Fig 1: Opal and CODEX multiplexed tissue imaging data validate increased M2-like macrophage infiltration and T cell exhaustion in the nonrejected SCCs. A, Representative Opal Multiplex IHC stains for F4/80 (red), CD204 (yellow), and CD206 (green) in tumors derived from SCC31, SCC40, SCC32, SCC35, and SCC37 on day 10 after inoculation. Four areas from each tumor and three tumors from each SCC were examined. Scale bars, 20 μm. DAPI staining is not shown. B, Representative CODEX multiplexed tissue images for CD3 (green), CD8 (red), TIM3 (magenta), and LAG3 (cyan blue) in tumors derived from SCC31, SCC40, SCC32, SCC35, and SCC37 on day 6 after inoculation. Five areas from each tumor and three tumors from each SCC were examined. Scale bars, 20 μm. C, Quantification of the percentage of F4/80+CD204+CD206+ cells described in A. Data are mean ± SEM. The rejected and nonrejected groups were compared using the Wilcoxon rank-sum test; W = 456; P = 2.55 × 10−8. D, Quantification of the percentage of CD3+CD8+ cells described in B. Data are mean ± SEM. P = 0.00055; t = −5.27; degrees of freedom (df) = 9, unpaired t test. E, Quantification of the percentage of TIM3+LAG3+/CD3+CD8+ cells described in B. Data are mean ± SEM. P = 0.02522; t = 2.6798; df = 9, unpaired t test. F, Boxplot quantifying the percentage of total cells for each CD8 cluster identified in CyTOF analysis. G, Boxplot quantifying the percentage of total cells for each macrophage cluster identified in CyTOF analysis. H, CFSE-based T cell proliferation assay after co-culture for 48 hours with nonrejected SCC35 tumor-derived macrophages isolated 10 days after inoculation. Representative histogram plot and quantification of CFSE intensity (n = 5). CD8+/CD4+ T cells isolated from healthy mouse spleens cultured without macrophages served as the controls. CD8: Kruskal–Wallis χ2 test, χ2 = 12.5; P = 0.001930454. Pairwise comparison by the Wilcoxon test with Bonferroni correction is shown in the figure. *, P value < 0.05. CD4: Kruskal–Wallis χ2 test, χ2 = 12.5; P = 0.00193. Pairwise comparison by the Wilcoxon test with Bonferroni correction is shown in the figure. *, P value < 0.05. ELISA measurement of IFNγ in culture media 48 hours after co-culture of T cells with or without tumor-derived macrophages. n = 6. Data are mean ± SEM. Kruskal–Wallis χ2 test, χ2 = 15.726; P = 0.0003847. Pairwise comparison by the Wilcoxon test with Bonferroni correction is shown. ***, P value < 0.01. MFI, mean fluorescence intensity.