Phenotype of Gli2ΔN₂ transgenic mice. (A) Relative cell number (calculated relative to the mean of WT littermates) in the thymus and spleen of WT (gray bars, n = 6) and Gli2ΔN₂ transgenic (□, n = 6). (B) CD4 and CD8 profiles in the Gli2ΔN₂ transgenic mouse. Thymocytes from Gli2ΔN₂ transgenic and WT littermates were analyzed for CD4 and CD8 expression in the thymus (top panels), spleen (middle panels), and lymph node (bottom panels). Differences between the percentage of populations between transgenic and littermate were statistically significant as given: (thymus) CD4SP, P = .005; DP, P = .014; (spleen) CD4, P = .002; CD8, P = .001; and (lymph node) CD4, P = .005; CD8, P = .041. (C) Bar chart to show change in CD4/CD8 ratio in thymus (WT [n = 6], Gli2ΔN₂ [n = 6], P = .032). (D) CD3 expression on CD4 (top histograms) and CD8 (bottom histograms) SP thymocytes. (E) HSA expression on CD4 (top) and CD8 (bottom) SP thymocytes. (F) CD2 expression on DP (top), CD4SP (middle), and CD8SP (bottom) cells in the Gli2ΔN₂ transgenic (right panels) and the WT (left panels). (G) CD5 expression on CD8SP cells in the Gli2ΔN₂ transgenic thymus (gray line) compared with the WT (black line). The bar chart shows mean MFI for WT (▩) and Gli2ΔN₂ transgenic (□). (H) CD69 expression on DP (top), CD4 (middle), and CD8 (bottom) SP thymocytes. Numbers in dot plots or histograms indicate the percentage of cells falling in the quadrant, region, or marker. Error bars show standard error (SE). Data are representative of at least 3 experiments.

Shh treatment reduced CD5 expression and the CD4/CD8 ratio in thymus explants. (A) CD4 and CD8 profiles of 7-day C57BL/6 E17.5 FTOC control and treated with 0.05 μg/mL r-Shh. The bar chart shows the mean ratio of mature (CD3^high) CD4/CD8 SP in 7-day FTOC for Shh^−/− and C57BL/6 E17.5 cultured with the stated concentration of recombinant Shh (r-Shh). For all treatments, n = 4. Mean cell recovery was as follows: control, 1.87 × 10⁵; 0.5 μg/mL r-Shh, 1.66 × 10⁵; 0.05 μg/mL r-Shh, 2.07 × 10⁵; 0.005 μg/mL r-Shh, 2.1 × 10⁵; and 0.0005 μg/mL r-Shh, 1.54 × 10⁵. (B) CD5 expression on r-Shh–treated FTOC. Overlaid histograms of CD5 expression gated on the subsets from the FACS plots above for control cultures (gray filled) and r-Shh–treated cultures (gray line only, no fill). MFIs were (control/+ r-Shh) as follows: DP, 424.5/219.6; CD4SP, 1197.7/586.6; and CD8SP, 425.3/220.8. The bar chart shows MFI for the control (▩) and Shh-treated (□) cultures. (C) CD4 and CD8 profiles of 10-day C57BL/6 E17.5 FTOC control and treated with 0.5 μg/mL r-Shh. Mean cell recovery was as follows: control, 1.34 × 10⁵; 0.5 μg/mL r-Shh, 1.44 × 10⁵. (D) Profiles of CD4 and CD8, gated on CD3^high of 10-day C57BL/6 FTOC control and treated with 0.5 μg/mL r-Shh.

