Cathepsin W restrains peripheral regulatory T cells for mucosal immune quiescence.

Peripheral regulatory T (pTreg) cells are a key T cell lineage for mucosal immune tolerance and anti-inflammatory responses, and interleukin-2 receptor (IL-2R) signaling is critical for Treg cell generation, expansion, and maintenance. The expression of IL-2R on pTreg cells is tightly regulated to ensure proper induction and function of pTreg cells without a clear molecular mechanism. We here demonstrate that Cathepsin W (CTSW), a cysteine proteinase highly induced in pTreg cells under transforming growth factor-ß stimulation is essential for the restraint of pTreg cell differentiation in an intrinsic manner. Loss of CTSW results in elevated pTreg cell generation, protecting the animals from intestinal inflammation. Mechanistically, CTSW inhibits IL-2R signaling in pTreg cells by cytosolic interaction with and process of CD25, repressing signal transducer and activator of transcription 5 activation to restrain pTreg cell generation and maintenance. Hence, our data indicate that CTSW acts as a gatekeeper to calibrate pTreg cell differentiation and function for mucosal immune quiescence.

Functional Analysis of Foxp3 and Its Mutants by Retroviral Transduction of Murine Primary CD4+ T Cells.

The transcription factor Foxp3/FOXP3 orchestrates regulatory T (Treg) cell development and function by interacting with numerous target genes and partner proteins. Functional analysis of naturally occurring or engineered Foxp3/FOXP3 mutations has provided important insights into how the complex Foxp3/FOXP3-centered molecular network operates. Here, we describe detailed protocols for retroviral transduction of murine primary conventional CD4+ T cells to determine the impacts of Foxp3 mutations on the Treg-cell-like phenotype and function conferred by Foxp3.

Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus.

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3-/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

Regulatory T-cells regulate neonatal heart regeneration by potentiating cardiomyocyte proliferation in a paracrine manner.

The neonatal mouse heart is capable of transiently regenerating after injury from postnatal day (P) 0-7 and macrophages are found important in this process. However, whether macrophages alone are sufficient to orchestrate this regeneration; what regulates cardiomyocyte proliferation; why cardiomyocytes do not proliferate after P7; and whether adaptive immune cells such as regulatory T-cells (Treg) influence neonatal heart regeneration have less studied. Methods: We employed both loss- and gain-of-function transgenic mouse models to study the role of Treg in neonatal heart regeneration. In loss-of-function studies, we treated mice with the lytic anti-CD25 antibody that specifically depletes Treg; or we treated FOXP3DTR with diphtheria toxin that specifically ablates Treg. In gain-of-function studies, we adoptively transferred hCD2+ Treg from NOD.Foxp3hCD2 to NOD/SCID that contain Treg as the only T-cell population. Furthermore, we performed single-cell RNA-sequencing of Treg to uncover paracrine factors essential for cardiomyocyte proliferation. Results: Unlike their wild type counterparts, NOD/SCID mice that are deficient in T-cells but harbor macrophages fail to regenerate their injured myocardium at as early as P3. During the first week of injury, Treg are recruited to the injured cardiac muscle but their depletion contributes to more severe cardiac fibrosis. On the other hand, adoptive transfer of Treg results in mitigated fibrosis and enhanced proliferation and function of the injured cardiac muscle. Mechanistically, single-cell transcriptomic profiling reveals that Treg could be a source of regenerative factors. Treg directly promote proliferation of both mouse and human cardiomyocytes in a paracrine manner; and their secreted factors such as CCL24, GAS6 or AREG potentiate neonatal cardiomyocyte proliferation. By comparing the regenerating P3 and non-regenerating P8 heart, there is a significant increase in the absolute number of intracardiac Treg but the whole transcriptomes of these Treg do not differ regardless of whether the neonatal heart regenerates. Furthermore, even adult Treg, given sufficient quantity, possess the same regenerative capability. Conclusion: Our results demonstrate a regenerative role of Treg in neonatal heart regeneration. Treg can directly facilitate cardiomyocyte proliferation in a paracrine manner.

Single-cell transcriptomics reveal that PD-1 mediates immune tolerance by regulating proliferation of regulatory T cells.

