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Donor-specific tolerance remains a goal in transplantation because it could improve graft survival and reduce morbidity. Cotransplantation of donor hematopoietic cells to achieve chimerism is a promising approach for tolerance induction, which was successfully tested in clinical trials. However, current protocols are associated with side effects related to the myelosuppressive recipient conditioning, which makes it difficult to introduce them as standard therapy. More recently, adoptive cell therapy with polyclonal or donor-specific regulatory T cells (Treg) proved safe and feasible in several transplant trials, but it is unclear whether it can induce tolerance on its own. The combination of both approaches-Treg therapy and hematopoietic cell transplantation-leads to chimerism and tolerance without myelosuppressive treatment in murine models. Treg therapy promotes engraftment of allogeneic hematopoietic cells, reducing conditioning requirements and enhancing regulatory mechanisms maintaining tolerance. This review discusses possible modes of action of transferred Treg in experimental chimerism models and describes translational efforts investigating the potent synergy of Treg and chimerism., Competing Interests: T.W. received speaker’s honoraria from eGenesis and Mallinckrodt/Therakos and is a DSMB member for Quell Therapeutics. The other author declares no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Hematopoietic chimerism remains the most promising strategy to bring transplantation tolerance into clinical routine. The concept of chimerism-based tolerance aims to extend the recipient's mechanisms of self-tolerance (ie, clonal deletion, anergy, and regulation) to include the tolerization of donor antigens that are introduced through the cotransplantation of donor hematopoietic cells. For this to be successful, donor hematopoietic cells need to engraft in the recipient at least temporarily. Three pioneering clinical trials inducing chimerism-based tolerance in kidney transplantation have been published to date. Within this review, we discuss the mechanisms of tolerance that are associated with the specific therapeutic protocols of each trial. Recent data highlight the importance of regulation as a mechanism that maintains tolerance. Insufficient regulatory mechanisms are also a likely explanation for situations of tolerance failure despite persisting donor chimerism. After decades of preclinical development of chimerism protocols, mechanistic data from clinical trials have recently become increasingly important. Better understanding of the required mechanisms for tolerance to be induced in humans will be a key to design more reliable and less invasive chimerism protocols in the future., Competing Interests: T.W. received speaker’s honoraria from eGenesis and Mallinckrodt/Therakos and is a Data and Safety Monitoring Board member for Quell Therapeutics. The other authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Infusion of recipient regulatory T (Treg) cells promotes durable mixed hematopoietic chimerism and allograft tolerance in mice receiving allogeneic bone marrow transplant (BMT) with minimal conditioning. We applied this strategy in a Cynomolgus macaque model., Methods: CD4 CD25 Treg cells that were polyclonally expanded in culture were highly suppressive in vitro and maintained high expression of FoxP3. Eight monkeys underwent nonmyeloablative conditioning and major histocompatibility complex mismatched BMT with or without Treg cell infusion. Renal transplantation (from the same BMT donor) was performed 4 months post-BMT without immunosuppression to assess for robust donor-specific tolerance., Results: Transient mixed chimerism, without significant T cell chimerism, was achieved in the animals that received BMT without Treg cells (N = 3). In contrast, 2 of 5 recipients of Treg cell BMT that were evaluable displayed chimerism in all lineages, including T cells, for up to 335 days post-BMT. Importantly, in the animal that survived long-term, greater than 90% of donor T cells were CD45RA CD31, suggesting they were new thymic emigrants. In this animal, the delayed (to 4 months) donor kidney graft was accepted more than 294 days without immunosuppression, whereas non-Treg cell BMT recipients rejected delayed donor kidneys within 3 to 4 weeks. Early CMV reactivation and treatment was associated with early failure of chimerism, regardless of Treg cell administration., Conclusions: Our studies provide proof-of-principle that, in the absence of early CMV reactivation (and BM-toxic antiviral therapy), cotransplantation of host Treg cell can promote prolonged and high levels of multilineage allogeneic chimerism and robust tolerance to the donor.

