Th differentiation from control (C) or posttransplantation patients (P) cells. CD4+ T cells were purified from PBMCs using CD4 magnetic beads (Miltenyi Biotech, Auburn, CA). CD4+ T cells (5 × 105/mL) were cultured with the Dynabeads CD3/CD28 T Cell Expander (Invitrogen, Carlsbad, CA) under Th1 (20 ng/mL hIL-12 (PeproTech, Rocky Hill, NJ) + 2.5 μg/mL anti–IL-4 mAb (R&D Systems, Minneapolis, MN)) or Th2 (20 ng/mL hIL-4 (PeproTech) + 2.5 μg/mL anti-IFNγ mAb (R&D Systems) differentiation conditions. After 4 days of culture, cells were expanded with human IL-2 (100 unit/mL). After a total of 7 days of culture, differentiated Th cells were analyzed or used for extended culture. Cells were analyzed for STAT4 expression (A,B) and IL12RB2 expression (D) using real-time PCR (A,D) or immunoblot (B). Cells differentiated in panel A were washed and stimulated with anti-CD3 for 1 day before IFNγ levels were measured using ELISA (C). (E) For long-term cultures, cells were resuspended at 106/mL, stimulated with plate-bound anti-CD3 (4 μg/mL) and soluble anti-CD28 (1 μg/mL) in the presence of Th1 or Th2 conditions at each weekly interval. After 3 weeks, cultures were washed and stimulated with anti-CD3 for 1 day. Levels of IFNγ, TNFα, IL-4, and IL-5 in the supernatant were determined using either ELISA or multiplex Fluorokine MultiAnalyte Profiling Kit in the Luminex 200 analyzer (Millipore). Data are presented as mean ± SD from a total of 4 healthy control and 9 patient samples (2-5 pooled/experiment) across 3 independent experiments. Statistical significance was evaluated with an independent Student t test using SPSS 16.0 program (SPSS, Chicago IL), and P < .05 was considered significant. *Significantly different from healthy controls (P < .05).
