α2β1 Integrin Is Required for Optimal NK Cell Proliferation during Viral Infection but Not for Acquisition of Effector Functions or NK Cell−Mediated Virus Control This information is current as of September 28, 2021. Colby Stotesbury, Pedro Alves-Peixoto, Brian Montoya, Maria Ferez, Savita Nair, Christopher M. Snyder, Shunchuan Zhang, Cory J. Knudson and Luis J. Sigal J Immunol published online 3 February 2020 http://www.jimmunol.org/content/early/2020/01/31/jimmun Downloaded from ol.1900927

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 3, 2020, doi:10.4049/jimmunol.1900927 The Journal of Immunology

a2b1 Integrin Is Required for Optimal NK Cell Proliferation during Viral Infection but Not for Acquisition of Effector Functions or NK Cell–Mediated Virus Control

Colby Stotesbury, Pedro Alves-Peixoto, Brian Montoya, Maria Ferez, Savita Nair, Christopher M. Snyder, Shunchuan Zhang, Cory J. Knudson, and Luis J. Sigal

NK cells play an important role in antiviral resistance. The integrin a2, which dimerizes with integrin b1, distinguishes NK cells from innate lymphoid cells 1 and other leukocytes. Despite its use as an NK cell marker, little is known about the role of a2b1in NK cell biology. In this study, we show that in mice a2b1 deficiency does not alter the balance of NK cell/ 1 generation and slightly decreases the number of NK cells in the bone marrow and spleen without affecting NK cell maturation.

NK cells deficient in a2b1 had no impairment at entering or distributing within the draining lymph node of ectromelia virus Downloaded from (ECTV)–infected mice or at becoming effectors but proliferated poorly in response to ECTV and did not increase in numbers following infection with mouse CMV (MCMV). Still, a2b1-deficient NK cells efficiently protected from lethal mousepox and controlled MCMV titers in the spleen. Thus, a2b1 is required for optimal NK cell proliferation but is dispensable for protection against ECTV and MCMV, two well-established models of viral infection in which NK cells are known to be important. The Journal of Immunology, 2020, 204: 000–000. http://www.jimmunol.org/ atural killer cells are leukocytes of the innate immune The phenotypes and functions of NK cells overlap extensively system that belong to the group 1 of innate lymphoid cells with those of ILC1s, the only other member of the group 1 ILCs. N (ILCs). After developing in the bone marrow, NK cells For example, both ILC1s and NK cells express the typical NK cell– circulate between blood and tissues patrolling the body for in- activating receptors NKp46 and, in B6 mice, NK1.1. Both ILC1s fections and tumors. Following viral infections, NK cells are and NK cells need the transcription factor T-bet for their devel- rapidly recruited from the blood to secondary draining lymph opment (11). Moreover, NK cells and ILC1s produce IFN-g upon nodes (dLNs) and other infected tissues, where they produce stimulation. Because of this, ILC1s can participate in virus control IFN-g, kill infected cells, and proliferate, playing a critical role in tissues (12). Yet, ILC1s and NK cells also exhibit differences as a first line of antiviral defense (1, 2). For example, C57BL/6 that make them unique. ILC1s are tissue resident and do not cir- by guest on September 28, 2021 (B6) mice, which are normally resistant to mouse CMV culate in the blood, whereas NK cells circulate in the blood and (MCMV) and ectromelia virus (ECTV), become susceptible are transient within tissues. NK cells but not ILC1s are cytolytic when NK cells are eliminated or dysfunctional (3–7). Also, in and express the transcription factor eomesodermin (Eomes) (13–16). humans, NK cell–deficient individuals become sick or succumb At the cell surface, the major differences between ILC1s and NK to normally non–life-threatening infections with human CMV cells is in the expression of integrins. ILC1s but not NK cells express or with varicella zoster (8–10). the integrin b3 and some but not all ILC1s express integrin a1(12). In contrast, NK cells but not ILC1s express the integrin aM (CD11b, Department of Microbiology and Immunology, Thomas Jefferson University, also expressed by other unrelated leukocytes) and the integrin a2(in Philadelphia, PA 19107 this study a2; also known as CD49b). ORCIDs: 0000-0002-6082-6838 (P.A.-P.); 0000-0002-1969-2911 (B.M.); 0000-0001- Integrins are cell surface heterodimeric receptors formed by an a- 6725-0459 (S.N.); 0000-0003-1370-7198 (C.M.S.); 0000-0001-6642-5472 (L.J.S.). and a b-chain (17). Integrins function in the adhesion of leukocytes Received for publication August 2, 2019. Accepted for publication January 5, 2020. to the endothelium and their extravasation into lymph nodes (LNs) This work was supported by grants from the National Institute of Allergy and Infec- and inflamed tissues. Integrins are also important for the displace- tious Diseases (NIAID) (R01AI110457 and R01AI065544) and the National Institute ment of leukocytes within tissues and can participate in their de- on Aging (AG048602 to L.J.S.). B.M. and C.J.K. were supported by Grant T32 AI134646 from the NIAID. P.A.-P. was partially supported by a Ph.D. fellowship (PD/ velopment and activation (18). a2 dimerizes with the integrin b1(in BD/128078/2016) from the M.D./Ph.D. Program of the University of Minho-School of this study b1; also known as CD29) to form a2b1 (also known as Medicine funded by the Fundac¸a˜oparaaCieˆncia e Tecnologia. Research reported in this VLA-2), a receptor for collagens type I and III. Of note, b1is publication used the Flow Cytometry and Animal Laboratory facilities at the Sidney Kimmel Cancer Center at Jefferson Health and was supported by the National Cancer thought to be the only partner of a2, whereas b1 can heterodimerize Institute of the National Institutes of Health under Award P30CA056036. with 12 different a-chains (19, 20), including a1(a1b1) in ILC1s. Address correspondence and reprint requests to Dr. Luis J. Sigal, Thomas Jefferson Notably, a2b1 is also expressed by human NK cells (21). University, 233 South 10th Street, Philadelphia, PA 19107. E-mail address: Despite a2 being a reliable NK cell marker, the role of a2b1in [email protected] the biology of NK cells is not well understood. When the Lanier The online version of this article contains supplemental material. group discovered a2 on murine NK cells, they also showed that Abbreviations used in this article: a2, integrin a2; b1, integrin b1; dLN, draining lymph node; dpi, day postinfection; ECTV, ectromelia virus; Eomes, eomesodermin; two different anti-a2 mAbs, DX5 and HMa2, do not block the GzmB, granzyme B; hpi, hour postinfection; ILC, innate lymphoid cell; LN, lymph binding of NK cells to collagen-coated plates, despite that HMa2 node; K181, MCMV K181; MCMV, mouse CMV; ndLN, nondraining LN; qRT-PCR, blocks the a2b1-dependent binding of platelets to collagen. In quantitative RT-PCR, . addition, the Lanier group demonstrated that immobilized anti-a2 Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 mAbs did not stimulate IFN-g production in NK cells, nor did

