The mouse NKR-P1B:Clr-b recognition system is a negative regulator of innate immune responses

The importance of natural killer (NK) cells in host defense against microbial infections and tumors has been highlighted in individuals lacking NK cells or NK cell functions; these individuals suffer from persistent and life-threatening infections of normally benign herpes viruses and tumors.^1-3  NK cell function is regulated by integrating activating and inhibitory signals from engaged NK cell receptors.⁴  The NK cell receptor repertoire in mice includes the Ly49, NKG2D, CD94/NKG2, and NKR-P1 families of receptors, all of which are encoded by genes in the NK gene complex (NKC) on chromosome 6.^5,6  The well-characterized Ly49 receptor family is the mouse functional equivalent of the human killer cell immunoglobulin-like receptor (KIR) family, which recognizes class I major histocompatibility complex (MHC-I) molecules.^5,6  This NK cell recognition system, termed “missing-self,” involves surveillance of host MHC-I molecules and the response to cells without MHC-I expression.⁷ 

NKR-P1 receptors are homodimeric type II transmembrane C-type lectinlike molecules^4,6,8  and are conserved across many species.⁹  This receptor family consists of 5 members in mice (NKR-P1A, NKR-P1B/D, NKR-P1C, NKR-P1F, and NKR-P1G; Nkrp1e is a pseudogene).^10-12  NKR-P1A and NKR-P1F are proposed to be activating and are expressed at low levels on all NK cells.¹³  The activating NK1.1 (NKR-P1C) receptor, a prototypical antigen defining mouse NK cells in the C57BL/6 (B6) mouse strain, is a product of the Nkrp1c^B6 gene.¹⁴  NKR-P1G has only recently been documented to be inhibitory and primarily involved in mucosal immunity,¹⁵  whereas NKR-P1B is a known inhibitory receptor first identified in the Swiss and SJL mouse strains.^10,11,16-18  At least 3 different Nkrp1b alleles have been described. The B6 allele has been variably termed Nkrp1d or Nkrp1b^B6, and encodes the NKR-P1B^B6 receptor, also known as NKR-P1D.¹¹  NKR-P1B is expressed only on a subset of NK cells.¹³  NKR-P1B⁺ and NKR-P1B⁻ NK cells differ in their expression of other NK cell receptors in both mice and rats.^13,19,20  Ligands for most NKR-P1 receptors have been identified as members of the C-type lectin-related (Clr) family of membrane glycoproteins encoded by the Clec2 genes, which are intermingled among the Nkrp1 (Klrb1) genes within the mouse NKC.^12,17,18  The currently known receptor-ligand pairs for these families include NKR-P1B:Clr-b; NKR-P1F:Clr-c,d,g; and NKR-P1G:Clr-d,f,g.^17,18,21,22 

We have generated an NKR-P1B-deficient B6 mouse strain to study the role of NKR-P1B in NK cell development and function in vivo. We chose to target NKR-P1B for the following reasons: (1) the NKR-P1B:Clr-b system is well characterized in mice, and appropriate reagents, such as specific monoclonal antibodies (mAbs) and a complementary Clr-b gene–deficient mouse strain, are available^13,23 ; (2) the NKR-P1B:Clr-b system is analogous to the inhibitory NKR-P1A:LLT1 system in humans, although their expression patterns may vary^24,25 ; (3) the existence of 3 significantly different Nkrp1b alleles suggests a possible divergence as a result of pathogen challenge (eg, rat cytomegalovirus encodes a C-type lectinlike protein with homology to rat Clr-11 [Clec2d11] that protects infected cells from NK recognition via the inhibitory rat NKR-P1B receptor)²⁶ ; and (4) in contrast to other, tissue-specific Clr family members, Clr-b, like MHC-I, is broadly expressed on hematopoietic cells, and its expression on transfected cells protects them from NK-mediated lysis.^12,17,18,27  In addition, Clr-b expression is often downmodulated on tumor cell lines after virus infection and during genotoxic and cellular stress in vitro.^17,26,28  Therefore, NKR-P1B:Clr-b interactions represent an MHC-I-independent missing-self recognition system to monitor cellular levels of Clr-b.¹⁷ 

C57BL/6 (B6), β₂m-deficient (B2m^−/−) (B6.129P2-B2m^tm1Unc/J), CMV-cre transgenic (Tg) (B6.C-Tg[CMV-cre]1Cgn/J), and Eµ-myc Tg (B6.Cg-Tg[IghMyc]22Bri/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Clr-b-deficient (Ocil^−/−) mice were previously described.²³  β₂m/Clr-b double-deficient (B2m^−/−Clrb^−/−) mice were produced by breeding Ocil^−/− mice with B2m^−/− mice. All mice were maintained in the Animal Care and Veterinary Service at the University of Ottawa (Ottawa, Ontario), Sunnybrook Research Institute, or the Donnelly Center for Cellular and Biomolecular Research, University of Toronto (Toronto, Ontario), in accordance with institutional guidelines.