Altered TCR repertoire selection in Shh mutant thymus. (A) Thymus phenotype of 4-week-old Shh^+/− HY-TCR female mice. CD4 and CD8 profiles of HY-TCR and Shh^+/− HY-TCR littermate mice (left dot plots). Mean thymus sizes were 3.7 × 10⁷ for HY-TCR and 4.8 × 10⁷ for Shh^+/− HY-TCR. CD4 and CD8 profiles gated on D^bSmcy-positive cells in the HY-TCR and Shh^+/− HY-TCR thymus (right dot plots). (B) Thymus phenotype of E17.5 Shh^−/− HY-TCR female FTOC cultured for 10 days. CD4 and CD8 profiles of HY-TCR and Shh^−/− HY-TCR littermate FTOC. Bar chart shows the relative percentage of transgenic (Tg) TCR⁺ CD8^bright SP cells for HY-TCR (n = 3) and Shh^−/− HY-TCR (n = 3); P = .027. The histograms show Vβ8 and T3.70 (transgenic TCRα chain) staining on CD8SP cells. (C) Thymus phenotype of 12-day-old Shh^+/− HY-TCR male. CD4 and CD8 profiles of HY-TCR and Shh^+/− HY-TCR littermates. Mean thymus sizes were 2.4 × 10⁷ for HY-TCR and 2.1 × 10⁷ for Shh^+/− HY-TCR. Bar charts show the relative percentage of transgenic (Tg) TCR⁺ CD8^bright SP or DP cells for HY-TCR (n = 3) and Shh^+/− HY-TCR (n = 3); P = .019. The histograms show Vβ8 and T3.70 expression on CD8SP cells. (D) Thymus phenotype of E19 Shh^−/− HY-TCR male. CD4 and CD8 profiles of HY-TCR, Shh^+/− HY-TCR, and Shh^−/− HY-TCR littermates. Mean thymus sizes were 2.5 × 10⁵ for HY-TCR, 2.4 × 10⁵ for Shh^+/− HY-TCR, and 1.1 × 10⁴ for Shh^−/− HY-TCR. Bar charts show the relative percentage of Tg TCR⁺ CD8^bright SP or DP cells for HY-TCR (n = 4), Shh^+/− HY-TCR (n = 4, P = .034), and Shh^−/− HY-TCR (n = 3, P = .018). The histograms show D^bSmcy and T3.70 staining on CD8SP cells. Numbers in dot plots indicate the percentage of cells falling in the quadrant. Error bars show SE. Data are representative of at least 3 experiments.

Expression of Gli1 and Gli2ΔN₂ in WT and Gli2ΔN₂ transgenic mice. (A) Hh activation status, shown by quantitative RT-PCR analysis of gli1 mRNA in FACS-sorted populations of the adult murine thymus. (B) Quantitative PCR of genomic Gli2ΔN₂, showing high copy number of the Gli2ΔN₂ transgene. (C) Relative Gli2ΔN₂ expression, by quantitative RT-PCR analysis, in thymus, lymph node, and spleen (D) and up-regulation of Gli1 transcription in Gli2ΔN₂ transgenic CD4SP thymocytes. Error bars show standard deviation (SD). Data are representative of at least 3 experiments.

Increased differentiation to CD4SP in the Shh^−/− thymus. (A) CD4 and CD8 staining of E14.5 Shh^−/−, Shh^+/−, and Shh^+/+ FTOC littermates, cultured for 6 days. Mean thymus sizes were 2.3 × 10⁴ for Shh^−/−, 1.6 × 10⁵ for Shh^+/−, and 1.8 × 10⁵ for Shh^+/+. (B-C) Bar charts to show the relative proportion of CD4SP thymocytes in E14.5 Shh^−/− and littermate FTOC after 3 (B) and 6 (C) days in culture. For each thymus, the proportion of CD4SP cells was calculated relative to the mean proportion in WT littermates. The difference in the relative proportion of CD4SP cells between Shh^−/− and Shh^+/+ thymi was statistically significant: P = .006 for 3-day cultures (WT [n = 7], Shh^−/− [n = 5]), P = .022 for 6-day cultures (WT [n = 7], Shh^−/− [n = 6]). (D) CD4 and CD8 staining of E17.5 Shh^−/−, Shh^+/−, and Shh^+/+ FTOC littermates, cultured for 7 days. The average percentage of CD4SPs in the Shh^−/− was increased 1.4 times compared with littermates (P = .046). The ratio of CD4/CD8 SP cells was 2.6 in Shh^+/+ FTOC and 3.4 in Shh^−/− FTOC. Bar charts to show CD3 and CD69 expression on CD4 and CD8 SP thymocytes in the Shh^−/− (□, n = 3) and littermates (▩, n = 14). Mean thymus sizes were 9 × 10⁴ for Shh^−/−, 1.3 × 10⁵ for Shh^+/−, and 1.9 × 10⁵ for Shh^+/+. (E) Bar chart to show mean CD4/CD8 ratio for the different culture conditions. (F) Bar chart to show mean SP/DP ratio for the different culture conditions. Error bars show SE. Data are representative of at least 3 experiments.