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900927 2 ROLE OF a2b1 INTEGRIN IN NK CELLS they block NK cell killing of YAC-1 cells (22, 23). Together, these (American Type Culture Collection; ATCC CRL-1972TM) with a multi- data suggested that a2b1 may not be essential for NK cell func- plicity of infection of 0.01 for 4–5 days postinfection (dpi) or until cyto- tion. Later, others showed that positive purification of NK cells pathic effect in culture was observed. Cells and supernatant were collected, centrifuged at 2600 3 g for 10 min, and resuspended in 10 ml of complete using DX5 mAb negatively affected their ability to produce IFN-g RPMI 1640 (Corning) supplemented with 10% FBS, 10 mM HEPES in vitro, kill target cells in vitro and in vivo, and impaired their (Corning), 1 mM sodium pyruvate (Corning), 100 U penicillin (Corning), motility in LNs (24). However, these data did not directly demon- and 100 mg/ml streptomycin (Corning). Debris were then lysed with a strate a physiological role for a2b1. More recently, it was shown Dounce homogenizer while on ice and spun. Supernatant was collected, transferred to ultracentrifuge tubes (Beckman Coulter), and spun at that NK cells interacted with collagen fibers in the dLNs of mice 50,000 3 g for 1 h at 4˚C. The supernatant was discarded, and the infected in the ear flaps with Toxoplasma gondii, and that admin- pellet was resuspended in 500 ml of complete RPMI 1640. V70 stocks istration of HMa2 in the ear flap decreased the presence of NK cells were produced by infecting BALB/c mice (.3 wk old) with 10 PFU of in T. gondii–infected foci in dLNs (25). This suggested that a2b1 tissue-cultured V70 for 21 d. Whole salivary glands were harvested and interactions with collagen might be involved in NK cell migration placed in 1 ml of sterile PBS (Corning), processed with a tissue grinder, and diluted to a 10% mixture of homogenate to media with a final to infected foci. Similarly, a study with human NK cells in vitro also concentration of 5% DMSO. Virus titers were determined by infecting suggested that a2b1 might participate in the interaction of NK cells M210-B4 monolayers for 4–5 d and counting the resulting plaques. with collagen, but the data were correlative (21). This protocol was adapted from a published protocol (27). To further understand the role of a2b1 in NK cell biology, we Cell depletions produced mice with specific genetic ablations of a2 in NKp46+ cells. Using these mice, we show that a2b1 affects but is not NK cells were depleted by i.p. inoculation of 100 mgNK1.1mAb (PK136; Bio X Cell) 1 d before injection and 1 dpi with ECTV. For critical for NK development and maturation. Moreover, we also MCMV infection, NK cells were depleted similarly 1 d before and 1 and Downloaded from show that during ECTV infection a2b1 is not necessary for NK 3 dpi with MCMV. recruitment to or distributing within the dLN of ECTV-infected Immunofluorescent microscopy mice or at becoming effectors after ECTV or MCMV infection. Notably, a2b1 is required for optimal NK proliferation in re- Preparation of nondraining LNs (ndLNs) and dLNs was performed as sponse to ECTV and increases in numbers in response to MCMV previously described (28). Ten-micrometer sections were stained with allophycocyanin–NK1.1 (PK136; BioLegend) and mounted in ProLong but is dispensable for survival to ECTV and the control of MCMV Diamond plus DAPI (Thermo Fisher Scientific). Images were collected http://www.jimmunol.org/ titers in the spleen. These results indicate that a2b1 is not es- with a Nikon A1R laser scanning microscope. For better visualization, sential for NK cell differentiation or effector function. Moreover, all the photographs were assembled in a single file, and the contrast and we demonstrate that NK cells with defective proliferation can still brightness was increased in unison using Adobe Photoshop. In addi- protect from ECTV lethality and can control MCMV replication. tion, allophycocyanin–NK1.1 is shown in the green channel to distin- guish NK cells more clearly from virally infected cells. Materials and Methods Cell isolation Mice Mice were euthanized by cervical dislocation. Single‐cell suspensions All the procedures involving mice were carried out in strict accordance were prepared from spleen and bone marrow and lysed for RBCs using ‐ ‐ by guest on September 28, 2021 with the recommendations in the eighth edition of the Guide for the Care ammonium chloride potassium (ACK) lysis buffer, and cells were washed with RPMI 1640 (Corning) supplemented with 5% FCS and later used for and Use of Laboratory Animals of the National Research Council of the National Academies. All protocols were approved by the Thomas Jefferson flow cytometric analysis. To obtain single-cell suspensions, LNs were first University Institutional Animal Care and Use Committee. All mice used incubated in Liberase TM (1.67 Wu¨nsch units/ml) (Sigma-Aldrich) in PBS in experiments were 6–12-wk-old Ncr1Cre+-Itgb1fl/fl or Ncr1Cre+-Itga2fl/fl with 25 mM HEPES for 30 min at 37˚C before adding PBS with 25 mM with appropriate littermate controls. No sex differences were observed. HEPES plus 10% FBS to halt the digestion process, followed by me- m C57BL/6 (B6) and B6.CD45.1 mice were purchased from Charles River chanical disruption of the tissue through a 70- m filter. Laboratories directly for experiments or as breeders. Aged B6 mice Flow cytometry (18–20 mo) were obtained from the National Institute of Aging aged colony at Charles River Laboratories. B6.Cg-Itga2tm1.1Tkun/J (a2fl/fl) and Flow cytometry to characterize NK cells in the bone marrow and spleen was B6;129-Itgb1tm1Efu/J (b1fl/fl) mice were purchased from Jackson Labora- performed as previously described (29). To determine NK cell responses in tories. Of note, the b1fl/fl mice originally obtained from Jackson were in the LNs, intact LNs were incubated at 37˚C for 1 h in media containing mixed 129/B6 background and carried the NK complex in six 10 mg/ml brefeldin A and then made into single-cell suspensions. The cells from the 129 strain. Therefore, they lacked NK1.1. To solve this problem, were then stained for cell surface molecules, fixed, permeabilized, and the original b1fl/fl mice were backcrossed to C57BL/6 (B6) mice for two stained for intracellular molecules using the Cytofix/Cytoperm Kit (BD generations to generate b1fl/fl mice expressing NK1.1 homozygously. Biosciences) or the eBioscience Foxp3/Transcription Factor Staining Kit C57BL/6-Ncr1tm1.1(iCre)Viv/Orl mice (Ncr1-Cre)wereagiftofDr.E.Vivier (Invitrogen) according to the manufacturer’s instructions. The following (Marseille, France). Colonies were bred at Thomas Jefferson University Abs were used: FITC–CD3ε (clone 145-2C11; BioLegend), allophycocyanin/ under specific pathogen–free conditions. Fire750-CD11b/BV605-CD11b (clone M1/70; BioLegend), PerCP/Cy5.5-CD27 (clone LG.3A10; BioLegend), PE/Cy7-CD29 (clone HMb1-1; BioLegend), Viruses and infection PE–CD45.1 (clone A20; BioLegend), Percp-cy5.5-CD45.2 (104; Bio- ECTV-Moscow strain (American Type Culture Collection; ATCC Legend), BV421–CD49b (clone RB6-8C5; BioLegend), AF488–Eomes g VR-1374) and ECTV-mCherry were propagated in tissue culture as (clone Dan11mag; eBioscience), PE/Cy7-IFN- (XMG1.2; BioLegend), previously described (26). Mice were infected in the footpad with 3000 allophycocyanin–Ki67 (16A8; BioLegend), PE/FITC-Ly49H (3D10; BioLegend), allophycocyanin–NK1.1/BV605-NK1.1 (PK136; BioLegend), or 100,000 PFUs of ECTV as indicated. For the determination of sur- allophycocyanin–NKp46 (clone 29A1.4; BioLegend), BV786–TCRb (clone vival, mice were monitored daily, and, to avoid unnecessary suffering, mice were euthanized and counted as dead when imminent death was H57-597; BD Biosciences), and either Pacific Blue–granzyme B (GzmB) certain as determined by lack of activity and unresponsiveness to (clone GB11; BioLegend) or PE-labeled anti–human GzmB (Thermo touch. Euthanasia was carried out according to the 2013 edition of the Fisher Scientific) that cross-reacts with mouse GzmB. For analysis, samples American Veterinary Medical Association Guidelines for the Euthanasia were acquired using a BD LSRFortessa flow cytometer (BD Biosciences), of Animals. For virus titers, the entire spleen or portions of the liver and data were analyzed with FlowJo software (Treestar). were homogenized in 2.5% FBS RPMI 1640 medium (Corning) using Adoptive transfers of lymphocytes a TissueLyser (QIAGEN). Virus titers were determined on BSC-1 cells as previously described (26). Splenocytes were obtained from the indicated strains of mice, mixed in a 1:1 For MCMV infections, indicated strains were infected i.p. with 2.5 3 105 ratio, then labeled with 4 mM CFSE (Thermo Fisher Scientific), and a total PFU of MCMV K181 (K181) or 1.0 3 104 PFU of MCMV V70 (V70) of 2 3 107 cells (1 3 107 cells each) in 0.2 ml of PBS were inoculated i.v. for 120 h. K181 stocks were produced by infecting 4 3 106 M210-B4 into the recipient mice. The Journal of Immunology 3