All genetic modifications were performed on the Nkrp1b^B6 allele. For clarity and simplicity, this allele will be referred to as Nkrp1b and the receptor as NKR-P1B in the remainder of this article. A targeting vector containing Nkrp1b genomic sequence with a floxed phosphoglycerate kinase (PGK)–neomycin cassette replacing exons 2 to 5 of Nkrp1b was created in a modified pBluescript-SK+ vector by bacterial artificial chromosome recombineering using clone RP23-127M20 in SW106 bacteria with an EcoRV-flanked galactokinase selection cassette, as previously described.²⁹  For a brief description of the strategy, please refer to the supplemental Materials and Methods on the Blood Web site. The pBluescript backbone was removed after AatII and SalI digestion before electroporation into C57BL/6 Bruce-4 embryonic stem (ES) cells, followed by selection in G418. Neomycin-resistant clones were screened by Southern blot analysis, and a targeting efficiency of ∼17% was observed. Chimeric Nkrp1b^neo founder mice were produced with Nkrp1b-targeted ES cells by the Clinical Research Institute of Montreal microinjection service (Montreal, Quebec). These mice were bred with B6 females to produce Nkrp1b^wt/neo heterozygous mice. Heterozygous mice were interbred to obtain Nkrp1b^neo/neo mice. To remove the neomycin cassette, Nkrp1b^neo/neo mice were bred with CMV-cre Tg mice on a B6 background (The Jackson Laboratory). The resulting Nkrp1b^lox/wt mice were interbred to produce Nkrp1b^lox/lox mice. Mice were genotyped regularly using specific primers (supplemental Materials and Methods). Wild-type (WT) and NKR-P1B-deficient littermates were used in all experiments unless otherwise indicated.

YAC-1 and CHO cells were purchased from the American Type Culture Collection. CHO cells were stably transfected with pcDNA3-Clr-b expression vector using Lipofectamine (Invitrogen). Lymphokine-activated killer (LAK) cells and bone marrow–derived dendritic cells (BM-DCs) were generated as previously described.^30,31 

For the source of commercially purchased antibodies, please refer to the supplemental Materials and Methods. Anti-Clr-b (4A6) and anti-NKR-P1B (2D9) antibodies have been previously described.^13,17,18  Anti-CRACC antibody and anti-NKR-P1B (2D12) hybridoma were kind gifts from Dr André Veillette (Clinical Research Institute of Montreal) and Dr. Koho Iizuka (University of Minnesota, Minneapolis, Minnesota), respectively. Antibody staining for flow cytometry was performed as previously described.³² 

NK cell cytotoxicity was measured using the standard 4-hour ⁵¹Cr-release assay as previously described.³³  For intracellular interferon (IFN)-γ measurement and CD107a staining, splenocytes from polyinosinic:polycytidylic acid (poly[I:C])-treated mice (150-µg intraperitoneal [IP] injection for 18 hours) or phosphate-buffered saline–treated mice were incubated with YAC-1 cells, plate-bound anti-NKR-P1C (NK1.1), and anti-NKR-P1B (2D12), or with phorbol 12-myristate 13-acetate (PMA; 10 ng/mL) and ionomycin (1 µg/mL) for 5 hours with brefeldin A and monensin (eBioscience). Intracellular IFN-γ staining using the Cytofix/Cytoperm kit (BD Biosciences) and CD107a staining were performed as previously described.³⁴ 

Splenocytes from WT B6, MHC-I-deficient, Clr-b-deficient, and MHC-I/Clr-b double-deficient mice were labeled with different concentrations of 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (Invitrogen) as previously described.³⁵  A 1:1 mixture of 1 × 10⁷ WT and various gene-deficient splenocytes were injected into the tail vein of recipient mice that were either untreated or treated with 150 µg of IP poly(I:C) for 24 hours or 5 µg of IV CpG-B (ODN 2006, Hycult Biotech) and 30 µg of IV dioleoyltrimethylammoniumpropane (DOTAP) (Invitrogen) for 6 hours prior to splenocyte injection. For NK cell depletion, mice were treated with 200 µg of IP anti-NKR-P1C (NK1.1) antibody 48 hours prior to splenocyte injection. Spleens of recipient mice were harvested 5 to 18 hours later and analyzed as previously described.³⁵ 

Statistical significance was determined by Student t test and log-rank test, where applicable, with a cut-off P value of .05.

