Bone Morphogenetic Proteins Shape Treg Cells.

The transforming growth factor-ß (TGF-ß) family includes cytokines controlling cell behavior, differentiation and homeostasis of various tissues including components of the immune system. Despite well recognized importance of TGF-ß in controlling T cell functions, the immunomodulatory roles of many other members of the TGF-ß cytokine family, especially bone morphogenetic proteins (BMPs), start to emerge. Bone Morphogenic Protein Receptor 1α (BMPR1α) is upregulated by activated effector and Foxp3+ regulatory CD4+ T cells (Treg cells) and modulates functions of both of these cell types. BMPR1α inhibits generation of proinflammatory Th17 cells and sustains peripheral Treg cells. This finding underscores the importance of the BMPs in controlling Treg cell plasticity and transition between Treg and Th cells. BMPR1α deficiency in in vitro induced and peripheral Treg cells led to upregulation of Kdm6b (Jmjd3) demethylase, an antagonist of polycomb repressive complex 2 (PRC2), and cell cycle inhibitor Cdkn1a (p21Cip1) promoting cell senescence. This indicates that BMPs and BMPR1α may represent regulatory modules shaping epigenetic landscape and controlling proinflammatory reprogramming of Th and Treg cells. Revealing functions of other BMP receptors and their crosstalk with receptors for TGF-ß will contribute to our understanding of peripheral immunoregulation.

Bone Morphogenic Protein Signaling and Melanoma.

OPINION STATEMENT: Malignant melanoma is a deadly form of skin cancer caused by neoplastic transformation of melanocytic cells. Despite recent progress in melanoma therapy, by inhibition of activated oncogenes or immunotherapy, survival rate for metastatic melanoma patients remains low. The remarkable phenotypic plasticity of melanoma cells allows for rapid development of invasive properties and metastatic tumors, the main cause of mortality in melanoma patients. Phenotypic and molecular analyses of developing tumors revealed that epithelial-mesenchymal transition (EMT), a cellular and molecular mechanism, controls transition from mature melanocyte to less differentiated melanocyte lineage progenitor cells forming melanoma tumors. This transition is facilitated by persistence of transcriptional regulatory circuit characteristic of embryonic stage in mature melanocytes. Switching of the developmental program of mature melanocyte to EMT is induced by accumulated mutations, especially targeting BRAF, N-RAS, or MEK1/2 signaling pathways, and further promoted by dynamic stimuli from local environment including hypoxia, interactions with extracellular matrix and growth factors or cytokines. Recent reports demonstrate that signaling mediated by transforming growth factor-ß (TGF-ß) and bone morphogenic proteins (BMPs) play critical roles in inducing EMT by controlling expression of critical transcription factors. BMPs are essential modulators of differentiation, proliferation, apoptosis, invasiveness, and metastases in developing melanoma tumors. They control transcription and epigenetic landscape of melanoma cells. Better understanding of the role of BMPs may lead to new strategies to control EMT processes in melanocyte cell lineage and to achieve clinical benefits for the patients.

Self and microbiota-derived epitopes induce CD4+ T cell anergy and conversion into CD4+Foxp3+ regulatory cells.

The physiological role of T cell anergy induction as a key mechanism supporting self-tolerance remains undefined, and natural antigens that induce anergy are largely unknown. In this report, we used TCR sequencing to show that the recruitment of CD4+CD44+Foxp3-CD73+FR4+ anergic (Tan) cells expands the CD4+Foxp3+ (Tregs) repertoire. Next, we report that blockade in peripherally-induced Tregs (pTregs) formation due to mutation in CNS1 region of Foxp3 or chronic exposure to a selecting self-peptide result in an accumulation of Tan cells. Finally, we show that microbial antigens from Akkermansia muciniphila commensal bacteria can induce anergy and drive conversion of naive CD4+CD44-Foxp3- T (Tn) cells to the Treg lineage. Overall, data presented here suggest that Tan induction helps the Treg repertoire to become optimally balanced to provide tolerance toward ubiquitous and microbiome-derived epitopes, improving host ability to avert systemic autoimmunity and intestinal inflammation.

Bone Morphogenic Proteins Are Immunoregulatory Cytokines Controlling FOXP3+ Treg Cells.

