Lineage Choice Research Articles

Critical Role for TCR Signal Strength and MHC Specificity in ThPOK-Induced CD4 Helper Lineage Choice.

Sustained TCR signaling is critical for ThPOK induction in MHC class II (MHCII)-signaled thymocytes leading to the CD4 helper lineage commitment. ThPOK suppresses the cytotoxic program in the signaled thymocytes and is shown to be necessary and sufficient for the CD4 helper lineage choice. Accordingly, loss and gain of ThPOK function redirects MHCII- and MHC class I (MHCI)-signaled thymocytes into the CD8 cytotoxic and CD4 helper lineage, respectively. However, the impact of a defined ThPOK level on the CD4 helper lineage choice of MHCII- and MHCI-specific thymocytes and the role of TCR signaling in this process is not evaluated. Equally, it is not clear if suppression of the cytotoxic program by ThPOK is sufficient in redirecting MHCI-restricted thymocytes into the CD4 helper lineage. In this study, we have investigated CD8 to CD4 helper lineage redirection in three independent ThPOK overexpressing transgenic mouse lines. Our analysis shows that one of the transgenic lines, despite overexpressing ThPOK compared with wild-type CD4 mature T cells and compromising cytotoxic program, failed to redirect all MHCI-signaled thymocytes into the CD4 helper lineage, resulting in the continued presence of CD8+ mature T cells and the generation of a large number of double negative mature T cells. Critically, the same ThPOK transgene completely restored the CD4 helper lineage commitment of MHCII-specific Thpok -/- thymocytes. Importantly, augmenting TCR signaling significantly enhanced the ThPOK-mediated CD4 helper lineage choice of MHCI-specific thymocytes but was still substantially less efficient than that of MHCII-specific thymocytes expressing the same amount of ThPOK. Together, these data suggest that the ThPOK-induced CD4 helper lineage commitment is strongly influenced by TCR signal strength and MHC specificity of developing thymocytes.

γδTCR-independent origin of neonatal γδ T cells prewired for IL-17 production.

A classical view of T cell lineages consists of two major clades of T cells expressing either the αβ or γδ T cell receptor (TCR). However, genome-wide assessments indicate molecular clusters segregating T cell subsets that are preprogrammed for effector function (innate) from those that mediate conventional adaptive response, regardless of the TCR types. Within this paradigm, γδ T cells remain the prototypic innate-like lymphocytes, many subsets of which are programmed during intrathymic development for committed peripheral tissue localization and effector responses. Emerging evidence for innate γδ T cell lineage choice dictated by developmental gene programs rather than the sensory TCR is discussed in this review.

The Hox transcription factor Ubx stabilizes lineage commitment by suppressing cellular plasticity in Drosophila.

During development cells become restricted in their differentiation potential by repressing alternative cell fates, and the Polycomb complex plays a crucial role in this process. However, how alternative fate genes are lineage-specifically silenced is unclear. We studied Ultrabithorax (Ubx), a multi-lineage transcription factor of the Hox class, in two tissue lineages using sorted nuclei and interfered with Ubx in mesodermal cells. We find that depletion of Ubx leads to the de-repression of genes normally expressed in other lineages. Ubx silences expression of alternative fate genes by retaining the Polycomb Group protein Pleiohomeotic at Ubx targeted genomic regions, thereby stabilizing repressive chromatin marks in a lineage-dependent manner. Our study demonstrates that Ubx stabilizes lineage choice by suppressing the multipotency encoded in the genome via its interaction with Pho. This mechanism may explain why the Hox code is maintained throughout the lifecycle, since it could set a block to transdifferentiation in adult cells.

