The clinical success of solid organ transplantation is still limited by the insufficiency of immunosuppressive regimens to control chronic rejection and late graft loss. Moreover, serious side ...effects caused by chronic immunosuppressive treatment increase morbidity and mortality in transplant patients. Regulatory T cells (Tregs) have proven to be efficient in the induction of allograft tolerance and prolongation of graft survival in numerous preclinical models, and treatment has now moved to the clinics. The results of the first Treg-based clinical trials seem promising, proving the feasibility and safety of Treg therapy in clinical organ transplantation. However, many questions regarding Treg phenotype, optimum dosage, antigen-specificity, adjunct immunosuppressants and efficacy remain open. This review summarizes the results of the first Treg-based clinical trials for tolerance induction in solid organ transplantation and recapitulates what we have learnt so far and which questions need to be resolved before Treg therapy can become part of daily clinical practice. In addition, we discuss new strategies being developed for induction of donor-specific tolerance in solid organ transplantation with the clinical aims of prolonged graft survival and minimization of immunosuppression.
The efficacy of costimulation blockade with CTLA4-Ig (belatacept) in transplantation is limited due to T cell-mediated rejection, which also persists after induction with anti-thymocyte globulin ...(ATG). Here, we investigate why ATG fails to prevent costimulation blockade-resistant rejection and how this barrier can be overcome. ATG did not prevent graft rejection in a murine heart transplant model of CTLA4-Ig therapy and induced a pro-inflammatory cytokine environment. While ATG improved the balance between regulatory T cells (Treg) and effector T cells in the spleen, it had no such effect within cardiac allografts. Neutralizing IL-6 alleviated graft inflammation, increased intragraft Treg frequencies, and enhanced intragraft IL-10 and Th2-cytokine expression. IL-6 blockade together with ATG allowed CTLA4-Ig therapy to achieve long-term, rejection-free heart allograft survival. This beneficial effect was abolished upon Treg depletion. Combining ATG with IL-6 blockade prevents costimulation blockade-resistant rejection, thereby eliminating a major impediment to clinical use of costimulation blockers in transplantation.The use of CTLA4-Ig fusion proteins in transplantation remains limited due to co-stimulation blockade-resistant rejection (CBRR). In this study, the authors demonstrate that IL-6 blockade reduces CBRR in murine cardiac transplants in the context of T cell-depleting induction regimens and CTLA4-Ig treatment.
The importance and exact role of graft-resident leucocytes (also referred to as passenger leucocytes) in transplantation is controversial as these cells have been reported to either initiate or ...retard graft rejection. T cell activation to allografts is mediated
recognition of intact or processed donor MHC molecules on antigen-presenting cells (APC) as well as through interaction with donor-derived extracellular vesicles. Reduction of graft-resident leucocytes before transplantation is a well-known approach for prolonging organ survival without interfering with the recipient's immune system. As previously shown by our group, injecting mice with IL-2/anti-IL-2 complexes (IL-2cplx) to augment expansion of CD4 T regulatory cells (Tregs) induces tolerance towards islet allografts, and also to skin allografts when IL-2cplx treatment is supplemented with rapamycin and a short-term treatment of anti-IL-6. In this study, we investigated the mechanisms by which graft-resident leucocytes impact graft survival by studying the combined effects of IL-2cplx-mediated Treg expansion and passenger leucocyte depletion. For the latter, effective depletion of APC and T cells within the graft was induced by prior total body irradiation (TBI) of the graft donor. Surprisingly, substantial depletion of donor-derived leucocytes by TBI did not prolong graft survival in naïve mice, although it did result in augmented recipient leucocyte graft infiltration, presumably through irradiation-induced nonspecific inflammation. Notably, treatment with the IL-2cplx protocol prevented early inflammation of irradiated grafts, which correlated with an influx of Tregs into the grafts. This finding suggested there might be a synergistic effect of Treg expansion and graft-resident leucocyte depletion. In support of this idea, significant prolongation of skin graft survival was achieved if we combined graft-resident leucocyte depletion with the IL-2cplx protocol; this finding correlated along with a progressive shift in the composition of T cells subsets in the grafts towards a more tolerogenic environment. Donor-specific humoral responses remained unchanged, indicating minor importance of graft-resident leucocytes in anti-donor antibody development. These results demonstrate the importance of donor-derived leucocytes as well as Tregs in allograft survival, which might give rise to new clinical approaches.
