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Signaling via the pre-T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (β-selection) of TCRβ(+) CD4(-)CD8(-) double-negative stage 3 (DN3) and DN4 progenitor cells that differentiate into CD4(+)CD8(+) double-positive (DP) thymocytes, which then rearrange the locus encoding the TCR α-chain (Tcra). Interleukin 7 (IL-7) promotes the survival of TCRβ(-) DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during β-selection have remained unclear. Here we found that IL-7 signaled TCRβ(+) DN3 and DN4 thymocytes to upregulate genes encoding molecules involved in cell growth and repressed the gene encoding the transcriptional repressor Bcl-6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly. Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not. Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during β-selection.

International audience; Intestinal CD8αα intraepithelial T lymphocytes (T-IELs) have a key role in mucosal immunity and, unlike other T cells, were proposed to differentiate locally. In apparent contradiction, these cells were also shown to originate from a wave of thymus migrants colonizing the gut in the first 3 weeks after birth. We here identify previously uncharacterized very immature CD4(-)CD8(-)CD3(-)CD44(+)CD25(int) thymocytes, which have not yet rearranged their T-cell antigen receptor (TCR), as having the capacity to leave the thymus, migrate to the blood, colonize the gut, and reconstitute CD8αα T-IEL, and show that this cell set is fully responsible for the generation of the CD8αα T-IEL pool. Thus, although the thymus may be fundamental for efficient T-cell commitment, CD8αα T-IEL' complete TCR rearrangements and TCR-αβ/γδ lineage commitment must occur in the gut. These results demonstrate a major role of the gut environment as a primary lymphoid organ.

In human B-acute lymphoblastic leukemia (B-ALL), RAG1-induced genomic alterations are important for disease progression. However, given that biallelic loss of the RAG1 locus is observed in a subset of cases, RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf−/−Rag1−/− mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model, we identified a new, Rag1-independent leukemia-initiating mechanism originating from a Sca1+CD19+ precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM, a similar CD34+CD19+ population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans.

The entry of T cell progenitors to the thymus marks the beginning of a multistage developmental process that culminates in the generation of self–MHC-restricted CD4+ and CD8+ T cells. Although multiple factors including the chemokine receptors CCR7 and CCR9 are now defined as important mediators of progenitor recruitment and colonization in both the fetal and adult thymi, the heterogeneity of thymus-colonizing cells that contribute to development of the T cell pool is complex and poorly understood. In this study, in conjunction with lineage potential assays, we perform phenotypic and genetic analyses on thymus-settling progenitors (TSP) isolated from the embryonic mouse thymus anlagen and surrounding perithymic mesenchyme, including simultaneous gene expression analysis of 14 hemopoietic regulators using single-cell multiplex RT-PCR. We show that, despite the known importance of CCL25-CCR9 mediated thymic recruitment of T cell progenitors, embryonic PIR+c-Kit+ TSP can be subdivided into CCR9+ and CCR9− subsets that differ in their requirements for a functional thymic microenvironment for thymus homing. Despite these differences, lineage potential studies of purified CCR9+ and CCR9− TSP reveal a common bias toward T cell-committed progenitors, and clonal gene expression analysis reveals a genetic consensus that is evident between and within single CCR9+ and CCR9− TSP. Collectively, our data suggest that although the earliest T cell progenitors may display heterogeneity with regard to their requirements for thymus colonization, they represent a developmentally homogeneous progenitor pool that ensures the efficient generation of the first cohorts of T cells during thymus development.

Au cours de la greffe de cellules souches hematopoietiques, la lignee des lymphocytes T est la derniere a etre generee. Ce processus est responsable de l’absence d’une composante majeure du systeme immunitaire chez ces patients pendant un laps de temps entrainant des complications souvent fatales. Accelerer la reconstitution de cette voie lymphoide permettrait de restaurer une immunite efficace et rapide. L’hematopoiese precoce des lymphocytes T est tout a fait particuliere, en ce sens que ces progeniteurs et leur descendance se situent sur plusieurs sites et que des intermediaires de differenciation dans plusieurs organes existent. Par ailleurs, les genes responsables de l’engagement irreversible dans cette lignee et leur coordination sont encore meconnus. En effet, contrairement a la lignee B, plus d’un gene est indispensable pour signifier la specificite T. Enfin, le branchement de la voie T dans le schema hematopoietique general est encore incertain, plusieurs options etant sans cesse remises en question. Ainsi, une meilleure comprehension des facteurs et mecanismes impliques dans la generation de la lignee T permettra de maitriser sa production et d’acceder a des therapies nouvelles.

Extrathymic T cell precursors can be detected in many tissues and represent an immediately competent population for rapid T cell reconstitution in the event of immunodeficiencies. Blood T cell progenitors have been detected, but their source in the bone marrow (BM) remains unclear. Prospective purification of BM-resident and circulating progenitors, together with RT-PCR single-cell analysis, was used to evaluate and compare multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). Molecular analysis of circulating progenitors in comparison with BM-resident progenitors revealed that CCR9+ progenitors are more abundant in the blood than CCR7+ progenitors. Second, although Flt3− CLPs are less common in the BM, they are abundant in the blood and have reduced Cd25+-expressing cells and downregulated c-Kit and IL-7Rα intensities. Third, in contrast, stage 3 MPP (MPP3) cells, the unique circulating MPP subset, have upregulated Il7r, Gata3, and Notch1 in comparison with BM-resident counterparts. Evaluation of the populations’ respective abilities to generate splenic T cell precursors (Lin−Thy1.2+CD25+IL7Rα+) after grafting recipient nude mice revealed that MPP3 cells were the most effective subset (relative to CLPs). Although several lymphoid genes are expressed by MPP3 cells and Flt3− CLPs, the latter only give rise to B cells in the spleen, and Notch1 expression level is not modulated in the blood, as for MPP3 cells. We conclude that CLPs have reached the point where they cannot be a Notch1 target, a limiting condition on the path to T cell engagement.

