Recent convincing data have shown that naturally occurring CD8+CD122+ T cells are also regulatory T cells. Paradoxically, CD8+CD122+ T cells have been well described as memory T cells. Given their critical role in tolerance versus long-term immunity, it is important to reconcile this profound dichotomy. In this study, we reported that CD8+CD122+ T cells contain both programmed death-1 (PD-1)− and PD-1+ populations. It was CD8+CD122+PD-1+ T cells, but not their PD-1− counterparts, that suppressed T cell responses in vitro and in vivo. This suppression was largely dependent on their production of IL-10. Moreover, the costimulatory signaling of both CD28 and PD-1 is required for their optimal IL-10 production. In contrast, Ag-specific CD8+CD122+PD-1− T cells were bona fide memory T cells. Thus, CD8+CD122+ T cells can be either regulatory T or memory T cells, depending on their PD-1 expression and Ag specificity. This study reconciles previously contradictory findings and has important implications for tolerance induction.

BackgroundInsulin-producing cell clusters (IPCCs) have recently been generated in vitro from adipose tissue-derived stem cells (ASCs) to circumvent islet shortage. However, it is unknown how long they can survive upon transplantation, whether they are eventually rejected by recipients, and how their long-term survival can be induced to permanently cure type 1 diabetes. IPCC graft survival is critical for their clinical application and this issue must be systematically addressed prior to their in-depth clinical trials.Methodology/Principal FindingsHere we found that IPCC grafts that differentiated from murine ASCs in vitro, unlike their freshly isolated islet counterparts, did not survive long-term in syngeneic mice, suggesting that ASC-derived IPCCs have intrinsic survival disadvantage over freshly isolated islets. Indeed, β cells retrieved from IPCC syngrafts underwent faster apoptosis than their islet counterparts. However, blocking both Fas and TNF receptor death pathways inhibited their apoptosis and restored their long-term survival in syngeneic recipients. Furthermore, blocking CD40-CD154 costimulation and Fas/TNF signaling induced long-term IPCC allograft survival in overwhelming majority of recipients. Importantly, Fas-deficient IPCC allografts exhibited certain immune privilege and enjoyed long-term survival in diabetic NOD mice in the presence of CD28/CD40 joint blockade while their islet counterparts failed to do so.Conclusions/SignificanceLong-term survival of ASC-derived IPCC syngeneic grafts requires blocking Fas and TNF death pathways, whereas blocking both death pathways and CD28/CD40 costimulation is needed for long-term IPCC allograft survival in diabetic NOD mice. Our studies have important clinical implications for treating type 1 diabetes via ASC-derived IPCC transplantation.

BackgroundMajor histocompatibility complex (MHC) antigens are important for alloimmune responses as well as immune tolerance. Previous studies have shown that presentation of donor MHC antigens by donor-specific transfusion prior to or upon transplantation promotes transplant tolerance induced by other agents. However, it is unclear whether presentation of donor MHC antigens by DNA vaccination induces long-term allograft survival.Methodology/Principal FindingsWe investigated whether presentation of MHC class-II and/or class-I donor antigens by DNA vaccination suppresses alloimmune responses and promotes long-term allograft acceptance. We initially found that presentation of both MHC donor antigens by DNA vaccination itself prior to transplantation fails to significantly prolong islet allograft survival in otherwise untreated mice. However, islet allograft survival was significantly prolonged when MHC class-II DNA vaccination was accompanied with IL-2 administration (MHCII + IL-2) while MHC class-I DNA vaccination was followed by IL-2 and subsequent neutralizing anti-IL-2 treatments (MHCI + IL-2/anti-IL-2). Especially, this protocol promoted long-term allograft survival in the majority of recipients (57%) when combined with low doses of rapamycin post-transplantation. Importantly, MHCII + IL-2 induced FoxP3+ Treg cells in both spleens and grafts and suppressed graft-infiltrating CD4+ cell proliferation, whereas MHCI + IL-2/anti-IL-2 mainly inhibited graft-infiltrating CD8+ cell proliferation and donor-specific CTL activity. The combined protocol plus rapamycin treatment further reduced both CD4+ and CD8+ T cell proliferation as well as donor-specific CTL activity but spared FoxP3+ Treg cells. Depleting CD25+ Treg cells or adoptive transfer of pre-sensitized CD8+ T cells abolished this long-term allograft survival.Conclusions/SignificanceManipulating IL-2 availability during presentation of MHC class-II and class-I donor antigens by DNA vaccination pre-transplantation induces Treg cells, suppresses alloimmune responses and promotes long-term allograft survival.

Immune responses are subdued in immune privileged sites, which provide a unique opportunity to prolong cellular allograft survival. We and others have previously shown that inducing tolerance of islet allografts transplanted in murine testes is easier than in conventional sites. However, it is not practical to transplant islets in human testes. Here we found that immunization of B6 recipients with irradiated Balb/C splenocytes through testicular injection, 7 and 14 days before islet transplantation under the kidney capsule, promotes long-term allograft survival induced by low doses of anti-CD40L (MR1) or CTLA4-Ig (0.1mg/day, days 0,2,4,6), with 75% islet allografts achieving long-term survival (>120 days). Same Ab treatment without the immunization failed to do so. Immunization without MR1 or CTLA4-Ig did not prolong allograft survival. We also found that islet transplantation under renal capsule of previously immunized recipients generates much less CD44+CD8+ memory T cells but induces more CD4+CD25+ Tregs than under renal capsule of unimmunized recipients. These Tregs exhibited donor-specific suppression of alloimmune responses in vitro. These findings reveal new mechanisms of immune privilege and may provide novel approaches to inducing long-term allograft survival or transplant tolerance in both immunologically privileged and unprivileged sites.