Fig. 4: T-DXd cytotoxicity induces immunogenic tumor cell death, activating nearby myeloid immune cells for antigen presentation.

a, b Au565 cells were treated with DXd, DM1, or HER2-ADCs for 3 days, and extracellular ATP was measured. c, d Au565 and KPL4 cells were treated for 3 days, and extracellular HMGB1 release was quantified by Lumit-Immunoassays normalized to cell viability. e Surface Calreticulin (CRT) on dying tumor cells was measured by flow cytometry on Au565 cells after treatment for 2 days. a–e Two-way ANOVA with Tukey’s multiple comparisons (n = 3). f–j Flow cytometry assessment of human macrophage surface expression of (f) HLA-A2, (g) HLA-DR, (h) CD80, (i) CD40, and (j) CCR7 after 2-days co-culturing with Au565 cells pre-treated with indicated antibodies or ADCs. Macrophage experiments derived from two representative PBMC donors are shown. MFI Mean Fluorescence Intensity. One-way ANOVA with Tukey’s multiple comparisons test (n = 3 per donor). k, l RNA-seq analysis of gene expression by human macrophages after one day co-culture with Au565 cells pre-treated with indicated antibodies/ADCs. Relative gene expression involved in (k) antigen presentation and (l) chemotaxis were assessed. Heat maps represent average gene counts (n = 3 per group) normalized with z-scores. All data is presented as mean ± SEM with p values.