivity in the mixed leukocyte culture (MLC) showed no correlation between the proliferative response of recipient T cells in vitro to either donor or third-party alloantigen and the survival of either heart of skin allografts. These results illustrate the significant challenge presented when attempting to define and assess accurately the state of transplantation tolerance.