Figure 3. Regulatory CD11c^loCD45RB^hi DCs induce proliferation and IL-10 expression in antigen-specific CD4 T cells.

Mice were injected i.p. with 10⁶ infected erythrocytes. At days 0 (naive mice) and 10 post-injection, DC subsets were sorted by FACS. DC subsets were then co-cultured with naive CD4 T cells isolated from DO11.10 mice in the presence of OVA peptide. (A) After 4 days of co-culture, T cell proliferation was determined using the incorporation of [H³]-thymidine for the final 16 hours of culture as a readout. Error bars represent standard deviation within co-cultures of cells sorted from 3 individual mice. (B-C) After 24 hours or 7 days of co-culture with DCs sorted from infected (B) or naive (C) mice, T cells were collected and assayed via flow cytometry for markers of T cell activation. FACS histograms show the expression of the surface marker when T cells are co-cultured with CD11c^hi DCs (thick line) or CD11c^loCD45RB^hi DCs (thin line). Stainings with isotype control antibodies are shown in the grey shaded histograms. Each FACS plot is representative of one out of three individual mice. (D) After 7 days of co-culture with sorted DCs from day 0 (naïve) or day 10-infected mice, cells were restimulated with PMA and ionomycin for 5 hours. CD4 T cells were stained intracellularly for cytokine expression. Isotype controls for the cytokine-specific antibodies are shown as FACS histograms, as detailed in B. Each FACS plot is representative of one out of three DC-T cell co-cultures, and are representative of two independent experiments.