Lymphoid Gene Upregulation on Circulating Progenitors Participates in Their T-Lineage Commitment.

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.

IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte ß-selection.

Signaling via the pre-T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (ß-selection) of TCRß(+) 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 TCRß(-) DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during ß-selection have remained unclear. Here we found that IL-7 signaled TCRß(+) 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 ß-selection.

Single-cell analysis of thymocyte differentiation: identification of transcription factor interactions and a major stochastic component in αß-lineage commitment.

T cell commitment and αß/γδ 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 αß/γδ lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not αß/γδ-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 αß-committed cells do not express the pre-TCR and revealed a major stochastic component in αß-lineage specification.

Loss of p19Arf in a Rag1(-/-) B-cell precursor population initiates acute B-lymphoblastic leukemia.

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.

Clonal analysis reveals uniformity in the molecular profile and lineage potential of CCR9(+) and CCR9(-) thymus-settling progenitors.

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.

Gene coexpression analysis in single cells indicates lymphomyeloid copriming in short-term hematopoietic stem cells and multipotent progenitors.

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 approximately 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.