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In this issue, Sobrino et al. present a case of hematopoietic chimerism and subsequent tolerance after isolated kidney transplantation in a pediatric patient with syndromic combined immune deficiency. This report not only highlights the chimerism approach for tolerance induction in transplantation, but it is also the first evidence of hematopoietic stem cells in human kidneys. This commentary discusses the potency and risks of the chimerism approach for tolerance induction after solid organ transplantation, especially in (pediatric) patients with immune deficiencies.

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.

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.

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.

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.

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 donor-specific 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.

CTLA4Ig has been shown to improve kidney allograft function, but an increased frequency of early rejection episodes poses a major obstacle for more widespread clinical use. The deleterious effect of CTLA4Ig on Treg numbers provides a possible explanation for graft injury. Therefore, we aimed at improving CTLA4Ig's efficacy by therapeutically increasing the number of Tregs. Murine cardiac allograft transplantation (BALB/c to B6) was performed under CTLA4Ig therapy modeled after the clinically approved dosing regimen and Tregs were transferred early or late after transplant. Neither early nor late Treg transfer prolonged allograft survival. Transferred Tregs were traceable in various lymphoid compartments but only modestly increased overall Treg numbers. Next, we augmented Treg numbers in vivo by means of IL2 complexes. A short course of IL2/anti-IL2-complexes administered before transplantation reversed the CTLA4Ig-mediated decline in Tregs. Of note, the addition of IL2/anti-IL2-complexes to CTLA4Ig therapy substantially prolonged heart allograft survival and significantly improved graft histology on day 100. The depletion of Tregs abrogated this effect and resulted in a significantly diminished allograft survival. The increase in Treg numbers upon IL2 treatment was associated with a decreased expression of B7 on dendritic cells. These results demonstrate that therapy with IL2 complexes improves the efficacy of CTLA4Ig by counterbalancing its unfavorable effect on Tregs.

Resistance to parental bone marrow (BM) grafts in F1 hybrid recipients is due to natural killer (NK) cell–mediated rejection triggered through “missing self” recognition. “Hybrid resistance” has usually been investigated in lethally irradiated F1 recipients in conjunction with pharmacological activation of NK cells. Here, we investigated BM‐directed NK‐cell alloreactivity in settings of reduced conditioning. Nonlethally irradiated (1‐3 Gy) or nonirradiated F1 (C57BL6 × BALB/c) recipient mice received titrated doses (5‐20 x 106) of unseparated parental BALB/c BM without pharmacological NK cell activation. BM successfully engrafted in all mice and multilineage donor chimerism persisted long‐term (24 weeks), even in the absence of irradiation. Chimerism was associated with the rearrangement of the NK‐cell receptor repertoire suggestive of reduced reactivity to BALB/c. Chimerism levels were lower after transplantation with parental BALB/c than with syngeneic F1 BM, indicating partial NK‐mediated rejection of parental BM. Activation of NK cells with polyinosinic–polycytidylic acid sodium salt poly(I:C), reduced parental chimerism in nonirradiated BM recipients but did not prevent hematopoietic stem cell engraftment. In contrast, equal numbers of parental lymph node cells were completely rejected. Hence, hybrid resistance leads to incomplete rejection of parental BM under reduced conditioning settings., Modifying conditioning in the hybrid resistance model reveals that lasting chimerism ensues after nonmyeloablative irradiation, even when no recipient irradiation is given, indicating that natural killer cell–mediated bone marrow rejection is incomplete in reduced conditioning settings.

Background The mixed chimerism approach for intentional induction of donor-specific tolerance was shown to be successful in various models from mice to humans. For transplant patients, the approach would obviate the need for long-term immunosuppression and associated side effects; moreover, it would preclude the risk of late graft loss due to chronic rejection. Widespread clinical application is hindered by toxicities related to recipient pre-conditioning. Herein we aimed to investigate a clinically relevant protocol for tolerance induction to cardiac allografts, sparing CD40 blockade or T-cell depletion. Methods B6 mice were conditioned with non-myeloablative total body irradiation, fully mismatched BALB/c bone marrow cells, and short-term therapy, based on either anti– lymphocyte function–associated antigen-1 (anti–LFA-1) or anti-CD40L. Multilineage chimerism was followed by flow-cytometric analysis, tolerance was assessed with skin and heart allografts from fully or major histocompatibility complex–mismatched donors. Mechanisms of tolerance were investigated by analysis of donor-specific antibodies (DSAs), mixed lymphocyte reaction (MLR) assays, and deletion of donor-reactive T cells. Results We found that the combination of cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and rapamycin with LFA-1 blockade enhanced bone marrow engraftment and led to more efficient T-cell engraftment and subsequent tolerization. Although fully mismatched skin grafts were chronically rejected, primarily vascularized heart allografts survived indefinitely and without signs of chronic rejection, independent of minor antigen mismatches. Conclusions We have demonstarted a robust protocol for the induction of tolerance for cardiac allografts in the absence of CD40 blockade. Our findings demonstrate the potential of a clinically relevant minimal conditioning protocol designed to induce lifelong immunologic tolerance toward cardiac allografts.

