ThPOK Expression Research Articles

Super-enhancer profiling reveals ThPOK/ZBTB7B, a CD4 + cell lineage commitment factor, as a master regulator that restricts breast cancer cells to a luminal non-migratory phenotype.

Despite efforts to understand breast cancer biology, metastatic disease remains a clinical challenge. Identifying suppressors of breast cancer progression and mechanisms of transition to more invasive phenotypes could provide game changing therapeutic opportunities. Transcriptional deregulation is central to all malignancies, highlighted by the extensive reprogramming of regulatory elements that underlie oncogenic programs. Among these, super-enhancers (SEs) stand out due to their enrichment in genes controlling cancer hallmarks. To reveal novel breast cancer dependencies, we integrated the analysis of the SE landscape with master regulator activity inference for a series of breast cancer cell lines. As a result, we identified T - h elper-inducing Poxviruses and Zinc-finger ( PO Z)/ K rüppel-like factor (ThPOK, ZBTB7B ), a CD4 + cell lineage commitment factor, as a breast cancer master regulator that is recurrently associated with a SE. ThPOK expression is highest in luminal breast cancer but is significantly reduced in the basal subtype. Manipulation of ThPOK levels in cell lines shows that its repressive function restricts breast cancer cells to an epithelial phenotype by suppressing the expression of genes involved in the epithelial-mesenchymal transition (EMT), WNT/β-catenin target genes, and the pro-metastatic TGFβ pathway. Our study reveals ThPOK as a master transcription factor that restricts the acquisition of metastatic features in breast cancer cells.

CD4, but not Cxcr6, is necessary for control of Pneumocystis murina infection

CD4+ T cells are critical to control of Pneumocystis infection, and Cxcr6 has been shown to be upregulated in these cells during infection, but the roles of CD4 and Cxcr6 in this setting are undefined. To explore this, mice deficient in CD4 or Cxcr6 expression were utilized in a co-housing mouse model that mimics the natural route of Pneumocystis infection. Organism load and anti-Pneumocystis antibodies were assayed over time, and immunohistochemistry, flow cytometry, and quantitative PCR were used to characterize host immune responses during infection. CD4 was found to be necessary for clearance of Pneumocystis murina, though partial control was seen in it's absence; based on ThPOK expression, double negative T cells with T helper cell characteristics may be contributing to this control. Using a Cxcr6 deficient mouse expressing gfp, control of infection in the absence of Cxcr6 was similar to that in heterozygous control mice. It is noteworthy that gfp + cells were seen in the lungs with similar frequencies between the 2 strains. Interferon-ɣ and chemokine/ligands Cxcr3, Cxcl9, and Cxcl10 increased during P. murina infection in all models. Thus, CD4, but not Cxcr6, is needed for clearance of P. murina infection.

CD4+CD8αα+ is the dominant phenotype of intraepithelial lymphocytes and regulated by ThPOK and Runx3 in oral lichen planus.

Oral lichen planus (OLP) is a common T cell-mediated oral mucosal immune inflammatory disease. Intraepithelial lymphocytes (IELs) are a unique subset of T cells that play an important role in regulating immune response. This study aims to investigate the phenotype and the differentiation mechanism of IELs in OLP. The expression of CD4, CD8α, CD8β, T-helper-inducing POZ/Krueppel-like factor (ThPOK), and RUNX family transcription factor 3 (Runx3) in the epithelium and peripheral blood mononuclear cells (PBMCs) of OLP was determined by immunofluorescence and immunohistochemistry. Then, the correlations among them were analyzed. Naïve CD4+ T cells were sorted from blood of OLP patients and stimulated with retinoic acid (RA) and transforming growth factor-β1 (TGF-β1). Then the expression of CD4, CD8α, CD8β, ThPOK, and Runx3 was investigated by immunocytochemistry. CD8α expression and CD8αα+ cells were upregulated in the epithelium of OLP, whereas they were downregulated in PBMCs of OLP. CD8β was not expressed in the epithelium of OLP. CD4, CD8α, and Runx3 expression and CD4+CD8α+ cells were increased, whereas ThPOK expression was decreased in the epithelium of OLP. CD8α expression was positively correlated with Runx3 expression, whereas ThPOK expression was negatively correlated with Runx3 expression. After RA and TGF-β1 stimulation, CD8α and Runx3 expression was upregulated, and ThPOK expression was downregulated in naïve CD4+ T cells. CD4+CD8αα+ IELs may be the dominant phenotype of IELs in OLP, and the differentiation of CD4+CD8αα+ IELs in OLP is negatively regulated by ThPOK and positively regulated by Runx3.

