Down-regulation of MHC II synthesis in DCs maturing in vivo in the steady state. (A) DC populations in the subcutaneous LN. A preparation of LN DCs was analyzed by FACS with the use of mAbs for CD11c, CD8, CD205, and MHC II. (i) The contour plot shows the expression of CD8 and CD205 in the CD11c⁺ cells. Three populations can be distinguished: CD8^–CD205^– DCs (which include the LN equivalent of the CD4⁺ and CD4^–CD8^– splenic DC populations); CD8⁺CD205⁺ DCs (the LN equivalent of the splenic CD8⁺ DCs); and the skin-derived CD8lowCD205⁺ DCs. (ii) The histograms show the expression level of MHC II in each of the 3 LN DC populations. Note that overlapping populations obscure the distinction between MHC II^low and MHC IIhigh DCs in the histograms. The dashed line represents an arbitrary division between MHCII^low and MHChigh expression labels. (B) The 3 LN DC populations were metabolically labeled for 60 minutes immediately after purification. MHC II (top panel) and MHC I (bottom) were immunoprecipitated with mAb N22 and Y3, respectively, and run in a 12.5% SDS-PAGE. The amount of immunoprecipitate loaded was normalized for the efficiency of incorporation of radioactivity in each sample, measured by TCA precipitation of the cell lysates.