Since the discovery of regulatory T cells (Tregs) as crucial regulators of immune tolerance against self-antigens, these cells have become a promising tool for the induction of donor-specific ...tolerance in transplantation medicine. The therapeutic potential of increasing in vivoTreg numbers for a favorable Treg to Teff cell ratio has already been demonstrated in several sophisticated pre-clinical models and clinical pilot trials. In addition to improving cell quantity, enhancing Treg function utilizing engineering techniques led to encouraging results in models of autoimmunity and transplantation. Here we aim to discuss the most promising approaches for Treg-enhancing therapies, starting with adoptive transfer approaches and ex vivoexpansion cultures (polyclonal vs. antigen specific), followed by selective in vivostimulation methods. Furthermore, we address next generation concepts for Treg function enhancement (CARs, TRUCKs, BARs) as well as the advantages and caveats inherit to each approach. Finally, this review will discuss the clinical experience with Treg therapy in ongoing and already published clinical trials; however, data on long-term results and efficacy are still very limited and many questions that might complicate clinical translation remain open. Here, we discuss the hurdles for clinical translation and elaborate on current Treg-based therapeutic options as well as their potencies for improving long-term graft survival in transplantation.
Prophylactic strategies to prevent the development of allergies by establishing tolerance remain an unmet medical need. We previously reported that the transfer of autologous hematopoietic stem cells ...(HSC) expressing the major timothy grass pollen allergen, Phl p 5, on their cell surface induced allergen-specific tolerance in mice. In this study, we investigated the ability of allergen-expressing immune cells (dendritic cells, CD4
T cells, CD8
T cells, and CD19
B cells) to induce allergen-specific tolerance in naive mice and identified CD19
B cells as promising candidates for allergen-specific cell therapy.
For this purpose, CD19
B cells were isolated from Phl p 5-transgenic BALB/c mice and transferred to naive BALB/c mice, pre-treated with a short course of rapamycin and an anti-CD40L antibody. Subsequently, the mice were subcutaneously sensitized three times at 4-week intervals to Phl p 5 and Bet v 1 as an unrelated control allergen. Allergen-expressing cells were followed in the blood to monitor molecular chimerism, and sera were analyzed for Phl p 5- and Bet v 1-specific IgE and IgG
levels by RBL assay and ELISA, respectively.
allergen-induced lung inflammation was measured by whole-body plethysmography, and mast cell degranulation was determined by skin testing.
The transfer of purified Phl p 5-expressing CD19
B cells to naive BALB/c mice induced B cell chimerism for up to three months and prevented the development of Phl p 5-specific IgE and IgG
antibody responses for a follow-up period of 26 weeks. Since Bet v 1 but not Phl p 5-specific antibodies were detected, the induction of tolerance was specific for Phl p 5. Whole-body plethysmography revealed preserved lung function in CD19
B cell-treated mice in contrast to sensitized mice, and there was no Phl p 5-induced mast cell degranulation in treated mice.
Thus, we demonstrated that the transfer of Phl p 5-expressing CD19
B cells induces allergen-specific tolerance in a mouse model of grass pollen allergy. This approach could be further translated into a prophylactic regimen for the prevention of IgE-mediated allergy in humans.
Injection of Interleukin-2 (IL-2) complexed with a particular anti–IL-2 monoclonal antibody (mab) JES6-1 has been shown to selectively expand CD4⁺Foxp3⁺ T regulatory T cells (Tregs) in vivo. Although ...the potency of this approach with regard to transplantation has already been proven in an islet transplantation model, skin graft survival could not be prolonged. Since the latter is relevant to human allograft survival, we sought to improve the efficiency of IL-2 complex (cplx) treatment for skin allograft survival in a stringent murine skin graft model. Here, we show that combining low doses of IL-2 cplxs with rapamycin and blockade of the inflammatory cytokine IL-6 leads to long-term (>75 d) survival of major histocompatibility complex-different skin allografts without the need for immunosuppression. Allograft survival was critically dependent on CD25⁺FoxP3⁺ Tregs and was not accompanied by impaired responsiveness toward donor alloantigens in vitro after IL-2 cplx treatment was stopped. Furthermore, second donor-type skin grafts were rejected and provoked rejection of the primary graft, suggesting that operational tolerance is not systemic but restricted to the graft. These findings plus the lack of donorspecific antibody formation imply that prolonged graft survival was largely a reflection of immunological ignorance. The results may represent a potentially clinically translatable strategy for the development of protocols for tolerance induction.
Numerous alterations in CD8+ T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell‐intrinsic dysfunctions and microenvironmental changes is ...challenging. TCR transgenic OT‐I mice are utilized to investigate CD8+ T‐cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT‐I model minimizes the current limitations and provides a valuable tool to assess antigen‐specific T‐cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8+ T cells from OT‐I+/+ and OT‐I+/− mice to prove the applicability of the heterozygous system. Our data reveal that OVA‐activated CD8+ T cells from adult OT‐I+/− mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age‐related alterations in CD8+ T cells, including naive T‐cell deterioration and decreased proliferative capacity, also occur in elderly OT‐I+/− mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT‐I+/− model to investigate cell‐intrinsic alterations affecting the cytotoxic behavior of aged CD8+ T cells after antigen‐specific in vitro activation. Time‐resolved analysis of antigen‐directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8+ T cells increases with age. Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8+ T cells shifts the mode of target cell death from granzyme‐mediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation.