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) can lead to donor-specific tolerance. Patients reported in the literature that underwent kidney transplantation (KT) after a previous HSCT from the same haploidentical donor typically received short-term immunosuppression, mainly for safety reasons and concerns of triggering graft-versus-host disease., Methods: We describe the case of a 22-year-old patient who developed chronic kidney failure after receiving haploidentical HSCT from his father for the treatment of metastatic rhabdomyosarcoma. Five years after HSCT, he received a preemptive kidney transplant from his father. Steroid treatment, which had been prescribed for the underlying kidney disease, was withdrawn within 2 months posttransplant, and no de novo immunosuppression was given. Donor-specific tolerance was assessed with mixed lymphocyte reaction and INF-γ ELISPOT before (D0) and after KT (D9). Furthermore, the exact level of donor-derived T cells was measured with real-time polymerase chain reaction before and 1 year after KT., Results: In vitro assays (mixed lymphocyte reaction and ELISPOT) revealed donor-specific tolerance before and after transplantation, respectively. The number of recipient-derived T cells was low before KT and virtually did not change over time (0.0139% ± 0.0039 and 0.0120% ± 0.0067; P = NS). Graft function was excellent throughout the follow-up (36 months post KT: serum creatinine, 1.18 mg/dL). Protocol biopsies performed 1 and 12 months after transplantation confirmed the absence of rejection., Conclusions: This is one of the first cases of kidney transplantation from the same donor after previous haploidentical HSCT with a corticosteroid taper alone. Our results suggest that immunosuppression can be avoided in such cases.

Background: CTLA-4 immunoglobulin fusion proteins (CTLA4-Ig) suppress immune reactions by blocking the T-cell costimulatory CD28-CD80-86 pathway and are used in clinical trials for diseases featuring exaggerated T-cell reactivity including autoimmune diseases and allograft rejection. However, because CTLA4-Ig has been suspected to interfere with T regulatory (Treg) cell homeostasis and function, recently, substantial concerns on CTLA4-Ig's potentially antitolerogenic effects have been raised., Methods: We tested immunoregulatory CTLA4-Ig explicitly for its effect on Treg cell numbers, frequencies and function in an in vitro murine major histocompatibility complex mismatched setting using C57BL/6 bone marrow-derived dendritic cells as stimulators of allogeneic Balb/c Foxp3 T cells, which allowed for tracing Treg cells in a straightforward fashion., Results: The presence of CTLA4-Ig in mixed leukocyte reactions-while dampening the global proliferative response of allostimulated Balb/c T cells-resulted in a relative increase of the frequency of thymus-derived CD4CD25Foxp3 Treg cells with intact suppressive activity. This relative increase was caused by a selective inhibitory effect of CTLA4-Ig on proliferating conventional T cells, whereas the proliferative capacity of Treg cells in cell cultures remained unaffected. Additionally, in the presence of CTLA4-Ig, the frequency of apoptosis was decreased in these cells., Conclusion: Our findings unequivocally demonstrate that CTLA4-Ig does not negatively affect Treg cell frequencies and function in vitro.

Background: Allogeneic bone marrow transplantation is under investigation for a range of nonmalignant indications, including tolerance induction through mixed chimerism. This strategy has so far been tested experimentally only in young recipients. Due to immunosenescence, older patients have an increase in memory T cells (TMEM) as well as other alterations to their immune system, which may influence the potential to induce tolerance. We therefore investigated the impact of immunosenescence on chimerism-based tolerance induction., Methods: Groups of young (2 months) and old (12 months) C57BL/6 recipients received BALB/c bone marrow under nonmyeloablative (3 Gy) and minimal (1 Gy) total body irradiation and treatment with costimulation blockade, T-cell depletion, or rapamycin. Multilineage chimerism, clonal deletion, and lymphocyte subsets were analyzed by flow cytometry. Tolerance was assessed by skin and heart grafts and enzyme-linked immunospot, intracellular cytokine, and mixed lymphocyte reaction assays., Results: Unexpectedly, chimerism and tolerance were established in old recipients with comparable-and in some cases increased-efficacy as in young recipients employing costimulation blockade-based or T-cell depletion-based conditioning with 1 or 3 Gy total body irradiation. TMEM reactivity in (naïve) old mice was augmented in response to polyclonal but not to allogeneic stimulation, providing a mechanistic underpinning for the susceptibility to chimerism induction despite increased TMEM frequencies. Tolerance in old recipients was associated with peripheral and central clonal deletion and a higher frequency of regulatory T cells., Conclusion: Advanced age does not impair bone marrow engraftment, thereby widening the clinical potential of experimental protocols inducing transplantation tolerance through mixed chimerism.

