ABSTRACT
Regulatory T (Treg) cells suppress the development of inflammatory disease, but our knowledge of transcriptional regulators that control this function remains incomplete. Here we show that expression of Id2 and Id3 in Treg cells was required to suppress development of fatal inflammatory disease. We found that T cell antigen receptor (TCR)-driven signaling initially decreased the abundance of Id3, which led to the activation of a follicular regulatory T (TFR) cell-specific transcription signature. However, sustained lower abundance of Id2 and Id3 interfered with proper development of TFR cells. Depletion of Id2 and Id3 expression in Treg cells resulted in compromised maintenance and localization of the Treg cell population. Thus, Id2 and Id3 enforce TFR cell checkpoints and control the maintenance and homing of Treg cells.
Subject(s)
Inflammation/immunology , Inhibitor of Differentiation Protein 2/immunology , Inhibitor of Differentiation Proteins/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Base Sequence , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Movement/immunology , Cell Proliferation , Female , Forkhead Transcription Factors/biosynthesis , Gene Expression Regulation/immunology , Green Fluorescent Proteins/genetics , Inflammation/genetics , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Proteins/biosynthesis , Inhibitor of Differentiation Proteins/genetics , Interleukin-10/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Antigen, T-Cell/immunology , Receptors, CXCR5/biosynthesis , Sequence Analysis, RNAABSTRACT
Foxp3(+) regulatory T cells (T(reg) cells) maintain immunological tolerance, and their deficiency results in fatal multiorgan autoimmunity. Although heightened signaling via the T cell antigen receptor (TCR) is critical for the differentiation of T(reg) cells, the role of TCR signaling in T(reg) cell function remains largely unknown. Here we demonstrated that inducible ablation of the TCR resulted in T(reg) cell dysfunction that could not be attributed to impaired expression of the transcription factor Foxp3, decreased expression of T(reg) cell signature genes or altered ability to sense and consume interleukin 2 (IL-2). Instead, TCR signaling was required for maintaining the expression of a limited subset of genes comprising 25% of the activated T(reg) cell transcriptional signature. Our results reveal a critical role for the TCR in the suppressor capacity of T(reg) cells.
Subject(s)
Cell Differentiation/immunology , Forkhead Transcription Factors/immunology , Interleukin-2/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Autoimmunity/immunology , Cell Adhesion/immunology , Diphtheria Toxin/administration & dosage , Female , Forkhead Transcription Factors/biosynthesis , Hyaluronan Receptors/biosynthesis , Immune Tolerance/immunology , Interferon-gamma/biosynthesis , Interleukin-13/biosynthesis , Interleukin-2/biosynthesis , Interleukin-4/biosynthesis , Ki-67 Antigen/biosynthesis , Male , Mice , Receptors, Interleukin-2/immunology , Signal Transduction/immunology , T-Lymphocytes, Regulatory/cytology , Tamoxifen/pharmacologyABSTRACT
Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.
Subject(s)
CRISPR-Cas Systems , Forkhead Transcription Factors/metabolism , T-Lymphocytes, Regulatory/metabolism , Animals , Autoimmunity/immunology , Cells, Cultured , Forkhead Transcription Factors/biosynthesis , Gene Editing , Gene Expression Regulation , Humans , Immunotherapy , Male , Mice , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/prevention & control , Protein Stability , Reproducibility of Results , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , Ubiquitin Thiolesterase/deficiency , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/metabolismABSTRACT
Regulatory T cells (Treg cells) are required for immune homeostasis. Chromatin remodeling is essential for establishing diverse cellular identities, but how the epigenetic program in Treg cells is maintained throughout the dynamic activation process remains unclear. Here we have shown that CD28 co-stimulation, an extracellular cue intrinsically required for Treg cell maintenance, induced the chromatin-modifying enzyme, Ezh2. Treg-specific ablation of Ezh2 resulted in spontaneous autoimmunity with reduced Foxp3(+) cells in non-lymphoid tissues and impaired resolution of experimental autoimmune encephalomyelitis. Utilizing a model designed to selectively deplete wild-type Treg cells in adult mice co-populated with Ezh2-deficient Treg cells, Ezh2-deficient cells were destabilized and failed to prevent autoimmunity. After activation, the transcriptome of Ezh2-deficient Treg cells was disrupted, with altered expression of Treg cell lineage genes in a pattern similar to Foxp3-deficient Treg cells. These studies reveal a critical role for Ezh2 in the maintenance of Treg cell identity during cellular activation.