A heterozygous OT‐I aging model reveals that the intrinsic cytotoxic capacity of antigen‐specific CD8+ T cells increases with age. High perforin expression in CD8+ T cells from elderly OT‐I mice results in target cells' membrane disruption, replacing granzyme‐mediated apoptosis by rapid necrosis induction. Our study further demonstrates that heterozygous OT‐I mice offer a widely applicable tool to assess age‐related alterations in antigen‐specific CD8+ Tcell response.
Ca
is a crucial second messenger for proper T cell function. Considering the relevance of Ca
signals for T cell functionality it is surprising that no mechanistic insights into T cell Ca
signals from ...elderly individuals are reported. The main Ca
entry mechanism in T cells are STIM-activated Orai channels. Their role during lymphocyte aging is completely unknown. Here, we report not only reduced Ca
signals in untouched and stimulated, but also in central and effector memory CD8
T cells from elderly (18-24 months) compared to adult (3-6 months) mice. Two mechanisms contribute to the overall reduction in Ca
signals of CD8
T cells of elderly mice: 1) Reduced Ca
currents through Orai channels due to decreased expressions of STIMs and Orais. 2) A faster extrusion of Ca
owing to an increased expression of PMCA4. The reduced Ca
signals correlated with a resistance of the cytotoxic efficiency of CD8
T cells to varying free Ca
with age. In summary, reduced STIM/Orai expression and increased Ca
clearing rates following enhanced PMCA4 expression contribute to reduced Ca
signals in CD8
T cells of elderly mice. These changes are apparently relevant to immune function as they reduce the Ca
dependency of CTL cytotoxicity.
Eliminating cytoreductive conditioning from chimerism‐based tolerance protocols would facilitate clinical translation. Here we investigated the impact of major histocompatibility complex (MHC) and ...minor histocompatibility antigen (MiHA) barriers on mechanisms of tolerance and rejection in this setting. Transient depletion of natural killer (NK) cells at the time of bone marrow (BM) transplantation (BMT) (20 × 106 BALB/c BM cells → C57BL/6 recipients under costimulation blockade CB and rapamycin) prevented BM rejection. Despite persistent levels of mixed chimerism, BMT recipients gradually rejected skin grafts from the same donor strain. Extending NK cell depletion did not improve skin graft survival. However, F1 (C57BL/6×BALB/c) donors, which do not elicit NK cell‐mediated rejection, induced durable chimerism and tolerance. In contrast, if F1 donors with BALB/c background only were used (BALB/c×BALB.B), no tolerance was observed. In the absence of MiHA disparities (B10.D2 donors, MHC‐mismatch only), temporal NK cell depletion established stable chimerism and tolerance. Conversely, MHC identical BM (BALB.B donors, MiHA mismatch only) readily engrafted without NK cell depletion but no skin graft tolerance ensued. Therefore, we conclude that under CB and rapamycin, MHC disparities provoke NK cell‐mediated BM rejection in nonirradiated recipients whereas MiHA disparities do not prevent BM engraftment but impede skin graft tolerance in established mixed chimeras.
This article shows that in nonirradiated mice, major histocompatibility antigen barriers are mainly responsible for NK cell–mediated bone marrow rejection under costimulation blockade and rapamycin while minor histocompatibility antigen barriers determine donor skin graft survival. See Sánchez‐Fueyo and Dazzi's editorial on page 919.
Summary
Cell therapy has emerged as an attractive therapeutic option in organ transplantation. During the last decade, the therapeutic potency of Treg immunotherapy has been shown in various ...preclinical animal models and safety was demonstrated in first clinical trials. However, there are still critical open questions regarding specificity, survival, and migration to the target tissue so the best Treg population for infusion into patients is still under debate. Recent advances in CAR technology hold the promise for Treg‐functional superiority. Another exciting strategy is the generation of B‐cell antibody receptor (BAR) Treg/cytotoxic T cells to specifically regulate or deplete alloreactive memory B cells. Finally, B cells are also capable of immune regulation, making them promising candidates for immunomodulatory therapeutic strategies. This article summarizes available literature on cell‐based innovative therapeutic approaches aiming at modulating alloimmune response for transplantation. Crucial areas of investigation that need a joined effort of the transplant community for moving the field toward successful achievement of tolerance are highlighted.
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