Blockade of the CD28:CD80/86 costimulatory pathway has been shown to be potent in blocking T cell activation in vitro and in vivo. The costimulation blocker CTLA4Ig has been approved for the treatment of autoimmune diseases and transplant rejection. The therapeutic application of regulatory T cells (Tregs) has recently gained much attention for its potential of improving allograft survival. However, neither costimulation blockade with CTLA4Ig nor Treg therapy induces robust tolerance on its own. Combining CTLA4Ig with Treg therapy would be an attractive approach for minimizing immunosuppression or for possibly achieving tolerance. However, since the CD28 pathway is more complex than initially thought, the question arose whether blocking CD80/86 would inadvertently impact immunological tolerance by interfering with Treg generation and function. We therefore wanted to investigate the compatibility of CTLA4Ig with regulatory T cells by evaluating direct effects of CTLA4Ig on murine Treg generation and function in vitro. For generation of polyclonal-induced Tregs, we utilized an APC-free in vitro system and added titrated doses of CTLA4Ig at different time points. Phenotypical characterization by flow cytometry and functional characterization in suppressor assays did not reveal negative effects by CTLA4Ig. The costimulation blocker CTLA4Ig does not impair but rather improves murine iTreg generation and suppressor function in vitro.

Therapeutic administration of regulatory T cells (Tregs) leads to engraftment of conventional doses of allogeneic bone marrow (BM) in non-irradiated recipient mice conditioned with costimulation blockade and mTOR inhibition. The mode of action responsible for this Treg effect is poorly understood but may encompass the control of costimulation blockade-resistant natural killer (NK) cells. We show that transient NK cell depletion at the time of BM transplantation (BMT) led to BM engraftment and persistent chimerism without Treg transfer, but failed to induce skin graft tolerance. In contrast, the permanent absence of anti-donor NK reactivity in mice grafted with F1 BM was associated with both chimerism and tolerance comparable to Treg therapy, implying that NK cell tolerization is a critical mechanism of Treg therapy. Indeed, NK cells of Treg-treated BM recipients reshaped their receptor repertoire in the presence of donor MHC in a manner suggesting attenuated donor-reactivity. These results indicate that adoptively transferred Tregs prevent BM rejection, at least in part, by suppressing NK cells and promote tolerance by regulating the appearance of NK cells expressing activating receptors to donor class I MHC. This article is protected by copyright. All rights reserved.

Purpose of Review The mixed chimerism approach is an exceptionally potent strategy for the induction of donor-specific tolerance in organ transplantation and so far the only one that was demonstrated to work in the clinical setting. Regulatory T cells (Tregs) have been shown to improve chimerism induction in experimental animal models. This review summarizes the development of innovative BMT protocols using therapeutic Treg transfer for tolerance induction. Recent Findings Treg cell therapy promotes BM engraftment in reduced conditioning protocols in both, mice and non-human primates. In mice, transfer of polyclonal recipient Tregs was sufficient to substitute cytotoxic recipient conditioning. Treg therapy prevented chronic rejection of skin and heart allografts related to tissue-specific antigen disparities, in part by promoting intragraft Treg accumulation. Summary Adoptive Treg transfer is remarkably effective in facilitating BM engraftment in reduced-intensity protocols in mice and non-human primates. Furthermore, it promotes regulatory mechanisms that prevent chronic rejection.