MTORC1 signaling mediates T cell lineage choices

Abstract The thymus supports and guides the generation of a diverse repertoire of mature T cells from precursors derived from the bone marrow. The interaction between extrinsic factors and intrinsic signal strength governs thymocyte development, but the mechanisms linking these two aspects of T cell development remain elusive. Capitalizing on genetic deletion of RAPTOR, here we report that RAPTOR-dependent mTORC1 signaling couples microenvironmental cues with metabolic programs in orchestrating reciprocal development of two fundamentally distinct T cell lineages: αβ and γδ T cells. Loss of RAPTOR impairs αβ but promotes γδ T cell development while disrupting metabolic remodeling of oxidative and glycolytic metabolism. Mechanistically, we identify mTORC1-dependent control of reactive oxygen species (ROS) production as a key metabolic signal that, upon perturbation of redox homeostasis, impinges upon T cell fate decision. Our results establish mTORC1-driven metabolic signaling as a fundamental mechanism underlying thymocyte lineage choices.

Quantitative Difference in PLZF Protein Expression Determines iNKT Lineage Fate and Controls Innate CD8 T Cell Generation

Zbtb16 encodes the zinc-finger protein PLZF, which is often used as a lineage marker for innate-like Tcells and is specifically required for the generation of invariant natural killer T (iNKT) cells in the thymus. Here, we report that not only PLZF expression itself but also the relative abundance of PLZF proteins plays critical roles in iNKT cell development. Utilizing a Zbtb16 hypomorphic allele, PLZFGFPCre, which produces PLZF proteins at only half of the level of the wild-type allele, we show that decreased PLZF expression results in a significant decrease in iNKT cell numbers, which is further associated with profound alterations in iNKT lineage choices and subset composition. These results document that there is a quantitative aspect of PLZF expression in iNKT cells, demonstrating that the availability of PLZF protein is a critical factor for both effective iNKT cell generation and subset differentiation.

Negative Co-stimulation Constrains T Cell Differentiation by Imposing Boundaries on Possible Cell States

Co-stimulation regulates Tcell activation, but it remains unclear whether co-stimulatory pathways also control Tcell differentiation. We used mass cytometry to profile Tcells generated in the genetic absence of the negative co-stimulatory molecules CTLA-4 and PD-1. Our data indicate that negative co-stimulation constrains the possible cell states that peripheral Tcells can acquire. CTLA-4 imposes major boundaries on CD4+ Tcell phenotypes, whereas PD-1 subtly limits CD8+ Tcell phenotypes. By computationally reconstructing Tcell differentiation paths, we identified protein expression changes that underlied the abnormal phenotypic expansion and pinpointed when lineage choice events occurred during differentiation. Similar alterations in Tcell phenotypes were observed after anti-CTLA-4 and anti-PD-1 antibody blockade. These findings implicate negative co-stimulation as a key regulator and determinant of Tcell differentiation and suggest that checkpoint blockade might work in part by altering the limits of Tcell phenotypes.

Blimp-1 Rather Than Hobit Drives the Formation of Tissue-Resident Memory CD8+ T Cells in the Lungs.

Tissue-resident memory CD8+ T (TRM) cells that develop in the epithelia at portals of pathogen entry are important for improved protection against re-infection. CD8+ TRM cells within the skin and the small intestine are long-lived and maintained independently of circulating memory CD8+ T cells. In contrast to CD8+ TRM cells at these sites, CD8+ TRM cells that arise after influenza virus infection within the lungs display high turnover and require constant recruitment from the circulating memory pool for long-term persistence. The distinct characteristics of CD8+ TRM cell maintenance within the lungs may suggest a unique program of transcriptional regulation of influenza-specific CD8+ TRM cells. We have previously demonstrated that the transcription factors Hobit and Blimp-1 are essential for the formation of CD8+ TRM cells across several tissues, including skin, liver, kidneys, and the small intestine. Here, we addressed the roles of Hobit and Blimp-1 in CD8+ TRM cell differentiation in the lungs after influenza infection using mice deficient for these transcription factors. Hobit was not required for the formation of influenza-specific CD8+ TRM cells in the lungs. In contrast, Blimp-1 was essential for the differentiation of lung CD8+ TRM cells and inhibited the differentiation of central memory CD8+ T (TCM) cells. We conclude that Blimp-1 rather than Hobit mediates the formation of CD8+ TRM cells in the lungs, potentially through control of the lineage choice between TCM and TRM cells during the differentiation of influenza-specific CD8+ T cells.

