Prevention of Allograft Rejection by Use of Regulatory T Cells With an MHC‐Specific Chimeric Antigen Receptor

CD4⁺CD25^highFOXP3⁺ regulatory T cells (Tregs) are involved in graft‐specific tolerance after solid organ transplantation. However, adoptive transfer of polyspecific Tregs alone is insufficient to prevent graft rejection even in rodent models, indicating that graft‐specific Tregs are required. We developed a highly specific chimeric antigen receptor that recognizes the HLA molecule A*02 (referred to as A2‐CAR). Transduction into natural regulatory T cells (nTregs) changes the specificity of the nTregs without alteration of their regulatory phenotype and epigenetic stability. Activation of nTregs via the A2‐CAR induced proliferation and enhanced the suppressor function of modified nTregs. Compared with nTregs, A2‐CAR Tregs exhibited superior control of strong allospecific immune responses in vitro and in humanized mouse models. A2‐CAR Tregs completely prevented rejection of allogeneic target cells and tissues in immune reconstituted humanized mice in the absence of any immunosuppression. Therefore, these modified cells have great potential for incorporation into clinical trials of Treg‐supported weaning after allogeneic transplantation.     

Central Role of CD45RA? Foxp3hi Memory Regulatory T Cells in Clinical Kidney Transplantation Tolerance

The role of Foxp3⁺ regulatory T cells (Tregs) in operational tolerance remains elusive, as initial results revealed an increased frequency of this subset in tolerant patients but no functional differences compared with immunosuppressed recipients. In addition, recent studies of regulatory B cells strongly suggest that Tregs may not have a central role in kidney transplantation tolerance. However, recent investigations of the crucial role of Foxp3 demethylation in Treg function and the possibility of identifying distinct Foxp3 T cell subsets prompted us to more thoroughly characterize Tregs in operationally tolerant patients. Thus, we studied the level of demethylation of the Foxp3 Treg-specific demethylated region (TSDR) in circulating CD4⁺ T cells and analyzed Treg subset frequency in tolerant patients, healthy volunteers, patients with stable graft function under immunosuppression, and chronically rejecting recipients. We observed a higher proportion of CD4⁺ T cells with demethylated Foxp3 and a specific expansion of CD4⁺ CD45RA⁻ Foxp3^hi memory Tregs exclusively in tolerant patients. The memory Tregs of tolerant recipients exhibited increased Foxp3 TSDR demethylation, expressed higher levels of CD39 and glucocorticoid-induced TNF-related receptor, and harbored greater suppressive properties than memory Tregs from patients with stable graft function. Taken together, our data demonstrate that operationally tolerant patients mobilize an array of potentially suppressive cells, including not only regulatory B cells but also Tregs. Our results also indicate that tolerant patients have potent CD4⁺CD45RA⁻ Foxp3^hi memory Tregs with a specific Foxp3 TSDR demethylation pattern, which may contribute to the maintenance of graft tolerance.     

Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney–Bone Marrow Transplantation to Induce Transplantation Tolerance

We examined tolerance mechanisms in patients receiving HLA‐mismatched combined kidney–bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high‐throughput sequencing of T cell receptor‐β hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non‐Tregs revealed marked early enrichment of Tregs (CD3⁺CD4⁺CD25^highCD127^lowFoxp3⁺) in blood that resulted from peripheral proliferation (Ki67⁺), possibly new thymic emigration (CD31⁺), and, in one tolerant subject, conversion from non‐Tregs. Among recovering conventional T cells, central memory CD4⁺ and CD8⁺ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor‐reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia‐driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.     

