Fig 2: Scheme indicates the proposed mechanism of combined NIR-PIT concurrently targeting hEGFR and CD25 against mEERL-hEGFR tumor. hEGFR-targeted NIR-PIT induces immunogenic cell death of mEERL-hEGFR cancer cells, promoting tumor-associated antigen presentation to dendritic cells. CD25-targeted NIR-PIT attenuates the intratumoral immunosuppression mediated by Tregs, inducing proliferation and activation of cytotoxic T cells.

Fig 3: PERK inhibition blocks SBA-induced OT-I T cell activation by DCs. OT-I T cell activation assay. GM–CSF-cultured BMDCs were treated with OVA protein, ISCOMs and/or the PERK inhibitor, washed, and co-cultured for 24 h or 72 h with CFSE-labeled CD8 + CD90.1 + T cells isolated from OT-I transgenic mice. Marker expression within CD8 + CD90.1 + T cells with CD69 and CD25 expression after 24 h and CD44 and CD62L after 72 h of co-culture (a). CFSE staining as read out for proliferation within CD8 + CD90.1 + T cells (left) and the percentage of T cells which proliferated four or more times (right) after 72 h of co-culture (b). IFN-? production measured in the supernatant after 72 h of co-culture (c). Assays were performed with 3 biological replicates. Significance is shown as: not significant p > 0.05, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001

Fig 4: In vivo efficacy of combined NIR-PIT targeting hEGFR and CD25. (a) Treatment schedule. (b) The fluorescence of the tumor decreased immediately after light exposure. White arrowheads represent the locations of tumors. A.U., arbitrary unit. (c) Tumor volume curves (n = 10; repeated measures two-way ANOVA followed by Tukey's test; ****, p < 0.0001). (d) Survival curves (n = 10; log-rank test with Bonferroni correction; *, p < 0.05, **, p < 0.01, ***, p < 0.001). The CD25-PIT and the combined PIT cleared the tumor in 1/10, 3/10 mice, respectively.

Fig 5: ATT activation capacity is dependent upon diet type.(A) Flow cytometry gating strategy used for ATT activation assays. Plot shows eWAT SVF after 3 days of coculture with aCD3/CD28 Dynabeads. Gating is representative of all ATT activation assays. (B) Frequency of CD25 expression on Tconv, CD8+, and Tregs after T cell activation assays. T cells from splenocytes, oWAT SVF, and eWAT SVF cultures taken from ND- and HFD-fed mice after 18 weeks of feeding. (C) Frequency of Ki67 expression on Tconv, CD8+, and Tregs from splenocytes and eWAT after T cell activation assays. Cell fractions were assessed with or without aCD3/CD28 Dynabead coculture for 3 days. n = 4 biological replicates/group, analyzed by 2-way ANOVA where *P < 0.05, **P < 0.01, ****P < 0.0001. (D and E) Heatmap of luminex assessment of supernatants taken from T cell activation cultures after 3 days (D) and bar graph representation of IL-2, IFN-?, IL-17, and IL-4 data (E) shown in D. n = 3 biological replicates/group, analyzed by 2-way ANOVA where *P < 0.05, **P < 0.01, ***P < 0.001.