IL-4Rα expression is not required for the development of thymic iNKT cells.

<p>(<b>A</b>) Flow-cytometric analysis of expression of CD1d-PBS-57 and TCRβ on total thymocytes from control and IL-4RαKO mice. Numbers adjacent to outlined areas and the graphs show percent and cell numbers of total iNKT cells as indicated. Data are representative of three independent analyses with total six mice per group. <b>(B)</b> Flow-cytometric analysis of expression of PLZF and T-bet on gated thymic iNKT cells from control and IL-4RαKO mice. Numbers adjacent to outlined areas and the graph show percent iNKT cells divided into subsets A (PLZF⁺) and B (T-bet⁺). Data are representative of three independent analyses with total six mice per group. <b>(C)</b> Flow-cytometric analysis of expression of CD44 and NK1.1 on gated thymic iNKT cells from control and IL-4RαKO mice. Numbers adjacent to outlined areas and graphs show percent of gated iNKT cells divided into maturation stages 1, 2 and 3. Data are representative of three independent analyses with total six mice per group. <b>(D)</b> Flow-cytometric analysis of expression of CD4 and CD8 on gated thymic iNKT cells from control and IL-4RαKO mice. Numbers in quadrants and graphs show percent of CD4⁺CD8⁻ and CD4⁻CD8⁻ iNKT cells. Data are representative of three independent analyses with total six mice per group.</p

IL-4 is not required for the development of iNKT cells in thymus.

<p>(<b>A</b>) Flow-cytometric analysis of expression of CD1d-PBS-57 and TCRβ on total thymocytes from control and IL-4KO mice. Numbers adjacent to outlined areas and the graphs show percent and cell numbers of total iNKT cells as indicated. Data are representative of three independent analyses with total six mice per group. <b>(B)</b> Flow-cytometric analysis of expression of CD44 and NK1.1 on gated thymic iNKT cells from control and IL-4KO mice. Numbers adjacent to outlined areas and graphs show percent of gated iNKT cells divided into maturation stages 1 (CD44^loNK1.1⁻), 2 (CD44^hiNK1.1⁻) and 3 (CD44^hiNK1.1⁺). Data are representative of three independent analyses with total six mice per group. <b>(C)</b> Flow-cytometric analysis of expression of PLZF and T-bet on gated thymic iNKT cells from control and IL-4KO mice. Numbers adjacent to outlined areas and the graph show percent iNKT cells divided into subsets A (PLZF⁺) and B (T-bet⁺). Data are representative of three independent analyses with total six mice per group. <b>(D)</b> Flow-cytometric analysis of expression of CD4 and CD8 on gated thymic iNKT cells from control and IL-4KO mice. Numbers in quadrants and graphs show percent of CD4⁺CD8⁻ and CD4⁻CD8⁻ iNKT cells. Data are representative of three independent analyses with total six mice per group.</p

Peripheral iNKT cells are not affected by IL-4 deficiency.

<p>(<b>A</b>) Flow-cytometric analysis of expression of CD1d-PBS-57 and TCRβ on total lymphocytes from spleen, lymph nodes (LNs) and liver of control and IL-4KO mice. Numbers adjacent to outlined areas and the graphs show percent of total iNKT cells for each organ as indicated. Data are representative of three independent analyses with total 4–6 mice per group. <b>(B)</b> Flow-cytometric analysis of expression of PLZF and T-bet on gated iNKT cells from spleen, LNs and liver of control and IL-4KO mice. Numbers adjacent to outlined areas and the graphs show percent iNKT cells divided into subsets A (PLZF⁺) and B (T-bet⁺). Data are representative of three independent analyses with total 4–6 mice per group. <b>(C)</b> Flow-cytometric analysis of expression of CD44 and NK1.1 on gated iNKT cells from spleen, LNs and liver of control and IL-4KO mice. Numbers adjacent to outlined areas and the graphs show percent of gated iNKT cells divided into maturation stages 1, 2 and 3. Data are representative of three independent analyses with total 4–6 mice per group.</p

Stimulated iNKT cells produce IFN-γregardless of IL4 or IL-4Rα deficiency.

<p>(<b>A–C</b>) Total thymocytes were left unstimulated or stimulated with PMA (50 ng/ml) and ionomycin (1 µM) (P+I) for 5 hours from control, IL-4KO and IL-4RαKO mice. Brefeldin A was added for the last 3.5 hours. Data are representative of total six mice per group. <b>(A)</b> Flow-cytometric analysis of size (FSC-A) and complexity (SSC-A) of total thymocytes unstimulated or stimulated with P+I from one representative mouse. <b>(B)</b> Graphic representation of percent of IFN-γ positive thymic iNKT cells from control, IL-4KO and IL-4RαKO mice, as indicated. Data are representative of six mice per group. <b>(C)</b> IFN-γ intracellular expression by thymic iNKT cells from unstimulated (shaded) or stimulated with P+I (open) of control, IL-4KO and IL-4RαKO mice. Numbers in plots indicate percent of IFN-γ positive cells. <b>D–E)</b> Control, IL-4KO and IL-4RαKO mice were analized 3 hours after i.p. injection of 3 µg αGalCer or PBS. Data are representative of total six mice per group. <b>(D)</b> Graphic representation of percent of IFN-γ positive splenic iNKT cells. <b>(E)</b> IFN-γ and IL-4 production detected by ELISA using serum of stimulated mice.</p

T cell development requires constraint of the myeloid regulator C/EBP-α by the Notch target and transcriptional repressor Hes1

International audienceNotch signaling induces gene expression of the T cell lineage and discourages alternative fate outcomes. Hematopoietic deficiency in the Notch target Hes1 results in severe T cell lineage defects; however, the underlying mechanism is unknown. We found here that Hes1 constrained myeloid gene-expression programs in T cell progenitor cells, as deletion of the myeloid regulator C/EBP-α restored the development of T cells from Hes1-deficient progenitor cells. Repression of Cebpa by Hes1 required its DNA-binding and Groucho-recruitment domains. Hes1-deficient multipotent progenitor cells showed a developmental bias toward myeloid cells and dendritic cells after Notch signaling, whereas Hes1-deficient lymphoid progenitor cells required additional cytokine signaling for diversion into the myeloid lineage. Our findings establish the importance of constraining developmental programs of the myeloid lineage early in T cell development