TRIB2 safeguards naive T cell homeostasis during aging

Naive CD4+ Tcells are more resistant to age-related loss than naive CD8+ Tcells, suggesting mechanisms that preferentially protect naive CD4+ Tcells during aging. Here, we show that TRIB2 is more abundant in naive CD4+ than CD8+ Tcells and counteracts quiescence exit by suppressing AKT activation. TRIB2 deficiency increases AKT activity and accelerates proliferation and differentiation in response to interleukin-7 (IL-7) in humans and during lymphopenia in mice. TRIB2 transcription is controlled by the lineage-determining transcription factors ThPOK and RUNX3. Ablation of Zbtb7b (encoding ThPOK) and Cbfb (obligatory RUNT cofactor) attenuates the difference in lymphopenia-induced proliferation between naive CD4+ and CD8+ cells. In older adults, ThPOK and TRIB2 expression wanes in naive CD4+ Tcells, causing loss of naivety. These findings assign TRIB2 a key role in regulating Tcell homeostasis and provide a model to explain the lesser resilience of CD8+ Tcells to undergo changes with age.

TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner.

TET proteins mediate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives. We have previously demonstrated a dynamic enrichment of 5hmC during T and invariant natural killer T cell lineage specification. Here, we investigate shared signatures in gene expression of Tet2/3 DKO CD4 single positive (SP) and iNKT cells in the thymus. We discover that TET proteins exert a fundamental role in regulating the expression of the lineage specifying factor Th-POK, which is encoded by Zbtb7b. We demonstrate that TET proteins mediate DNA demethylation - surrounding a proximal enhancer, critical for the intensity of Th-POK expression. In addition, TET proteins drive the DNA demethylation of site A at the Zbtb7b locus to facilitate GATA3 binding. GATA3 induces Th-POK expression in CD4 SP cells. Finally, by introducing a novel mouse model that lacks TET3 and expresses full length, catalytically inactive TET2, we establish a causal link between TET2 catalytic activity and lineage specification of both conventional and unconventional T cells.

ThPOK inhibits the immune escape of gastric cancer cells by inducing STPG1 to inactivate the ERK pathway

BackgroundGastric cancer is the second most frequently diagnosed cancer worldwide. Weak immunogenicity helps cancer cells escape from immune elimination and grow into predominant subpopulations. This study aimed to investigate the effect of Zinc finger and BTB domain containing 7B (Zbtb7b, Alias ThPOK) on T cell activation after coculture with gastric cancer cells.MethodsCell Counting Kit-8 assay (CCK-8) was performed to explore the viability of gastric cancer cells. Flow cytometry analysis was used to measure CD3+ T cell proliferation and the ratio of activated IFN-γ+ T cells which were co-incubated with gastric cancer cells (HGC-27, SNU-1). The binding between ThPOK and the promoter of its target sperm tail PG-rich repeat containing 1 (STPG1) was explored using ChIP and luciferase reporter assays. Relative gene expression was quantified using RT-qPCR.ResultsThPOK was expressed at a low level in gastric cancer tissues and cells at mRNA and protein levels. Gastric cancer patients with lower ThPOK expression had poorer prognosis. ThPOK overexpression suppressed gastric cancer cell viability and increased T cell activation. ThPOK served as a transcription factor for STPG1. STPG1 expression was also at a low level in the tissues and cells of gastric cancer. ThPOK positively regulated the mRNA and protein levels of STPG1 in gastric cancer cells. Moreover, ThPOK was demonstrated to bind with STPG1 promoter. STPG1 upregulation also exerted inhibitory effects on gastric cancer cell viability and T cell activation. Additionally, ThPOK and STPG1 were revealed to inactivate the ERK pathway in gastric cancer cells.ConclusionThPOK inhibits gastric cancer cell viability and increases T cell activation by inducing STPG1 to inactivate the ERK pathway.

ThPOK Inhibits Osteoclast Formation Via NFATc1 Transcription and Function.