The histone demethylase UTX/KDM6A in cancer: Progress and puzzles.

The lysine-specific demethylase 6A/UTX (gene name KDM6A) acts as a component of the COMPASS complex to control gene activation. UTX demethylates H3K27me2/3 at genes and enhancers. Deleterious mutations in KDM6A are found in many cancer types, prominently urothelial carcinoma and certain T-cell leukemias. In certain cancers, however, UTX supports oncogenic transcription factors, e.g. steroid hormone receptors in breast and prostate cancer. In fetal development, UTX regulates lineage choice and cell differentiation. Analogously, loss of UTX function in cancer may lead to metaplasia or impede differentiation. Likely because its function is contingent on its interacting transcription factors, the effects of UTX inactivation are not uniform and require detailed investigation in each cancer type. In urothelial carcinoma, in particular, the functional consequences of the frequent mutations in KDM6A and other COMPASS component genes are poorly understood. Nevertheless, UTX inactivation appears to sensitize many cancers to inhibitors of the H3K27 methyltransferase EZH2. Conversely, inhibitors of UTX enzymatic activity may be applicable in cancers with an oncogenic UTX function. Intriguingly, the fact that KDM6A is localized on the X-chromosome, but both copies are expressed, may account for gender-specific differences in cancer susceptibility. In conclusion, despite recent progress, many open questions need to be addressed, most importantly, the detailed mechanisms by which KDM6A inactivation promotes various cancers, but also with which proteins UTX interacts in and apart from the COMPASS complex, and to which extent its catalytic function is required for its tumor-suppressive function.

MiR-149-3p Regulates the Switch Between Adipogenic and Osteogenic Differentiation of BMSCs by Targeting FTO

Bone marrow-derived mesenchymal stem cells (BMSCs) have been suggested to have the capacity to differentiate into different cell lineages. Maintaining a balanced stem cell differentiation program is crucial to the bone microenvironment and bone development. MiRNAs have played a critical role in regulating the differentiation of BMSCs into particular lineage. However, the role of miR-149-3p in the adipogenic and osteogenic differentiation of BMSCs has not been extensively discovered. In this study, we aimed to detect the expression levels of miR-149-3p during the differentiation of BMSCs and investigate whether miR-149-3p participated in the lineage choice of BMSCs or not. Compared to mimic-NC, miR-149-3p mimic made the adipogenic differentiation potential of BMSCs decreased and the osteogenic differentiation potential increased. Luciferase reporter assay further revealed that miR-149-3p repressed the expression of fat mass and obesity-associated (FTO) gene through binding to the 3ʹ-untranslated region (3ʹ-UTR) of the FTO mRNA. And the role of miR-149-3p in inhibiting adipogenic lineage differentiation and potentiating osteogenic lineage differentiation were mainly through targeting FTO, which also played an important role in regulating body weight and fat mass. We found that miR-149-3p regulated the differentiation of BMSCs by repressing FTO. In addition, BMSCs treated with miR-149-3p AMO exhibited higher potential to differentiate into adipocytes and lower tendency to differentiate into osteoblasts compared to BMSCs transfected with negative control. In summary, our results detected the effects of miR-149-3p in cell fate specification of BMSCs, and revealed that miR-149-3p inhibited the adipogenic differentiation of BMSCs via a miR-149-3p/FTO regulatory axis. This study provides cellular and molecular insights into the observation that miR-149-3p is a prospective candidate gene for BMSCs-based bone tissue engineering in treating osteoporosis. Funding Statement: This work was supported by grants from the Natural Science Foundation of China (81501920/81573434/81800784), the Postdoctoral Science Foundation of China (2018M630370)，the Heilongjiang Postdoctoral Fund (LBH-Z17134) Declaration of Interests: The authors indicate no potential conflicts of interest Ethics Approval Statement: All animal procedures were approved by the Animal Experimental Ethic Committee of Harbin Medical University