Subject(s)
CD28 Antigens/immunology , Lymphocyte Activation/immunology , Polycomb Repressive Complex 2/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Autoimmunity/genetics , Autoimmunity/immunology , CD8-Positive T-Lymphocytes/immunology , Chromatin Assembly and Disassembly , Encephalomyelitis, Autoimmune, Experimental/immunology , Enhancer of Zeste Homolog 2 Protein , Female , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Gene Expression Regulation , Heparin-binding EGF-like Growth Factor/genetics , Immune Tolerance/genetics , Immune Tolerance/immunology , Lymphocyte Depletion , Mice , Mice, Inbred C57BL , Mice, Transgenic , Polycomb Repressive Complex 2/genetics , Promoter Regions, Genetic/genetics , T-Lymphocytes, Regulatory/cytologyABSTRACT
The critical role of IL-10-producing B cells (B10 cells) with a unique CD1dhiCD5+ phenotype in suppressing autoimmune responses and relieving inflammation has been demonstrated in several models of autoimmune diseases. However, the regulatory role of B10 cells in T cell-mediated autoimmune responses during the natural history of type 1 diabetes is unclear. In this study, we used the NOD mouse model of autoimmune diabetes to clarify the changes and potential mechanisms of B10 cells for disease. Compared with B10 cells present in the 4-wk-old normoglycemic NOD mice, the frequency of B10 cells was increased in the insulitis and diabetic NOD mice, with the highest proportion in the insulitis NOD mice. The changes in the relative number of B10 cells were most pronounced in the pancreas-draining lymph nodes. The pathogenic T cells, including Th1 and Th17 cells, remarkably increased. The assays in vitro showed that B10 cells in the NOD mice did not inhibit the proliferation of CD4+CD25- T cells. They also had no regulatory effect on IFN-γ and IL-4 secretion or on Foxp3 expression of T cells. B10 cells suppressed T cell-mediated autoimmune responses via an IL-10-dependent pathway. In contrast, B10 cells in the NOD mice exhibited a significant reduction in IL-10 production. In summary, a defect in the number and function of B10 cells may participate in the development and progression of type 1 diabetes.
Subject(s)
B-Lymphocytes, Regulatory/immunology , Diabetes Mellitus, Type 1/immunology , Interleukin-10/immunology , Lymphocyte Activation/immunology , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Cell Proliferation/physiology , Cells, Cultured , Cellular Microenvironment/immunology , Diabetes Mellitus, Type 1/pathology , Disease Models, Animal , Female , Forkhead Transcription Factors/biosynthesis , Homeostasis/immunology , Interferon-gamma/biosynthesis , Interleukin-10/biosynthesis , Interleukin-4/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Th1 Cells/immunology , Th17 Cells/immunologyABSTRACT
T follicular regulatory (Tfr) cells are a subset of CD4+ T cells that express CXCR5 and migrate into germinal centers (GCs). They regulate GC reactions by communicating with T follicular helper (Tfh) and B cells. TNF inhibitors are used in inflammatory diseases; however, the generation of autoantibodies or anti-drug Abs sometimes causes problems. Because TNFR2 signaling is important for suppressive functions of regulatory T cells, we investigated the role of TNFR2 on human Tfr cells. Tfr cells stimulated with MR2-1 (an anti-TNFR2 agonistic Ab) were analyzed for cell proliferation, Foxp3 expression, and surface molecules. Tfh/B cell proliferation, IgM production, and differentiation in cocultures with MR2-1-stimulated Tfr cells were examined. Tfr cells express a high level of TNFR2. MR2-1 stimulation altered the gene expression profile of Tfr cells. Cell proliferation and Foxp3 expression of Tfr cells were enhanced by MR2-1. MR2-1-stimulated Tfr cells expressed ICOS and Programmed cell death protein 1 and significantly suppressed Tfh/B cell proliferation, IgM production, and B cell differentiation. TNFR2-stimulated Tfr cells retained the migration function according to the CXCL13 gradient. In conclusion, we showed that TNFR2-stiumulated Tfr cells can regulate Tfh and B cells. Aberrant antibody production during TNF inhibitor treatment might be, at least in part, associated with TNFR2 signaling inhibition in Tfr cells. In addition, expansion and maturation of Tfr cells via TNFR2 stimulation in vitro may be useful for a cell-based therapy in inflammatory and autoimmune diseases to control GC reactions.
