Figure S4.

Loss of sGSN does not impact tumor antigen uptake and activation status of cDC1s, related to Figures 4 and 5

(A) OVA-specific IgG antibody response in WT and sGsn^−/−mice injected with MCA-205 LA-OVA-mCherry cells expressing cGSN as in (Figure 3F) on day 30 post-tumor inoculation. EC₅₀ titer (left) is shown as mean ± SEM from two experiments. Representative serum titrations from one experiment are shown on the right.

(B) Representative dot plot and gating strategy for CD8⁺ OVA-specific pentamer⁺ cells in tumor samples at day 16 post-tumor inoculation as in Figure 4F.

(C and D) Quantification of cDC1 in tumors (left) and migratory cDC1 in tdLNs (right) of WT (n = 8 or 10), sGsn^−/− (n = 9 or 9) or sGsn^−/−Clec9a^gfp/gfp (n = 7 or 9) mice injected with (C) B16F10 LA-OVA-mCherry at day 15 or (D) MCA-205 LA-OVA-mCherry tumor cells analyzed at day 26 post-inoculation. Data (C, D) are presented as mean frequency (top) or number of cDC1 cells per gram of tumor (bottom) ± SEM and are representative of two experiments (C) and one experiment (D).

(E) Representative histogram of tumor-derived mCherry across the indicated immune populations in B16F10 LA-OVA-mCherry tumors (left) and tdLNs (right) at day 15 post-inoculation.

(F) Representative histograms of mCherry fluorescence in WT, sGsn^−/− or sGsn^−/−Clec9a^gfp/gfp cDC1 or mig cDC1 intratumorally (left) and in the tdLN (right) of B16F10 LA-OVA-mCherry tumors at day 15 post-inoculation as in Figure 5A.

(G) Quantification of mCherry⁺ cDC1 or mig cDC1 in WT (n = 10), sGsn^−/− (n = 9) or sGsn^−/−Clec9a^gfp/gfp (n = 9) intratumorally (left) and in the tdLN (right) of MCA-205 LA-OVA-mCherry tumors at day 26 post-inoculation. Data are mean ± SEM and are representative of one experiment..

(H) Quantification of geometric mean fluorescent intensity of CD86 and MHC class II staining of cDC1 or mig cDC1 intratumorally (left) and in the tdLN (right) at day 26 post-tumor inoculation with MCA-205 LA-OVA-mCherry into WT (n = 9 or n = 9), sGsn^−/− (n = 7 or n = 9) or sGsn^−/−Clec9a^gfp/gfp (n = 8 or n = 9). Data are mean ± SEM and are representative of one experiment.

(I) Representative flow cytometric plot of naive OT-I proliferation as measured by dilution of VPD450 dye at 72 h following co-culture with the tdLN mig cDC1 derived from WT, sGsn^−/− or sGsn^−/−Clec9a^gfp/gfp at day 14 post-inoculation (B16F10 LA-OVA-mCherry) as in Figure 5C.

(J) Quantification of naive OT-I proliferation following ex vivo co-culture with sorted mig cDC1 as in Figure 5C from WT (n = 13), sGsn^−/− (n = 12) or sGsn^−/−Clec9a^gfp/gfp (n = 10) in the presence of 10 pM SIINFEKL peptide. Data are mean of relative units (% OT-I proliferated cells normalized to WT) and are representative of one experiment.

(K and L) Growth profile of tumors formed following subcutaneous inoculation of (K) 0.3 × 10⁶ B16F10 cancer cells expressing LA-OVA-mCherry implanted in WT (n = 7) or Clec9a^cre/cre (n = 6) co-housed mice or (L) 0.5 × 10⁶ MCA-205 cancer cells expressing LA-OVA-mCherry into WT (n = 10) or Clec9a^cre/cre (n = 10) co-housed mice.

Groups in (C, D, G, H) were compared using Bonferroni-corrected one-way ANOVA. Tumor growth profiles (K, L) are presented as tumor volume (mm³) ± SEM, are representative of one experiment. and were compared using Bonferroni-corrected two-way ANOVA. ns, not significant.