ABSTRACTBoth LRF (Zbtb7a) and ThPOK (Zbtb7b) belong to the POK (BTB/POZ and Kruppel) family of transcription repressors that participate in development, differentiation, and oncogenesis. Although LRF mediates osteoclast differentiation by regulating NFATc1 expression, the principal established function of ThPOK is transcriptional control of T‐cell lineage commitment. Whether ThPOK affects osteoclast formation or function is not known. We find that marrow macrophage ThPOK expression diminishes with exposure to receptor activator of NF‐kB ligand (RANKL), but ThPOK deficiency does not affect osteoclast differentiation. On the other hand, enhanced ThPOK, in macrophages, substantially impairs osteoclastogenesis. Excess ThPOK binds the NFATc1 promoter and suppresses its transcription, suggesting a mechanism for its osteoclast inhibitory effect. Despite suppression of osteoclastogenesis by excess ThPOK being associated with diminished NFATc1, osteoclast formation is not rescued by NFATc1 overexpression. Thus, ThPOK appears to inhibit NFATc1 transcription and its osteoclastogenic capacity, while its depletion has no effect on the bone‐resorptive cell. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

ThPOK inhibits osteoclast formation via NFATc1 transcription and function

Both LRF (Zbtb7a) and ThPOK (Zbtb7b) belong to the POK (BTB/POZ and Kruppel) family of transcription repressors which participate in development, differentiation and oncogenesis. While LRF mediates osteoclast differentiation by regulating NFATc1 expression, the principal established function of ThPOK is transcriptional control of T-cell lineage commitment. Whether ThPOK affects osteoclast formation or function, is not known.We find that marrow macrophage ThPOK expression diminishes with exposure to RANKL but ThPOK deficiency does not affect osteoclast differentiation. On the other hand, enhanced ThPOK, in macrophages, substantially impairs osteoclastogenesis. Excess ThPOK binds the NFATc1 promoter and suppresses its transcription suggesting a mechanism for its osteoclast inhibitory effect. Despite suppression of osteoclastogenesis by excess ThPOK being associated with diminished NFATc1, osteoclast formation is not rescued by NFATc1 overexpression. Thus, ThPOK appears to inhibit NFATc1 transcription and its osteoclastogenic capacity while its depletion has no effect on the bone resorptive cell. This article is protected by copyright. All rights reserved.

An autonomous TCR signal-sensing switch influences CD4/CD8 lineage choice in mice

How multipotential cells initiate distinct gene expression programs in response to external cues to instruct cell fate choice remains a fundamental question in biology. Establishment of CD4 and CD8 T cell fates during thymocyte development is critically regulated by T cell receptor (TCR) signals, which in turn control expression of the CD4-determining transcription factor ThPOK. However, the mechanism whereby differential TCR signals are molecularly interpreted to promote or antagonize ThPOK expression, and thereby CD4 versus CD8 lineage fates remains unknown. Here we show, using reverse genetic and molecular approaches that an autonomous, position-independent TCR-sensing switch is embedded within the ThPOK locus. Further, using an in vivo mutagenesis approach, we demonstrate that differential TCR signals are interpreted during lineage commitment by relative binding of EGR, NFAT and Ebox factors to this bistable switch. Collectively our study reveals the central molecular mechanism whereby TCR signaling influences differential lineage choice. Ultimately, these findings may provide an important new tool for skewing T cell fate to treat cancer and autoimmune diseases.

Essential role of a ThPOK autoregulatory loop in the maintenance of mature CD4+ T cell identity and function.

The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4+ intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (SilThPOK). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the SilThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (Treg) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITRloPD-1loCD25lo (Triplelo) Treg cells and conversion to CD4+ IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance.

Twist2 promotes CD8+ T-cell differentiation by repressing ThPOK expression.

CD4/CD8 T-cell lineage differentiation is a key process in immune system development; however, a defined regulator(s) that converts the signal from T-cell receptor and co-receptor complexes into lineage differentiation remains unclear. Here, we show that Twist2 is a critical factor in CD4/CD8 thymocyte differentiation. Twist2 expression is differentially regulated by T-cell receptor signaling, leading to differentiation into the CD4 or CD8 lineage. Forced Twist2 expression perturbed CD4+ thymocyte differentiation while enhancing CD8+ thymocyte differentiation. Furthermore, Twist2 expression produced mature CD8+ thymocytes in B2m-/- mice, while its deficiency significantly impaired CD8+ cells in MHC class-II-/- and TCR transgenic mice, favoring CD8 T-cell differentiation. During CD8 lineage differentiation, Twist2 interacted with Runx3 to bind to the silencer region of the ThPOK locus, thereby blocking ThPOK expression. These findings indicate that Twist2 is a part of the transcription factor network controlling CD8 lineage differentiation.