Bone morphogenic proteins (BMPs) are members of the transforming growth factor ß (TGF-ß) cytokine family promoting differentiation, homeostasis, and self-renewal of multiple tissues. We show that signaling through the bone morphogenic protein receptor 1α (BMPR1α) sustains expression of FOXP3 in Treg cells in peripheral lymphoid tissues. BMPR1α signaling promotes molecular circuits supporting acquisition and preservation of Treg cell phenotype and inhibiting differentiation of pro-inflammatory effector Th1/Th17 CD4+ T cell. Mechanistically, increased expression of KDM6B (JMJD3) histone demethylase, an antagonist of the polycomb repressive complex 2, underlies lineage-specific changes of T cell phenotypes associated with abrogation of BMPR1α signaling. These results reveal that BMPs are immunoregulatory cytokines mediating maturation and stability of peripheral FOXP3+ regulatory T cells (Treg cells) and controlling generation of iTreg cells. Thus, we establish that BMPs, a large cytokine family, are an essential link between stromal tissues and the adaptive immune system involved in sustaining tissue homeostasis by promoting immunological tolerance.

Dormant pathogenic CD4+ T cells are prevalent in the peripheral repertoire of healthy mice.

Thymic central tolerance eliminates most immature T cells with autoreactive T cell receptors (TCR) that recognize self MHC/peptide complexes. Regardless, an unknown number of autoreactive CD4+Foxp3- T cells escape negative selection and in the periphery require continuous suppression by CD4+Foxp3+ regulatory cells (Tregs). Here, we compare immune repertoires of Treg-deficient and Treg-sufficient mice to find Tregs continuously constraining one-third of mature CD4+Foxp3- cells from converting to pathogenic effectors in healthy mice. These dormant pathogenic clones frequently express TCRs activatable by ubiquitous autoantigens presented by class II MHCs on conventional dendritic cells, including self-peptides that select them in the thymus. Our data thus suggest that identification of most potentially autoreactive CD4+ T cells in the peripheral repertoire is critical to harness or redirect these cells for therapeutic advantage.

Cell-based high throughput screening identified a novel compound that promotes regulatory T cells and prevents autoimmune colitis.

Regulatory T cells (TR) show great promise for treating autoimmune diseases, allergies and preventing transplant rejection; however, their clinical application has been hampered by the lack of efficient ex vivo or in vivo expansion strategies. Here we report screening data on 130,000 low molecular weight (LMW) compounds for their TR promoting potential using a self-developed high-throughput cell-based assay. One of the lead compounds, an isoxazolecarboxamide designated as TRP38, efficiently converts naïve CD4+ T cells to TR cells in vitro and protects mice from autoimmune colitis in vivo. In addition, TRP38 can synergize with other compounds and/or cytokines such as rapamycin and TGFß for TR conversion, probably via directly inhibiting P70s6 phosphorylation without affecting mTOR expression, underscoring the importance of complementary and coordinated activity of multiple signaling pathways for the increased level of stable TR cell production.

TGF-ß-mediated enhancement of TH17 cell generation is inhibited by bone morphogenetic protein receptor 1α signaling.

The cytokines of the transforming growth factor-ß (TGF-ß) family promote the growth and differentiation of multiple tissues, but the role of only the founding member, TGF-ß, in regulating the immune responses has been extensively studied. TGF-ß is critical to prevent the spontaneous activation of self-reactive T cells and sustain immune homeostasis. In contrast, in the presence of proinflammatory cytokines, TGF-ß promotes the differentiation of effector T helper 17 (TH17) cells. Abrogating TGF-ß receptor signaling prevents the development of interleukin-17 (IL-17)-secreting cells and protects mice from TH17 cell-mediated autoimmunity. We found that the receptor of another member of TGF-ß family, bone morphogenetic protein receptor 1α (BMPR1α), regulates T helper cell activation. We found that the differentiation of TH17 cells from naive CD4+ T cells was inhibited in the presence of BMPs. Abrogation of BMPR1α signaling during CD4+ T cell activation induced a developmental program that led to the generation of inflammatory effector cells expressing large amounts of IL-17, IFN-γ, and TNF family cytokines and transcription factors defining the TH17 cell lineage. We found that TGF-ß and BMPs cooperated to establish effector cell functions and the cytokine profile of activated CD4+ T cells. Together, our data provide insight into the immunoregulatory function of BMPs.

