Nuclear transfer nTreg model reveals fate-determining TCR-β and novel peripheral nTreg precursors.
ABSTRACT: To study the development and function of "natural-arising" T regulatory (nTreg) cells, we developed a novel nTreg model on pure nonobese diabetic background using epigenetic reprogramming via somatic cell nuclear transfer. On RAG1-deficient background, we found that monoclonal FoxP3(+)CD4(+)Treg cells developed in the thymus in the absence of other T cells. Adoptive transfer experiments revealed that the thymic niche is not a limiting factor in nTreg development. In addition, we showed that the T-cell receptor (TCR) β-chain of our nTreg model was not only sufficient to bias T-cell development toward the CD4 lineage, but we also demonstrated that this TCR β-chain was able to provide stronger TCR signals. This TCR-β-driven mechanism would thus unify former per se contradicting hypotheses of TCR-dependent and -independent nTreg development. Strikingly, peripheral FoxP3(-)CD4(+)T cells expressing the same TCR as this somatic cell nuclear transfer nTreg model had a reduced capability to differentiate into Th1 cells but were poised to differentiate better into induced nTreg cells, both in vitro and in vivo, representing a novel peripheral precursor subset of nTreg cells to which we refer to as pre-nTreg cells.
Project description:To study the development and function of “natural-arising” T regulatory (nTreg) cells, we developed a novel nTreg model on pure nonobese diabetic background using epigenetic reprogramming via somatic cell nuclear transfer. On RAG1-deficient background, we found that monoclonal FoxP3+ CD4+ Treg cells developed in the thymus in the absence of other T cells. Adoptive transfer experiments revealed that the thymic niche is not a limiting factor in nTreg development. In addition, we showed that the T-cell receptor (TCR) β-chain of our nTreg model was not only sufficient to bias T-cell development toward the CD4 lineage, but we also demonstrated that this TCR β-chain was able to provide stronger TCR signals. This TCR-β–driven mechanism would thus unify former per se contradicting hypotheses of TCR-dependent and -independent nTreg development. Strikingly, peripheral FoxP3− CD4+ T cells expressing the same TCR as this somatic cell nuclear transfer nTreg model had a reduced capability to differentiate into Th1 cells but were poised to differentiate better into induced nTreg cells, both in vitro and in vivo, representing a novel peripheral precursor subset of nTreg cells to which we refer to as pre-nTreg cells. We performed RNA-Seq analysis to determine the transcriptional differences between monoclonal FoxP3GFP-positive and -negative CD4+ T cells from NOD.TCRab.FoxP3GFP.Rag-/- and compared it with polyclonal FoxP3GFP-positive and -negative CD4+ T cells from NOD.FoxP3GFP mice
Project description:Fc receptor-like 3 (FCRL3) is a cell surface protein homologous to Fc receptors. The FCRL3 gene is present in humans but not in mice. We found that FCRL3 protein is expressed on 40% of human naturally occurring CD4(+) regulatory T (nTreg) cells (CD4(+)CD25(+)CD127(low)). Sorted nTreg cells with the surface phenotype FCRL3(+) and FCRL3(-) were both hypoproliferative to TCR stimulation and both suppressive on proliferation of conventional T cells (CD4(+)CD25(-)) in vitro. They both expressed forkhead box p3 (Foxp3) protein, the intracellular regulatory T cell marker. However, in contrast to FCRL3(-) nTreg cells, FCRL3(+) nTreg cells were not stimulated to proliferate by the addition of exogenous IL-2. In addition, Foxp3(+) cells induced from conventional T cells by TGF-beta treatment did not exhibit FCRL3 expression. These results suggest that the FCRL3(+) subset of human nTreg cells identified in this study arise in vivo and Foxp3 expression alone is not sufficient to induce FCRL3 expression. FCRL3 may be involved in human-specific mechanisms to control the generation of nTreg cells.
Project description:Thymus-derived Foxp3(+) natural regulatory CD4 T cells (nTregs) prevent autoimmunity through control of pathogenic, autoreactive T cells and other immune effector cells. Using T cell receptor (TCR) transgenic models, diversity within this lineage has been found to be similar to that of conventional CD4 T cells. To determine whether balanced TCR diversity may be perturbed in autoimmunity, we have analyzed receptor composition in C57BL/6 and autoimmune non-obese diabetic (NOD) mice. The natural regulatory and conventional CD4 repertoires of C57BL/6 had similar diversities. Despite the apparently normal thymic development of the NOD nTreg lineage, TCR diversity within the selected repertoire was markedly restricted. Detailed analysis of TCRalpha and -beta chain composition is consistent with positive selection into the natural regulatory lineage being under stringent audition for interaction with MHC class II/self-peptide. The NOD MHC region, including the unique H2-A(g7) class II molecule, partly accounts for the reduction in diversity, but additional NOD genetic contribution(s) are required for complete repertoire compaction. Mechanistic links between MHC, autoimmunity, and nTreg diversity identified in this study are discussed.
Project description:Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo-derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential nonredundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.
