Fig. 2: Multi-omic analyses across treatment timepoints reveals immediate and persistent loss of ICRhigh TILs after radiotherapy in HNSCC.
a Integrated single-cell datasets of 11 separate HyPR-HN biopsies with T cells extracted and re-clustered from four patients over three timepoints with UMAP profiling demonstrating global loss of TILs post-radiation and revealing persistent loss of exhausted CD8 T cells. b Analysis across timepoints confirms immediate T cell depletion on the last day of radiation quantitatively as a percent of the T cell transcriptome. c Characterization of T cells co-expressing multiple ICRs (PDCD1, HAVCR2, TIGIT, and LAG3) demonstrates immediate and persistent loss of ICR expression in response to radiotherapy. d mFC confirms immediate loss of ICRhigh TILs as a percentage of viable cells in response to radiation, despite (e) preserved expression as a percentage of CD8 T cells. Representative mFC plots of CD8 T cell infiltration and ICR expression are shown for HyPR-HN Patient 02 (f) pre-treatment and (g) on the last day of radiation. Gating is provided as a percentage of total live cells. Pre-ex Pre-exhausted T cells, Reg Regulatory T cells, TRM Tissue-resident memory T cells, Prolif Proliferative T cells, ExCD8 Exhausted CD8 T cells, ExCD4 Exhausted CD4 T cells, IFN Type I interferon-responsive T cells. T-tests with Holm-Sidak’s multiple comparison correction were used to analyze differences between groups. Flow cytometry gating strategy demonstrated in Supplementary Fig. 4.
