CD3/CD28 stimulation induces PILAR up-regulation. (A) CD3/CD28 (100 ng/mL each) stimulation of peripheral immunopurified CD8+ (left) or CD4+ (right) T cells (106/mL) resulted in a transient up-regulation of PILAR mRNA, with maximum levels between 9 and 24 hours. These data are representative of 3 independent experiments (total of 6 samples). Error bars indicate standard error. (B) Peripheral CD3+ T cells stimulated for different periods with aAPCs coated with agonistic anti-CD3/CD28 Abs (100 ng/mL) up-regulated PILAR at the protein level. This staining was performed with a PILAR-specific polyclonal Ab. These data, representative of 3 independent experiments, were confirmed using a monoclonal Ab (see also Figure S1). NTC indicates irrelevant rabbit Ab (Lab Vision, Freemont, CA) plus anti–rabbit Ig-FITC (Biomeda; top). (C) CD4 T cells contained in peripheral blood mononuclear cells stimulated for 7 days with aAPCs coated with agonistic CD3/CD28 Abs (100 ng/mL) up-regulated PILAR at the protein level in another independent experiment. (D) More than 40% of proliferating CD4 T cells from a different donor, but not cells that did not proliferate, expressed detectable levels of PILAR using the polyclonal Ab after 7 days of stimulation under identical conditions. (E) Up-regulation of CD161 on the peripheral T cells of a different donor after 3 and 7 days of CD3/CD28 stimulation using aAPCs (see also Figure S1F). Numbers in quadrants indicate percentage of gated cells in that quadrant (panels C and D, percentage of total CD4 T cells; panel E, percentage of total T cells).