We generated NKR-P1B-deficient mice on a B6 background to study this receptor’s role in NK cell function. A floxed neomycin cassette was inserted into the Nkrp1b gene by homologous recombination, replacing exons 2 to 5 in B6-background ES cells (Figure 1A). Founder mice carrying the Nkrp1b^neo allele were bred to WT B6 females. The resulting Nkrp1b^wt/neo offspring were genotyped by Southern blot analysis and PCR, and interbred to produce Nkrp1b^neo/neo mice (Figure 1B-C). Nkrp1b^neo/neo mice were then bred with CMV-cre Tg mice on a B6 background to remove the neomycin cassette through Cre-mediated recombination. The resulting Nkrp1b^wt/lox mice were genotyped by PCR and interbred to obtain Nkrp1b^lox/lox mice (Figure 1C). We confirmed Nkrp1b deletion by flow cytometric analysis using 2 different NKR-P1B-specific mAbs, 2D9 and 2D12.^13,18  DX5⁺TCRβ⁻ NK cells from Nkrp1b^lox/lox mice lacked any detectable NKR-P1B surface expression (Figure 1D) and are, therefore, referred to as Nkrp1b^−/− or NKR-P1B-deficient mice hereafter. NKR-P1B-deficient progeny from heterozygous parents were born with expected Mendelian frequency and without any gross pathological signs. These mice display normal immune development, including normal numbers of T, B, dendritic, NK, and NK T (NKT) cells in central and peripheral immune organs, comparable to B6 WT mice (Table 1).

NK cells develop in the bone marrow with sequential acquisition of surface markers at different stages of differentiation.^36-38  We determined whether NK cell development and differentiation was affected by the lack of NKR-P1B. We first analyzed the expression of multiple NKC-encoded NK cell receptors in NKR-P1B-deficient mice by flow cytometry and found them expressed at normal levels and percentages on NK cells (Figure 2A). Interestingly, this finding included Ly49 receptors and CD94-NKG2 receptors that are expressed at variable frequencies on NKR-P1B⁺ and NKR-P1B^– NK cell subsets in rodents.^13,19,20  One exception was NKG2D, whose expression was moderately downmodulated on NK (NK1.1⁺TCRβ⁻), but not NKT (NK1.1⁺TCRβ⁺), cells from NKR-P1B-deficient mice (Figure 2B-C). Moreover, NKR-P1B⁺ and NKR-P1B^– NK cells from WT mice had similar surface expression levels of NKG2D (Figure 2D), indicating that NKG2D downregulation was specific to NK cells from NKR-P1B-deficient mice. However, these cells were not impaired in their ability to kill targets expressing NKG2D ligands (data not shown). Interestingly, the percentage of NKR-P1B⁺ NK cells in heterozygous mice (∼45%) was higher than that deduced from the product rule (∼32%), as was observed for Ly49 expression in heterozygous NKC^KD mice.³²  This result is consistent with previous studies showing a more codominant vs purely stochastic expression of NKR-P1B in NK cells.¹³ 

The numbers and frequencies of NK cells in the spleen, lung, and liver were similar in WT and NKR-P1B-deficient mice (Figure 3A; Table 1). Additionally, markers associated with mature NK cells were normally expressed on splenic NK cells (Figure 3B). When cultured with interleukin-2, NKR-P1B-deficient NK cells produced granzyme A and upregulated CD69 and KLRG1 activation markers comparable to WT NK cells (Figure 3C). In addition, IP injection of poly(I:C) resulted in similar CD69 upregulation on NK cells from NKR-P1B-deficient and WT mice (Figure 3D). However, we observed a lower percentage of NK cells expressing KLRG1 in naïve NKR-P1B-deficient mice compared with WT mice (Figure 3D), similar to Ly49-deficient (NKC^KD) and MHC-I-deficient mice.^32,39,40  Apart from the slight downregulation of NKG2D and KLRG1, development and differentiation of NK cells appears normal in NKR-P1B-deficient mice.

