Antigen‐specific regulatory T cells can induce tolerance to immunogenic grafts without the need for chronic immunosuppression

Regulatory CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) play an important role in the induction of allospecific tolerance. However tolerance in solid organ transplantation by mere transfer of Tregs has been difficult. Besides this the stability of the differentiation phenotype of Tregs has recently been questioned. We therefore aimed in generating large numbers of stable allospecific Tregs from naïve T cells by retroviral transduction with Foxp3. These were tested in an immunogenic skin transplantation model (C57BL/6→BALB/c). We established a system of transduction of mouse T cells with ecotropic retroviruses expressing Foxp3 and Thy1.1 as a surface marker to follow up transduced T cells. Alloantigen‐specific Tregs were generated by stimulating naïve recipient CD4⁺ T cells with irradiated donor splenocytes. CD25⁺ and/or CD69⁺ allospecific recipient CD4⁺ T cells were isolated and transduced with Foxp3. Alloantigen‐specific Foxp3 T cells (iTregs) showed high expression for the Treg markers Foxp3, CTLA4 and GITR. They could suppress a MLR in an alloantigen‐specific manner. Furthermore, they could be expanded up to 18 fold in vitro while maintaining their Treg phenotype and expression of lymph node homing markers like CCR7 and CD62L. iTregs prevented skin graft rejection without the need for chronic immunosuppression and recipients showed systemic allospecific allotolerance. Alloantigen‐specific Tregs were far more potent than polyspecific Tregs. Mechanisms of tolerance were graft specific homing, expansion and long‐term persistence of Tregs within the graft (>100 days, 90% of intragraft Tregs were alloantigen‐specific). In fact, tolerance could be transferred with re‐transplantation of the tolerant graft onto secondary recipients. Third party grafts were readily rejected demonstrating specificity of tolerance. Due to the Foxp3 transduction, iTregs did not lose their Treg phenotype. The results prove that large numbers of stable alloantigen‐specific Tregs can be generated from a polyclonal repertoire of naïve T cells. This is the first time that allotolerance was achieved in a non‐lymphopenic transplant model using skin grafts in an immunogenic strain combination. Therefore, antigen‐specific Tregs might have a huge therapeutic potential after solid organ transplantation.     

Both CD45RA+ and CD45RO+ human CD4+ T cells drive direct xenogeneic T‐cell responses against porcine aortic endothelial cells

Kim CH, Oh K, Kim D‐E, Lee SB, Yang JH, Lee G, Cho J, Lee D‐S. Both CD45RA⁺ and CD45RO⁺ human CD4⁺ T cells drive direct xenogeneic T‐cell responses against porcine aortic endothelial cells. Xenotransplantation 2010; 17: 224–232. © 2010 John Wiley & Sons A/S. Abstract: Background: Xenogeneic cellular immune responses are mediated by either direct or indirect pathways depending on the participation of donor or host antigen presenting cells, respectively. The contribution of direct response of human T cells, especially memory T cells, to porcine antigen presenting cells is currently unknown. Here, we sought to determine whether human peripheral blood memory/activated phenotype T cells are directly responsive to porcine endothelial cells. Methods: Porcine aortic endothelial cells (PAECs) were prepared from Yorkshire or miniature pigs. Highly purified human T cells, including naïve and memory/activated phenotype cells, were incubated with PAECs with or without the addition of exogenous cytokines. T‐cell proliferation and T‐cell receptor (TCR) Vβ usage in response to PAECs were analyzed. Results: Both CD8⁺ and CD4⁺ T cells responded directly to PAECs and exhibited exclusive responsiveness to SLA class I and class II molecules, respectively. Naïve and memory/activated phenotype CD4⁺ T cells responded against PAECs, whereas only naïve phenotype CD8⁺ T cells contributed to such a response. In addition, both populations of xenogeneic human CD4⁺ T cells exhibited similar and diverse Vβ usage. Conclusion: Due to the considerable contribution of human CD45RO⁺CD4⁺ T cells to the xenoreactivity against PAECs, effective control of xenogeneic memory/activated T‐cell responses would significantly affect long‐term survival of transplanted grafts.     

