Increasingly, roles are emerging for C-type lectin receptors in immune regulation. One receptor whose function has remained largely enigmatic is human NKR-P1A (CD161), present on NK cells and subsets of T cells. In this study, we demonstrate that the lectin-like transcript-1 (LLT1) is a physiologic ligand for NKR-P1A. LLT1-containing liposomes bind to NKR-P1A+ cells, and binding is inhibited by anti-NKR-P1A mAb. Additionally, LLT1 activates NFAT-GFP reporter cells expressing a CD3ζ-NKR-P1A chimeric receptor; reciprocally, reporter cells with a CD3ζ-LLT1 chimeric receptor are stimulated by NKR-P1A. Moreover, LLT1 on target cells can inhibit NK cytotoxicity via interactions with NKR-P1A.
Rodents have several Klrb1 (also named Nkrp1) genes encoding either activating or inhibitory NK receptors of the C-type lectin superfamily, including Nkr-p1c, the NK1.1 Ag defining mouse NK cells (1). By contrast, only a single, nonpolymorphic gene in the Nkrp1 family, KLRB1, exists in humans (2). KLRB1 encodes a type II disulfide-linked homodimer, named CD161 or NKR-P1A, which is expressed on most NK cells. Human NKR-P1A is on a subset of peripheral T cells, including CD4+ and CD8+ T cells (mostly effector/memory phenotype), invariant NKT cells, and γδ-TCR+ T cells, and on a subset of CD3+ thymocytes (2).
NKR-P1A is expressed on immature human NK cells, before acquisition of CD16 or CD56 (3), and its expression is up-regulated on mature NK cells by IL-12 (4, 5). NKR-P1A on CD4+ T cells and γδ-TCR+ T cells has been implicated in transendothelial migration (6, 7). Additionally, anti-NKR-P1A mAbs costimulate the anti-CD3 mAb-induced proliferation of CD1d-specific NK T cells (8) and induce proliferation of immature thymocytes (9). Cross-linking with an anti-NKR-P1A mAb inhibits human NK cell-mediated cytotoxicity against FcR+ target cells (4, 5).
Recently, ligands for two mouse Klrb1 family members have been identified (10, 11). The activating Nkr-p1f receptor recognizes Clr-g (encoded by Clec2i) (10), and the inhibitory Nkr-p1d recognizes Clr-b (encoded by Clec2d) (11). Another name for Clr-b is osteoclast inhibitory lectin, because it was also identified as an osteoblast-derived glycoprotein, which inhibits in vitro osteoclastogenesis (12, 13). Although mice have multiple Clr family genes, only one ortholog, CLEC2D (also named lectin-like transcript-1 (LLT1)4), exists in humans. Like mouse Clr-b, human LLT1 blocks osteoclast differentiation (12, 13). Mouse and human Clec2 gene products are type II proteins of the C-type lectin superfamily.
The ability of mouse Nkrp1 family receptors to recognize Clr ligands prompted the question of whether this interaction is conserved in humans. In this study, we have examined whether LLT1 serves as a ligand of human NKR-P1A and have described the functional consequences of this novel receptor-ligand interaction.
Materials and Methods
A cDNA encoding the extracellular domain of LLT1 with a N-terminal 6-His tag was cloned into a pFASTBac vector, modified to encode a signal sequence for ecdysteroid UDP glucosyltransferase, and expressed in the Bac-to-Bac system (Invitrogen). Recombinant protein was purified by Ni-NTA affinity chromatography. The preparation of LLT1-fluorescent liposome complexes and the procedure for binding to cells was described previously (14, 15, 16). To ensure binding was specific, cells were incubated with an anti-CD161 mAb or an isotype-matched control Ig (10 μg/ml) before incubating with the liposome-rLLT1 complexes.