NKR-P1B interacting with Clr-b has been shown to inhibit NK cells in vitro.^13,17,18  We performed NK cell cytotoxicity assays against stably transfected CHO target cells expressing high and low levels of Clr-b (Figure 4A). In contrast to the WT LAK cells, which were inhibited by Clr-b-expressing CHO cells (Figure 4B), the NKR-P1B-deficient LAK cells were not inhibited (Figure 4C). Notably, these LAK cells were generated from whole splenocytes, thus the WT LAK cells contained a mixture of NKR-P1B⁺ (∼60%) and NKR-P1B^– (∼40%) cells. For a better comparison, we sorted for WT NKR-P1B⁺ LAK (NK1.1⁺TCRβ⁻) cells on day 3 of culture and further expanded them with interleukin-2 for 6 days. NKR-P1B-deficient LAK (NK1.1⁺TCRβ⁻) cells were treated similarly. The sorted WT NKR-P1B⁺ LAKs maintained NKR-P1B surface expression in culture (Figure 4D) and were inhibited by Clr-b-expressing CHO cells, whereas the NKR-P1B-deficient LAK cells were not inhibited (Figure 4E-F). The sorted WT NKR-P1B^– LAK cells often reverted to a mixed population resembling the presort cells (data not shown), suggesting that some of these cells might have expressed NKR-P1B at low levels or induced NKR-P1B during LAK culture. We also performed NK cell cytotoxicity assays using Clr-b-deficient LAK cells, which expressed comparable levels of NKR-P1B (Figure 4G). Similar to WT LAK cells, Clr-b-deficient LAK cells were inhibited by Clr-b-expressing CHO cells (Figure 4H-I), suggesting normal NKR-P1B function despite the absence of its ligand (Clr-b) in these mice.⁴¹ 

Intracellular IFN-γ production and degranulation potential of NKR-P1B-deficient NK cells were normal in response to YAC-1 cells, activating NKR-P1C receptor (NK1.1) cross-linking, and PMA/ionomycin treatment, compared to WT NK cells (Figure 4J and supplemental Figure 1). Engagement of NKR-P1B using 2D12 mAb inhibited IFN-γ production by WT, but not NKR-P1B-deficient, NK cells on simultaneous stimulation via NK1.1 cross-linking or PMA/ionomycin (Figure 4J). Additionally, in WT mice, a higher proportion of IFN-γ⁺ NK cells coexpressed NKR-P1B and Ly49C/I, as compared to those expressing either or none of the inhibitory receptors (Figure 4K). In contrast to previous studies,⁴²  we also observed hyporesponsiveness of NK cells lacking NKR-P1B and Ly49C/I receptors when treated with PMA/ionomycin, perhaps due to our use of a lower concentration of PMA/ionomycin; this difference was abolished on stimulation with higher concentrations of PMA/ionomycin (data not shown). Together, these data reaffirm the inhibitory nature of NKR-P1B and demonstrate that, like the MHC-I-specific Ly49 receptors, NKR-P1B contributes to NK cell functional responsiveness and tolerance.

Clr-b is expressed at high levels on mouse hematopoietic cells and frequently downregulated on tumor cell lines cultured in vitro.¹⁷  NKR-P1B is thought to be involved in MHC-independent missing-self recognition by monitoring Clr-b expression on host cells. To test this hypothesis in vivo, we performed acute hematopoietic cell rejection assays using Clr-b^−/− splenocytes. Splenocytes from Clr-b^−/− mice were rejected very weakly within 18 hours by naïve WT and NKR-P1B-deficient mice (Figure 5A), likely a consequence of having normal expression of MHC-I (data not shown). This result is similar to the lower rejection of H-2K^b or H-2D^b single-deficient cells compared to that of H-2K^b/D^b double-deficient cells.^32,35  However, when mice were treated with the Toll-like receptor (TLR) agonists, poly(I:C) or CpG-B ODN, Clr-b^−/− splenocytes were efficiently rejected by WT recipient mice, particularly in the CpG-B ODN-treated group, but not by NKR-P1B-deficient mice (Figure 5A). CpG ODN has been shown to directly activate NK cell cytokine responses via activation of TLR9.⁴³  Because Clr-b^−/− splenocytes are MHC-I sufficient, their rejection by WT NK cells was due solely to the lack of Clr-b (missing-self), and failure of NKR-P1B-deficient NK cells to reject these cells indicates an inability to detect the presence or absence of Clr-b. Similarly, Clr-b-deficient mice, in which NK cells develop in the absence of the ligand for NKR-P1B, failed to reject Clr-b^−/− splenocytes (Figure 5B). Rejection of Clr-b^−/− splenocytes by WT mice is mediated by NK cells, because NK cell depletion abrogated this response (Figure 5B).⁴¹ 