The mechanisms shaping the repertoire of CD4+ Foxp3+ regulatory T cells.

Regulatory T (Treg) cells expressing Foxp3 transcription factor control homeostasis of the immune system, antigenic responses to commensal and pathogenic microbiota, and immune responses to self and tumour antigens. The Treg cells differentiate in the thymus, along with conventional CD4+ T cells, in processes of positive and negative selection. Another class of Treg cells is generated in peripheral tissues by inducing Foxp3 expression in conventional CD4+ T cells in response to antigenic stimulation. Both thymic and peripheral generation of Treg cells depends on recognition of peptide/MHC ligands by the T-cell receptors (TCR) expressed on thymic Treg precursors or peripheral conventional CD4+ T cells. This review surveys reports describing how thymus Treg cell generation depends on the selecting peptide/MHC ligands and how this process impacts the TCR repertoire expressed by Treg cells. We also describe how Treg cells depend on sustained signalling through the TCR and how they are further regulated by Foxp3 enhancer sequences. Finally, we review the impact of microbiota-derived antigens on the maintenance and functionality of the peripheral pool of Treg cells.

Coordination of different ligands to copper(II) and cobalt(III) metal centers enhances Zika virus and dengue virus loads in both arthropod cells and human keratinocytes.

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100µM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen)2]Cl2, (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen)3]Cl3, (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl2·2H2O) or cobalt(II) chloride hexahydrate (CoCl2·6H2O) alone had no effects as "free" cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes. IMPORTANCE: Mosquito-borne diseases are of great concern to the mankind. Use of chemicals/repellents against mosquito bites and transmission of microbes has been the topic of interest for many years. Here, we show that thiosemicarbazone ligand(s) derived from 2-acetylethiazole or citral or 1,10-phenanthroline upon conjugation with copper(II) or cobalt(III) metal centers enhances dengue virus (serotype 2; DENV2) and/or Zika virus (ZIKV) infections in mosquito, mouse and human cells. Enhanced ZIKV/DENV2 capsid mRNA or envelope protein loads were evident in mosquito cells and human keratinocytes, when treated with compounds before/after infections. Also, treatment with copper(II) or cobalt(III) conjugated compounds increased viral titers and number of plaque formations. These studies suggest that conjugation of compounds in repellents/essential oils/natural products/food additives with copper(II) or cobalt(III) metal centers may not be safe, especially in tropical and subtropical places, where several dengue infection cases and deaths are reported annually or in places with increased ZIKV caused microcephaly.

Bone Morphogenetic Protein Signaling Regulates Development and Activation of CD4(+) T Cells.

Bone morphogenetic proteins (BMPs) are growth factors belonging to the TGF-ß (transforming growth factor ß) superfamily. BMPs were found to regulate multiple cell processes such as proliferation, survival, differentiation, and apoptosis. They were originally described to play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites but were found to play a significant role in embryogenesis and development of multiple tissues and organs. Activities of BMPs are regulated by a number of secreted proteins, which modulate their availability to bind cellular receptors. The functions of individual BMPs are highly redundant due to binding the same receptors and inducing overlapping signal transduction pathways. Recently, BMPs were found to regulate cells of the innate and adaptive immune system. BMPs are involved in thymic development of T cells at the early, double negative, as well as later, double positive, stages of thymopoesis. They specifically modulate thymic development of regulatory T cells (T(reg)). In the periphery, BMPs affect T cell activation, promoting generation of T(reg) cells. We found that mice deficient for one of the receptors activated by BMPs demonstrated slower growth of transplantable melanoma tumors.

Cx3cr1 deficiency in mice attenuates hepatic granuloma formation during acute schistosomiasis by enhancing the M2-type polarization of macrophages.