Nonchimeric HLA‐Identical Renal Transplant Tolerance: Regulatory Immunophenotypic/Genomic Biomarkers

We previously described early results of a nonchimeric operational tolerance protocol in human leukocyte antigen (HLA)‐identical living donor renal transplants and now update these results. Recipients given alemtuzumab, tacrolimus/MPA with early sirolimus conversion were multiply infused with donor hematopoietic CD34⁺ stem cells. Immunosuppression was withdrawn by 24 months. Twelve months later, operational tolerance was confirmed by rejection‐free transplant biopsies. Five of the first eight enrollees were initially tolerant 1 year off immunosuppression. Biopsies of three others after total withdrawal showed Banff 1A acute cellular rejection without renal dysfunction. With longer follow‐up including 5‐year posttransplant biopsies, four of the five tolerant recipients remain without rejection while one developed Banff 1A without renal dysfunction. We now add seven new subjects (two operationally tolerant), and demonstrate time‐dependent increases of circulating CD4⁺CD25⁺⁺⁺CD127^?FOXP3⁺ Tregs versus losses of Tregs in nontolerant subjects (p < 0.001). Gene expression signatures, developed using global RNA expression profiling of sequential whole blood and protocol biopsy samples, were highly associative with operational tolerance as early as 1 year posttransplant. The blood signature was validated by an external Immune Tolerance Network data set. Our approach to nonchimeric operational HLA‐identical tolerance reveals association with Treg immunophenotypes and serial gene expression profiles.

     

Mechanisms of Donor‐Specific Tolerance in Recipients of Haploidentical Combined Bone Marrow/Kidney Transplantation

We recently reported long‐term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor‐specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory‐type cell predominance and an increased proportion of CD4⁺CD25⁺CD127^?FOXP3⁺ Treg during the lymphopenic period. Complete donor‐specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL‐2‐producing and cytotoxic cells, developed and persisted for the 3‐year follow‐up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long‐term, systemic donor‐specific unresponsiveness in patients with HLA‐mismatched allograft tolerance. While regulatory cells may play an early role, long‐term tolerance appears to be maintained by a deletion or anergy mechanism.     

Clonal Deletion Established via Invariant NKT Cell Activation and Costimulatory Blockade Requires In Vivo Expansion of Regulatory T Cells

Recently, the immune‐regulating potential of invariant natural killer T (iNKT) cells has attracted considerable attention. We previously reported that a combination treatment with a liposomal ligand for iNKT cells and an anti‐CD154 antibody in a sublethally irradiated murine bone marrow transplant (BMT) model resulted in the establishment of mixed hematopoietic chimerism through in vivo expansion of regulatory T cells (Tregs). Herein, we show the lack of alloreactivity of CD8⁺T cells in chimeras and an early expansion of donor‐derived dendritic cells (DCs) in the recipient thymi accompanied by a sequential reduction in the donor‐reactive Vβ‐T cell receptor repertoire, suggesting a contribution of clonal deletion in this model. Since thymic expansion of donor DCs and the reduction in the donor‐reactive T cell repertoire were precluded with Treg depletion, we presumed that Tregs should preform before the establishment of clonal deletion. In contrast, the mice thymectomized before BMT failed to increase the number of Tregs and to establish CD8⁺T cell tolerance, suggesting the presence of mutual dependence between the thymic donor–DCs and Tregs. These results provide new insights into the regulatory mechanisms that actively promote clonal deletion.     

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.     

Hyperlipidemia Alters Regulatory T Cell Function and Promotes Resistance to Tolerance Induction Through Costimulatory Molecule Blockade

Recent work from our laboratory has shown that hyperlipidemia promotes accelerated rejection of vascularized cardiac allografts in mice by inducing anti‐donor Th17 reactivity and production of IL‐17. Here, we show that hyperlipidemia also affects FoxP3⁺ regulatory T cells (Tregs). Hyperlipidemia promotes the development of Tregs that express low levels of CD25. Hyperlipidemia also promotes a decrease in central Tregs and an increase in effector Tregs that appears to account for the increase in the frequency of CD25^low Tregs. Alterations in Treg subsets also appear to lead to alterations in Treg function. The ability of FoxP3⁺, CD25^high_, CD4⁺ Tregs from hyperlipidemic mice to inhibit proliferation of effector T cells stimulated with anti‐CD3 and CD28 was reduced when compared with Tregs from control mice. Regulatory T cells isolated from hyperlipidemic recipients exhibit increased activation of Akt, and a reduction in Bim levels that permits the expansion of FoxP3⁺CD25^lowCD4⁺ T cells. Hyperlipidemic mice were also resistant to tolerance induction using costimulatory molecule blockade consisting of anti‐CD154 and CTLA4Ig, a strategy that requires Tregs. Together, our data suggest that hyperlipidemia profoundly affects Treg subsets and function as well as the ability to induce tolerance.     

