Fig. 3: Multiplex IHC analysis reveals changes in lymphocyte infiltrates in tumor and stroma after anti-OX40.

Multiplex IHC was performed on FFPE specimens from N = 15 patients to determine the composition and changes of the immune infiltrate in tumor and stroma. For each patient, on each slide, six regions of interest (ROI) were analyzed for tumor and stroma. a Fold change of CD8+ CD103+ CD39+ Ki-67+ T cells for all patients as determined by flow cytometric analysis. Filled orange circles indicate patients with an activation index above 1. Filled black circles indicate patients with an activation index below 1. IR immune responder, INR immune non-responders. b Total number of CD3+ and Foxp3+ T cells among tumor and stroma in N = 15 patients pre- and post anti-OX40. Error bars indicate mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant. P values were determined by paired two-tailed Student’s t test between pre and post samples and between tumor and stroma. c, d Representative multiplex IHC of two HPV+ (c) and two HPV− (d) head and neck squamous cell carcinoma (HNSCC) patients pre and post anti-OX40, identified by the response (IR and INR) as determined in (a). e Magnified view (bottom) of cells expressing CD8, CD103, and Ki-67 in the tumor area (top). f Summary of the numbers of Ki-67+ CD103+ CD8+ cells in the tumor and stroma pre- and post anti-OX40, in patients stratified by the immunologic response. N = 4 IR, N = 10 INR. Error bars indicate mean ± SEM. The values presented in (b) and (f) represent the mean of the ROI analysis for each patient. g Comparison of the numbers of Ki-67+ CD103+ CD8+ cells enumerated by multiplex IHC in the tumor and stroma, with the percentages of the same subset determined by flow cytometry. Black dots represent the immune non-responders. Red dots represent the immune responders. Source data are provided as Source Data file.