Fig. 9: Radiorecurrent HNSCCs after conventionally fractionated radiation are relatively CD8 TIL depleted, including ICRhigh TILs.
a 67 samples from 61 HNSCC patients included in a prospective tumor registry underwent multiplex immunofluorescence (mIF) demonstrating lower CD8 and CD4 T cell populations in radiation-recurrent compared to previously untreated disease. Six patients are represented twice due to analysis of matched tumor samples obtained both pre-treatment and at the time of radiation recurrence from the same patients in the same anatomic location. Therefore, each sample represents an independent biological replicate. b The finding of decreased T cells in radio-recurrent samples was consistent across both stromal and cancer compartments. c Analysis of patient-matched biopsies obtained pre-treatment and at the time of in-field radiation recurrence confirms relative CD8 TIL depletion of radiation recurrent tumors compared to matched pre-treatment levels, d an effect not seen other immune cell subsets. Representative PET/CT, mIF, and multiplex flow cytometry (mFC) images are shown for (e) a patient-matched previously untreated and (f) radiorecurrent HNSCC. g mFC with a T cell exhastion panel was also performed from adjacent tissue fragements in 32 of the samples, which also underwent mIF. For these patients, exhausted T cell populations were particularly diminished in radiorecurrent disease as measued by mFC. Gate frequencies are provided as percentage of live cells. 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. 4.