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.

Background: Mixed chimerism is an effective strategy for the induction of transplantation tolerance but the toxicity of recipient conditioning makes current bone marrow (BM) transplantation (BMT) protocols unsuitable for widespread clinical application. Therapies promoting BM engraftment under minimal conditioning would facilitate translation of this concept to the clinic. Recently, we have shown that regulatory T cell (Treg) therapy has potent engraftment-enhancing effects in an irradiation-free noncytotoxic BMT protocol, but only if it is combined with rapamycin treatment., Methods: Here, we investigated whether polyclonal Treg therapy is effective in promoting chimerism and tolerance in an otherwise unsuccessful BMT protocol using low-dose total body irradiation (1 Gy) and costimulation blockade and determined whether Tregs do so on their own without rapamycin., Results: The application of polyclonal FoxP3-transduced recipient Tregs led to durable multilineage chimerism and donor-specific skin graft tolerance whereas recipients receiving costimulation blockade alone or green flourescent protein (GFP)-transduced cells failed to develop chimerism. Infused Tregs had a limited life span as indicated by polymerase chain reaction analysis but rather contribute to de novo induction of subsequent Treg generations. Deletion of donor-reactive T cells was observed but progressed more slowly over time compared with recipients of a nonmyeloablative BMT protocol using 3 Gy total body irradiation., Conclusions: In conclusion, Treg therapy promotes BM engraftment on its own in a low-dose irradiation BMT protocol, leading to chimerism and tolerance maintained through deletional and nondeletional mechanisms.

The following position paper summarizes the recommendations for early clinical trials and ongoing basic research in the field of mesenchymal stem cell-induced solid organ graft acceptance--agreed upon on the first meeting of the Mesenchymal Stem Cells In Solid Organ Transplantation (MISOT) study group in late 2008.

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.

Background: The purpose of this study was to analyze the impact of extended donor criteria (EDC) and of changes in the Model for End-Stage Liver Disease (MELD) score while waiting for liver-transplantation (Delta-MELD) on patient survival and initial graft function., Methods: We included 386 consecutive patients with end-stage liver disease who underwent orthotopic liver transplantation at the Medical University Vienna between 1997 and 2003. Primary outcome was patient survival and secondary outcome was initial graft function. EDC included: age >60 years, >4 days intensive medical care, cold ischemia time >10 hr, need for noradrenalin >0.2 microg/kg/min or doputamin >6 microg/kg/min, a donor peak serum sodium >155 mEq/L, a donor serum creatinine >1.2 mg/100 mL, and a body mass index >30., Results: Delta-MELD was significantly higher in the nonsurvivor population (P=0.01) and EDC showed a significant influence on initial graft function (P=0.01). Worsening in either Delta-MELD or the presence of at least two EDC was not associated with an increased risk of primary graft dysfunction and death. Worsening in Delta-MELD and the presence of at least two EDC was significantly associated with primary graft dysfunction (P=0.01) and death (P=0.008)., Conclusion: The combination of a liver recipient with worsening Delta-MELD and a potential donor with at least two EDC should be avoided.

Bone marrow transplantation (BMT) under costimulation blockade induces mixed chimerism and tolerance in rodent models. Recent data, predominantly from in vitro studies, suggest that in addition to blocking the CD28 costimulation pathway CTLA4Ig also acts through upregulating the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Here we demonstrate that even though CTLA4Ig is critically required for the induction of chimerism and tolerance in a murine model of nonmyeloablative BMT, IDO activity is not. No significant differences were detectable in the kynurenine to tryptophan ratios (indicative of IDO activity) in sera of BMT recipients treated with CTLA4Ig (tolerant group) versus BMT recipients treated without CTLA4Ig (nontolerant group) versus naïve controls. In vivo inhibition of IDO immediately after BMT with CTLA4Ig or several months thereafter did not block achievement of chimerism and tolerance. Thus, IDO does not play a critical role in the induction or maintenance of chimerism and tolerance in a CTLA4Ig-based BMT model.