B7.1/2-targeted costimulation blockade (CTLA4 immunoglobulin [CTLA4-Ig]) is available for immunosuppression after kidney transplantation, but its potentially detrimental impact on regulatory T cells (Tregs) is of concern. We investigated the effects of CTLA4-Ig monotherapy in a fully mismatched heart transplant model (BALB/c onto C57BL/6). CTLA4-Ig was injected chronically (on days 0, 4, 14, and 28 and every 4 weeks thereafter) in dosing regimens paralleling clinical use, shown per mouse: low dose (LD), 0.25 mg (≈10 mg/kg body weight); high dose (HD), 1.25 mg (≈50 mg/kg body weight); and very high dose (VHD), 6.25 mg (≈250 mg/kg body weight). Chronic CTLA4-Ig therapy showed dose-dependent efficacy, with the LD regimen prolonging graft survival and with the HD and VHD regimens leading to >95% long-term graft survival and preserved histology. CTLA4-Ig's effect was immunosuppressive rather than tolerogenic because treatment cessation after ≈3 mo led to rejection. FoxP3-positive Tregs were reduced in naive mice to a similar degree, independent of the CTLA4-Ig dose, but recovered to normal values in heart recipients under chronic CTLA4-Ig therapy. Treg depletion (anti-CD25) resulted in an impaired outcome under LD therapy but had no detectable effect under HD therapy. Consequently, the immunosuppressive effect of partially effective LD CTLA4-Ig therapy is impaired when Tregs are removed, whereas CTLA4-Ig monotherapy at higher doses effectively maintains graft survival independent of Tregs.

SummaryBackground IgE-mediated allergy is a common disease characterized by a harmful immune response towards otherwise harmless environmental antigens. Induction of specific immunological non-responsiveness towards allergens would be a desirable goal. Blockade of costimulatory pathways is a promising strategy to modulate the immune response in an antigen-specific manner. Recently, OX40 (CD134) was identified as a costimulatory receptor important in Th2-mediated immune responses. Moreover, synergy between OX40 blockade and ‘conventional’ costimulation blockade (anti-CD40L, CTLA4Ig) was observed in models of alloimmunity. Objective We investigated the potential of interfering with OX40 alone or in combination with CD40/CD28 signals to influence the allergic immune response. Methods The OX40 pathway was investigated in an established murine model of IgE-mediated allergy where BALB/c mice are repeatedly immunized with the clinically relevant grass pollen allergen Phl p 5. Groups were treated with combinations of anti-OX40L, CTLA4Ig and anti-CD40L. In selected mice, Tregs were depleted with anti-CD25. Results Blockade of OX40L alone at the time of first or second immunization did not modulate the allergic response on the humoral or effector cell levels but slightly on T cell responses. Administration of a combination of anti-CD40L/CTLA4Ig delayed the allergic immune response, but antibody production could not be inhibited after repeated immunization even though the allergen-specific T cell response was suppressed in the long run. Notably, additional blockade of OX40L had no detectable supplementary effect. Immunomodulation partly involved regulatory T cells as depletion of CD25+ cells led to restored T cell proliferation. Conclusions and Clinical Relevance Collectively, our data provide evidence that the allergic immune response towards Phl p 5 is independent of OX40L, although reduction on T cell responses and slightly on the asthmatic phenotype was detectable. Besides, no relevant synergistic effect of OX40L blockade in addition to CD40L/CD28 blockade could be detected. Thus, the therapeutic potential of OX40L blockade for IgE-mediated allergy appears to be ineffective in this setting.

The mixed chimerism approach achieves donor-specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti-CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor-specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC-mismatched/minor antigen-matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non-MHC antigens cause graft rejection and split tolerance.

Molecular chimerism is a promising strategy to induce tolerance to disease-causing antigens expressed on genetically modified haematopoietic stem cells. The approach was employed successfully in models of autoimmunity and organ transplantation. Recently, we demonstrated that molecular chimerism induces robust and lasting tolerance towards the major grass pollen allergen Phl p 5. Since allergens are a group of antigens differing widely in their function, origin and structure we further examined the effectiveness of molecular chimerism using the Phl p 5-unrelated major birch pollen allergen Bet v 1, co-expressed with the reporter GFP. Besides, inhibition of CD26 was used to promote engraftment of modified stem cells. Retrovirus VSV-Betv1-GFP was generated to transduce 5-FU-mobilized BALB/c hematopoietic cells to express membrane-bound Bet v 1 (VSV-GFP virus was used as control). Myeloablated BALB/c mice received Betv1-GFP or GFP expressing bone marrow cells, pre-treated with a CD26 inhibitor. Chimerism was followed by flow cytometry. Tolerance was assessed by measuring allergen-specific isotype levels in sera, RBL assays and T-cell proliferation assays. Mice transplanted with transduced BMC developed multi-lineage molecular chimerism which remained stable long-term (>8 months). After repeated immunizations with Bet v 1 and Phl p 5 serum levels of Bet v 1-specific antibodies (IgE, IgG1, IgG2a, IgG3 and IgA) remained undetectable in Betv1-GFP chimeras while high levels of Phl p 5-specific antibodies developed. Likewise, basophil degranulation was induced in response to Phl p 5 but not to Bet v 1 and specific non-responsiveness to Bet v 1 was observed in proliferation assays. These data demonstrate successful tolerization towards Bet v 1 by molecular chimerism. Stable long-term chimerism was achieved under inhibition of CD26. These results provide evidence for the broad applicability of molecular chimerism as tolerance strategy in allergy.