Subject(s)
B-Lymphocytes/immunology , Receptors, Tumor Necrosis Factor, Type II/metabolism , T Follicular Helper Cells/immunology , T-Lymphocytes, Regulatory/immunology , Autoimmune Diseases/therapy , B-Lymphocytes/cytology , B7-H1 Antigen/metabolism , Cell Differentiation/immunology , Cell Movement/immunology , Cell Proliferation , Chemokine CXCL13/metabolism , Forkhead Transcription Factors/biosynthesis , Gene Expression Profiling , Germinal Center/cytology , Humans , Immunoglobulin M/biosynthesis , Inducible T-Cell Co-Stimulator Protein/biosynthesis , Lymphocyte Activation/immunology , Programmed Cell Death 1 Receptor/metabolism , Receptors, CXCR5/metabolism , Receptors, Tumor Necrosis Factor, Type II/antagonists & inhibitors , Signal Transduction/immunology , Tumor Necrosis Factors/metabolismABSTRACT
In this issue of Immunity, Wu et al. (2014) report that galectin-9 is required for the formation and stability of iTreg cells. Galectin-9 interacts with CD44 in association with TGF-ß receptors to drive both Foxp3 and galectin-9 expression in a positive-feedforward loop involving Smad3 activation.
Subject(s)
Forkhead Transcription Factors/biosynthesis , Galectins/immunology , Hyaluronan Receptors/immunology , T-Lymphocytes, Regulatory/immunology , AnimalsABSTRACT
The ß-galactoside-binding protein galectin-9 is critical in regulating the immune response, but the mechanism by which it functions remains unclear. We have demonstrated that galectin-9 is highly expressed by induced regulatory T cells (iTreg) and was crucial for the generation and function of iTreg cells, but not natural regulatory T (nTreg) cells. Galectin-9 expression within iTreg cells was driven by the transcription factor Smad3, forming a feed-forward loop, which further promoted Foxp3 expression. Galectin-9 increased iTreg cell stability and function by directly binding to its receptor CD44, which formed a complex with transforming growth factor-ß (TGF-ß) receptor I (TGF-ßRI), and activated Smad3. Galectin-9 signaling was further found to regulate iTreg cell induction by dominantly acting through the CNS1 region of the Foxp3 locus. Our data suggest that exogenous galectin-9, in addition to being an effector molecule for Treg cells, acts synergistically with TGF-ß to enforce iTreg cell differentiation and maintenance.
Subject(s)
Forkhead Transcription Factors/biosynthesis , Galectins/immunology , Hyaluronan Receptors/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cell Differentiation/immunology , Colitis/genetics , Colitis/immunology , Galectins/genetics , Hepatitis A Virus Cellular Receptor 2 , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Serine-Threonine Kinases/immunology , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/immunology , Receptors, Virus/immunology , Signal Transduction/immunology , Smad3 Protein/immunology , Transforming Growth Factor beta/immunologyABSTRACT
Regulatory T (Treg) cells maintain immune homeostasis and prevent inflammatory and autoimmune responses. During development, thymocytes bearing a moderately self-reactive T cell receptor (TCR) can be selected to become Treg cells. Several observations suggest that also in the periphery mature Treg cells continuously receive self-reactive TCR signals. However, the importance of this inherent autoreactivity for Treg cell biology remains poorly defined. To address this open question, we genetically ablated the TCR of mature Treg cells in vivo. These experiments revealed that TCR-induced Treg lineage-defining Foxp3 expression and gene hypomethylation were uncoupled from TCR input in mature Treg cells. However, Treg cell homeostasis, cell-type-specific gene expression and suppressive function critically depend on continuous triggering of their TCR.
