Figure S2.
Elevated cDC2 cross priming activity, cell counts, and MHC-I expression in H2-Aacit/citmice. (A) In vitro cross-priming by macrophages. Representative flow cytometric histogram plots of CTV-labeled naïve OT-I CD8 T cells after co-culture (3 days) with macrophages purified from draining lymph nodes on day 6 after inoculation of mice with B16F10-OVA tumors. Macrophages were sorted as Lin− CD45+ CD11b+ F4/80+. Inset: Ratio of peak area for G0, G1, G2, or G3/total peak area (G0+G1+G2+G3). (B and C) In vitro cross-priming by cDC2. CTV-labeled naïve OT-I CD8 T cells were co-cultured (3 days) with OVA (100 μg/ml) and cDC2 purified from Flt3L (100 ng/ml) induced WT or H2-Aacit/cit BMDCs. (B) Representative flow cytometric histogram plots of CTV-labeled naïve OT-I CD8 T cells after co-culture. cDC2 were sorted as Lin− CD45+ Ly6C− CD11c+ CD11bhi Xcr1lo. Right: Ratio of peak area for G0, G1, G2, G3, or G4/total peak area (G0+G1+G2+G3+G4). (C) ELISA analysis of IFNγ secreted by OT-I CD8 T cells after co-culture. (D) The total number of cDC1 in spleens of WT and H2-Aacit/cit mice. (E) The frequencies of splenic cDC1 in mice of the indicated genotypes. (F) Representative flow cytometry plots of pre-cDC in the bone marrow. Bone marrow cells were gated on Lin− CD11c+ CD172a− Flt3+. Unc pre-cDC, uncommitted pre-cDC. (G) Intracellular Irf8 mean fluorescence intensity (MFI) in splenic cDC1. (H) Representative flow cytometry plots of the indicated cDC populations in mesenteric lymph nodes (MLN) of WT and H2-Aacit/cit mice. Cells were gated on Lin− CD45+ Ly6C− CD11c+. (I) The total number of cDC2 in MLN of WT and H2-Aacit/cit mice. (J) MHC-I and CD80 MFI on cDC2. (K) Frequency of H2-Kb-SIINFEKL tetramer positive tumor infiltrated CD8 T cells in WT or H2-Aacit/cit mice on day 9 after s.c inoculation with B16F10-OVA cells. Data points represent individual mice (A–E, G, and I–K). Data are representative of two independent experiments (A–K). WT littermates (A–D and F–K) and WT C57BL/6J (E) from JAX were used as controls. Error bars indicate SD (A–E, G, and I–K). P values were determined by Student’s t test (A–E, G, and I–K). n = 3 per group (A–C, F, and G), n = 4 per group (D, E, and H−K). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Elevated cDC2 cross priming activity, cell counts, and MHC-I expression in H2-Aa cit/cit mice. (A) In vitro cross-priming by macrophages. Representative flow cytometric histogram plots of CTV-labeled naïve OT-I CD8 T cells after co-culture (3 days) with macrophages purified from draining lymph nodes on day 6 after inoculation of mice with B16F10-OVA tumors. Macrophages were sorted as Lin− CD45+ CD11b+ F4/80+. Inset: Ratio of peak area for G0, G1, G2, or G3/total peak area (G0+G1+G2+G3). (B and C) In vitro cross-priming by cDC2. CTV-labeled naïve OT-I CD8 T cells were co-cultured (3 days) with OVA (100 μg/ml) and cDC2 purified from Flt3L (100 ng/ml) induced WT or H2-Aacit/cit BMDCs. (B) Representative flow cytometric histogram plots of CTV-labeled naïve OT-I CD8 T cells after co-culture. cDC2 were sorted as Lin− CD45+ Ly6C− CD11c+ CD11bhi Xcr1lo. Right: Ratio of peak area for G0, G1, G2, G3, or G4/total peak area (G0+G1+G2+G3+G4). (C) ELISA analysis of IFNγ secreted by OT-I CD8 T cells after co-culture. (D) The total number of cDC1 in spleens of WT and H2-Aacit/cit mice. (E) The frequencies of splenic cDC1 in mice of the indicated genotypes. (F) Representative flow cytometry plots of pre-cDC in the bone marrow. Bone marrow cells were gated on Lin− CD11c+ CD172a− Flt3+. Unc pre-cDC, uncommitted pre-cDC. (G) Intracellular Irf8 mean fluorescence intensity (MFI) in splenic cDC1. (H) Representative flow cytometry plots of the indicated cDC populations in mesenteric lymph nodes (MLN) of WT and H2-Aacit/cit mice. Cells were gated on Lin− CD45+ Ly6C− CD11c+. (I) The total number of cDC2 in MLN of WT and H2-Aacit/cit mice. (J) MHC-I and CD80 MFI on cDC2. (K) Frequency of H2-Kb-SIINFEKL tetramer positive tumor infiltrated CD8 T cells in WT or H2-Aacit/cit mice on day 9 after s.c inoculation with B16F10-OVA cells. Data points represent individual mice (A–E, G, and I–K). Data are representative of two independent experiments (A–K). WT littermates (A–D and F–K) and WT C57BL/6J (E) from JAX were used as controls. Error bars indicate SD (A–E, G, and I–K). P values were determined by Student’s t test (A–E, G, and I–K). n = 3 per group (A–C, F, and G), n = 4 per group (D, E, and H−K). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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