Progress in single-cell analysis of hematopoiesis

The mechanism underlying production of various types of blood cells from hematopoietic stem and progenitor cells has been a central theme in hematology. Conventionally, hematopoietic cell populations are analyzed by cell surface markers to judge cell types and differentiation stages, and by transplantation assays to assess differentiation potential. Recently, however, next-generation sequencing technology has enabled single-cell transcriptome and epigenome analyses and cell barcoding-based lineage tracing during unperturbed hematopoiesis. These innovative assays revealed that each cell population is extensively heterogenous. Many cells within hematopoietic stem cell populations may not be multipotent, and conversely, hematopoietic progenitor cells often display self-renewal capacity. Moreover, cells tend to make their lineage choice much earlier than previously thought. Altogether, these results challenge the current hierarchical differentiation models and propose new continuous models. Single-cell analyses are expected to greatly contribute to our understanding of normal and abnormal hematopoiesis and to the development of new therapies for blood disorders.

Genome Engineering for Stem Cell Transplantation.

To avoid the ethical issues of embryonic stem cells, genome engineering has focused on inducible pluripotent stem cells, which can develop into all 3 germ layers. The ability to detect methylation patterns in these cells allows research into pluripotency markers. The recently developed CRISPR system has allowed widespread application of genome engineering techniques. The CRISPR-Cas9 system, a potent system for genome editing, can be used for gene knockout or knock-in genome manipulations through substitution of a target genetic sequence with a desired donor sequence. Two types of genome engineering can be initiated: homologous or nonhomologous DNA repair by the Cas9 nuclease. Delivery of the CRISPR-Cas9 and target donor vectors in human pluripotent stem cells can be accomplished via viral and nonviral delivery methods. Nonviral delivery includes lipid-mediated transfection and electroporation. It has become the most common and efficient in vitro delivery method for human pluripotent stem cells. The CRISPR-Cas9 system can be combined with inducible pluripotent stem cells to generate single or multiple gene knockouts, correct mutations, or insert reporter transgenes. Knockouts can also be utilized to investigate epigenetic roles and targets, such as investigation of DNA methylation. CRISPR could be combined with human pluripotent stem cells to explore genetic determinants of lineage choice, differentiation, and stem cell fate, allowing investigators to study how various genes or noncoding elements contribute to specific processes and pathways. The CRISPR-Cas9 system can also be used to create null or nucleasedead Cas9, which has no enzymatic activity but has been utilized through fusion with other functional protein domains. In conclusion, RNA-guided genome targeting will have broad implications for synthetic biology, direct perturbation of gene networks, and targeted ex vivo and in vivo gene therapy.

MiR-21 Participates in the PD-1/PD-L1 Pathway-Mediated Imbalance of Th17/Treg Cells in Patients After Gastric Cancer Resection.