BACKGROUND: We have previously reported an antigen-specific protocol to induce transplant tolerance and linked suppression to human embryonic stem cell (hESC)-derived tissues in immunocompetent mice through coreceptor and costimulation blockade. However, the exact mechanisms of acquired immune tolerance in this model have remained unclear. METHODS: We utilize the NOD.Foxp3hCD2 reporter mouse line and an ablative anti-hCD2 antibody to ask if CD4+FOXP3+ regulatory T cells (Treg) are required for coreceptor and costimulation blockade-induced immune tolerance. We also perform genome-wide single-cell RNA-sequencing to interrogate Treg during immune rejection and tolerance and to indicate possible mechanisms involved in sustaining Treg function. RESULTS: We show that Treg are indispensable for tolerance induced by coreceptor and costimulation blockade as depletion of which with an anti-hCD2 antibody resulted in rejection of hESC-derived pancreatic islets in NOD.Foxp3hCD2 mice. Single-cell transcriptomic profiling of 12,964 intragraft CD4+ T cells derived from rejecting and tolerated grafts reveals that Treg are heterogeneous and functionally distinct in the two outcomes of transplant rejection and tolerance. Treg appear to mainly promote chemotactic and ubiquitin-dependent protein catabolism during transplant rejection while seeming to harness proliferative and immunosuppressive function during tolerance. We also demonstrate that this form of acquired transplant tolerance is associated with increased proliferation and PD-1 expression by Treg. Blocking PD-1 signaling with a neutralizing anti-PD-1 antibody leads to reduced Treg proliferation and graft rejection. CONCLUSIONS: Our results suggest that short-term coreceptor and costimulation blockade mediates immune tolerance to hESC-derived pancreatic islets by promoting Treg proliferation through engagement of PD-1. Our findings could give new insights into clinical development of hESC-derived pancreatic tissues, combined with immunotherapies that expand intragraft Treg, as a potentially sustainable alternative treatment for T1D.

Deregulated Mucosal Immune Surveillance through Gut-Associated Regulatory T Cells and PD-1+ T Cells in Human Colorectal Cancer.

Disturbed balance between immune surveillance and tolerance may lead to poor clinical outcomes in some malignancies. In paired analyses of adenocarcinoma and normal mucosa from 142 patients, we found a significant increase of the CD4/CD8 ratio and accumulation of regulatory T cells (Tregs) within the adenocarcinoma. The increased frequency of Tregs correlated with the local infiltration and extension of the tumor. There was concurrent maturation arrest, upregulation of programmed death-1 expression, and functional impairment in CD8+ T cells (CTLs) isolated from the adenocarcinoma. Adenocarcinoma-associated Tregs directly inhibit the function of normal human CTLs in vitro. With histopathological analysis, Foxp3+ Tregs were preferentially located in stroma. Concurrent transcriptome analysis of epithelial cells, stromal cells, and T cell subsets obtained from carcinomatous and normal intestinal samples from patients revealed a distinct gene expression signature in colorectal adenocarcinoma-associated Tregs, with overexpression of CCR1, CCR8, and TNFRSF9, whereas their ligands CCL4 and TNFSF9 were found upregulated in cancerous epithelium. Overexpression of WNT2 and CADM1, associated with carcinogenesis and metastasis, in cancer-associated stromal cells suggests that both cancer cells and stromal cells play important roles in the development and progression of colorectal cancer through the formation of a tumor microenvironment. The identification of CTL anergy by Tregs and the unique gene expression signature of human Tregs and stromal cells in colorectal cancer patients may facilitate the development of new therapeutics against malignancies.

Attenuation of CD4+CD25+ Regulatory T Cells in the Tumor Microenvironment by Metformin, a Type 2 Diabetes Drug.

CD4+CD25+ regulatory T cells (Treg), an essential subset for preventing autoimmune diseases, is implicated as a negative regulator in anti-tumor immunity. We found that metformin (Met) reduced tumor-infiltrating Treg (Ti-Treg), particularly the terminally-differentiated CD103+KLRG1+ population, and also decreased effector molecules such as CTLA4 and IL-10. Met inhibits the differentiation of naïve CD4+ T cells into inducible Treg (iTreg) by reducing forkhead box P3 (Foxp3) protein, caused by mTORC1 activation that was determined by the elevation of phosphorylated S6 (pS6), a downstream molecule of mTORC1. Rapamycin and compound C, an inhibitor of AMP-activated protein kinase (AMPK) restored the iTreg generation, further indicating the involvement of mTORC1 and AMPK. The metabolic profile of iTreg, increased Glut1-expression, and reduced mitochondrial membrane-potential and ROS production of Ti-Treg aided in identifying enhanced glycolysis upon Met-treatment. The negative impact of Met on Ti-Treg may help generation of the sustained antitumor immunity.

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.

T follicular helper and T follicular regulatory cells have different TCR specificity.

