TIGIT inhibits YTS killing. (A) Flow cytometry analysis of the human NK cell leukemia cell line, YTS, transfected with ecotropic receptor only (YTS/eco, Upper) or with TIGIT attached to HA tag (Lower) stained with anti-HA mAb (left histograms). The same cells TIGIT were stained with seven different antibodies directed against TIGIT. Gray filled histograms, background staining with the secondary fluorescein-conjugated antibody only. Numbers indicate median fluorescence intensity. (B) Killing of 721.221 cells or 721.221 cells expressing PVR (721.221/PVR), by YTS/TIGIT preincubated with mAb #4 directed against TIGIT (black column) or a control anti-CD99 mAb (white column). The effector-to-target (E:T) ratio was 4:1.

The ITIM of TIGIT delivers the inhibitory signal. Killing of 721.221 cells or 721.221/PVR transfectants by the various YTS cells. The E:T ratios are indicated in the x axis.

Biochemical characterization of TIGIT. YTS/eco cells and YTS-transfectants expressing HA-tagged TIGIT were lysed with either 1% Nonidet P-40 or RIPA lysis buffer. Immunoprecipitation of TIGIT was performed by adding either anti-HA-agarose (A) or anti-TIGIT mAb (B). Membranes were immunoblotted with POD-labeled anti-HA mAbs (Roche).

TIGIT function on primary immune cells. (A) TIGIT is expressed by NK, NKT, Treg, CD8⁺ T cells and memory CD4⁺ T cells. FACS analysis of freshly isolated peripheral blood lymphocytes subsets (indicated in the histograms) stained for TIGIT expression. NK cells were characterized by CD3⁻CD56⁺, NKT cells by CD3⁺CD56⁺, T cells either by CD8⁺CD56⁻ or by CD4⁺CD56⁻, Treg cells are CD4⁺CD25⁺FOXp3⁺ and memory CD4⁺ T cells were characterized by CD45RO staining (right histogram). (B) Flow cytometry of freshly isolated or IL-2 -activated NK cells stained for TIGIT, DNAM1, and CD96 expression. Gray shaded histograms, background staining with the secondary fluorescein-conjugated antibody only. (C) DNAM1 and CD96 are costimulatory receptors. P815 cells were incubated either with no mAb or with the combinations of anti-2B4 or anti-NKp30 mAbs (indicated in the x axis) and anti-DNAM1 or with anti-CD96 mAb (indicated by the different column colors). Redirected NK cytotoxicity against P815 cells was determined at E:T ratio of 3:1. (D) P815 cells were incubated either with no mAb or with combinations of anti-2B4 or anti-NKp30 mAbs (indicated in the x axis) with and without anti-TIGIT, or with anti-CD99 (indicated by the different column colors) to redirect NK cytotoxicity. Effector IL-2 activated bulk NK cells were then added in E:T ratio of 5:1. The two redirected killing assays presented in C and D were done independently. (E) Killing of the target cells 721.221 and 721.221/PVR by primary NK cells at E:T ratio of 5:1. NK cells were preincubated with the various mAb indicated in the x axis.

PVR and PVRL2 but not PVRL3 are ligands for TIGIT. (A) Flow cytometry analysis of 721.221 parental cell line and the various transfectants stained with the indicated mAbs and with the fusion proteins TIGIT-Ig and DNAM1-Ig (black lines). Gray filled histograms are the background staining with the secondary fluorescein- (for mAbs) or phycoerythrin- (for fusion proteins) conjugated antibody only. The parental 721.221 cells were stained by all three anti-PVR, PVRL2 and PVRL3 antibodies (B) Mouse IL-2 secretion from parental BW cells or BW cells expressing the TIGIT ζ chimeric protein, incubated for 48 h with the 721.221 transfectants presented in A. Mouse IL-2 secretion was measured by ELISA. (C) Killing of the indicated 721.221 transfectant cells by YTS/TIGIT and YTS/TIGIT Y231stop cells. The E:T ratios are indicated in the x axis.

TIGIT protects normal cells expressing PVR and PVR-like proteins from NK cell-mediated killing. (A) Primary fibroblasts characterization. Flow cytometry analysis of primary human foreskin fibroblast (HFF) cells stained with the indicated mAbs and fusion proteins (black lines). Gray filled histograms are the background staining with the secondary fluorescein- (for mAbs) or phycoerythrin- (for fusion proteins) conjugated antibody only. (B) Killing of the primary human foreskin fibroblasts by primary NK cells at an E:T ratio of 20:1. NK cells were either preincubated or not with anti-TIGIT mAb #5 or with no mAb. Fibroblasts were preincubated either with no fusion proteins or with the indicated fusion proteins.