Jiang Zhang

Mélanie Wencker

Quentin Marliac

Aurore Berton

Uzma Hasan

Raphaël Schneider

Daphné Laubreton

Dylan E Cherrier

Anne-Laure Mathieu

Amaury Rey

Wenzheng Jiang

Julie Caramel

Laurent Genestier

Antoine Marçais

Jacqueline Marvel

Yad Ghavi-Helm

Thierry Walzer
email: thierry.walzer@inserm.fr

Zeb1 represses TCR signaling, promotes the proliferation of T cell progenitors and is essential for NK1.1+ T cell development

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

T cell development occurs in the thymus and starts from immature thymocytes that are double negative (DN) for Cd4 and Cd8 expression. The DN population can be subdivided into four subsets, DN1-DN4, depending on the expression of the cell surface molecules Cd44 and Cd25 (for a review, see [START_REF] Shah | An Overview of the Intrathymic Intricacies of T Cell Development[END_REF]). DN1 cells (Cd44 + Cd25 -) are the most immature progenitors and retain the ability to differentiate into non-T cell lineages. In DN2 cells (Cd44 + Cd25 + ), the expression of RAG1/2 is induced, which promotes the rearrangements of gene segments encoding for TCR-β, TCR-γ, and TCR-δ subunits. In DN3 cells (Cd44 -Cd25 + ) the TCR β-chain associates with the pre-TCR α-chain and Cd3 subunits to form the pre-TCR complex; the pre-TCR allows βselection to occur. During β-selection, DN3 cells with productive TCRβ rearrangements receive survival and proliferative signals and mature into the DN4 (Cd44 -Cd25 -) stage. DN4 thymocytes then develop into Cd4 + Cd8 + double-positive (DP) stages [START_REF] Rothenberg | Launching the T-cell-lineage developmental programme[END_REF].

At the DP stage, a succession of events takes place and determines the fate of developing T cells; including rearrangement of the T cell antigen receptor (TCR) alpha locus, association of the αβ T cell receptor, and subsequent thymic selection.

In general, high affinity interactions between the αβTCR and self-peptide-MHC complexes (pMHC) presented by different thymic cells lead to negative selection and elimination of self-reactive thymocytes while low affinity interactions result in positive selection and development of Cd4 or Cd8 single positive (SP) T cells [START_REF] Kurd | T-cell selection in the thymus: a spatial and temporal perspective[END_REF][START_REF] Hogquist | The self-obsession of T cells: how TCR signaling thresholds affect fate "decisions" and effector function[END_REF][START_REF] Gascoigne | TCR Signal Strength and T Cell Development[END_REF]. In spite of this general rule, regulatory T cells (Tregs) and invariant NKT cells (iNKT) receive stronger TCR signals than conventional T cells during their development [START_REF] Moran | T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse[END_REF], as a result of selection by agonist self-antigens. iNKT cells are a subset of innate like T cells with a single invariant TCRα chain (Vα14-Jα18 in mice) and a limited repertoire of TCRβ chains (Vβ8.2, Vβ7, or Vβ2) that recognize glycolipid antigens bound to Cd1d, a non-polymorphic MHC molecule [START_REF] Godfrey | The burgeoning family of unconventional T cells[END_REF]. Their development includes discrete stages (stages 0-3) that can be discriminated by Cd44 and NK1.1 expression [START_REF] Kronenberg | Innate-like recognition of microbes by invariant natural killer T cells[END_REF].

Three functionally distinct iNKT cell subsets have also been identified; iNKT1 cells express T-bet and mainly secrete IFN-γ; iNKT2 cells express Gata3 and Plzf and secrete IL-4 and IL-13; iNKT17 express Rorγt and secrete IL-17. The TCR signal strength during selection governs the development of iNKT cell subsets, with strong signals promoting iNKT2 and iNKT17 development [START_REF] Tuttle | TCR signal strength controls thymic differentiation of iNKT cell subsets[END_REF][START_REF] Zhao | Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70[END_REF]. A large number of molecules regulate the strength of the TCR-derived signaling cascade. TCR signal strength can also be modulated at the transcriptional level by transcription factors (TFs) such as Sox4 [START_REF] Malhotra | SOX4 controls invariant NKT cell differentiation by tuning TCR signaling[END_REF], or at the post-transcriptional level by miR-181 [START_REF] Ziętara | Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells[END_REF][START_REF] Henao-Mejia | The microRNA miR-181 is a critical cellular metabolic rheostat essential for NKT cell ontogenesis and lymphocyte development and homeostasis[END_REF]. The loss of either blocks iNKT cell development. Mechanistically, miR-181a regulates the expression of multiple phosphatases and other proteins to boost TCR signaling as well as cell metabolism [START_REF] Ziętara | Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells[END_REF][START_REF] Henao-Mejia | The microRNA miR-181 is a critical cellular metabolic rheostat essential for NKT cell ontogenesis and lymphocyte development and homeostasis[END_REF]. Interestingly, mice expressing a hypomorphic form of Zap70, a major TCR-proximal kinase, also have impaired developmental maturation of γδ T cells, suggesting that innate-like T cell subsets are particularly dependent on a tight regulation of TCR signal strength for their development [START_REF] Wencker | Innate-like T cells straddle innate and adaptive immunity by altering antigen-receptor responsiveness[END_REF].

A dense network of TFs have been shown to regulate T cell development [START_REF] Seo | Transcriptional regulation of early T-cell development in the thymus[END_REF].

Early commitment is dependent on Notch signals [START_REF] Maillard | Regulation of lymphoid development, differentiation, and function by the Notch pathway[END_REF], which induce many TFs and maintain their expression throughout T cell development. Among those factors, the E protein family factors E2a, Tcf1 (encoded by Tcf7) and Heb (encoded by Tcf12) [START_REF] Murre | Helix-loop-helix proteins and lymphocyte development[END_REF] induce the expression of TCR components and balance the survival and proliferation of thymocytes [START_REF] Hosokawa | Cytokines, Transcription Factors, and the Initiation of T-Cell Development[END_REF]. Many other TF such as Gata3, Myb, Runx1, and Bcl11b also cooperate with E proteins at different developmental stages and further establish T cell identity [START_REF] Seo | Transcriptional regulation of early T-cell development in the thymus[END_REF][START_REF] Hosokawa | Cytokines, Transcription Factors, and the Initiation of T-Cell Development[END_REF].

