Alloantigen-specific Tregs Research Articles

Polyclonally Derived Alloantigen-Specific T Regulatory Cells Exhibit Target-Specific Suppression and Capture MHC Class II from Dendritic Cells.

Foxp3+ T regulatory (Treg) cells prevent allograft rejection and graft-versus-host disease. Although polyclonal Tregs have been used both in animal models and in humans, the fine specificity of their suppressive function is poorly defined. We have generated mouse recipient-derived alloantigen-specific Tregs invitro and explored the fine specificity of their suppressive function and their mechanism of action invitro and invivo. In vitro, when alloantigen and peptide Ag were both presented on the same dendritic cell, both responses were suppressed by iTregs specific either for the alloantigen or for the peptide Ag. In vivo, iTreg suppression was limited to the cognate Ag, and no bystander suppression was observed when both allo-antigen and peptide Ag were present on the same dendritic cell. In vitro, alloantigen-specific Tregs captured cognate MHC but failed to capture noncognate MHC. Our results demonstrate that a polyclonal population of iTregs generated from naive T cells can mediate highly specific function invivo and support the view that Treg therapy, even with unselected polyclonal populations, is likely to be target antigen-specific and that bystander responses to self-antigens or to infectious agents are unlikely.

Nanoparticles loaded with IL-2 and TGF-β promote transplantation tolerance to alloantigen.

We have previously reported that nanoparticles (NPs) loaded with IL-2 and TGF-β and targeted to T cells induced polyclonal T regulatory cells (Tregs) that protected mice from graft-versus-host disease (GvHD). Here, we evaluated whether administration of these NPs during alloantigen immunization could prevent allograft rejection by converting immunogenic responses to tolerogenic ones. Using C57BL/6 mice and BALB/c mice as either donors or recipients of allogeneic splenocytes, we found that treatment with the tolerogenic NPs in both strains of mice resulted in a marked inhibition of mixed lymphocyte reaction (MLR) to donor cell alloantigen but not to third-party control mouse cells after transfer of the allogeneic cells. The decreased alloreactivity associated with a four- to fivefold increase in the number of CD4+ and CD8+ T regulatory cells (Tregs) and the acquisition of a tolerogenic phenotype by recipient dendritic cells (DCs) in NP-treated mice. As allogeneic cells persisted in NP-treated mice, these findings suggest that tolerogenic NPs can induce alloantigen-specific Tregs and tolerogenic DCs promoting tolerogenic responses to alloantigen. By inhibiting reactivity to allotransplant, this approach could help reduce the need for immune suppression for the maintenance of allografts.

Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines.

The adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited due to their low frequency in peripheral blood and the scarce development of efficient protocols to ensure their purity, expansion, and stability. Here, we describe a new experimental protocol that allows the long-term expansion of highly purified allospecific natural Tregs (nTregs) from both healthy controls and chronic kidney disease (CKD) patients, which maintain their phenotype and suppressive function under inflammatory conditions. Firstly, we co-cultured CellTrace Violet (CTV)-labeled Tregs from CKD patients or healthy individuals with allogeneic monocyte-derived dendritic cells in the presence of interleukin 2 (IL-2) and retinoic acid. Then, proliferating CD4+CD25hiCTV− Tregs (allospecific) were sorted by fluorescence-activated cell sorting (FACS) and polyclonally expanded with anti-CD3/CD28-coated beads in the presence of transforming growth factor beta (TGF-β), IL-2, and rapamycin. After 4 weeks, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded alloTregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of inflammatory cytokines (IFN-γ, IL-4, IL-6, or TNF-α). Unexpectedly, the long-term expansion resulted in an increased methylation of the specific demethylated region of Foxp3. Interestingly, alloTregs from both normal individuals and CKD patients maintained their immunosuppressive phenotype and function after being expanded for two additional weeks under an inflammatory microenvironment. Finally, phenotypic and functional evaluation of cryopreserved alloTregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients.

Alloactivation of Naïve CD4+CD8-CD25+T Regulatory Cells: Expression of CD8α Identifies Potent Suppressor Cells That Can Promote Transplant Tolerance Induction.