Donor‐Specific CD8+Foxp3+ T Cells Protect Skin Allografts and Facilitate Induction of Conventional CD4+Foxp3+ Regulatory T Cells

CD4⁺ regulatory T cells play a critical role in tolerance induction in transplantation. CD8⁺ suppressor T cells have also been shown to control alloimmune responses in preclinical and clinical models. However, the exact nature of the CD8⁺ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen‐specific CD8⁺Foxp3⁺ T cells can be induced and significantly expanded by stimulating naïve CD8⁺ T cells with donor dendritic cells in the presence of IL‐2, TGF‐β1 and retinoic acid. These CD8⁺Foxp3⁺ T cells express enhanced levels of CTLA‐4, CCR4 and CD103, inhibit the up‐regulation of costimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor‐specific and contact‐dependent manner. Importantly, upon adoptive transfer, the induced CD8⁺Foxp3⁺ T cells protect full MHC‐mismatched skin allografts. In vivo, the CD8⁺Foxp3⁺ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4⁺Foxp3⁺ T cells and concurrently suppress effector T cell expansion. We conclude that donor‐specific CD8⁺Foxp3⁺ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.     

Differential Role of Naïve and Memory CD4+ T‐Cell Subsets in Primary Alloresponses

The T cell response to major histocompatibility complex (MHC) alloantigens occurs via two main pathways. The direct pathway involves the recognition of intact allogeneic MHC:peptide complexes on donor cells and provokes uniquely high frequencies of responsive T cells. The indirect response results from alloantigens being processed like any other protein antigen and presented as peptide by autologous antigen‐presenting cells. The frequencies of T cells with indirect allospecificity are orders of magnitude lower and comparable to other peptide‐specific responses. In this study, we explored the contributions of naïve and memory CD4⁺ T cells to these two pathways. Using an adoptive transfer and skin transplantation model we found that naive and memory CD4⁺ T cells, both naturally occurring and induced by sensitization with multiple third‐party alloantigens, contributed equally to graft rejection when only the direct pathway was operative. In contrast, the indirect response was predominantly mediated by the naïve subset. Elimination of regulatory CD4⁺CD25⁺ T cells enabled memory cells to reject grafts through the indirect pathway, but at a much slower tempo than for naïve cells. These findings have implications for better targeting of immunosuppression to inhibit immediate and later forms of alloimmunity.     

Relative Resistance of Human CD4+ Memory T Cells to Suppression by CD4+CD25+ Regulatory T Cells

Successful expansion of functional CD4⁺CD25⁺ regulatory T cells (T_reg) ex vivo under good manufacturing practice conditions has made T_reg‐cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, T_reg cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4⁺CD25^? T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti‐CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that T_reg cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human T_reg‐cell therapy, it is important to define the effectiveness of T_reg cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded T_reg cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that T_reg cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of T_reg cells.     

Siplizumab selectively depletes effector memory T cells and promotes a relative expansion of alloreactive regulatory T cells in vitro

Siplizumab, a humanized anti‐CD2 monoclonal antibody, has been used in conditioning regimens for hematopoietic cell transplantation and tolerance induction with combined kidney‐bone marrow transplantation. Siplizumab‐based tolerance induction regimens deplete T cells globally while enriching regulatory T cells (Tregs) early posttransplantation. Siplizumab inhibits allogeneic mixed‐lymphocyte reactions (MLRs) in vitro. We compared the impact of siplizumab on Tregs versus other T cell subsets in HLA‐mismatched allogeneic MLRs using PBMCs. Siplizumab predominantly reduced the percentage of CD4⁺ and CD8⁺ effector memory T cells, which express higher CD2 levels than naïve T cells or resting Tregs. Conversely, siplizumab enriched proliferating CD45RA^? FoxP3^HI cells in MLRs. FoxP3 expression was stable over time in siplizumab‐containing cultures, consistent with enrichment for bona fide Tregs. Consistently, high‐throughput TCRβ CDR3 sequencing of sorted unstimulated and proliferating T cells in MLRs revealed selective expansion of donor‐reactive Tregs along with depletion of donor‐reactive CD4⁺ effector/memory T cells in siplizumab‐containing MLRs. These results indicate that siplizumab may have immunomodulatory functions that may contribute to its success in tolerance‐inducing regimens. Our studies also confirm that naïve in addition to effector/memory T cells contribute to the allogeneic MLR and mandate further investigation of the impact of siplizumab on alloreactive naïve T cells.     