Zeb family of TFs consist of Zeb1 and Zeb2. They are best known for their role in epithelial-to-mesenchymal transition (EMT). EMT programs operate at different stages of embryonic development and are downstream of Wnt, TGF-β, Bmp, Notch, and other signaling pathways [START_REF] Gheldof | Evolutionary functional analysis and molecular regulation of the ZEB transcription factors[END_REF]. Zeb1 -/-mice exhibit multiple developmental defects and die at birth [START_REF] Takagi | DeltaEF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages[END_REF]. In pathological settings, activation of EMT programs contributes to fibrosis and cancer metastases [START_REF] Caramel | Pleiotropic Roles for ZEB1 in Cancer[END_REF]. Zeb1 and Zeb2 are highly homologous and are characterized by two clusters of zinc fingers expressed on the protein extremities. They also contain a homeodomain, a Smad-binding domain and can interact with many other TFs [START_REF] Conidi | Few Smad proteins and many Smadinteracting proteins yield multiple functions and action modes in TGFβ/BMP signaling in vivo[END_REF]. Zeb1 and Zeb2 are also expressed in a tightly regulated manner in the immune system and regulate cell differentiation [START_REF] Scott | ZEBs: Novel Players in Immune Cell Development and Function[END_REF]. We and others have previously shown that Zeb2 regulated terminal NK cell [START_REF] Van Helden | Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection[END_REF] and effector Cd8 T cell differentiation [START_REF] Dominguez | The transcription factors ZEB2 and T-bet cooperate to program cytotoxic T cell terminal differentiation in response to LCMV viral infection[END_REF][START_REF] Omilusik | Transcriptional repressor ZEB2 promotes terminal differentiation of CD8+ effector and memory T cell populations during infection[END_REF]. Mutated mice expressing a truncated form of Zeb1, that removes the C-terminal zinc finger clusters at C727, have a small and hypocellular thymus caused by a reduction in early T-cell precursors [START_REF] Higashi | Impairment of T cell development in deltaEF1 mutant mice[END_REF]. In Cellophane mutant mice, a T→A mutation in the seventh exon of Zeb1 replaces the tyrosine at position 902 with a premature stop codon [START_REF] Arnold | A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity[END_REF]. The resulting mRNA encodes a truncated protein lacking the C-terminal zinc finger domain, which is predicted to be hypomorphic. Cellophane homozygote mice have small hypocellular thymi with fewer DP thymocytes. However, the mechanism of Zeb1 action during T cell development, and its role in mature T cell subsets remain unclear. Here, we show that Cellophane homozygous mice virtually lack several peripheral T cell subsets including iNKT cells, NK1.1 + γδ T cells and Ly49 expressing Cd8 T cells. This specific defect in innate-like T cells is caused by a cell-intrinsic role of Zeb1 in T cell development. We show that Zeb1 expression is maximal at the DN2 and DP stages of T cell development. Furthermore Zeb1 regulates the transition to the SP stage by promoting cell proliferation, survival and repressing the expression of various molecules involved in the strength of TCR signaling. Therefore, we propose that Zeb1 is a key regulator of thymocyte selection, essential for the development and survival of innate-like T cell subsets undergoing agonist-type selection.

Results

Zeb1 is highly expressed at the DN2 and DP stages of T cell development

To study the role of Zeb1 in T cell development, we sorted thymocyte subsets and measured Zeb1 transcript levels by semi-quantitative (Q) RT-PCR (reverse transcription polymerase chain reaction). As shown in Figure 1A, Zeb1 transcript levels were low in DN1, started to increase at the DN2 stage and were maximal in DP thymocytes. This expression then decreased as T cells underwent selection and matured into either conventional T cells, or iNKT cells. Interestingly, the expression of Zeb2 was somewhat reciprocal to that of Zeb1, with high expression in early thymic progenitors (DN1 to DN4) and lowest expression in DP (Figure 1B). This pattern of expression was matched by data from the Immgen consortium [START_REF] Heng | The Immunological Genome Project: networks of gene expression in immune cells[END_REF] (Figure S1A). Thus, as seen for memory T cells [START_REF] Guan | ZEB1, ZEB2, and the miR-200 family form a counterregulatory network to regulate CD8+ T cell fates[END_REF], Zeb1 and Zeb2 have reciprocal patterns of expression in thymocytes. We used the Immgen web browser to search for coregulated genes across different immune subsets. The E-protein Heb (encoded by Tcf12) was in the top 3 genes found to be co-regulated with Zeb1 (Figure S1B) [START_REF] Heng | The Immunological Genome Project: networks of gene expression in immune cells[END_REF].

Heb is well known for its important role throughout T cell development [START_REF] Jones | Stage-specific functions of E-proteins at the β-selection and T-cell receptor checkpoints during thymocyte development[END_REF], and especially at the DP stage [START_REF] Emmanuel | TCF-1 and HEB cooperate to establish the epigenetic and transcription profiles of CD4+CD8+ thymocytes[END_REF], further pointing to Zeb1 as a potential regulator of this developmental stage. We also analyzed the expression of Zeb1 protein in total thymocytes (80% of which are composed of DP). Zeb1 was strongly expressed in WT but not Cellophane thymocytes. Mutant mice only expressed reduced quantities of a truncated form of Zeb1 (Figure 1C).

Impaired development of both conventional and unconventional T cells in

Cellophane mice

To define the impact of the Cellophane mutation on T cell development, we analyzed the T cell composition in the thymus, spleen and lymph nodes of Cellophane mice.

The cell numbers in spleen, and liver were normal while the number of lymphocytes was reduced in lymph nodes (LN) (Figure 2A). As shown by previous observations [START_REF] Arnold | A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity[END_REF] we also observed a strong decrease in cell numbers from thymus in Zeb1mutated mice (Figure 2A). This decreased number affected all subsets defined by Cd4, Cd8, Cd44 and Cd25 expression (Figure 2B-C). The Cd4 + Cd8 + DP thymocytes and DN2 populations also decreased in frequency within Cellophane thymocytes (Figure 2B-C). In the LN and spleen, the percentage of Cd4 T cells, and Cd8 T cells were reduced (Figure 2D-E) and the proportion of memory-phenotype Cd44 + T cells from Cd8 T cells was decreased by nearly 30% (Figure 2F).

We then investigated the development of unconventional T cell subsets. We observed a drastic decrease in the frequency and in the number of iNKT cells as well as NK1.1 + γδ T cells in Cellophane mice compared to littermate controls (Figure 3A-D). This decrease affected all organs from Cellophane mice (Figure 3A-D). iNKT cells were mainly affected at stage 3 (Figure 3E). To complete our analysis, we also studied memory-phenotype Ly49 + Cd8 T cells which are thought to arise "naturally" in the thymus, without antigenic experience [START_REF] Rahim | Ly49 Receptors: Innate and Adaptive Immune Paradigms[END_REF]. All Ly49 + Cd8 T cell populations were also strongly decreased in LN and spleen from Cellophane mice, both in terms of frequencies and numbers, and irrespective of the inhibitory Ly49 receptor analyzed (Ly49A, Ly49F or Ly49G2) (Figure 3F-G).

Altogether, these data confirm the important role of Zeb1 in early T cell development. We also demonstrate an essential and specific role of Zeb1 in the development of peripheral T cell subsets expressing NK cell markers such as iNKT cells, NK1.1 + γδ T cells and Ly49 + Cd8 T cells.

