PILAR is expressed on T cells in inflammatory synovial fluid. Proportions of PILAR⁺CD3⁺CD8⁻ (CD4⁺) T cells in synovial fluid specimens from rheumatoid arthritis and gout patients using rabbit polyclonal or mouse monoclonal PILAR-specific Abs. Staining with an irrelevant mouse IgG (BD PharMingen, San Diego, CA) is additionally shown.

PILAR encodes a new lymphocyte transmembrane molecule. (A) PILAR gene spans 5 exons encoding a predicted transmembrane molecule that contains a truncated C-type lectin domain in the extracellular region. (B) PILAR alignment with EU095394 and the 2 closest human molecules: Leukocyte markers CD69 and CLEC2D. (C) TaqMan analysis of the expression of PILAR in 10 different human tissues. (D) PILAR is predominantly expressed by naive lymphocyte, but not by myeloid cells. These data are representative of DCs from 2 different donors.

PILAR is a ligand for CD161. (A) 293 cells transfected with HA-PILAR and K562 cells transduced with CD161 are identified as “+,” whereas “−” denotes the parental cell line. Cell extracts were incubated for 2 hours and immunoprecipitation (IP) was performed with either anti-HA Ab (HA11; left; Covance, Berkeley, CA) or anti-CD161 Ab (right), plus protein G/protein A agarose beads. The immunoprecipitates were run in a Western blot and analyzed for their reactivity against the opposite Ab. A positive control including either CD161⁺ cells (left) or PILAR⁺ cells (right) without IP was also included. (B) Coincubated PILAR⁺ and CD161⁺ cell extracts immunoprecipitated with an irrelevant Ab (iAb; left), or coincubated lysates from K562 cells ectopically expressing CD161 plus 293 cells expressing an irrelevant HA-tagged sequence (right) did not contain CD161. A positive control including CD161⁺ cells was included. (C) Immunoprecipitates of coincubated lysates from CLEC2D⁺ and CD161⁺ cells performed with an anti-CD161 Ab contained the expected approximately 25-kDa CLEC2D band. A positive control, including CLEC2D⁺ cells (left) without IP, was included. (D) Pilar transduced/untransduced (+/−) K562 cells were incubated with a CD161-Myc (left) or a control-Myc (GenBank no. NM_007048; right) protein (0.5 μg/mL, 45 minutes). Specific binding was confirmed 3 times with an anti–Myc-FITC Ab (Sigma-Aldrich, St Louis, MO).

PILAR, but not CD161, is expressed by a proportion of both CD4⁺ and CD8⁺ tumor-infiltrating T cells. (A) CD161 is expressed by individual cells in the ovarian carcinoma microenvironment. Data are representative of 20 immunostained specimens. Original magnification, ×100 (left); ×200 (right). Photomicrographs were taken with a Nikon Eclipse 801 (Nikon, Melville, NY) at 10× (left) or 20× (right) 0.30 plan Fluor objective. Images were acquired using Micropublisher v. 5.0 (QImaging, Surrey, BC) and processed using Image ProPlus v. 5.1.1 (Media Cybernetics, Bethesda, MD). (B) PILAR (polyclonal Ab) is expressed by tumor-associated CD4 and CD8 T cells, whereas CD161 is only found on CD4 T lymphocytes. Gated on viable cells. Numbers in quadrants indicate percentage of CD3⁺ T cells in that quadrant. (C) Expression of both PILAR and CD161 can be recovered on tumor-associated cytotoxic T cells ex vivo by CD3/CD28 (100 ng/mL each) stimulation. Dotted, tumor-derived unstimulated CD8⁺ T cells; thick, the same cells after stimulation with CD3/CD28 for 5 days. Gated on viable cells.

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).

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.

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.