(A) Supernatant HSP70 and caspase-1 p20 Western blot analysis following treatment of human WM266 melanoma cells with IFN-γ with or without anti–PD-L1 Ab. Blots are representative of 3 independent experiments. (B) Wnt5A Western blot analysis of HSP70-treated human WM266 melanoma cells with or without TLR4 inhibitor. Blots are representative of 3 independent experiments. (C) HSP70 and caspase-1 p20 Western blot analysis following treatment of human WM266 melanoma cells with ATP in the absence and presence of MCC950. Blots are representative of 2 independent experiments. (D) Cytolytic T cell markers correlated with ITGAM (CD11B), CD33, and NLRP3 gene expression in the melanoma TCGA-SKCM database. (E) RNA-Seq analysis of human melanoma tissue specimens collected before anti–PD-1 Ab therapy and at the time of disease progression on anti–PD-1 Ab therapy. TPM, transcripts per million. (F) Plasma HSP70 ELISA at week 0 and week 12 in patients with advanced melanoma undergoing anti–PD-1 Ab immunotherapy. (G) Change in HSP70 plasma levels following anti–PD-1 Ab immunotherapy in patients with advanced melanoma who were responders (R) or nonresponders (NR). The response was based on week-12 CT imaging. HSP70 changes were normalized to target tumor burden based on week-12 CT imaging. In the box-and-whisker plots, the central line represents the median, the box represents the first and third quartiles, and the error bars represent the data range. *P < 0.05 and **P < 0.005, by Student’s t test (E and G).