Inhibition of exposed PS with unconjugated annV modulates clustering of T cells. Melan-A–specific CTLs were stimulated in Ca2+-containing medium with Melan-A–loaded T2 stimulator cells in the presence or absence of 50 μg/mL unconjugated blocking annV during the assay. (A) After 5 hours of coculture, supernatants were harvested and the amount of indicated cytokines was determined by a cytokine bead array. Shown are mean values of annV-blocked samples normalized to unblocked control ± SEM; n = 5; **P < .001. (B) Cells were harvested at indicated time points, stained with Melan-A tetramer (TM), CD8-APC, annV-FITC and analyzed by FACS. Shown are mean values of blocked and unblocked CTL normalized to unstimulated CTL ± SEM; n = 4; *P < .08, **P < .01. (C) After a coculture of 24 hours, images were taken from representative unblocked (middle images) or annV-blocked (lower images) cell pellets in 96-well round bottom plates. Unstimulated CTLs (upper images) served as a control. One representative experiment of 5 is shown. Images were taken on an Axiovert25 microscope at a × 20 (insert) and × 50 magnification.
