Fig. 6.

Cellophane DP thymocytes display increased TCR signaling. a Flow cytometry analysis of Cd69, Cd25, Cd5, Nur77, Cd4, Cd8, and Tcrβ expression (mean fluorescence intensity) in DP thymocytes of wild-type and Cellophane mice. b Left panels: Representative flow cytometry analysis of iNKT1 (Plzf lo, Rorγt-, T-bet+), iNKT2 (Plzf hi, Rorγt-, T-bet-), and iNKT17 (Plzf int, Rorγt+, T-bet-) cell subsets in wild-type and Cellophane mice (as defined by the gating strategy shown in the upper panel). Right panel: graphs of the percentages of iNKT cell subsets within total iNKT cells. c The TCRVβ repertoire of thymic iNKT cells was analyzed by flow cytometry. Bar graphs show the mean ± SD frequencies of individual Vβ chains within iNKT cells. d Expression of Cd4, T-bet, Egr2, and Plzf in iNKT cells as measured by flow cytometry (mean fluorescence intensity). e Phosphorylation levels of Erk, S6, and Akt (S473) in resting DP thymocytes from WT and Cellophane mice. f Phosphorylation levels of Erk, S6, and Akt (Ser473) in DP cells stimulated with anti-CD3 antibody for the indicated times. The results are normalized to the nonstimulated condition (i.e., basal level is 100%) for each group. g Representative histogram overlay showing the Ca²⁺ flux in thymic DP cells from wild-type or Cellophane mice stimulated with anti-CD3 antibodies, as measured by flow cytometry. Thymocytes were activated following incubation with biotinylated anti-CD3 (arrow) followed by cross-linking with streptavidin (arrowhead). h Dot plots showing the AUC (area under the curve) and Ca²⁺ peak. Statistical analysis was performed using an unpaired Student’s t test. Each dot represents an individual mouse. Data are pooled from three experiments with a total of eight mice in each group (a), three experiments with six mice (b), three mice (c), two experiments with six mice (d–f) or three experiments with three mice (g, h)