The liver has the outstanding ability to regenerate itself and restore parenchymal tissue after injury. The most common cell source in liver growth/regeneration is replication of preexisting hepatocytes although liver progenitor cells have been postulated to participate in liver regeneration in cases of massive injury. Bone marrow derived hematopoietic stem cells (BM-HSC) have the formal capacity to act as a source for hepatic regeneration under special circumstances; however, the impact of this process in liver tissue maintenance and regeneration remains controversial. Whether BM-HSC are involved in liver regeneration or not would be of particular interest as the cells have been suggested to be an alternative donor source for the treatment of liver failure. Data from murine models of liver disease show that BM-HSC can repopulate liver tissue and restore liver function; however, data obtained from human liver transplantation show only little evidence for liver regeneration by this mechanism. The cell source for liver regeneration seems to depend on the nature of regeneration process and the extent of injury; however, the precise mechanisms still need to be resolved. Current data suggest, that in human orthotopic liver transplantation, liver regeneration by BM-HSC is a rather rare event and therefore not of clinical relevance.

Central clonal deletion has been considered the critical factor responsible for the robust state of tolerance achieved by chimerism-based experimental protocols, but split-tolerance models and the clinical experience are calling this assumption into question. Although clone-size reduction through deletion has been shown to be universally required for achieving allotolerance, it remains undetermined whether it is sufficient by itself. Therapeutic Treg treatment induces chimerism and tolerance in a stringent murine BM transplantation model devoid of myelosuppressive recipient treatment. In contrast to irradiation chimeras, chronic rejection (CR) of skin and heart allografts in Treg chimeras was permanently prevented, even in the absence of complete clonal deletion of donor MHC-reactive T cells. We show that minor histocompatibility antigen mismatches account for CR in irradiation chimeras without global T cell depletion. Furthermore, we show that Treg therapy–induced tolerance prevents CR in a linked suppression–like fashion, which is maintained by active regulatory mechanisms involving recruitment of thymus-derived Tregs to the graft. These data suggest that highly efficient intrathymic and peripheral deletion of donor-reactive T cells for specificities expressed on hematopoietic cells preclude the expansion of donor-specific Tregs and, hence, do not allow for spreading of tolerance to minor specificities that are not expressed by donor BM.

BACKGROUND: Bone marrow-derived hematopoietic stem cells (BM-HSCs) have been shown to act as source for hepatic regeneration in rodent models; however, their ability to participate in human liver regeneration remains controversial. The aim of this study was to investigate the origin of hepatocytes in sex-mismatched cases of orthotopic liver transplantation in longitudinally performed liver biopsies. METHODS: Paraffin-embedded liver biopsy samples of 14 patients after sex-mismatched (female-to-male) liver transplantation were investigated. Biopsies were taken at multiple time points and subjected to histologic examination. Immunohistochemical staining with a hepatocyte-specific antibody and fluorescent in situ hybridization for visualization of Y chromosomes were performed to analyze the presence of recipient-derived hepatocytes. RESULTS: We analyzed 30 liver biopsy samples ranging from 1 week to more than 3 years after transplantation. There was no evidence for recipient-derived hepatocytes in liver transplants at any time point. We were able to detect recipient-specific chromosomal status in inflammatory cells within the liver but not within hepatocytes. Results were independent of liver injury at the time of biopsy, caused by hepatitis C recurrence or rejection episodes. CONCLUSIONS: Our results show no evidence for involvement of BM-HSCs in liver regeneration after orthotopic liver transplantation. We think that recipient BM-HSC-derived hepatocyte repopulation is a very rare event at best and is not of clinical relevance.

Purpose of review Organ transplantation is the state-of-the-art treatment for end-stage organ failure; however, long-term graft survival is still unsatisfactory. Despite improved immunosuppressive drug therapy, patients are faced with substantial side effects and the risk of chronic rejection with subsequent graft loss. The transplantation of donor bone marrow for the induction of mixed chimerism has been recognized to induce donor-specific tolerance a long time ago, but safety concerns regarding toxicities of current bone marrow transplantation (BMT) protocols impede widespread application. Recent findings Recent studies in nonhuman primates and kidney transplant patients have demonstrated successful induction of allograft tolerance even though--in contrast to murine models--only transient chimerism was achieved. Progress toward the development of nontoxic murine BMT protocols revealed that Treg therapy is a potent therapeutic adjunct eliminating the need for cytotoxic recipient conditioning. Furthermore, new insight into the mechanisms underlying tolerization of CD4 and CD8 T cells in mixed chimeras has been gained and has identified possible difficulties impeding clinical translation. Summary This review will address the recent advances in murine models as well as findings from the first clinical trials for the induction of tolerance through mixed chimerism. Both the potential for more widespread clinical application and the remaining hurdles and challenges of this tolerance approach will be discussed.