Background: Induction of mixed chimerism and tolerance usually requires cytoreduction or transplantation of high numbers of bone marrow cells (BMC). However, such protocols have only a suboptimal success rate and, more importantly, equivalent numbers of BMC cannot be routinely obtained in the clinical setting. The authors therefore evaluated whether a short-course of immunosuppression (IS) given in addition to co-stimulation blockade would facilitate chimerism induction and allow reduction of the minimally required number of BMC without cytoreduction., Methods: B6 mice received 200, 100, or 50 x 10 unseparated BMC from Balb/c donors plus an anti-CD40L monoclonal antibody (mAb) and CTLA4Ig (without irradiation or cytotoxic drugs). Some groups were treated additionally with IS (rapamycin, methylprednisolone, and mycophenolate mofetil for 4 weeks after bone marrow transplantation), donor-specific transfusion (DST), or anti-OX40L mAb, as indicated., Results: IS led to long-term multilineage chimerism in 9 of 10 mice receiving 200 x 10 BMC (without IS, 1 of 4; P<0.05), in all mice (n=10) receiving 100 x 10 (without IS, 6 of 9; P<0.05), and notably in 9 of 10 mice treated with 50 x 10 BMC (without IS, 4 of 10; P<0.05). With transient IS, donor skin grafts were accepted longer than 170 days in 9 of 10 mice receiving 200 x 10 (without IS, 0 of 5 mice; P<0.05), all mice receiving 100 x 10 (without IS, 6 of 9; P<0.05), and 6 of 11 mice receiving 50 x 10 BMC (without IS, 4 of 10). The use of DST or anti-OX40L mAb had no beneficial effect., Conclusions: Transient IS significantly improves rates of chimerism and donor skin graft survival, and allows lasting mixed chimerism after transplantation of only 50 x 10 BMC. Thus, IS might help in the further development of noncytoreductive chimerism protocols.

Background: Costimulatory blockade has been shown to allow long-term survival of xenogeneic islets. The aim of the present study was to analyze the possibility of xenogeneic islet retransplantation using costimulatory blockade., Methods: Streptozotocin-induced diabetic C57/BL6 mice were transplanted with 1000 human islet equivalents. After 14 days, mice were nephrectomized (graftectomy) and retransplanted with human leukocyte antigen (HLA)-mismatched human islets under contralateral kidney capsule. Four groups were performed: I: all transplants (Tx) without MR1; II: first Tx without MR1, second Tx with MR1; III: first Tx with MR1, second Tx without MR1; and IV: all Tx with MR1. Recipient serums were analyzed by cross-match for serum-mediated cytotoxicity against human lymphocytes and islets., Results: In group I, the second graft rejection was accelerated (graft survival, 5 +/- 3 days) compared with the first graft without MR1 (13 +/- 7 days). In groups II and III, second graft survivals were 16 +/-1 3 and 62 +/- 15 days, respectively. In group IV, second graft function was maintained for >100 days. Pretransplant cross-matches were all negative. Post-second Tx cross-matches were positive in groups I and II and negative in group IV. In group III, post-second Tx cross-matches were negative only for cells with HLA molecules present in the first donor., Conclusions: MR1 was unable to induce tolerance after sensitization. MR1 given at the first Tx only allowed prolonged survival of the second Tx, but rejection still occurred with development of antibodies against molecules not present on first donor cells, indicating that costimulatory blockade does not induce linked-suppression against species-specific antigens of xenografts but can induce donor-specific unresponsiveness. MR1 given for all sequential transplantation allowed long-term regraft survival and prevented occurrence of antidonor antibodies.

