Figure 5. Dox-treated autophagy-deficient tumors elicit equivalent T cell responses despite altered secretion of immunomodulatory factors.

(A) Autophagy-competent (shCTL) and -deficient (shAtg7 or shAtg12) B16 cells treated with 10 μM Dox or vehicle control. ATP and HMGB1 secretion measured in conditioned medium and surface PD-L1 measured by flow cytometry. Data points represent biological replicates; bars represent mean values. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; not significant (NS) using 2-way ANOVA multiplicity-adjusted P values (Dunnett’s correction). (B) Autophagy-competent (shCTL) and -deficient (shAtg7) B16 cells were treated for 24 hours with 8.8 μM and 7.5 μM Dox, respectively, and subsequently injected into WT C57BL/6 mice (n = 5). Control mice were injected with Matrigel (n = 10). Mice were rechallenged with WT B16 cells 1 week later; tumor incidence was monitored daily. (C) Autophagy-competent and -deficient B16 cells were injected into GREAT reporter mice; mice were then treated with 5 mg/kg Dox weekly. Tumor growth curves: arrows indicate Dox treatment; error bars represent SD (B16-shCTL: n = 6; B16-shATG7: n = 7; B16-shCTL + Dox: n = 8; B16-shATG7 + Dox: n = 8). Resected tumor mass: box and whisker plots indicate minimum, median, and maximum values. Untreated tumors are also included in Figure 1A. (D) Resected and digested tumors were immunoblotted for LC3 (autophagy deficiency) and γH2AX (DNA damage). Expression quantified normalized to GAPDH. Error bars represent SD. *P < 0.05; NS, not significant, using unpaired t test. (E) T cell infiltration and functional status (CD44, PD-1, eYFP) in control and Dox-treated autophagy-competent and -deficient B16 tumors from 2 experimental cohorts. Each data point represents a distinct tumor from an individual host mouse. Bars represent mean values. *P < 0.05; ***P < 0.001; ****P < 0.0001, using 2-way ANOVA multiplicity-adjusted P values (Tukey’s correction). Measurements of untreated tumors are also included in Figures 2B and 3B.