The programmed cell death-1/programmed cell death-ligand1 (PD-1/PD-L1) pathway has been shown to be involved in trauma-induced immunosuppression and to influence CD4+ T cell differentiation. MicroRNA (miR)-21 is a critical player in immune responses. However, it remains largely unknown whether miR-21 is regulated by PD-1 and influences CD4+ T-cell lineage choice after gastric cancer resection. In the present study, we analyzed the percentages of T-helper (Th)-17/regulatory T (Treg) cells and PD-1/PD-L1 expression on peripheral blood mononuclear cells (PBMCs) during the perioperative period. We also detected the secretion of interleukin (IL)-17 and transforming growth factor (TGF)-β1 using enzyme-linked immunosorbent assays (ELISAs). Furthermore, PBMCs isolated from patients were transfected with or without adenovirus-short hairpin-PD-1 (Ad-sh-PD1), pre-miR-21 or adenovirus-green fluorescent protein (Ad-GFP), and the percentages of Th17/Treg cells and related transcription factors were measured. In patients who underwent gastric cancer resection, the number of Th17 cells decreased, whereas the number of Treg cells increased, accompanied by an increased expression of PD-1/PD-L1. In addition, the expression of RORγt and IL-17 decreased, whereas the expression of Foxp3 and TGF-β1 increased. In vitro, silencing PD-1 via Ad-sh-PD1 promoted the expression of miR-21 and increased the percentage of Th17 cells, but decreased the percentage of Treg cells. The overexpression of miR-21 increased the percentage of Th17 cells but decreased the percentage of Treg cells. Our study demonstrated that gastric cancer resection altered the balance of Th17/Treg cells and increased PD-1/PD-L1 expression. In the in vitro experiments, the transfection of Ad-sh-PD1 ameliorated Th17/Treg cell imbalance partially by increasing the expression of miR-21.

Cell Cycle Heterogeneity Can Generate Robust Cell Type Proportioning

SummaryCell-cell heterogeneity can facilitate lineage choice during embryonic development because it primes cells to respond to differentiation cues. However, remarkably little is known about the origin of heterogeneity or whether intrinsic and extrinsic variation can be controlled to generate reproducible cell type proportioning seen in vivo. Here, we use experimentation and modeling in D. discoideum to demonstrate that population-level cell cycle heterogeneity can be optimized to generate robust cell fate proportioning. First, cell cycle position is quantitatively linked to responsiveness to differentiation-inducing signals. Second, intrinsic variation in cell cycle length ensures cells are randomly distributed throughout the cell cycle at the onset of multicellular development. Finally, extrinsic perturbation of optimal cell cycle heterogeneity is buffered by compensatory changes in global signal responsiveness. These studies thus illustrate key regulatory principles underlying cell-cell heterogeneity optimization and the generation of robust and reproducible fate choice in development.

Functional Conservation of a Developmental Switch in Mammals since the Jurassic Age.

ThPOK is a "master regulator" of T lymphocyte lineage choice, whose presence or absence is sufficient to dictate development to the CD4 or CD8 lineages, respectively. Induction of ThPOK is transcriptionally regulated, via a lineage-specific silencer element, SilThPOK. Here, we take advantage of the available genome sequence data as well as site-specific gene targeting technology, to evaluate the functional conservation of ThPOK regulation across mammalian evolution, and assess the importance of motif grammar (order and orientation of TF binding sites) on SilThPOK function in vivo. We make three important points: First, the SilThPOK is present in marsupial and placental mammals, but is not found in available genome assemblies of nonmammalian vertebrates, indicating that it arose after divergence of mammals from other vertebrates. Secondly, by replacing the murine SilThPOK in situ with its marsupial equivalent using a knockin approach, we demonstrate that the marsupial SilThPOK supports correct CD4 T lymphocyte lineage-specification in mice. To our knowledge, this is the first in vivo demonstration of functional equivalency for a silencer element between marsupial and placental mammals using a definitive knockin approach. Finally, we show that alteration of the position/orientation of a highly conserved region within the murine SilThPOK is sufficient to destroy silencer activity in vivo, demonstrating that motif grammar of this "solid" synteny block is critical for silencer function. Dependence of SilThPOK function on motif grammar conserved since the mid-Jurassic age, 165Ma, suggests that the SilThPOK operates as a silenceosome, by analogy with the previously proposed enhanceosome model.

Cell Lineage Choice during Haematopoiesis: In Honour of Professor Antonius Rolink

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Glutathione de novo synthesis but not recycling process coordinates with glutamine catabolism to control redox homeostasis and directs murine T cell differentiation.