Cell intrinsic role of Zeb1 in thymic progenitors and T cell development

Zeb1 is also required for the development and expression of non-hematopoeitic tissues and cell types [START_REF] Takagi | DeltaEF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages[END_REF]. To test if Zeb1 had an intrinsic role in T cell development, we generated chimeric mice by reconstituting sub-lethally irradiated Ly5a (Cd45.1) mice with the BM from Cellophane (Cd45.2) or "WT" Ly5a x C57BL/6 (Cd45.1/2) mice. In the thymus of chimeric mice, the frequency of DN2 and DP was strongly decreased for Cellophane BM-reconstituted mice compared to WT BM-reconstituted mice, while the proportion of other populations defined by Cd4 and Cd8 was increased (Figure 4A). iNKT cells and Ly49 + T cell subsets were also strongly reduced in the peripheral organs of CellophaneLy5a BM chimeric mice compared to WTLy5a chimera indicating that Zeb1 regulated T cell development intrinsically (Figure 4B-C). Cell numbers for all thymic T cell subsets and for peripheral iNKT cells and Ly49+ T cells were decreased in CellophaneLy5a BM chimeric mice compared to WTLy5a chimera (Figure S2). NK1.1 + γδ T cells were not analyzed because many of them are derived from fetal precursors [START_REF] Grigoriadou | Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors[END_REF], and are not reconstituted in BM chimera mice. To further test the role of Zeb1 in the environment of developing T cells, we also made different BM chimeric mice using WT and Cellophane mice as both recipients and BM donors (WTWT, WTCellophane, CellophaneWT and CellophaneCellophane). As shown in Figure S3A-B, the frequencies and numbers of DN and DP subsets were determined by the genotype of the BM donor and not by that of the host genotype. Similar conclusions could be reached upon examination of the frequency and number of NKT cells in the thymus and in the liver (Figure S3C).

We then generated mixed BM chimera by reconstituting lethally irradiated Ly5a mice with a 1:1 mixture of BM from Cellophane and Ly5a x C57BL/6 (WT) mice.

Cellophane T cell progenitors had a poor competitive fitness in BM chimeric mice (Figure 4D). Indeed, the percentage of cells originating from the Cellophane BM progenitors was already low in DN and further decreased when transitioning between DN and DP stages (Figure 4D). In the periphery of mixed BM chimeric mice, we found that the frequency of iNKT and Ly49 + Cd8 T cells was strongly reduced amongst Cellophane compared to WT lymphocytes (Figure 4E-F), thus revealing the role of Zeb1 in T cell development is cell-intrinsic, and not due to a defective stromal environment. Of note, we also analyzed the reconstitution of myeloid cells as control.

In the spleen, on average 20% of macrophages, 25% of dendritic cells and 28% of neutrophils were of Cellophane origin (Figure S3D), suggesting that Zeb1 regulated the development of all hematopoietic subsets, perhaps by regulating multipotent progenitors. However, the most important effects were observed for thymocytes and peripheral T cell subsets expressing NK cell markers.

Reduced survival and proliferation of Cellophane DN2 and DP cells

The decreased cellularity of the Cellophane thymi could be due to a reduced proliferation or increased apoptosis of thymocytes. To address this point, we first compared the survival of WT and Cellophane thymocytes upon ex vivo culture. We found that Cellophane DN2, DN3 and DN4 had a reduced ex vivo viability compared to their WT counterparts (Figure 5A). Moreover, after 24 or 48 h in culture, Cellophane DN2, DN3, DN4 and DP were also less viable than controls (Figure 5A).

Next, we compared in vivo proliferation of WT and Cellophane thymocytes, as measured by EdU incorporation. Cellophane DN2 and DP proliferated less than their WT counterpart, while Cellophane proliferated more than WT DN3 (Figure 5B). Ki67 staining corroborated our data obtained with EdU incorporation (Figure 5C-D). As all DP cells were Ki67 positive, we reported only changes in the MFI (Figure 5D). Thus, the Cellophane mutation affects both the survival and proliferation of developing DN2 and DP thymocytes, which could account for the decreased number of cells in the Cellophane thymi.

Zeb1 tunes TCR signal strength

To gain insight in the mechanism of Zeb1 function, we focused on DP as they expressed the highest level of Zeb1 among thymocytes (Figure 1A). We first compared the expression of membrane proteins involved in thymocyte selection in Cellophane vs WT DP cells by flow cytometry. Cellophane DP expressed higher levels of Cd69, Cd25, and Cd5 compared to WT thymocytes (Figure 6A). Nur77 is an early response gene expressed in T cells within hours after TCR stimulation. We observed an increase in the intracellular expression for Nur77 which correlated to TCRβ levels in Cellophane compared to WT DP thymocytes (Figure 6A). Of note similar levels of Cd4 and Cd8 were measured (Figure 6A).

Next, we focused our attention on thymic iNKT cell subsets. The mouse thymus is known to contain at least three iNKT subsets, ie iNKT1, iNKT2 and iNKT17 that are thought to have distinct roles in the immune response [START_REF] Gapin | iNKT cell autoreactivity: what is "self" and how is it recognized?[END_REF]. NKT1 cells correspond mainly to stage 3 NKT cells. TCR signal strength governs the development of iNKT cell subsets in the thymus, in which high signal strength is necessary for iNKT2 and iNKT17 development [START_REF] Tuttle | TCR signal strength controls thymic differentiation of iNKT cell subsets[END_REF][START_REF] Zhao | Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70[END_REF]. We examined iNKT cell subsets by staining for Plzf and Rorγt [START_REF] Tuttle | Characterization of Thymic Development of Natural Killer T Cell Subsets by Multiparameter Flow Cytometry[END_REF] in Cellophane mice vs WT mice. Results in Figure 6B show a significant increase in the representation of iNKT2 and iNKT17 and a decrease in that of iNKT1 in Cellophane compared to control mice. These data evocated an increase in the TCR signal from DP progenitors of iNKT cells in Cellophane mice. The change in iNKT subsets was associated with subtle changes in the TCR repertoire as assessed by measuring the frequency of Vβ8, Vβ7 and Vβ2 positive cells amongst iNKT cells of each genotype. We observed a 2-fold increase in Vβ7 usage in Cellophane mice (Figure 6C). This could reflect the increase in iNKT2 cells as a previous article showed that Vβ7 was more often associated with iNKT2 cells [START_REF] Tuttle | TCR signal strength controls thymic differentiation of iNKT cell subsets[END_REF]. Thymic Cellophane iNKT cells expressed normal levels of transcription factors T-bet and Egr2 but strongly reduced levels of Cd4 (Figure 6D). Since Cd4 is known to sustain TCR signal strength [START_REF] Gascoigne | TCR Signal Strength and T Cell Development[END_REF], the selection of Cd4 low iNKT cells in Cellophane mice could reflect an adaptation to overt TCR signaling in Cellophane DP .

We then analyzed specifically TCR signaling in developing thymocytes. We started by measuring the phosphorylation (p) level of a series of signaling proteins involved in TCR-mediated activation, either at steady-state in freshly isolated thymocytes, or following TCR engagement by crosslinking anti-Cd3 antibodies. To minimize the experimental variation, we used a barcoding strategy that allowed We also measured preTCR signaling in DN cells following stimulation with anti-CD3 antibodies. This analysis did not reveal any difference between WT and Cellophane DN cells (data not shown).

Zeb1 broadly shapes transcription at the transition DPSP to promote proliferation and repress TCR signaling

To further uncover the mechanisms of Zeb1 function during T cell development, we performed RNA-seq to compare WT and Cellophane DP transcriptomes. We found 538 differentially expressed genes (DEGs, p-value<0.05, Log2 Fold change>1). 204 genes were increased and 334 were down regulated in Cellophane DP compared to WT DP. These data reveal that Zeb1 broadly shapes the genetic program of developing thymocytes (Figure 7A and Table S1). For some of the DEGs identified, antibodies were available, and we were thus able to confirm higher expression of Foxo1, Ms4a4b, Itgb7, Ccr7 and Ccr4 in Cellophane compared to control DP cells and reciprocally we observed lower Cd81 expression in Cellophane DP vs WT (Figure 7B).