RNA and DNA preparation and reverse transcriptase quantitative PCR Total RNA from LNs was obtained with the RNeasy Mini Kit (QIAGEN), as previously described (30, 31). First-strand cDNA was synthesized with High Capacity cDNA Reverse Transcription Kit (Life Technologies). For EVM003 and Gapdh, quantitative RT-PCR (qRT-PCR) was performed using iTaq Universal SYBR Green with the following primers: Gapdh forward: 59-TGTCCGTCGTGGATCTGAC-39, reverse: 59-CCTGCTTC- ACCACCTTCTTG-39; EVM003 forward: 59-TCTGTCCTTTAACAGCA- TAGATGTAGA-39,reverse:59-TGTTAACTCGGAAGTTGATATGGTA-39. For viral loads, RNA (ECTV) from naive mice was used as control, and no amplification was observed. Thus, for quantification purposes, their cycle threshold values were adjusted to 40. Total DNA was extracted from the spleen using the Gentra Puregene Tissue Kit (QIAGEN), following the manufacturer’s instructions for ex- traction of DNA from tissues. The spleen was homogenized in 10 ml of 2.5% FBS RPMI (Corning), and 1 ml was taken for DNA extraction. RNA andproteinwereremovedbyaddingRNaseASolutionandProteinase K. Extracted DNA was quantified by nanodrop, and 2 ml of DNA was used as a template in each qRT-PCR reaction using the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) with the following