Upon antigen stimulation, T lymphocytes undergo dramatic changes in metabolism to fulfill the bioenergetic, biosynthetic and redox demands of proliferation and differentiation. Glutathione (GSH) plays an essential role in controlling redox balance and cell fate. While GSH can be recycled from Glutathione disulfide (GSSG), the inhibition of this recycling pathway does not impact GSH content and murine T cell fate. By contrast, the inhibition of the de novo synthesis of GSH, by deleting either the catalytic (Gclc) or the modifier (Gclm) subunit of glutamate-cysteine ligase (Gcl), dampens intracellular GSH, increases ROS, and impact T cell differentiation. Moreover, the inhibition of GSH de novo synthesis dampened the pathological progression of experimental autoimmune encephalomyelitis (EAE). We further reveal that glutamine provides essential precursors for GSH biosynthesis. Our findings suggest that glutamine catabolism fuels de novo synthesis of GSH and directs the lineage choice in T cells.

Dual use of hematopoietic and mesenchymal stem cells enhances engraftment and immune cell trafficking in an allogeneic humanized mouse model of head and neck cancer.

In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.

TET mediated epigenetic regulation of iNKT cell lineage fate choice and function

During the last years, intensive research has shed light in the transcriptional networks that shape the invariant NKT (iNKT) cell lineage and guide the choices towards functionally distinct iNKT cell subsets (Constantinides and Bendelac, 2013; Engel and Kronenberg, 2014; Gapin, 2016; Kim et al., 2015). However, the epigenetic players that regulate gene expression and orchestrate the iNKT cell lineage choices remain poorly understood. Here, we summarize recent advances in our understanding of epigenetic regulation of iNKT cell development and lineage choice. Particular emphasis is placed on DNA modifications and the Ten Eleven Translocation (TET) family of DNA demethylases.

3030 - Functional Dominance of CHIP-Mutated Hematopoietic Stem Cells in Patients Undergoing Autologous Stem Cell Transplantations

Hematopoiesis is maintained by a polyclonal pool of hematopoietic stem cells (HSCs). Single somatic mutations in recurrent genes cause the emergence of expanded clones in elderly persons, a phenomenon called clonal hematopoiesis of indeterminate potential (CHIP). While the multilineage differentiation of these clones suggests an involvement of HSCs in CHIP, direct evidence of the fitness of CHIP-mutated human HSCs in blood reconstitution is lacking. Interestingly, previous studies showed a constant clone size for decades under steady-state conditions. Myeloablative treatments and stem cell transplantation put enforced stress on HSCs to reconstitute the blood system, and the fitness of HSCs is challenged. To assess whether human CHIP-mutated HSCs outcompete their wildtype counterparts in stress hematopoiesis, we took advantage of a well-characterized cohort of currently 81 patients undergoing high-dose chemotherapy conditioning and autologous stem cell transplantation (autoPBSCT) for the treatment of solid tumors or lymphoid diseases in our center. Using deep next generation sequencing of 56 myeloid cancer genes, we found that 17 patients (21%) are affected by CHIP (variant allele frequency >2%) in their blood at a time 6–102 months after autoPBSCT, with a mean variant allele burden of 4.5%. To explore whether the mutations had been present before the high-dose chemotherapy, and whether the CHIP-mutated HSCs expanded after autoPBSCT, we sequenced the frozen samples of the transplanted grafts. Importantly, the mutations can be detected in the majority of patients already in the transplanted cells, suggesting that they are not induced by high dose chemotherapy; however, the allele burden in the graft is significantly lower, often below 0.5%, than in the blood months to years after autoPBSCT suggesting a selective advantage of mutated HSCs outcompeting normal HSCs upon stress hematopoiesis. Ongoing functional evaluations will reveal CHIP-related differences in HSC behavior and lineage choice at single HSC resolution using time-lapse microscopy-based cell tracking and in vivo competitive transplantations, to elucidate the causative of CHIP-related disorders.

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.