Alefacept Promotes Immunosuppression‐Free Renal Allograft Survival in Nonhuman Primates via Depletion of Recipient Memory T Cells

Renal allograft tolerance has been achieved in MHC‐mismatched primates via nonmyeloablative conditioning beginning 6 days prior to planned kidney and donor bone marrow transplantation (DBMT). To extend the applicability of this approach to deceased donor transplantation, we recently developed a novel‐conditioning regimen, the “delayed protocol” in which donor bone marrow (DBM) is transplanted several months after kidney transplantation. However, activation/expansion of donor‐reactive CD8⁺ memory T cells (TMEM) occurring during the interval between kidney and DBM transplantation impaired tolerance induction using this strategy. In the current study, we tested whether, Alefacept, a fusion protein which targets LFA‐3/CD2 interactions and selectively depletes CD2^highCD8⁺ effector memory T cells (TEM) could similarly induce long‐term immunosuppression‐free renal allograft survival but avoid the deleterious effects of anti‐CD8 mAb treatment. We found that Alefacept significantly delayed the expansion of CD2^high cells including CD8⁺ TEM while sparing naïve CD8⁺ T and NK cells and achieved mixed chimerism and long‐term immunosuppression‐free renal allograft survival. In conclusion, elimination of CD2^high T cells represents a promising approach to prevent electively the expansion/activation of donor‐reactive TEM and promotes tolerance induction via the delayed protocol mixed chimerism approach.

     

The Role of Regulatory Cells in Miniature Swine Rendered Tolerant to Cardiac Allografts by Donor Kidney Cotransplantation

To determine the mechanism by which cotransplantation of a kidney allograft induces tolerance to a donor heart in miniature swine, we examined the role of CD25⁺ cells in heart/kidney recipients. Tolerance was induced to class‐I MHC mismatched hearts by cotransplanting a donor‐specific kidney with a 12‐day course of cyclosporine. Peripheral blood leukocytes (PBL) were isolated from tolerant heart/kidney recipients and used in cell‐mediated lympholysis (CML) coculture assays as either unmodified PBL, PBL enriched for CD25⁺ cells or PBL depleted of CD25⁺ cells to assess their ability to suppress CML responses of naïve recipient‐matched leukocytes against mismatched target cells. Primed PBL from tolerant heart/kidney recipients completely suppressed lysis by naïve cells. Complete suppression of the response of naïve recipient‐matched leukocytes against donor‐matched target cells was lost following the depletion of CD25⁺ cells from tolerant heart/kidney animal PBL, but it was reestablished by incubation of naïve cells with small populations of CD25⁺ cells from tolerant heart/kidney animals. These data suggest that peripheral blood from tolerant heart/kidney recipients contains regulatory cells that, upon priming, can suppress the response of naïve‐matched PBL in coculture CML assays, and that suppression appears to be dependent on cells expressing CD25.     

Uremia‐associated immunological aging is stably imprinted in the T‐cell system and not reversed by kidney transplantation

The uremia‐induced inflammatory environment in end‐stage renal disease (ESRD) patients is associated with premature T‐cell aging resulting in a defective T‐cell immunity. As kidney transplantation (KTx) reduces the pro‐inflammatory environment, we hypothesized that KTx would rejuvenate the aged T‐cell system. As aging parameters, we determined in 70 KTx recipients the differentiation status by immunophenotyping, thymic output by the T‐cell receptor excision circle (TREC) content together with CD31⁺ naïve T‐cell numbers and the relative telomere length (RTL) as a measure for proliferative history at pre‐KTx, 3, 6 and 12 months post‐KTx. In addition, T‐cell function was determined by measuring the proliferative capacity and percentages of cytokine‐producing cells. Directly post‐KTx, memory T‐cell numbers were diminished but restored to pre‐KTx values at 12 months, except for CD4⁺EM T cells. The RTL of (memory) CD4⁺ and CD8⁺ T cells did not change. In contrast, TREC content and CD31⁺ naïve T‐cell numbers were stable post‐KTx although the RTL of naïve CD4⁺ and CD8⁺ T cells decreased implying homeostatic proliferation of naïve cells, in response to a temporary decrease in memory cells. The T‐cell function was not improved post‐KTx. Our findings demonstrate that the uremia‐associated aged phenotype is stably imprinted in the T‐cell system and not reversed by KTx.     