We next performed in vivo rejection assays using MHC-I-deficient cells. Strikingly, MHC-I-deficient (B2m^−/−) splenocytes were more efficiently rejected by NKR-P1B-deficient mice compared with WT mice (Figure 5C, left). However, WT and NKR-P1B-deficient mice were equally efficient at rejecting MHC-I/Clr-b double-deficient (B2m^−/−Clr-b^−/−) splenocytes, suggesting that the reduced rejection of MHC-I-deficient splenocytes in WT mice was due to inhibitory NKR-P1B:Clr-b interactions (Figure 5C, right). This finding was also recapitulated in cytotoxicity assays in vitro using B2m^−/− and B2m^−/−Clr-b^−/− BM-DCs as target cells (Figure 5D). Moreover, rejection of B2m^−/−Clr-b^−/− splenocytes was significantly more efficient than rejection of B2m^−/− splenocytes by both WT and NKR-P1B-deficient mice (Figure 5C, left vs right), suggesting that loss of Clr-b synergizes with MHC-I deficiency to activate NK cell killing. Furthermore, this finding may also suggest differential dependence on MHC-I-dependent missing-self recognition by WT and NKR-P1B-deficient NK cells due to altered NK cell education and subset/repertoire formation in the absence of NKR-P1B. Together, these results demonstrate a loss of Clr-b-dependent inhibitory signals and Clr-b-dependent missing-self responses in NKR-P1B-deficient mice.

Because NKR-P1B is involved in missing-self recognition of Clr-b, which is often lost or downregulated on tumor cell lines in vitro, we next investigated the role of NKR-P1B in tumor development in vivo using the Eµ-myc Tg mouse model. Eµ-myc Tg mice develop spontaneous B-cell lymphomas due to transgenic expression of the c-myc oncogene in B cells.⁴⁴  We bred B6-background Eµ-myc Tg mice with NKR-P1B-deficient mice to obtain Eµ-myc⁺Nkrp1b^−/−, Eµ-myc⁺Nkrp1b^+/−, and Eµ-myc⁺Nkrp1b^+/+ littermates. The 3 groups of mice were monitored for 20 weeks for the appearance of palpable tumors (enlarged lymph nodes) and sickness, most often respiratory distress due to an enlarged thymus. The onset of B-cell lymphoma was significantly delayed in NKR-P1B-deficient mice, and these mice stayed tumor-free and healthy longer compared with WT and heterozygous littermates (Figure 6A). Eventually, most Eµ-myc⁺ NKR-P1B-deficient mice and all of their WT and heterozygous littermates developed tumors by 20 weeks of age. Affected lymphoid organs (thymus, spleen, and lymph nodes) were enlarged and composed mainly of B cells, as reported previously.^44,45  Representative examples of immature pre-B cell (B220⁺IgM^–) and mature B cell (B220⁺IgM⁺) lymphomas, and Clr-b expression patterns, are shown in Figure 6B. Both the mature B and immature pre-B cell lymphomas from NKR-P1B-deficient mice displayed significantly reduced expression of Clr-b compared to normal mature B cells, whereas lymphomas from WT littermate controls maintained higher Clr-b levels (Figure 6C). Together, these results show that, in NKR-P1B-deficient mice, oncogene-transformed cells may spontaneously lose Clr-b compared to healthy cells (as observed in vitro), yet malignant tumor cells in WT mice may selectively maintain high Clr-b levels to escape immune surveillance by inhibiting NK cells via NKR-P1B.

NKR-P1B is an inhibitory receptor on ∼60% of NK cells in B6 mice that recognizes the Clr-b ligand.^13,17,18  Using a gene-knockout model, we show a loss of Clr-b-mediated inhibition in NKR-P1B-deficient mice. Although the WT NK cells are inhibited by cell surface Clr-b, NKR-P1B-deficient NK cells efficiently kill CHO target cells expressing Clr-b. Additionally, inhibition of IFN-γ production by NKR-P1B receptor cross-linking in WT NK cells is abolished in NKR-P1B-deficient NK cells. Notably, NK-dependent in vivo rejection of MHC-I-deficient cells is elevated in NKR-P1B-deficient mice compared with WT mice, due to the lack of Clr-b-mediated inhibition.