Therapy with alloantigen-specific CD4+CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are not alloantigen-specific and are weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells in the presence of rIL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rβ2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined additional markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+CD25+Foxp3+Treg. After culture of naïve DA CD4+CD8−CD25+T cells with rIL-2 and PVG alloantigen, or rIL-2 without alloantigen, CD8α was expressed on 10–20% and CD8β on <5% of these cells. These cells expressed ifngr and Il12rb2. CD8α+ cells had increased Ifngr that characterizes Ts1 cells as well was Irf4, a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4+CD8α+CD25+Treg consistent with the CD8α+ cells expressing IL-12R. In MLC, the CD8α+ fraction suppressed responses against allogeneic stimulators more than the mixed Ts1 population, whereas the CD4+CD8−CD25+T cells were less potent. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8α+ cells were much less effective. This study demonstrated that expression of CD8α by rIL-2 and alloantigen activation of CD4+CD8−CD25+Foxp3+T cells was a marker of activated and potent Treg that included alloantigen-specific Treg.

Recently alloactivated CD4+CD8−CD25+T regulatory cells express CD8alpha and are potent suppressor cells

Abstract Therapy with antigen-specific CD4+CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are non-antigen specific and weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells and IL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rb2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined other markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+CD25+Foxp3+Treg. After culture of naïve DA CD4+CD8−CD25+T cells with rIL-2 and PVG alloantigen, CD8a was expressed on 10–20% and CD8b on &amp;lt;5% of these cells. RT-PCR showed the CD8+ cells had increased Ifngr that characterizes Ts1 cells as well was Irf4, a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4+CD8+CD25+Treg consistent with the CD8a+ cells expressing IL-12R. In MLC, the CD8+ fraction had greater suppression than the mixed Ts1 population, whereas the CD4+CD8−CD25+T cells had less suppression. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8a cells did not prevent rejection. This study showed the expression of CD8alpha by rIL-2 and alloantigen activation of CD4+CD8−CD25+Foxp3+T cells was a marker of alloantigen-specific Treg and may be of use to enrich alloantigen-specific Treg.

Alloactivation of Human CD4+CD25+CD127loCD45RA-Fox3hi Treg Population to Induce Potential Alloantigen Specific Treg

Alloactivation of Human CD4+CD25+CD127loCD45RA-Fox3hi Treg Population to Induce Potential Alloantigen Specific Treg

Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells.

Regulatory T cells (Tregs) are potential immunotherapeutic candidates to induce transplantation tolerance. However, stability of Tregs still remains contentious and may potentially restrict their clinical use. Recent work suggested that epigenetic imprinting of Foxp3 and other Treg-specific signature genes is crucial for stabilization of immunosuppressive properties of Foxp3+ Tregs, and that these events are initiated already during early stages of thymic Treg development. However, the mechanisms governing this process remain largely unknown. Here we demonstrate that thymic antigen-presenting cells (APCs), including thymic dendritic cells (t-DCs) and medullary thymic epithelial cells (mTECs), can induce a more pronounced demethylation of Foxp3 and other Treg-specific epigenetic signature genes in developing Tregs when compared to splenic DCs (sp-DCs). Transcriptomic profiling of APCs revealed differential expression of secreted factors and costimulatory molecules, however neither addition of conditioned media nor interference with costimulatory signals affected Foxp3 induction by thymic APCs in vitro. Importantly, when tested in vivo both mTEC- and t-DC-generated alloantigen-specific Tregs displayed significantly higher efficacy in prolonging skin allograft acceptance when compared to Tregs generated by sp-DCs. Our results draw attention to unique properties of thymic APCs in initiating commitment towards stable and functional Tregs, a finding that could be highly beneficial in clinical immunotherapy.

