Splenic DC populations and maturation in culture. (A) Mouse spleens contain 3 populations of CD11c⁺ DCs that can be distinguished by their expression of CD4 and CD8: CD4⁺ DCs, CD8⁺ DCs, and CD4^–CD8^– DCs. (B) FACS analysis of MHC II and CD86 expression in splenic DCs freshly isolated (f, thin continuous line) or after overnight culture (o/n, thick line). The dashed line corresponds to unlabeled cells (b, background). The histograms correspond to the whole spleen DC preparation; the results were similar for each of the 3 splenic DC subtypes. (C) Immunofluorescence confocal microscopy (ICM) analysis of I-A^b (red) and lysosome-associated membrane protein–1 (Lamp 1) (green) localization in splenic CD8⁺ DCs freshly isolated (left panels) or after culture for 18 hours (right panels). The dye DAPI (4′,6 diamidino-2-phenylindole) (blue) was included to label the nuclei. Original magnification, × 63. Similar results were obtained with CD4⁺ DCs and CD4^–CD8^– DCs (not shown). (D) Left bar: 5 × 10³ purified CD8⁺ DCs were incubated with 50 μg/mL OVA for 45 minutes, washed, and then incubated with naive OVA-specific OT-II cells labeled with CFSE. After 2.5 days in culture, the number of proliferating OT-II cells (propidium iodide [PI]^– CFSElow) was determined by FACS analysis. Central bar: after the incubation with OVA, the CD8⁺ DCs were washed, cultured for 16 hours, and then incubated with OT-II cells for 2.5 days. Right bar: the CD8⁺ DCs were first cultured for 16 hours and then incubated with OVA for 45 minutes, washed, and incubated with the OT-II cells for 2.5 days. Similar results were obtained with CD8^– DCs (not shown).25 (E) Splenic DCs were fixed in PFA immediately after purification (dashed line) or after overnight culture (continuous line) and then incubated with CFSE-labeled OT-II cells in the presence of 0.1 μg/mL OVA323-339 peptide. OT-II proliferation was determined 2.5 days later. Data for experiments in D and E were obtained in duplicate; bars represent the range of the values obtained.