Deliberate establishment of donor-specific immunologic tolerance is considered to be the “Holy Grail” in transplantation medicine, but clinical tolerance protocols for routine organ transplantation are still an unmet need. Mixed hematopoietic chimerism is an attractive tolerance strategy with considerable potential. Recent pilot trials provide proof-of-principle that mixed chimerism can induce tolerance in renal transplant recipients. Routine clinical translation, however, is impeded by the side effects of the cytotoxic recipient conditioning necessary for the transient engraftment of HLA-mismatched BM. In murine studies recently published in The American Journal of Transplantation, we demonstrated that the therapeutic application of polyclonal recipient regulatory T cells (Tregs) leads to engraftment of practicable doses of fully allogeneic BM and to donor-specific tolerance without any cytotoxic conditioning, thereby eliminating a major impediment for the clinical translation of the mixed chimerism strategy in the experimental setting. The background and the implications of these findings are discussed.

Distinct lymphocyte populations have been identified that either promote or impede the establishment of chimerism and tolerance through allogeneic bone marrow transplantation (BMT). Natural killer T (NKT) cells have pleiotropic regulatory properties capable of either augmenting or downmodulating various immune responses. We investigated in this study whether NKT cells affect outcome in mixed chimerism models employing fully mismatched nonmyeloablative BMT with costimulation blockade (CB). The absence of NKT cells had no detectable effect on chimerism or skin graft tolerance after conditioning with 3Gy total body irradiation (TBI), and a limited positive effect with 1Gy TBI. Stimulation of NKT cells with alpha-galactosylceramide (alpha-gal) at the time of BMT prevented chimerism and tolerance. Activation of recipient (as opposed to donor) NKT cells was necessary and sufficient for the alpha-gal effect. The detrimental effect of NKT activation was also observed in the absence of T cells after conditioning with in vivo T-cell depletion (TCD). NKT cells triggered rejection of BM via NK cells as chimerism and tolerance were not abrogated when NKT cells were stimulated in the absence of both NK cells and T cells. Thus, activation of NKT cells at the time of BMT overcomes the effects of CB, inhibiting the establishment of chimerism and tolerance.

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-beta induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

Organ transplant recipients require life-long treatment with immunosuppressive drugs. Currently available immunosuppression is associated with substantial morbidity and mortality, and is ineffective in inhibiting chronic rejection and graft loss. Therefore, a permanent state of donor-specific tolerance remains a primary goal for transplantation research. The induction of mixed hematopoietic chimerism is an attractive concept in this regard. Hematopoietic chimerism modulates the immunologic repertoire by extending the mechanisms of self-tolerance to donor-specific allotolerance. Despite recent progress in developing nontoxic bone marrow transplantation protocols for rodents, translation to large animals has remained difficult. Here, we outline the concept of tolerance via mixed chimerism, and review recent progress and remaining challenges in bringing this approach to the clinical setting.

Bone marrow (BM) transplantation under costimulation blockade induces chimerism and tolerance. Cotransplantation of donor T cells (contained in substantial numbers in mobilized peripheral blood stem cells and donor lymphocyte infusions) together with donor BM paradoxically triggers rejection of donor BM through undefined mechanisms. Here, nonmyeloablatively irradiated C57BL/6 recipients simultaneously received donor BM (BALB/c) and donor T cells under costimulation blockade (anti-CD154 and CTLA4Ig). Donor CD4, but not CD8 cells, triggered natural killer-independent donor BM rejection which was associated with increased production of IL-6, interferon gamma (IFN-γ) and IL-17A. BM rejection was prevented through neutralization of IL-6, but not of IFN-γ or IL-17A. IL-6 counteracted the antiproliferative effect of anti-CD154 in vitro. Rapamycin and anti-lymphocyte function-associated antigen 1 negated this effect of IL-6 in vitro and prevented BM rejection in vivo. Simultaneous cotransplantation of (BALB/cxB6)F1, recipient or irradiated donor CD4 cells, or late transfer of donor CD4 cells did not lead to BM rejection, whereas cotransplantation of third party CD4 cells did. Transferred donor CD4 cells became activated, rapidly underwent apoptosis and triggered activation and proliferation of recipient T cells. Collectively, these results provide evidence that donor T cells recognizing the recipient as allogeneic lead to the release of IL-6, which abolishes the effect of anti-CD154, triggering donor BM rejection through bystander activation.

