Expansion and Protection by a Virus-Specific NK Cell Subset

Expansion and Protection by a Virus-Specific NK Cell Subset Lacking Expression of the Inhibitory NKR-P1B Receptor during Murine Cytomegalovirus Infection This information is current as of September 25, 2021. Mir Munir A. Rahim, Andrew Wight, Ahmad Bakur Mahmoud, Oscar A. Aguilar, Seung-Hwan Lee, Silvia M. Vidal, James R. Carlyle and Andrew P. Makrigiannis J Immunol 2016; 197:2325-2337; Prepublished online 10 August 2016; Downloaded from doi: 10.4049/jimmunol.1600776 http://www.jimmunol.org/content/197/6/2325 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2016/08/10/jimmunol.160077 Material 6.DCSupplemental References This article cites 71 articles, 42 of which you can access for free at: http://www.jimmunol.org/content/197/6/2325.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Expansion and Protection by a Virus-Speciﬁc NK Cell Subset Lacking Expression of the Inhibitory NKR-P1B Receptor during Murine Cytomegalovirus Infection

Mir Munir A. Rahim,* Andrew Wight,* Ahmad Bakur Mahmoud,*,† Oscar A. Aguilar,‡ Seung-Hwan Lee,* Silvia M. Vidal,x James R. Carlyle,‡ and Andrew P. Makrigiannis*,1

NK cells play a major role in immune defense against human and murine CMV (MCMV) infection. Although the MCMV genome encodes for MHC class I–homologous decoy ligands for inhibitory NK cell receptors to evade detection, some mouse strains have evolved activating receptors, such as Ly49H, to recognize these ligands and initiate an immune response. In this study, we demonstrate that approximately half of the Ly49H-expressing (Ly49H+) NK cells in the spleen and liver of C57BL/6 mice also 2 + express the inhibitory NKR-P1B receptor. During MCMV infection, the NKR-P1B Ly49H NK cell subset proliferates to con- Downloaded from stitute the bulk of the NK cell population. This NK cell subset also confers better protection against MCMV infection compared with the NKR-P1B+Ly49H+ subset. The two populations are composed of cells that differ in their surface expression of receptors such as Ly49C/I and NKG2A/C/E, as well as developmental markers, CD27 and CD11b, and the high-afﬁnity IL-2R (CD25) following infection. Although the NKR-P1B+ NK cells can produce effector molecules such as IFNs and granzymes, their proliferation is inhibited during infection. A similar phenotype in MCMV-infected Clr-b–deﬁcient mice, which lack the ligand for NKR-

P1B, suggests the involvement of ligands other than the host Clr-b. Most interestingly, genetic deﬁciency of the NKR-P1B, but not http://www.jimmunol.org/ Clr-b, results in accelerated virus clearance and recovery from MCMV infection. This study is particularly signiﬁcant because the mouse NKR-P1B:Clr-b receptor:ligand system represents the closest homolog of the human NKR-P1A:LLT1 system and may have a direct relevance to human CMV infection. The Journal of Immunology, 2016, 197: 2325–2337.