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. Interleukin-4 does not maintain alloantigen specific CD4+CD25+ Treg

IL-4 is thought to promote induction of transplantation tolerance and alloantigen-specific CD4⁺CD25⁺ T regulatory cells (Treg). This study examined the effect of IL-4 on the induction and maintenance of the CD4⁺ T regulatory cells (Treg) that mediate transplantation tolerance. Tolerance was induced in DA rats with PVG heterotopic cardiac allografts by a short course of cyclosporine. Naïve and tolerant lymphocytes, including the CD4⁺ and CD4⁺CD25⁺ T cell subsets, were assayed in mixed lymphocyte cultures with or without recombinant (r)IL-4 or other cytokines. The proliferation, cell surface and cytokine phenotype of these cells was examined, as was their capacity to adoptively transfer tolerance. rIL-4 enhanced the proliferation of naïve and tolerant lymphoid cells, including CD4⁺ and CD4⁺CD25⁺ T cells, but this was not alloantigen specific. Naïve or tolerant CD4⁺ T cells cultured with rIL-4 and donor PVG antigen effected rapid graft rejection, even though before culture tolerant CD4⁺ T cells transferred antigen-specific tolerance. These rIL-4 cultured CD4⁺ T cells had a phenotype consistent with activated CD4⁺CD25⁺FoxP3⁻ Th2 cells. While naïve natural CD4⁺CD25⁺ T cells (nTreg) cultured with alloantigen and rIL-4 had enhanced proliferation and capacity to suppress rejection in vivo, the culture of tolerant CD4⁺CD25⁺ T cells with alloantigen and rIL-4 could not sustain their proliferation against specific donor, nor their capacity to transfer tolerance to specific donor allograft. Thus, IL-4 promotes both regulatory and effector T cells early in the immune response, but once alloimmune tolerance is established, IL-4 promoted the activation of effector cells to mediate rejection and did not support alloantigen-specific Treg that could transfer specific tolerance.     

Human CD45RA− FoxP3hi Memory‐Type Regulatory T Cells Show Distinct TCR Repertoires With Conventional T Cells and Play an Important Role in Controlling Early Immune Activation

Adoptive immunotherapy with regulatory T cells (Treg) is a new option to promote immune tolerance following solid organ transplantation (SOT). However, Treg from elderly patients awaiting transplantation are dominated by the CD45RA^?CD62L⁺ central memory type Treg subset (TregCM), and the yield of well‐characterized and stable naïve Treg (TregN) is low. It is, therefore, important to determine whether these TregCM are derived from the thymus and express high stability, suppressive capacity and a broad antigen repertoire like TregN. In this study, we showed that TregCM use a different T cell receptor (TCR) repertoire from conventional T cells (Tconv), using next‐generation sequencing of all 24 Vβ families, with an average depth of 534 677 sequences. This showed almost no contamination with induced Treg. Furthermore, TregCM showed enhanced suppressive activity on Tconv at early checkpoints of immune activation controlling activation markers expression and cytokine secretion, but comparable inhibition of proliferation. Following in vitro expansion under mTOR inhibition, TregCM expanded equally as well as TregN without losing their function. Despite relatively limited TCR repertoire, TregCM also showed specific alloresponse, although slightly reduced compared to TregN. These results support the therapeutic usefulness of manufacturing Treg products from CD45RA^?CD62L⁺ Treg‐enriched starting material to be applied for adoptive Treg therapy.

     

Anti‐TCRβ mAb Induces Long‐Term Allograft Survival by Reducing Antigen‐Reactive T Cells and Sparing Regulatory T Cells

TCR specific antibodies may modulate the TCR engagement with antigen–MHC complexes, and in turn regulate in vivo T cell responses to alloantigens. Herein, we found that in vivo administration of mAbs specific for mouse TCRβ (H57–597), TCRα or CD3 promptly reduced the number of CD4⁺ and CD8⁺ T cells in normal mice, but H57–597 mAb most potently increased the frequency of CD4⁺Foxp3⁺ Treg cells. When mice were injected with staphylococcal enterotoxin B (SEB) superantigen and H57–597 mAb, the expansion of SEB‐reactive Vβ8⁺ T cells was completely abrogated while SEB‐nonreactive Vβ2⁺ T cells remained unaffected. More importantly, transient H57–597 mAb treatment exerted long‐lasting effect in preventing T cell responses to alloantigens, and produced long‐term cardiac allograft survival (>100 days) in 10 out of 11 recipients. While Treg cells were involved in maintaining donor‐specific long‐term graft survival, T cell homeostasis recovered over time and immunity was retained against third party allografts. Moreover, transient H57–597 mAb treatment significantly prolonged survival of skin allografts in naïve recipients as well as heart allografts in skin‐sensitized recipients. Thus, transient modulation of the TCRβ chain by H57–597 mAb exhibits potent, long‐lasting therapeutic effects to control alloimmune responses.     