Background The mixed chimerism approach induces donor-specific tolerance in both pre-clinical models and clinical pilot trials. However, chronic rejection of heart allografts and acute rejection of skin allografts were observed in some chimeric animals despite persistent hematopoietic chimerism and tolerance toward donor antigens in vitro. We tested whether additional cell therapy with regulatory T cells (Tregs) is able to induce full immunologic tolerance and prevent chronic rejection. Methods We recently developed a murine “Treg bone marrow (BM) transplantation (BMT) protocol” that is devoid of cytoreductive recipient pre-treatment. The protocol consists of a moderate dose of fully mismatched allogeneic donor BM under costimulation blockade, together with polyclonal recipient Tregs and rapamycin. Control groups received BMT under non-myeloablative irradiation and costimulation blockade without Treg therapy. Multilineage chimerism was followed by flow cytometry, and tolerance was assessed by donor-specific skin and heart allografts. Results Durable multilineage chimerism and long-term donor skin and heart allograft survival were successfully achieved with both protocols. Notably, histologic examination of heart allografts at the end of follow-up revealed that chronic rejection is prevented only in chimeras induced with the Treg protocol. Conclusions In a mouse model of mixed chimerism, additional Treg treatment at the time of BMT prevents chronic rejection of heart allografts. As the Treg-chimerism protocol also obviates the need for cytoreductive recipient treatment it improves both efficacy and safety over previous non-myeloablative mixed chimerism regimens. These results may significantly impact the development of protocols for tolerance induction in cardiac transplantation.

The transplantation of allergens (e.g. Phl p 5 or Bet v 1) expressed on BM cells as membrane-anchored full-length proteins leads to permanent tolerance at the T-cell, B-cell, and effector-cell levels. Since the exposure of complete allergens bears the risk of inducing anaphylaxis, we investigated here whether expression of Phl p 5 in the cytoplasm (rather than on the cell surface) is sufficient for tolerance induction. Transplantation of BALB/c BM retrovirally transduced to express Phl p 5 in the cytoplasm led to stable and durable molecular chimerism in syngeneic recipients (∼20% chimerism at 6 months). Chimeras showed allergen-specific T-cell hyporesponsiveness. Further, Phl p 5-specific TH 1-dependent humoral responses were tolerized in several chimeras. Surprisingly, Phl p 5-specific IgE and IgG1 levels were significantly reduced but still detectable in sera of chimeric mice, indicating incomplete B-cell tolerance. No Phl p 5-specific sIgM developed in cytoplasmic chimeras, which is in marked contrast to mice transplanted with BM expressing membrane-anchored Phl p 5. Thus, the expression site of the allergen substantially influences the degree and quality of tolerance achieved with molecular chimerism in IgE-mediated allergy.

Development of antigen-specific preventive strategies is a challenging goal in IgE-mediated allergy. We have recently shown in proof-of-concept experiments that allergy can be successfully prevented by induction of durable tolerance via molecular chimerism. Transplantation of syngeneic hematopoietic stem cells genetically modified to express the clinically relevant grass pollen allergen Phl p 5 into myeloablated recipients led to high levels of chimerism (i.e. macrochimerism) and completely abrogated Phl p 5-specific immunity despite repeated immunizations with Phl p 5.It was unclear, however, whether microchimerism (drastically lower levels of chimerism) would be sufficient as well which would allow development of minimally toxic tolerance protocols.Bone marrow cells were transduced with recombinant viruses integrating Phl p 5 to be expressed in a membrane-anchored fashion. The syngeneic modified cells were transplanted into non-myeloablated recipients that were subsequently immunized repeatedly with Phl p 5 and Bet v 1 (control). Molecular chimerism was monitored using flow cytometry and PCR. T cell, B-cell and effector-cell tolerance were assessed by allergen-specific proliferation assays, isotype levels in sera and RBL assays.Here we demonstrate that transplantation of Phl p 5-expressing bone marrow cells into recipients having received non-myeloablative irradiation resulted in chimerism persisting for the length of follow-up. Chimerism levels, however, declined from transient macrochimerism levels to persistent levels of microchimerism (followed for 11 months). Notably, these chimerism levels were sufficient to induce B-cell tolerance as no Phl p 5-specific IgE and other high affinity isotypes were detectable in sera of chimeric mice. Furthermore, T-cell and effector-cell tolerance were achieved.Low levels of persistent molecular chimerism are sufficient to induce long-term tolerance in IgE-mediated allergy. These results suggest that it will be possible to develop minimally toxic conditioning regimens sufficient for low level engraftment of genetically modified bone marrow.