Subject(s)
Autoimmunity/immunology , Forkhead Transcription Factors/biosynthesis , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cell Differentiation/immunology , Cell Lineage/immunology , DNA Methylation/immunology , DNA-Binding Proteins/genetics , Forkhead Transcription Factors/genetics , Inflammation/immunology , Interferon Regulatory Factors/biosynthesis , Mechanistic Target of Rapamycin Complex 1 , Mechanistic Target of Rapamycin Complex 2 , Mice , Mice, Inbred C57BL , Mice, Knockout , Multiprotein Complexes/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , Signal Transduction/immunology , TOR Serine-Threonine Kinases/metabolism , Thymocytes/cytologyABSTRACT
Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the upregulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8⺠T cells from proliferating and upregulating Granzyme-B and interferon-γ in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors and promoted protection against tumor rechallenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in preactivated CD8⺠T cells in response to microenvironmental transforming growth factor-ß (TGF-ß), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-ß-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-ß signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation.
Subject(s)
Forkhead Transcription Factors/immunology , Neoplasms/immunology , Repressor Proteins/immunology , T-Lymphocytes, Cytotoxic/immunology , Transforming Growth Factor beta/immunology , Tumor Escape/immunology , Adoptive Transfer , Animals , Antigens, Neoplasm/immunology , CD4-Positive T-Lymphocytes/immunology , Cell Proliferation , Female , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Gene Expression Regulation , Granzymes/biosynthesis , Interferon-gamma/biosynthesis , JNK Mitogen-Activated Protein Kinases/biosynthesis , JNK Mitogen-Activated Protein Kinases/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-myc/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Signal Transduction/immunology , Smad2 Protein/immunology , Smad3 Protein/immunology , T-Lymphocytes, Cytotoxic/transplantation , Transcription, Genetic , Transcriptional Activation , Tumor Microenvironment/immunologyABSTRACT
Protein kinase B (also known as AKT) and the mechanistic target of rapamycin (mTOR) are central regulators of T cell differentiation, proliferation, metabolism, and survival. Here, we show that during chronic murine lymphocytic choriomeningitis virus infection, activation of AKT and mTOR are impaired in antiviral cytotoxic T lymphocytes (CTLs), resulting in enhanced activity of the transcription factor FoxO1. Blockade of inhibitory receptor programmed cell death protein 1 (PD-1) in vivo increased mTOR activity in virus-specific CTLs, and its therapeutic effects were abrogated by the mTOR inhibitor rapamycin. FoxO1 functioned as a transcriptional activator of PD-1 that promoted the differentiation of terminally exhausted CTLs. Importantly, FoxO1-null CTLs failed to persist and control chronic viral infection. Collectively, this study shows that CTLs adapt to persistent infection through a positive feedback pathway (PD-1?FoxO1?PD-1) that functions to both desensitize virus-specific CTLs to antigen and support their survival during chronic viral infection.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Forkhead Transcription Factors/immunology , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/immunology , Programmed Cell Death 1 Receptor/biosynthesis , T-Lymphocytes, Cytotoxic/immunology , Animals , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , CD28 Antigens/immunology , Cell Differentiation/immunology , Cell Line, Tumor , Chronic Disease , Forkhead Box Protein O1 , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Granzymes/biosynthesis , Humans , Interferon-gamma/biosynthesis , Jurkat Cells , Lymphocyte Activation/immunology , Lymphocytic Choriomeningitis/virology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Programmed Cell Death 1 Receptor/immunology , Proto-Oncogene Proteins c-akt/biosynthesis , Receptors, Antigen, T-Cell/immunology , Sirolimus/pharmacology , T-Lymphocytes, Cytotoxic/cytology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/biosynthesisABSTRACT
Colorectal cancer (CRC) is a worldwide disease with worse survival. Our objective is to identify previously unrecognized prognostic factors to better evaluate disease progression. Seven GEO datasets were collected and analysed using R software, followed by KEGG enrichment analysis and TFs network construction. LASSO-COX analysis was performed to select the most useful prognostic features. COX model was used to analyse prognostic factors associated with OS. The survival curve was constructed using Kaplan-Meier analysis. A Nomogram model was also constructed to predict prognosis. A total of 3559 differentially expressed genes (DEGs) and 66 differentially expressed transcription factors were identified. FOXD1 was identified as the most differentially expressed factor of TFs covering the most downstream DEGs and independent risk prognostic factor. Next, FOXD1 expression was detected using immunohistochemical staining in 131 CRC patients' tissue and the association between FOXD1 expression and clinicopathologic features was analysed. High expression of FOXD1 was correlated with TNM stage and pathological differentiation. Multivariate COX regression analyses confirmed that FOXD1 high-expression, TNM stage and tumour differentiation were independent prognostic risk factor of OS and DFS. Patients with high expression of FOXD1 were more likely to have poor overall survival and disease-free survival. The combination of FOXD1 and Plk2 which we have previously reported allowed us to predict the survival of post-surgical CRC patients more accurately, adding to the former prognostic model based on the TNM Stage. The results showed that patients with high expression of both FOXD1 and Plk2 have the worst survival. A combination of FOXD1 and Plk2 can better evaluate patients' survival.
