Rejuvenated by the seminal findings of Qin et al.  in 1993, understanding the process of induced regulatory tolerance in immunologically mature animals has been a major goal in experimental transplantation. While there is great ongoing interest in the nature and function of donor-reactive regulatory T cells, uncertainty remains concerning exactly what type of regulatory activity is required to mediate tolerance and when and where such control of allograft reactivity occurs. The majority of transplant tolerance studies—as well as in the general study of peripheral tolerance—arguably tend to focus on CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) as the hallmark mediators of immune regulation. However, it is essential to consider that several types of antigen-specific T cells with differing forms of regulatory function have been described . Thus, immune ‘regulation’ is a biological activity and is not necessarily a specific cellular or molecular phenotype. In this issue of the American Journal of Transplantation, Gagliani et al.  present very intriguing results suggesting that Foxp3+ Tregs and IL-10-producing T regulatory type 1 (Tr1) cells each play obligate and cooperative roles in inducing and maintaining allograft tolerance.
The authors use a pancreatic islet allograft model in which tolerance is induced with a combinational therapeutic regimen consisting of anti-CD45RB monoclonal antibodies plus adjunct rapamycin and IL-10 administration. To monitor the development and anatomical location of regulatory T cells, this study makes elegant use of a double transgenic reporter model in which both Foxp3+ and/or IL-10-producing cells can be visualized ex vivo. In this way, the induction and function of such regulatory T cells can be interrogated during either the initiation (early) or maintenance (late) phase of induced allograft tolerance. There are two primary types of results generated from this study: Firstly, early tolerance induction is predominately associated with Foxp3+ Tregs within the allograft and draining lymph nodes with little contribution of IL-10+ Tr1 cells. However, over time there is a pronounced accumulation of Tr1 cells within the host spleen. Striking results show that transfer of these early graft-infiltrating Foxp3+ Tregs following tolerance therapy can recapitulate tolerance in secondary wild-type animals, but only if co-transplanted locally with a donor-type islet graft. Importantly, the long-term maintenance of tolerance requires active participation of splenic cells; late splenectomy of the host (>150 days posttransplant) results in rapid rejection of the established allograft. Moreover, transfer of Tr1-containing spleen cells into secondary animals was sufficient to produce tolerance. Taken together, these studies suggest that there is both a temporal and anatomical compartmentalization of regulatory activity during the generation and maintenance of allograft tolerance. That is, while initial tolerance induction involves Foxp3+ Treg activity within the allograft micro-environment, a secondary peripheral response evolves within the spleen predominated by IL-10-producing Tr1 cells. Under appropriate circumstances, either of these regulatory compartments is capable of re-creating the tolerant state in secondary animals.
This study has very important implications regarding both the study and potential therapeutic application of regulatory T cells to control allograft immunity and possibly autoimmunity. For example, there is considerable evidence that Foxp3+ Tregs play a key role in allograft tolerance and may be essential throughout the course of allograft tolerance . However, while Foxp3+ Tregs may be necessary for allograft tolerance, they may not always be sufficient to account for the entire tolerogenic response. The additional requirement for splenic cells (presumably Tr1 cells) to actively maintain tolerance over time as shown in the current study may explain why studies sometimes show late allograft failure after initial treatment with tolerance-promoting therapies. While Foxp3+ Tregs may play a key role during early tolerance induction, the subsequent generation of Tr1 cells may be essential for the ongoing persistence of the tolerant state. This study shows empirical evidence that graft-destructive T cells persist in functionally tolerant animals since late splenectomy unveils their activity, a result consistent with other studies showing that tolerance can sometimes be broken even during the later maintenance phase [4, 5]. So, if allograft tolerance requires the summation of differing regulatory activities acting at different sites, then the therapeutic administration of only one type of regulatory T cell phenotype may not be sufficient to achieve the robust and durable control of immunity desired.
One caveat of this study is whether the specific tolerance-promoting protocol itself contributed to the regulatory activities observed. That is, this study utilized a somewhat uncommon treatment regimen consisting of ant-CD45RB monoclonal antibody therapy plus rapamycin and IL-10 administration. The IL-10 component of this protocol could potentially be responsible for generating exaggerated Tr1 activity. It is certainly conceivable that the roles for Foxp3+ Tregs versus IL-10 producing Tr1 cells, or even other potential regulatory activities, may vary according to the specific tolerance-promoting intervention and/or the type of tissue or organ transplanted. Nevertheless, this study provides a significant proof of principle that a ‘division of labor’ can occur in which differing regulatory T cell phenotypes cooperate in an orchestrated process resulting in the initiation and maintenance of induced allograft tolerance.