Fig. 5

CTLA-4 is expressed by both human NK cell lines and healthy human donor-derived NK cells. A UMAP-dimension reduction with cells colored by single-cell gene expression for CTLA-4 and representative immune activation genes in mouse (left) and human (right) intratumoral NK cells. The pattern of CTLA-4 expression is consistent with the reduced ability of scRNA-seq to capture low to moderately expressed genes. B Western blot demonstrating CTLA-4 expression in human NK cell lines. Representative of two independent experiments. C Quantitative real-time PCR (qRT-PCR) analysis of total CTLA-4 expression (both isoforms) in a CTLA-4 null line (PANC-1), T cell lines (Jurkat, CEM, HuT78), and NK cell lines (NK92, NKL, YT, KHYG-1). p-value < 0.001 = **** as determined by unpaired, two-tailed t-test. D qRT-PCR demonstrating CTLA-4 expression in CD56+ selected ex vivo unstimulated NK cells derived from healthy human donors. E Western blot of CTLA-4 expression in CD56+ selected ex vivo unstimulated NK cells derived from healthy human donors. F Western blot of total protein (T) and intracellular (IC) protein isolated from human NK cell line NK-92 and unstimulated primary human NK cells using cell surface protein biotinylation for exclusion of surface proteins demonstrating surface expression of CTLA-4 dimers and intracellular expression of CTLA-4 monomers. G Flow cytometry demonstrating NK-92 does not express antibody receptor CD16. Positive control was the NK-92 line that had been transfected with a CD16 expressing plasmid, NK-92-CD16v. H Immunofluorescent images of NK-92 cells stained with Dylight550-labeled ipilimumab demonstrating that ipilimumab binds to the NK cell surface. Blue staining indicates DAPI. Shown are representative images of a single field of view taken via confocal microscopy (magnification, 63×; zoom, 3×)