The game-changing contribution submitted by Singh et al was the amalgamation of ethylcarbodiimide-treated splenocytes (providing ADL) with a novel non myeloablative induction therapy.
Donor-specific tolerance is a long-held, elusive goal for transplantation. While robust dominant tolerance across major histocompatibility complex (MHC) barriers has been achieved by numerous manipulations in rodents, translation into nonhuman primate (NHP) models or clinical practice has been challenging. Apart from the induction of hematopoietic chimerism,1,2 there are very few examples of allograft tolerance in clinical practice. Whether persistent chimerism (versus that of a transient nature) is an absolute requirement for tolerance has been debated, however without resolution. Furthermore, the induction of chimerism requires extensive preconditioning and is associated with significant toxicity, particularly graft versus host disease, factors which have limited its clinical feasibility.
The article by Singh et al3 in Nature Communications is thus of considerable interest as it describes a method for inducing donor-specific tolerance in NHP. The authors use a preconditioning strategy of apoptotic donor lymphocytes in association with a short-term immune-modulating strategy that does not rely on T-cell depletion. Strict selection criteria were required: while donor and recipient could be mismatched at MHC class I, they were required to share 1 MHC class II DRB allele. Full mismatches and the use of sensitized recipients limited the efficacy of apoptotic donor lymphocytes. The induction protocol took 8 days to complete before transplantation, involving administration of anti-CD40, soluble tumor necrosis factor receptor (etanercept), anti-interleukin (IL)-6 receptor (tocilizumab) monoclonal antibody (mAb), and rapamycin, with cessation of all immunosuppression at day 7 posttransplantation.
Donor-specific transfusion as a mechanism for inducing tolerance was first described >30 years ago.4,5 A more sophisticated, mechanistic understanding of professional antigen-presenting cell (APC) subsets, in addition to APC-T-cell interactions, has helped understand these initial observations. Transfusion of donor leukocytes, specifically APC,6 is effective at inducing tolerance in rodents and improving allograft survival in NHP.7 The mechanism of action is not fully understood. Internalization of donor cells by recipient APC providing a negative costimulatory signal may lead to maturation arrest and abortive T-cell activation.8 Moreover, the use of ethylcarbodiimide-treated splenocytes (a combination of T and B lymphocytes, natural killer T cells, dentritic cells, monocytes, and neutrophils9) has been suggested to delete allospecific T cells that are typically activated via indirect antigen presentation.10 In addition, nonphagocytosed apoptotic donor lymphocytes (ADL) interacting directly with allospecific T cells have been linked to incomplete activation and subsequent anergy. As a result of this approach, both direct and indirect allospecific T-cell activation pathways have been effectively inhibited with the induction of CD4+FoxP3+ regulatory T cells.
The game-changing contribution submitted by Singh et al3 was the amalgamation of ethylcarbodiimide-treated splenocytes (providing ADL) with a novel nonmyeloablative induction therapy. This approach appeared effective at inducing allospecific tolerance in outbred NHP. The authors combined costimulation blockade with specific therapies known to promote the expansion of allospecific regulatory T cells. Moreover, they included additional strategies that inhibited effector T-cell activation while preventing T-cell–mediated B cell help blocking the development of donor specific antibody.
In prior NHP studies, costimulation blockade and subsequent long-term islet allograft survival have been achieved with anti-CD154 mAb.11 However, this agent has not been approved for clinical use because of safety concerns. Singh et al3 replaced anti-CD154 mAb by an anti-CD40 mAb (CD40 being the receptor for anti-CD154). Rapamycin suppresses the activation and proliferation of immune cells, including T cells, by blocking a component of mammalian target of rapamycin function.12 Rapamycin inhibits IL-2–induced phosphorylation and subsequent T-cell proliferation while impairing CD8+ T-cell immune responses. In experimental studies, rapamycin has been protolerogenic, expanding naturally occurring regulatory T cells (Treg),13 depleting CD4+ T effector cells13 while promoting apoptosis. The investigators also included a soluble inhibitor of tumor necrosis factor and anti-IL-6R mAb into their approach facilitating the inhibition of IL-6–mediated Th1 and Th17 responses. Concurrent blockade of tumor necrosis factor signaling provides additional effector T-cell functional suppression, without directly targeting IL-2 or interferon (IFN)-γ, both required for Treg expansion. In addition, anti-IL6R mAb has been shown in clinical studies to be effective at inhibiting chronic antibody-mediated rejection14 and may thus prove important in suppression of T-cell–mediated de novo B-cell responses.
Interestingly, the tolerogenic effects of Singh et al’s3 approach seem to focus on the development of a broad immune regulatory framework rather than ongoing deletion of alloreactive T cells, as one might have predicted from the known mechanisms of action of ADL. While T follicular helper, CD4+, and CD8+ T effector memory were decreased in tolerant animals, the authors observed increased numbers of PD1+ T cells, Treg, Tr1 regulatory cells, Breg, and B10 cells in the peripheral blood, lymph node, and liver (the site of the islet graft). These cells demonstrated donor-specific suppression in vitro, and, in the case of Tr1 cells, this seemed to be mediated by IL-10.
An important part of the protocol was the sharing of at least 1 MHC class II molecule, as animals that were completely mismatched did not achieve tolerance. While the mechanisms involved in class II–matched tolerance success were not explored in this article, the authors hypothesized that presentation of MHC class II self-peptides may be important for the expansion of Treg, a potential source of IL-10 for the production and maintenance of Tr1 cells.
Overall, the authors present an impressive advance to the understanding of immune regulation with clinical relevance. As suggested by the authors, further studies are required to better delineate the precise mechanism of action.
Several challenges remain before this protocol is ready for prime-time clinical practice. The requirement for shared MHC class II DR molecule between donor and recipient and the 8-day preconditioning makes the protocol more suitable for live (as distinct from deceased) donor kidney transplant rather than islet transplantation, where other logistic issues limit donor organ availability. The dose of 0.25 × 109 ADL/kg is large and comes with translational challenges to apply the approach outside the deceased donor setting. Other issues to consider include the clinical availability of anti-CD40 mAb, the lack of a reliable biomarker for tolerance or allograft acceptance. Moreover, it may be challenging to deal with the recurrence of autoimmune disease, particularly type I diabetes mellitus.
Despite these obstacles, this study has rekindled our interest in donor lymphocyte transfusion promoting clinical donor-specific tolerance or unresponsiveness. Finally, given the potential importance of liver mononuclear cells for the promotion of tolerance, future studies using a similar approach in liver transplantation may be of interest.
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