Figure 1.
cDC1 are necessary and sufficient for priming CD8+T cells with cell-associated antigen. (A) Principal component analysis by all expressed mRNAs of splenic cDC1s and cDC2s from WT, Δ32, and Δ1+2+3 mice. PC1: 0.68585 of variance. PC2: 0.24495 of variance. Volcano plots of differentially expressed genes as the Log2 (fold change) over −Log10 (P value). (Left) cDC1 and cDC2 in WT mice. Data are represented as two biologically independent samples. (Middle) cDC1 in WT and Δ1+2+3 mice. (Right) cDC2 in WT and Δ32 mice. Transcripts with significant changes in expression are highlighted in red (increased) or blue (decreased). (B) In vitro proliferation of 2.5 × 104 OT-I cells cultured with 104 splenic cDC1 or cDC2 alone or with 2.5 × 104 Abelson-mOVA cells, 108 HKLM-OVA, or sOVA (100 μg/ml) as indicated. Numbers represent the percent of cells in the indicated gates. (C) Percent OT-I proliferation from B averaged for two independent experiments. Data are represented as mean values ± SD. Multiple t tests. ****P < 0.0001. (D and E) In vivo proliferation of OT-I in WT, Δ32, Δ1+2+3, and Wdfy4−/− mice on day 3 after immunization with 3.3 × 105 Abelson-mOVA cells. Data are represented as mean values ± SD of pooled biologically independent samples from two independent experiments. n = 4 for WT, Δ32, and Δ1+2+3; n = 3 for Wdfy4−/− mice. ns = not significant; *P < 0.05. (F) Two-color histograms for splenocytes from WT, Δ32, and Δ1+2+3 mice on day 12 after injection of 106 1956-mOVA cells. CD8+ T cells were analyzed for the frequency of Kb-SIINFEKL tetramer staining. Pregating is indicated above the histograms. Numbers indicate the percentage of cells in the indicated gates. (G) Frequencies of Kb-SIINFEKL tetramer–positive CD8+ T cells from mice described in F. n = 6 for WT and Δ32; n = 5 for Δ1+2+3 mice. (H and I) Individual or averaged tumor growth in WT, Δ32, and Δ1+2+3 mice implanted with 106 1956-mOVA cells. n = 6 for each genotype. (E and H) Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. **P < 0.01; ***P < 0.001. (I) Two-way ANOVA with Tukey’s multiple comparisons test. ns = not significant; WT versus Δ1+2+3. ****P < 0.0001; WT versus Δ32. ****P < 0.0001; Δ32 versus Δ1+2+3. PCA: Principal component analysis. Refer to the image caption for details.

cDC1 are necessary and sufficient for priming CD8 + T cells with cell-associated antigen. (A) Principal component analysis by all expressed mRNAs of splenic cDC1s and cDC2s from WT, Δ32, and Δ1+2+3 mice. PC1: 0.68585 of variance. PC2: 0.24495 of variance. Volcano plots of differentially expressed genes as the Log2 (fold change) over −Log10 (P value). (Left) cDC1 and cDC2 in WT mice. Data are represented as two biologically independent samples. (Middle) cDC1 in WT and Δ1+2+3 mice. (Right) cDC2 in WT and Δ32 mice. Transcripts with significant changes in expression are highlighted in red (increased) or blue (decreased). (B) In vitro proliferation of 2.5 × 104 OT-I cells cultured with 104 splenic cDC1 or cDC2 alone or with 2.5 × 104 Abelson-mOVA cells, 108 HKLM-OVA, or sOVA (100 μg/ml) as indicated. Numbers represent the percent of cells in the indicated gates. (C) Percent OT-I proliferation from B averaged for two independent experiments. Data are represented as mean values ± SD. Multiple t tests. ****P < 0.0001. (D and E) In vivo proliferation of OT-I in WT, Δ32, Δ1+2+3, and Wdfy4−/− mice on day 3 after immunization with 3.3 × 105 Abelson-mOVA cells. Data are represented as mean values ± SD of pooled biologically independent samples from two independent experiments. n = 4 for WT, Δ32, and Δ1+2+3; n = 3 for Wdfy4−/− mice. ns = not significant; *P < 0.05. (F) Two-color histograms for splenocytes from WT, Δ32, and Δ1+2+3 mice on day 12 after injection of 106 1956-mOVA cells. CD8+ T cells were analyzed for the frequency of Kb-SIINFEKL tetramer staining. Pregating is indicated above the histograms. Numbers indicate the percentage of cells in the indicated gates. (G) Frequencies of Kb-SIINFEKL tetramer–positive CD8+ T cells from mice described in F. n = 6 for WT and Δ32; n = 5 for Δ1+2+3 mice. (H and I) Individual or averaged tumor growth in WT, Δ32, and Δ1+2+3 mice implanted with 106 1956-mOVA cells. n = 6 for each genotype. (E and H) Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. **P < 0.01; ***P < 0.001. (I) Two-way ANOVA with Tukey’s multiple comparisons test. ns = not significant; WT versus Δ1+2+3. ****P < 0.0001; WT versus Δ32. ****P < 0.0001; Δ32 versus Δ1+2+3. PCA: Principal component analysis.

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