Natural regulatory T cells from patients with end-stage renal disease can be used for large-scale generation of highly suppressive alloantigen-specific Tregs

Natural occurring regulatory T cells (nTregs) have the potential to offer a targeted approach of immunosuppression and are the cell type of interest for inducing tolerance in kidney transplantation. End-stage renal disease (ESRD) profoundly affects the composition and function of circulating T cells but little is known with respect to how nTreg potential is affected. To address this, nTregs of patients with ESRD (on dialysis or not) and healthy individuals were isolated, expanded using allogeneic mature monocyte-derived dendritic cells followed by anti-CD3/anti-CD28-coated beads and the different nTregs were extensively characterized by the demethylation status of the Treg-specific demethylated region within FOXP3 and expression of typical nTreg markers. Additionally, the suppressive capacity as well as cytokine producing cells were analyzed for allogeneic mature monocyte-derived dendritic cell-expanded nTregs. Compared to age- and gender-matched healthy individuals, similar frequencies of nTregs were present within the circulation of patients with ESRD either on dialysis or not. The isolated nTregs could be equally well or even better expanded using allogeneic mature monocyte-derived dendritic cells and extensive characterization did not reveal significant differences. The demethylation status of the Treg-specific demethylated region was maintained or even further promoted as was the expression of markers characteristic for nTregs. Moreover, suppressive capacity and the cytokine profile of allogeneic mature monocyte-derived dendritic cell-expanded nTregs was similar to that of healthy individuals. Thus, circulating nTregs of patients with ESRD can effectively be expanded to stable allo antigen-specific nTregs with potential clinical applicability.

Allospecific Tregs Expanded After Anergization Remain Suppressive in Inflammatory Conditions but Lack Expression of Gut-homing Molecules.

Cell therapy with antigen-specific regulatory T-cells (Treg) has great potential to selectively control unwanted immune responses after allogeneic stem-cell or solid organ transplantation and in autoimmune diseases. Ex vivo allostimulation with costimulatory blockade (alloanergization) of human T-cells expands populations of alloantigen-specific Treg, providing a cellular strategy to control donor T-cell alloresponses causing graft-versus-host disease after allogeneic hematopoietic stem-cell transplantation. Crucially, it is not known if Treg expanded in this way are stable in proinflammatory conditions encountered after transplantation, or if they possess capacity to migrate to key target organs. Using an in vitro model to functionally characterize human Treg expanded after alloanergization, we now show that these cells remain potently allosuppressive in the presence of relevant exogenous inflammatory signals. Expanded allospecific Treg retained expression of molecules conferring migratory capacity to several organs but small intestine-specific chemotaxis was markedly impaired, in keeping with the preponderance of gut graft-versus-host disease in previous clinical studies using this strategy. Importantly, impaired gut-specific chemotaxis could be partially corrected by pharmacological treatment. These findings will facilitate more effective application of this cellular approach to limit T-cell alloresponses after hematopoietic stem-cell transplantation and the wider application of the strategy to other clinical settings.

Generation of alloantigen-specific T regulatory cells via a chimeric antigen receptor targeting HLA-A2

Abstract CD4+ T regulatory cells (Tregs) are of clinical interest due to their potent immunosuppressive activity in both transplantation and autoimmunity models. Clinical efficacy in humans has been limited by aspects of Treg biology, including the difficulty of isolating sufficient numbers for potency without sacrificing purity and thus function. Enriching for antigen-specific Tregs greatly increases potency, however, antigen-specific Tregs are extremely rare. The success of chimeric antigen receptor (CAR) technology in generating antigen-specific effector T cells suggested that a similar approach could be used to generate alloantigen-specific Tregs for use in transplantation. We generated a new CAR specific for HLA-A2 (A2-CAR) and tested its application in the generation of alloantigen-specific human Tregs. Naïve Tregs were sorted from human peripheral blood, activated, and transduced with A2-CAR lentivirus. Stimulation through the A2-CAR upregulated Treg activation markers and functional molecules, and resulted in proliferation. Importantly, in vitro studies showed that A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function, before, during and after HLA-A2-mediated stimulation. In vivo, A2-CAR Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic graft-versus-host disease caused by HLA-A2+ T cells. Together, these data suggest that alloantigen-specific Tregs generated by the introduction of an A2-CAR may be a potent and specific immunosuppressive treatment in transplantation.

Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

Generation of Human Alloantigen-Specific Regulatory T Cells Under Good Manufacturing Practice-Compliant Conditions for Cell Therapy.

Natural regulatory T cells (Tregs) may have a great therapeutic potential to induce tolerance in allogeneic cells and organ transplantations. In mice, we showed that alloantigen-specific Tregs (spe-Tregs) were more efficient than polyclonal Tregs (poly-Tregs) in controlling graft-versus-host disease (GVHD). Here we describe a clinical-grade compliant method for generating human spe-Tregs. Tregs were enriched from leukapheresis products with anti-CD25 immunomagnetic beads, primed twice by allogeneic mature monocyte-derived dendritic cells (mDCs), and cultured during 3 weeks in medium containing interleukin 2 (IL-2), IL-15, and rapamycin. After 3 weeks of culture, final cell products were expanded 8.3-fold from the initial CD25(+) purifications. Immunophenotypic analyses of final cells indicate that they were composed of 88 ± 2.6% of CD4(+) T cells, all expressing Treg-specific markers (FOXP3, Helios, GARP, LAP, and CD152). Spe-Tregs were highly suppressive in vitro and also in vivo using a xeno-GVHD model established in immunodeficient mice. The specificity of their suppressive activity was demonstrated on their ability to significantly suppress the proliferation of autologous effector T cells stimulated by the same mDCs compared to third-party mDCs. Our data provide evidence that functional alloantigen Tregs can be generated under clinical-grade compliant conditions. Taking into account that 130 × 10(6) CD25(+) cells can be obtained at large scale from standard leukapheresis, our cell process may give rise to a theoretical final number of 1 × 10(9) spe-Tregs. Thus, using our strategy, we can propose to prepare spe-Tregs for clinical trials designed to control HLA-mismatched GVHD or organ transplantation rejection.

Alloreactive Regulatory T Cells Allow the Generation of Mixed Chimerism and Transplant Tolerance.

The induction of donor-specific transplant tolerance is one of the main goals of modern immunology. Establishment of a mixed chimerism state in the transplant recipient has proven to be a suitable strategy for the induction of long-term allograft tolerance; however, current experimental recipient preconditioning protocols have many side effects, and are not feasible for use in future therapies. In order to improve the current mixed chimerism induction protocols, we developed a non-myeloablative bone-marrow transplant (NM-BMT) protocol using retinoic acid (RA)-induced alloantigen-specific Tregs, clinically available immunosuppressive drugs, and lower doses of irradiation. We demonstrate that RA-induced alloantigen-specific Tregs in addition to a NM-BMT protocol generates stable mixed chimerism and induces tolerance to allogeneic secondary skin allografts in mice. Therefore, the establishment of mixed chimerism through the use of donor-specific Tregs rather than non-specific immunosuppression could have a potential use in organ transplantation.

Alloantigen-specific CD4+ regulatory T cells induced in vivo by ultraviolet irradiation after alloantigen immunization require interleukin-10 for their induction and activation, and flexibly mediate bystander immunosuppression of allograft rejection

Ultraviolet (UV) irradiation prior to antigen immunization is employed to induce antigen-specific regulatory T cells (Tregs). UV-induced Tregs demonstrate unique bystander suppression, although antigen-specific activation is required initially. We previously reported the phenotype of alloantigen-specific transferable Tregs induced by UV-B irradiation after immunization was the same as T regulatory type 1-like CD4+ T cells, with antigen-specific interleukin (IL)-10 production. Here, by using semi-allogeneic transplantation models in vivo, we investigated the role of IL-10 in the induction and activation of these Tregs, and the possibility of bystander suppression of third-party allograft rejection. Naïve mice (H-2b) were immunized with alloantigen (H-2b/d), and received UV-B irradiation (40kJ/m2) 1week later. Four weeks afterwards, splenic CD4+ T cells were purified from the UV-irradiated immunized mice, and were transferred into naïve mice (H-2b). Allografts expressing the same alloantigen as T-cell donors were immunized against (H-2b/d) or an irrelevant alloantigen (H-2b/k) were transplanted to CD4+ T-cell-transferred mice, and an alloantigen-specific prolongation of allograft survival observed. Experiments where IL-10 was neutralized by monoclonal antibody in the induction or effector phase revealed that IL-10 is critical, not only for induction but also for immunosuppressive function of CD4+ Tregs induced by UV irradiation after alloantigen immunization. Third-party allografts (H-2d/k) were transplanted to CD4+ T-cell-transferred mice, and graft survival was also prolonged. Even a graft only partially compatible with immunized alloantigen worked well in vivo to activate CD4+ Tregs induced by UV irradiation after alloantigen immunization, which resulted in the bystander suppression of third-party allograft rejection.

