Figure 4
Figure 4. The influence of modulated SOCS3 expression on cytokine expression of CD4+ T cells. (A) Intracellular cytokine expression of in vitro restimulated CD4+ T cells (PMA/ionomycin) 2 days after ectopic expression of SOCS3 (black) or control vector (gray) by lentiviral transduction. Relative cytokine expression values compared with cells without transduction (dotted line) are shown as box plots. Box plots and error bars represent 5, 25, 50, 75, and 95 percentiles; n = 10. *P < .05 (paired t test). (B) Cytokine expression in “fast” and “slow” proliferating T cells on day 5 after transduction is shown for SOCS3 (black boxes), control vector (gray boxes), and nontransduced (open boxes) T cells, restimulated with PMA/ionomycin. We calculated cytokine expression ratios of “fast” versus “slow” proliferating T cells. Equal expression between “fast” and “slow” proliferating T cells is indicated as a dotted line. Box plots and error bars represent 5, 25, 50, 75, and 95 percentiles; n = 5. *P < .05 (Student t test). **P < .01 (Student t test). ***P < .001 (Student t test). (C) Cytokine concentration determined in the supernatant of SOCS3 (black bars), control vector (gray bars), and nontransduced (open bars) T cells. Before in vitro restimulation with PMA/ionomycin (day 3), cells were enriched according to eGFP expression. Cytokine concentrations normalized to cell numbers of nontransduced cells are shown. Bars represent mean ± SD of 2 replicates. One representative experiment of 3 is shown. (D) SOCS3 expression of CD4+ T cells after 5 days in vitro stimulation with αCD3/CD28 in the presence or absence of recombinant IL-7. Bars represent mean ± SD of 2 replicates. One representative experiment of 4 is shown. *P < .05 (paired t test). (E) Cytokine expression of IL-7–treated cells. IL-7 was added on the day of transduction, and T cells were then restimulated with PMA/ionomycin on 3 days after transduction. Ratios of TNF-α, IFN-γ, and IL-17-expressing T-cell proportions (IL-7–treated/non-IL-7–treated) are shown on the y-axis. The dotted line indicates equal proportions of cytokine-expressing T cells between treatments. Box plots and error bars represent 5, 25, 50, 75, and 95 percentiles; n = 5. *P < .05 (Student t test). **P < .01 (Student t test). ***P < .001 (Student t test).

The influence of modulated SOCS3 expression on cytokine expression of CD4+ T cells. (A) Intracellular cytokine expression of in vitro restimulated CD4+ T cells (PMA/ionomycin) 2 days after ectopic expression of SOCS3 (black) or control vector (gray) by lentiviral transduction. Relative cytokine expression values compared with cells without transduction (dotted line) are shown as box plots. Box plots and error bars represent 5, 25, 50, 75, and 95 percentiles; n = 10. *P < .05 (paired t test). (B) Cytokine expression in “fast” and “slow” proliferating T cells on day 5 after transduction is shown for SOCS3 (black boxes), control vector (gray boxes), and nontransduced (open boxes) T cells, restimulated with PMA/ionomycin. We calculated cytokine expression ratios of “fast” versus “slow” proliferating T cells. Equal expression between “fast” and “slow” proliferating T cells is indicated as a dotted line. Box plots and error bars represent 5, 25, 50, 75, and 95 percentiles; n = 5. *P < .05 (Student t test). **P < .01 (Student t test). ***P < .001 (Student t test). (C) Cytokine concentration determined in the supernatant of SOCS3 (black bars), control vector (gray bars), and nontransduced (open bars) T cells. Before in vitro restimulation with PMA/ionomycin (day 3), cells were enriched according to eGFP expression. Cytokine concentrations normalized to cell numbers of nontransduced cells are shown. Bars represent mean ± SD of 2 replicates. One representative experiment of 3 is shown. (D) SOCS3 expression of CD4+ T cells after 5 days in vitro stimulation with αCD3/CD28 in the presence or absence of recombinant IL-7. Bars represent mean ± SD of 2 replicates. One representative experiment of 4 is shown. *P < .05 (paired t test). (E) Cytokine expression of IL-7–treated cells. IL-7 was added on the day of transduction, and T cells were then restimulated with PMA/ionomycin on 3 days after transduction. Ratios of TNF-α, IFN-γ, and IL-17-expressing T-cell proportions (IL-7–treated/non-IL-7–treated) are shown on the y-axis. The dotted line indicates equal proportions of cytokine-expressing T cells between treatments. Box plots and error bars represent 5, 25, 50, 75, and 95 percentiles; n = 5. *P < .05 (Student t test). **P < .01 (Student t test). ***P < .001 (Student t test).

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