PILAR engagement of CD161 enhances T-cell proliferation. (A) CD3 (100 ng/mL)–mediated proliferation of naive T cells is dramatically enhanced at day 7 in the presence of ectopically expressed PILAR (dotted line), compared with mocked transduced aAPCs (thick line). (B) Ectopic expression of PILAR increases antiapoptotic Bcl-xL on T cells after 24 hours of stimulation with aAPCs coated with 100 ng/mL anti-CD3 Ab. (C) Comparison of the cytokine profiles of T cells activated for 7 days with 100 ng/mL anti-CD3 Ab, in the presence or the absence of ectopic PILAR. Levels are normalized by final cell numbers. Error bars indicate SE. (D top) Increasing PILAR availability by ectopic expression on the aAPCs in the presence of CD28 costimulation (100 ng/mL) resulted in a decrease of the CD3 (50 ng/mL)–mediated proliferation of naive T cells at day 7. Dotted line denotes cells incubated in the presence of ectopic PILAR, compared with mock transduced aAPCs (thick line). (D bottom) Final T-cell counts after 7 days of stimulation under the conditions detailed in panel A or D. Error bars indicate SE. (E) T cells expanded at a comparable rate in the presence of CD3 plus CD28 costimulation (50 and 100 ng/mL of each Ab) and in the presence of CD3 (50 ng/mL of Ab) plus ectopic PILAR expressed on the aAPCs. These results are representative of 2 independent experiments (total of 6 observations). (F) All data are representative of at least 3 experiments. (Top) Blockade of CD161 with 10 μg/mL Ab (B199.2) but not incubation with an identical concentration of an irrelevant IgG induces apoptosis on T cells activated with 100 ng/mL anti-CD3 Ab for 24 hours only in the presence of ectopic PILAR (1, 2, and 3). (Bottom) CD161-blocked T cells stimulated for 3 days also initiated apoptosis.