Subject(s)
Colorectal Neoplasms , Forkhead Transcription Factors , Protein Serine-Threonine Kinases , Biomarkers, Tumor/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Disease-Free Survival , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Humans , Kaplan-Meier Estimate , Predictive Value of Tests , Prognosis , Proportional Hazards Models , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
BACKGROUND: Foxp3+RORγt+ T cells possess both characteristics of regulatory T cells and T helper 17 cells and show significant immunoregulatory functions in autoimmune diseases. However, the role and clinical significance of Foxp3+RORγt+ T cells in gastric cancer remains unclear. METHODS: We enrolled 452 gastric cancer tissue microarray samples and 60 fresh tumor tissue samples from Zhongshan Hospital. The infiltration of Foxp3+RORγt+ T cells and immune contexture were examined by immunohistochemistry and flow cytometry. Survival analyses of patient subgroups were conducted by Kaplan-Meier curves, log-rank test and Cox proportional model. RESULTS: High infiltration of Foxp3+RORγt+ T cells predicted poor overall survival (P = 0.0222 and 0.0110) and inferior therapeutic response (P = 0.003 for interaction) in gastric cancer. Foxp3+RORγt+ T cells were associated with impaired effective function of CD8+ T cells featured by decreased interferon-γ, granzyme B and CD107a expression. Co-evaluation of Foxp3+RORγt+ T cells and CD8+ T cells could predict survival outcomes and chemotherapeutic responsiveness more precisely. CONCLUSIONS: We found that Foxp3+RORγt+ T cells could potentially attenuate effective functions of CD8+ T cells and led to adverse survival outcomes and inferior chemotherapeutic responsiveness. Moreover, the novel co-evaluation system might be useful for prognosis prediction for appropriate treatment in gastric cancer. NOVELTY AND IMPACT STATEMENTS: Clinical significance of Foxp3+RORγts+ T cells has not been studied in gastric cancer. Herein, we investigated the prognostic value of Foxp3+RORγt+ T cells in 452 patients. We demonstrated that intratumoral Foxp3+RORγt+ T cell infiltration was a prognostic biomarker for overall survival and the identification of patients might benefit from post-gastrectomy 5-fluorouracil. These findings allow a more precise stratification upon the co-evaluation with CD8+ T cells to better clinical management for patients who would benefit from 5-fluorouracil.
Subject(s)
Forkhead Transcription Factors/biosynthesis , Gene Expression Regulation, Neoplastic , Nuclear Receptor Subfamily 1, Group F, Member 3/biosynthesis , Stomach Neoplasms/immunology , Stomach Neoplasms/metabolism , T-Lymphocytes/cytology , Aged , Autoimmune Diseases/immunology , CD8-Positive T-Lymphocytes/cytology , Chemotherapy, Adjuvant , Female , Humans , Kaplan-Meier Estimate , Lymphocytes, Tumor-Infiltrating , Male , Middle Aged , Prognosis , Proportional Hazards Models , Retrospective Studies , Tissue Array Analysis , Treatment OutcomeABSTRACT
CD4(+) regulatory T cells (T(reg) cells) characterized by expression of the transcription factor Foxp3 have a pivotal role in maintaining immunological tolerance. Here we show that mice with T cell-specific deletion of both the Foxo1 and Foxo3 transcription factors (collectively called 'Foxo proteins' here) developed a fatal multifocal inflammatory disorder due in part to T(reg) cell defects. Foxo proteins functioned in a T(reg) cell-intrinsic manner to regulate thymic and transforming growth factor-beta (TGF-beta)-induced Foxp3 expression, in line with the ability of Foxo proteins to bind to Foxp3 locus and control Foxp3 promoter activity. Transcriptome analyses showed that Foxo proteins regulated the expression of additional T(reg) cell-associated genes and were essential for inhibiting the acquisition of effector T cell characteristics by T(reg) cells. Thus, Foxo proteins have crucial roles in specifying the T(reg) cell lineage.