ECDI-fixed allogeneic splenocytes combined with α1-antitrypsin prolong survival of rat renal allografts.

Pre- and post-transplant infusions of donor splenocytes treated with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI-SPs) induce donor-specific tolerance and prolong rat renal allograft survival. However, proinflammatory cytokine production during peritransplantation negates the effects of ECDI-SPs. Therefore, we reasoned that blocking proinflammatory cytokines would promote long-term ECDI-SP-induced allograft survival. We therefore examined the effects of infusing ECDI-SPs alone or in combination with a short course of α1-Antitrypsin (AAT) on the long-term outcomes of a rat kidney allograft model. The data showed that ECDI-SPs+AAT promote renal allograft survival compared with ECDI-SPs alone. This effect was accompanied by expansion of Foxp3+ Tregs, enhanced alloantigen-specific Treg function, and modulation of expression levels of proinflammatory cytokines IL-1β, IL-6, TNF-α, and the anti-inflammatory cytokine IL-10. In conclusion, our strategy of combining ECDI-SPs and AAT provides a promising approach for inducing specific transplant tolerance.

Ultraviolet-induced alloantigen-specific immunosuppression in transplant immunity.

After the first observation of the immunosuppressive effects of ultraviolet (UV) irradiation was reported in 1974, therapeutic modification of immune responses by UV irradiation began to be investigated in the context immunization. UV-induced immunosuppression is via the action of regulatory T cells (Tregs). Antigen-specific Tregs were induced by high-dose UV-B irradiation before antigen immunization in many studies, as it was considered that functional alteration and/or modulation of antigen-presenting cells by UV irradiation was required for the induction of antigen-specific immunosuppression. However, it is also reported that UV irradiation after immunization induces antigen-specific Tregs. UV-induced Tregs are also dominantly transferable, with interleukin-10 being important for UV-induced immunosuppression. Currently, various possible mechanisms involving Treg phenotype and cytokine profile have been suggested. UV irradiation accompanied by alloantigen immunization induces alloantigen-specific transferable Tregs, which have potential therapeutic applications in the transplantation field. Here we review the current status of UV-induced antigen-specific immunosuppression on the 40(th) anniversary of its discovery.

Overexpression of Jagged-1 combined with blockade of CD40 pathway prolongs allograft survival.

Dendritic cells (DCs) have the tolerogenic potential to regulate adaptive immunity and induce allografts acceptance. Here we investigated whether blockade of the CD40 pathway could enhance the immune tolerance induced by DC2.4 cells modified to express Jagged-1 (JAG1-DC) in heart transplantation. Results showed that JAG1-DC treatment combined with anti-CD40L monoclonal antibody (mAb) administration significantly prolonged cardiac allograft survival in mice, with long-term survival (>110 days) of 50% of the allografts in the recipients. The therapy specifically inhibited the immune response, induced alloantigen-specific T-cell hyporesponsiveness, upregulated transforming growth factor-β synthesis and increased the population of regulatory T cells (Tregs) driven by Jagged-1-Notch activation. These results highlight the potential application of gene therapy to induce alloantigen-specific Tregs effectively by providing the Jagged-1 stimulation.

Expansion of Functionally Active Alloantigen-Specific Regulatory T Cells Using Activated B Cells.