Project description:The relationship between the TCR repertoires of natural regulatory T cells (nTregs) and conventional CD4(+) T cells (Tconv) capable of responding to the same antigenic epitope is unknown. In this study, we used TCR?-chain transgenic mice to generate polyclonal nTreg and Tconv populations specific for a foreign Ag. CD4(+) T cells from immunized 3.L2?(+/-) TCR?(+/-) Foxp3(EGFP) mice were restimulated in culture to yield nTregs (EGFP(+)) and Tconv (EGFP(-)) defined by their antigenic reactivity. Relative to Tconv, nTreg expansion was delayed, although a higher proportion of viable nTregs had divided after 72 h. Spectratype analysis revealed that both the nTreg and Tconv responses were different and characterized by skewed distributions of CDR3 lengths. CDR3 sequences from nTregs displayed a divergent pattern of J? usage, minimal CDR3 overlap (3.4%), and less diversity than did CDR3 sequences derived from Tconv. These data indicate that foreign Ag-specific nTregs and Tconv are clonally distinct and that foreign Ag-specific nTreg populations are constrained by a limited TCR repertoire.
Project description:The Wiskott-Aldrich syndrome, a primary human immunodeficiency, results from defective expression of the hematopoietic-specific cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASP). Because CD4(+)CD25(+)Foxp3(+) naturally occurring regulatory T (nTreg) cells control autoimmunity, we asked whether colitis in WASP knockout (WKO) mice is associated with aberrant development/function of nTreg cells. We show that WKO mice have decreased numbers of CD4(+)CD25(+)Foxp3(+) nTreg cells in both the thymus and peripheral lymphoid organs. Moreover, we demonstrate that WKO nTreg cells are markedly defective in both their ability to ameliorate the colitis induced by the transfer of CD45RB(hi) T cells and in functional suppression assays in vitro. Compared with wild-type (WT) nTreg cells, WKO nTreg cells show significantly impaired homing to both mucosal (mesenteric) and peripheral sites upon adoptive transfer into WT recipient mice. Suppression defects may be independent of antigen receptor-mediated actin rearrangement because both WT and WKO nTreg cells remodeled their actin cytoskeleton inefficiently upon T cell receptor stimulation. Preincubation of WKO nTreg cells with exogenous interleukin (IL)-2, combined with antigen receptor-mediated activation, substantially rescues the suppression defects. WKO nTreg cells are also defective in the secretion of the immunomodulatory cytokine IL-10. Overall, our data reveal a critical role for WASP in nTreg cell function and implicate nTreg cell dysfunction in the autoimmunity associated with WASP deficiency.
Project description:Regulatory T cells (Treg) that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs - selective accumulation or proliferation of natural thymus-derived Treg (nTreg) or induced Treg (iTreg) converted in the periphery from naïve T cells - is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL) and splenocytes (SPL) in B16 murine melanoma-bearing C57BL/6 Foxp3(EGFP) mice. OT-II Foxp3(EGFP) mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA). Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3(EGFP) mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations - wild-type CD4, pmel CD8 and OTII CD4 - could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR) and exposure to transforming growth factor ? (TGF?). B16 melanoma produces TGF? and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment.
Project description:Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFβ, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts.
Project description:The development of natural Foxp3(+) CD4 regulatory T cells (nTregs) proceeds via two steps that involve the initial antigen dependent generation of CD25(+)GITR(hi)Foxp3(-)CD4(+) nTreg precursors followed by the cytokine induction of Foxp3. Using mutant mouse models that lack c-Rel, the critical NF-?B transcription factor required for nTreg differentiation, we establish that c-Rel regulates both of these developmental steps. c-Rel controls the generation of nTreg precursors via a haplo-insufficient mechanism, indicating that this step is highly sensitive to c-Rel levels. However, maintenance of c-Rel in an inactive state in nTreg precursors demonstrates that it is not required for a constitutive function in these cells. While the subsequent IL-2 induction of Foxp3 in nTreg precursors requires c-Rel, this developmental transition does not coincide with the nuclear expression of c-Rel. Collectively, our results support a model of nTreg differentiation in which c-Rel generates a permissive state for foxp3 transcription during the development of nTreg precursors that influences the subsequent IL-2 dependent induction of Foxp3 without a need for c-Rel reactivation.
Project description:Natural regulatory T (nT(reg)) cells are important for maintaining tolerance to self- and foreign antigens, and they are thought to develop from thymocytes that receive strong T cell receptor (TCR)-mediated signals in the thymus. TCR engagement leads to the activation of phospholipase C-?1, which generates the lipid second messenger diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate. We used mice that lack the ? isoform of DAG kinase (DGK?), which metabolizes DAG to terminate its signaling, to enhance TCR-mediated signaling and identify critical signaling events in nT(reg) cell development. Loss of DGK? resulted in increased numbers of thymic CD25(+)Foxp3(-)CD4(+) nT(reg) cell precursors and Foxp3(+)CD4(+) nT(reg) cells in a cell-autonomous manner. DGK?-deficient T cells exhibited increased nuclear translocation of the nuclear factor ?B subunit c-Rel, as well as enhanced extracellular signal-regulated kinase (ERK) phosphorylation in response to TCR stimulation, suggesting that these downstream pathways may contribute to nT(reg) cell development. Indeed, reducing c-Rel abundance or blocking ERK phosphorylation abrogated the increased generation of nTreg cells by DGK?-deficient thymocytes. The extent of ERK phosphorylation correlated with TCR-mediated acquisition of Foxp3 in immature thymocytes in vitro. Furthermore, the development of nT(reg) cells was augmented in mice in which ERK activation was selectively enhanced in T cells. Together, these data suggest that DGK? regulates the development of nT(reg) cells by limiting the extent of activation of the ERK and c-Rel signaling pathways.