Pancreatic islets engineered with a FasL protein induce systemic tolerance at the induction phase that evolves into long‐term graft‐localized immune privilege

We have previously shown that pancreatic islets engineered to transiently display a modified form of FasL protein (SA‐FasL) on their surface survive indefinitely in allogeneic recipients without a need for chronic immunosuppression. Mechanisms that confer long‐term protection to allograft are yet to be elucidated. We herein demonstrated that immune protection evolves in two distinct phases; induction and maintenance. SA‐FasL‐engineered allogeneic islets survived indefinitely and conferred protection to a second set of donor‐matched, but not third‐party, unmanipulated islet grafts simultaneously transplanted under the contralateral kidney capsule. Protection at the induction phase involved a reduction in the frequency of proliferating alloreactive T cells in the graft‐draining lymph nodes, and required phagocytes and TGF‐β. At the maintenance phase, immune protection evolved into graft site‐restricted immune privilege as the destruction of long‐surviving SA‐FasL‐islet grafts by streptozotocin followed by the transplantation of a second set of unmanipulated islet grafts into the same site from the donor, but not third party, resulted in indefinite survival. The induced immune privilege required both CD4⁺CD25⁺Foxp3⁺ Treg cells and persistent presence of donor antigens. Engineering cell and tissue surfaces with SA‐FasL protein provides a practical, efficient, and safe means of localized immunomodulation with important implications for autoimmunity and transplantation.     

The Effect of Costimulatory and Interleukin 2 Receptor Blockade on Regulatory T Cells in Renal Transplantation

Regulatory T cells (Treg) are critical regulators of immune tolerance. Both IL‐2 and CD28‐CD80/CD86 signaling are critical for CD4⁺CD25⁺FOXP3⁺ Treg survival in mice. Yet, both belatacept (a second‐generation CTLA‐4Ig) and basiliximab (an anti‐CD25 monoclonal antibody) are among the arsenal of current immunotherapies being used in kidney transplant patients. In this study, we explored the direct effect of basiliximab and belatacept on the Tregs in peripheral blood both in the short term and long term and in kidney biopsies of patients with acute rejection. We report that the combined belatacept/basiliximab therapy has no long‐term effect on circulating Tregs when compared to a calcineurin inhibitor (CNI)‐treated group. Moreover, belatacept‐treated patients had a significantly greater number of FOXP3⁺ T cells in graft biopsies during acute rejection as compared to CNI‐treated patients. Finally, it appears that the basiliximab caused a transient loss of both FOXP3⁺ and FOXP3^? CD25⁺ T cells in the circulation in both treatment groups raising important questions about the use of this therapy in tolerance promoting therapeutic protocols.     

CMV‐infected kidney grafts drive the expansion of blood‐borne CMV‐specific T cells restricted by shared class I HLA molecules via presentation on donor cells

We aimed to determine the role of cytomegalovirus (CMV)‐infected donor cells in the development of a CMV‐specific immune response in kidney transplant recipients. We assessed the CMV pp65‐specific immune response by using interferon‐? ELISPOT and dextramers in peripheral blood mononuclear cells from 115 recipients (D+R? 31, D+R + 44, D?R + 40) late after transplantation (mean 59 ± 42 months). Receiving a kidney from a D+ donor resulted in a higher number of IFN‐?‐producing anti‐CMV T cells (P = .004). This effect disappeared with the absence of shared HLA class I specificities between donors and recipients (P = .430). To confirm the role of donor cells in stimulating the expansion of newly developed CMV‐specific CD8⁺ T cells after transplantation, we compared the number of HLA‐A2–restricted CMV‐specific CD8⁺ T cells in primo‐infected recipients who received an HLA‐A2 or non–HLA‐A2 graft. The median of anti‐CMV pp65 T cells restricted by HLA‐A2 was very low for patients who received a non–HLA‐A2 graft vs an HLA‐A2 graft (300 [0‐14638] vs. 17972 [222‐85594] anti‐CMV pp65 CD8⁺ T cells/million CD8⁺ T cells, P = .001). This adds new evidence that CMV‐infected kidney donor cells present CMV peptides and drive an inflation of memory CMV‐specific CD8⁺ T cells, likely because of frequent CMV replications within the graft.     

