Fig. 7: Concomitant and neoadjuvant draining lymph node irradiation induce prolonged lymphopenia in the irradiated lymph node.

A All B16F10-Luc tumor-bearing mice received α-CTLA-4. Sham IR + ICI” group (gray) was sham-irradiated, “TM IR + ICI” group (blue) received tumor IR, “TM + C-DLN IR + ICI” group (orange) received DLN IR concomitantly to the tumor IR, and “TM + NEO-DLN IR + ICI” group (purple) received DLN IR 7 days prior to tumor IR. Immune cell composition of the DLNs was analyzed at different timepoints (indicated by arrows). Gating strategy is shown in Supplementary Fig. 4A. B–D Immune cell composition of the DLN in response to IR. B Absolute cell counts of all CD45⁺ cells. C, D CD8⁺ T cells, regulatory T cells (CD4⁺ FOXP3⁺) and helper T cells (CD4⁺ FOXP3⁻) represented by cell counts (C) and as a percentage of CD45⁺ cells (D). E, F Expression of various activation and exhaustion markers on day 4 after tumor IR, expressed as the geometric mean of the fluorescence intensity (MFI), normalized to the average MFI value of the “Sham IR + ICI” group. E Helper T cells. F CD8⁺ T cells. G Percentage of CD8⁺ T cells in the DLN positive for Ki67. Each dot represents an individual mouse. Floating bars span from the minimal to the maximal value of each group. Line indicates the mean. n ≥ 8 for B–D, and n ≥ 3 mice per groups for E–G (exact numbers provided in Source Data file). Data were tested for normality using the Shapiro–Wilk test. For data following a normal distribution (all data except as specified below), treatment groups were compared using the two-sided unpaired t test or one-way ANOVA with Holm–Sidak’s multiple comparisons test. For non-normally distributed data, comparisons were performed using the two-sided Mann–Whitney test (helper T cells in C, day 0; regulatory T cells in D, day 0; G, day 0) or the Kruskal–Wallis test (CD8⁺ T cells in C, day 4; TIM-3 and CTLA-4 in E and F), with Dunn’s multiple comparisons test. 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.