Figure S2. IC antigens are efficiently... | Rockefeller University Press

Figure S2.

IC antigens are efficiently cross-presented in vivo by both cDC1 and cDC2. (A) In vitro proliferation of OT-I cultured with 1.25 × 104 cDC1 or cDC2 generated from BM in the presence of Flt3L, either alone or with 2.5 × 104 Abelson-mOVA cells, 108 HKLM-OVA, or sOVA (100 μg/ml). Numbers represent the percent of cells in the indicated gates. (B) Percent OT-I proliferation from A averaged for two independent experiments. Multiple t tests. (C and D) WT and Δ32 mice were injected with OT-I and immunized with Abelson-mOVA 1 day after RRV infection. 3 days later, spleen and draining inguinal LN were analyzed for DCs and OT-I populations. Data are the mean ± SD for technical replicates of two independent experiments. Two-way ANOVA with Tukey’s multiple comparisons test. (C) Percentage of live CD11c+ MHCII+ MAR-1+ CD64+ cells in popliteal LN (left) and in spleen (right). (D) Percentage OT-I proliferation in spleen. (E) Percentage (left) and the representative plots (right) of IgG+ Abelson-mOVA after preincubation either with isotype IgG or with anti-OVA IgG from two independent experiments. Unpaired t test. (F) Representative plot of Fig. 2 E. (G) Frequency of proliferating OT-I cells from Fig. 1, D and E shown as a percentage of total splenocytes. (H) Frequency of proliferating OT-1 cells from Fig. 2, A and B shown as a percentage of total splenocytes. (I) Frequency of proliferating OT-II cells from Fig. 4, A and B shown as a percentage of total splenocytes. (J) Frequency of proliferating OT-II cells from Fig. 4, C and D shown as a percentage of total splenocytes. (F and H) Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. (G and I) Two-way ANOVA with Tukey’s multiple comparisons test. ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MAR-1: FceR1 α monoclonal antibody. Refer to the image caption for details.

IC antigens are efficiently cross-presented in vivo by both cDC1 and cDC2. (A) In vitro proliferation of OT-I cultured with 1.25 × 10⁴ cDC1 or cDC2 generated from BM in the presence of Flt3L, either alone or with 2.5 × 10⁴ Abelson-mOVA cells, 10⁸ HKLM-OVA, or sOVA (100 μg/ml). Numbers represent the percent of cells in the indicated gates. (B) Percent OT-I proliferation from A averaged for two independent experiments. Multiple t tests. (C and D) WT and Δ32 mice were injected with OT-I and immunized with Abelson-mOVA 1 day after RRV infection. 3 days later, spleen and draining inguinal LN were analyzed for DCs and OT-I populations. Data are the mean ± SD for technical replicates of two independent experiments. Two-way ANOVA with Tukey’s multiple comparisons test. (C) Percentage of live CD11c⁺ MHCII⁺ MAR-1⁺ CD64⁺ cells in popliteal LN (left) and in spleen (right). (D) Percentage OT-I proliferation in spleen. (E) Percentage (left) and the representative plots (right) of IgG⁺ Abelson-mOVA after preincubation either with isotype IgG or with anti-OVA IgG from two independent experiments. Unpaired t test. (F) Representative plot of Fig. 2 E. (G) Frequency of proliferating OT-I cells from Fig. 1, D and E shown as a percentage of total splenocytes. (H) Frequency of proliferating OT-1 cells from Fig. 2, A and B shown as a percentage of total splenocytes. (I) Frequency of proliferating OT-II cells from Fig. 4, A and B shown as a percentage of total splenocytes. (J) Frequency of proliferating OT-II cells from Fig. 4, C and D shown as a percentage of total splenocytes. (F and H) Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. (G and I) Two-way ANOVA with Tukey’s multiple comparisons test. ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MAR-1: FceR1 α monoclonal antibody.