atural killer cells are a crucial component of an antiviral for NK cells in providing antiviral immunity has been shown immune response. NK cells mediate resistance to viruses against murine CMV (MCMV) infection in mice. Resistance to N in at least three different ways. These include direct MCMV infection is associated with a robust MCMV-specific NK recognition and lysis of virus-infected cells (1–4), production of cell response in particular strains of mice (17). Depletion of NK by guest on September 25, 2021 antiviral cytokines such as IFN-g (5–8), and modulation of the cells by Ab treatment abolishes resistance to MCMV in the pre- adaptive antiviral immune response (9–13). NK cell deficiency viously resistant mouse strains (18, 19). Similarly, an unusually and functional abnormalities in humans correlate with an in- severe and life-threatening human CMV (HCMV) infection may creased susceptibility to virus infections (14–16). A definitive role be attributed to a complete lack of NK cells and their functions, for example, in patients carrying GATA2 mutations associated with *Department of Biochemistry, Microbiology and Immunology, University of Ottawa, monocytopenia with Mycobacterium avium complex/dendritic cell, † Ottawa, Ontario K1H 8M5, Canada; College of Applied Medical Sciences, Taibah monocyte, B and NK lymphoid deficiency syndrome (16, 20). University, 30001 Madinah Munawwarah, Saudi Arabia; ‡Department of Immunol- ogy, Sunnybrook Research Institute, University of Toronto, Toronto, Ontario M4N NK cell function is tightly regulated by the integration of signals 3M5, Canada; and xDepartment of Human Genetics, McGill University, Montreal, generated through the engagement of an array of activating and Quebec H3G 0B1, Canada inhibitory cell surface receptors. NK cell receptors in mice include, 1 Current address: Department of Microbiology and Immunology, Dalhousie Univer- but are not restricted to, members of the Ly49, NKR-P1, and CD94/ sity, Halifax, NS, Canada. NKG2 receptor families (21). Ligands for the inhibitory receptors ORCID: 0000-0002-4069-4276 (S.M.V.). are generally host-derived molecules such as the MHC class I Received for publication May 4, 2016. Accepted for publication July 8, 2016. (MHC-I) and C-type lectin-related (Clr) ligands, which interact This work was supported by Canadian Institutes of Health Research Grant CIHR with the inhibitory members of Ly49 and NKR-P1 receptors, re- 86630 (to A.P.M. and J.R.C.) and an Early Researcher Award from the Ontario Ministry of Research and Innovation (to J.R.C.). A.P.M. holds a Canada Research spectively (22). In contrast, the ligands for the activating receptors Chair in Innate Pathogen Resistance. J.R.C. holds a Canadian Institutes of Health include tumor-associated and pathogen-derived molecules such as Research New Investigator Award and an Investigator in the Pathogenesis of Infec- tious Disease Award from the Burroughs Wellcome Fund, USA. the MCMV m157 protein recognized by the activating Ly49H Address correspondence and reprint requests to Dr. Mir Munir A. Rahim at the receptor (23, 24). The m157 protein is a structural homolog of current address: Department of Microbiology and Immunology, Dalhousie Univer- MHC-I and is proposed to act as a viral decoy to evade the NK cell sity, 5850 College Street, Halifax, NS B3H 4R2, Canada, or Dr. James R. Carlyle, response by engaging the inhibitory Ly49I receptor in the sus- Department of Immunology, Sunnybrook Research Institute, University of Toronto, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada. E-mail addresses: ceptible 129 mouse strain (23). The activating Ly49H receptor has [email protected] (M.M.A.R.) or [email protected] (J.R.C.) evolved in the mouse as a host adaptation mechanism, where it has The online version of this article contains supplemental material. been maintained in the C57BL/6 (B6) strain to enable the specific Abbreviations used in this article: B6, C57BL/6; Clr, C-type lectin-related; HCMV, recognition of the MCMV m157 gene product, in turn leading to human CMV; MCMV, murine CMV; MHC-I, MHC class I; p.i., postinfection; WT, NK cell activation and resistance to MCMV (23–26). wild-type. Activated NK cells undergo rapid proliferation and expansion Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 of their numbers during viral infections (27–29). In healthy www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600776 2326 NEGATIVE IMPACT OF NKR-P1B ON INNATE IMMUNITY TO MCMV individuals, an HCMV seropositive status is strongly associated mouse embryonic fibroblast cells as described previously (45). B6, NKR-P1B– 4 with increased proportions of circulating CD94/NKG2C+ NK cells deficient, and Clr-b–deficient mice were infected by i.p. injection of 2 3 10 PFU of MCMV. Ly49H-deficient mice were infected i.p. with 3 3 103 PFU (30). It is proposed that HCMV promotes the expansion of CD94/ 3 + of MCMV. Newborn BALB/c mice (4–5 d old) were infected i.p. with 5 NKG2C NK cells in healthy carrier individuals (30). Similarly, 102 PFU of MCMV. MCMV Dm157 strain was previously generated by a MCMV infection in B6 mice has been shown to promote expan- deletion of the m157 gene in a bacterial artificial chromosome–derived sion of Ly49H+ NK cells (28, 29). Following infection of B6 mice MCMV MW97.01 strain (46). B6 and NKR-P1B–deficient mice were in- 3 5 3 6 with MCMV, NK cells undergo two distinct phases of prolifera- fected i.p. with 2 10 and 2 10 PFU of MCMV Dm157 strain. All breeding and manipulations performed on animals were approved by tion (28). The initial phase, which occurs within the first 2 d the University of Ottawa animal care committee in accordance with the following infection, is characterized by a general activation and principles published in the Canadian Council on Animal Care’s Guide to proliferation of the bulk NK cells independent of Ly49H expres- the Care and Use of Experimental Animals and with the Animals for sion. This is subsequently followed by a preferential proliferation Research Act, R.S.O. 1990, c.22, s. 17(1-3). and expansion of the MCMV-specific Ly49H+ NK cell subset (28). Abs and flow cytometry Expression of the inhibitory Ly49C/I receptors on Ly49H+ NK cells has been shown to limit the proliferation of this NK cell The following fluorochrome-conjugated Abs were purchased from com- subset during MCMV infection (31). Variants of MCMV m157 mercial sources: anti-TCRb (H57-597), anti-CD49b (DX5), anti-NKp46 (29A1.4), anti-CD27 (LG.7F9), anti-CD11b (M1/70), anti-Ly49H (3D10), protein have been shown to interact with the inhibitory Ly49C anti–IFN-g (XMG1.2), anti-CD107a (eBio1D4B), and anti–Ki-67 (SolA15) receptor from B6, BALB, and NZB mouse strains (32). It has been Abs were purchased from eBioscience. Anti-NKR-P1C (NK1.1), anti- shown that simultaneous interaction of m157 variants with Ly49C Ly49C/I (5E6), and anti-NKG2A/C/E (20d5) were purchased from BD and H can disrupt NK cell activation; however, because most Biosciences. Anti-CD16/CD32 (2.4G2) for FcR blocking was purchased Downloaded from + from BD Biosciences. Anti–NKR-P1B (2D12) hybridoma was a gift from Ly49H NK cells do not express Ly49C, resistance to MCMV is Dr. K. Iizuka (University of Minnesota, Minneapolis, MN). Ab staining for flow not disrupted in B6 mice (32, 33). It remains to be determined how cytometry was performed as described previously (47). 7-Aminoactinomycin the expression of other inhibitory NK cell receptors may influence D (eBioscience) staining was performed following the manufacturer’s in- the Ly49H-mediated NK cell response during MCMV infection. structions. Dead cells were excluded from analysis using the Fixable NKR-P1B, a member of the NKR-P1 receptor family on NK cells, Viability Dye (eBioscience). Sample acquisition was performed on a CyAN-ADP instrument using Summit software and data were analyzed is an inhibitory type II transmembrane C-type lectin–like receptor in using Kaluza software (Beckman Coulter). http://www.jimmunol.org/ mice (34–36). NKR-P1B is expressed on ∼60% of total NK cells in the spleen of B6 mice (37, 38). Engagement of NKR-P1B by its Adoptive transfer natural ligand, a member of the C-type lectin–related ligand family, For adoptive transfer into Ly49H-deficient mice, NK cells were enriched from Clr-b leads to NK cell inhibition (38–40). In rodents, the Nkrp1b spleens of B6 mice using an NK Cell Isolation Kit (Miltenyi Biotec), followed gene exhibits allelic polymorphisms among different strains, which by staining with fluorochrome-conjugated Abs against NK1.1, TCRb, Ly49H, may have evolved under selective pressure imposed by the pathogen and NKR-P1B and sorting using a MoFlo Astrios Sorter. Enriched NK cells (1–2 3 106 cells) or sorted NK cell subsets (1–2 3 105 cells) were trans- (35, 41, 42). At least three different alleles of the Nkrp1b gene have ferred into recipient mice with i.v. injections. Prior to transfer into recipient been reported in different mouse strains (22, 34–36). Similarly, the mice, NK cells were stained with Cell Proliferation Dye (eBioscience) fol- Nkrp1c gene, which is tightly linked to Nkrp1b, also shows allelic lowing the manufacturer’s instructions. Recipient mice were infected i.p. by guest on September 25, 2021 3 polymorphism (22, 35). The Nkrp1c gene encodes for the activating with 3 3 10 PFU of MCMV the following day. For adoptive transfer to NKR-P1C (NK1.1) receptor whose ligand has not yet been de- newborn BALB/c mice, NK cells were enriched from spleens of adult B6 mice using an NK Cell Isolation Kit (Miltenyi Biotec). Enriched NK cells scribed. However, it appears to have coevolved along with the were stained with fluorochrome-conjugated Abs against NK1.1, TCRb, Nkrp1b locus, possibly because of a selective pressure imposed by a Ly49H, and NKR-P1B and sorted using a MoFlo Astrios Sorter. Sorted NK pathogenic challenge (22). The rat CMV genome encodes for a cells (5–7 3 104 cells) were transferred i.p. into 4- to 5-d-old mice. Mice 3 2 C-type lectin–like protein with homology to Clr-b (42). Its interaction were infected i.p. with 5 10 PFU of MCMV the following day. with NKR-P1B inhibits NK cells and increases rat CMV virulence In vitro NK cell assays in rats (42). The MCMV genome also appears to encode for Clr-b– independent ligands, which can functionally engage NKR-P1B re- For intracellular IFN-g measurement and CD107a staining, splenocytes were incubated with plate-bound anti–NKR-P1C (NK1.1), IL-12 (5 ng/ml) ceptor in reporter cell assays (43). However, their identity, and and IL-18 (5 ng/ml), or PMA (10 ng/ml) and ionomycin (1 mg/ml) (PMA/ whether they bear homology to Clr-b, remains to be determined. iono) for 5 h. Brefeldin A and monensin (eBioscience) were added for the In the current study, we show that NKR-P1B negatively regulates the last 4 h of incubation. Intracellular IFN-g staining using Cytofix/Cytoperm NK cell–mediated immune response during MCMV infection. Ap- kit (BD Biosciences) and CD107a staining were performed as described proximately 50% of Ly49H+ NK cells in the spleen and liver of B6 mice previously (48). For the NK cell proliferation assay, splenocytes were in- + cubated with IL-2 or IL-15 (0, 10, and 100 ng/ml) for 48 h. Ki-67 staining express NKR-P1B. We observed a preferential expansion of the Ly49H was performed with anti–K-i67 Ab using Foxp3 staining buffer (eBio- NK cell subset, which does not express NKR-P1B during MCMV in- science) following the manufacturer’s instructions. fection. This subset also confers better protection against MCMV infection compared with the Ly49H+ NK cell subset which expresses Statistical analysis NKR-P1B. In addition, NKR-P1B deficiency in B6 mice results in Statistical significance was determined by the Student t test and the Mann– better virus clearance and faster recovery from MCMV infection. Whitney U test, where applicable, with a cutoff p value of 0.05. Results Materials and Methods Phenotypic and functional differences between NK cell subsets Mice and infections in steady-state condition C57BL/6 (B6) and BALB/c mice were purchased from The Jackson Labo- NK cells express multiple activating and inhibitory receptors in ratory (Bar Harbor, ME). NKR-P1B–deficient mice have been previously an overlapping fashion (21). The integration of signals from 2/2 described (38). Clr-b–deficient (Ocil )micewereagiftfromDrs. these receptors regulates NK cell function. We determined the V. Kartsogiannis and M. T. Gillespie (Monash Medical Centre, Clayton, VIC, Australia). Ly49H-deficient (Ly49h2/2) mice have been described expression profiles of the activating Ly49H and the inhibitory previously (44). MCMV Smith strain was passaged once in BALB/c mice, NKR-P1B receptors on splenic NK cells in B6 mice by flow and salivary gland–isolated virus titers were determined by plaque assay on cytometry analysis. On the basis of the expression of these two The Journal of Immunology 2327 receptors, four phenotypically distinct NK cell subsets were Increased proliferation of Ly49H+ NK cells that lack NKR-P1B identified (Fig. 1A). Analysis of CD11b and CD27 expression during MCMV infection + associated with NK cell maturation showed that the NKR-P1B Activated NK cells undergo rapid proliferation during a viral NK cells, in general, appear to be more mature compared with 2 infection (27–29). MCMV infection of B6 mice induces two the NKR-P1B NK cells (Fig. 1B). Approximately 40% of distinct phases of NK cell proliferation (28). The initial phase, Ly49H2NKR-P1B2 NK cells exhibit an immature CD11b2 2 which occurs within the first 2 d following infection, involves CD27 phenotype (Fig. 1B, compare R4 with R1–3). Among the the bulk of NK cells regardless of Ly49H expression. This is Ly49H+ NK cells, those that express NKR-P1B (Ly49H+NKR- + followed by a subsequent proliferation phase that involves P1B ) represent a higher proportion of cells exhibiting a fully + specifically the Ly49H NK cells (28). We determined the pro- mature CD11b+CD272 phenotype compared with those that lack portion of proliferating cells among the different NK cell sub- expression of NKR-P1B (Ly49H+NKR-P1B2) (Fig. 1B, com- sets in the spleen and liver of infected mice on days 1.5, 3, and pare R2 and R3). The Ly49H+NKR-P1B+ NK cell subset also 5p.i.byflowcytometryanalysisofKi-67expression,whichis has a higher representation of cells expressing NKG2A/C/E and 2 associated with cell proliferation (49). Maximum proliferation Ly49C/I, compared with the Ly49H+NKR-P1B NK cell subset of NK cells was observed on day 3 p.i. in the spleens and livers (Fig. 1C, compare R2 and R3). Although the majority of the 2 + of the infected mice (Fig. 3A, 3B). In the spleen, the highest Ly49H NKR-P1B NK cells express NKG2A/C/E, most of the + 2 2 frequency of proliferating cells was detected among the Ly49H Ly49H NKR-P1B NK cells do not express Ly49C/I, consis- 2 NKR-P1B NK cell subset on days 1.5 and 3 p.i. (Fig. 3A, tent with their immature phenotype (Fig. 1C). compare R3 with R1, 2, and 4). In the liver, an equally high Functional heterogeneity among the NK cell subsets was Downloaded from frequency of proliferating cells was detected among the Ly49H+ observed regarding IFN-g production, degranulation, and pro- 2 2 2 2 NKR-P1B and the Ly49H NKR-P1B NK cell subsets on liferation upon in vitro stimulation. Although the Ly49H NKR- 2 days 1.5 and 3 p.i., which was significantly higher than the P1B subset exhibited the highest level of degranulation remaining two subsets (Fig. 3B, compare R3 and 4 with R1 and (CD107a+) (Fig. 1D, compare R4 with R1–3), IFN-g production + 2). In both organs, the frequency of proliferating cells was was a property of the NKR-P1B NK cells with the highest + 2 + 2 + significantly higher among the Ly49H NKR-P1B subset representation of IFN-g cells among the Ly49H NKR-P1B + + http://www.jimmunol.org/ subset (Fig. 1D, compare R1 with R2–4). In contrast, highest compared with the Ly49H NKR-P1B subset, reflecting the proliferation capacity upon stimulation with IL-2 or IL-15 was increased prevalence of the former subset following MCMV exhibited by the Ly49H+NKR-P1B2 NK cell subset (Fig. 1E, 1F, infection (Fig. 2B). These results were further confirmed by an in vivo proliferation dye dilution assay to assess cell prolifera- compare R3 with R1, 2, and 4). These data together demonstrate 2 + the phenotypic and functional heterogeneity among NK cell tion. B6 splenic NK cells (CD45.1 CD45.2 ) were stained with a cell proliferation dye and adoptively transferred into Ly49H- subsets expressing different combinations of the activating + 2 Ly49H and the inhibitory NKR-P1B receptors, which may pro- deficient mice on a CD45.1 CD45.2 congenic background prior to MCMV infection. On day 3 post-MCMV infection, foundly influence their function during immune responses to + pathogens. proliferating donor CD45.2 NK cells in the spleen were de- by guest on September 25, 2021 tected as a function of reduced fluorescence intensity of the Expansion of Ly49H+ NK cells that lack NKR-P1B during proliferation dye. In agreement with the Ki-67 expression data, a MCMV infection significantly higher frequency of proliferating cells was detected + 2 NK cells are crucial for virus clearance and resistance to MCMV among the Ly49H NKR-P1B NK cells compared with the infection (25, 26). In B6 mice, the activating Ly49H receptor on other three subsets (Fig. 3C, compare R3 with R1, 2 and 4). NK cells recognizes the MCMV m157 protein resulting in NK Taken together, these data demonstrate an increased prolifera- + cell activation and elimination of the infected cells (23, 24). We tion of Ly49H NK cells that do not express the inhibitory NKR- assessed the distribution of NK cell subsets identified based on P1B receptor, thus leading to their expansion in the spleen and the expression of Ly49H and NKR-P1B in the spleen and liver of liver of MCMV-infected B6 mice. B6 mice prior and subsequent to MCMV infection. NK cell We also pursued two other possible scenarios that may lead to + 2 numbers rapidly decrease in the spleen, concurrent with an in- the expansion of the Ly49H NKR-P1B NK cell subset during crease in the liver following infection (Fig. 2A). As was shown MCMV infection. These are as follows: 1) increased cell death of + + previously by others, the Ly49H+ NK cell compartment expands the Ly49H NKR-P1B NK cell subset, thereby decreasing their over time following MCMV infection (28, 29). Interestingly, frequency and numbers; and 2) loss of NKR-P1B receptor ex- most of this increase was apparent among the Ly49H+NKR- pression during MCMV infection resulting in differentiation of + + + 2 P1B2 NK cell subset (Fig. 2B). Although the frequency of the Ly49H NKR-P1B NK cells to Ly49H NKR-P1B NK cells. All Ly49H2NKR-P1B+ subset decreases and the Ly49H+NKR- of the four NK cell subsets, described above (Fig. 1A), showed P1B2 subset increases dramatically during the course of MCMV comparable levels of cellular death as revealed by flow cytometry + infection, the Ly49H+NKR-P1B+ subset remains more or less analysis of 7AAD apoptotic/dead cells following MCMV unchanged in the spleen and liver (Fig. 2C, 2D). We also ob- infection (Supplemental Fig. 1). Adoptive transfer of sorted 2 2 served an increase in the frequency of the Ly49H2NKR-P1B2 Ly49H+NKR-P1B+ and Ly49H+NKR-P1B NK cells (CD45.1 NKcellsubsetinthespleenofinfectedmiceondays1.5and3 CD45.2+ background) into adult Ly49H-deficient recipients 2 postinfection (p.i.); however, this was highly variable between (CD45.1+CD45.2 background) followed by MCMV infection the animals (Fig. 2C). On day 5 p.i., Ly49H+NKR-P1B2 NK showed that loss of NKR-P1B expression could not explain the 2 cells are the predominant NK cell subset, both in frequency and preferential expansion of the Ly49H+NKR-P1B NK cell subset, in overall numbers, in the spleen and liver of the infected mice which was also evident in these experiments (Supplemental Fig. (Fig. 2C, 2D). These data demonstrate that the increase in the 2A, 2B). Although a major proportion of the donor cells corre- frequency of Ly49H+ NK cells during MCMV infection in B6 sponded to the sorted input phenotype following MCMV mice is restricted to the subset that does not express the inhib- infection, plasticity of NKR-P1B expression appeared to be itory NKR-P1B receptor. comparable in both populations (Supplemental Fig. 2A, 2B). 2328 NEGATIVE IMPACT OF NKR-P1B ON INNATE IMMUNITY TO MCMV Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Phenotypic and functional characterization of NK cell subsets in the spleen of B6 mice. Spleen leukocytes were analyzed by ﬂow cytometry ex vivo and following in vitro stimulations. (A) Four different NK cells subsets (R1: Ly49H2NKR-P1B+, R2: Ly49H+NKR-P1B+, R3: Ly49H+NKR-P1B2, and R4: Ly49H2NKR-P1B2) are detected based on the expression of Ly49H and NKR-P1B. (B) Frequency of expression of CD11b and CD27 on different NK cell subsets. (C) Frequency of expression of NKG2A/C/E and Ly49C/I on different NK cell subsets. (D) IFN-g production and degranulation (CD107a expression) by different NK cell subsets following in vitro simulation with the indicated stimuli. (E and F) Frequencies of proliferating NK cell subsets measured by ﬂow cytometry analysis of intracellular Ki-67 expression following stimulation with different concentrations of IL-2 (E) and IL-15 (F). Data in panels (B) and (C) are pooled from two to three independent experiments. Data in panels (A), (D), (E), and (F) are representative of at least two independent experiments. Statistical analysis was performed by Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 2329 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. NK cell subset distribution in the spleen and liver of MCMV-infected B6 mice. Mice were infected with 2 3 104 PFUMCMVSmithstraini.p.Spleen and liver leukocytes were isolated and analyzed by ﬂow cytometry. (A) NK cell numbers in the spleen and liver at different times p.i. (B) Distribution of NK cell subsets based on the expression of Ly49H and NKR-P1B in the spleen and liver of infected mice. (C and D) Frequency (left panel) and numbers (right panel) of NK cell subsets (R1: Ly49H2NKR-P1B+, R2: Ly49H+NKR-P1B+, R3: Ly49H+NKR-P1B2, and R4: Ly49H2NKR-P1B2) in the spleen (C)andliver(D) at different time points p.i. Data are pooled from at least two independent experiments. Statistical analysis was performed by Student t test. *p , 0.05, **p , 0.01, ***p , 0.001.

