Gli2\#916;N₂ expression impairs TCR-induced activation and proliferation of peripheral T cells and reduces ERK phosphorylation. (A) Quantitative RT-PCR analysis of Gli2\#916;N₂ transgene expression in FACS-sorted peripheral T-cell populations (left graph). The right graph shows up-regulation of Gli1 transcription on CD4 and CD8 T cells in the Gli2\#916;N₂ (\#9641;) compared with the WT (\#9633;). Error bars represent SD. (B) Histograms showing the early activation marker CD69 (top panels) and the later activation marker CD25, on CD4 and CD8 SP cells after 24 hours in culture stimulated with 0.01 \#956;g/mL of each of anti-CD3 and anti-CD28. Gray fill indicates WT; no fill, Gli2\#916;N₂. Numbers indicate the percentage of cells falling within the marker. (In all unstimulated controls, CD69 expression was approximately 5%, and CD25 expression was approximately 8%; data not shown). (C) Bar graphs to show cell divisions using CFSE staining in CD4 cells cultured for 96 hours with 0.01 \#956;g/mL of each of anti-CD3 and anti-CD28 with (middle panel) and without (left panel) 20 IU/mL IL-2. The numbers 0-7 represent the number of divisions for cells falling in the marker. Gray indicates WT; white, Gli2\#916;N₂ transgenic. The bar graph on the far right shows the percentage of CFSE-labeled cells falling in the markers for 5 to 8 divisions for the WT and the Gli2\#916;N₂ transgenic with and without IL-2. (D) Bar graph shows proliferation for WT and the Gli2\#916;N₂ transgenic cultured in 0.1, 0.01, and 0.001 \#956;g/mL of each of anti-CD3 and anti-CD28. (E) Western blot showing the levels of phosphorylated (upper panel) Erk1 and Erk2 kinases in WT and Gli\#916;N₂ transgenic splenocytes stimulated with 1 \#956;g/mL of each of anti-CD3 and anti-CD28 for the indicated period of time. For comparison, the total levels of Erk kinases expressed in the cytoplasm of stimulated cells is shown (lower panel). (F) Intracellular phosphorylated ERK (p-Erk) on CD4⁺ cells. Top left histogram shows the CD4⁺ gate. The right histograms show intracellular p-Erk levels in unactivated CD4⁺ (dashed line) and CD4⁺ activated with 1 \#956;g/mL of each of anti-CD3 and anti-CD28 for 2 minutes (gray line). The bar chart shows the MFI (unactivated/activated) for WT (10.0/19.1) and Gli\#916;N₂ (7.4/11.5). (G) Histograms to show basal intracellular p-Erk in WT (top, MFI = 45.2) and Gli\#916;N₂ (bottom, MFI = 29.0) thymocytes.