Acute schistosomiasis is characterized by pro-inflammatory responses against tissue- or organ-trapped parasite eggs along with granuloma formation. Here, we describe studies in Cx3cr1(-/-) mice and demonstrate the role of Cx3cr1 in the pathoetiology of granuloma formation during acute schistosomiasis. Mice deficient in Cx3cr1 were protected from granuloma formation and hepatic injury induced by Schistosoma japonicum eggs, as manifested by reduced body weight loss and attenuated hepatomegaly along with preserved liver function. Notably, S. japonicum infection induced high levels of hepatic Cx3cr1 expression, which was predominantly expressed by infiltrating macrophages. Loss of Cx3cr1 rendered macrophages preferentially towards M2 polarization, which then led to a characteristic switch of the host immune defense from a conventional Th1 to a typical Th2 response during acute schistosomiasis. This immune switch caused by Cx3cr1 deficiency was probably associated with enhanced STAT6/PPAR-γ signaling and increased expression of indoleamine 2,3-dioxygenase (IDO), an enzyme that promotes M2 polarization of macrophages. Taken together, our data provide evidence suggesting that CX3CR1 could be a viable therapeutic target for treatment of acute schistosomiasis.

Altered connexin 43 expression underlies age-dependent decrease of regulatory T cell suppressor function in nonobese diabetic mice.

Type 1 diabetes is one of the most extensively studied autoimmune diseases, but the cellular and molecular mechanisms leading to T cell-mediated destruction of insulin-producing ß cells are still not well understood. In this study, we show that regulatory T cells (T(regs)) in NOD mice undergo age-dependent loss of suppressor functions exacerbated by the decreased ability of activated effector T cells to upregulate Foxp3 and generate T(regs) in the peripheral organs. This age-dependent loss is associated with reduced intercellular communication mediated by gap junctions, which is caused by impaired upregulation and decreased expression of connexin 43. Regulatory functions can be corrected, even in T cells isolated from aged, diabetic mice, by a synergistic activity of retinoic acid, TGF-ß, and IL-2, which enhance connexin 43 and Foxp3 expression in T(regs) and restore the ability of conventional CD4(+) T cells to upregulate Foxp3 and generate peripherally derived T(regs). Moreover, we demonstrate that suppression mediated by T(regs) from diabetic mice is enhanced by a novel reagent, which facilitates gap junction aggregation. In summary, our report identifies gap junction-mediated intercellular communication as an important component of the T(reg) suppression mechanism compromised in NOD mice and suggests how T(reg) mediated immune regulation can be improved.

Modulation of bone morphogenic protein signaling in T-cells for cancer immunotherapy.

Immunotherapy is becoming an increasingly attractive therapeutic alternative for conventional cancer therapy. In recent years Foxp3(+) regulatory T-cells (T(R)) were identified as the major obstacle to effective cancer immunotherapy. The abundance of these cells in peripheral blood is increased in patients with multiple types of cancer and their prevalence among tumor-infiltrating lymphocytes correlated with poor clinical prognosis. In contrast, removal or inactivation of T(R) cells led to enhanced anti-tumor immune response and better efficacy of cancer vaccines. This study reports that Bone Morphogenic Protein Receptor 1α (BMPR1α, Alk-3) is expressed by activated effector CD4(+) and T(R) cells and modulates functions of both cell types. Bone Morphogenic Proteins (BMPs) belong to the transforming growth factor (TGF)-ß family of cytokines that also include TGFß and activins. BMPs play crucial roles in embryonic development, tissue differentiation and homeostasis, and development of cancer. It was demonstrated that BMPs and activins synergize with TGFß to regulate thymic T-cell development, maintain T(R) cells, and control peripheral tolerance. Inactivation of BMPR1α in T-cells results in impaired thymic and peripheral generation of T(R) cells. BMPR1α-deficient activated T-cells produced a higher level of interferon (IFN)-γ than BMPR1α-sufficient T-cells. Moreover, transplanted B16 melanoma tumors grew smaller in mice lacking expression of BMPR1α in T-cells and tumors had few infiltrating TR cells and a higher proportion of CD8(+) T-cells than wild-type mice.

SUMO1 regulates endothelial function by modulating the overall signals in favor of angiogenesis and homeostatic responses.

