Fig. 3: Concomitant draining lymph node irradiation abrogates the beneficial effect of radioimmunotherapy.

A B16F10-Luc tumor-bearing mice received α-CTLA-4 and tumor IR, with or without concomitant DLN IR. All groups received α-CTLA-4. “Sham IR + ICI” group (gray) was sham-irradiated, “TM IR + ICI” group (blue) received tumor IR, and “TM + C-DLN IR + ICI” group (orange) received DLN IR concomitantly to the tumor IR. Tumor growth was followed over 60 days. Immune cell composition was analyzed at different timepoints, as indicated. Treatment response represented by individual tumor growth curves (B) and a waterfall plot derived from the mRECIST analysis (C). Each line in B and each bar in C represents an individual mouse. Best average response value in C is described in the Methods section. Number of mice is indicated in B. D–H Tumor immune microenvironment. Gating strategy is shown in Supplementary Fig. 1A. Each dot represents an individual mouse. Floating bars span from the minimal to the maximal value of each group. Line indicates the mean. D Tumor-infiltrating T cells (TCRβ⁺). Left: cell count per mg tumor, right: TCRβ⁺ cells as a percent of CD45⁺ cells. E Tumor-infiltrating CD8⁺ T cells. Left: cell count per mg tumor, right: CD8⁺ cells as a percent of CD45⁺ cells. F Representative plots on day 7 after tumor IR. Numbers indicate the percentages of CD8⁺ and CD4⁺ T cells within the T cell compartment. G PD-1 expression on CD8⁺ T cells, expressed as the geometric mean of the fluorescence intensity (MFI), normalized to the average MFI value of the “Sham IR + ICI” group. H CD8⁺ PD-1⁺ CD39⁺ T cells. Left: expressed as a percent of all PD-1⁺ CD8⁺ T cells, right: CD8⁺ PD-1⁺ CD39⁺ T cell count per mg tumor. n ≥ 4 for D, n ≥ 5 for E and G, and n = 3 mice per group for H (exact numbers provided in Source Data file). Data were tested for normality using the Shapiro–Wilk test. For data following a normal distribution, treatment groups were compared using the two-sided unpaired t test (D) or one-way ANOVA with Holm–Sidak’s multiple comparisons test (E, left, G and H). For non-normally distributed data, the comparison was performed using the Kruskal–Wallis test with Dunn’s multiple comparisons test (E, right). All p values are displayed, with *, ** and *** indicating p < 0.05, p < 0.01 and p < 0.001, respectively. Source data are provided as a Source Data file. Figure A, created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.