Reduced proliferation of Ly49H+NKR-P1B+ NK cells is MCMV-infected Clr-b–deficient mice (Fig. 4A). We also observed a independent of the Clr-b ligand interaction variable expansion of Ly49H2NKR-P1B2 NK cells in the spleen NKR-P1B is an inhibitory receptor that can negatively influence NK but not the liver of MCMV-infected Clr-b–deficient mice on days + 2 cell function when engaged by its Clr-b ligand (34, 38–40). To 1.5 and 3; however, the Ly49H NKR-P1B NK cells were the determine whether the reduced proliferation of Ly49H+NKR-P1B+ predominant NK cell subset on day 5 p.i. (Fig. 4A). Flow cytometry NK cells compared with Ly49H+NKR-P1B2 NK cells is due to the analysis of Ki-67 expression revealed a significant increase in the + 2 inhibitory signals delivered through NKR-P1B:Clr-b interaction, we frequency of proliferating cells among the Ly49H NKR-P1B NK 2 analyzed NK cell proliferation upon MCMV infection in Clr-b– cell subset compared with the Ly49H+NKR-P1B+ and the Ly49H deficient mice. Similar to the B6 mice, we observed expansion of NK cell subsets in the spleen and liver of the infected Clr-b– the Ly49H+NKR-P1B2 NK cells in the spleen and the liver of deficient mice (Fig. 4B, 4C, compare R3 with R1, 2, and 4). We also 2330 NEGATIVE IMPACT OF NKR-P1B ON INNATE IMMUNITY TO MCMV Downloaded from http://www.jimmunol.org/

