Purified T cells from the indicated strains of mice were labeled with CFSE and stimulated with plate-bound anti-TCRβ at (A) the indicated concentrations; (B) 100 μg/mL; and (C) 30 μg/mL. Proliferation was assessed every 24 h by flow cytometry. (D) Primary CFSE histogram data from the experiment shown in (C). Overlays show the dilution of CFSE in CD4⁺ and CD8⁺ T cells over time. (E) Total numbers of live T cells from the experiment in (C) were quantified by Trypan Blue staining. Data in panel A are representative of 2 experiments performed at similar doses of anti-TCRβ stimulation, while data in panels B-E are representative of 4 experiments performed with 30-100 μg/mL anti-TCRβ stimulation.

(A) IL-2 ELISAs of supernatants from T cells stimulated with 100 μg/mL of anti-TCRβ(detection limit=570 pg/mL). Data are representative of 3 experiments; bars indicate SEM. (B) IL-2 ELISAs of total T cells or purified CD4⁺ or CD8⁺ T cells from WT or Malt1^-/- mice stimulated with 100 μg/mL anti-TCRβ (detection limit=340 pg/mL). Data are representative of 2 biological replicates; bars indicate SEM. (C) FACS analysis showing the percentage of CD4⁺ and CD8⁺ T cells upregulating both CD44 and CD25 in response to 100 μg/mL anti-TCRβ. Data are representative of 3 experiments at doses ranging from 10-100 μg/mL anti-TCRβ. (D) Cells were stimulated with the indicated concentrations of plate-bound anti-TCRβ and stained every 24 h with antibodies to CD25 and CD44. Histogram overlays show CD25 expression on CD4⁺ and CD8⁺ T cells at 0 and 72 h post-TCR ligation.

(A) T cells from the experiment in Fig. 4A were stained with anti-CD4 and anti-CD8 antibodies and with PI and Hoechst 33342 to measure apoptosis. Data are representative of 2 experiments performed with 30-100 μg/mL anti-TCRβ and 1-50 ng/mL rmIL-2. (B) Quantification of apoptosis in parent (undivided) and divided CD8⁺ T cells from WT, PKCθ^-/-, and Bcl10^-/- mice in the presence or absence of exogenous IL-2. A second experiment using PI to quantify apoptotic cells yielded highly similar results.

(A) Epifluorescence microscopy images showing nuclear localization of p65 (red) at 0 h, 24 h, and 48 h post-TCR ligation for WT and PKCθ^-/- CD4⁺ (green) and CD8⁺ (blue) cells. (B and C) Quantification of levels of nuclear p65 relative to levels of cytoplasmic p65 at 0, 24, and 48 h post-TCR ligation for WT and PKCθ^-/- CD4⁺ and CD8⁺ T cells. The box indicates the distribution of values around the median (solid line), while the whiskers indicate the highest and lowest value for each time point. The data analysis strategy is described in detail in Materials and Methods. For data analysis, approximately 30 images were analyzed for each genotype at each time point. Statistical comparisons between WT and PKCθ^-/- and between time points are shown in (B) and (C), respectively. ^†p<0.0001, *p<0.0005, ^‡p<0.01, ^§p<0.05.

CFSE-labeled lymphocytes from the indicated strains of mice were incubated with mitomycin C-inactivated C57BL/6 splenocytes plus the indicated concentrations of stimulatory peptide or 5 μg/ml of a null variant. Proliferation was assessed at the indicated times by flow cytometry.

(A) CFSE-labeled T cells were stimulated with 30 μg/mL plate-bound anti-TCRβ and were supplemented with the indicated concentrations of recombinant murine IL-2 at t=24 h. Proliferation was assessed by FACS at the indicated times post-stimulation. (B) Primary CFSE histogram data from Figure 4A, showing the dilution of CFSE signal in CD4⁺ and CD8⁺ T cells at 0 and 72 h post-TCR ligation. (C) Total cell number data from Figure 4A. Cells were counted every 24 h, and live cell numbers determined by Trypan Blue staining. Data are representative of 2 experiments performed with 30-100 μg/mL anti-TCRβ and 1-50 ng/mL rmIL-2.

(A) Western blots of whole cell lysates prepared from WT and PKCθ^-/- T cells stimulated with the indicated concentrations of plate-bound anti-CD3ε plus soluble anti-CD28 (1 μg/mL). Data are representative of 2 experiments. (B) Validation of the phospho-IκBα flow assay. Purified WT T cells were stimulated with 100 μg/mL plate-bound anti-TCRβ plus 1 μg/mL soluble anti-CD28. Media, DMSO, or IKK inhibitors 82 (BAY 117-7082) or 85 (BAY 11-7085) were added after 48 h, and cells were harvested at 30 min and 2 h. Phospho-IκBα levels were assessed as described in Materials and Methods. Data are representative of 3 experiments. (C-E) Intracellular FACS analysis of phospho-IκBα. Purified T cells from the indicated strains of mice were stimulated with (C) 100 μg/mL plate-bound anti-TCRβ plus 1 μg/mL soluble anti-CD28, (D) 100 μg/mL plate-bound anti-TCRβ, or (E) 30 μg/mL plate-bound anti-TCRβ plus 1 μg/mL soluble anti-CD28. Error bars in (C) indicate standard deviation of data from 3 parallel samples from 3 mice. Data in (D) are representative of 3 experiments. Data in (E) are representative of at least 3 experiments per phenotype performed between 30-100 anti-TCRβ. (F) Primary histogram data from stimulation experiment in (E), showing 0 h (no fill) and 48 h (grey fill) post-TCR ligation. Dashed line indicates the peak level of IκBα phosphorylation at t=48 h in WT cells. (G) Primary histogram overlays showing phospho-IκBα levels in parent and divided CD8⁺ T cells at t=0 h (no fill) and t=48 h (grey fill) post-TCR ligation. (H) Quantification of phospho-IκBα levels in parent and divided CD8⁺ T cells. CFSE labeled cells were stimulated with the indicated concentration of anti-TCRβ. Parent and divided cells were identified using a similar gating strategy as in Fig. 5B, and phospho-IκBα levels were determined in each population.