Gli2ΔN₂ expression influences TCR repertoire selection. (A) Thymus phenotype of male mice with the transgenic HY-TCR. CD4 and CD8 expression in HY-TCR–positive mice with (Gli2ΔN₂HY-TCR) and HY-TCR littermates without the Gli2ΔN₂ transgene. Mean thymus sizes were 2.4 × 10⁷ for HY-TCR and 2.3 × 10⁷ for Gli2ΔN₂HY-TCR. The histograms show D^bSmcy tetramer, Vβ8, and CD69 staining on CD8SP cells from the Gli2ΔN₂HY-TCR. The bar chart shows the percentage of CD8 D^bSmcy-positive cells in the HY-TCR (n = 12) and Gli2ΔN₂HY-TCR (n = 8) thymus (P = < .001). (B) Peripheral phenotype of male mice with the HY transgenic TCR. CD4 and CD8 profile of a Gli2ΔN₂HY-TCR lymph node. The bar chart shows the percentage of CD8 D^bSmcy-positive cells in HY-TCR (n = 12) and Gli2ΔN₂HY-TCR (n = 8) lymph node (P = < .001). The histograms show D^bSmcy and Vβ8 expression on CD8 T cells from the Gli2ΔN₂HY-TCR. (C) Lymph node of male mice with the B6.2.16β transgenic TCR. Bar chart to show the relative number of CD8⁺ D^bSmcy^bright cells in B6.2.16β-positive mice (B6) and B6.2.16βGli2ΔN₂-positive mice (Gli2ΔN₂B6) (P = .021, n = 4, n = 5, respectively). The histograms show Vβ8 expression on CD8 T cells. (D) Thymus phenotype of HY-TCR female mice. CD4 and CD8 profiles of HY-TCR and Gli2ΔN₂HY-TCR mice (left dot plots). Mean thymus sizes were 8.7 × 10⁷ for HY-TCR and 8.4 × 10⁷ for Gli2ΔN₂HY-TCR. CD4 and CD8 profiles gated on D^bSmcy-positive cells in the HY-TCR and Gli2ΔN₂HY-TCR thymus (right dot plots). The percentages of cells falling in the D^bSmcy-positive gate were typically 9.6% in the HY-TCR and 9.4% in the Gli2ΔN₂HY-TCR. (E) Peripheral phenotype of HY-TCR female mice. CD4 and CD8 profiles in HY-TCR and Gli2ΔN₂HY-TCR mice. Numbers in dot plots or histograms indicate the percentage of cells falling in the quadrant, region, or marker. Error bars show SE. Data are representative of at least 3 experiments.

Gli2ΔN₂ expression impairs TCR-induced activation and proliferation of peripheral T cells and reduces ERK phosphorylation. (A) Quantitative RT-PCR analysis of Gli2ΔN₂ 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 Gli2ΔN₂ (▩) compared with the WT (□). 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 μg/mL of each of anti-CD3 and anti-CD28. Gray fill indicates WT; no fill, Gli2ΔN₂. 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 μg/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, Gli2ΔN₂ 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 Gli2ΔN₂ transgenic with and without IL-2. (D) Bar graph shows proliferation for WT and the Gli2ΔN₂ transgenic cultured in 0.1, 0.01, and 0.001 μg/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 GliΔN₂ transgenic splenocytes stimulated with 1 μg/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 μg/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 GliΔN₂ (7.4/11.5). (G) Histograms to show basal intracellular p-Erk in WT (top, MFI = 45.2) and GliΔN₂ (bottom, MFI = 29.0) thymocytes.