Next, we queried the Immgen database to retrieve the expression profile of the Zeb1-regulated geneset (induced or repressed) across all thymocyte subsets. Interestingly, genes down regulated in Cellophane DP (ie normally induced by Zeb1) correspond to genes that are normally expressed at high levels in early T cell progenitors and low levels in mature T cells (Figure 7C). Their expression level normally drops at the DP to SP transition, at the time when Zeb1 is highly expressed.

Genes up regulated in Cellophane DP correspond to genes that follow the reciprocal pattern of expression (Figure 7C). This pattern of expression also correlates with cell proliferation and TCR responsiveness in thymocytes. Indeed, irrespective of mouse genotype, SP T cells are much more responsive to TCR signaling mediated calcium responses than DP, but reciprocally do not cycle as much as DP (data not shown).

This suggests that Zeb1 promotes cell proliferation and represses TCR signaling specifically at the DP stage, presumably to ensure proper selection.

A functional annotation analysis of DEGs in our RNAseq analysis using "Metascape" [START_REF] Zhou | Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[END_REF] highlighted the cell cycle as the most down regulated biological process in Cellophane DP compared to control DP (Figure 7D, and Table S2), confirming findings in Figure 3. Pathways linked to Ifnγ (but also type I-Ifn, Table S2), antigen presentation, leukocyte differentiation and apoptosis were significantly associated with genes up regulated in Cellophane DP compared to WT (Figure 7D).

Of note, a modest but significant enrichment for genes involved in the calcium response was also associated with these genes, corroborating our data in Figure 6.

To further annotate this dataset, we also performed individual Pubmed searches, looking for connections between genes up regulated in Cellophane DP compared to control DP and "T cell activation" or "TCR signaling" or "T cell development". Interestingly this analysis showed more than 25% of the genes in the list had a known role in T cell activation or TCR signaling; and 10% had a role in T cell development, as defined using loss-of-function mouse strains (Table S3). Moreover, we used the STRING database of physical and functional protein interactions [START_REF] Szklarczyk | STRING v10: protein-protein interaction networks, integrated over the tree of life[END_REF] to further annotate genes up or down regulated in Cellophane DP compared to control DP. In particular, we used the Pubmed module that searches for enrichment for gene lists in articles in Pubmed. This unbiased analysis showed that genes up regulated in Cellophane DP were significantly enriched for genes involved in negative selection [START_REF] Liston | Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling[END_REF] or T cell maturation regulated by Bcl11b [START_REF] Kastner | Bcl11b represses a mature T-cell gene expression program in immature CD4(+)CD8(+) thymocytes[END_REF] (Table S4), which corroborated with our manual PubMed searches.

Chromatin regions remodeled at the DP stage contain Zeb1 binding motifs

Next, we wanted to determine if Zeb1 could regulate chromatin remodeling at the DP stage of T cell development. For this, we took advantage of a recently published large-scale analysis of chromatin accessibility and gene expression across 86 immune subsets including T cell developmental stages in the thymus [START_REF] Yoshida | The cis-Regulatory Atlas of the Mouse Immune System[END_REF]. In this study, in silico predictions pointed to Zeb1 as one of the few TF whose expression correlated with modifications of chromatin accessibility during thymic T cell development, and for which the corresponding chromatin regions contained sites predicted to be bound by Zeb1. Other TFs in this category included Gata3, Tcf7, Lef1, Tcf12 and Zfp740 (Figure S4A), and especially Tcf7 and Tcf12 whose role in T cell development has been well established [START_REF] Emmanuel | TCF-1 and HEB cooperate to establish the epigenetic and transcription profiles of CD4+CD8+ thymocytes[END_REF]. We retrieved open chromatin regions (OCRs) for which Zeb1 motifs were discovered in this study and whose accessibility changed during T cell development (see corresponding clusters in Figure S4B) and compared the list of corresponding genes with DEGs identified in our own study between Cellophane and WT DP. We found an important overlap between both lists that included many of the genes previously highlighted in our analysis (Figure 7E, p-value=1.839413e-46). Altogether, these data suggest that Zeb1 is a direct transcriptional regulator of T cell development, especially operating at the DP to SP transition to promote proliferation and ensure proper selection.

Discussion

Here, we demonstrated that Zeb1 is essential for the transition through the DN2 and DP stages of T cell development as well as for the differentiation of iNKT cells, NK1.1 + γδ T cells and Ly49 + Cd8 T cells. Mechanistically, Zeb1 regulates the expression of a number of genes that were notably involved in cell proliferation or in TCR signaling at the DP stage. In Cellophane mice, these events may perturb thymic development and selection in a way that does not allow the production of NK1.1 + and Ly49 + T cell subsets.

Zeb1 expression was found to increase at the DN2 stage and to be maximal at the DP stage of T cell development. Accordingly, we found a decrease in frequency of DN2 and DP thymocytes in Cellophane mice. This could be accounted for by a cell-intrinsic role for Zeb1 in DN2 and DP proliferation. A number of genes involved in the cell cycle were differentially expressed between WT and Cellophane DP as revealed by our RNAseq analysis. There is also a strong link in the literature between Zeb1 and cell proliferation in cancer. In particular, Zeb1 interacts with many TFs involved in the regulation of cell growth such as Smad TFs which is downstream of several growth factor pathways [START_REF] Conidi | Few Smad proteins and many Smadinteracting proteins yield multiple functions and action modes in TGFβ/BMP signaling in vivo[END_REF]. Moreover, Zeb1 is known to repress cyclindependent kinases during EMT [START_REF] Caramel | Pleiotropic Roles for ZEB1 in Cancer[END_REF]. However, Cdkn2c and Cdkn3 were decreased by Zeb1 in DP thymocytes, suggesting differential roles of Zeb1 in epithelial versus lymphoid cells. The decreased proliferation of DN2 and DP is expected to have important consequences on the general thymic output. Indeed, we found decreased numbers of peripheral T cells in Cellophane mice. However, this defect was much more pronounced for iNKT cells, NK1.1 + γδ T cells and Ly49 + Cd8 T cells. In particular, iNKT cells were virtually absent from the periphery. This altered development was associated with an increased TCR signaling at the DP stage, which was verified by increased basal levels of Cd5 and Nur77 but also increased mTOR activity and calcium flux upon Cd3 engagement. iNKT cells are known to receive stronger TCR signals than conventional T cells during their development [START_REF] Moran | T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse[END_REF]. Thus, increased TCR signaling in Cellophane DP could trigger cell death i.e. via negative selection of iNKT precursors. The increased negative selection of iNKT cells has already been shown to occur in mice in which transgenic TCR-β chain conferred high affinity for self-lipid/Cd1d complexes when randomly paired with Vα14-Jα18 rearrangements [START_REF] Bedel | Effective functional maturation of invariant natural killer T cells is constrained by negative selection and T-cell antigen receptor affinity[END_REF]. Thus, increased negative selection could impair the development of iNKT cells in Cellophane mice, and perhaps that of other T cell subsets expressing NK cell markers. Indeed, strong TCR-mediated signals are also important for γδ T cell development [START_REF] Hayes | Strength of signal: a fundamental mechanism for cell fate specification[END_REF][START_REF] Haks | Attenuation of gammadeltaTCR signaling efficiently diverts thymocytes to the alphabeta lineage[END_REF]. In particular, NK1.1 + γδ T cells have an oligoclonal TCR repertoire and accumulate in mouse models of decreased TCR signaling [START_REF] Alonzo | Development of promyelocytic zinc finger and ThPOK-expressing innate gamma delta T cells is controlled by strength of TCR signaling and Id3[END_REF], suggesting that this subset of γδ cells can also be negatively selected.

