Fig. 3

Ability of DC produced in co-cultures to induce T cell responses. Co-cultures were established by overlay of Lin⁻ BM from Act-mOVA mice above 5G3 stroma and non-adherent cells collected after 21 days. Cells were stained for CD11b, CD11c and MHC-II expression and subsets of L-DC and cDC-like cells sorted and then incubated with CD8⁺ T cells purified from OT-I TCR-tg mice specific for OVA_257–264/H-2K^b or OT-II TCR-tg mice specific for OVA_323–339/H-2IA^b at APC:T cell ratios of 1:10, 1:100 and 1:1000. Controls included T cells only with no APC. Control APC included CD11c⁺ DC from spleen. (a) Cells for analysis of OT-I responses were collected from cultures and gated as live (PI⁻) Thy1.2⁺Vα2⁺CD8⁺ T cells. T cell activation was measured at 24 h in terms of % cells expressing CD69. T cell proliferation was measured after 4 days in terms of a reduction in CFSE staining. Data represent mean ± S.E. of three replicate co-cultures. L-DC gave statistically greater T cell activation across all three APC:T cell ratios, while statistically greater T cell proliferation was noted only at an APC:T cell ratio of 10:1 (p ≤ 0.05). (b) Cells for analysis of OT-II responses were collected from cultures and T cells gated as live (PI⁻) Thy1.2⁺Vα2⁺CD4⁺ cells. T cell activation was measured at 24 h as % cells expressing CD69. T cell proliferation was measured at 4 days in terms of reduction in CFSE staining. Production of regulatory T cells was assessed through intracellular Foxp3 expression in CD4⁺ T cells analysed after 4 days by flow cytometry