FIGURE 3. Differential proliferation of NK cell subsets in the spleen and liver of MCMV-infected B6 mice. Mice were infected with 2 3 104 PFU MCMV Smith strain i.p. Spleen and liver leukocytes were isolated and analyzed by flow cytometry. (A and B) Frequency of Ki-67+ NK cells in the spleen (A)andliver 2 + + + + 2 2 2 (B) of infected mice at different time points p.i. (R1: Ly49H NKR-P1B , R2: Ly49H NKR-P1B , R3: Ly49H NKR-P1B ,andR4:Ly49H NKR-P1B ).(C) by guest on September 25, 2021 Flow cytometry analysis of adoptively transferred NK cells (CD45.2+) in CD45.1+ congenic Ly49H-deficient recipients infected with 3000 PFU of MCMV Smith strain for 3 d (left panel). Frequency of NK cells that have undergone cell division in the spleen of infected mice at day 3 p.i. (right panel). Data are pooled from at least two independent experiments. Statistical analysis was performed by Student t test. *p , 0.05, ***p , 0.001. observed robust proliferation of NK cells in livers from Clr-b– between the expression of maturation markers (CD11b and CD27), as deficient mice on day 5 p.i. in contrast to wild-type (WT) livers where well as NK cell receptors (Ly49C/I and NKG2A/C/E), with the level NK cell proliferation decreased after day 3 p.i. (compare Fig. 4C of proliferation in in vitro stimulations and during MCMV infection. with Fig. 3B). Ly49H+NKR-P1B2 NK cells also exhibited higher Analysis of in vitro proliferation of cells within the Ly49H+NKR- proliferation capacity than the other NK cell subsets when splenic P1B+ and Ly49H+NKR-P1B2 NK cell compartments showed that the NK cells from Clr-b–deficient mice were stimulated in vitro with majority of proliferating cells exhibited a less mature CD11b+CD27+ IL-15 or IL-2 (Fig. 4D, 4E, compare R3 with R1, 2, and 4). Both the phenotype. This was true for NK cells obtained from both the WT in vivo and in vitro cell proliferation properties of NK cell subsets in (Fig. 5A) and Clr-b–deficient (Fig. 5B) mice. However, during Clr-b–deficient mice appear to be similar to what we have observed MCMV infection, a correlation between higher proliferation and less in B6 mice, wherein the Ly49H+NKR-P1B2 NK cell subset dem- mature phenotype was observed only in the Ly49H+NKR-P1B+ but onstrates a higher proliferative capacity than the Ly49H+NKR-P1B+ not the Ly49H+NKR-P1B2 NK cell compartment in both the WT NK cell subset. These data together demonstrate that the differences (Fig. 5A) and Clr-b–deficient (Fig. 5B) mice. In contrast to in vitro in cell proliferation capacity of NKR-P1B+ and NKR-P1B2 subsets stimulation, a large proportion of Ly49H+NKR-P1B2 NK cells with a of the Ly49H+ NK cells during MCMV infection is independent of mature CD11b+ CD272 phenotype proliferated efficiently during an the interactions between NKR-P1B and Clr-b. MCMV infection (Fig. 5A, 5B). We also observed that NK cells

