Dendritic cells (DCs) of the immune system have the dual capacity to promote T cell immunity or tolerance. Two decades ago, this led to the idea that donor- or recipient-derived DCs generated in vitro and rendered maturation-resistant and tolerogenic by pharmacologic or genetic manipulation could be used as a novel approach to restrain the adaptive immune response against allografts in a donor-specific manner .
Initially, experimental therapy with regulatory DC (DCreg) vaccines was based on the administration of donor-derived DCregs . In murine models, the best results were obtained when the donor-derived DCregs were administered intravenously (i.v.) 1 week before transplantation. More recently, this approach has been shown to prolong renal allograft survival in a clinically relevant nonhuman primate model . However, use of donor-derived DCregs is restricted to live donor transplantation because of the time window (1 week) required for generation of DCregs from donor DC precursors, plus the time necessary for the donor-derived DCregs to “condition” the anti-donor T cell response in vivo following their administration to the prospective graft recipient.
These limitations led to the alternative strategy of using recipient-derived DCregs pulsed with donor antigen (Ag) in the form of soluble peptides or particles, the latter as donor leukocyte sonicates, apoptotic leukocyte-derived vesicles or leukocyte-derived exosomes . Although in this alternative approach to DCreg therapy, the DCregs can be generated from the recipient's blood-borne DC precursors in advance of transplantation, and kept frozen until use, it still requires a source of donor Ag that can again present a problem with use of deceased donors. Besides, standardization of the dose/format of donor Ag and the methodology used by different laboratories to pulse the DCregs in vitro is likely to prove challenging. Moreover, in mouse models, there is evidence that both i.v.-injected donor-derived DCregs on the one hand and recipient-derived DCregs pulsed with donor Ag on the other are short-lived and function as Ag-transporting cells by conveying donor Ag to recipient conventional DCs in secondary lymphoid organs . Presentation of the transferred donor Ag to host T cells by the quiescent conventional DCs of the recipient, instead of by the infused DCregs, down-regulates the anti-donor adaptive immune response and prolongs graft survival in mice .
In 2005, Cuturi's group made the unexpected finding  that in vitro-generated autologous (AT) DCregs not pulsed with donor Ag, and administered systemically, just 1 day before transplantation, prolonged heart allograft survival in rats. Combination of AT DCreg therapy with the immunosuppressant LF 15-0195 (an NFκβ inhibitor) led to donor-specific graft acceptance in most recipients. Further studies demonstrated that the regulatory effects of AT DCregs were associated with interferon-γ secretion by a subset of CD4 CD8 double negative regulatory T cells in the spleen , and expression of the cytokine chain Epstein–Barr virus-induced gene 3, an IL-12 family member, by the AT DCregs . It is important to note that, in these experiments, the AT DCregs were not exposed to donor Ag in vitro. Therefore, if the AT DCregs were not pulsed with donor Ag before injection, how did they induce donor-specific T cell unresponsiveness in vivo?
In this issue of the American Journal of Transplantation, Segovia et al  from the Cuturi Laboratory extend their previous findings by unveiling the mechanism by which i.v.-administered AT DCregs, not exposed to donor Ag before injection, prolong graft survival in a donor-specific manner. The authors demonstrate that systemic administration of female AT DCregs 1 day before transplantation, in combination with peri-transplant anti-CD3 antibody therapy, significantly prolongs the survival of minor histocompatibility mismatched male skin grafts in female mice. The administration of AT DCregs plus CD3 antibody was associated with an increase in the percentage and absolute number of donor male peptide-specific CD8+CD11c+ cells in graft-draining lymph nodes. These CD8+CD11c+ T cells exhibited regulatory function, as shown by the fact that, when transferred to otherwise untreated female recipients, they significantly prolonged male skin allograft survival . They were not, however, characterized further in the study.
Segovia et al  demonstrate that the injected AT DCregs can be detected in the skin graft and later in graft-draining lymphoid organs, suggesting that they take up donor Ags in the transplant before homing to the lymph nodes. Interestingly, administration of AT DCregs deficient in TMEM176B, a protein that controls phagosomal pH, failed to promote expansion of donor-specific CD8+ CD11c+ regulatory T cells and to prolong skin graft survival under the same experimental conditions. In vitro, TMEM176B-deficient AT DCregs were severely impaired in their ability to cross-present male Ag or ovalbumin to CD8 TCR transgenic T cells. Taken together, these results suggest that AT DCregs require TMEM176B for optimal endosome/phagosome function and therefore, cross-presentation of internalized donor Ag to donor-specific CD8+CD11c+ regulatory T cells. Indeed, the authors demonstrate that phagosomes of TMEM176B-deficient AT DCregs exhibit excessive alkalinization, which may impair Ag degradation. However, although the authors demonstrate that the absence of TMEM176B deprives AT DCregs of their immunosuppressive activity in vivo, the in vitro experiments only indicate that TMEM176B-deficient AT DCregs are unable to cross-present Ag to CD8 T cells, without providing an explanation of the active mechanism of down-regulation of the T cell response.
In summary, the authors provide evidence that the beneficial effect of AT DCregs in transplantation across a minor–MHC mismatch requires cross-presentation of in vivo internalized donor Ag by the injected DCs. Although the influence of unpulsed AT DCregs under more stringent conditions (i.e. across a full MHC barrier, or in sensitized recipients) remains to be tested, the potential use of non-pulsed AT DCregs in the clinic may be highly relevant. AT DCregs can be generated from human blood-borne DC precursors and may have potential to induce donor-specific immune suppression, even when injected 1 day before transplantation. Significantly, DCreg and other forms of regulatory innate and adaptive immune cell therapy are quickly approaching clinical testing in organ transplantation, such as in the European consortium “One Study.”