Inhibitory receptors are required for the education (licensing) of developing NK cells to acquire the ability to respond to activation signals. NK cell responsiveness is proportional to the number of self-MHC-specific inhibitory receptors expressed; that is, NK cells that express more inhibitory Ly49 receptors are also more responsive toward stimulation.^42,46  Our study demonstrates a higher responsiveness of NK cells expressing the inhibitory receptors NKR-P1B and Ly49C/I, in contrast to those expressing either or none of the receptors. Therefore, like the Ly49 receptors, NKR-P1B seems to contribute to NK cell education and hence higher responsiveness.

It has been shown that lack of MHC-I-dependent NK cell education in full MHC-I-deficient mice results in hyporesponsiveness of the uneducated/unlicensed NK cells toward activating stimuli.^47,48  Because our data support a role of the NKR-P1B receptor in NK cell tolerance, one might expect NKR-P1B-deficient NK cells to be hyporesponsive. However, in our study, IFN-γ production and degranulation in response to general activation stimuli appear to be normal in NKR-P1B-deficient NK cells. In contrast, NK cells from Clr-b-deficient mice, like full MHC-I-deficient mice, appear to be hyporesponsive to activating receptor cross-linking and cytokine stimulation,⁴¹  despite a complementarity of deficiencies in either the Clr-b ligand or the NKR-P1B receptor. We have encountered a similar situation in Ly49-deficient mice, in which NK cell responses to various general activating stimuli were preserved.³²  Similarly, NKR-P1B-deficient mice are as efficient as WT mice regarding in vivo rejection of hematopoietic cells lacking both Clr-b and MHC-I, despite an expected hyporesponsiveness of NKR-P1B-deficient NK cells. This finding may suggest a dominant effect of MHC-I-dependent NK cell education and missing-self responses over those mediated via the single NKR-P1B:Clr-b interaction. Alternatively, a qualitative skewing of NK cell subsets during NK cell education and repertoire formation may result in increased dependence on, and a stronger magnitude of, MHC-I-dependent missing-self responses in NKR-P1B-deficient vs WT mice. In addition, other Clr-b interactions, such as with alternative inhibitory or stimulatory NK receptors recognizing Clr-b alone or in complex with other Clr family members, may impart some education on these cells. Notably, NKR-P1G is another inhibitory member of the NKR-P1 receptor family, and NKR-P1F shares overlapping ligand specificity with NKR-P1G.^{11,15,21,22,49}  A full analysis of Clr-b interactions with other Clr family members remains to be tested.

Educated/licensed NK cells play a role in missing-self recognition, where target cells with reduced expression of inhibitory self-ligands are specifically recognized and eliminated by NK cells.⁷  Clr-b is broadly expressed on hematopoietic cells and at least some nonhematopoietic tissues, thus overlapping the expression of MHC-I ligands for the inhibitory Ly49 receptors.^17,27,50  Clr-b recognition by NKR-P1B provides an MHC-I-independent missing-self recognition mechanism. Although Clr-b-deficient splenocytes are acutely rejected in WT mice treated with TLR agonists, they are not rejected in NKR-P1B-deficient mice in vivo, providing a genetic proof-of-principle for MHC-independent missing-self recognition by NK cells (this study and Chen et al⁴¹ ). This complementation resembles the inability of Ly49-deficient mice to reject MHC-I-deficient cells.³²  Similarly, Clr-b-deficient mice are unable to reject Clr-b-deficient splenocytes due to self-tolerance mechanisms influencing NKR-P1B:Clr-b-dependent NK cell education.⁴¹  Note that MHC-dependent NK cell education and missing-self responses are intact in NKR-P1B-deficient mice, as demonstrated by enhanced rejection of MHC-I-deficient cells. Thus, NKR-P1B:Clr-b-dependent missing-self recognition appears to function independently and in parallel with missing-self recognition of MHC-I ligands by Ly49 receptors.