+ + expressing the inhibitory Ly49C/I receptor proliferated less than those Reduced proliferation of Ly49H NKR-P1B NK cells during devoid of this receptor in the Ly49H+NKR-P1B+ cell compartment MCMV infection is confined to the subset exhibiting a mature + 2 (Fig. 5C, 5D), but this difference was less significant compared with CD11b CD27 phenotype the difference in proliferation of cells at different maturation stages. NK cells with an immature phenotype (CD11b+CD27+ or CD11b2 No correlation between the expression of NKG2A/C/E receptors and CD27+) tend to have a higher homeostatic proliferative capacity NK cell proliferation was observed during both in vitro stimulation than the ones which have a mature (CD11b+CD272) phenotype and in vivo MCMV infection (Supplemental Fig. 3). The data col- (50, 51). Because we observed that a significantly higher pro- lectively indicate that during in vitro stimulation, fully mature NK portion of cells within the NKR-P1B+ NK cell subset exhibit a cells exhibit less proliferative capacity than the less mature NK cell mature phenotype compared with the NKR-P1B2NK cell subset subsets; however, during MCMV infection, mature NK cells lacking (Fig. 1B), we decided to determine whether a correlation exists NKR-P1B expression are highly proliferative. In contrast, proliferation The Journal of Immunology 2331 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Differential proliferation of NK cell subsets during MCMV infection is independent of NKR-P1B:Clr-b interactions. Clr-b–deficient mice were infected with 2 3 104 PFU of MCMV Smith strain i.p. (A) Distribution of NK cell subsets based on the expression of Ly49H and NKR-P1B in the spleen and liver of infected mice. (B and C) Frequencies of Ki-67+ NK cells in the spleen (B) and liver (C) of infected mice at different time points p.i. (D and E) Frequencies of Ki-67+ NK cells following in vitro stimulation of splenocytes with different concentrations of IL-2 (E) and IL-15 (D). Panels (B and C) represent data pooled from at least two independent experiments. R1: Ly49H2NKR-P1B+, R2: Ly49H+NKR-P1B+, R3: Ly49H+NKR-P1B2, and R4: Ly49H2NKR-P1B2. Statistical analysis was performed by Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. of NKR-P1B+ NK cells with a mature phenotype is inhibited in a Clr- by different NK cell subsets in the spleen and liver of MCMV-infected b–independent manner during MCMV infection. mice by intracellular staining and flow cytometry. Following MCMV infection, production of IFN-g and granzyme B by NK cells peaks on Functional differences between NK cell subsets in the liver and day 1.5 p.i. in the spleen (Fig. 6A, 6B). Althpugh NK cell production of spleen of MCMV-infected mice IFN-g drops after day 1.5, granzyme B production persists longer. Both NK cell–mediated cytotoxicity and cytokine production are two im- the NKR-P1B+ and NKR-P1B2 subsets of the Ly49H+ NK cells are portant functions of NK cells in response to an infection (52). The equally good producers of IFN-g and granzyme B, which is signifi- phenotypic and proliferative differences between different NK cell cantly higher than the Ly49H2 subsets (Fig. 6A, 6B, compare R2 and 3 subsets described above may influence the way NK cells perform these with R1 and 4). In contrast to the splenic NK cells, liver NK cells do not functions. Therefore, we analyzed IFN-g and granzyme B production appear to be good producers of IFN-g, whereas all NK cell subsets 2332 NEGATIVE IMPACT OF NKR-P1B ON INNATE IMMUNITY TO MCMV

