Antigen presentation and T-cell stimulation by the lymphoid organ DCs. (A) FACS analysis of proliferation of CFSE-labeled OT-II cells. DCs were incubated without (top) or with (center) OVA for 45 minutes, washed, and then incubated with CFSE-labeled OT-II cells for 2.5 days. The number of live (PI^–), proliferating OT-II cells (region R1) was then determined by FACS analysis. The histogram at the the bottom shows the number of dividing OT-II cells obtained in response to DCs that had been incubated with the indicated concentrations of OVA. (B) Induction of OT-II proliferation by the spleen, thymus, and subcutaneous LN DC populations. DCs were incubated for 45 minutes with the indicated concentrations of OVA protein (left) or OVA_323-339 peptide (right), washed, and then incubated with CFSE-labeled OT-II T cells for 2.5 days. The amount of proliferating OT-II cells was determined as in panel A. The data shown are the average of duplicate determinations. The results in each panel were obtained in a single experiment in which all the DC populations described in Figure 1A were purified and analyzed in parallel. The number of thymic DCs (_TDC) in this particular experiment allowed us to analyze only one concentration of OVA. Multiple experiments have been performed comparing only the populations from a single organ or 2 organs, and the results were consistent with the data shown. (C) Presentation of OVA and induction of OT-II proliferation by CD8⁺CD205⁺ DCs and epDCs purified from mesenteric LNs. The experiment was performed as described in panel B, left panel. (D) Antigen presentation of hen egg lysozyme (HEL) to the hybridoma BO4H9.1. The indicated number of the splenic and subcutaneous LN DC populations was incubated for 18 hours with the BO4H9.1 T-cell hybridoma in the presence of 0.1 mM HEL. The amount of IL-2 released was then determined using a standard CTLL2 proliferation assay. The data shown were obtained in triplicate and are representative of 2 experiments.