primer for MCMV-E1 forward: 59-TCGCCCATCGTTTCGAGA-39, Downloaded from reverse: 59-TCTCGTAGGTCCACTGACGGA-39. Genome copy numbers were calculated based on a standard curve of a plasmid containing the MCMV-E1 . Statistical analysis Statistical analysis was performed with Prism Software (GraphPad Soft-

ware, La Jolla, CA). For survival studies, p values were obtained with http://www.jimmunol.org/ the log‐rank (Mantel–Cox) test. The p values were determined using the Mann–Whitney U test, and when multiple groups had to be compared, we used one‐way ANOVA and Fisher least significant difference test for multiple comparisons.

Results Generation and characterization of mice with NK cells deficient in a2b1

To investigate the role of a2b1 in NK cell maturation and function, by guest on September 28, 2021 we crossed Ncr1Cre+ mice (Ncr1 encodes NKp46) (32) with either Itgb1fl/fl or Itga2fl/fl mice to, respectively, generate mice with NK cells deficient in b1(Ncr1Cre+-Itgb1fl/fl,inthisstudyCre+-Itgb1fl/fl) or a2(Ncr1Cre+-Itga2fl/fl, in this study Cre+-Itga2fl/fl). Flow cytometry analysis of bone marrow and spleen cells showed that most but not all CD32 NK1.1+ cells in Cre2-Itgb1fl/fl littermate control mice but only a few Cre+-Itgb1fl/fl mice expressed a2and b1. This indicates that in NK1.1+ cells, a2 is only expressed at the cell surface if b1 is also expressed. T cells (CD3e+ NK1.12) did not express a2 and had low levels of b1ineitherCre2- or Cre1-Itgb1fl/fl mice (Fig. 1A). Meanwhile, a2inCD32 NK1.1+ was expressed at high levels in Cre2-Itga2fl/fl mice but was absent in Cre+-Itga2fl/fl mice, which mostly maintained b1. In contrast, a2 was not expressed by T cells in either Cre2- or Cre1-Itga2fl/fl mice (Fig. 1B). This demonstrates that a2 is efficiently deleted in NK1.1+ cells of Cre+-Itga2fl/fl mice. Thus, Cre+-Itga2fl/fl mice have a2b1 deficiency at the surface of NK1.1+ cells. However, because b1was still expressed, b1 likely pairs with other integrins at the surface of NK1.1+ cells. Thus, subsequent experiments are

side scatter–height. From these plots, expression of a2 and b1 was 2 FIGURE 1. Generation and characterization of mice with NK cells determined on gated NK cells (CD3e NK1.1+, gray histogram) or T cells 2 deficient in a2b1. Ncr1Cre+ mice were crossed with Itgb1fl/fl or Itga2fl/fl (CD3e+ NK1.1 , white histograms). These were used to establish the mice to generate Cre+-Itgb1fl/fl, Cre+-Itga2fl/fl,andCre2 littermate con- definitive gates for NK cells shown as contour plots on the right. (C) trols. (A and B) Representative flow cytometry analysis of the indicated Representative flow plots showing the gating strategy to identify ILC1s tissues and mouse strains: NK1.1 and T cells were distinguished by and NK cells in the indicated tissues based on NK1.1 and Eomes and CD3e and NK1.1 staining (left panels) on cells previously gated on stacked columns depicting the frequencies of ILC1s and NK cells as forward light scatter–area versus forward light scatter–height to elimi- mean 6 SEM in different tissues. Data correspond to two independent nate doublets and on lymphocytes by forward light scatter–height and experiments combined with a total of six to eight mice per group. 4 ROLE OF a2b1 INTEGRIN IN NK CELLS

FIGURE 2. a2b1 affects but not critically the development of NK cells. The indicated tissues from Cre+- and Cre2-Itga2fl/fl mice were analyzed for the frequency of NK1.1+ cells, and their maturation stages (R1, R2, and R3) according to CD27 and CD11b expression. (A) Representative flow cytometry plots indicating the gating strategy. (B–D) Summary graphs indicating the frequency of NK1.1+ cells in individual mice with mean 6 SEM (left panels) and the mean 6 SEM frequencies of R1 (CD27+ CD11b2), R2 (CD27+ CD11b+), and R3 (CD272 CD11b+) cells within the NK1.1+ CD3e2 gate in the indicated tissues and mice. Data correspond to three or four independent experiments combined with a total of 11–27 mice per group. The p values were calculated Downloaded from using the Mann–Whitney U statistical test or ANOVA as necessary. *p , 0.05, **p , 0.01. described with Cre+-andCre2-Itga2fl/fl mice, as in addition to a2b1 accumulation of NK cells in the dLN of Cre+ mice resulted in a loss, NK1.1+ cells in Cre+-Itgb1fl/fl have deficiencies in additional b1 relative increase in the frequency of mature R3 NK cells and a pairs. Also, Itgb1fl/fl mice are in a B6;129 mixed background, which concomitant decrease in immature R1 NK cells (Fig. 3A, 3B). + fl/fl