Immunization leads to the formation of germinal centres (GCs) that contain both T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Whether T-cell receptor (TCR) specificity defines the differential functions of Tfh and Tfr cells is unclear. Here we show that antigen-specific T cells after immunization are preferentially recruited to the GC to become Tfh cells, but not Tfr cells. Tfh cells, but not Tfr cells, also proliferate efficiently on restimulation with the same immunizing antigen in vitro. Ex vivo TCR repertoire analysis shows that immunization induces oligoclonal expansion of Tfh cells. By contrast, the Tfr pool has a TCR repertoire that more closely resembles that of regulatory T (Treg) cells. Our data thus indicate that the GC Tfh and Tfr pools are generated from distinct TCR repertoires, with Tfh cells expressing antigen-responsive TCRs to promote antibody responses, and Tfr cells expressing potentially autoreactive TCRs to suppress autoimmunity.

Helios Enhances Treg Cell Function in Cooperation With FoxP3.

OBJECTIVE: Helios+FoxP3+CD4+ (Helios+) Treg cells are believed to be involved in the regulation of various autoimmune diseases; however, the regulatory mechanisms underlying the development of Helios+ Treg cells remain uncertain. This study was undertaken to elucidate the regulatory mechanisms of Helios expression in CD4+ T cells and its roles in transforming growth factor ß (TGFß)-induced Treg cell function. METHODS: We examined the expression of Helios in CD4+ T cells in patients with rheumatoid arthritis by DNA microarray analysis before and after treatment with biologic agents. We also examined the effect of interleukin-6 (IL-6) and TGFß on Helios expression in CD4+ T cells in humans and mice. The effect of forced expression of Helios on murine induced Treg cell function was also examined. The role of FoxP3 in the induction and function of Helios was assessed by using CD4+ T cells from FoxP3-deficient scurfy mice. RESULTS: Tocilizumab, but not tumor necrosis factor (TNF) inhibitors or abatacept, increased Helios expression in CD4+ T cells in patients with a good response. IL-6 inhibited the TGFß-induced development of Helios+ induced Treg cells in both humans and mice. Both cell-intrinsic FoxP3 expression and TGFß signaling were required for Helios induction in murine induced Treg cells. The forced expression of Helios enhanced the expression of various Treg cell-related molecules and the suppressive function in murine induced Treg cells. Helios-mediated enhancement of the suppressive function of induced Treg cells was obvious in FoxP3-sufficient CD4+ T cells but not in FoxP3-deficient CD4+ T cells. CONCLUSION: Our findings indicate that Helios enhances induced Treg cell function in cooperation with FoxP3.

Sphingosine-1-phosphate receptor 2 is critical for follicular helper T cell retention in germinal centers.

Follicular helper T (Tfh) cells access the B cell follicle to promote antibody responses and are particularly important for germinal center (GC) reactions. However, the molecular mechanisms of how Tfh cells are physically associated with GCs are incompletely understood. We report that the sphingosine-1-phosphate receptor 2 (S1PR2) gene is highly expressed in a subpopulation of Tfh cells that localizes in GCs. S1PR2-deficient Tfh cells exhibited reduced accumulation in GCs due to their impaired retention. T cells deficient in both S1PR2 and CXCR5 were ineffective in supporting GC responses compared with T cells deficient only in CXCR5. These results suggest that S1PR2 and CXCR5 cooperatively regulate localization of Tfh cells in GCs to support GC responses.

Prostaglandin E2-prostaglandin E receptor subtype 4 (EP4) signaling mediates UV irradiation-induced systemic immunosuppression.

UV radiation induces systemic immunosuppression. Because nonsteroidal anti-inflammatory drugs suppress UV-induced immunosuppression, prostanoids have been suspected as a crucial mediator of this UV effect. However, the identity of the prostanoid involved and its mechanism of action remain unclear. Here, we addressed this issue by subjecting mice deficient in each prostanoid receptor individually or mice treated with a subtype-specific antagonist to UV irradiation. Mice treated with an antagonist for prostaglandin E receptor subtype 4 (EP4), but not those deficient in other prostanoid receptors, show impaired UV-induced immunosuppression, whereas administration of an EP4 agonist rescues the impairment of the UV-induced immunosuppression in indomethacin-treated mice. The EP4 antagonist treatment suppresses an increase in the number of CD4(+)/forkhead box P3-positive (Foxp3(+)) regulatory T cells (Treg cells) in the peripheral lymph nodes (LNs) and dendritic cells expressing DEC205 in the LNs and the skin after UV irradiation. Furthermore, the EP4 antagonist treatment down-regulates UV-induced expression of receptor activator of NF-κB ligand (RANKL) in skin keratinocytes. Finally, administration of anti-RANKL antibody abolishes the restoration of UV-induced immunosuppression by EP4 agonism in indomethacin-treated mice. Thus, prostaglandin E(2) (PGE(2))-EP4 signaling mediates UV-induced immunosuppression by elevating the number of Treg cells through regulation of RANKL expression in the epidermis.

