Fig. 2: Elevated HSP70 expression in T cells from BMs is associated with poor prognosis in patients undergoing immunotherapy.

A Volcano plots showing differentially expressed genes between primary and brain metastatic NSCLC in CD4⁺ and CD8⁺ T cells. B Boxplots comparing the mean HSP70 score between primary and brain metastatic samples. P-values from two-sided Wilcoxon Rank Sum tests are shown. CD4⁺ T cells: Primary n = 8 and Brain n = 10; CD8⁺ T cells: Primary n = 8 and Brain n = 11. C Representative multiplex immunofluorescent images of NSCLC sections stained for CD4 (green), CD8 (red), and HSP70 (yellow); PTs and BMs are compared. Scale bars, 50 μm (main panels) and 10 μm (insets). Images are representative of n = 34 PT and n = 26 BM patients. D, E Box plots quantifying HSP70⁺ CD4⁺ T (D) and HSP70⁺ CD8⁺ T E cells from multiplex immunofluorescence images. (n = 34 Primary and n = 26 Brain samples). P-values from two-sided Wilcoxon Rank Sum tests are shown. F A dotplot illustrating average score of curated gene signatures in HSP70-High, HSP70-Medium, and HSP70-Low CD4⁺ (left) and CD8⁺ (right) T cells. G, H Enriched Gene Ontology (GO) terms in HSP70-High CD8⁺ T (G) and HSP70-High CD4⁺ T (H) cells. I, J Kaplan–Meier survival curves for patients with NSCLC in the Ravi (n = 122 patients) and Kim cohorts (n = 27 patients), stratified by HSP70-High CD8⁺ T cell (I) and HSP70-High CD4⁺ T cell (J) signature scores. Optimal cutoffs were determined using “surv_cutpoint” within each cohort. Hazard ratios (HRs) with 95% confidence intervals (CIs) and P-values from two-sided log-rank tests are shown. Survival curves represent Kaplan–Meier estimates; +, censored observations. In box plots (B, D, E), center line represents the median, box bounds indicate the 25th and 75th percentiles (IQR), whiskers extend to 1.5× IQR from box bounds, and outliers are shown as individual points.