Fig. 1: Multi-omic analyses across treatment timepoints reveals immediate and profound TIL depletion after radiotherapy in HNSCC.

a Schematic of trial design including timepoints and translational correlative analyses. b Combined integrated single-cell datasets including 11 separate HyPR-HN biopsies from four patients over three timepoints. Analysis across timepoints reveals immediate T cell depletion on the last day of radiation both (c) quantitatively as a percent of the single-cell transcriptome and (d) visually across UMAP profiles. e mFC confirms immediate TIL depletion in response to radiation, despite (f) preserved overall immune infiltrate and increased intra-tumoral myeloid cells. g Representative mFC plots of CD8 and CD4 T cell infiltrate and (h) mIF with corresponding clinical MR (pre-treatment and last day of radiation) and CT (6 weeks post-radiation) imaging are shown for HyPR-HN Patient 01 serially across treatment timepoints. Antibody fluorescent conjugates include CD8: light blue, CD4: green, CD20: red, Foxp3: yellow, CD68: orange, Pan-CK: magenta, DAPI: dark blue. Scale bar = 50 µM. T-tests with Holm-Sidak’s multiple comparison correction were used to analyze differences between groups. Flow cytometry gating strategy demonstrated in Supplementary Fig. 1. Graphic created in BioRender. Zenga, J. (2025) https://BioRender.com/zu8pe9n.