could complicate the analysis. Of note, the deficiency of a2in It was possible that in Cre -Itga2 mice NK cells deficient in http://www.jimmunol.org/ NK1.1+ cells did not redirect NK cells to the ILC1 linage be- a2b1 migrated normally to the dLN because of a lack of com- cause the frequencies of NK cells (Eomes+)andILC1s(Eomes2) petition. Thus, splenocytes from Cre+- and Cre2-Itga2fl/fl mice within group 1 ILCs (NK1.1+ CD32)wassimilarinthebone were mixed in a 1:1 ratio, labeled with CFSE, and transferred into marrow, spleen, and peripheral LN of Cre2-andCre+-Itga2fl/fl B6 mice, which were subsequently infected with ECTV in the (Fig. 1C), where NK cells were the predominant population. footpad. At 48 hpi, the ratios of Cre+/Cre2Itga2fl/fl NK cells in the a2b1 affects but not critically the development of NK cells ndLN and the dLN were similar, confirming that deficiency in a2b1 does not affect the constitutive or virus-induced entry of NK a b We next investigated whether intrinsic 2 1 deficiency affects cells into the dLN (Fig. 3C). NK cell development. Expression of CD27 and CD11b defines by guest on September 28, 2021 + 2 Next, we looked at NK cell localization within LNs. As men- different maturational stages of NK cells in which CD27 CD11b tioned previously, Ab blockade suggested that a2b1 directs NK (in this study R1) are immature, CD27+ CD11b+ (in this study R2) 2 + cells to sites of bacterial infection within the dLN via interactions are transitional but with effector capacity, and CD27 CD11b (in with collagen (24). Thus, we analyzed whether absence of a2b1 this study R3) are the most mature and have the strongest cytolytic + fl/fl results in NK cell mislocalization within the dLN during a viral function (33) (Fig. 2A). We found that in Cre -Itga2 mice, the + 2 fl/fl + infection. Cre - and Cre -Itga2 mice were infected with ECTV frequency of NK1.1 cells was decreased in the bone marrow expressing mCherry, and ndLNs and dLNs were visualized by (Fig. 2B) and in the spleen (Fig. 2C) but not in the blood confocal microscopy after staining with anti-NK1.1 mAb. The (Fig. 2D). However, the frequencies of the different maturation analysis confirmed recruitment because there was an increase in stages (R1, R2, and R3) were not altered in any of the organs NK1.1+ cells (green) in the dLN versus the ndLN of both Cre+ and a b 2 2 + (Fig.2B–D).Thus,theabsenceof 2 1 seems to slightly de- Cre mice. Furthermore, similar to Cre controls, NK1.1 cells in crease the frequency of NK cells in the bone marrow and spleen + Cre mice distributed throughout the dLN, including infected and but has no effect on NK cell maturation. uninfected areas (Fig. 3D). Response to viral infection We also analyzed effector functions. At 60 hpi, the NK cells in Deficiency in a2b1 does not impair the constitutive or ECTV- the dLN of B6 mice are highly activated as determined by GzmB induced accumulation of NK cells in LNs, their distribution and IFN-g expression, and these effector molecules are critical for within the LN, or the acquisition of an effector phenotype. NK the control of ECTV dissemination from the dLN to the liver and cells are present in LNs at low numbers at steady state and increase the spleen (7). Thus, we tested whether a2b1 deficiency affects 2 their numbers by migrating from the circulation during inflam- expression of GzmB and IFN-g in NK cells. We found that Cre - + fl/fl mation. We have previously shown that mature R3 NK cells rapidly and Cre -Itga2 had similarly increased frequencies of both + + enter the dLN after ECTV infection (7). Given the importance of IFN-g and GzmB NK cells in the dLN at 60 hpi with ECTV integrins in lymphocyte adhesion and migration it was possible when compared with the ndLN (Fig. 3E). that a2b1 was required in this process. Cre+- and Cre2-Itga2fl/fl a2b1 is necessary for optimal NK cell proliferation in the spleen mice were challenged with ECTV and the recruitment of NK during ECTV and MCMV infections but is not required for the cells into the dLNs was measured by comparing frequencies of acquisition of effector functions. In addition to curbing virus NK cells in the ndLN (i.e., at steady state) and dLN at 48 h spread from LNs, NK cells play an important role in the control of postinfection (hpi). We observed that Cre+-Itga2fl/fl mice pref- ECTV by becoming activated in the spleen and liver until the T cell erentially recruited NK cells to the dLN but slightly significantly response develops (3, 7). Thus, we also looked at NK cell re- less than Cre2-Itga2fl/fl mice, which could be an effect of the sponses in the spleen. At 120 hpi with ECTV, which is the peak decreased number of NK cells in the bone marrow and spleen of the NK response, the frequency and absolute numbers of NK of Cre+-Itga2fl/fl. Nevertheless, similar to Cre2 controls, the cells in the spleens of Cre+ mice were significantly lower than in The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Deficiency in a2b1 does not impair the constitutive or ECTV-induced accumulation of NK cells in LNs, their distribution within the LN, or the acquisition of an effector phenotype. (A–E) Cre+- and Cre2-Itga2fl/fl mice were infected with 3000 PFU wild-type (WT) ECTV or ECTV-mCherry and their ndLN and dLNs were analyzed at 48–72 hpi. (A) Representative flow cytometry plots showing the gating strategy. (B) Summary graphs indicating the frequency of NK1.1+ cells in individual mice with mean 6 SEM and frequencies of R1 and R3 cells in individual mice with mean 6 SEM in Cre2- and Cre+-Itga2fl/fl mice. In (A)and (B), data correspond to three individual experiments combined with a total (n of 11–15) mice per group. (C) Splenocytes from Cre+-Itga2fl/fl and Cre2- Itga2fl/fl were mixed at a 1:1 ratio, labeled with 4 mM CFSE, and transferred into recipient B6 mice. The mice were infected with 3000 PFU WT ECTV 1 d posttransfer, and their ndLN and dLNs were analyzed at 48 hpi. Representative flow plots show the gating strategy for iden- tification of adoptively transferred cells and WT and a2b1-deficient NK cells. Dot plots show the ratio of a2/WT NK cells in the ndLNs and dLNs of individual mice with mean 6 SEM. All experiments were repeated two to three times. Data are displayed as a combination of all the repeats with a total (n of 17 or 18) mice per group. (D) Cre+- and Cre2-Itga2fl/fl mice were infected with ECTV-mCherry. At 72 hpi, their ndLN and dLN were visualized by confocal microscopy with original magnification 310. Blue: DAPI; green: NK1.1 staining; red: mCherry. Pictures are from the ndLN or dLN of ECTV- infected mice. Experiments were repeated two times with a total (n = 6) mice per group. Representative lymph nodes from (Figure legend continues) 6 ROLE OF a2b1 INTEGRIN IN NK CELLS