FIGURE 5. Reduced proliferation of Ly49H+ NKR-P1B+ NK cells exhibiting a mature CD11b+ CD272 phenotype. In vitro stimulation was performed using splenic NK cells and IL-15 at 100 ng/ ml. MCMV infection was performed i.p. with 2 3 104 PFU of MCMV Smith strain. (A and B) Fre- quencies of proliferating (Ki-67+) cells among the + 2 +

mature CD11b CD27 and immature CD11b Downloaded from CD27+ NK cell subsets measured by flow cytometry analysis in B6 (A) and Clr-b–deficient (B) mice. (C and D) Frequencies of proliferating (Ki- 67+) cells among Ly49C/I+ and Ly49C/I2 NK cell subsets measured by flow cytometry analysis in B6 (C) and Clr-b–deficient (D) mice. Data are pooled

from two independent experiments. Statistical http://www.jimmunol.org/ analysis was performed by Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 25, 2021

analyzed in the liver were efficient producers of granzyme B (Fig. 6C, greater proliferative capacity and increased numbers of the NKR- 6D). We also observed that ∼40% of Ly49H2NKR-P1B+ NK cells P1B2 subset during an MCMV infection may confer greater constitutively produced granzyme B in the liver of naive B6 mice (Fig. protection to the host. To determine whether this is indeed the 6D). The differences in the NK cell response against MCMV infection case, we adoptively transferred sorted Ly49H+NKR-P1B+ and in the spleen and the liver perhaps reflects differences in the patho- Ly49H+NKR-P1B2 NK cells isolated from the spleen of B6 mice genesis of MCMV infection in the two anatomically distinct organs. into MCMV-susceptible BALB/c neonate mice (Fig. 7A). Recip- MCMV infection induces expression of the IL-2R a-chain (CD25) ient mice were then infected with MCMV and monitored for and formation of a high-affinity IL-2R on NK cells, which can in- survival. Adoptive transfer of Ly49H+ NK cells into BALB/c fluence their proliferation in response to IL-2 stimulation (53). We neonate mice have been shown to protect them against MCMV determined the surface expression of CD25 induced on splenic NK infection (54, 55). We observed better survival of the mice that cell subsets following MCMV infection. On day 3 p.i., CD25 was received Ly49H+NKR-P1B2 NK cells as compared with those expressed at significantly higher levels on the Ly49H+ NKR-P1B2 that received Ly49H+NKR-P1B+ NK cells at different cell num- NKcellsubsetcomparedwiththeLy49H+NKR-P1B+ and the bers (Fig. 7B). In fact, transfer of 5 3 104 Ly49H+NKR-P1B2 NK Ly49H2 NK cell subsets (Fig. 6E, compare R3 with R2 and 4). The cells seemed to provide the same level of protection as the transfer reducedlevelsofCD25onLy49H+NKR-P1B+ NK cells may have of 7 3 104 Ly49H+NKR-P1B+ NK cells. These data show that the contributed to their lower proliferation capacity compared with the Ly49H+ NK cell subset which lacks the expression of NKR-P1B is Ly49H+KR-P1B2 NK cell subset. Therefore, despite the differences more protective against MCMV infection compared with those in the proliferative capacity between the Ly49H+NKR-P1B2 and the that express NKR-P1B. Ly49H+NKR-P1B+ NK cell subsets, both seem to be equally effi- Genetic deficiency of NKR-P1B, but not Clr-b, results in faster cient producers of IFN-g and granzyme B during MCMV infection. recovery from an MCMV infection + Better protection against MCMV infection by Ly49H NK cells As demonstrated above, the absence of NKR-P1B expression on a that lack NKR-P1B subset of Ly49H+ NK cells is associated with faster proliferation Although both the NKR-P1B+ and NKR-P1B2 subsets of the Ly49H+ and better protection against MCMV infection. To further deter- NK cells are efficient producers of effector molecules, the mine whether NKR-P1B:Clr-b recognition plays a role in NK The Journal of Immunology 2333 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. IFN-g and granzyme B (GzmB) production, and CD25 upregulation by NK cell subsets in spleen and liver of MCMV-infected B6 mice. Mice were infected with 2 3 104 PFU of MCMV Smith strain i.p. Intracellular levels of IFN-g and GzmB in NK cells were measured by flow cytometry at different time points p.i. (A) Frequencies of IFN-g+ NK cells in the spleen. (B) Frequencies of GzmB+ NK cells in the spleen. (C) Frequencies of IFN-g+ NK cells in the liver. (D) Frequencies of GzmB+ NK cells in the liver. (E) Fold increase in the surface expression of CD25 on NK cell subsets following MCMV infection, shown as the relative mean fluorescence intensity (MFI) measured by flow cytometry. Data are pooled from at least two independent experiments. R1: Ly49H2NKR-P1B+, R2: Ly49H+NKR-P1B+, R3: Ly49H+NKR-P1B2, and R4: Ly49H2NKR-P1B2. Statistical analysis was performed by Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. cell–mediated response against MCMV infection, we used mice detected in spleens from NKR-P1B–deficient mice compared with with genetic deficiency of either NKR-P1B (Nkrp1b2/2) or Clr-b WT mice on day 3 p.i. (Fig. 8B), which also corresponds to the (Clr-b2/2) (38, 56). We infected groups of age- and gender- time when the NKR-P1B–deficient mice start to recover body matched WT, NKR-P1B–deficient, and Clr-b–deficient mice weight (Fig. 8A). On day 8 p.i., MCMV was undetectable in with 2 3 104 PFU MCMV (i.p.) and measured their body weight spleens from ∼50% of NKR-P1B–deficient mice, whereas all of daily. WT and Clr-b–deficient mice lost body weight until day 5 p.i., the WT- and Clr-b–deficient spleens still harbored the virus following which the body-weight recovered (Fig. 8A). In contrast, (Fig. 8C). Similarly, MCMV was not detectable in the livers from NKR-P1B–deficient mice started recovering on day 3 p.i. ∼50% of WT and NKR-P1B–deficient mice but could be detected (Fig. 8A). Body weight loss was most severe in Clr-b–deficient in the livers from all of the infected Clr-b–deficient mice on day 8 mice, followed by the WT mice, and least severe in the NKR- p.i. (Fig. 8C). Analysis of NK cell numbers, Ly49H, CD11b, and P1B–deficient mice. Significantly lower titers of MCMV were CD27 expression, and IFN-g production by NK cells revealed no 2334 NEGATIVE IMPACT OF NKR-P1B ON INNATE IMMUNITY TO MCMV

