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. Author manuscript; available in PMC: 2013 Apr 15.
Published in final edited form as: J Immunol. 2012 Mar 19;188(8):3639–3647. doi: 10.4049/jimmunol.1101580

Figure 2.

Figure 2

CD8+ T cells induce IL-12 production from DCs. (A) B6 mice were OVA vaccinated and CD8depleted as depicted. CD11c+ DCs were magnetically purified from inguinal DLNs and pooled (n=10).DCs were stimulated with LPS (1μg/mL) and IFN-γ (100 ng/mL) for 20 hours, and analyzed in triplicate. For analysis, DCs were defined as Thy1.2−/IgM−/CD3−/CD11c+/MHCII+/CD11b+. (B) Purified CD11c+ DCs (105) were loaded with OVA257-264 or hgp10025-33 (Irr. pep.) peptide and co-cultured with purified OT-I CD8+ T cells (103–105, as indicated) or DCs in the absence of OT-I CD8+ T cells (DCs). Representative flow plots showing intracellular IL-12p40 production by DCs following 24 hours of co-culture. The IL-12 gate was set by using MHCII− cells for comparison. For the blockade experiment, 1μg/ml of IFN-γ blocking antibody (XMG1.2) or control IgG antibody (LTF-2) was added to the culture. (C) CD11c+ DCs were magnetically purified from inguinal DLNs from B6 mice treated with either IgG, CD8 depletion, or anti-IFN-γ (IFN-γ blockade; 500μg at day 0 and day 5) and analyzed for IL-12p40 production as in (A). Cumulative bar graph of IL-12p40 production by DCs. (D)Thymectomized C57BL/6 mice were CD8+ T cell depleted (500μg) (day -14), rested for 14 days, and reconstituted with PBS(No Txfr; no CD8+ T cells), IFN-γ+/+ (C57BL/6), or IFN-γ−/−CD8+ T cells (4*106). One day following reconstitution mice were OVA vaccinated (days 1 and 6). IL-12p40 production was analyzed from DCs isolated from the draining inguinal lymph nodes (day 7). IL-12p40 production induced by vaccination was compared relative to non-vaccinated reconstituted mice receiving the same source of CD8+ T cells to account for intrinsic differences of DCs to produce IL-12 and thus, demonstrate IL-12 production induced by vaccination. Average percentages for IL-12p40 positive for non-vaccinated groups (No Txfr= 3.73%, IFN-γ+/+ = 2.52%,and IFN-γ−/− =6.53%) and for vaccinated groups (No Txfr= 6.00%, IFN-γ+/+ = 6.87%,and IFN-γ−/− = 7.52%).All data shown are representative of results from 2–3 independent experiments with n=3–5 per group. *p < 0.05.