Purpose of review Blockade of costimulatory signalling is a promising approach to inhibit T-cell responses and consequently allograft rejection. The last decade was marked by progress in understanding the details of various costimulatory pathways and by the development of biologicals targeting these pathways with the aim of selectively and efficiently modulating T-cell responses. Recent findings Here we focus on the clinically relevant costimulatory pathways CD28:CD80/86, CD40:CD154 (CD40L), CD2:LFA-3 and ICAM:LFA-1. We will give a short overview of the physiologic function of these pathways and discuss results from preclinical and clinical studies of costimulation blockers targeting these pathways. Summary The development of costimulation blockers for clinical application in the field of organ transplantation was delayed by several setbacks. However, belatacept has recently been approved as first in class for renal transplantation. Several additional costimulation blockers are under development with some having already entered into clinical trials. Costimulation blockers are a new class of rationally designed immunosuppressive drugs with considerable potential for improving outcome of organ transplantation.

Pharmacologic immunosuppression was originally thought of as the global inhibition of effector T-cell responses. With the emerging understanding of the role of Tregs in the acceptance of allografts, this concept had to be revisited and the potential effects of immunosuppressants on regulatory mechanisms to be considered. The costimulation blocker belatacept (Nulojix, Bristol-Myers Squibb; an antiB7 compound designed to block CD28) has recently been approved and provides an alternative to calcineurin inhibitors in renal transplant recipients. The now-recognized complexities of the CD28–CTLA-4–B7 costimulation pathway urge the question as to whether belatacept affects not only effector T cells but also Tregs. Here, we provide a personal perspective on the current insight of possible interactions of belatacept and Tregs and their potential clinical relevance.

Summary While costimulation blockade-based mixed chimerism protocols work well for inducing tolerance in rodents, translation to preclinical large animal/nonhuman primate models has been less successful. One recognized cause for these difficulties is the high frequency of alloreactive memory T cells (Tmem) found in the (pre)clinical setting as opposed to laboratory mice. In the present study, we therefore developed a murine bone marrow transplantation (BMT) model employing recipients harboring polyclonal donor-reactive Tmem without concomitant humoral sensitization. This model was then used to identify strategies to overcome this additional immune barrier. We found that B6 recipients that were enriched with 3 × 107 T cells isolated from B6 mice that had been previously grafted with Balb/c skin, rejected Balb/c BM despite costimulation blockade with anti-CD40L and CTLA4Ig (while recipients not enriched developed chimerism). Adjunctive short-term treatment of sensitized BMT recipients with rapamycin or anti-LFA-1 mAb was demonstrated to be effective in controlling Tmem in this model, leading to long-term mixed chimerism and donor-specific tolerance. Thus, rapamycin and anti-LFA-1 mAb are effective in overcoming the potent barrier that donor-reactive Tmem pose to the induction of mixed chimerism and tolerance despite costimulation blockade.

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.

Deliberateinductionofdonor-specificimmunologicaltol-eranceisstilltheultimategoalintransplantationmedicineasitwouldobviatetheneedforlife-longimmunosuppres-sive therapy and as it has the potential to improve long-term graft survival. Although the development of newimmunosuppressive drugs led to substantial progress intheinhibitionofacuterejectionandimprovementofshort-term graft survival after organ transplantation, unexpect-edlyhowever,long-termoutcomedidnotimproveaccord-ingly [1]. Through extensive research over the past dec-ades, numerous tolerance protocols were developed inrodents; however, the majority of them failed when trans-latedtothelargeanimalsetting[2].Themixedchimerismapproach is a notable exception as its effectiveness wasdemonstrated in several large animal models [3–6] andrecently in clinical proof-of-concept trials of combinedkidney and bone marrow transplantation (BMT) [7,8,9 ].Mixed chimerism is defined as a state of coexistence ofdonor and host hematopoietic cells after the transplan-tation of donor hematopoietic stem cells (HSCs) into apreconditioned host. Since the introduction of the mixedchimerism concept with myeloablative conditioning bytotal body irradiation (TBI) [10], step-wise progress hasbeen achieved toward the development of less toxicBMT protocols more suitable for clinical application[11 ,12,13]. Intrathymic deletion of donor-reactive Tcells was revealed to be the dominant tolerance mech-anism in early mixed chimerism protocols involving theglobal destruction of the recipient’s T-cell repertoire[14,15]. Intrathymic deletion is also a critical tolerancemechanism in regimens relying on costimulation block-ade [16], but the situation is more complex, withadditional mechanisms also playing a crucial role.