Donor‐specific chimeric antigen receptor Tregs limit rejection in naive but not sensitized allograft recipients

Cell therapy with autologous donor‐specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor‐specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR‐Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor‐specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti‐HLA‐A2‐specific CAR were administered to Bl/6 mice at the time of transplanting an HLA‐A2⁺ Bl/6 skin graft. Donor‐specific CAR‐Tregs, but not irrelevant‐CAR Tregs, significantly delayed skin rejection and diminished donor‐specific antibodies (DSAs) and frequencies of DSA‐secreting B cells. Donor‐specific CAR‐Treg–treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen‐specific suppression. When donor‐specific CAR Tregs were tested in HLA‐A2‐sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor‐specific CAR‐Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation.     

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self‐apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self‐tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN‐α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid‐derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft‐infiltrating CD4⁺CD25⁺Foxp3⁺ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN‐α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.     

Induction of bona fide regulatory T cells after liver transplantation - the potential influence of polyclonal antithymocyte globulin

T‐cell‐depleting strategies are an integral part of immunosuppressive regimens used in the hematological and solid organ transplant setting. Besides prevention of alloreactivity, treatment with rabbit antithymocyte globulin (rATG) has been related to the induction of immunoregulatory T cells (Treg) in vitro and in vivo. To investigate Treg induced by rATG, we prospectively studied the effect of rATG induction therapy in liver‐transplanted recipients in vivo (n = 28). Treg induction was further evaluated by means of Treg‐specific demethylation region (TSDR) analysis within the FOXP3 locus. Whereas no induction of CD4⁺ CD25^highCD127^? Treg could be observed by phenotypic analysis, we could demonstrate an induction of TSDR⁺ T cells within CD4⁺ T cells exclusively for rATG‐treated patients in the long‐term (day 540) compared with controls (P = NS). Moreover, although in vitro experiments confirm that rATG primarily led to a conversion of CD4⁺ CD25^? into CD4⁺ CD25⁺ T cells displaying immunosuppressive capacities, these cells cannot be classified as bona fide Treg based on their FOXP3 demethylation pattern. Consequently, the generation of Treg after rATG co‐incubation in vitro does not reflect the mechanisms of Treg induction in vivo and therefore the potential clinical relevance of these cells for transplant outcome remains to be determined.     

Association of Higher CD4+CD25highCD127low,FoxP3+, and IL‐2+ T Cell Frequencies Early After Lung Transplantation With Less Chronic Lung Allograft Dysfunction at Two Years

Regulatory T cells (Treg) can regulate alloantigens and may counteract chronic lung allograft dysfunction (CLAD) in lung transplantation. We analyzed Treg in peripheral blood prospectively and correlated percentages of subpopulations with the incidence of CLAD at 2 years. Among lung‐transplanted patients between January 2009 and July 2011, only patients with sufficient Treg measurements were included into the study. Tregs were measured immediately before lung transplantation, at 3 weeks and 3, 6, 12, and 24 months after transplantation and were defined as CD4⁺CD25^high T cells and further analyzed for CTLA4, CD127, FoxP3, and IL‐2 expressions. Between January 2009 and July 2011, 264 patients were transplanted at our institution. Among the 138 (52%) patients included into the study, 31 (22%) developed CLAD within 2 years after transplantation. As soon as 3 weeks after lung transplantation, a statistically significant positive association was detected between Treg frequencies and later absence of CLAD. At the multivariate analysis, increasing frequencies of CD4⁺CD25^highCD127^low, CD4⁺CD25^highFoxP3⁺ and CD4⁺CD25^highIL‐2⁺ T cells at 3 weeks after lung transplantation emerged as protective factors against development of CLAD at 2 years. In conclusion, higher frequencies of specific Treg subpopulations early after lung transplantation are protective against CLAD development.     

