Figure 3. ITK-SYK mimics a TCR signal in... | Rockefeller University Press

$Figure 3. ITK-SYK mimics a TCR signal in vivo. (A) ITK-SYK expression in the thymus of ITK-SYKCD4-Cre animals. Thymocytes were stained against CD4 and CD8 and analyzed by FACS. eGFP fluorescence indicative of ITK-SYK expression was measured in the CD4+CD8+ DP and in the CD4+ or CD8+ SP populations of 4–5-wk-old ITK-SYKCD4-Cre or control (CD4-Cre transgenic) mice. (B and C) ITK-SYK induces DP thymocyte deletion. (B) Thymocytes were stained as in A and analyzed for the expression of CD4 or CD8. The frequencies of individual thymocyte subsets are indicated. (C) The total DP or SP thymocyte cell numbers from ITK-SYKCD4-Cre (n = 9) or control (n = 6) mice are shown. (D) Total splenic B or T cell numbers from ITK-SYKCD4-Cre (n = 10) or control (n = 9) mice are indicated. Each symbol in C and D represents an individual mouse. Statistical significance was analyzed using the unpaired two-tailed Student’s t test. ***, statistically significant (P < 0.0001); ns, not statistically significant (P ≥ 0.05). Horizontal bars indicate the means. (E and F) Single cell suspensions from lymph node (LN) or spleen (SPL) from 4–5-wk-old ITK-SYKCD4-Cre or control mice were stained against CD4 and CD8. (E) CD4 and CD8 expression was analyzed by FACS. The frequencies of individual T cell subsets are indicated. (F) eGFP fluorescence indicative of ITK-SYK expression was determined in peripheral CD4+ or CD8+ T cells of ITK-SYKCD4-Cre or control mice. Data from spleen are shown. (G) ITK-SYK expressing T cells exhibit an activated phenotype in vivo. Peripheral lymphocytes from 4–5-wk-old ITK-SYKCD4-Cre or control mice were stained against CD4, CD8, CD44, CD62L, and TCR-β. Forward scattering (FCS) as a parameter for cell size and expression of CD44, CD62L, and TCR-β was analyzed in the CD4+ and CD8+ T cell compartments using FACS. Data shown in A, B, and E–G are representative of five independent experiments with a total number of at least 10 mice analyzed per genotype. (H) ITK-SYK induces PLCγ1 phosphorylation in primary T cells in vivo. Splenic T cells were isolated from control or ITK-SYKCD4-Cre mice that were >12 wk old. Cells were left unstimulated and control cells additionally stimulated with 5 µg/ml of anti-CD3 and 1 µg/ml of anti-CD28. Lipid raft fractions were prepared and subjected to Western blot analysis with an anti–phospho-PLCγ1 antibody. Dot blots for GM1 show successful raft preparation and equal loading. Data shown are representative of three independent experiments. Black lines indicate that intervening lanes have been spliced out.$

Figure 3.

ITK-SYK mimics a TCR signal in vivo. (A) ITK-SYK expression in the thymus of ITK-SYK^CD4-Cre animals. Thymocytes were stained against CD4 and CD8 and analyzed by FACS. eGFP fluorescence indicative of ITK-SYK expression was measured in the CD4⁺CD8⁺ DP and in the CD4⁺ or CD8⁺ SP populations of 4–5-wk-old ITK-SYK^CD4-Cre or control (CD4-Cre transgenic) mice. (B and C) ITK-SYK induces DP thymocyte deletion. (B) Thymocytes were stained as in A and analyzed for the expression of CD4 or CD8. The frequencies of individual thymocyte subsets are indicated. (C) The total DP or SP thymocyte cell numbers from ITK-SYK^CD4-Cre (n = 9) or control (n = 6) mice are shown. (D) Total splenic B or T cell numbers from ITK-SYK^CD4-Cre (n = 10) or control (n = 9) mice are indicated. Each symbol in C and D represents an individual mouse. Statistical significance was analyzed using the unpaired two-tailed Student’s t test. ***, statistically significant (P < 0.0001); ns, not statistically significant (P ≥ 0.05). Horizontal bars indicate the means. (E and F) Single cell suspensions from lymph node (LN) or spleen (SPL) from 4–5-wk-old ITK-SYK^CD4-Cre or control mice were stained against CD4 and CD8. (E) CD4 and CD8 expression was analyzed by FACS. The frequencies of individual T cell subsets are indicated. (F) eGFP fluorescence indicative of ITK-SYK expression was determined in peripheral CD4⁺ or CD8⁺ T cells of ITK-SYK^CD4-Cre or control mice. Data from spleen are shown. (G) ITK-SYK expressing T cells exhibit an activated phenotype in vivo. Peripheral lymphocytes from 4–5-wk-old ITK-SYK^CD4-Cre or control mice were stained against CD4, CD8, CD44, CD62L, and TCR-β. Forward scattering (FCS) as a parameter for cell size and expression of CD44, CD62L, and TCR-β was analyzed in the CD4⁺ and CD8⁺ T cell compartments using FACS. Data shown in A, B, and E–G are representative of five independent experiments with a total number of at least 10 mice analyzed per genotype. (H) ITK-SYK induces PLCγ1 phosphorylation in primary T cells in vivo. Splenic T cells were isolated from control or ITK-SYK^CD4-Cre mice that were >12 wk old. Cells were left unstimulated and control cells additionally stimulated with 5 µg/ml of anti-CD3 and 1 µg/ml of anti-CD28. Lipid raft fractions were prepared and subjected to Western blot analysis with an anti–phospho-PLCγ1 antibody. Dot blots for GM1 show successful raft preparation and equal loading. Data shown are representative of three independent experiments. Black lines indicate that intervening lanes have been spliced out.

This Feature Is Available To Subscribers Only