Fig 2: Absence of IDO contributed to increased CD8+T infiltration in colon. (A) Absolute numbers of CD4+ and CD8+ T cells infiltrated in tumor were measured by flow cytometry (n = 6). (B) The percentage of CD4+ and CD8+ T cells in spleen lymphocytes was analyzed by flow cytometry (n = 6). (C, D) Absolute numbers of CD4+ and CD8+ T cells in spleen lymphocytes were detected by flow cytometry (n = 6). (E, F) The frequency of intratumoral CD8+/ CD69+ T cells was determined (n = 6). (G, H) The percentage of CD8+ GZB+ T cells was detected (n = 6). (I, J) The expression of Ki67 and its mean fluorescence intensity (MFI) of CD8+ T were shown (n = 6). (K, L) Annexin V expression on intratumoral CD8+ T cells and its mean fluorescence intensity were determined by flow cytometry (n = 6). (M, N) Serial sections of colon tissues were immunostained with DAPI (blue), anti F4/80 (green), and CD8 (red). They were observed by confocal laser scanning microscope (scale bar = 50 µm, 10 µm). Expressions of CD11b (red) and CD4 (green) were analyzed by immunofluorescence cytochemistry (scale bar = 50 µm, 10 µm). The results are representative of at least 6 independent experiments and expressed as mean ± SD. *P < .05, **P < .01 indicates significant difference.

Fig 3: ENI ablates the immune response to combined radiation and immunotherapy.A Schematic of the experimental design for gross tumor irradiation with or without elective nodal irradiation (ENI). Mice were implanted both in the buccal and in the flank on day 0 post-implantation (DPI). Stereotactic body irradiation (SBRT) was given when tumors reached ~150mm3 and anti-CD25 was given a day before SBRT. Created with BioRender.com. B Tumor growth curves, from the experiment depicted in (A), Buccal tumor (top) and flank tumor growth curves (bottom) for mice treated with anti-CD25 (n = 5), anti-CD25 and tumor only SBRT (n = 7), anti-CD25 and ENI (n = 7), and tumor only SBRT alone (n = 5). C Buccal tumor growth curves for mice implanted with the P029 cell line (n = 10 per group). Mice were implanted in the buccal on day 0 post-implantation (DPI). SBRT was given when tumors reached ~150mm3 and anti-CD25 was given a day before SBRT and once a week thereafter. The doses of SBRT were spaced by 4–5 days. D Quantification of the percentage of mice with P029 tumors that had radiographically detectable lung metastases at days 41 (ENI, n = 10; tumor only, n = 7) and 47 (ENI, n = 9; tumor only, n = 7) post-tumor cell implantation. Lung metastases were evaluated by microCT images. E A representative microCT image of a lung metastasis identified in a mouse treated with ENI in the P029 model. A metastasis is highlighted with a white circle. F Flow cytometry analysis of blood taken from mice at day 24 DPI in the experiment depicted in (A) (ENI, n = 4; tumor only, n = 4). CD8 T cells were defined as CD45+CD3+CD8+ and CD4 T cells were defined as CD45+CD3+CD4+. For tumor growth at different time points, 3 or more groups differences were determined by a One-Way ANOVA test with Tukey’s post hoc comparisons, with only 2 groups a Two-Way ANOVA was used. To test if there is a difference between tumor only SBRT and ENI treatment groups in reducing the number of mice that grew flank tumors, we used a Fischer’s Exact test. For the flow cytometry analysis, a two-tailed student’s t-test was used. Significance was determined if the p-value was <0.05* and <0.01**. The error bars represent the standard error of the mean (± SEM). Source data are provided as a Source Data file. p-values are indicated for figures B. buccal tumor only **0.0035, buccal ENI **0.0032, *0.0278, (C) *0.0371, and (F) CD69 *0.0222, IL-2 *0.0284, CCR7 *0.0366.