Reconstitution of STAT4 and IL-12Rβ2 expression in differentiated Th1 cells. Th1 cells after 1 week of differentiation from control and posttransplantation patients were transiently transfected with plasmids encoding human STAT4¹⁰  (S4), IL-12Rβ2¹¹  (β2), or vector alone using a Human T cell Nucleofector Kit (Amaxa). Protein expression was analyzed by Western blot 3 days after transfection from total cell populations using anti-STAT4 (C-20; Santa Cruz Biotechnology) and antihuman IL12Rβ2 (R&D Systems) antibodies. (A) Representative immunoblot of STAT4 protein expression. (B) Immunoblot band intensity was quantified by densitometry, normalized to GAPDH (Cell Signaling, Beverly, MA), and expressed as the ratios of STAT4 or IL-12Rβ2 to GAPDH. The protein levels are expressed in arbitrary units and presented as mean plus or minus SD of 4 independent experiments. (C) One day after transient transfection, as in panels A and B, cells were washed and stimulated with anti-CD3 plus IL-12 (2 ng/mL). After 2 days of stimulation, IFNγ levels in supernatants were measured using ELISA and presented as mean ± SD of 4 independent experiments. In panels B and C, there was a total of 5 healthy controls and 12 patient samples (2-5 pooled/experiment) across 4 independent experiments. (D) Th1 cells cultured for 1 week were transduced with retroviruses encoding STAT4 and EGFP or EGFP alone. At the end of the second week of differentiation, EGFP-positive cells were sorted and total RNA was extracted to analyze IFNG and IL12RB2 gene expression by real-time PCR.⁹  Results shown are mean ± SD from a total of 2 healthy control and 7 patient samples (2-5 pooled/experiment) across 2 independent experiments. (E) Intracellular cytokine staining was performed on transduced cells in panel D with anti-CD3 (4 μg/mL) plus IL-12 (2 ng/mL) for 5 hours of stimulation in the presence of monensin at the last 2 hours of incubation. Expression of IFNγ was evaluated on 5000 events of GFP-positive cells by flow cytometry and presented as mean fluorescence intensity (MFI). Values are presented as mean MFI ± SD from a total of 4 healthy control and 6 patient samples (2 pooled/experiment) across 3 independent experiments. For all panels: *Significantly different from healthy controls (P < .05); **significantly different from patients (P < .05).