Subject(s)
Cell Differentiation/immunology , Forkhead Transcription Factors/metabolism , T-Lymphocytes, Regulatory/metabolism , Thymus Gland/metabolism , Animals , CD4 Antigens/biosynthesis , Cell Lineage , Cells, Cultured , Forkhead Box Protein O1 , Forkhead Box Protein O3 , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Gene Expression Profiling , Gene Expression Regulation, Developmental , Inflammation , Mice , Mice, Knockout , Mice, Mutant Strains , Mutation/genetics , Protein Binding , Regulatory Elements, Transcriptional/genetics , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Thymus Gland/immunology , Thymus Gland/pathology , Transforming Growth Factor beta/metabolismABSTRACT
Stable Foxp3 expression is crucial for regulatory T (Treg) cell function. We observed that antigen-driven activation and inflammation in the CNS promoted Foxp3 instability selectively in the autoreactive Treg cells that expressed high amounts of Foxp3 before experimental autoimmune encephalitis induction. Treg cells with a demethylated Treg-cell-specific demethylated region in the Foxp3 locus downregulated Foxp3 transcription in the inflamed CNS during the induction phase of the response. Stable Foxp3 expression returned at the population level with the resolution of inflammation or was rescued by IL-2-anti-IL-2 complex treatment during the antigen priming phase. Thus, a subset of fully committed self-antigen-specific Treg cells lost Foxp3 expression during an inflammatory autoimmune response and might be involved in inadequate control of autoimmunity. These results have important implications for Treg cell therapies and give insights into the dynamics of the Treg cell network during autoreactive CD4(+) T cell effector responses in vivo.
Subject(s)
Autoantigens/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Forkhead Transcription Factors/physiology , Gene Expression Regulation/immunology , T-Lymphocytes, Regulatory/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Cell Lineage , Central Nervous System/immunology , DNA Methylation , Down-Regulation/immunology , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Genes, Reporter , Lymph Nodes/immunology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Myelin-Oligodendrocyte Glycoprotein/immunology , Peptide Fragments/immunology , Receptors, Interleukin-2/physiology , Recombinant Fusion Proteins/immunology , Regulatory Sequences, Nucleic Acid , Specific Pathogen-Free OrganismsABSTRACT
Regulatory T (Treg) cells expressing the transcription factor Foxp3 have a pivotal role in maintaining immunological self-tolerance; yet, excessive Treg cell activities suppress anti-tumour immune responses. Compared to the resting Treg (rTreg) cell phenotype in secondary lymphoid organs, Treg cells in non-lymphoid tissues exhibit an activated Treg (aTreg) cell phenotype. However, the function of aTreg cells and whether their generation can be manipulated are largely unexplored. Here we show that the transcription factor Foxo1, previously demonstrated to promote Treg cell suppression of lymphoproliferative diseases, has an unexpected function in inhibiting aTreg-cell-mediated immune tolerance in mice. We find that aTreg cells turned over at a slower rate than rTreg cells, but were not locally maintained in tissues. aTreg cell differentiation was associated with repression of Foxo1-dependent gene transcription, concomitant with reduced Foxo1 expression, cytoplasmic localization and enhanced phosphorylation at the Akt sites. Treg-cell-specific expression of an Akt-insensitive Foxo1 mutant prevented downregulation of lymphoid organ homing molecules, and impeded Treg cell homing to non-lymphoid organs, causing CD8(+) T-cell-mediated autoimmune diseases. Compared to Treg cells from healthy tissues, tumour-infiltrating Treg cells downregulated Foxo1 target genes more substantially. Expression of the Foxo1 mutant at a lower dose was sufficient to deplete tumour-associated Treg cells, activate effector CD8(+) T cells, and inhibit tumour growth without inflicting autoimmunity. Thus, Foxo1 inactivation is essential for the migration of aTreg cells that have a crucial function in suppressing CD8(+) T-cell responses; and the Foxo signalling pathway in Treg cells can be titrated to break tumour immune tolerance preferentially.