International audience; T cell commitment and $\alpha\beta$/$\gamma\delta$ lineage specification in the thymus involves interactions between many different genes. Characterization of these interactions thus requires a multiparameter analysis of individual thymocytes. We developed two efficient single-cell methods: (i) the quantitative evaluation of the co-expression levels of nine different genes, with a plating efficiency of 99-100% and a detection limit of 2 mRNA molecules/cell; and (ii) single-cell differentiation cultures, in the presence of OP9 cells transfected with the thymus Notch1 ligand DeltaL4. We show that during T cell commitment, Gata3 has a fundamental, dose-dependent role in maintaining Notch1 expression, with thymocytes becoming T-cell-committed when they co-express Notch1, Gata3 and Bc11b. Of the transcription factor expression patterns studied here, only that of Bcl11b was suggestive of a role in Pu1 down-regulation. Individual thymocytes became $\alpha\beta$$\gamma\delta$-lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not $\alpha\beta$/$\gamma\delta$ lineage committed and TN4 cells comprise two main subpopulations with different degrees of maturity. The existence of a correlation between differentiation potential and expression of the pre-TCR showed that 83% of $\alpha\beta$-committed cells do not express the pre-TCR and revealed a major stochastic component in $\alpha\beta$-lineage specification.

Progressive restriction to a differentiation pathway results from both activation and silencing of particular gene expression programs. To identify the coexpression and the expression levels of regulatory genes during hematopoietic stem cell (HSC) differentiation toward the T cell branch, we applied a new single-cell RT-PCR technique to analyze the simultaneous expression of 13 genes in 9 functionally purified populations from the bone marrow and the thymus. We report in this paper that Lin−Sca1+ckit+ HSCs display, at the single-cell level, a homogeneous and high transcriptional activity as do early thymic progenitors. Moreover, the coexpression of lymphoid and myeloid genes is an early event detected in ∼30% of short-term HSC and most multipotent progenitors, suggesting novel sources for the generation of early thymic progenitors, common lymphoid progenitors (CLPs), and common myeloid progenitors. Loss of multipotency in Lin−Sca1+ckit+ cells directed to the lymphoid branch is characterized by Lmo2 and Gata2 gene expression downregulation. Indeed, highest levels of Gata2 expression are detected only in long-term and short-term HSC populations. Complete shutdown of Pu1 gene expression in all triple-negative (TN)3 stage thymic pre-T cells is indicative of total T cell commitment. Interestingly, this is also observed in 30% of TN2 cells and 25% of CLP in the bone marrow, suggesting a possible initiation of T cell engagement in TN2 and CLP. Also, our strategy highlights similar gene patterns among HSCs and intrathymic progenitors, proposing, therefore, that identical activation signals are maintained until further maturation and generation of CD4 and CD8 coreceptors bearing thymocytes.

Abstract 1475 Poster Board I-498 We have recently described the hematopoietic potential of ckit+ Lin- cells from the murine and human amniotic fluid (1). These cells were able to generate all types of blood cells in vitro and as far as mice are concerned, to generate a complete hematopoietic system once transplanted to immunodeficient recipients. This strong hematopoietic potential was accompanied by a molecular signature measured by unicellular RT-PCR, characteristic of fetal hematopoietic progenitors. Indeed coexpression of Gata2, Lmo2 and Aml1 was found in 28% of ckit+Lin- AF cells. Intriguingly, murine ckit+Lin- AF cells can be subdivided into two fractions depending on the level of ckit expression (low or high). In in vitro assays, we demonstrated that hematopoietic potential was strictly restricted to the ckit high expressing fraction. The expansion of these cells would have great impact even in the clinical field as AF could be seen as a source of transplantable hematopoietic stem cells (HSCs). Many of the early studies that documented some expansion ability of HSCs included fetal calf serum in the protocol. Given the poorly defined combination of factors in serum and the variability between different serum lots, these protocols were often difficult to reproduce. Serum-free media supplied with specific inducers have been shown to bring several advances in driving direct differentiation of embryonic stem cells. Murine AF Lin-ckitlo and ckithi were cultured in serum- and feeder layer-free culture conditions. Lin-ckitlo AF cells died within a few days. Conversely, Lin-ckithi AF cells were maintained for up to 6 weeks, with a proliferation rate of more than 100 during the first three weeks. Their phenotype remained stable, ckithi CD45+ and Lin-. The hematopoietic potential tested in methylcellulose assays showed an increased frequency of mixed CFU-GEMM (from 24% to 84%). In vivo, CFU-S12 composed of erythroid, myeloid and Lin-ckit+Sca1+ progenitor cells were observed after the injection of AF ckithi in lethally irradiated recipients. Gene expression profile analyzed by single cell multiplex RT-PCR analysis correlated with the in vitro and in vivo results of differentiation. LMO2 was coexpressed with Gata2 and Aml1 in 66% of expanded cells, demonstrating the maintenance of an overall pattern of expression. Collectively, our results indicate that the hematopoietic potential of AF resides in the ckithi fraction and that these cells can be expanded in serum-free condition for prolonged periods of time without reduction of their hematopoietic potential. This strongly supports the idea that AF may be an excellent source of cells for therapeutic applications. 1. Ditadi 2009 Disclosures: No relevant conflicts of interest to declare.