Figure 3. Increased immunogenicity of Bax^−/−Bak^−/− DC.

(A) Draining lymph node cells from 6-week-old DC-cre mice as wild type (WT) control, DC-DKO, Bak^−/−, DC-Bax^−/− mice immunized with OVA (5 mice per group) were re-stimulated in vitro with OVA. Cell proliferation was measured by ³H-thymidine incorporation 4 days later. WT versus DKO: **P<0.01. (B) CD4⁺CD25⁻ OT2 T cells from OT2 transgenic mice crossed with CD45.1 congenic mice (CD45.1×CD45.2 OT2 F1) were sorted, labeled with CFSE and injected intravenously into recipient C57BL6 mice. Recipient mice (5 mice/group) were then immunized with bone marrow-derived DCs (BMDCs) from DC-DKO or control mice pulsed with OVA_323–339 peptide the next day. Proliferation of transferred CD4⁺CD45.1⁺ T cells by CFSE dilution in draining lymph nodes was analyzed 4 days later. The numbers of cell cycle (average ± SD) are shown. WT versus DKO: P<0.01, with 65% increased in cell cycles induced by DKO DCs. (C) BMDCs from DC-DKO or control mice with or without OVA_323–339 pulsing were incubated with CD4⁺CD25⁻ OT2 T cells. ³H-thymidine incorporation was measured on day 4. WT versus DKO: *P<0.05, **P<0.01. (D) CFSE-labeled OT2 T cells (5×10⁴/well) were incubated with BMDCs from DC-DKO or control mice pulsed with OVA_323–339 peptide (300 DCs/well) in 96-well plates. Cell proliferation was measured by CFSE dilution 4 days later. The numbers of cell cycle (average ± SD) are shown. WT versus DKO: P<0.05 (n=4), with 13.9% increase in cell cycles induced by DKO DCs. (E) BMDCs from DC-DKO or control mice pulsed with OVA_323–339 peptide were cultured with CD4⁺CD25⁻ T cells from OT2 mice, in the absence or presence of T_reg cells sorted and expanded from FoxP3^GFP mice. Proliferation of OT2 T cells was measured by ³H-thymidine incorporation 4 days later. Statistic comparison with control group containing no T_reg cells: **P<0.01 (n=3). (F) CD4⁺CD25⁻ OT2 T cells as in Fig. 3B were labeled with CFSE and injected intravenously into recipient C57BL6 mice. Recipient mice were then immunized at the footpad with BMDCs from DC-DKO or control mice pulsed with OVA_323–339 peptide together with T_reg cells stimulated with anti-CD3- anti-CD28-coated Dynabeads in the presence of IL-2 for 3 days (T_reg:DC: 0.3:1). The proliferation of CD45.1⁺ OT2 T cells in the draining popliteal lymph nodes was quantitated by CFSE dilution 4 days later. The numbers of cell cycle (average ± SD) are shown (n=4). Statistic comparison between groups with or without T_reg cells: P<0.01 (WT DC), not significant (DKO DC).