The Potency of Allospecific Tregs Cells Appears to Correlate With T Cell Receptor Functional Avidity

CD4⁺CD25⁺ regulatory T cells (T_reg cells) are an attractive adoptive cell therapy in mediating transplantation tolerance. T‐cell receptor (TcR) activation is critical for T_reg function, suggesting that the TcR avidity of T_reg cells used in therapy may affect the therapeutic outcome. To address this, we compared the regulatory capacity of T_reg lines expressing TcRs derived from two TcR transgenic mice shown to have the same specificity but different functional avidities. T_reg lines generated from CD4⁺CD25⁺ T cells from C57BL/6 mice were transduced with one of either of these TcRs. The antigen specificity of the transduced T_reg lines was confirmed in vitro. T_reg lines expressing the TcR with higher functional avidity showed stronger suppressive capacity in a linked suppression model in vitro. Furthermore, the same T_reg lines demonstrated a stronger proliferation in vivo following antigen exposure. Pretreatment of recipient BL/6 mice with these T_reg cells, together with anti‐CD8 antibody and Rapamycin therapies, prolonged survival of BALB/c skins, as compared with mice that received T_reg lines with lower TcR avidity. Taken together, these data suggest that the TcR functional avidity may be important for T_reg function. It highlights the fact that strategies to select T_reg with higher functional avidity might be beneficial for immunotherapy 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.     

Low‐Dose Rapamycin Treatment Increases the Ability of Human Regulatory T Cells to Inhibit Transplant Arteriosclerosis In Vivo

Regulatory T cells (T_reg) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote T_reg expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human T_reg to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low‐dose rapamycin and subtherapeutic T_reg numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2^?/?Il2rg^?/? mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T‐cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4⁺ but not CD8⁺ T lymphocytes were sensitive to T_reg and rapamycin‐induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of T_reg‐based immunosuppressive protocols in clinical practice. By inhibiting TA, T_reg and rapamycin may prevent chronic transplant dysfunction and improve long‐term allograft survival.     

Isolated CD39 Expression on CD4+ T Cells Denotes both Regulatory and Memory Populations

Foxp3⁺ regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4⁺CD39⁺ T‐cell pool contains two roughly equal size Foxp3⁺ and Foxp3^? populations. While Foxp3⁺CD39⁺ cells are CD73^bright and are the bone fide Tregs, Foxp3^?CD39⁺ cells do not have suppressive activity and are CD44⁺CD62L^?CD25^?CD73^dim/?, exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP‐induced apoptosis. Compared with Foxp3^?CD39^? naïve T cells, Foxp3^?CD39⁺ cells freshly isolated from non‐immunized mice express at rest significantly higher levels of mRNA for T‐helper lineage‐specific cytokines IFN‐γ (Th1), IL‐4/IL‐10 (Th2), IL‐17A/F (Th17), as well as pro‐inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3^?CD39⁺ cells inhibits TGF‐β induction of Foxp3 in Foxp3^?CD39^? cells. Furthermore, when transferred in vivo, Foxp3^?CD39⁺ cells rejected MHC‐mismatched skin allografts in a much faster tempo than Foxp3^?CD39^? cells. Thus, besides Tregs, CD39 is also expressed on pre‐existing memory T cells of Th1‐, Th2‐ and Th17‐types with heightened alloreactivity.     

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.     

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.     

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.     

Allergic Conjunctivitis Renders CD4+ T Cells Resistant to T Regulatory Cells and Exacerbates Corneal Allograft Rejection

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T‐cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL‐4, but not IL‐5 or IL‐13, prevented Treg suppression of CD4⁺ effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4⁺ effector T‐cell proliferation. In addition, IL‐4 did not inhibit Treg suppression of IL‐4Rα^?/? CD4⁺ T‐cell responses, suggesting that IL‐4 rendered effector T cells resistant to Tregs. SRW‐sensitized IL‐4Rα^?/? mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL‐4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti‐IL‐4 antibody. Thus, allergy‐induced exacerbation of corneal graft rejection is due to the production of IL‐4, which renders effector T cells resistant to Treg suppression of alloimmune responses.     

Rapamycin‐Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF‐β1 for Islet Transplantation

Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin‐conditioned donor DCs could effectively induce CD4⁺CD25⁺Foxp3⁺ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro‐differentiated CD4⁺CD25⁺Foxp3⁺ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow‐derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4⁺CD25⁺Foxp3⁺ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro‐induced CD4⁺CD25⁺Foxp3⁺ iTregs exerted donor‐specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG^?/‐ hosts upon coadoptive transfer with T‐effector cells. The CD4⁺CD25⁺Foxp3⁺ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4⁺CD25⁺Foxp3⁺ iTregs were further able to induce endogenous naïve T cells to convert to CD4⁺CD25⁺Foxp3⁺ T cells. We conclude that rapamycin‐conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen‐specific CD4⁺CD25⁺Foxp3⁺ iTregs. Such in vitro‐generated donor‐specific CD4⁺CD25⁺Foxp3⁺ iTregs are able to effectively control allogeneic islet graft rejection.     

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 of CD39 by Human Peripheral Blood CD4+CD25+ T Cells Denotes a Regulatory Memory Phenotype

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4⁺Foxp3⁺ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood‐derived human CD4⁺CD25⁺CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4⁺CD39⁺ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4⁺ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL‐17. These latter cell populations are increased, with a concomitant decrease in the CD4⁺CD25⁺CD39⁺ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell‐populations to allow tracking of these in health and disease, as in renal allograft rejection.     