Low‐Dose IL‐2 for In Vivo Expansion of CD4+ and CD8+ Regulatory T Cells in Nonhuman Primates

IL‐2 is a known potent T cell growth factor that amplifies lymphocyte responses in vivo. This capacity has led to the use of high‐dose IL‐2 to enhance T cell immunity in patients with AIDS or cancer. However, more recent studies have indicated that IL‐2 is also critical for the development and peripheral expansion of regulatory T cells (Tregs). In the current study, low‐dose IL‐2 (1 million IU/m² BSA/day) was administered to expand Tregs in vivo in naïve nonhuman primates. Our study demonstrated that low‐dose IL‐2 therapy significantly expanded peripheral blood CD4⁺ and CD8⁺ Tregs in vivo with limited expansion of non‐Treg cells. These expanded Tregs are mainly CD45RA^? Foxp3 ^high activated Tregs and demonstrated potent immunosuppressive function in vitro. The results of this preclinical study can serve as a basis to develop Treg immunotherapy, which has significant therapeutic potential in organ/cellular transplantation.     

Expression profiles of adhesion molecules on naïve T cells in bone marrow grafts of healthy donors treated with granulocyte colony-stimulating factor

Background and objectivesAdhesion molecules on T cells were implicated in the process of leukocyte migration and GVHD. The aim of this study was to investigate the profiles of adhesion molecule expression on naïve T cells in bone marrow grafts of healthy donors treated with G-CSF.MethodsThe expression of four adhesion molecules, including VLA-4, ICAM-1, L-selectin, and LFA-1, on naïve T cells in G-CSF-primed bone marrow grafts (G-BM) from 35 healthy donors was analyzed using flow cytometry.ResultsG-CSF administration significantly decreased the expression of VLA-4, ICAM-1, L-selectin, and LFA-1 on naïve CD4⁺ and CD8⁺ T cells in bone marrow grafts. Treating healthy donors with G-CSF significantly increased the percentages of interleukin-4 (IL-4) positive cells in naïve CD4⁺ and CD8⁺ T cell subsets, leading to polarization of bone marrow naïve CD4⁺ and CD8⁺ T cells from Th1 to Th2 phenotype.ConclusionsOur results suggest that CD4⁺ and CD8⁺ naïve T cells in G-BM have lower intensities of expression of several adhesion molecules. The clinical significance of these alteration warrants further study.     

Morphological reseach on expression of inflammatory mediators in murine models of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) induced by T2 antigen

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common clinical syndrome with unknown aetiology. In this study, we used the T2 peptide in C57BL/6 (B6) mice and Sprague Dawley (SD) rats model during different stages. We sought to understand the role of CD4⁺ T cells and macrophages in CP/CPPS. A total of 16 B6 mice and 18 SD rats were divided into five groups: B6‐naïve (n = 6), B6 model (n = 10), SD‐naïve (n = 6), SD‐45‐day model (n = 6) and SD‐56‐day model (n = 6). The B6 model group was subcutaneously injected with 0.2 ml of (225μg/ml) T2 peptide on 0 and 14th day and was finally sacrificed on 28th day. The SD‐45‐ and SD‐56‐day model groups were subcutaneously injected with 1ml of (50 μg/ml) T2 peptide on 0 and 14th day and were finally sacrificed on 45th and 56th day respectively. An equivalent volume of normal saline (NS) solution was injected to the naïve groups and analysed the pain and voiding behaviour. We have calculated the prostate index, H&E staining and immunofluorescence of CD4⁺ T cells and macrophages (CD68) in each group. T2 peptide immunization in B6 mice and SD rats caused severe prostatitis and cell infiltration, mainly composed of CD4⁺ T cells and macrophages. The SD‐56‐day model group showed more severe inflammatory cells infiltration than SD‐45‐day model group. Moreover, inflammatory cells infiltration and red secretions in B6 model were less than SD model. Expression of CD4⁺ T cells and macrophages was also consistent with H&E results. These results indicated that different stages of CP/CPPS, inflammatory response, and the inflammation of the rat were stronger than the mouse. Our study suggests that CD4⁺ T cells and macrophages are key factors in the development of CP/CPPS.     

