H2-Aa ^cit/cit cDC2 have increased cross-priming activity. (A–D) Antigen uptake assays of BMDC (n = 4 or 5 per group). Frequency of FITC-positive BMDC (A and B) or CellTrace Violet (CTV)-positive BMDC (C and D) after incubation with FITC-labeled OVA (A and B) or after co-culture with CTV-labeled B16F10 cells (C and D). BMDC were induced from bone marrow cells by GM-CSF (A and C) or Flt3L (B and D). (E and F) In vitro cross-priming by cDC2 (E) and cDC1 (F). Representative flow cytometric histogram plots of CTV-labeled naïve OT-I CD8 T cells after co-culture (3 days) with DC purified from draining lymph nodes on day 6 after inoculation of mice with B16F10-OVA tumors. cDC2 cells were sorted as Lin− CD45⁺ Ly6C− CD11c+ Xcr1− CD11b+ and cDC1 cells were sorted as Lin− CD45⁺ Ly6C− CD11c+ Xcr1+ CD11b−. Inset, ratio of peak area for G0, G1, G2, or G3/total peak area (G0+G1+G2+G3). (G) In vivo cross-priming by DC. WT and H2-Aa^cit/cit mice were injected intravenously with equal amounts of CTV-labeled OT-I CD8 T cells, and 1 day later, recipients received OVA by i.p. injection. 4 days after OVA injection, splenocytes were collected and analyzed by flow cytometry. CTV mean fluorescence intensity (MFI, left), and the total number of OT-I CD8 T cells in the spleen (right). Three mice per group are shown (E–G). Data points represent individual mice (A–G). Data are representative of two independent experiments (A–G). WT littermates were used as controls (A–G). Error bars indicate SD (A–G). P values were determined by Student’s t test (A–G); no differences between genotypes were found in A–D and F. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.