The ontogeny of Ly49 + Cd8 T cells is not very well known, but our data suggest that their development and selection could share common mechanisms with that of NK1.1 + T cells. How does Zeb1 regulate TCR signal strength? Cellophane DP thymocytes expressed higher levels of TCRβ than control DP, and this could certainly lead to higher TCR signaling. Moreover, the RNAseq analysis we performed suggested multiple connections between Zeb1 and signal transduction through the TCR. For example, multiple members of the GTPases of the IMmunityAssociated Proteins (GIMAP) family (GIMAP5 and GIMAP8) were up regulated in Cellophane DP. Interestingly, Gimap5 enhances calcium influx following TCR stimulation [START_REF] Ilangumaran | Loss of GIMAP5 (GTPase of immunity-associated nucleotide binding protein 5) impairs calcium signaling in rat T lymphocytes[END_REF].

Several members of the family of four-pass membrane receptors Ms4a are also up regulated in Cellophane DP and could reinforce TCR signaling. Indeed, transduction of Ms4a4b into naive T cells can heighten their sensitivity to antigen through a process that could involve association with costimulatory molecules [START_REF] Howie | MS4A4B is a GITR-associated membrane adapter, expressed by regulatory T cells, which modulates T cell activation[END_REF]. Several phosphatases and kinases are also deregulated in Cellophane DP and could perhaps alter TCR signaling. In particular the expression of Pyk2 (encoded by Ptk2b) or that of Rasgrp4, Rasl1 and Rasa3 could all contribute to higher TCR signaling via the calcium flux or the Mapk pathway.

A series of TF were also deregulated in Cellophane DP, with a notable up regulation of JunB, Jun, Atf6, Foxo1, Stat4 or Irf7/9. JunB and Jun are essential components of AP-1 TFs and are typically induced downstream of TCR stimulation.

Similar to Nur77, they could represent surrogate markers of increased TCR signaling in Cellophane DP. The derepression of Foxo1 could in part account for altered T cell development as it regulates Ccr7, Cd62L and S1pr1 via Klf2 [START_REF] Carlson | Kruppel-like factor 2 regulates thymocyte and T-cell migration[END_REF]. The lack of Klf2 control could perturb, perhaps accelerate, the normal migration of developing thymocytes in the medullar region where negative selection occurs. Foxo1 deletion in thymocytes was reported to decrease the number of DP thymocytes, and Foxo1 deficient peripheral T cells seem to be refractory to TCR stimulation through unknown mechanisms [START_REF] Bupp | T cells require Foxo1 to populate the peripheral lymphoid organs[END_REF]. Moreover, up regulation of Foxo1 in Cellophane DP could in part explain the opposite changes in cell proliferation observed in these cells compared to control DP, since Foxo TFs are known to promote stem cell quiescence [START_REF] Li | Molecular Pathways: Stem Cell Quiescence[END_REF] and clearly contribute to the regulation of cell division, survival and metabolism in T cells [START_REF] Hedrick | FOXO transcription factors throughout T cell biology[END_REF]. A recent study showed that the transcriptional repressor Gfi1 is important to maintain Foxo1 expression at low levels in DP thymocytes [START_REF] Shi | Gfi1-Foxo1 axis controls the fidelity of effector gene expression and developmental maturation of thymocytes[END_REF]. In the absence of Gfi1, there was premature expression of genes normally expressed in mature T cells and accelerated maturation of DP to SP thymocytes, largely attributable to Foxo1 derepression. Zeb1 and Gfi1 could therefore cooperate to repress Foxo1.

There are many similarities in the phenotype of Tcf12 deficient [START_REF] Cruz | An essential role for the transcription factor HEB in thymocyte survival, Tcra rearrangement and the development of natural killer T cells[END_REF] and Cellophane mutant mice, in particular susceptibility of DP to cell death and impaired development of iNKT cells. Moreover, microarray data from the Immgen consortium suggest that Tcf12 and Zeb1 are strongly co-regulated and ATAC-seq data predict that they control chromatin accessibility during thymic T cell development together with Gata3, Tcf1, Lef1 and Zfp740 (Figure S4 and [START_REF] Yoshida | The cis-Regulatory Atlas of the Mouse Immune System[END_REF]). Altogether these data suggest a strong functional link between Zeb1 and Heb (encoded by Tcf12), and perhaps Tcf1, which acts in coordination with Heb [START_REF] Emmanuel | TCF-1 and HEB cooperate to establish the epigenetic and transcription profiles of CD4+CD8+ thymocytes[END_REF]. The fact that Zeb1 is known to bind tandem E-box motifs, suggests a possible competition between Zeb members and E-proteins for those genes regulated by tandem E-boxes. Such a competition has been previously established at least in the context of the CD4 enhancer which is repressed by Zeb1, through a competition with Heb for E-box binding [START_REF] Brabletz | Negative regulation of CD4 expression in T cells by the transcriptional repressor ZEB[END_REF]. Moreover, Zfh-1 and Daugtherless, the drosophila homologs of Zeb1 and Tcf12, are also known to compete for the same genomic sites [START_REF] Postigo | zfh-1, the Drosophila Homologue of ZEB, Is a Transcriptional Repressor That Regulates Somatic Myogenesis[END_REF]. The deletion of Tcf12 and Tcf1 in thymocytes has the opposite phenotype as Cellophane Zeb1 mutation in terms of DP proliferation [START_REF] Emmanuel | TCF-1 and HEB cooperate to establish the epigenetic and transcription profiles of CD4+CD8+ thymocytes[END_REF]. This suggests that Heb and Zeb1 could have partially antagonistic activities in the regulation of genes bearing tandem E-box elements. A competition between Zeb1 and E-proteins has already been suggested in the control of GATA3 expression in human CD4 T cells [START_REF] Grégoire | T-cell expression of the human GATA-3 gene is regulated by a non-lineage-specific silencer[END_REF]. Cellophane mice express a truncated form of Zeb1 that is also expressed at lower levels than WT Zeb1. As the phenotype of these mice is milder than that of Zeb1 -/-mice, we assumed that the Cellophane mutation was hypomorphic. However, we cannot exclude that Cellophane Zeb1 can retain some DNA binding and therefore act as a dominant negative molecule by preventing binding of E-box proteins. Further work will be needed to precisely map the interactions between Zeb TFs and E proteins. This regulatory network may also include inhibitor of differentiation genes Id2 and Id3, as these TFs bind and inactivate E-proteins, thereby regulating their function. Moreover, the deficiency in Id3 has the same impact on NK1.1+ γδ T cells as a deficiency in TCR signaling [START_REF] Alonzo | Development of promyelocytic zinc finger and ThPOK-expressing innate gamma delta T cells is controlled by strength of TCR signaling and Id3[END_REF], which links both events. Zeb1 genomic region is frequently deleted in cutaneous T cell lymphomas (CTCL) [START_REF] Wang | Genomic profiling of Sézary syndrome identifies alterations of key T cell signaling and differentiation genes[END_REF]. These deletions are often associated with genetic mutations in components of the TCR signaling machinery (recurrent alterations in Card11, Plcg1, Lat, Rac2, Prkcq, Cd28 and genes that encode calcium channel subunits). This observation, together with our own data showing a role for Zeb1 in repressing TCR signaling, suggest that Zeb1 deletion could promote lymphomagenesis by releasing TCR signaling natural brakes. Of note, a previous study proposed an essential role of IL-15 in CTCL development, and showed that IL-15 expression was depressed in patients with CTCL due to promoter hypermethylation and in the failure of Zeb1 to have access to and repress the IL-15 regulatory region [START_REF] Mishra | Mechanism, Consequences, and Therapeutic Targeting of Abnormal IL15 Signaling in Cutaneous T-cell Lymphoma[END_REF]. However, IL-15 expression was not detected in developing thymocytes in our RNAseq analysis, excluding the possibility that the IL-15 pathway could play a role in the Zeb1 mechanism of action in T cell development. Yet, we found that the transcriptional response to different cytokines such as interferons or IL-6 was increased in Cellophane DP (table S2). Zeb1 may therefore normally repress the response to these cytokines, presumably to ensure proper selection. TGF-β is a known regulator of iNKT cell development [START_REF] Doisne | iNKT cell development is orchestrated by different branches of TGF-signaling[END_REF] and that it promotes early differentiation and prevents apoptosis of developing iNKT cells. A recent study showed that Zeb1 was induced by TGF-β in conventional Cd8 T cells stimulated through the TCR and was essential for memory T cell survival and function [START_REF] Guan | ZEB1, ZEB2, and the miR-200 family form a counterregulatory network to regulate CD8+ T cell fates[END_REF]. Although we failed to detect any effect of recombinant TGF-β on Zeb1 expression in thymocytes (data not shown), it would be interesting to address this question in vivo using appropriate genetic models.