Cre2-Itga2fl/fl and naive mice (Fig. 4A, 4B). A similar effect was mice were resistant, indicating that a2b1 is not required for NK observed in the liver and blood (Supplemental Fig. 1). However, cell–mediated resistance to lethal mousepox (Fig. 5C). Moreover, the frequency of GzmB+ NK cells was not significantly different Cre+-Itga2fl/fl mice were resistant when challenged with a signif- between Cre+ and Cre2 mice and was significantly higher than in icantly higher dose of ECTV, but control aged B6 mice, which naive mice (Fig. 4A, 4C), indicating that NK cells do not need have immature and dysfunctional NK cells, succumbed to ECTV a2b1to acquire effector functions in the spleen or liver during infection (Fig. 5D). Also, at 5 dpi with tissue-cultured (K181) and ECTV infection. salivary gland passaged (V70) MCMV, Cre2 and Cre+ mice had To determine whether reduced numbers of NK cells with efficient similar virus loads in the spleen, which increased similarly after effector differentiation was a characteristic of the ECTV NK cell NK cell depletion (Fig. 5E, 5F). These results indicate that the response or could be extended to other viral infections, we a2b1 in NK cells and optimal NK cell proliferation are dispens- infected Cre+-andCre2-Itga2fl/fl mice with MCMV. As with able for effective NK cell–mediated control of two mouse-specific ECTV, Cre+-Itga2fl/fl mice had reduced frequencies and num- viruses in the acute phase of infection. bers of NK cells than Cre2-Itga2fl/fl mice at 120 hpi. Compared 2 with naive mice, MCMV-infected Cre -Itga2fl/fl had an increased Discussion total number (but not frequency) of NK cells (Fig. 4D, 4E). As In this study, we show that a2b1 expression by NK cells is not + fl/fl with ECTV, the upregulation of GzmB in Cre -Itga2 mice essential for their maturation or commitment to the NK cell remained intact (Fig. 4D, 4F). lineage, entry to and distribution within LNs, or the induction of During MCMV infection of B6 mice, NK cells expand in two an anti-ECTV effector response. However, a2b1 is required for phases (the early phase occurs within the first 2 d of infection), is optimal NK cell proliferation during ECTV infection and for Downloaded from dependent on cytokines, and most NK cells expand (34, 35). The increase in NK cell during MCMV infection. Despite this, a2b1- + late phase occurs ∼120 hpi and happens mostly in Ly49H NK deficient NK cells were still able to protect from ECTV and cells that recognize the viral m157 at the surface of infected control MCMV titers in the spleen, demonstrating that mature + 2 cells (36–40). Analysis of Ly49H and Ly49H NK cells revealed and functional NK cells can mediate protection without exten- + + fl/fl that Ly49H NK cells from Cre -Itga2 mice did not increase sive proliferation in two viral infection models in which the + 2 in number, whereas the Ly49H NK cells from Cre mice did antiviral role of NK cells is very well established. http://www.jimmunol.org/ + + (Fig. 4G, 4H). Yet, similar frequencies of NK1.1 Ly49H cells Previous research has shown that the VLA family of integrin 2 + fl/fl produced GzmB in Cre -andCre -Itga2 mice (Fig. 4G, 4I). heterodimers has roles in metastasis of cancer cells, immuno- + Therefore, during MCMV infection, Ly49H NK cells deficient modulation, and leukocyte extravasation and retention in tissue in a2b1failedtoincreaseinnumbers,butMCMV-specificNK (42–47). Whereas a2b1 is used as a marker to identify NK cells + cells (i.e., Ly49H ) still acquired an effector phenotype. across mouse strains as well as to distinguish NK cells from Given the reduced NK cell numbers, we next used the ECTV ILC1s, its role in NK cell biology remains unclear. Bone modeltotestwhethera2b1 is necessary for NK cell proliferation. At marrow stromal cells synthesizetype1and3collagenfibers + fl/fl 120 hpi with ECTV, Cre -Itga2 mice had a significantly lower (48), and it has been shown that their reduction in bone marrow