FIGURE 7. Better protection of BALB/c pups by MCMV-speciﬁc NK cells that lack expression of NKR-P1B. Splenic NK cells were sorted and adoptively transferred to 4- to 5-d-old BALB/c pups i.p. Pups were infected (i.p.) with 500 PFU of MCMV Smith strain the following day. (A) Sorted Ly49H+ NK cell subsets, with and without NKR-P1B expression, analyzed by ﬂow cytometry prior to adoptive transfers to the recipient pups. (B) Survival of BALB/c pups infected with MCMV following adoptive transfer of sorted Ly49H+ NK cell subsets, with (gray) and without (black) NKR-P1B expression (NKR- P1B+ and NKR-P1B2, respectively) at different cell numbers. The data are representative

of 10–11 pups in each group. Statistical anal- Downloaded from ysis was performed by log-rank test. *p , 0.05. http://www.jimmunol.org/ major differences between the NKR-P1B–deficient, Clr-b–deficient, and granzyme B, the greater protection offered by the Ly49H+NKR- and WT mice upon MCMV infection (data not shown). Interest- P1B2 NK cells is due to their higher in vivo proliferative capacity ingly, the enhanced protection of NKR-P1B–deficient mice was not and increased numbers compared with the Ly49H+NKR-P1B+ statistically observed upon infection using a Dm157-mutant strain, NK cells during MCMV infection. Interestingly, the expanded which resulted in a similar virus titer and disease course in both the NKG2C+ NK cells in humans infected with HCMV has been WT and NKR-P1B–deficient mice (Supplemental Fig. 4); however, shown to also exhibit a reduced expression of CD161 (NKR-P1A), we cannot exclude the possibility that this may be due to the distinct the closest homolog to mouse NKR-P1B (57). Whether signals background from which the Dm157-mutant was derived (i.e., the delivered through the engagement of NKR-P1A also have a neg- by guest on September 25, 2021 parental MW97.01 strain) versus the Smith strain. We are currently ative impact on NK cell response against HCMV infection in investigating this further. humans has not yet been established. Our study has provided Taken together, these data suggest that NKR-P1B, but not Clr-b, evidence in support of at least three possible and overlapping can negatively influence immune responses against MCMVand that mechanisms to account for the increased proliferation and better a genetic deficiency of NKR-P1B results in faster virus clearance and protection by the NKR-P1B2 NK cell subset against MCMV. recovery from MCMV-Smith infection. These are as follows: 1) greater intrinsic proliferative capacity of the Ly49H+NKR-P1B2 NK cells compared with the Ly49H+NKR- Discussion P1B+ NK cells, 2) increased inhibitory signals through NKR- Resistance to MCMV infection is associated with a robust MCMV- P1B and other inhibitory receptors on Ly49H+NKR-P1B+ NK cells specific NK cell response in mice (17). In MCMV-resistant B6 during infection, and 3) inhibition of NKR-P1B+ NK cells by ligands mice, the activating Ly49H receptor on NK cells recognizes other than Clr-b. 2 MCMV m157 protein, which otherwise binds to the inhibitory Ly49H+NKR-P1B NK cells have a higher proliferative ca- Ly49I receptor in the MCMV-susceptible 129 mouse strain to pacity than Ly49H+NKR-P1B+ NK cells during MCMV infection. evade recognition by NK cells (23–26). Once activated, the However, this is also true when NK cells are stimulated with Ly49H+ NK cells undergo rapid proliferation and expansion in cytokines in vitro. Moreover, this appears to be independent of MCMV-infected B6 mice to control the virus infection (28, 29). NKR-P1B:Clr-b interactions because Clr-b–deficient NK cell Our studies demonstrate that ∼50% of Ly49H+ NK cells in the subsets show a differential proliferative capacity in vitro similar to 2 spleen and liver of B6 mice also express the NKR-P1B receptor. the WT NK cell subsets. Therefore, the Ly49H+NKR-P1B NK NKR-P1B is an inhibitory type II transmembrane C-type lectin– cell subset appears to have a higher intrinsic proliferative capacity like receptor expressed on ∼60% of total NK cells in B6 mice than the Ly49H+NKR-P1B+ NK cell subset in the absence of an 2 (34–38). We have found that the NKR-P1B2 subset of Ly49H+ infection. Within the Ly49H+NKR-P1B NK cell compartment, a NK cells proliferate rapidly to become the predominant NK cell significantly higher proportion of cells exhibited a less mature 2 subset in the spleen and liver of MCMV-infected B6 mice and are CD11b CD27+ or CD11b+CD27+ phenotype, and fewer cells 2 also highly protective against MCMV infection. Moreover, genetic displayed a mature CD11b+CD27 phenotype. NK cells at dif- deficiency of NKR-P1B results in lower virus titer and faster re- ferent maturation stages have distinct tissue distributions, recovery from an MCMV infection. Signaling through the inhibitory sponsiveness, and homeostatic proliferative capacities (50, 51, 2 NKR-P1B receptor, therefore, appears to have a negative impact 58). The CD11b CD27+ and CD11b+CD27+ NK cells in the on the NK cell–mediated immune response against MCMV. Be- spleen and bone marrow represent the majority of proliferating NK cause both the NKR-P1B+ and NKR-P1B2 subsets of the Ly49H+ cells, whereas CD11b+CD272 NK cells represent the senescent NK cells are efficient producers of effector molecules such as IFN-g subset of NK cells (50). A higher representation of CD11b2CD27+ The Journal of Immunology 2335

large proportion of CD11b+CD272 NK cells in the Ly49H+NKR- P1B2 NK cell compartment was able to proliferate. Only the CD11b+CD272 NK cells in the Ly49H+NKR-P1B+ NK cell compartment showed a profound reduction in proliferative capacity. Therefore, although the mature CD11b+CD272 NK cells have a lower intrinsic proliferative capacity compared with the CD11b+CD27+ NK cells in vitro, they seem to proliferate efficiently during MCMV infection, although their proliferation is reduced if they express NKR-P1B receptor. This reduction appears to be independent of NKR-P1B:Clr-b interactions. During an MCMV infection, the Ly49H+NKR-P1B2 NK cell subset was found to express significantly higher levels of CD25, the high- affinity IL-2Ra-chain, compared with the Ly49H+NKR-P1B+ NK cell subset. Cytokines such as IL-2, IL-12, and IL-18 have been shown to be important for NK cell expansion during MCMV infection (53, 59). IL-12–dependent upregulation of the high- affinity IL-2R on NK cells during MCMV infection makes them highly responsive to low levels of IL-2 for proliferation (53).