As a versatile regulatory mechanism, sumoylation has been found to be essential for ordered diverse cellular processes. However, the exact impact of sumoylation on endothelial function largely remained elusive. Here we investigated the role of small ubiquitin-like modifier 1 (SUMO1) mediated sumoylation in the regulation of endothelial function by examining its effect on angiogenesis and homeostatic responses. Adenoviral-mediated SUMO1 expression in porcine aortic endothelial cells (PAECs) dose-dependently promoted proliferation, migration and tube formation. In line with these results in PAECs, Matrigel plug assays in SUMO1 transgenic mice demonstrated a significant higher capacity for vascular neogenesis as compared with that of control littermates. Moreover, SUMO1 expression protected PAECs from serum starvation or H2O2-induced apoptosis. Mechanistic studies demonstrated that SUMO1 sumoylation modulates ERK1/2 activation and MMP13 expression as well as Jak2/STAT5 signaling to promote angiogenesis. SUMO1 sumoylation also suppressed NFκB and c-JUN transcriptional activity to provide protection for PAECs against oxidative stress-induced apoptosis. Given that sumoylation is a reversible process, dynamic regulation of the sumoylation function could be a novel strategy to modulate endothelial function in disease states.

Thymus-derived regulatory T cells contribute to tolerance to commensal microbiota.

Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4(+) Foxp3(+) regulatory T (Treg) cells generated in the thymus or extrathymically by induction of naive CD4(+) Foxp3(-) T cells. Previous studies suggested that the T-cell receptor repertoires of thymic Treg cells and induced Treg cells are biased towards self and non-self antigens, respectively, but their relative contribution in controlling immunopathology, such as colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved. The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which Treg cells and other T-cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage and favour tolerogenic presentation of antigens to naive CD4(+) T cells, suggesting that intestinal homeostasis depends on microbiota-specific induced Treg cells. Here, to identify the origin and antigen-specificity of intestinal Treg cells, we performed single-cell and high-throughput sequencing of the T-cell receptor repertoires of CD4(+) Foxp3(+) and CD4(+) Foxp3(-) T cells, and analysed their reactivity against specific commensal species. We show that thymus-derived Treg cells constitute most Treg cells in all lymphoid and intestinal organs, including the colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that thymic Treg cells, and not induced Treg cells, dominantly mediate tolerance to antigens produced by intestinal commensals.

Systematic evaluation of 640 FDA drugs for their effect on CD4⁺Foxp3⁺ regulatory T cells using a novel cell-based high throughput screening assay.

Regulatory T cells (Treg), which play a pivotal role in maintaining immune homeostasis by suppressing the proliferation of effector T cells, have great therapeutic potential for autoimmune diseases and transplantation. However, progress on their clinical application has been hampered by the lack of high throughput screening (HTS) strategies for the systematic and rapid evaluation of existing drugs and the identification of novel drug candidates. In this report, we present an innovative in vitro HTS assay using CD4⁺ T cells from Foxp3-GFP transgenic mice that specifically express the GFP signal in Foxp3⁺ Treg cells detectable by FACS analysis in a high throughput manner. Systematic evaluation of 640 FDA-approved drugs revealed that 70 drugs increased the number of Treg cells with suppression function only in the presence of TGFß, 75 drugs increased Treg numbers even in the absence of TGFß, and 32 drugs increased Treg numbers synergistically with TGFß. The identified Treg-promoting drugs include those previously known to induce Treg (rapamycin and retinoic acid), statins, glucocorticoids and drugs in many other categories. Furthermore, Treg cells cultured with the identified drugs possess surface and intracellular markers characteristic of natural Treg cells and possess suppressive function. These results suggest that this Treg HTS assay can be used to screen compound libraries to identify novel chemical entities for Treg-based immune therapies.

Connexin 43 signaling enhances the generation of Foxp3+ regulatory T cells.

Despite their importance for the functioning of the immune system, thymic development and peripheral maintenance of Foxp3(+) regulatory T (T(R)) cells are poorly understood. We have found that connexin 43 (Cx43), expressed by thymic T(R) cells progenitors, supports T(R) development. Mice with deletion of the Cx43 gene induced in T cells produce only few T(R) cells and had increased proportion of activated T cells in the lymph nodes, suggesting impaired peripheral tolerance. Reduction of the T(R) cell numbers was accompanied by increased presence of CD4(+)CD25(+)GITR(+)Foxp3(-) T cells, which did not produce inflammatory cytokines and lost suppressor function. These results strongly argue that we have discovered a novel signaling pathway, controlled by Cx43, that enhances the generation of T(R) cells. We propose that a possible mechanism of Cx43 activity is by regulating Foxp3 expression in T(R) lineage cells.

Intratumoral convergence of the TCR repertoires of effector and Foxp3+ CD4+ T cells.

