Phenotypes and cytokines of NK cells in triple-negative breast cancer resistant to checkpoint blockade immunotherapy.

Neoadjuvant checkpoint blockade immunotherapy (NATI) significantly prolonged outcomes for triple-negative breast cancer (TNBC). Residual tumor cells that survive NATI represent high-risk cell populations with metastatic potential and usually evade immunosurveillance by NK cells. Using an 82-protein panel, we here profiled single-cell membrane proteomics of CD56+ (NCAM1+) NK cells from tumor, peri-cancerous tissue, as well as peripheral blood from 28 TNBC patients post-NATI of residual cancer burden II/III. Unsupervised clustering resulted in several distinct clusters: 2 tumor-infiltrating NK (TINK) clusters with divergent functions of immune activation (TNFRSF7+) and suppression (SELL+); 2 immuno-suppressive peri-cancerous clusters; and 1 periphery-specific cluster. Considering the contradiction of the 2 TINK clusters, we further tested cytokine functions of SELL + and TNFRSF7 + TINKs by single-cell secreting proteomics using a 32-cytokine panel. Consistently, SELL + TINK clusters were characterized by immuno-suppressive secretion patterns (IL10+). A low proportion of SELL + TINK cluster and low proportion of IL10 + secreting SELL + TINK cluster (single-cell secreting proteomics) were both associated with better progression-free survival time. These findings were validated in an independent cohort of 15 patients during 16-month follow-up. Overall, we identified a distinct immuno-suppressive TINK cell group, featuring IL10 + secreting and SELL expression with a strong relation to poor survival prognosis in TNBC patients post-NATI.