+ + 2 fl/fl by guest on September 28, 2021 frequency of Ki67 NK1.1 cells in the spleen than Cre -Itga2 stromal cells leads to decreased collagen synthesis (49, 50). mice, indicating that in the absence of a2b1, fewer NK cells entered Previously, we have shown that the NK cells in the bone marrow into G1 (Fig. 4J). To determine whether reduced Ki67 expression and other organs of aged mice are immature with increased R1 and translated to impaired cell division, we adoptively transferred decreased R3 populations (29, 51). We also showed that NK cells + 2 fl/fl CFSE-labeled splenocytes from Cre -andCre -Itga2 mice in in the bone marrow of aged mice have lower expression of a2b1 a 1:1 ratio into a CD45.1 host, which was then infected with (51) and that NK cell extrinsic factors in the bone marrow stroma + 2 ECTV in the footpad. Proliferation of Cre and Cre NK cells are responsible for the defective maturation of NK cells in aged (identified by a2 staining) was assessed by CFSE dilution at 144 hpi. mice, leading us to hypothesize that a2b1 and its interaction with + The results indicated significantly decreased proliferation in Cre - collagen are necessary for NK cell maturation. Thus, we also 2 fl/fl compared with Cre -Itga2 mice (Fig. 4K). This indicates that hypothesized that expression of a2b1 on developing NK cells and a2b1 is required for optimal proliferation of NK cells during ECTV their interaction with collagen could be required for their proper infection. linage commitment and maturation in the bone marrow. However, a2b1-deficient NK cells protect from lethal mousepox and control we found that a2b1 does not influence lineage commitment of the MCMV titers in the spleen. B6 mice are naturally resistant to lethal common innate lymphoid progenitor to NK cells versus ILC1s. mousepox but become highly susceptible when depleted of NK Also, although a2b1 deficiency slightly decreases the frequency cells with anti-NK1.1 or Asialo GM Ab or when NK cells are of NK cells in the bone marrow and spleen, it does not affect NK dysfunctional, as demonstrated by increased virus titers in spleen cell maturation because the proportions of R1s and R3s were and liver at 3 and 5 dpi, and by high lethality (29, 41). When similar within the bone marrow, peripheral blood, and spleen of 2 challenged with ECTV, Cre - and Cre+-Itga2fl/fl had similar levels Cre+- and Cre-Itga2fl/fl mice. Thus, the data strongly suggest that of viral gene expression in the dLNs at 72 hpi (Fig. 5A) and the deficient NK cell maturation in aged mice (51) is not because similar titers of replicating virus in the spleen and liver at 120 hpi, of deficient a2b1 expression. as determined by plaque assay (Fig. 5B). Treatment with anti-NK1.1 It is known that CXCR3 directs homing of NK cells to the dLN 2 mAb resulted in a significant increase in lethality in both Cre -and during viral infection (28, 52), but the identity of the integrin(s) 2 Cre+-Itga2fl/fl mice, whereas undepleted Cre - and Cre+-Itga2fl/fl involved in entry into the dLN remains unknown. We show that

one mouse in each group are shown. (E) Cre+- and Cre2-Itga2fl/fl were infected with 3000 PFU WT ECTVand their ndLN and dLNs were analyzed at 48 hpi. Representative flow cytometry plots showing the gating strategy for expression of intracellular IFN-g or GzmB. Dot plots depict the frequencies with mean 6 SEM of NK1.1+ cells that were IFN-g+ (left) or GzmB+ (right) in the ndLN and dLN of individual Cre2-orCre+-Itga2fl/fl mice as indicated. Data correspond to three individual experiments combined with a total (n of 11–15) mice per group. The p values were calculated using the Mann–Whitney U statistical test or ANOVA when necessary. *p , 0.05, **p , 0.01, ****p , 0.0001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. a2b1 is necessary for optimal NK cell proliferation in the spleen during ECTV and MCMV infections but is not required for the acquisition of NK cell effector functions. Cre+-andCre2-Itga2fl/fl mice were infected with either 3000 PFU of wild-type (WT) ECTV or 250,000 PFU of K181 (MCMV), and their spleens were analyzed at 120 hpi. Naive B6 mice were used as an additional control. (A) Representative flow cytometry plots showing the gating strategy for identification of total NK cells in indicated mice infected with ECTV as well as intracellular expression of GzmB in total NK1.1+ cells. (B) Dot plots depict the frequencies and total numbers with mean 6 SEM of NK1.1+ cells that were in the spleen of ECTV-infected individual Cre2-orCre+-Itga2fl/fl mice as indicated. Data are displayed as a combination of two individual experiments with a total of (n = 5–7) mice per group. (C) Dot plots depict the frequency with mean 6 SEM of GzmB+ NK1.1+ cells that were in the spleen of ECTV-infected individual Cre2-orCre+-Itga2fl/fl mice as indicated. Data correspond to two individual experiments combined with a total of (n = 5–7) mice per group. (D) Representative flow cytometry plots showing the gating strategy for identification of total NK cells in indicated mice infected with MCMV as well as intracellular expression of GzmB in total NK1.1+ cells. (E) Dot plots depict the frequencies and total numbers with mean 6 SEM of NK1.1+ cells in the spleen of MCMV-infected individual Cre2-orCre+-Itga2fl/fl mice as indicated. Data are displayed as a combination of three individual experiments with a total of (n = 8–10) mice per group. (F) Dot plots depict the frequency with mean 6 SEM of GzmB+ NK1.1+ cells in the spleen of MCMV-infected individual Cre2-orCre+-Itga2fl/fl mice as indicated. Data are displayed as a combination of three individual experiments with a total of (n = 8–10) mice per group. (G) Representative flow cytometry plots showing the gating strategy for identification of total NK cells as well as intracellular expression of GzmB in Ly49H+ or Ly49H2 cells within the NK1.1+ population in mice infected with MCMV. (H) Bar graphs depict the number of Ly49H+ NK1.1+ or Ly49H2 NK1.1+ cells with mean 6 SEM of NK1.1+ in the spleen of MCMV-infected individual Cre2-or Cre+-Itga2fl/fl mice as indicated. (I) Bar graphs depict the frequency of GzmB+ cells with mean 6 SEM in NK1.1+ Ly49H+ or Ly49H2 cells in the spleen of MCMV-infected individual Cre2-orCre+-Itga2fl/fl mice as indicated. Data are displayed as a combination of three individual experiments with a total of (n = 8–10) mice per group. (J) Dot plots depict the frequency with mean 6 SEM of Ki67+ NK1.1+ cells in the spleen of ECTV-infected individual Cre2-orCre+-Itga2fl/fl mice as indicated. Data are displayed as a combination of two individual experiments with a total of (n = 5–7) mice per group. (K) Splenocytes from Cre+-Itga2fl/fl and Cre2-Itga2fl/fl were mixed at a 1:1 ratio, labeled with 4 mM CFSE, and transferred into recipient CD45.1 mice. Recipient mice were either naive or infected with 3000 PFU WT ECTV 1 d posttransfer, and their spleens were analyzed at 120 hpi. Representative flow plots show the gating strategy for identification of adoptively transferred cells, either WT and a2b1-deficient NK cells, and the dilution of CFSE by either WT or a2b1-deficient NK cells. Bar graphs show the frequency of WT or a2b1-deficient NK cells that diluted their CFSE in the spleen of individual mice with mean 6 SEM. Data correspond to three individual experiments combined with a total of (n = 8–11 [naive] and 10–12 [infected] mice per group. The p values were calculated using the Mann–Whitney U statistical test or ANOVA when necessary. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 8 ROLE OF a2b1 INTEGRIN IN NK CELLS

