Involvement of the Fas (CD95) system in peripheral cell death and lymphoid organ development

International audienceFas‐mediated apoptosis is a form of cell death that operates through a Fas‐Fas ligand (FasL) interaction. In this study we investigated the role of the Fas system during development of normal and Fas‐mutated lymphocytes. Irradiated RAG2–/– recipients were reconstituted with bone marrow cells from B6 and lpr mice (Fas defective) or from B6 and gld mice (FasL defective), and analyzed for long‐term development. The results showed a primary role of the Fas system in peripheral cell death and thymic colonization. In the periphery, the interaction in vivo between Fas+ and Fas– T cell populations indicated that cellular homeostasis was defective. Indeed, we observed a FasL‐mediated cytotoxic effect on normal‐derived T cells, explaining the dominance of lpr T cells in the mixed chimeras. The Fas mutation affected neither cell activation nor cell proliferation, as the effector (Fas–) and target (Fas+) cells behaved similarly with regard to activation marker expression and cell cycle status. However, Fas– T cells failed to seed the periphery and the thymus in the long term. We suggest that this could be due to the fact that FasL is involved in the structural organization of the lymphoid compartment

The thymus exports long-lived fully committed T cell precursors that can colonize primary lymphoid organs

International audienceThymic export of cells is believed to be restricted to mature T cells. Here we show that the thymus also exports fully committed T cell precursors that colonize primary lymphoid organs. These precursor cells exited the thymus before T cell receptor rearrangements and colonized lymphoid organs such as the thymus and the gut. Migration of the thymic T cell–committed precursors led to permanent colonization of the gut precursor compartment, improved the capacity of gut precursors to further differentiate into T cells and was sufficient for the generation of 'euthymic like' CD8αα+ intraepithelial lymphocytes. These data demonstrate a new function for the thymus in peripheral seeding with T cell precursors that become long lived after thymus export

Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers

BACKGROUND INFORMATION: In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. RESULTS: Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. CONCLUSIONS: This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential

Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers

none11BACKGROUND INFORMATION: In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. RESULTS: Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. CONCLUSIONS: This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential.noneFranzin, Chiara; Piccoli, Martina; Serena, Elena; Bertin, Enrica; Urbani, Luca; Luni, Camilla; Pasqualetto, Valerie; Eaton, Simon; Elvassore, Nicola; De Coppi, Paolo; Pozzobon, Michela*Franzin, Chiara; Piccoli, Martina; Serena, Elena; Bertin, Enrica; Urbani, Luca; Luni, Camilla; Pasqualetto, Valerie; Eaton, Simon; Elvassore, Nicola; De Coppi, Paolo; Pozzobon, Michel

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

International audienceExtrathymic 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

A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127.

International audienceNatural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets