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IMMUNOBIOLOGY

The mouse NKR-P1B:Clr-b recognition system is a negative regulator ofinnate immune responsesMir Munir A. Rahim,1 Peter Chen,2 Amelia N. Mottashed,1 Ahmad Bakur Mahmoud,1,3 Midhun J. Thomas,1 Qinzhang Zhu,4

Colin G. Brooks,5 Vicky Kartsogiannis,6 Matthew T. Gillespie,6,7 James R. Carlyle,2 and Andrew P. Makrigiannis1

1Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada; 2Department of Immunology, University of

Toronto, Sunnybrook Research Institute, Toronto, ON, Canada; 3College of Applied Medical Sciences, Taibah University, Madinah Munawwarah, Kingdom

of Saudi Arabia; 4Transgenic Core Facility, Clinical Research Institute of Montreal, Montreal, QC, Canada; 5Institute of Cell and Molecular Biosciences, The

Medical School, Newcastle, United Kingdom; 6Prince Henry’s Institute, Monash Medical Centre, Clayton, VIC, Australia; and 7Department of Biochemistry

and Molecular Biology, Monash University, Clayton, VIC, Australia

Key Points

• NKR-P1B is involved in NKcell tolerance and MHC-I-independent missing-selfrecognition of Clr-b-deficienttarget cells.

• The NKR-P1B:Clr-b systemplays a role in tumorsurveillance and immuneescape in the Em-myctransgenic mouse model ofB-cell lymphoma.

NKR-P1B is a homodimeric type II transmembrane C-type lectinlike receptor that inhibits

natural killer (NK) cell function upon interaction with its cognate C-type lectin-related

ligand, Clr-b. The NKR-P1B:Clr-b interaction represents a major histocompatibility

complex class I (MHC-I)-independent missing-self recognition system that monitors

cellular Clr-b levels. We have generated NKR-P1BB6-deficient (Nkrp1b2/2) mice to study

the role of NKR-P1B in NK cell development and function in vivo. NK cell inhibition by

Clr-b is abolished in Nkrp1b2/2 mice, confirming the inhibitory nature of NKR-P1BB6.

Inhibitory receptors also promote NK cell tolerance and responsiveness to stimulation;

hence,NKcells expressingNKR-P1BB6 andLy49C/I display augmented responsiveness

to activating signals vs NK cells expressing either or none of the receptors. In addition,

Nkrp1b2/2 mice are defective in rejecting cells lacking Clr-b, supporting a role for

NKR-P1BB6 in MHC-I-independent missing-self recognition of Clr-b in vivo. In contrast,

MHC-I-dependentmissing-self recognition is preserved inNkrp1b2/2mice. Interestingly,

spontaneous myc-induced B lymphoma cells may selectively use NKR-P1B:Clr-b in-

teractions to escape immune surveillance by wild-type, but not Nkrp1b2/2, NK cells.

These data provide direct genetic evidence of a role for NKR-P1B in NK cell tolerance and MHC-I-independent missing-self

recognition. (Blood. 2015;125(14):2217-2227)

Introduction

The importance of natural killer (NK) cells in host defense againstmicrobial infections and tumors has been highlighted in individualslacking NK cells or NK cell functions; these individuals suffer frompersistent and life-threatening infections of normally benign herpesviruses and tumors.1-3 NK cell function is regulated by integratingactivating and inhibitory signals from engaged NK cell receptors.4 TheNK cell receptor repertoire inmice includes the Ly49, NKG2D,CD94/NKG2, and NKR-P1 families of receptors, all of which are encodedby genes in the NK gene complex (NKC) on chromosome 6.5,6 Thewell-characterized Ly49 receptor family is themouse functional equiv-alent 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 andthe response to cells without MHC-I expression.7

NKR-P1 receptors are homodimeric type II transmembrane C-typelectinlike molecules4,6,8 and are conserved across many species.9 This

receptor family consists of 5members inmice (NKR-P1A,NKR-P1B/D,NKR-P1C, NKR-P1F, and NKR-P1G; Nkrp1e is a pseudogene).10-12

NKR-P1AandNKR-P1F are proposed to be activating and are expressedat low levels on all NK cells.13 The activating NK1.1 (NKR-P1C)receptor, a prototypical antigen definingmouseNKcells in theC57BL/6(B6) mouse strain, is a product of the Nkrp1cB6 gene.14 NKR-P1G hasonly recently beendocumented to be inhibitory andprimarily involved inmucosal immunity,15 whereas NKR-P1B is a known inhibitory receptorfirst identified in the Swiss and SJL mouse strains.10,11,16-18 At least 3different Nkrp1b alleles have been described. The B6 allele has beenvariably termed Nkrp1d or Nkrp1bB6, and encodes the NKR-P1BB6

receptor, also known as NKR-P1D.11 NKR-P1B is expressed only ona subset of NK cells.13 NKR-P1B1 and NKR-P1B2 NK cells differ intheir expression of other NK cell receptors in both mice and rats.13,19,20

Ligands formostNKR-P1 receptorshavebeen identified asmembers ofthe C-type lectin-related (Clr) family of membrane glycoproteinsencoded by the Clec2 genes, which are intermingled among the

Submitted February 12, 2014; accepted January 6, 2015. Prepublished online

as Blood First Edition paper, January 22, 2015; DOI 10.1182/blood-2014-02-

556142.

J.R.C. and A.P.N. contributed equally to this study.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked “advertisement” in accordance with 18 USC section 1734.

© 2015 by The American Society of Hematology

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Nkrp1 (Klrb1) genes within the mouse NKC.12,17,18 The currentlyknown 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 strainto study the role of NKR-P1B in NK cell development and functionin vivo.We chose to target NKR-P1B for the following reasons: (1)the NKR-P1B:Clr-b system is well characterized in mice, and ap-propriate reagents, such as specific monoclonal antibodies (mAbs)and a complementary Clr-b gene–deficient mouse strain, areavailable13,23; (2) the NKR-P1B:Clr-b system is analogous to the in-hibitory NKR-P1A:LLT1 system in humans, although their expres-sionpatternsmayvary24,25; (3) the existenceof 3 significantly differentNkrp1b alleles suggests a possible divergence as a result of pathogenchallenge (eg, rat cytomegalovirus encodes a C-type lectinlike proteinwith homology to rat Clr-11 [Clec2d11] that protects infected cellsfrom 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 itsexpression on transfected cells protects them from NK-mediatedlysis.12,17,18,27 In addition, Clr-b expression is often downmodulatedon tumor cell lines after virus infection and during genotoxic andcellular stress in vitro.17,26,28 Therefore, NKR-P1B:Clr-b interactionsrepresent an MHC-I-independent missing-self recognition system tomonitor cellular levels of Clr-b.17

Materials and methods

Mice

C57BL/6 (B6), b2m-deficient (B2m2/2) (B6.129P2-B2mtm1Unc/J), CMV-cretransgenic (Tg) (B6.C-Tg[CMV-cre]1Cgn/J), and Em-myc Tg (B6.Cg-Tg[IghMyc]22Bri/J) mice were purchased from The Jackson Laboratory(Bar Harbor, ME). Clr-b-deficient (Ocil2/2) mice were previously de-scribed.23b2m/Clr-b double-deficient (B2m2/2Clrb2/2) micewere producedby breedingOcil2/2micewithB2m2/2mice.Allmiceweremaintained in theAnimal Care and Veterinary Service at the University of Ottawa (Ottawa,Ontario), Sunnybrook Research Institute, or the Donnelly Center for Cellularand Biomolecular Research, University of Toronto (Toronto, Ontario), inaccordance with institutional guidelines.

Generation of NKR-P1B-deficient mice

All geneticmodificationswere performed on theNkrp1bB6 allele. For clarity andsimplicity, this allele will be referred to asNkrp1b and the receptor as NKR-P1Bin the remainder of this article. A targeting vector containing Nkrp1b genomicsequence with a floxed phosphoglycerate kinase (PGK)–neomycin cassette re-placing exons 2 to 5 of Nkrp1b was created in a modified pBluescript-SK1vector by bacterial artificial chromosome recombineering using clone RP23-127M20 in SW106 bacteria with an EcoRV-flanked galactokinase selectioncassette, as previously described.29 For a brief description of the strategy, please

Figure 1. Generation of NKR-P1B-deficient mice. (A) Nkrp1b deletion strategy. Exons 2 to 5 were replaced with a floxed neomycin (neor) cassette by homologous

recombination in ES cells of B6 background. Correctly targeted ES cell clones were selected by Southern blot analysis and used to generate Nkrp1bneo mice. These mice

were bred with CMV-cre Tg mice to produce Nkrp1blox mice. Filled boxes denote exons (numbered), and arrowheads represent PCR primers (P) described in the “Materials

and methods” section. The location of 59 and 39 Southern probes is underlined, and EcoRV (E) and BamHI (B) restriction enzyme sites are shown. (B) Southern blot of EcoRV-

digested genomic DNA from mice of the indicated genotypes using the 59 probe. (C) PCR analysis of tail DNA from Nkrp1bneo and Nkrp1blox mice. (D) Surface expression of

NKR-P1B is absent on NK cells from NKR-P1B-deficient mice. Splenocytes from WT and Nkrp1blox/lox mice were stained with mAbs to DX5, TCRb, and NKR-P1B (2D9 and

2D12). The percentage of NKR-P1B1 NK cells (DX51TCRb–) is indicated. PCR, polymerase chain reaction.

2218 RAHIM et al BLOOD, 2 APRIL 2015 x VOLUME 125, NUMBER 14




refer to the supplemental Materials and Methods on the BloodWeb site. ThepBluescript backbone was removed after AatII and SalI digestion beforeelectroporation into C57BL/6 Bruce-4 embryonic stem (ES) cells, followedby selection in G418. Neomycin-resistant clones were screened by Southernblot analysis, and a targeting efficiency of ;17% was observed. ChimericNkrp1bneo founder mice were produced withNkrp1b-targeted ES cells by theClinical Research Institute of Montreal microinjection service (Montreal,Quebec). These mice were bred with B6 females to produce Nkrp1bwt/neo

heterozygous mice. Heterozygous mice were interbred to obtain Nkrp1bneo/neo

mice. To remove the neomycin cassette, Nkrp1bneo/neo mice were bred withCMV-cre Tg mice on a B6 background (The Jackson Laboratory). The resultingNkrp1blox/wt mice were interbred to produce Nkrp1blox/lox mice. Mice weregenotyped regularly using specific primers (supplemental Materials andMethods). Wild-type (WT) and NKR-P1B-deficient littermates were used inall experiments unless otherwise indicated.

Cells

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

Flow cytometry

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

In vitro NK cell assays

NK cell cytotoxicity was measured using the standard 4-hour 51Cr-release assayas previously described.33 For intracellular interferon (IFN)-gmeasurement andCD107a staining, splenocytes from polyinosinic:polycytidylic acid (poly[I:C])-treated mice (150-mg intraperitoneal [IP] injection for 18 hours) or phosphate-buffered saline–treated mice were incubated with YAC-1 cells, plate-boundanti-NKR-P1C (NK1.1), and anti-NKR-P1B (2D12), or with phorbol 12-myristate 13-acetate (PMA; 10 ng/mL) and ionomycin (1 mg/mL) for 5 hourswith brefeldin A andmonensin (eBioscience). Intracellular IFN-g staining usingthe Cytofix/Cytoperm kit (BD Biosciences) and CD107a staining were per-formed as previously described.34

In vivo splenocyte rejection assay

Splenocytes from WT B6, MHC-I-deficient, Clr-b-deficient, and MHC-I/Clr-b double-deficient mice were labeled with different concentrations of5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (Invitrogen) aspreviously described.35 A 1:1 mixture of 1 3 107 WT and various gene-deficient splenocytes were injected into the tail vein of recipient mice thatwere either untreated or treated with 150 mg of IP poly(I:C) for 24 hours or5 mg of IV CpG-B (ODN 2006, Hycult Biotech) and 30 mg of IV dioleoyl-trimethylammoniumpropane (DOTAP) (Invitrogen) for 6 hours prior tosplenocyte injection. For NK cell depletion, mice were treated with 200 mgof IP anti-NKR-P1C (NK1.1) antibody 48 hours prior to splenocyte in-jection. Spleens of recipient mice were harvested 5 to 18 hours later andanalyzed as previously described.35

Statistical analysis

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

Results

Targeted deletion of the Nkrp1b gene

We generated NKR-P1B-deficient mice on a B6 background to studythis receptor’s role inNKcell function.Afloxed neomycin cassettewasinserted into theNkrp1b gene by homologous recombination, replacingexons 2 to 5 in B6-background ES cells (Figure 1A). Founder micecarrying the Nkrp1bneo allele were bred to WT B6 females. Theresulting Nkrp1bwt/neo offspring were genotyped by Southern blotanalysis and PCR, and interbred to produce Nkrp1bneo/neo mice(Figure 1B-C).Nkrp1bneo/neomice were then bred with CMV-cre Tgmice on a B6 background to remove the neomycin cassette throughCre-mediated recombination. The resulting Nkrp1bwt/lox mice weregenotyped by PCR and interbred to obtain Nkrp1blox/lox mice(Figure 1C). We confirmed Nkrp1b deletion by flow cytometricanalysis using 2 different NKR-P1B-specific mAbs, 2D9 and2D12.13,18 DX51TCRb2 NK cells from Nkrp1blox/lox mice lackedany detectable NKR-P1B surface expression (Figure 1D) and are,therefore, referred to as Nkrp1b2/2 or NKR-P1B-deficient micehereafter. NKR-P1B-deficient progeny from heterozygous parentswere bornwith expectedMendelian frequency andwithout any grosspathological signs. These mice display normal immune develop-ment, including normal numbers of T, B, dendritic, NK, and NK T(NKT) cells in central and peripheral immune organs, comparable toB6 WT mice (Table 1).

Developmental and activation marker expression on

NKR-P1B-deficient NK cells

NK cells develop in the bone marrow with sequential acquisition ofsurface markers at different stages of differentiation.36-38 We deter-mined whether NK cell development and differentiation was affectedby the lack of NKR-P1B. We first analyzed the expression of multiple

Table 1. Immune cell populations in lymphoid and nonlymphoidorgans from NKR-P1B-deficient mice

Organ Cell populations

Cell numbers

Nkrp1b1/1 Nkrp1b2/2

Thymus (3106) Total 131.6 6 27.3 118.5 6 32

CD4 SP 9.9 6 4.2 11.4 6 3.4

CD8 SP 4.63 6 3.3 4.0 6 1.1

CD4 CD8 DP 112.3 6 22 98.9 6 27.7

NKT 0.45 6 0.22 0.5 6 0.04

Spleen (3106) Total 87.1 6 10.4 83.4 6 15.4

CD4 19.0 6 2.8 18.2 6 5.3

CD8 12.2 6 1.3 11.3 6 2.7

B 45.1 6 12.7 44.1 6 14.6

NK 3.99 6 0.8 3.68 6 1.3

NKT 1.27 6 0.24 1.29 6 0.32

DC 2.5 6 0.7 2.7 6 0.7

pDC 0.17 6 0.08 0.17 6 0.06

Lungs (3105) Total 17.0 6 3.4 18.4 6 2.4

NK 0.60 6 0.1 0.85 6 0.3

NKT 0.054 6 0.01 0.072 6 0.01

Liver (3105) Total 26.2 6 8 25.0 6 6.2

NK 1.9 6 0.3 2.0 6 0.4

NKT 1.11 6 0.4 1.13 6 0.39

Single-cell suspensions from different organs were stained with antibodies for

cell-specific markers and analyzed by flow cytometry. Values are presented as the

mean 6 SD from 6 to 7 mice of each genotype.

DC, dendritic cells; DP, double positive; SP, single positive; pDC, plasmacytoid

dendritic cells.

BLOOD, 2 APRIL 2015 x VOLUME 125, NUMBER 14 ROLE OF NKR-P1B IN NATURAL KILLER CELL FUNCTION 2219




NKC-encoded NK cell receptors in NKR-P1B-deficient mice by flowcytometry and found them expressed at normal levels and percentageson NK cells (Figure 2A). Interestingly, this finding included Ly49receptors and CD94-NKG2 receptors that are expressed at variablefrequencies on NKR-P1B1 and NKR-P1B– NK cell subsets inrodents.13,19,20 One exception was NKG2D, whose expression wasmoderately downmodulated on NK (NK1.11TCRb2), but not NKT(NK1.11TCRb1), cells fromNKR-P1B-deficientmice (Figure 2B-C).Moreover, NKR-P1B1 and NKR-P1B– NK cells from WT mice hadsimilar surface expression levels of NKG2D (Figure 2D), indicatingthatNKG2Ddownregulationwas specific toNKcells fromNKR-P1B-deficient mice. However, these cells were not impaired in their ability tokill targets expressing NKG2D ligands (data not shown). Interestingly,the percentage of NKR-P1B1NK cells in heterozygous mice (;45%)was higher than that deduced from the product rule (;32%), as wasobserved for Ly49 expression in heterozygous NKCKD mice.32 Thisresult is consistent with previous studies showing a more codominantvs purely stochastic expression of NKR-P1B in NK cells.13

The numbers and frequencies of NK cells in the spleen, lung, andliver were similar in WT and NKR-P1B-deficient mice (Figure 3A;Table 1). Additionally, markers associated with mature NK cells werenormally expressed on splenic NK cells (Figure 3B). When culturedwith interleukin-2,NKR-P1B-deficientNKcells producedgranzymeAand upregulated CD69 and KLRG1 activation markers comparable toWTNKcells (Figure 3C). In addition, IP injection of poly(I:C) resultedin similar CD69 upregulation on NK cells from NKR-P1B-deficientand WT mice (Figure 3D). However, we observed a lower percent-age of NK cells expressing KLRG1 in naıve NKR-P1B-deficient micecompared with WT mice (Figure 3D), similar to Ly49-deficient(NKCKD) and MHC-I-deficient mice.32,39,40 Apart from the slight

downregulation of NKG2D and KLRG1, development and differ-entiation of NK cells appears normal in NKR-P1B-deficient mice.

NKR-P1B:Clr-b inhibitory signals are abolished in

NKR-P1B-deficient NK cells

NKR-P1B interacting with Clr-b has been shown to inhibit NK cells invitro.13,17,18 We performed NK cell cytotoxicity assays against stablytransfected CHO target cells expressing high and low levels of Clr-b(Figure 4A). In contrast to theWTLAK cells, which were inhibited byClr-b-expressingCHOcells (Figure 4B), theNKR-P1B-deficient LAKcells were not inhibited (Figure 4C). Notably, these LAK cells weregenerated from whole splenocytes, thus the WT LAK cells containeda mixture of NKR-P1B1 (;60%) and NKR-P1B– (;40%) cells. Fora better comparison, we sorted for WT NKR-P1B1 LAK (NK1.11

TCRb2) cells on day 3 of culture and further expanded them withinterleukin-2 for 6 days. NKR-P1B-deficient LAK (NK1.11TCRb2)cells were treated similarly. The sorted WT NKR-P1B1 LAKs main-tained NKR-P1B surface expression in culture (Figure 4D) and wereinhibited by Clr-b-expressing CHO cells, whereas the NKR-P1B-deficient LAK cells were not inhibited (Figure 4E-F). The sorted WTNKR-P1B– LAKcells often reverted to amixed population resemblingthe presort cells (data not shown), suggesting that some of these cellsmight have expressed NKR-P1B at low levels or induced NKR-P1Bduring LAK culture. We also performed NK cell cytotoxicity assaysusing Clr-b-deficient LAK cells, which expressed comparable levelsof NKR-P1B (Figure 4G). Similar to WT LAK cells, Clr-b-deficientLAKcellswere inhibited byClr-b-expressingCHOcells (Figure 4H-I),suggesting normal NKR-P1B function despite the absence of its ligand(Clr-b) in these mice.41

Figure 2. NKC gene expression in NKR-P1B-deficient mice. (A) Splenic NK cells (DX51TCRb–) from WT, NKR-P1B-deficient, and heterozygous littermate mice were

analyzed for expression of the indicated NK cell receptors encoded in the NKC. Representative plots from 1 of 6 to 7 mice are shown. The percentage of positively stained NK

cells is indicated. (B) Downmodulation of NKG2D on splenic NK cells (NK1.11TCRb–) but not NKT cells (NK1.11TCRb1) from NKR-P1B-deficient mice. (C) Graphical

representation of relative median fluorescence intensity (MFI) of NKG2D on NK cells from WT and NKR-P1B-deficient littermate mice (n 5 6 mice). Statistical analysis was

performed by Student t test, and the P value is indicated. (D) NKG2D expression on NKR-P1B1 and NKR-P1B2 subsets of NK cells from the spleen of a WT B6 mouse.





Intracellular IFN-g production and degranulation potential ofNKR-P1B-deficient NK cells were normal in response to YAC-1cells, activating NKR-P1C receptor (NK1.1) cross-linking, andPMA/ionomycin treatment, compared to WT NK cells (Figure 4Jand supplemental Figure 1). Engagement of NKR-P1B using 2D12mAb inhibited IFN-g 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-g1 NK cells coexpressed NKR-P1Band Ly49C/I, as compared to those expressing either or none ofthe inhibitory receptors (Figure 4K). In contrast to previousstudies,42 we also observed hyporesponsiveness of NK cellslacking NKR-P1B and Ly49C/I receptors when treated with PMA/ionomycin, perhaps due to our use of a lower concentration ofPMA/ionomycin; this differencewas abolished on stimulationwithhigher concentrations of PMA/ionomycin (data not shown).Together, these data reaffirm the inhibitory nature of NKR-P1Band demonstrate that, like the MHC-I-specific Ly49 receptors,NKR-P1B contributes to NK cell functional responsiveness andtolerance.

NKR-P1B-deficient mice exhibit decreased rejection of

Clr-b-deficient splenocytes and increased rejection

of MHC-I-deficient splenocytes

Clr-b is expressed at high levels on mouse hematopoietic cells andfrequently downregulated on tumor cell lines cultured in vitro.17 NKR-P1B is thought to be involved in MHC-independent missing-selfrecognition by monitoring Clr-b expression on host cells. To test thishypothesis in vivo, we performed acute hematopoietic cell rejectionassays using Clr-b2/2 splenocytes. Splenocytes from Clr-b2/2 micewere rejected very weakly within 18 hours by naıve WT and NKR-P1B-deficientmice (Figure 5A), likely a consequence of havingnormalexpression of MHC-I (data not shown). This result is similar to thelower rejection of H-2Kb or H-2Db single-deficient cells compared tothat ofH-2Kb/Dbdouble-deficient cells.32,35However,whenmiceweretreated with the Toll-like receptor (TLR) agonists, poly(I:C) or CpG-BODN, Clr-b2/2 splenocytes were efficiently rejected by WT recipientmice, particularly in the CpG-B ODN-treated group, but not byNKR-P1B-deficient mice (Figure 5A). CpGODNhas been shown todirectly activate NK cell cytokine responses via activation of TLR9.43

Figure 3. Expression of developmental and activation markers on NK cells from NKR-P1B-deficient mice. (A) Similar proportions of NK cells in the spleen, lungs, and

liver of WT and NKR-P1B-deficient littermate mice were observed. A representative plot from each group of mice is shown. The NK cell percentage is indicated. (B) Graphical

representation of cell surface marker expression on NK cells from WT and NKR-P1B-deficient littermate mice (n 5 6 mice). Splenocytes were stained with mAbs against

NK1.1, TCRb, and various cell surface markers. The mean 6 SD of the percentage of positively staining NK cells is depicted. (C) Expression of intracellular granzyme A and

activation markers (CD69 and KLRG-1) on NK cells from WT and NKR-P1B-deficient littermates following interleukin-2 treatment in vitro for 3 days. The percentage of

positively stained NK cells is indicated. Gray line represents staining with an isotype antibody. (D) Expression of CD69 and KLRG-1 on NK cells from naıve and poly(I:C)-

treated WT and NKR-P1B-deficient littermates. The percentage of positively stained NK cells is indicated. Statistical analysis was performed by Student t test, and P values

are indicated.SD, standard deviation.





Figure 4. NKR-P1B-deficient NK cells are not inhibited by Clr-b on target cells. (A) Flow cytometric analysis of Clr-b expression on Clr-b-transfected CHO cells. Two

clones with high (HI) and low expression levels are shown. (B-C) Ability of LAK cells from WT and NKR-P1B-deficient mice to kill CHO target cells was tested by 51Cr-release

assay. Data are represented as mean 6 SD of percent killing measured in triplicate wells at different effector cell (E) to target cell (T) ratios. (D) NKR-P1B expression on

sorted WT NKR-P1B1 and NKR-P1B-deficient LAK cells (NK1.11TCRb2), which were further expanded in culture with interleukin-2. (E-F) Ability of sorted WT NKR-P1B1

and NKR-P1B-deficient LAK cells to mediate cytotoxicity toward CHO target cells was tested by 51Cr-release assay. Data are represented as the mean6 SD of percent killing

measured in triplicate. (G) NKR-P1B expression on LAK cells from WT, Clr-b-deficient, and NKR-P1B-deficient mice. (H-I) Ability of LAK cells from WT, Clr-b-deficient, and

NKR-P1B-deficient mice to kill CHO and Clr-b-expressing CHO (CHO-Clrb) target cells was tested by 51Cr-release assay. Data are represented as mean 6 SD of percent

killing measured in triplicate wells at different E:T ratios. (J) Splenocytes from NKR-P1B-deficient and WT mice, pretreated with poly(I:C), were incubated with plate-bound

isotype control antibody (Isotype Ab), anti-NKR-P1C antibody (NK1.1), or PMA/ionomycin for 5 hours in the presence or absence of plate-bound anti-NKR-P1B antibody

(2D12). Intracellular IFN-g in NK cells was analyzed by flow cytometry, and the mean percentage6 SD of IFN-g1 NK cells for each stimulation is shown. (K) Splenocytes from

WT mice were incubated with YAC-1 cells, plate-bound anti-NKR-P1C antibody (NK1.1), isotype control antibody, or PMA/ionomycin for 5 hours. Intracellular IFN-g in NK cell

subsets, based on the expression of NKR-P1B and Ly49C/I receptors, was analyzed by flow cytometry, and the mean percentage 6 SD of IFN-g1 NK cells for each

stimulation is shown. Data in panels A-K are representative data from 1 of multiple independent experiments. Statistical analysis was performed by Student t test, and

P values are indicated where applicable. Ab, antibody; PMA1Iono, PMA/ionomycin.





BecauseClr-b2/2 splenocytes areMHC-I sufficient, their rejection byWT NK cells was due solely to the lack of Clr-b (missing-self), andfailure ofNKR-P1B-deficientNKcells to reject these cells indicates aninability to detect the presence or absence of Clr-b. Similarly, Clr-b-deficient mice, in which NK cells develop in the absence of the ligandfor NKR-P1B, failed to reject Clr-b2/2 splenocytes (Figure 5B).Rejection of Clr-b2/2 splenocytes by WT mice is mediated by NKcells, becauseNK cell depletion abrogated this response (Figure 5B).41

We next performed in vivo rejection assays usingMHC-I-deficientcells. Strikingly, MHC-I-deficient (B2m2/2) splenocytes were moreefficiently rejected by NKR-P1B-deficient mice compared with WTmice (Figure 5C, left). However, WT and NKR-P1B-deficient micewere equally efficient at rejecting MHC-I/Clr-b double-deficient(B2m2/2Clr-b2/2) splenocytes, suggesting that the reduced rejectionof MHC-I-deficient splenocytes in WT mice was due to inhibitoryNKR-P1B:Clr-b interactions (Figure 5C, right). This finding was alsorecapitulated in cytotoxicity assays in vitro using B2m2/2 and B2m2/2

Clr-b2/2 BM-DCs as target cells (Figure 5D). Moreover, rejection ofB2m2/2Clr-b2/2 splenocytes was significantly more efficient thanrejection of B2m2/2 splenocytes by bothWT and NKR-P1B-deficientmice (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-deficientNK cells due to altered NK cell education and subset/repertoire forma-tion in the absence of NKR-P1B. Together, these results demonstratea loss of Clr-b-dependent inhibitory signals and Clr-b-dependentmissing-self responses in NKR-P1B-deficient mice.

Involvement of NKR-P1B in tumor surveillance and

immune escape

Because NKR-P1B is involved in missing-self recognition of Clr-b,which is often lost or downregulatedon tumor cell lines in vitro,wenextinvestigated the role of NKR-P1B in tumor development in vivo usingthe Em-myc Tg mouse model. Em-myc Tg mice develop spontaneousB-cell lymphomas due to transgenic expression of the c-myc oncogenein B cells.44 We bred B6-background Em-myc Tg mice with NKR-P1B-deficientmice to obtainEm-myc1Nkrp1b2/2,Em-myc1Nkrp1b1/2,and Em-myc1Nkrp1b1/1 littermates. The 3 groups of mice weremonitored for 20 weeks for the appearance of palpable tumors(enlarged lymph nodes) and sickness, most often respiratory distress

Figure 5. NKR-P1B-deficient mice show impaired

acute rejection of Clr-b-deficient splenocytes but

enhanced rejection of MHC-I-deficient splenocytes.

(A) Rejection of 5-(and 6)-carboxyfluorescein diacetate

succinimidyl ester (CFSE)-labeled splenocytes from

Clr-b2/2 mice 18 hours after injection. Some mice were

treated with poly(I:C) or CpG-B ODN for 24 or 6 hours,

respectively, before CFSE-labeled splenocyte injection.

Each symbol represents an individual mouse. Small hori-

zontal bars represent mean values. Data are pooled

from multiple experiments. (B) Rejection of CFSE-labeled

splenocytes from Clr-b2/2 mice 18 hours after injection.

Some of the WT mice were treated with anti-NKR-P1C

antibody (NK1.1) to deplete NK cells (NK-depleted). All

mice were treated with CpG-B ODN for 6 hours before

CFSE-labeled splenocyte injection. Each symbol repre-

sents an individual mouse. Small horizontal bars repre-

sent mean values. Data are pooled from 2 independent

experiments. (C) Rejection of CFSE-labeled splenocytes

from B2m2/2 and B2m2/2Clr-b2/2 mice at different time

points after injection. Mice were not stimulated before

splenocyte injection. Mean 6 SD of percent rejection at

each time point is shown. Data are pooled from multiple

experiments. Statistical analysis was performed by

Student t test, and P values are indicated. (D) Ability

of LAK cells from WT and NKR-P1B-deficient mice to

mediate cytotoxicity toward B2m2/2 or B2m2/2Clr-b2/2

BM-DCs was tested by 51Cr-release assay. Data are

represented as the mean 6 SD of percent killing

measured in triplicate. Statistical analysis was per-

formed by Student t test, and P values are indicated.





due to an enlarged thymus. The onset of B-cell lymphoma wassignificantly delayed in NKR-P1B-deficient mice, and these micestayed tumor-free and healthy longer compared with WT andheterozygous littermates (Figure 6A). Eventually, most Em-myc1

NKR-P1B-deficient mice and all of their WT and heterozygouslittermates developed tumors by 20 weeks of age. Affected lym-phoid organs (thymus, spleen, and lymph nodes) were enlargedand composed mainly of B cells, as reported previously.44,45 Rep-resentative examples of immature pre-B cell (B2201IgM–) andmature B cell (B2201IgM1) lymphomas, and Clr-b expressionpatterns, are shown in Figure 6B. Both the mature B and immaturepre-B cell lymphomas from NKR-P1B-deficient mice displayedsignificantly reduced expression ofClr-b compared to normalmatureB cells, whereas lymphomas fromWT littermate controlsmaintainedhigher Clr-b levels (Figure 6C). Together, these results show that, inNKR-P1B-deficient mice, oncogene-transformed cells may sponta-neously lose Clr-b compared to healthy cells (as observed in vitro),yet malignant tumor cells inWTmice may selectively maintain highClr-b levels to escape immune surveillance by inhibiting NK cellsvia NKR-P1B.

Discussion

NKR-P1B is an inhibitory receptor on;60% of NK cells in B6 micethat 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-deficientmice. Although the WT NK cells are inhibited by cell surface Clr-b,

NKR-P1B-deficient NK cells efficiently kill CHO target cells ex-pressing Clr-b. Additionally, inhibition of IFN-g production byNKR-P1B receptor cross-linking in WT NK cells is abolished inNKR-P1B-deficient NK cells. Notably, NK-dependent in vivorejection ofMHC-I-deficient cells is elevated in NKR-P1B-deficientmice compared with WT mice, due to the lack of Clr-b-mediatedinhibition.

Inhibitory receptors are required for the education (licensing) ofdeveloping NK cells to acquire the ability to respond to activationsignals. NK cell responsiveness is proportional to the number of self-MHC-specific inhibitory receptors expressed; that is, NK cells thatexpress more inhibitory Ly49 receptors are also more responsivetoward stimulation.42,46 Our study demonstrates a higher responsive-ness of NK cells expressing the inhibitory receptors NKR-P1B andLy49C/I, in contrast to those expressing either or none of the receptors.Therefore, like the Ly49 receptors, NKR-P1B seems to contribute toNK cell education and hence higher responsiveness.

It has been shown that lack ofMHC-I-dependent NK cell educationin full MHC-I-deficient mice results in hyporesponsiveness of theuneducated/unlicensed NK cells toward activating stimuli.47,48 Be-cause our data support a role of the NKR-P1B receptor in NK celltolerance, one might expect NKR-P1B-deficient NK cells to behyporesponsive. However, in our study, IFN-g production anddegranulation in response to general activation stimuli appear to benormal in NKR-P1B-deficient NK cells. In contrast, NK cells fromClr-b-deficient mice, like full MHC-I-deficient mice, appear to behyporesponsive to activating receptor cross-linking and cytokinestimulation,41 despite a complementarity of deficiencies in eitherthe Clr-b ligand or the NKR-P1B receptor. We have encountered

Figure 6. Delayed onset of B lymphoma in NKR-P1B-

deficient mice expressing the Em-myc transgene. (A)

Kaplan-Meier representation of palpable tumor appear-

ance in Em-myc.Nkrp1b1/1, Em-myc.Nkrp1b1/2, and

Em-myc.Nkrp1b2/2 mice. Statistical analysis was per-

formed by log-rank test, and the P value is indicated. (B)

Flow cytometric analysis of B220, IgM, and Clr-b expres-

sion on B lymphoma cells from affected organs (lymph

nodes, spleen, and thymus) from Em-myc.Nkrp1b1/1

and Em-myc.Nkrp1b2/2 mice after disease onset and

from healthy WT mice. The percentage of gated cells

from each organ is indicated. Clr-b expression on gated

B lymphoma cells is represented on the histograms by

a dark line; isotype control by a gray line. Clr-b median

fluorescence intensity (MFI) values are normalized to

isotype control IgM MFI (Clr-b/isotype Ig MFI), and are

shown on respective plots. (C) Graphical representation

of Clr-b expression levels on mature (IgM1), immature

(IgM–), and all B lymphoma cells taken together, in the

spleen and lymph nodes of Em-myc.Nkrp1b1/1 and

Em-myc.Nkrp1b2/2 mice relative to the expression of

Clr-b on mature B cells in WT mice. Vertical columns

represent Clr-b/isotype Ig MFI ratio, and error bars

represent SD. The horizontal dotted line represents the

Clr-b/isotype Ig MFI ratio on mature B cells (B2201IgM1)

from WT mice (arbitrarily set to 1.0). Statistical analysis

was performed by Student t test, and P values are

indicated. Ig, immunoglobulin.





a similar situation in Ly49-deficient mice, in which NK cellresponses to various general activating stimuli were preserved.32

Similarly, NKR-P1B-deficient mice are as efficient as WT miceregarding 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 effectof MHC-I-dependent NK cell education and missing-self responsesover those mediated via the single NKR-P1B:Clr-b interaction.Alternatively, a qualitative skewing of NK cell subsets during NKcell education and repertoire formation may result in increaseddependence on, and a stronger magnitude of, MHC-I-dependentmissing-self responses in NKR-P1B-deficient vs WT mice. Inaddition, other Clr-b interactions, such as with alternative inhibitoryor stimulatory NK receptors recognizing Clr-b alone or in complexwith other Clr family members, may impart some education on thesecells. Notably, NKR-P1G is another inhibitory member of the NKR-P1 receptor family, and NKR-P1F shares overlapping ligandspecificity with NKR-P1G.11,15,21,22,49 A full analysis of Clr-binteractions with other Clr family members remains to be tested.

Educated/licensed NK cells play a role in missing-self recogni-tion, 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 somenonhematopoietic tissues, thus overlapping the expression of MHC-Iligands for the inhibitory Ly49 receptors.17,27,50 Clr-b recognition byNKR-P1B provides an MHC-I-independent missing-self recognitionmechanism. Although Clr-b-deficient splenocytes are acutely rejectedin WT mice treated with TLR agonists, they are not rejected in NKR-P1B-deficient mice in vivo, providing a genetic proof-of-principle forMHC-independent missing-self recognition by NK cells (this studyand Chen et al41). This complementation resembles the inability ofLy49-deficientmice to rejectMHC-I-deficient cells.32 Similarly, Clr-b-deficient mice are unable to reject Clr-b-deficient splenocytes due toself-tolerance mechanisms influencing NKR-P1B:Clr-b-dependent NKcell education.41 Note that MHC-dependent NK cell education andmissing-self responses are intact in NKR-P1B-deficient mice, as dem-onstrated by enhanced rejection ofMHC-I-deficient cells. Thus, NKR-P1B:Clr-b-dependent missing-self recognition appears to functionindependently and in parallel with missing-self recognition of MHC-Iligands by Ly49 receptors.

Clr-b is frequently downregulated on tumor cell lines in vitro.17

However, myc-induced B lymphoma cells in Em-myc Tg mice re-producibly maintain high-level expression of Clr-b comparable tonormal B cells from WT mice. In contrast, Clr-b was found to befrequently and spontaneously downregulated on B lymphoma cellsfrom NKR-P1B-deficient mice. Taken together, these results suggestthat oncogene-transformed cells may face selective pressure from NKcell–mediated immunosurveillance to maintain high Clr-b expressionlevels, which may in turn represent an effective escape mechanism toinhibitWT (NKR-P1B1), but notNKR-P1B-deficient,NK cells. Thus,NKR-P1B-deficient NK cells, which are resistant to Clr-b inhibition,eliminate tumor cells more efficiently, thereby significantly delayingkinetics and decreasing penetrance of myc-induced spontaneous lym-phomas in NKR-P1B-deficient vs WT mice.

In summary, this study provides in vivo evidence of a negativeregulatory role of the NKR-P1B:Clr-b recognition axis in innateimmune responses. Figure 7 demonstrates 4 different scenarios and theconsequences of NKR-P1B:Clr-b recognition by NK cells: (1) normalhealthy cells express bothMHC-I andClr-b and are spared byNK cells(Figure 7A); (2)Clr-b-deficient cells are killed byWTNKcells throughanMHC-I-independentmissing-self response, andNKR-P1B-deficientNK cells cannot sense the absence of Clr-b, resulting in a loss of Clr-b-

dependent missing-self recognition (Figure 7B); (3) MHC-I-deficientcells are killed byWTNKcells through anMHC-I-dependentmissing-self response, and NKR-P1B-deficient NK cells are more efficient inkilling MHC-I-deficient cells, possibly due to a lack of Clr-b-mediatedinhibition and/or a higher dependence on MHC-I-mediated education(Figure 7C); and (4) B lymphoma cells maintain normal surfaceexpression of MHC-I and Clr-b to inhibit NK cells, and NKR-P1B-deficient NK cells are resistant to tumor immune evasion via inhibitoryClr-b ligand (Figure 7D). This latter scenario has potential therapeutic

Figure 7. Schematic representation of the role of NKR-P1B:Clr-b recognition in

NK cell function. Four different scenarios depicting NK cell function in the presence

or absence of NKR-P1B:Clr-b recognition are shown. (A) Normal healthy cells

express MHC-I and Clr-b, which are recognized by inhibitory Ly49 and NKR-P1B

receptors on NK cells, respectively, resulting in their protection from NK cells.

Normal MHC-I levels are sufficient to protect these cells from NKR-P1B-deficient NK

cells. (B) WT NK cells kill Clr-b-deficient target cells through an MHC-I-independent

missing-self response. NKR-P1B-deficient NK cells cannot sense Clr-b-deficiency

on target cells. (C) WT NK cells efficiently kill MHC-I-deficient target cells through

MHC-I-dependent missing-self recognition. NKR-P1B-deficient NK cells are more

efficient in killing MHC-I-deficient cells, possibly due to lack of Clr-b-mediated in-

hibition and a higher dependence on MHC-I-mediated education. (D) B lymphoma

cells express normal levels of MHC-I and Clr-b. Tumor cells also express ligands for

activating NK cell receptors, making them susceptible to NK cells. In the absence of

inhibitory NKR-P1B receptor signals, NK cells have improved immunosurveillance

capacity against Clr-b-expressing tumor cells, which can escape immune detection

by WT NK cells.





implications. Tumors and infected cells employ variousmechanisms toescape immune detection. Expression of ligands specific for inhibitoryNK receptorsmay render such pathological target cells resistant to lysisby NK cells. One could envisage a scenario whereby therapeuticblockade of inhibitoryNKreceptorswould result in augmentedNKcellactivity. This effect has been demonstrated in the case of T cells byblocking cytotoxic T lymphocyte–associated antigen-4, and in NKcells by blocking inhibitory Ly49C/I, both of which led to augmentedantitumor effects.51,52Our study shows that the absenceofNKR-P1B ingene-knockoutmice results in a significant advantage overWTmice incontrolling myc-induced B-cell lymphoma. This finding lends supportto the idea of suppressing inhibitory signals in immune cells to achieveoptimal antitumor and antipathogen immunity. NKR-P1B-deficientmice thus represent a useful model for the human NKR-P1A:LLT1receptor-ligand system to study the role of these receptors on immunecells and their contribution to immunity against tumors and pathogens.

Acknowledgments

This work was supported by an Operating Grant from the CanadianInstitutes of Health Research (CIHR 86630) (A.P.M. and J.R.C.);an Early Researcher Award from the Ontario Ministry of Researchand Innovation (J.R.C.); the Victorian Government OperationalInfrastructure support program (M.T.G.); and grants from the Bio-technology and Biological Sciences Research Council and Medical

Research Council (C.G.B.). A.P.M. holds a Canada Research Chairin Innate Pathogen Resistance; J.R.C. held a Canadian Institutes ofHealth Research New Investigator Award and holds an Investigatorin the Pathogenesis of Infectious Disease Award from the BurroughsWellcome Fund, USA.

Authorship

Contribution: M.M.A.R., P.C., J.R.C., and A.P.M. designed theresearch; 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 vitalnew 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. performedthe statistical analysis; and M.M.A.R., J.R.C., and A.P.M. wrote themanuscript.

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

Correspondence: Andrew P. Makrigiannis, Department of Bio-chemistry, Microbiology, and Immunology, University of Ottawa,Guindon Hall, Room 4226, 451 Smyth Rd, Ottawa, ON, CanadaK1H 8M5; e-mail: [email protected]; and James R. Carlyle,Department of Immunology, University of Toronto, SunnybrookResearch Institute, 2075 Bayview Ave, Toronto, ON, Canada M4N3M5; e-mail: [email protected].

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online January 22, 2015 originally publisheddoi:10.1182/blood-2014-02-556142

2015 125: 2217-2227

Andrew P. MakrigiannisQinzhang Zhu, Colin G. Brooks, Vicky Kartsogiannis, Matthew T. Gillespie, James R. Carlyle and Mir Munir A. Rahim, Peter Chen, Amelia N. Mottashed, Ahmad Bakur Mahmoud, Midhun J. Thomas, innate immune responsesThe mouse NKR-P1B:Clr-b recognition system is a negative regulator of

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