CD4 Cytotoxic T cells play a critical role in anti-tumor protection

Abstract The CD4 T cell lineage is established in the thymus by the transcription factor, ThPOK. In the periphery, ThPOK maintains the CD4 T helper commitment and allows the differentiation to different helper fates in response to stimulation with cognate antigens. We demonstrated that in the intestine, chronic stimulation of CD4 T cells results in the loss of ThPOK expression leading to the reprogramming of the T helper cells to cytotoxic T lymphocytes (CTL), expressing notably CD8 T cells attributes. Continuous activation in a non-immunogenic environment is also a hallmark of tumor progression. We investigated if the tumor environment supports the generation of CD4 CTL and moreover if they play essential roles in anti-tumor protection. Using the well-defined melanoma B16 model, we observed that CD4 T cells responding to the tumor uniquely lose ThPOK expression. To address the importance of the CD4 CTL in the tumor immune surveillance, we engineered two unique mouse models, in which either all the CD4 T cells are forced to reprogram to CTL or else, prevented from reprogramming to CTL. Using these models, we observed that the forced CD4 CTL eradicate melanoma cells, whereas the CD4 cells which cannot lose ThPOK and reprogram to CTL are ineffective against the tumor growth. These findings underscore the importance of CD4 CTL reprogramming in protective immunity. Finally, we developed a culture system to induce the reprograming of CD4 Th to CTL in vitro. We injected in vitro-generated CD4 CTL alone or with a co-transfer of CD8 T cells into Rag deficient hosts bearing tumor. As compared to the transfer of the CD4 CTL or CD8 CTL alone, the cooperation of the cytotoxic effector cells displayed a strong synergized effect on long term protection against tumor growth.

Thpok suppresses pathogenic cytokine production by Th17 cells in autoimmune disease

Abstract CD4+ T cells, including Th17 cells, are instrumental in the development and progression of autoimmune diseases such as multiple sclerosis. While pathogenic Th17 cells drive inflammation in autoimmunity, the molecular programming underlying their pathogenicity remains insufficiently understood. Through RNA-sequencing, we have identified the transcription factor Thpok (Zbtb7b), also known as the CD4 lineage determination factor, as an apparent negative regulator of Th17 pathogenicity. Thpok expression is maintained in peripheral CD4+ T cells and assists in formation of memory CD4+ T cells. Furthermore, Thpok expression in CD4+ T cells represses cytotoxic genes and contributes to the Th17 response during intestinal inflammation. Herein we show Thpok is upregulated in pathogenic Th17 cells both in vitro and in vivo. In addition, we show increased expression of Thpok results in reduced production of IL-17, while conversely, loss of Thpok results in higher inflammatory potential of Th17 cells. Moreover, we have demonstrated that Thpok expression in Th17 cells in vivo functions to inhibit the development of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Altogether this study establishes Thpok as a suppressor of the autoimmune inflammatory function of Th17 cells.

Cytotoxic CD4 T cells control host immunity during viral persistence

Abstract The CD4 T helper cell lineage is initially established and controlled in the thymus by the transcription factor, ThPOK. Sustained expression of ThPOK by mature T cells in the periphery maintains the CD4 T helper commitment and allows for plasticity and differentiation to different helper fates (Th1, Th2, Th17...) in response to stimulation with cognate antigens. Our group demonstrated before that in the intestine, repeated stimulation of mature CD4 T cells results in the loss of ThPOK expression followed by the reprogramming of the T helper cells to CD4 cytotoxic T lymphocytes (CTL), expressing notably CD8 T cells attributes (such as Runx3, granzyme B). Continuous activation is also characteristic of chronic viral infections. We therefore investigated if viral infections lead to the generation of reprogrammed CD4 CTL and moreover if these reprogrammed CD4 CTL play essential roles for the organism’s protection. Using the well-defined lymphocytic choriomeningitis virus (LCMV) acute (strain LCMV-Amstrong) versus chronic (LCMV-Clone 13) infection model, we observed that solely chronic infection enables activated CD4 T helper cells to lose ThPOK and convert to CTL with potent killer activity. Moreover, viral-induced CD4 CTL precursors produce various cytokines, notably Il-10, which is required for the induction of the reprogramming process. Finally, we engineered two unique mouse model systems, in which either all the CD4 T cells are forced to reprogram to CTL (gain of function) or else, prevented from reprogramming to CTL (loss of function). Using these models, we show that the reprogrammed CD4 CTL generated under chronic infection conditions, play critical roles in protective immunity.