Alloreactive natural killer cells promote haploidentical hematopoietic stem cell transplantation by expansion of recipient‐derived CD4+CD25+ regulatory T cells

Alloreactive NK cells (Allo‐NKs) have been shown to exert advantageous effects on the outcomes of haploidentical hematopoietic stem cell transplantation (Haplo‐HSCT) for cancer treatment. However, the mechanisms of action of Allo‐NKs remain unclear. We established a novel Haplo‐HSCT conditioning regimen composed of Allo‐NKs and a low dose of immunosuppressive drugs (Allo‐NKs + Chemo) to investigate alternative mechanisms besides direct cytotoxicity. The inhibitory effects of different cell subsets on the donor–recipient mixed lymphocyte reactions (MLRs) were evaluated after Haplo‐HSCT. The quantities and functions of CD4⁺CD25⁺ regulatory T cells (Tregs) and dendritic cells (DCs) in the spleen and the thymus were examined. Our results showed that the Allo‐NKs + Chemo regimen induced systemic tolerance, and that CD4⁺CD25⁺ Tregs played a significant role in inducing and maintaining systemic tolerance after Haplo‐HSCT. Alloreactive NK cells promoted the expansion of recipient‐derived CD4⁺CD25⁺CD127^? Tregs in the thymus and the spleen which could be amplified in vitro by the immature donor‐derived DC subset isolated from the thymus of Allo‐NKs + Chemo‐treated mice. Our findings suggested that Allo‐NKs are capable of inducing systemic tolerance after Haplo‐HSCT by assembling donor‐derived immature DCs to expand recipient‐derived Treg cells in the thymus.     

Selective CD28 Blockade Results in Superior Inhibition of Donor‐Specific T Follicular Helper Cell and Antibody Responses Relative to CTLA4‐Ig

Donor‐specific antibodies (DSAs) are a barrier to improved long‐term outcomes after kidney transplantation. Costimulation blockade with CTLA4‐Ig has shown promise as a potential therapeutic strategy to control DSAs. T follicular helper (Tfh) cells, a subset of CD4⁺ T cells required for optimal antibody production, are reliant on the CD28 costimulatory pathway. We have previously shown that selective CD28 blockade leads to superior allograft survival through improved control of CD8⁺ T cells relative to CTLA4‐Ig, but the impact of CD28‐specific blockade on CD4⁺ Tfh cells is unknown. Thus, we identified and characterized donor‐reactive Tfh cells in a murine skin transplant model and then used this model to evaluate the impact of selective CD28 blockade with an anti‐CD28 domain antibody (dAb) on the donor‐specific Tfh cell–mediated immune response. We observed that the anti‐CD28 dAb led to superior inhibition of donor‐reactive CXCR5⁺PD‐1^high Tfh cells, CD95⁺GL7⁺ germinal center B cells and DSA formation compared with CTLA4‐Ig. Interestingly, donor‐reactive Tfh cells differentially upregulated CTLA4 expression, suggesting an important role for CTLA4 in mediating the superior inhibition observed with the anti‐CD28 dAb. Therefore, selective CD28 blockade as a novel approach to control Tfh cell responses and prevent DSA after kidney transplantation warrants further study.     

Characterization,biology, and expansion of regulatory T cells in the Cynomolgus macaque for preclinical studies

Reliable in vitro expansion protocols of regulatory T cells (Tregs) are needed for clinical use. We studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed four in vitro Treg expansion protocols for translational studies. Tregs expanded 3000‐fold when artificial antigen presenting cells (aAPCs) expressing human CD80, CD58 and CD32 were used throughout the culture. When donor peripheral blood mononuclear cells (PBMCs) were used as the single source of APCs followed by aAPCs, Tregs expanded 2000‐fold. Tregs from all protocols suppressed the proliferation of anti‐CD2CD3CD28 bead‐stimulated autologous PBMCs albeit with different potencies, varying from 1:2‐1:4 Treg:PBMC ratios, up to >1:32. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed greater suppression of stimulation by anti‐CD2CD3CD28 beads of PBMCs from the same donor used for stimulation during the Treg cultures and of autologous cells than of third‐party PBMC responders. Similar to humans, the Treg–specific demethylated region (TSDR) within the Foxp3 locus correlated with suppressive activity and expression of Foxp3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression. In summary, we have characterized MCM Tregs and developed four Treg expansion protocols that can be used for preclinical applications.