TRAF6 directs commitment to regulatory T cells in thymocytes.

Regulatory T cells (Tregs), a subset of CD4(+) helper T cells, are crucial for immunological self-tolerance. Defect in development or function of Tregs results in autoimmune disease in human and mice. Whereas it is known that Tregs mainly develop in the thymus, the molecular mechanism underlying development of Treg is not fully understood. TRAF6-deficient mice showed a severe defect in the Treg development in thymus. In vitro fetal thymic organ culture experiments indicated that the defect is ascribed to the absence of TRAF6 in thymic cells. Moreover, mixed fetal liver transfer experiments revealed that the development of Foxp3(+) cells differentiated from Traf6(-/-) hematopoietic cells was specifically impaired in the thymus, indicating cell-intrinsic requirement for TRAF6 in the Treg development. On the other hand, TRAF6 is not required for the development of conventional CD4(+) T cell. In addition, TGFß-dependent induction of Foxp3 in CD4(+) T cells in vitro was not impaired by the absence of TRAF6. Overall, our data indicate that TRAF6 plays an essential role on the commitment of immature thymocytes to thymic Tregs in cell-intrinsic fashion.

c-Rel: a pioneer in directing regulatory T-cell lineage commitment?

The transcription factor Foxp3 controls the differentiation and function of Treg, but the molecular mechanisms that regulate Foxp3 transcription remain elusive. In particular, signals and factors that open and remodel the Foxp3 locus and imprint developing Treg with a stable Foxp3 phenotype are largely unknown. Two reports in this issue of the European Journal of Immunology, together with recent reports published elsewhere, demonstrate that a member of the NF-kappaB family transcription factors, c-Rel, is required for thymic differentiation of Foxp3(+) Treg. Moreover, c-Rel is shown to regulate Foxp3 transcription directly by binding to cis-regulatory elements at the Foxp3 locus upon TCR/CD28 stimulation, including the promoter and the newly identified conserved non-coding DNA sequence harboring a "permissive" chromatin status in Treg precursors. These findings collectively suggest that c-Rel may act as a pioneer transcription factor in initiating Foxp3 transcription in Treg precursors in the thymus.

Exacerbation of delayed-type hypersensitivity responses in EBV-induced gene-3 (EBI-3)-deficient mice.

Epstein-Barr virus-induced gene-3 (EBI-3) associates with p28 to form interleukin-27 (IL-27) or with IL-12p35 to form IL-35. Both IL-27 and IL-35 have immunosuppressive functions and especially IL-35 has been implicated in the suppressive function of regulatory T cells (Treg). To address the role of EBI-3 in immune regulation, delayed-type hypersensitivity (DTH) responses were examined in EBI-3-deficient (EBI-3(-/-)) mice. EBI-3(-/-) mice developed deteriorated DTH responses as shown by the enhanced footpad swelling and augmented infiltration of inflammatory cells into the antigen-challenged footpads as compared with wild-type (WT) mice. While EBI-3-deficiency showed little effects on antigen-specific IFN-gamma production of lymph node cells, IL-17 production was drastically augmented in EBI-3(-/-) cells as compared with in WT cells. In addition, reduced IL-10 production was also evident in EBI-3(-/-) CD4(+) T cells. Interestingly, the development and suppressive function of Treg to inhibit effector T cell proliferation was not affected by EBI-3-deficiency. These data clearly demonstrated the immunosuppressive function of EBI-3 and provided complementary evidence that EBI-3 and EBI-3-containing cytokines might be taken into consideration as potential targets for some immune-related diseases.

Cutting edge: depletion of Foxp3+ cells leads to induction of autoimmunity by specific ablation of regulatory T cells in genetically targeted mice.