Clr-b is frequently downregulated on tumor cell lines in vitro.¹⁷  However, myc-induced B lymphoma cells in Eµ-myc Tg mice reproducibly maintain high-level expression of Clr-b comparable to normal B cells from WT mice. In contrast, Clr-b was found to be frequently and spontaneously downregulated on B lymphoma cells from NKR-P1B-deficient mice. Taken together, these results suggest that oncogene-transformed cells may face selective pressure from NK cell–mediated immunosurveillance to maintain high Clr-b expression levels, which may in turn represent an effective escape mechanism to inhibit WT (NKR-P1B⁺), but not NKR-P1B-deficient, NK cells. Thus, NKR-P1B-deficient NK cells, which are resistant to Clr-b inhibition, eliminate tumor cells more efficiently, thereby significantly delaying kinetics and decreasing penetrance of myc-induced spontaneous lymphomas in NKR-P1B-deficient vs WT mice.

In summary, this study provides in vivo evidence of a negative regulatory role of the NKR-P1B:Clr-b recognition axis in innate immune responses. Figure 7 demonstrates 4 different scenarios and the consequences of NKR-P1B:Clr-b recognition by NK cells: (1) normal healthy cells express both MHC-I and Clr-b and are spared by NK cells (Figure 7A); (2) Clr-b-deficient cells are killed by WT NK cells through an MHC-I-independent missing-self response, and NKR-P1B-deficient NK cells cannot sense the absence of Clr-b, resulting in a loss of Clr-b-dependent missing-self recognition (Figure 7B); (3) MHC-I-deficient cells are killed by WT NK cells through an MHC-I-dependent missing-self response, and NKR-P1B-deficient NK cells are more efficient in killing MHC-I-deficient cells, possibly due to a lack of Clr-b-mediated inhibition and/or a higher dependence on MHC-I-mediated education (Figure 7C); and (4) B lymphoma cells maintain normal surface expression of MHC-I and Clr-b to inhibit NK cells, and NKR-P1B-deficient NK cells are resistant to tumor immune evasion via inhibitory Clr-b ligand (Figure 7D). This latter scenario has potential therapeutic implications. Tumors and infected cells employ various mechanisms to escape immune detection. Expression of ligands specific for inhibitory NK receptors may render such pathological target cells resistant to lysis by NK cells. One could envisage a scenario whereby therapeutic blockade of inhibitory NK receptors would result in augmented NK cell activity. This effect has been demonstrated in the case of T cells by blocking cytotoxic T lymphocyte–associated antigen-4, and in NK cells by blocking inhibitory Ly49C/I, both of which led to augmented antitumor effects.^51,52  Our study shows that the absence of NKR-P1B in gene-knockout mice results in a significant advantage over WT mice in controlling myc-induced B-cell lymphoma. This finding lends support to the idea of suppressing inhibitory signals in immune cells to achieve optimal antitumor and antipathogen immunity. NKR-P1B-deficient mice thus represent a useful model for the human NKR-P1A:LLT1 receptor-ligand system to study the role of these receptors on immune cells and their contribution to immunity against tumors and pathogens.

This work was supported by an Operating Grant from the Canadian Institutes of Health Research (CIHR 86630) (A.P.M. and J.R.C.); an Early Researcher Award from the Ontario Ministry of Research and Innovation (J.R.C.); the Victorian Government Operational Infrastructure support program (M.T.G.); and grants from the Biotechnology and Biological Sciences Research Council and Medical Research Council (C.G.B.). A.P.M. holds a Canada Research Chair in Innate Pathogen Resistance; J.R.C. held a Canadian Institutes of Health Research New Investigator Award and holds an Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, USA.

Contribution: M.M.A.R., P.C., J.R.C., and A.P.M. designed the research; M.M.A.R., P.C., A.N.M., A.B.M., M.J.T., and Q.Z. performed the research; C.G.B., V.K., and M.T.G. contributed vital new reagents; M.M.A.R., P.C., A.N.M., A.B.M., and M.J.T. collected the data; M.M.A.R., P.C., A.N.M., J.R.C., and A.P.M. analyzed and interpreted the data; M.M.A.R. and A.N.M. performed the statistical analysis; and M.M.A.R., J.R.C., and A.P.M. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Andrew P. Makrigiannis, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Guindon Hall, Room 4226, 451 Smyth Rd, Ottawa, ON, Canada K1H 8M5; e-mail: amakrigi@uottawa.ca; and James R. Carlyle, Department of Immunology, University of Toronto, Sunnybrook Research Institute, 2075 Bayview Ave, Toronto, ON, Canada M4N 3M5; e-mail: james.carlyle@utoronto.ca.