Reporter cell assays
An NFAT-GFP reporter construct was stably transduced into 2B4 T cell hybridoma cells (provided by H. Arase, Osaka University, Osaka, Japan). NFAT-GFP reporter cells were transduced with CD3ζ-LLT1 or CD3ζ-NKR-P1A chimeric receptors and analyzed as described (17, 18). Briefly, reporter and stimulator cells were cocultured at a 1:4 ratio for 16 h and then analyzed for GFP expression by flow cytometry.
Constructs and transductions
cDNAs for human NKp80 (provided by J. P. Houchins, R&D Systems, Minneapolis, MN), human CD69, LLT1 with a C-terminal Flag-epitope, human NKR-P1A, and the reporter constructs were subcloned into pMXs-puro vectors (provided by T. Kitamura, University of Tokyo, Tokyo, Japan) (19). Chimeric receptors were generated by fusing the extracellular and transmembrane domains of either LLT1 or NKR-P1A to the intracellular domain of human CD3ζ. Plasmid constructs were transfected into Phoenix packaging cells (20) to produce retroviruses (18, 21). Infected cells were selected in medium containing 1 μg/ml puromycin.
Mouse anti-human NKR-P1A mAbs DX1 and DX12 were produced in our laboratory, and HP3G10 was purchased from MBL International.
Primary cell cultures and cytotoxicity assays
Venous blood was obtained from healthy volunteers, under procedures approved by the Human Ethics Committees of the Australian Capital Territory Department of Health and Community Care and the Australian National University (Canberra, Australia) and the University of California, San Francisco, Committee on Human Research. Polyclonal NK cells were generated and tested in cytotoxicity assays as described previously (22, 23, 24).
Results and Discussion
LLT1 liposomes bind human NKR-P1A
We generated fluorescent-labeled liposomes containing human LLT1 and evaluated their staining of the mouse Ba/F3 pro-B cell line and BaF/3 cells stably transduced with human NKR-P1A (BaF/3-NKR-P1A). LLT1-liposome complexes bound specifically to BaF/3-NKR-P1A, but not parental BaF/3 (Fig. 1,A). This interaction was specifically blocked by anti-NKR-P1A mAb (dashed line), but not control Ig (solid). LLT1-liposomes also bound to the human NK cell line YT transfected with NKR-P1A (YT-NKR-P1A), but not to untransfected YT cells (Fig. 1,B), and the interaction was prevented by all anti-NKR-P1A mAbs tested (i.e., DX1, DX12, HP3G10), but not by control Ig (Fig. 1,B). Furthermore, LLT1-liposomes recognized endogenous NKR-P1A expressed by in vitro-activated human peripheral blood NK cells, and the binding was inhibited by anti-NKR-P1A mAbs, but not by control Ig (Fig. 1 C).
LLT1 reporter cells recognize NKR-P1A
We created mouse 2B4 NFAT-GFP reporter cells (18) expressing a chimeric protein consisting of the extracellular and transmembrane domains of LLT1 and the intracellular domain of CD3ζ. Receptor engagement of the CD3ζ-LLT1 chimera resulted in signaling from the CD3ζ ITAMs, activation of NFAT, and the expression of GFP. Coculture of the LLT1 reporter cells with BaF/3-NKR-P1A cells resulted in robust NFAT-GFP activation, whereas coculture with parent BaF/3 or BaF/3-NKp80 cells did not (Fig. 2,A). Moreover, the activation of CD3ζ-LLT1 reporter cells by BaF/3-NKR-P1A was specifically blocked by anti-NKR-P1A mAb, but not by control Ig (Fig. 2 B).
Similarly, we created reporter cells expressing a CD3ζ-NKR-P1A chimera and tested the ability of these cells to recognize LLT1-bearing cells. NKR-P1A reporter cells expressed GFP when cocultured with LLT1-transduced BaF/3 (BaF/3-LLT1), but not with untransfected BaF/3 or BaF/3 expressing human CD69 (BaF/3-CD69) (Fig. 2,C). LLT1-induced activation of the NKR-P1A reporter cells was inhibited by anti-NKR-P1A mAbs, but not control Ig (Fig. 2 D).