We have recently described two independent mouse models in which the administration of diphtheria toxin (DT) leads to specific depletion of regulatory T cells (Tregs) due to expression of DT receptor-enhanced GFP under the control of the Foxp3 promoter. Both mouse models develop severe autoimmune disorders when Foxp3(+) Tregs are depleted. Those findings were challenged in a recent study published in this journal suggesting the expression of Foxp3 in epithelial cells as the cause for disease development. By using genetic, cellular, and immunohistochemical approaches, we do not find evidence for Foxp3-expression in nonhematopoietic cells. DT injection does not lead to a loss of epithelial integrity in our Foxp3-DTR models. Instead, Foxp3 expression is Treg-specific and ablation of Foxp3(+) Tregs leads to the induction of fatal autoimmune disorders. Autoimmunity can be reversed by the adoptive transfer of Tregs into depleted hosts, and the transfer of Foxp3-deficient bone marrow into T cell-deficient irradiated recipients leads to full-blown disease development.

Preferential generation of follicular B helper T cells from Foxp3+ T cells in gut Peyer's patches.

Most of the immunoglobulin A (IgA) in the gut is generated by B cells in the germinal centers of Peyer's patches through a process that requires the presence of CD4+ follicular B helper T(TFH) cells. The nature of these T(FH) cells in Peyer's patches has been elusive. Here, we demonstrate that suppressive Foxp3+CD4+ T cells can differentiate into TFH cells in mouse Peyer's patches. The conversion of Foxp3+ T cells into TFH cells requires the loss of Foxp3 expression and subsequent interaction with B cells. Thus, environmental cues present in gut Peyer's patches promote the selective differentiation of distinct helper T cell subsets, such as TFH cells.

Full restoration of peripheral Foxp3+ regulatory T cell pool by radioresistant host cells in scurfy bone marrow chimeras.

Mutations in the gene encoding the transcription factor Foxp3 lead to fatal autoimmune pathology in mice and humans, which is associated with a deficiency in Foxp3(+) regulatory T cells (T(reg)). It has also been proposed that Foxp3 inactivation in nonhematopoietic tissues, particularly in thymic epithelium, is required for the pathogenesis, because Foxp3 mutant scurfy bone marrow cells fail to transmit the disease to lethally irradiated WT hosts. We demonstrate here that the lack of pathology in these radiation chimeras is due to the presence of radioresistant endogenous Foxp3(+) T(reg) of the host. In addition, chimeras carrying the scurfy mutation only in nonhematopoietic cells exhibit no evidence of autoimmune pathology. Thus, Foxp3 deficiency in nonhematopoietic cells does not contribute to the scurfy disease. Furthermore, our analyses of radiation chimeras revealed that the peripheral T(reg) pool is fully and specifically restored and maintained by radioresistant endogenous T(reg) or adoptively transferred exogenous T(reg) through "homeostatic" proliferation in the absence of T(reg) production from scurfy donor bone marrow cells. These results thus provide evidence that the autoimmune pathology in scurfy mice results indeed from a T(reg) deficiency and illustrate a robust homeostatic mechanism that strictly controls the size of peripheral T(reg) pool by fine-tuning of homeostatic proliferation.

Regulatory T cells, derived from naïve CD4+CD25- T cells by in vitro Foxp3 gene transfer, can induce transplantation tolerance.

BACKGROUND: Regulatory T (Treg) cells, generated in vitro by Foxp3 gene transfer into naive CD4+25- T cells, have been shown to inhibit the development of inflammation and autoimmune disease, but it is not known whether they are able to prevent allograft rejection. This study investigated whether Treg cells generated from naive CD4+ T cells by Foxp3 gene transfer could induce transplantation tolerance. METHODS: HY-specific, T-cell receptor (TCR)-transgenic CD4+25- T cells were retrovirally transduced with the Foxp3 gene. The phenotype, function, and cytokine profiles of the transduced cells were examined in vitro by fluorescence-activated cell sorter, T-cell proliferation assays, enzyme-linked immunosorbent assay, and intracellular cytokine staining. Adoptive transfer and skin grafting experiments were conducted to assess whether Foxp3-transduced HY-specific T cells could prevent the rejection of syngeneic male grafts. RESULTS: CD4+25- T cells retrovirally transduced with Foxp3 express a panel of cell surface and intracellular molecules closely associated with Treg activity. This Treg phenotype was stable during in vitro culture with some further maturation. In vitro, Foxp3-transduced cells were functionally anergic and suppressive T cells. In vivo adoptive transfer of Foxp3-transduced HY-specific TCR-transgenic CD4+ T cells protected male skin grafts from rejection by syngeneic females. Retroviral transduction of the Foxp3 gene into non-TCR-transgenic CD4+25- T cells, however, had no influence on male skin graft rejection. CONCLUSION: This study provides the first evidence that Foxp3-transduced T cells can control the rejection of an allogeneic transplant and suggests that T-cell Foxp3 gene transfer may have therapeutic value in clinical transplantation.