Ectopic expression of Bcl-x_L in developing TCF-1-deficient thymocytes rescues Vα14-Jα18 rearrangements and NKT cells.

<p>(<b>A</b>) Flow cytometry of thymocytes from WT, TCF-1-KO, Bcl-x_L-Tg and TCF-1-KO Bcl-x_L-Tg mice showing CD4⁻CD8⁻ (double-negative, DN), CD4⁺CD8⁺ (double positive, DP), CD4 single-positive (SP) and CD8SP thymocytes. <b>Top</b>, dot plots are representative of at least 4 different experiments. <b>Bottom</b>, graphs with DP cell percentages and cell numbers from 4 mice per group are shown (mean and s.e.m.). Numbers over dot plots refer to total thymocyte cell numbers. (<b>B</b>) Flow cytometry of thymocytes showing percent of gated CD1d-tetramer+ TCRβ+ NKT cells from WT, Bcl-x_L-Tg and TCF-1-KO Bcl-x_L-Tg mice. <b>Top</b>, dot plots are representative of at least 4 different experiments. <b>Bottom</b>, graphs with NKT cell percentages and cell numbers from 4 mice per group are shown (mean and s.e.m.). (<b>C</b>) Relative expression of Vα14-Jα18 rearrangements from WT and TCF-1-KO DP cells (n = 3). (<b>D</b>) Semiquantitative PCR of cDNA (1∶1, 1∶2, 1∶4 dilutions) from DP cells of WT, TCF-1-KO, Bcl-x_L-Tg and TCF-1-KO Bcl-x_L-Tg mice showing Vα14-Jα18 and control Vα14-Cα rearrangements (n = 3). *<i>P</i><.05; ***, <i>P</i><.001.</p

TCF-1-deficient mice have reduced proportion and number of NKT cells.

<p>Flow cytometry of thymocytes showing percent of gated CD1d-tetramer+ TCRβ+ NKT cells from wild-type (WT) and TCF-1 knock-out (KO) mice (<b>A</b>) and from WT, Vα14-Tg and TCF-1-KO Vα14-Tg mice (<b>B</b>). Representative dot plots and graphs with cell percentages and cell numbers from 4–6 mice per group are shown (mean and s.e.m.). Numbers over dot plots refer to total thymocyte cell numbers; *<i>P</i><.05; **, <i>P</i><.01; ***, <i>P</i><.001.</p

TCF-1 is expressed in NKT cells in a developmentally relevant manner.

<p>(<b>A</b>) Flow cytometry of thymocytes (<b>top</b>) and hepatic lymphocytes (<b>bottom</b>) from wild-type (WT) C57BL/6 mice showing TCF-1 intracellular expression versus the control antibody (<b>right</b>) in NKT cells (gated in <b>left</b> as CD1d-tetramer+ TCRβ+ cells, numbers indicate percentage of NKT cells). (<b>B</b>) Flow cytometry of CD1d-tetramer positive thymocytes (<b>top</b>) and hepatic lymphocytes (<b>bottom</b>) from WT mice stained with anti-CD44 and anti-NK1.1 to assess NKT developmental stages 1–3, as depicted in <b>left</b>. <b>Right</b>, TCF-1 intracellular expression among stages. Data are from a representative experiment out of four WT mice analyzed. (<b>C</b>) Flow cytometry of CD1d-tetramer positive thymocytes (<b>top</b>) and hepatic lymphocytes (<b>bottom</b>) from WT mice showing TCF1 intracellular expression versus the control antibody (<b>right</b>) in NKT1 and NKT2 NKT cells (gated in <b>left</b>, numbers indicate percentage of NKT cells). Data are from a representative experiment out of four WT mice analyzed.</p

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