LLT1 inhibits NK cytotoxicity via NKR-P1A
Prior studies have shown that anti-NKR-P1A mAb inhibits NK cell-mediated lysis of FcR-bearing cells (2). Now with a defined ligand for NKR-P1A, we examined the function of NKR-P1A in NK cells using target cells expressing its physiological ligand. EBV-transformed 721.221 B lymphoblastoid cells express LLT1 mRNA, as detected by PCR (not shown). In accordance, we found that 721.221 activated CD3ζ-NKR-P1Α reporter cells, an effect that was blocked by anti-NKR-P1A, but not control Ig (Fig. 3 A).
We tested the cytolytic ability of YT and YT-NKR-P1A against 721.221 targets. YT-NKR-P1A cells had diminished cytotoxic capacity against 721.221 compared with untransfected YT (Fig. 3,B); equivalent killing by YT and YT-NKR-P1A was seen against CD48-transduced BaF/3 targets, indicating that both YT and YT-NKR-P1A have similar lytic potential (not shown). Furthermore, the NKR-P1A-mediated inhibition of 721.221 lysis by YT-NKR-P1A cells was reversed by anti-NKR-P1A F(ab′)2, but not control F(ab′)2 (Fig. 3 C).
NK cells can be stimulated through several activating receptors that use biochemically distinct signaling pathways, for example, human NKG2D, which recognizes MHC class I chain-related A (MICA), and human CD244, which recognizes CD48 (25). We examined the effects of NKR-P1A on these activation pathways by transducing LLT1 into BaF/3 target cells expressing MICA or CD48 and determining the effect of NKR-P1A engagement on NK cell-mediated cytotoxicity. LLT1 expression by these targets diminished NK cell-mediated cytotoxicity induced by either the CD48/CD244 pathway (Fig. 3,D, left) or the MICA/NKG2D pathway (Fig. 3,D, right), an effect which was mediated through interactions with NKR-P1A and was reversed by anti-NKR-P1A F(ab′)2, but not control F(ab′)2 (Fig. 3 D).
These data prove that LLT1 is the ligand for human NKR-P1A, and this interaction inhibits NK cytotoxicity. In mice, the LLT1 ortholog Clr-b, like MHC class I, is widely expressed (11). Recently, potential orthologs for NKR-P1A and LLT1, B-NK and B-lec, respectively, have been described in the chicken MHC region (26). This raises the questions of whether B-NK and B-lec interact, and whether NKR-P1A-LLT1-like interactions have been preserved through evolution. LLT1 is transcribed in T cells, B cells, and NK cells (27) and in osteoblast cell lines (13).
Collectively, our findings provide another example of a functional interaction between two genomically linked C-type lectin proteins encoded by genes in the “NK complex.” This raises the question of whether other C-type lectins within this genomic region are interacting, and what the functional consequences may be. Additionally, the existence of the LLT1-NKR-P1A interaction in humans provides yet-another mechanism for the fine-tuning of NK cell and T cell responses using an inhibitory receptor that recognizes a non-MHC ligand.
We thank Joseph Altin for providing liposomes, Mark Hulett for advice on the Bac-to-Bac system, and Margaret Hilton, Taian Chen, and Allen Sun for technical assistance.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by National Institutes of Health Grant AI068129 and National Health and Medical Research Council Project Grant 151304. H.S.W. is a Senior Research Fellow of the National Health and Medical Research Council of Australia, and a Visiting Fellow of the Australian National University Medical School and The Canberra Hospital. L.L.L. is an American Cancer Society Research Professor. D.B.R. is supported by a Genentech Graduate Student Fellowship.
Abbreviations used in this paper: LLT1, lectin-like transcript-1; MICA, MHC class I chain-related A.