Subject(s)
Autoimmunity/immunology , CD8-Positive T-Lymphocytes/immunology , Forkhead Transcription Factors/metabolism , Immune Tolerance/immunology , Neoplasms/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Animals , Cell Differentiation , Cell Movement/immunology , Down-Regulation , Female , Forkhead Box Protein O1 , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Lymphocyte Activation , Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Male , Mice , Mutation , Phosphorylation , Signal Transduction/immunology , T-Lymphocytes, Regulatory/cytology , Transcription, GeneticABSTRACT
Chronic obstructive pulmonary disease (COPD) is an inflammatory condition associated with abnormal immune responses, leading to airflow obstruction. Lungs of COPD subjects show accumulation of proinflammatory T helper (Th) 1 and Th17 cells resembling that of autoreactive immune responses. As regulatory T (Treg) cells play a central role in the control of autoimmune responses and their generation and function are controlled by the adipocytokine leptin, we herein investigated the association among systemic leptin overproduction, reduced engagement of glycolysis in T cells, and reduced peripheral frequency of Treg cells in different COPD stages. These phenomena were also associated with an impaired capacity to generate inducible Treg (iTreg) cells from conventional T (Tconv) cells. At the molecular level, we found that leptin inhibited the expression of forkhead-boxP3 (FoxP3) and its splicing variants containing the exon 2 (FoxP3-E2) that correlated inversely with inflammation and weakened lung function during COPD progression. Our data reveal that the immunometabolic pathomechanism leading to COPD progression is characterized by leptin overproduction, a decline in the expression of FoxP3 splicing forms, and an impairment in Treg cell generation and function. These results have potential implications for better understanding the autoimmune-like nature of COPD and the pathogenic events leading to lung damage.
Subject(s)
Alternative Splicing/immunology , Forkhead Transcription Factors , Leptin , Pulmonary Disease, Chronic Obstructive , T-Lymphocytes, Regulatory , Female , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/immunology , Humans , Leptin/biosynthesis , Leptin/immunology , Male , Middle Aged , Pulmonary Disease, Chronic Obstructive/immunology , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathology , Th1 Cells/immunology , Th1 Cells/metabolism , Th1 Cells/pathology , Th17 Cells/immunology , Th17 Cells/metabolism , Th17 Cells/pathologyABSTRACT
BACKGROUND: Characterization of markers of both immune suppression and activation may provide more prognostic information than assessment of single markers in localized prostate cancer. We therefore sought to determine the association between CD8 and PD-L1 expression in localized prostate tumors and biochemical recurrence (BCR) and metastasis-free survival (MFS). METHODS: Tissue microarrays were constructed on 109 men undergoing radical prostatectomy (RP) for localized prostate cancer at Dana-Farber Cancer Institute between 1991 and 2008. Fluorescence immunohistochemistry was used to evaluate the expression of six immune markers (CD3, CD4, CD8, PD-1, PD-L1, FOXP3). Quantitative multispectral imaging analysis was used to calculate the density of each marker, which was dichotomized by the median as "high" or "low." Cox proportional hazards regression models and Kaplan-Meier analyses were used to analyze associations between immune marker densities and time to BCR and MFS. RESULTS: Over a median follow-up of 8.1 years, 55 (51%) and 39 (36%) men developed BCR and metastases, respectively. Median time to BCR was shorter in men with low CD8 (hazard ratio [HR] = 2.27 [1.27-4.08]) and high PD-L1 expression (HR = 2.03 [1.17-3.53]). While neither low CD8 or high PD-L1 alone were independent predictors of BCR or MFS on multivariable analysis, men with low CD8 and/or high PD-L1 had a significantly shorter time to BCR (median 3.5 years vs. NR) and MFS (median 10.8 vs. 18.4 years) compared to those with high CD8 and low PD-L1 expression. The main limitation is the retrospective and singe-center nature of the study. CONCLUSION: The presence of higher CD8 and lower PD-L1 expression in prostatectomy specimens was associated a low risk of biochemical relapse and metastatic disease. These findings are hypothesis-generating and further study is needed.