Therefore, a higher level of CD25 expression on Ly49H+NKR- Downloaded from P1B2 NK cells may contribute to their increased proliferative capacity compared with the Ly49H+NKR-P1B+ NK cell subset. Signals delivered through the engagement of inhibitory receptors, such as NKG2A, Ly49C and I, and NKR-P1B, can inhibit NK cell activation and proliferation. We have observed an unequal + + distribution of NKG2A/C/E and Ly49C/I cells among the NK http://www.jimmunol.org/ cell subsets. A larger proportion of cells within the Ly49H+NKR- P1B+ NK cell compartment express NKG2A/C/E and Ly49C/I, compared with the Ly49H+NKR-P1B2 NK cell subset. NKG2A and Ly49C/I receptors recognize nonclassical and classical MHC- I molecules, respectively (60–62). HCMV infection has been shown to increase the expression of HLA-E on infected cells through association with leader peptide derived from HCMV UL40 protein (63). Engagement of NKG2A on human NK cells by the overexpressed HLA-E results in disruption of NK cell acti- by guest on September 25, 2021 vation (64, 65). In mice, salivary gland infection by MCMV results in the upregulation of NKG2A and its ligand Qa-1b, which could have a negative impact on NK cell function (66). We did not observe a correlation between the expression of NKG2A/C/E receptors and NK cell proliferation during MCMV infection. Al- though we did not distinguish between NK cell subsets expressing the inhibitory NKG2A or activating NKG2C and E receptors in our assays, the data agree with the CD94 gene-deletion study, which demonstrated no adverse effect of the disruption of NKG2A/C/E expression on NK cell–mediated control of MCMV infection (67). The Ly49C/I receptor has been shown to inhibit NK cell pro- FIGURE 8. Faster recovery of NKR-P1B–deficient mice from MCMV liferation during MCMV infection (31). Ly49H+Ly49C/I+ NK cells infection. Groups of age- and gender-matched WT (Nkrp1b+/+), NKR- proliferate less than Ly49H+Ly49C/I2 NK cells, and their fre- 2/2 2/2 P1B–deficient (Nkrp1b ), and Clr-b–deficient (Clr-b ) mice were quency decreases during MCMV infection, which is dependent on 3 4 A infected i.p. with 2 10 PFU MCMV Smith strain. ( ) Body weight was the interaction between Ly49C/I and MHC-I molecules (31). In 6 measured daily and is shown as percentage (mean SD) of initial body addition, simultaneous interaction of Ly49C and H with m157 weight before infection. (B) MCMV titer in the spleen of WT and NKR- variants from different MCMV isolates can disrupt NK cell acti- P1B–deficient mice was measured by plaque assay on MEF cells at different times p.i. (C) MCMV titer in spleen and liver from WT, NKR- vation (32, 33). In all of these studies, the inhibitory Ly49C/I P1B–deficient, and Clr-b–deficient mice measured on day 8 p.i. Data are receptor appears to negatively regulate NK cell function during + pooled from two to three independent experiments. Statistical analysis was MCMV infection. In our assays, the Ly49C/I NK cells in the + + performed by Student t test (A and B) and Mann–Whitney U test (C). *p , Ly49H NKR-P1B NK cell compartment show reduced prolifer- 2 0.05, **p , 0.01. ative capacity compared with the Ly49C/I NK cells. Therefore, a higher representation of Ly49C/I+ cells among the Ly49H+NKR- P1B+ NK cell subset may result in reduced activation and slower and CD11b+CD27+ NK cells among the Ly49H+NKR-P1B2 NK proliferation of this subset compared with the Ly49H+NKR-P1B2 cell subset than the Ly49H+NKR-P1B+ NK cell subset would be NK cell subset. expected to result in an increased proliferative capacity for the NKR-P1B interaction with its Clr-b ligand leads to NK cell former subset. We found this to be true when NK cells were inhibition (39, 40). Although the slower proliferation of Ly49H+ stimulated in vitro; however, during MCMV infection, a fairly NKR-P1B+ NK cells compared with Ly49H+NKR-P1B2 NK cells 2336 NEGATIVE IMPACT OF NKR-P1B ON INNATE IMMUNITY TO MCMV during MCMV infection is independent of NKR-P1B:Clr-b in- 9. Robbins, S. H., G. Bessou, A. Cornillon, N. Zucchini, B. Rupp, Z. Ruzsics, T. Sacher, E. Tomasello, E. Vivier, U. H. Koszinowski, and M. Dalod. 2007. teractions, the presence of other inhibitory ligands, which are ei- Natural killer cells promote early CD8 T cell responses against cytomegalovirus. ther induced by the infection or encoded by the viral genome, with PLoS Pathog. 3: e123. specificity for NKR-P1B remain a possibility. The NKR-P1B re- 10. Mitrovic´, M., J. Arapovic´, S. Jordan, N. Fodil-Cornu, S. Ebert, S. M. Vidal, A. Krmpotic´, M. J. Reddehase, and S. Jonjic´. 2012. The NK cell response to ceptor appears to be negative regulator NK cell responses to mouse cytomegalovirus infection affects the level and kinetics of the early CD8+ MCMV infection because mice with NKR-P1B deficiency are T-cell response. J. Virol. 86: 2165–2175. more resistant to MCMV infection compared with the WT mice. 11. Su, H. C., K. B. Nguyen, T. P. Salazar-Mather, M. C. Ruzek, M. Y. Dalod, and + ∼ + C. A. Biron. 2001. NK cell functions restrain T cell responses during viral in- However, NKR-P1B NK cells constitute 50% of Ly49H NK fections. Eur. J. Immunol. 31: 3048–3055. cells; hence, resistance to MCMV is not disrupted in WT mice. 12. Waggoner, S. N., M. Cornberg, L. K. Selin, and R. M. Welsh. 2011. Natural This could also explain why a genetic deficiency of NKR-P1B killer cells act as rheostats modulating antiviral T cells. Nature 481: 394–398. 13. Lang, P. A., K. S. Lang, H. C. Xu, M. Grusdat, I. A. Parish, M. 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