Contrasting Effects of Cyclosporine and Rapamycin in De Novo Generation of Alloantigen-Specific Regulatory T Cells

The outcome of T‐cell‐mediated responses, immunity or tolerance, critically depends on the balance of cytopathic versus regulatory T (T_reg) cells. In the creation of stable tolerance to MHC incompatible allografts, reducing the unusually large mass of donor‐reactive cytopathic T effector (T_eff) cells via apoptosis is often required. Cyclosporine (CsA) blocks activation‐induced cell death (AICD) of T_eff cells, and is detrimental to tolerance induction by costimulation blockade, whereas Rapamycin (RPM) preserves AICD, and augments the potential of costimulation blockade to create tolerance. While differences between CsA and RPM in influencing apoptosis of activated graft‐destructive T_eff cells are apparent, their effects on graft‐protective T_reg cells remain enigmatic. Moreover, it is unclear whether tolerizing regimens foster conversion of naïve peripheral T cells into alloantigen‐specific T_reg cells for graft protection. Here we show, using reporter mice for T_reg marker Foxp3, that RPM promotes de novo conversion of alloantigen‐specific T_reg cells, whereas CsA completely inhibits this process. Upon transfer, in vivo converted T_reg cells potently suppress the rejection of donor but not third party skin grafts. Thus, the differential effects of RPM and CsA on T_eff and T_reg cells favor the use of RPM in shifting the balance of aggressive to protective type alloimmunity.     

The Changed Balance of Regulatory and Naive T Cells Promotes Tolerance after TLI and Anti‐T‐Cell Antibody Conditioning

The goal of the study was to determine how the changed balance of host naïve and regulatory T cells observed after conditioning with total lymphoid irradiation (TLI) and antithymocyte serum (ATS) promotes tolerance to combined organ and bone marrow transplants. Although previous studies showed that tolerance was dependent on host natural killer T (NKT) cells, this study shows that there is an additional dependence on host CD4⁺CD25⁺ Treg cells. Depletion of the latter cells before conditioning resulted in rapid rejection of bone marrow and organ allografts. The balance of T‐cell subsets changed after TLI and ATS with TLI favoring mainly NKT cells and ATS favoring mainly Treg cells. Combined modalities reduced the conventional naïve CD4⁺ T cells 2800‐fold. The host type Treg cells that persisted in the stable chimeras had the capacity to suppress alloreactivity to both donor and third party cells in the mixed leukocyte reaction. In conclusion, tolerance induction after conditioning in this model depends upon the ability of naturally occurring regulatory NKT and Treg cells to suppress the residual alloreactive T cells that are capable of rejecting grafts.     

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.     

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.

     

The Jak Inhibitor CP‐690,550 Preserves the Function of CD4+CD25brightFoxP3+ Regulatory T Cells and Inhibits Effector T Cells

The Jak inhibitor CP‐690,550 inhibits alloreactivity and is currently being investigated for prevention of allograft rejection after transplantation. In this study, we examined the effect of CP‐690,550 on IL‐2‐mediated Jak/STAT5 phosphorylation by CD4⁺CD25^brightFoxP3⁺CD127^?/low T cells (Treg) and CD4⁺CD25^neg effector T cells (Teff) in kidney transplant (KTx) patients. Phosphospecific flow cytometry was used to study the effect of CP‐690,550 on IL‐2‐induced intracellular STAT5‐phosphorylation. IL‐2‐induced phosphorylation of STAT5 (P‐STAT5) in both Treg and Teff, which was significantly higher for CD4⁺CD25^bright Treg (increased by 71%, mean) than for CD4⁺CD25^neg Teff (increased by 42%). In the presence of 100 ng/mL CP‐690,550, a clinically relevant exposure, IL‐2‐induced P‐STAT5 was partially inhibited in CD4⁺CD25^brightTreg (% inhibition; 51%), while almost completely blocked in Teff (%inhibition; 84%, p = 0.03). The IC₅₀ was 2–3 times higher for Treg (104 ng/mL) than for Teff (40 ng/mL, p = 0.02). In the presence of CP‐690,550, Treg exhibited additional suppressive activities on the alloactivated proliferation of Teff (56%, mean). In addition, CD4⁺CD25^bright Treg from KTx‐patients receiving CP‐690,550 vigorously suppressed the proliferation of Teff (87%, mean). Our findings show that CP‐690,550 effectively inhibits Teff function but preserves the suppressive activity of CD4⁺CD25^bright regulatory T cells.     

Regulatory T Cells Exhibit Decreased Proliferation but Enhanced Suppression After Pulsing With Sirolimus

Although regulatory T cells (Tregs) suppress allo‐immunity, difficulties in their large‐scale production and in maintaining their suppressive function after expansion have thus far limited their clinical applicability. Here we have used our nonhuman primate model to demonstrate that significant ex vivo Treg expansion with potent suppressive capacity can be achieved and that Treg suppressive capacity can be further enhanced by their exposure to a short pulse of sirolimus. Both unpulsed and sirolimus‐pulsed Tregs (SPTs) are capable of inhibiting proliferation of multiple T cell subpopulations, including CD4⁺ and CD8⁺ T cells, as well as antigen‐experienced CD28⁺CD95⁺ memory and CD28^?CD95⁺ effector subpopulations. We further show that Tregs can be combined in vitro with CTLA4‐Ig (belatacept) to lead to enhanced inhibition of allo‐proliferation. SPTs undergo less proliferation in a mixed lymphocyte reaction (MLR) when compared with unpulsed Tregs, suggesting that Treg‐mediated suppression may be inversely related to their proliferative capacity. SPTs also display increased expression of CD25 and CTLA4, implicating signaling through these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue to enhance Treg‐based suppression of allo‐immunity, in a manner amenable to large‐scale ex vivo expansion and combinatorial therapy with novel, costimulation blockade‐based immunosuppression strategies.     

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.     

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.