Functional Consequences of Interactions between Human NKR-P1A and Its Ligand LLT1 Expressed on Activated Dendritic Cells and B Cells1

Lectin-like transcript-1 (LLT1) (also named osteoclast inhibitory lectin or CLEC2D) is a ligand for the human NKR-P1A (CD161) receptor, present on NK cells and T cells. To further understand the physiological relevance of this interaction, we developed mAbs against LLT1, characterized the expression pattern of LLT1, and explored the functional consequence of LLT1 engagement of the NKR-P1A receptor on NK cells and T cells. LLT1 is expressed on TLR-activated plasmacytoid dendritic, TLR-activated mono-cyte-derived dendritic cells, and on B cells stimulated through TLR9, surface Ig, or CD40. Interactions between NKR-P1A on NK cells and LLT1 on target cells inhibit NK cell-mediated cytotoxicity and cytokine production and can inhibit TNF- (cid:1) production by TCR-activated NKR-P1A (cid:2) CD8 (cid:2) T cells. In contrast, NKR-P1A failed to inhibit or augment the TCR-dependent activation of NKR-P1A-bearing CD4 (cid:2) T cells. Expression of LLT1 on activated dendritic cells and B cells suggests that it might regulate the cross-talk between NK cells and APCs. The Journal of Immunology, 2008, 180: 6508–6517. were subsequently screened by using a cell-based ELISA for recognition of LLT1-transfected P815 cells, but not an irrelevant P815 transfectant. Hybridomas showing the desired reactivity were subcloned by limiting dilution and further screened on the LLT1- positive B cell lines 721.221 and Raji. Ab was purified from hybridoma supernatants by using Protein G affinity chromatography. We tested whether the anti-LLT1 mAbs (clones 4C7, 40262, or 402659) were capable of blocking the interaction with NKR-P1A by using our previously de- scribed NKR-P1A reporter cells (10) and found these mAbs lacked blocking activity (data not shown). Anti-LLT1 antisera was made by immunizing rabbits with a fusion protein of GST and the intracellular domain of LLT1 (aa 1–33). Abs were affinity purified on a fusion protein of Mannose-binding protein and the intracellular domain of LLT1, covalently attached to CNBr-activated sepharose (Sigma-Aldrich). Cells were on ice for 30 min with mAb, and subse- quently stained with secondary Abs. Cells were paraformal-dehyde data FlowJo (TreeStar).