Regulatory T cells in the induction and maintenance of peripheral transplantation tolerance

It is now possible to induce donor-specific transplantation tolerance in adult rodents using non-depleting monoclonal antibodies against T cell co-receptor and co-stimulation molecules or by immunisation with tolerogenic antigen-presenting cells. It is a common finding of all these models of peripheral tolerance, as well as of various mouse models of autoimmune disease, that regulatory CD4⁺ T cells are the principal mediators. There are currently no specific markers for regulatory T cells, but in some autoimmune models their activity has been associated with the expression of activation markers such as CD25 and CTLA4, or anti-inflammatory cytokines such as IL-10 and TGF-β. CD4⁺CD25⁺ T cells from both naïve and tolerised donors are able to transfer tolerance to grafts in lymphopenic recipients, and this may be directly applicable to bone-marrow transplantation. The challenge is now to understand the biological principles that allow such immune re-programming so that they can be safely applied to clinical organ grafting.     

Amnion‐Derived Multipotent Progenitor Cells Support Allograft Tolerance Induction

Donor‐specific immunological tolerance using high doses of bone marrow cells (BMCs) has been demonstrated in mixed chimerism‐based tolerance induction protocols; however, the development of graft versus host disease remains a risk. Here, we demonstrate that the co‐infusion of limited numbers of donor unfractionated BMCs with human amnion‐derived multipotent progenitor cells (AMPs) 7 days post–allograft transplantation facilitates macrochimerism induction and graft tolerance in a mouse skin transplantation model. AMPs + BMCs co‐infusion with minimal conditioning led to stable, mixed, multilineage lymphoid and myeloid macrochimerism, deletion of donor‐reactive T cells, expansion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_regs) and long‐term allograft survival (>300 days). Based on these findings, we speculate that AMPs maybe a pro‐tolerogenic cellular therapeutic that could have clinical efficacy for both solid organ and hematopoietic stem cell transplant applications.     

Different Mechanisms Control Peripheral and Central Tolerance in Hematopoietic Chimeric Mice

Regulatory T cells (Treg) are important in peripheral tolerance, but their role in establishing and maintaining hematopoietic mixed chimerism and generating central tolerance is unclear. We now show that costimulation blockade using a donor‐specific transfusion and anti‐CD154 antibody applied to mice given bone marrow and simultaneously transplanted with skin allografts leads to hematopoietic chimerism and permanent skin allograft survival. Chimeric mice bearing intact skin allografts fail to generate effector/memory T cells against allogeneic targets as shown by the absence of IFNγ‐producing CD44^highCD8⁺ T cells and in vivo cytotoxicity. Depletion of Tregs by injection of anti‐CD4 or anti‐CD25 antibody prior to costimulation blockade prevents chimerism, shortens skin allograft survival and leads to generation of effector/memory cytotoxic T cells. Depletion of Tregs by injection of anti‐CD4 or anti‐CD25 antibody two months after transplantation leads to loss of skin allografts even though mice remain chimeric and exhibit little in vivo cytotoxicity. In contrast, chimerism is lost, but skin allografts survive following naïve T‐cell injection. We conclude that hematopoietic chimerism and peripheral tolerance may be maintained by different mechanisms in mixed hematopoietic chimeras.     

Permanent CNI Treatment for Prevention of Renal Allograft Rejection in Sensitized Hosts Can Be Replaced by Regulatory T Cells

Recent data suggest that donor‐specific memory T cells (T_mem) are an independent risk factor for rejection and poor graft function in patients and a major challenge for immunosuppression minimizing strategies. Many tolerance induction protocols successfully proven in small animal models e.g. costimulatory blockade, T cell depletion failed in patients. Consequently, there is a need for more predictive transplant models to evaluate novel promising strategies, such as adoptive transfer of regulatory T cells (Treg). We established a clinically more relevant, life‐supporting rat kidney transplant model using a high responder (DA to LEW) recipients that received donor‐specific CD4⁺/ 8⁺ GFP⁺ T_mem before transplantation to achieve similar pre‐transplant frequencies of donor‐specific T_mem as seen in many patients. T cell depletion alone induced long‐term graft survival in naïve recipients but could not prevent acute rejection in T_mem⁺ rats, like in patients. Only if T cell depletion was combined with permanent CNI‐treatment, the intragraft inflammation, and acute/chronic allograft rejection could be controlled long‐term. Remarkably, combining 10 days CNI treatment and adoptive transfer of Tregs (day 3) but not Treg alone also induced long‐term graft survival and an intragraft tolerance profile (e.g. high TOAG‐1) in T_mem⁺ rats. Our model allows evaluation of novel therapies under clinically relevant conditions.