Tle corepressors are differentially partitioned to instruct CD8+ T cell lineage choice and identity.

Tle/Groucho proteins are transcriptional corepressors interacting with Tcf/Lef and Runx transcription factors, but their physiological roles in T cell development remain unknown. Conditional targeting of Tle1, Tle3 and Tle4 revealed gene dose-dependent requirements for Tle proteins in CD8+ lineage cells. Upon ablating all three Tle proteins, generation of CD8+ T cells was greatly diminished, largely owing to redirection of MHC-I-selected thymocytes to CD4+ lineage; the remaining CD8-positive T cells showed aberrant up-regulation of CD4+ lineage-associated genes including Cd4, Thpok, St8sia6, and Foxp3 Mechanistically, Tle3 bound to Runx-occupied Thpok silencer, in post-selection double-positive thymocytes to prevent excessive ThPOK induction and in mature CD8+ T cells to silence Thpok expression. Tle3 also bound to Tcf1-occupied sites in a few CD4+ lineage-associated genes, including Cd4 silencer and St8sia6 introns, to repress their expression in mature CD8+ T cells. These findings indicate that Tle corepressors are differentially partitioned to Runx and Tcf/Lef complexes to instruct CD8+ lineage choice and cooperatively establish CD8+ T cell identity, respectively.

An Immunotherapeutic CD137 Agonist Releases Eomesodermin from ThPOK Repression in CD4 T Cells.

Agonists to the TNF/TNFR costimulatory receptors CD134 (OX40) and CD137 (4-1BB) elicit antitumor immunity. Dual costimulation with anti-CD134 plus anti-CD137 is particularly potent because it programs cytotoxic potential in CD8+ and CD4+ T cells. Cytotoxicity in dual-costimulated CD4 T cells depends on the T-box transcription factor eomesodermin (Eomes), which we report is induced via a mechanism that does not rely on IL-2, in contrast to CD8+ CTL, but rather depends on the CD8 T cell lineage commitment transcription factor Runx3, which supports Eomes expression in mature CD8+ CTLs. Further, Eomes and Runx3 were indispensable for dual-costimulated CD4 T cells to mediate antitumor activity in an aggressive melanoma model. Runx3 is also known to be expressed in standard CD4 Th1 cells where it fosters IFN-γ expression; however, the CD4 T cell lineage commitment factor ThPOK represses transcription of Eomes and other CD8 lineage genes, such as Cd8a Hence, CD4 T cells can differentiate into Eomes+ cytotoxic CD4+CD8+ double-positive T cells by terminating ThPOK expression. In contrast, dual-costimulated CD4 T cells express Eomes, despite the continued expression of ThPOK and the absence of CD8α, indicating that Eomes is selectively released from ThPOK repression. Finally, although Eomes was induced by CD137 agonist, but not CD134 agonist, administered individually, CD137 agonist failed to induce CD134-/- CD4 T cells to express Eomes or Runx3, indicating that both costimulatory pathways are required for cytotoxic Th1 programming, even when only CD137 is intentionally engaged with a therapeutic agonist.

Th-POK regulates mammary gland lactation through mTOR-SREBP pathway.

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation.

Murine CD8+ Invariant Natural Killer T Cells are Negatively Selected by CD1d Expressed on Thymic Epithelial Cells and Dendritic Cells