Subject(s)
B7-H1 Antigen/biosynthesis , CD8 Antigens/biosynthesis , Prostatic Neoplasms/immunology , B7-H1 Antigen/immunology , CD3 Complex/biosynthesis , CD3 Complex/immunology , CD8 Antigens/immunology , Cohort Studies , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/immunology , Humans , Immunohistochemistry , Male , Middle Aged , Neoplasm Metastasis , Neoplasm Recurrence, Local/immunology , Programmed Cell Death 1 Receptor/biosynthesis , Programmed Cell Death 1 Receptor/immunology , Proportional Hazards Models , Prostatectomy , Prostatic Neoplasms/surgery , Retrospective Studies , Tissue Array AnalysisABSTRACT
We characterised patients with mantle cell lymphoma (MCL) with poor prognosis based on differences in immune infiltration. Different expressions of the tumour cell markers Cyclin D1 and sex-determining region Y-box transcription factor 11 (SOX11), and the immune markers cluster of differentiation 3 (CD3), CD4, CD8, CD25, forkhead box protein P3 (FoxP3), T-box transcription factor TBX21 (T-bet), programmed cell death protein 1 (PD-1), programmed-death ligand 1 (PD-L1) and CD163 were investigated for all-cause mortality in 282 patients with MCL and time-to-progression (TTP) in 106 clinical trial patients. With increasing age, a significantly lower infiltration of CD3+ T lymphocytes was seen. T-cell infiltration was independent of cellular tumour antigen p53 (p53) expression, Ki-67, morphology and frequency of tumour cells. The all-cause mortality was higher in patients with PD-L1-expression above cut-off [hazard ratio (HR) 1·97, 95% confidence interval (CI) 1·18-3·25, adjusted for sex and MCL International Prognostic Index (MIPI)] and a higher frequency of CD163+ cells (continuously, HR 1·51, 95% CI 1·03-2·23, adjusting for age, sex, morphology, Ki-67 and p53). In patients treated within the Nordic Lymphoma Group MCL2/3 trials, TTP was shorter in patients with a higher frequency of FoxP3+ cells (HR 3·22, 95% CI 1·40-7·43) and CD163+ cells (HR 6·09, 95% CI 1·84-20·21), independent of sex and MIPI. When combined a higher frequency of CD163+ macrophages and PD-L1+ cells or high CD163+ macrophages and FoxP3+ regulatory T cells indicated worse outcome independent of established risk factors. The T-cell infiltrate was in turn independent of molecular characteristics of the malignant cells and decreased with age.
Subject(s)
Aging/metabolism , Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , B7-H1 Antigen/biosynthesis , Forkhead Transcription Factors/biosynthesis , Lymphoma, Mantle-Cell , Neoplasm Proteins/biosynthesis , Receptors, Cell Surface/biosynthesis , Aged , Female , Humans , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/mortality , Male , Middle Aged , Risk FactorsABSTRACT
During forebrain development, a telencephalic organizer called the cortical hem is crucial for inducing hippocampal fate in adjacent cortical neuroepithelium. How the hem is restricted to its medial position is therefore a fundamental patterning issue. Here, we demonstrate that Foxg1-Lhx2 interactions are crucial for the formation of the hem. Loss of either gene causes a region of the cortical neuroepithelium to transform into hem. We show that FOXG1 regulates Lhx2 expression in the cortical primordium. In the absence of Foxg1, the presence of Lhx2 is sufficient to suppress hem fate, and hippocampal markers appear selectively in Lhx2-expressing regions. FOXG1 also restricts the temporal window in which loss of Lhx2 results in a transformation of cortical primordium into hem. Therefore, Foxg1 and Lhx2 form a genetic hierarchy in the spatiotemporal regulation of cortical hem specification and positioning, and together ensure the normal development of this hippocampal organizer.