PILAR blockade impairs T-cell proliferation and IFN-γ production. (A) Miltenyi bead–purified, CFSE-labeled peripheral T cells were stimulated for 5 days with aAPCs coated with the following agonistic Abs: (left) αCD3 (100 ng/mL), (center) αCD3 plus αCD28 (100 ng/mL, each), (right) αCD3 plus αCD28 (0.5 and 100 ng/mL, respectively), in the presence of 20 μg/mL of either blocking anti-PILAR Abs (solid or dotted line) or an irrelevant rabbit Ab (open thick line). These results are representative of 4 independent experiments. (B) These results are representative of at least 2 independent experiments performed in quadruplicate. (left) IFN-γ ELISPOT analysis of PBMCs from an A2⁺ donor, stimulated for 7 days with A2⁺CD80⁺ aAPCs (10:1 ratio) pulsed with the CEF peptide pool (2 μg/mL of each peptide; Mabtech, Nacka Strand, Sweden), in the presence of20 μg/mL of anti-PILAR or an irrelevant Ab. (Center) CFSE-labeled PBMCs (2 × 10⁶/mL) were incubated for 7 days with the CEF peptide pool in the presence of 20 μg/mL of anti-PILAR Abs (solid) or an irrelevant Ab (open). (Right) PBMCs stimulated for 7 days with autologous monocyte-derived dendritic cells (10:1 ratio, 10⁶ total cells/mL), pulsed with pool of 138 Cytomegalovirus peptides (2 μg/mL; pp65 sequence, strain AD169; BD Bioscences), in the presence of 20 μg/mL of anti-PILAR (opened) or an irrelevant Ab (solid). Error bars indicate SE; *P = .02. (C) CD3/CD28 stimulation of peripheral T cells for 2 days in the presence of 20 μg/mL of anti-PILAR Abs (opened) resulted in a decrease in the expression of CD28 and CD62L, compared with the same lymphocytes stimulated with an irrelevant Ab (solid). CD28 and CD62L signal detected after staining with isotype control Ig is indicated. The experiment was repeated 3 times with similar results.