The presence of Foxp3(+) regulatory CD4(+) T cells in tumor lesions is considered one of the major causes of ineffective immune response in cancer. It is not clear whether intratumoral T(reg) cells represent T(reg) cells pre-existing in healthy mice, or arise from tumor-specific effector CD4(+) T cells and thus representing adaptive T(reg) cells. The generation of T(reg) population in tumors could be further complicated by recent evidence showing that both in humans and mice the peripheral population of T(reg) cells is heterogenous and consists of subsets which may differentially respond to tumor-derived antigens. We have studied T(reg) cells in cancer in experimental mice that express naturally selected, polyclonal repertoire of CD4(+) T cells and which preserve the heterogeneity of the T(reg) population. The majority of T(reg) cells present in healthy mice maintained a stable suppressor phenotype, expressed high level of Foxp3 and an exclusive set of TCRs not used by naive CD4(+) T cells. A small T(reg) subset, utilized TCRs shared with effector T cells and expressed a lower level of Foxp3. We show that response to tumor-derived antigens induced efficient clonal recruitment and expansion of antigen-specific effector and T(reg) cells. However, the population of T(reg) cells in tumors was dominated by cells expressing TCRs shared with effector CD4(+) T cells. In contrast, T(reg) cells expressing an exclusive set of TCRs, that dominate in healthy mice, accounted for only a small fraction of all T(reg) cells in tumor lesions. Our results suggest that the T(reg) repertoire in tumors is generated by conversion of effector CD4(+) T cells or expansion of a minor subset of T(reg) cells. In conclusion, successful cancer immunotherapy may depend on the ability to block upregulation of Foxp3 in effector CD4(+) T cells and/or selectively inhibiting the expansion of a minor T(reg) subset.

TCR repertoire and Foxp3 expression define functionally distinct subsets of CD4+ regulatory T cells.

Despite extensive research efforts to characterize peripheral regulatory T (T(reg)) cells expressing transcription factor Foxp3, their subset complexity, phenotypic characteristics, TCR repertoire and Ag specificities remain ambiguous. In this study, we identify and define two subsets of peripheral T(reg) cells differing in Foxp3 expression level and TCR repertoires. T(reg) cells expressing a high level of Foxp3 and TCRs not used by naive CD4(+) T cells present a stable suppressor phenotype and dominate the peripheral T(reg) population in unmanipulated mice. The second T(reg) subset, expressing a lower level of Foxp3 and using TCRs shared with naive CD4(+) T cells constitutes a small fraction of all T(reg) cells in unmanipulated mice and enriches T(reg) population with the same Ag specificities as expressed by activated/effector T cells. This T(reg) subset undergoes extensive expansion during response to Ag when it becomes a major population of Ag-specific T(reg) cells. Thus, T(reg) cells expressing TCRs shared with naive CD4(+) T cells have a flexible phenotype and may down-regulate Foxp3 expression which may restore immune balance at the conclusion of immune response or convert these cells to effector T cells producing inflammatory cytokines.

Foxp3-deficient regulatory T cells do not revert into conventional effector CD4+ T cells but constitute a unique cell subset.

Homeostasis in the immune system is maintained by specialized regulatory CD4(+) T cells (T(reg)) expressing transcription factor Foxp3. According to the current paradigm, high-affinity interactions between TCRs and class II MHC-peptide complexes in thymus "instruct" developing thymocytes to up-regulate Foxp3 and become T(reg) cells. However, the loss or down-regulation of Foxp3 does not disrupt the development of T(reg) cells but abrogates their suppressor function. In this study, we show that Foxp3-deficient T(reg) cells in scurfy mice harboring a null mutation of the Foxp3 gene retained cellular features of T(reg) cells including in vitro anergy, impaired production of inflammatory cytokines, and dependence on exogenous IL-2 for proliferation and homeostatic expansion. Foxp3-deficient T(reg) cells expressed a low level of activation markers, did not expand relative to other CD4(+) T cells, and produced IL-4 and immunomodulatory cytokines IL-10 and TGF-beta when stimulated. Global gene expression profiling revealed significant similarities between T(reg) cells expressing and lacking Foxp3. These results argue that Foxp3 deficiency alone does not convert T(reg) cells into conventional effector CD4(+) T cells but rather these cells constitute a distinct cell subset with unique features.