Background: T cells and macrophages play a major role in the rejection of xenografted islets. Depending on the phylogenetic disparity, direct or indirect antigen presentation is predominant. The aim of this study was to analyze in vitro the predominance of direct or indirect presentation by depleting antigen-presenting cells in concordant and discordant xenogeneic combinations. In vivo, we analyzed the effect of macrophage depletion on concordant and discordant islet xenograft survival to assess in which combination this strategy can be used as therapeutic tool., Materials and Methods: In vitro, we performed mouse anti-rat and anti-human mixed lymphocyte reactions (MLR) after depletion of responder or stimulator antigen-presenting cells by magnetic sorting. In vivo, streptozotocin-induced diabetic C57BL/6 mice were treated by gadolinium chloride (GdCl) to deplete macrophages, and rat or human islets were transplanted under the kidney capsule. Islet function was followed by glycemia, and xenografts were analyzed at regular intervals for histology and immunohistochemistry., Results: Mouse anti-rat MLR showed a predominant direct antigen presentation pathway, whereas in mouse anti-human MLR, direct and indirect pathways were similarly involved. Survival of rat islets was not modified by GdCl therapy. In contrast, survival of human islets was significantly prolonged in GdCl-treated mice. Macrophage infiltration was decreased in concordant and discordant GdCl-treated xenografts at day 4 compared with controls. At day 15, macrophage, CD4+, and CD8+ cell infiltration was similar in all groups., Conclusions: Our results indicate that direct antigen presentation is dominant in the rejection mechanism of concordant islet xenografts and cannot be influenced by host macrophage depletion. Both direct and indirect antigen presentation are involved in rejection of discordant xenogeneic islets. Macrophage depletion should only be considered as therapeutic tool for discordant islet xenotransplantation.

The transplantation of donor hematopoietic stem cells has been used successfully in numerous experimental settings to induce donor-specific tolerance. After appropriate host conditioning, hematopoietic stem-cell transplantation leads to a lasting state of donor macrochimerism that is associated with a robust form of tolerance. One of the key factors in the success of this approach is its reliance on intrathymic clonal deletion to ensure lifelong tolerization of newly developing T cells. Evidence for ongoing central deletion comes from studies following superantigen-reactive T cells and from experiments using mice transgenic for an alloreactive T-cell receptor. In protocols inducing tolerance through macrochimerism, the preexisting mature T-cell repertoire is controlled by either globally destroying all T cells before the hematopoietic cell transplantation or, in more recent models, by tolerizing it through co-stimulation blockade. The peripheral mechanisms induced by hematopoietic stem-cell transplantation and co-stimulation blockade include both extrathymic clonal deletion and the nondeletional mechanisms anergy, suppression, or both. In addition to these immunologic hurdles, a physiologic engraftment barrier has to be surmounted for the successful induction of mixed chimerism. This can be achieved by cytoreductive host treatment or by the infusion of high numbers of donor hematopoietic cells. A detailed delineation of the mechanisms responsible for tolerance induction after hematopoietic stem-cell transplantation is expected to help in the translation of these experimental protocols to clinical organ transplantation.

Background: Thymic irradiation (TI) or repeated administration of T cell-depleting monoclonal antibodies (TCD mAbs) is required in a previously described non-myeloablative regimen allowing allogeneic marrow engraftment with stable mixed chimerism and tolerance. As both treatments might be associated with toxicity in the clinical setting, we evaluated whether T-cell costimulatory blockade could be used to replace them., Methods: C57BL/6 mice received depleting anti-CD4 and anti-CD8 mAbs on day -5, 3 Gy whole body irradiation (day 0), and 15x10(6) fully MHC-mismatched, B10.A bone marrow cells. In addition, hosts were injected with an anti-CD154 mAb (day 0) and/or CTLA4Ig (day +2). Chimerism in peripheral blood was followed by flow cytometric (FACS) analysis, and tolerance was assessed by skin grafting, and also by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. The frequency of certain Vbeta families was determined by FACS to assess deletion of donor-reactive T cells., Results: Chimerism was transient and tolerance was not present in animals receiving TCD mAbs on day -5 without costimulatory blockade. The addition of anti-CD154 and CTLA4Ig, alone or in combination, reliably permitted induction of high levels of stable (>6 months) multi-lineage chimerism, with specific tolerance to skin grafts and donor antigens by MLR and CML assays. Long-term chimeras showed deletion of donor-reactive CD4+ peripheral blood lymphocytes, splenocytes, and mature thymocytes. Administration of TCD mAbs only 1 day before bone marrow transplantation plus anti-CD154 also allowed induction of permanent chimerism and tolerance., Conclusions: One injection of anti-CD154 or CTLA4Ig overcomes the need for TI or prolonged host TCD in a preclinical model for the induction of mixed chimerism and deletional tolerance and thus further decreases the toxicity of this protocol. Achievement of tolerance with conditioning given over 24 hr suggests applicability to cadaveric organ transplantation.