Gray filled histograms are the background staining with the secondary fluorescein- (for mAbs) or phycoerythrin- (for fusion proteins) conjugated antibody only. of the inhibitory receptors is critical for their inhibitory activity (20, 21). To test whether the ITIM of TIGIT is responsible for its inhibitory activity, we generated a truncated form of the TIGIT receptor at position 231 in the ITIM motif (named Y231stop) and also a point mutation in the 231 tyrosine reside mutating it into alanine (named Y231A). These TIGIT proteins were expressed in YTS cells, and in addition, we generated control-transfected YTS cells expressing GFP. Expression levels of all TIGIT proteins were similar. The various YTS transfectants were next assayed for killing of RSK4 721.221 and 721.221/PVR cells. Importantly, although a strong PVR-mediated inhibition was observed with YTS/TIGIT, no major difference in the killing of 721.221 and 721.221/PVR was noticed when the control YTS/GFP, YTS/TIGIT Y231stop and YTS/TIGIT Y231A were used (Fig. 2). Thus TIGIT is a direct inhibitory receptor for NK cells and its inhibitory activity is depending on its ITIM. Open in a separate window Fig. 2. 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 axis. Biochemical Characterization of TIGIT. Next, we used our YTS transfectants for biochemical analysis of TIGIT and immunoprecipitated TIGIT from YTS/TIGIT-HA cells by using anti-HA-agarose beads, followed by immunobloting with anti-HA antibodies. Two protein bands in sizes of 30 and 34 kDa, probably representing different glycosylation forms of TIGIT, were noticed in the YTS/TIGIT and in the YTS/TIGIT Y231A cells, whereas, as expected, lower-weight protein bands Sitaxsentan were observed in the YTS/TIGIT Y231stop cells (Fig. 3axis) 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. (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 and were done independently. (axis. Next, we studied the TIGIT activity as part of the complex killing machinery of NK cells when encountering tumor cells expressing PVR. IL-2-activated bulk NK cell cultures were incubated with 721.221 and 721.221/PVR, and, as demonstrated in Fig. 4E, the killing of 721.221/PVR was only slightly induced, indicating that the PVR-CD96/DNAM-1 interactions are too weak in the context of 721.221/PVR cells to strongly up-regulate NK cytotoxicity. Importantly, blocking TIGIT-PVR interaction by mAb #4 and #5, but not with #1 (which did not bind the NK cells, Fig. S4), resulted in Sitaxsentan a significantly increased killing of the PVR expressing 721.221 cells, indicating that TIGIT inhibition is indeed dominant over the coactivation of CD96 and DNAM-1. PVR and PVRL2 but Not PVRL3 Are Ligands for TIGIT. We next assayed whether other PVR-like proteins, PVRL2 (CD112) and PVRL3 (CD113), would be recognized by TIGIT and whether such recognition will lead to inhibition of NK Sitaxsentan cell killing. PVRL2 or PVRL3 proteins were expressed in 721.221 cells (Fig. 5and in agreement with Yu et al. observations (18), TIGIT-Ig bound PVRL2 but with much lower affinity as compared to PVR. Surprisingly, in contrast to the results of Yu et al., TIGIT-Ig did not interact with PVRL3, despite the fact that several PVRL3 transfectants were used (Fig. 5axis. Finally, we tested the practical relevance of the PVR-like.