In summary, Zeb1 is an essential member of the TF network that regulates T cell development and selection at the DN2 and DP stages. Furthermore, we have also shown that Zeb1 allows the development of iNKT cells and other T cell subsets expressing NK cell markers by regulation of cell cycle and TCR signaling in developing thymocytes.

495

Material and Methods

Mice

Mice at 8 to 24 weeks were used. Wild-type C57BL/6 mice were purchased from Charles River Laboratories (L'Arbresle). Cellophane mice were previously described [START_REF] Arnold | A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity[END_REF], littermate mice were used as controls. This study was carried out in accordance with the French recommendations in the Guide for the ethical evaluation of experiments using laboratory animals and the European guidelines 86/609/CEE. All experimental studies were approved by the bio-ethic local committee CECCAPP.

Mice were bred in the Plateau de Biologie Expérimentale de la Souris (ENS, Lyon).

Bone marrow chimeric mice

8-to 10-week-old Ly5a mice were anesthetized with ketamine/xylazine before irradiation at 9 Gray with a X-ray irradiator XRAD-320. After irradiation, they were intravenously injected with 2-5x10^6 cells collected from either wild type or mutant murine bone marrow, or a mix of both (as indicated in the figures). Immune cell reconstitution was analyzed 8 weeks post BM injection.

Flow cytometry

Single-cell suspensions of the thymus, spleen and liver were used for flow cytometry.

Cell viability was measured using Annexin-V (BD Biosciences)/live-dead fixable (eBiosciences) staining. Intracellular stainings for TFs were performed using Foxp3 kit (ebioscience). Lyse/Fix and PermIII buffers (BD Biosciences) were used for intracellular staining of phosphorylated proteins. Flow cytometry was carried out on a FACS Canto, a FACS LSR II, or a FACS Fortessa (Becton-Dickinson). Data were analysed using FlowJo (Treestar). Antibodies were purchased from eBioscience, BD biosciences, R&D Systems, Beckman-Coulter, Miltenyi or Biolegend. We used the

Measurement of in vivo cell proliferation and ex vivo survival

Mice were given one intraperitoneal injection of 0.2 mg EdU (BD Bioscience). 12 hours after EdU injection, mice were sacrificed and organs harvested. Cells derived from the thymus were stained with antibodies specific for cell surface antigens as described above. After fixation and permeabilization, cells stained with FITC anti-EdU antibody and 7-AAD (BD Pharmingen), according to manufacturer instructions. EdU incorporation for different cell populations was measured by flow cytometry.

For the measurement of viability, we stained thymocyte suspensions with Annexin-V (BD Biosciences), 7-AAD and coupled with other surface markers Cd4, Cd8, Cd69, TCRβ, Cd25, Cd44, either ex vivo or 24, 48 or 72 h after in vitro culture in complete medium.

Cell sorting and RNA preparation

Lymphocytes were obtained from the thymus. Immune cell population including DN1-4, DP, single positive CD4 + and CD8 + , iNKT cells were stained in combination with other cell specific markers Cd4, Cd8, Cd69, TCRβ, Cd25, Cd44 and mCd1d/PBS-57 and subsequently sorted into different subsets using a FACSAria Cell Sorter (Becton-Dickinson, San Jose, USA). Purity of sorted cell populations was over 98% as validated by flow cytometry. Sorted cells were lysed using Trizol reagent (Invitrogen) or RLT buffer from the RNeasy Micro kit (Qiagen) and RNA was extracted according to the manufacturer's instructions.

Quantitative RT-PCR

We used High capacity RNA-to-cDNA kit (applied biosystem, Carlsbad, USA) or iScript cDNA synthesis kit (BioRad) to generate cDNA for RT-PCR. PCR was carried out with a SybrGreen-based kit (FastStart Universal SYBR Green Master, Roche, Basel, Switzerland) or SensiFast SYBR No-ROX kit (Bioline) on a StepOne plus instrument (Applied biosystems, Carlsbad, USA) or a LightCycler 480 system (Roche). Primers were designed using the Roche software. We used the following primers for mouse QPCR: Zeb1 forward primer, 5'-GCCAGCAGTCATGATGAAAA-3'; Zeb1 reverse primer, 5'-TATCACAATACGGGCAGGTG-3'; Zeb2 forward primer, 5'-CCAGAGGAAACAAGGATTTCAG-3'; Zeb2 reverse primer, 5'-AGGCCTGACATG TAGTCTTGTG-3'; Gapdh forward primer, 5'-GCATGGCCTTCCGTGTTC-3'; Gapdh reverse primer, 5'-TGTCATCATACTTGGCAGGTTTCT-3'. The relative expression of Zeb1 and Zeb2 were normalized to Gapdh expression.

Western blotting

Cells were lysed in NP40 lysis buffer (20mM Tris, HCl pH7.4; 150mM NaCl; 2mM EDTA; 1% NP40) containing protease inhibitors for 30min on ice. Supernatant was collected following 10min centrifugation at 12 000g at 4°C and protein concentration was quantified by μBCA quantification kit (Thermo Fisher scientific). Fifity μg of total cellular protein from thymus were incubated during 5 minutes at 95°C. Protein samples were separated by electrophoresis using Novex 4-12% Tris-Glycine gels (Life Technologies) for 1 hour at 120V. Proteins then were transferred on a PVDF membrane (Bio-rad). After blocking with PBS 0.1% tween and 5% milk for 1 hour, membranes were probed with the following primary antibodies: anti-Gapdh (Cell Signaling Technology, 2118), anti-Zeb1 (Cell Signaling Technology, 3396, raised against a peptide around Asp846, the Cellophane mutation truncating the protein after Tyr902) over night at 4°C. Membranes were washed three times with PBS 0.1% tween, secondary antibodies were added for one hour at RT. Anti-Rabbit and antimouse HRP conjugate secondary antibodies were provided by Jacson Immunoresearch. Proteins were revealed with Chemiluminescence Western Lightening Plus kit (Perkin-Elmer).

