VA Qu Spez-treated DCs do not induce CD4+ T cell anergy. The 4-day-old DCs (0.5 × 106) were treated with medium alone (Control) or with 15 ng/ml TNF-α or with 15 μg/ml of VA Qu Spez for 48 hours. The DCs were then co-cultured with the allogeneic CD4+ T cells at 1:10 ratio in a round bottom 96-welled plate for 72 hrs for the first cycle of stimulation. The T cells from in the co-cultures were then purified and were rested for 24 hrs in the presence of 2 IU/ml of IL-2. These CD4+ T cells were then subjected for second cycle of stimulation with similiarly treated DCs from same donor. After 48 hrs of co-culture, the cells were pulsed overnight with 0.5 μCi (0.037 MBq) of (3H)thymidine to quantify T-cell proliferation (Panel A, filled bars). Radioactive incorporation was measured by standard liquid scintillation counting, and the results were expressed as counts per minute (mean ± SEM of triplicate values). DC-T cell co-cultures of first cycle of stimulation that were maintained for 7 days were used for the comparison (Panel A, open bars). Statistical significance (*, p < 0.05) as analysed by Mann-Whitney test is indicated. The level of T cell cytokines IFNγ (Panels B and D) and TNF-α (Panels C and E) in the cell-free supernatants from above cultures were analysed by cytokine bead array. Panels B and C indicate the level of cytokines in DC-T cell co-cultures of first cycle of stimulation that were maintained for 7 days. Panels D and E present the level of cytokines in DC-T cell co-cultures of second cycle of stimulation.