Effects of hAAT therapy on T-cells and B-cells. (A) Proliferative response of splenocytes after stimulation with bovine type II collagen (bCII, 10 μg/ml). Splenocytes (4 × 105 cells/well, in 96-well plate) were isolated on day 28 after rAAV8-hAAT injection. Black bar, AAT gene therapy group (n = 6); open bar, control group (n = 4). Data are expressed as the stimulation index, determined by calculating the ratio of cell proliferation with antigen (measured in counts per minute, cpm) relative to that with medium alone (mean+SD). (B) Cytokine production from bCII-stimulated (100 μg/ml) splenocytes. Values are the mean+SD of each group (n = 6 for rAAV8-hAAT group, black bars; n = 4 for saline group, open bars). (C) Serum level of BAFF in hAAT treated mice (black bar, n = 9, day 35) and control mice (open bar, n = 7). Data is expressed as mean+SD. (D) BAFF serum level in rAAV8-hAAT treated mice (black bar, n = 10, day 28) and control (open bar, n = 10). In vitro effect of hAAT on (E) BAFF secretion into culture medium measured by ELISA and (F) BAFF gene expression determined by real-time PCR. Murine macrophages (RAW 264.7) were treated with hAAT (0.5mg/ml, black bar). Culture medium served as control (open bar). Both experiments were performed in quadruplicates and repeated twice. Data is expressed as mean+SD. *p < 0.05, **p < 0.01.