Figure 4

CCL21-DC are efficient APC in the absence of KLH. (A) Allogeneic MLR: Briefly, culture medium mock-transduced DC (DC) and CCL21-DC (AdDC) were analyzed for their ability to induce allogeneic T cell proliferation. The effect of pulsing 100% (DC KLH 100 and AdDC KLH 100) or 30% of cells (AdDC KLH 30) with 10 μg/ml KLH prior to initiating the MLR assay was assessed. After pre-treatment with mitomycin C and KLH, where indicated, DC and CCL21-DC were mixed with allogeneic T cells at several ratios, as shown. Cell proliferation was assessed by BrdU incorporation following 5 days incubation at 37°C. One representative experiment of three is shown. (B-G) Autologous T cell proliferation assay: T cells were cultured for 6 days with IL-2 (15 U/ml) prior to the TT presentation assay. After mitomycin-C treatment and KLH pulsing where indicated, DC (B, E), CCL21-DC (C, F), or CV-DC (D, G) were mixed at 1:10 ratio to autologous T cells in the presence of TT (2 μg/ml) or BSA (2 μg/ml). In (B-D), IFN-γ production was measured in the cell supernatant collected after 48 hours of cell proliferation. The measured values of IFN-γ detected by ELISA were within the linear portion of the IFN-γ concentration curve. The concentration of IFN-γ is expressed in pg/ml with basal IFN-γ production subtracted. In (E-G), T cell proliferation in response to TT and BSA was measured by BrdU incorporation following 5 days co-culture at 37°C and is expressed as a percentage of proliferating cells. One representative experiment of at least three is shown. P value ≤ 0.05 was considered significant.