The major factors that limit the success of organ transplantation are the host immune response to the foreign graft and the adverse effects of the chronic immunosuppressive therapy required to suppress this immune response. Deliberately establishing tolerance towards the donor tissue by reprogramming the immune system of the recipient thus holds great promise in improving organ transplant survival and eliminating the untoward effects of chronic drug therapy. The transplantation of donor bone marrow into recipients who are appropriately conditioned to allow development of either full or mixed chimerism has long been recognized to effectively induce donor-specific tolerance. Despite the demonstrated effectiveness of this technique, use of the mixed chimerism strategy in regular clinical practice has been hampered by the toxic side effects inherent to conventional bone marrow transplantation protocols. This Review addresses recent advances in preclinical and clinical studies inducing transplantation tolerance through mixed chimerism and discusses both the potential and the challenges of this approach.

To date, organ transplant patients have to deal with the numerous side effects of life-long dependence on immunosuppressive drugs, whereas at the same time these drugs fail to prevent chronic rejection in many cases. Finding ways to establish donor-specific immunological tolerance thus remains one of the major goals in transplantation medicine. Tolerance through mixed chimerism can be achieved in rodents and in humans by the transplantation of hematopoietic stem cells. Widespread clinical application of this tolerance approach is, however, prevented by the toxicities of current bone marrow transplantation protocols in humans. Cytotoxic recipient conditioning and the hazard of graft-versus-host disease are unacceptable risks for organ transplant recipients. However, considerable progress has been made toward nontoxic conditioning regimens in animal studies. Translation of these findings into large animal models and the clinical setting is expected to be an important step toward broad clinical application of the mixed chimerism approach in organ transplantation.

The only way to overcome the need for life-long immunosuppression in a transplant recipient is to induce tolerance. Deletional tolerance can be reliably achieved with the induction of mixed chimerism through transplantation of donor bone marrow (BM). Despite the development of increasingly milder BM transplantation (BMT) animal models, BM engraftment in humans still requires considerably toxic conditioning and puts patients at risk for the development of GVHD. However, in a proof-of-concept trial, mixed chimerism and tolerance have been successfully induced in highly selected patients suffering from both end-stage renal disease and multiple myeloma. Meanwhile, there has been notable progress in developing advanced experimental BMT regimens, in particular through the use of costimulation blockers. Costimulation blockade in rodent models allowed the design of BMT protocols entirely devoid of irradiation. Costimulation blockers have also succeeded in more complex protocols in non-human primates. They are under clinical evaluation in renal transplantation as immunosuppressive therapy. Costimulation blockade may lead the way for the development of milder BMT protocols and broader application of mixed chimerism in organ transplantation.

Purpose The mixed chimerism approach has outstanding potency in the induction of donor-specific tolerance in both preclinical animal models and clinical pilot trials. However, some chimeras develop a state of “split tolerance”, an incompletely understood phenomenon. Cardiac allograft vasculopathy (CAV) and skin graft rejection were observed in chimeric animals that are tolerant to donor antigen in vitro . We hypothesized that Tregs therapy is able to prevent “split tolerance” and chronic rejection. Methods and Materials Groups of B6 mice received 2x10 7 unseparated Balb/c BMC (d0, iv) and costimulation blockade consisting of antiCD40LmAb (1mg, d0) and CTLA4Ig (0.5mg, d2). Control groups received 3 GyTBI to facilitate BM engraftment, whereas the “Treg protocol” received 5x10 6 Tregs (d0) and a short course of rapamycin (0.1mg/mouse, d-1/d0/d2). Multilineage chimerism was followed by flowcytometry, heterotopic heart transplantation was used to assess donor-specific tolerance. Results Treg therapy reliably induced longterm multilineage chimerism without the need for recipient TBI (8/8 chimeras Treg protocol vs 13/18 control group, p=0.28). Heart allografts survived longterm in both groups (>100 days, Treg group n=4; control group n=7), whereas third-party allografts were promptly rejected. Histological examination of donor heart allografts revealed that Treg treatment potently avoids CAV (all grafts were scored with ISHLT grade 0 or 1; 0: n=2, 1: n=2). In the control group 4/7 mice were classified with a rejection rate 2 or higher (0: n=1, 1: n=2, 2:n=2, 3:n=2). Conclusions In this study we demonstrate that Treg treatment enhances the potency of the mixed chimerism approach for the induction of donor-specific tolerance. Treg treatment obviates the need for cytoreductive recipient pretreatment and prevents CAV. Thus, both efficiency and safety of the mixed chimerism approach are improved. We think that these results will have significant impact on the development of new protocols for tolerance induction in cardiac transplantation.