Background: Achieving transplant tolerance to eliminate chronic immunosuppression is the holy grail of transplantation. Polyclonally expanded regulatory T cells (Tregs) prolong graft survival in animal models without immunosuppression, but antigen-specific Tregs may have more potency and selectivity. B cells can be activated to serve as antigen presenting cells to stimulate Tregs in culture, and multimeric CD40L (Ultra-CD40L) may improve B cell expansion compared to monomeric CD40L. The purpose of the present study was to expand and activate B cells with Ultra-CD40L and use these to expand antigen-specific Tregs and test their functional capabilities. Methods & Results: Peripheral blood mononuclear cells (PBMC) were isolated from healthy laboratory volunteers and cultured under B cell-promoting conditions with Ultra-CD40L, IL-4, and cyclosporine A for 21 days (n=5). The B cells expanded 100-1,000 fold by day 14-21 and displayed increased expression of CD80, CD86, and HLA-DR, the hallmark of mature antigen presenting cells. Next, CD4+CD127-CD25+ Tregs were isolated from PBMCs of a different volunteer and stimulated with irradiated allogeneic B cells under Treg-promoting conditions with exogenous IL-2 and sirolimus (SRL) for 28 days. These cultured Tregs expanded 13-fold when SRL was removed after 21 days (n=5) and up to 28-fold when SRL was removed after 14 days (n=2). Cultured Tregs maintained the CD4+CD127-CD25+Foxp3+ phenotype and demonstrated dose-dependent inhibition of mixed lymphocyte reactions (MLR) of autologous responders in an antigen-specific manner (p<0.01). These Tregs also displayed antigen-specific infectious tolerance in MLR by generating and amplifying new Tregs in CFSE-labeled autologous responder cells stimulated with the specific allostimulator used in expanding the Tregs (n=4). Conclusions: B cells display robust expansion using Ultra-CD40L and can stimulate production of alloantigen-specific Tregs ex vivo. These Tregs retain antigen specificity and functionally behave like Tregs in vitro by inhibiting MLR proliferation and infectiously generating new Tregs. Upon further development such Tregs might be effectively utilized clinically to achieve transplant tolerance. DISCLOSURE:Kornbluth, R.: Other, Multimeric Biotherapeutics, President and Chief Scientific Officer.

Interleukin-12 (IL-12p70) Promotes Induction of Highly Potent Th1-Like CD4(+)CD25(+) T Regulatory Cells That Inhibit Allograft Rejection in Unmodified Recipients.

In rat models, CD4+CD25+ T regulatory cells (Treg) play a key role in the induction and maintenance of antigen-specific transplant tolerance, especially in DA rats with PVG cardiac allografts (1, 2). We have previously described generation of alloantigen-specific Treg (Ts1), by culture of naïve natural CD4+CD25+ Treg (nTreg) with specific alloantigen and IL-2 for 4 days. These cells express mRNA for IFN-γ receptor (ifngr) and suppress donor but not third party cardiac allograft rejection mediated by alloreactive CD4+ T cells at ratios of <1:10. Here, we show that Ts1 also expressed the IL-12p70 specific receptor (il-12rβ2) and that rIL-12p70 can induce their proliferation. Ts1 cells re-cultured with rIL-12p70 alone or rIL-12p70 and recombinant interleukin-2 (rIL-2), suppressed proliferation of CD4+ T cells in mixed lymphocyte culture at <1:1024, whereas Ts1 cells re-cultured with rIL-2 and alloantigen only suppressed at 1:32–64. The rIL-12p70 alloactivated Ts1 cells markedly delayed PVG, but not third party Lewis, cardiac allograft rejection in normal DA recipients. Ts1 cells re-cultured for 4 days with rIL-12p70 alone, but not those re-cultured with rIL-12p70 and rIL-2, expressed more il-12rβ2, t-bet, and ifn-γ, and continued to express the markers of Ts1 cells, foxp3, ifngr, and il-5 indicating Th1-like Treg were induced. Ts1 cells re-cultured with rIL-2 and alloantigen remained of the Ts1 phenotype and did not suppress cardiac graft rejection in normal DA rats. We induced highly suppressive Th1-like Treg from naïve nTreg in 7 days by culture with alloantigen, first with rIL-2 then with rIL-12p70. These Th1-like Treg delayed specific donor allograft rejection demonstrating therapeutic potential.

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.