RNAseq analysis

Thymic suspensions were stained in combination with anti-Cd3, anti-Cd4, anti-Cd8, anti-Cd69, anti-TCRβ and subsequently sorted into different subsets using a FACSAria Cell Sorter (Becton-Dickinson, San Jose, USA). Purity of sorted cell populations was over 98% as measured by flow cytometry. RNA libraries were prepared as previously described [START_REF] Picelli | Fulllength RNA-seq from single cells using Smart-seq2[END_REF]. Briefly, total RNA was purified from 5x10 4 sorted thymocytes using the Direct-Zol RNA microprep kit (Zymo Research) according to manufacturer instructions and was quantified using QuantiFluor RNA system (Promega). 1µl of 10µM Oligo-dT primer and 1µl of 10mM dNTPs mix were added to 0.15ng of total RNA in a final volume of 2.3µl. Oligo-dT were hybridized 3min at 72°C and reverse transcription (11 cycles) was performed. PCR preamplification was then conducted using 16 cycles. cDNA were purified on AmpureXP beads (Beckman Coulter) and cDNA quality was checked on D5000 screen tape and analysed on Tape station 4200 (Agilent). Three ng of cDNA were tagmented using Nextera XT DNA sample preparation kit (Illumina). Tagged fragments were further amplified and purified on AmpureXP beads (Beckman Coulter). Tagged library quality was checked on D1000 screen tape and analyzed on Tape station 4200 (Agilent).

Sequencing was performed by the GenomEast platform, a member of the "France Génomique" consortium (ANR-10-INBS-0009), on an Illumina HiSeq 4000 sequencing machine (read length 1x50nt).

Measurements of TCR signaling

Calcium response

Thymocytes were first barcoded with anti-Cd45 coupled with different fluorochromes for WT and Cellophane respectively and then stained at RT with fluorescent anti-Cd4 and anti-Cd8, anti-Cd69, anti-TCRβ, anti-Cd25 and anti-Cd44 antibodies, followed by a staining with Indo-1 (1µM, Life Technologies) at 1x10 7 cells/ml for 30 min at 37°C and two times' washing at 4°C. Cells were resuspended in the RPMI medium (0.2% BSA and 25mM Hepes) and were placed at 37°C for 5-10 min prior to acquisition.

Samples were acquired on a LSRII (BD) as following steps: 15 s baseline acquisition, addition of anti-Cd3 biotin (2C11, 10µg/ml), acquisition for 1 min 30 s, addition of Streptavidin (Life Technologies, 10µg/ml) and, acquisition for another 3-5 min.

Phosphorylation events

Different samples corresponding to the different mice were barcoded by labeling them with a series of anti-Cd45 antibodies coupled with different fluorochromes. For phospho-flow stainings, 3x10 6 mixed thymocytes were stained using biotinylated Cd3 (2C11, 5µg/ml) and other surface markers for 15 min, followed by streptavidin (Life Technologies, 10µg/ml) stimulation and fixed by addition of 10 volumes of Lyse/Fix at the indicated time point. The level of pErk, pS6 or pAkt were normalized by the mean fluorescence intensity (MFI) that was detected in the non-stimulated condition (regarded as 100%) for each mouse.

In silico analyses

Functional annotations of DEGs were performed using Metascape [START_REF] Zhou | Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[END_REF] or STRING [START_REF] Szklarczyk | STRING v10: protein-protein interaction networks, integrated over the tree of life[END_REF] using default parameters. Additionally, we used several functionalities of the Immgen database browsers [START_REF] Heng | The Immunological Genome Project: networks of gene expression in immune cells[END_REF] to generate some of the figures in the supplementary information.

Statistical analysis

Statistical analyses were performed using Prism 5 (Graph-Pad Software). Two tailed unpaired t-test, paired t-test, and ANOVA tests with Bonferroni correction were used as indicated. We used the hypergeometric test and Benjamini-Hochberg p-value correction algorithm to calculate if the enrichment of the overlap between gene lists were statistically significant.

  stimulating and then staining WT and Cellophane thymocytes simultaneously (see methods). Results in Figure 6E-F showed an increase in pAkt (Ser473), ribosomal protein pS6, and to a lesser extent of pErk, either at steady state or following TCR engagement in Cellophane DP compared to WT DP. Thus, the Mapk and PI3K/Akt pathways are more active in thymocytes undergoing selection in Cellophane compared to control mice. To complement this analysis, we also assessed the calcium response of DP from both genotypes in response to TCR engagement. As shown in Figure 6G-H, this response was stronger for Cellophane DP compared to control DP both in terms of intensity (peak) and duration (area under the curve, AUC). Thus, Zeb1 tunes signal strength downstream TCR engagement at the DP stage and Cellophane DP have increased TCR signaling. The latter may increase negative selection and therefore account for defective T cell development in Cellophane mice.

  following antibodies: anti-mouse Cd3 (clone 145-2C11), anti-mouse Cd4 (clone GK1.5), anti-mouse Cd8 (clone 53-6.7), anti-mouse TCRβ (clone H57-597), antimouse Cd69 (clone H1.2F3), anti-mouse TCRγδ (clone GL3), anti-mouse NK1.1 (clone PK136), anti-mouse Cd24 (clone M1/69), anti-mouse Cd44 (clone IM7), antimouse Cd27 (clone LG.7F9), anti-mouse TCRVβ2 (clone B20.6), anti-mouse TCRVβ7 (clone TR310), anti-mouse TCRVβ8.1/8.2 (clone KJ16-133), anti-mouse Ly49A (clone A1), anti-mouse Ly49E/F (clone REA218), anti-mouse Ly49G2 (clone 4D11), anti-mouse Cd45.1 (clone A20), anti-mouse Cd45.2 (clone 104), anti-mouse Nur77 (clone 12.14), anti-mouse Ccr7 (clone 4B12), anti-mouse Cd5 (clone 53-7.3), anti-mouse Cd81 (clone Eat-2), anti-mouse Cd53 (clone OX79), anti-mouse Lpam-1 (clone DATK-32), anti-mouse Foxo1 (clone C29H4), anti-mouse Ms4a4b (clone 444008), anti-mouse Cd74 (clone In1/Cd74), anti-mouse Tbet (clone 4B10), antimouse Egr2 (clone erongr2), anti-mouse Plzf (clone Mags.21F7), anti-mouse Rorүt (clone AFKJS-9), anti-mouse pErk (clone 20A), anti-mouse pAkt (Ser473) (clone M89-61), anti-mouse pS6 (clone D57.2.2E). For staining of iNKT cells, phycoerythrin (PE)-conjugated PBS-57 loaded on mouse Cd1d tetramers (mCd1d/PBS-57) were obtained from Tetramer Core Facility of the National Institute of Health.

