SOCS3 protein expression analysis by flow cytometry. SOCS3 expression of PBMCs and activated T cells was determined by staining with a polyclonal SOCS3-specific antibody (αSOCS3 Ab). (A) Flow cytometric histogram showing SOCS3 expression (MFI; x-axis) of PBMCs with SOCS3-specific antibodies (10 μg/mL; black curve) or without (gray curve). (B) SOCS3 staining of PBMCs after preincubation of antibodies with different concentrations of SOCS3 peptide (gray bars) or control peptide (open bars). MFIs for SOCS3 are shown on the y-axis. Bars represent mean ± SD of 2 replicates. One representative experiment of 3 is shown. (C-D) SOCS3 expression analysis of CD4⁺ T cells after in vitro culture with αCD3/CD28 or without for 2 days is shown. SOCS3 Ab with or without SOCS3 peptide preincubation was performed. (C) Western blot analysis indicates SOCS3 protein expression. β-Actin was used as a control. (D) SOCS3 expression analysis by flow cytometry. SOCS3 expression is indicated with (open bars) or without SOCS3 peptide (black filled bars). (E) SOCS3 expression of in vitro activated T cells at different time points is shown as relative expression of CD40L^high (activated) cells compared with CD40L^low (nonactivated) cells (dotted lines). Stimulations with PPD of M tuberculosis or αCD3/CD28 are depicted. Bars represent mean ± SD of 2 replicates; n = 4. *P < .05 (paired t test). **P < .01 (paired t test). (F) SOCS3 expression in PPD-specific T cells, cocultured with BCG-GFP–infected autologous MDMs at different time points. The relative expression of T cells, cocultured with infected MDMs to T cells, cocultured with noninfected MDMs is shown (dotted line). Bars represent mean ± SD of 2 replicates (n = 3).