ABSTRACTBackground: CD1d-dependent invariant natural killer (iNKT) cells are found as either CD4 single positive (SP) or CD4/CD8 double negative (DN) cells in mice. The size of the CD8+ iNKT population is extremely small. It is known that CD1d expression on developing thymocytes is sufficient for iNKT development and co-receptor choice, which is driven by Th-POK expression. This study aimed to examine the factors involved in the CD4/CD8 co-receptor choice of iNKT cells in addition to Th-POK-driven silencing of CD8 expression. Methods: In this study, we compared iNKT cells of wild-type (WT) mice with those of transgenic mice in which CD1d expression is restricted to developing thymocytes by the proximal Lck (pLCK) promoter. CD8 positive iNKT cell population were analyzed by flow cytometry. Results: We found that there was a substantial population of CD8+ iNKT cells in the thymus and spleen of transgenic mice, and these cells are negatively selected in between Stage 2 and Stage 3 of their developmental program by the CD1d expressed on Thymic epithelial cell (TEC) and Dendritic cells in WT mice. Conclusion: We conclude that TEC expression of CD1d in the murine thymus contributed to co-receptor choice of iNKT cells, in addition to Th-POK-driven silencing of CD8. Therefore, mostly CD4 SP and DN iNKT cells are produced under normal physiological conditions in mice.

Natural ThPOK− CD4 cytotoxic T lymphocytes : Thymic development of a new distinct self-specific CD4 T cell population.

Abstract The lineage commitment to either CD4 or CD8 T cells is made in the thymus, where thymocytes with specificity for MHC-II molecules differentiates into the CD4 “helper” linage whereas ones specific for MHC-I molecules commit to the CD8 “cytotoxic” lineage. The molecular regulation is controlled by the action of key transcription factors. Notably ThPOK, which promotes the CD4 fate and suppress the cytotoxic fate of CD4 T cells. Recently, we made a major breakthrough and showed that these lineages is not fixed and the mature peripheral CD4 T cells have the plasticity to lose ThPOK expression and reprogram to ThPOKneg CD4 CTL (cytotoxic T lymphocytes) marked by the expression of CD8a, Granzyme B and potent killer capacity. Investigating the origin of CD4 CTL, we observed in ThPOK fate maping mouse a strong proportion of CD4 CTL that never expressed ThPOK, suggesting that some CD4 CTL are natural cytotoxic cells that develop in the thymus. Based on a recent works allowing the analysis of mature CD4 cells in the thymus, we observed a substantial number of mature CD4 T cells that do not express ThPOK and display an active-like phenotype. Moreover, once injected, thymic ThPOKneg mature CD4 T cells remain ThPOKneg in the periphery and start to acquire CD8a expression demonstrating that natural CD4 CTL arise from the thymus. We also demonstrated that this population requires a strong TCR signal during their development, suggesting that natural CD4 CTL have a repertoire specific for self-antigens. We have identified a new distinct population of mature CD4 T cells that develop in the thymus: the natural cytotoxic CD4 T lymphocytes. We also established mechanism underlying these process by demonstrating that CD4 CTL needs a strong TCR signal for their differentiation.

MicroRNA-133b Regulation of Th-POK Expression and Dendritic Cell Signals Affect NKT17 Cell Differentiation in the Thymus.

NKT17 cells represent a functional subset of Vα14 invariant NKT (iNKT) cells with important effector functions in infections and autoimmune diseases. The mechanisms that drive NKT17 cell differentiation in the thymus are still largely unknown. The percentage of NKT17 cells has a high variability between murine strains due to differential thymic differentiation. For example, the NOD strain carries a high percentage and absolute numbers of NKT17 cells compared with other strains. In this study, we used the NOD mouse model to analyze what regulates NKT17 cell frequency in the thymus and peripheral lymphoid organs. In accordance with previous studies showing that the zinc finger transcription factor Th-POK is a key negative regulator of thymic NKT17 cell differentiation in the thymus, our data indicate that excessive NKT17 cell frequency in NOD mice correlates with defective Th-POK expression by thymic Vα14iNKT cells. Moreover, we found that Th-POK expression is under epigenetic regulation mediated by microRNA-133b whose expression is reduced in Vα14iNKT cells of NOD mice. We also demonstrated in a conditional knockout model of dendritic cell (DC) depletion (CD11cCreXDTA.B6 and CD11cCreRosa26DTA.NOD mice) that DCs play a crucial role in regulating Vα14iNKT cell maturation and their acquisition of an NKT17 cytokine secretion phenotype in the thymus. Overall, our data show that mechanisms regulating NKT17 cell differentiation are unique and completely different from those of Vα14iNKT cells. Specifically, we found that epigenetic regulation through microRNA-133b-regulated Th-POK expression and signals provided by DCs are fundamental for thymic NKT17 cell differentiation.