Figure 1 :

 1 Figure 1: Zeb1 expression in WT and Cellophane thymocytes (A-B) RT-PCR analysis of RNA from sorted thymocyte subsets isolated from C57BL/6 mice, as indicated. Results are presented relative to expression of the control gene Gapdh. (C) WB analysis of Zeb1 expression in total thymocytes of WT and Cellophane mice, as indicated. Data are representative of three independent experiments with 3 to 6 mice (A-B) or three independent experiments with three mice (C).

Figure 2 :

 2 Figure 2: Cellularity and proportion of T cell subsets in Cellophane mice. (A) Total cell number from the thymus, spleen, liver and LN. (B) Percentages and absolute numbers of indicated T cell subsets (DN, DP, Cd4 SP and Cd8 SP) in the thymus. (C) Percentages and absolute numbers of indicated DN subsets defined by Cd44 and Cd25 expression in the thymus. (D-E) Percentages and absolute numbers of (D) Cd4 T cells and (E) Cd8 T cells. (F) Percentages and absolute numbers of CD8 + Cd44 + memory T cell in LN and spleen. Each dot represents an individual mouse. Data are pooled from 7 to 8 mice in four experiments (A-E) or 3 to 6 mice in two experiments (F). The statistical analysis was performed using unpaired Student t test.

Figure 3 :

 3 Figure 3: Cellophane mice have decreased numbers of thymic DP and lack iNKT cells, NK1.1 + γδ T cells and Ly49 + Cd8 cells. (A) Flow cytometry analysis of Cd1d-tet + iNKT cells (black gate) from thymus, spleen, and liver in wild-type (WT) and Cellophane (Cello) homozygous mice as indicated. (B) Percentage and absolute numbers of iNKT cells (mCd1d/PBS-57 + TCRβ + ) in thymus, spleen, and liver. (C) Flow cytometry analysis of NK1.1 -and NK1.1 + γδ T cells (black square gates) from the spleen of WT and Cellophane mice. (D) Percentage within total γδ T cells and absolute numbers of NK1.1 -and NK1.1 + γδ T cells in spleen and LN. (E) Percentage within total thymic iNKT of stages 0-3, as defined by Cd24, Cd44 and NK1.1 expression. (F) Flow cytometry analysis of Cd8 + Cd44 + Ly49A + / Ly49F + / Ly49G2 + T cells (black square gates) from LN and spleen in WT and Cellophane mice. (G) Percentage within Cd8 + Cd44 + T cells and absolute numbers of Cd8 + Cd44 + Ly49 + T cells in spleen and LN. Each dot represents an

Figure 4 :

 4 Figure 4: The role of Zeb1 in T cell development is cell-intrinsic. (A) Flow cytometry analysis of the indicated thymocyte subsets from BM chimeras. Frequencies are shown for each subset. Recipients were Ly5a mice (Cd45.1), and donor BM was either from Ly5a x C57BL/6 (Cd45.1/2) or Cellophane (Cd45.2). (B) iNKT cell and (C) Cd8 + Ly49 + T cell frequencies in the spleen, liver or LN from the same BM chimera described in A. (D) DN1-4, DP, Cd4+ and Cd8+ cell reconstitution in thymus of competitive BM chimeras. Recipients were Ly5a mice (Cd45.1) and donor BM was a 1:1 mix of Ly5a x C57BL/6 (Cd45.1/2) and Cellophane (Cd45.2). 8 weeks after BM transplantation, cells were analyzed by flow cytometry. The number above the red bar shows the percentage of indicated cells originating from the Cellophane BM. (E-F) Frequency of iNKT cells (E) and Ly49 + cells within Cd8 + Cd44 + T cells in spleen, liver or LN from BM chimera described in D. Each dot represents an individual mouse. Results show the pooled data from two to three independent experiments for a total of 2 to 6 mice per group in each panel. Statistical analyses were performed using unpaired Student t test.

Figure 5 :

 5 Figure 5: Cellophane DP cells have reduced survival and proliferation. (A) Percentages of Annexin V-negative cells (live cells) by wild-type and Cellophane thymocytes cultured for 0-72h. Each graph shows a different subset, as indicated (B) EdU incorporation of wild-type and Cellophane thymocytes after a 12-hour in vivo pulse of 0.2 mg EdU. (C) Frequencies of Ki67+ cells in DN1-4 subsets from WT and Cellophane mice. (D) Ki67 nuclear staining in DP thymocytes from WT and Cellophane mice. Bar graphs (right panel) show mean+/-SD fluorescence intensity (MFI). Each dot represents an individual mouse. Data are pooled from three independent experiments with three mice in each group (A-D). Statistical analysis was performed using unpaired Student t test.

Figure 6 :

 6 Figure 6: Cellophane DP thymocytes display increased TCR signaling.

Figure 7 :

 7 Figure 7: RNAseq analysis of Zeb1-regulated genes. (A)Volcano-plot representing -log10 (adjusted p-value) as a function of the estimated log2 fold-change. Significant genes were selected using the following thresholds: adjusted p-value lower than 0.05 and absolute value of log2 Fold-Change greater than 1. For significant genes (plotted in red), a selection of gene names is displayed. Results are representative of biological replicates in each group. (B) Flow cytometry analysis of several genes identified by RNA-seq either up-regulated (Foxo1, Ms4a4b, integrin α4:β7, Ccr7, Ccr4, Cd74, Cd53,) or downregulated (Cd81) in Cellophane. Data are pooled from two independent experiments with a total of 6 mice. (C) Expression level of two

Figure S4 :

 S4 Figure 1

  Figure 3

Acknowledgements

The authors acknowledge the contribution of SFR Biosciences (UMS3444/CNRS, ENSL, UCBL, US8/INSERM) facilities, in particular the Plateau de Biologie Expérimentale de la Souris, and the flow cytometry facility. We thank Bruce Beutler for sharing the Cellophane mutant mice. We also thank Andrew Griffiths and Kiyoto Kurima for discussions regarding Twirler mutant mice and Fotini Gounari and Christophe Benoist for providing RNAseq/Chipseq data of T cell development. The TW lab is supported by the Agence Nationale de la Recherche (ANR GAMBLER to TW and ANR JC BaNK to AM), the Institut National du Cancer, and receives institutional grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Université Claude Bernard Lyon1 and ENS de Lyon, Joint Research Institute for Science and Society (JORISS). JZ is the recipient of a fellowship from the China Scholarship Council (CSC). RS and YGH were funded by a FRM grant (AJE20161236686) to YGH.

Author contributions

JZ, AB, MW, DL, DEC, ALM, AR and AM performed experiments. RS and QM performed in silico analyses. JC, LG, YGH provided reagents, conceptual insight and helped writing the paper. TW wrote the paper and supervised the work.

Conflict of interest

The authors declare no conflict of interest Supplementary information accompanies the manuscript on Cellular & Molecular Immunology's website (http://www.nature.com/cmi/) selected gene list (200 genes for each list) -either upregulated (WT<Cello) or S1: Genes differentially expressed between control and Cellophane DP. Filters: Adjusted p-value<0.05, Fold change>2. Table S2: Pathway analysis of genes up regulated or down regulated in Cellophane DP compared to control DP, using "metascape". Table S3: Functional annotation of genes up regulated in Cellophane DP compared to control DP. Individual Pubmed queries were performed to search for association between corresponding genes and T cell activation or T cell development. A summary of the association and the reference of the article are provided. Table S4: Functional annotation of genes up regulated in Cellophane DP compared to control DP using the Pubmed module search in "String".