Cre+-Itga2fl/fl NK cells enter ndLNs and dLNs normally whether or not they are in competition with Cre2-Itga2fl/fl NK cells. This indicates that a2b1 is not required for NK cell constitutive entry into the ndLN and ECTV-induced entry into the dLN. Also, using confocal microscopy, we found that a2b1-deficient NK cells distribute similarly within the ndLN and dLN, including areas of virus infection, indicating that a2b1 is not required for intranodal displacement. Thus, adhesion molecules other than a2b1 may be responsible for the NK cell extravasation into and distribution within ndLNs and dLNs. It has been reported that a2 in mouse NK cells and b1 in human NK cells are needed to control T. gondii and Cryptococcus neo- formans infections, respectively, via direct or indirect activation of NK cells (25, 53). In this study, we show that during viral infections a2b1 is not required for NK cell activation because Cre+-Itga2fl/fl NK cells upregulated IFN-g or GzmB normally during ECTV and MCMV infections.Furthermore,Cre2-andCre+-Itga2fl/fl mice controlled ECTV and MCMV similarly, whereas depletion of

a2b1 NK cells resulted in high susceptibility to lethal mousepox Downloaded from and higher MCMV titers in spleen, indicating a2b1 is dispensable at conferring NK cell–mediated control against ECTV and MCMV. Interactions with b1 and collagen have previously been shown to affect the expansion of pancreas b cells as well as the mor- phogenesis of submandibular epithelial cells (54, 55). Thus, we

believe a2b1 induces proliferation of NK cells but does not affect http://www.jimmunol.org/ their trafficking or localization during viral infections because of the global reduction in NK cells within the blood, liver, and spleen of infected Cre+-Itga2fl/fl mice. It remains possible, however, that NK cells are increased at other sites that we did not test. Impor- tantly, we showed that a2b1 is required for optimal NK cell proliferation. Future experiments should identify the ligands and signaling mechanisms of this a2b1-dependent proliferation and whether b1 plays a role in the proliferation of other leukocytes. It is well established that survival to ECTV and efficient control by guest on September 28, 2021 of MCMV requires NK cells and that the NK cell responses to both viruses include acquisition of effector functions and proliferation. Our results show that the NK cell–mediated resistance to ECTV and control of MCMV virus loads can still occur with signifi- cantly decreased proliferation. It is also known that innate immune mechanisms, including NK cell effector functions in the dLN, play a critical role in protection against lethal ECTV infection (29, 56).

the virus loads in spleens or livers of individual mice with mean 6 SEM were determined by plaque assay. Data correspond to two individual ex- periments combined with a total of six to nine mice per group. The limit of detection is indicated by the dotted line. (C) The indicated mice were depleted or not of NK cells with anti-NK1.1 mAb PK136 1 d before infection and 1 d postinfection with 3000 PFU ECTV in the footpad. Survival was monitored. Data are displayed as a combination of two independent experiments with a total of 3–10 mice per group. Statistical analysis was performed by log- test. (D) The indicated mice were infected with 100,000 PFU ECTV in the footpad. Survival was monitored. Data are displayed as a combination of two independent experiments with a total of 5–10 mice per group Statistical analysis was performed by log- rank test. (E and F) The indicated mice were depleted or not of NK cells with anti-NK1.1 mAb PK136 1 d before and 1, 3, and 5 d postinfection with 250,000 PFU of tissue-cultured K181 (E) or 10,000 PFU of salivary gland–passaged MCMV V70 (F) i.p. spleens were collected at 120 hpi, and FIGURE 5. a2b1-deficient NK cells protect from lethal mousepox and genome copies of MCMV E1 gene (E) or PFU (F) were calculated. Data from MCMV. (A) The indicated mice were infected with ECTV and at are displayed as a combination of two or three independent experiments 72 hpi the expression of the viral gene EVM003 in individual mice with with a total of 3–11 mice per group. The limit of detection for PFU is mean 6 SEM was determined in the dLN by qRT-PCR. Data correspond to indicated by the dotted line. The p values were calculated using the two individual experiments combined with a total of six to eight mice per Mann–Whitney U statistical test or ANOVA when necessary. *p , 0.05, group. (B) The indicated mice were infected with ECTV, and at 120 hpi, **p , 0.01, ***p , 0.001. The Journal of Immunology 9

Notably, at 48–72 hpi, the frequency of NK cells in the dLN of 15. Pikovskaya, O., J. Chaix, N. J. Rothman, A. Collins, Y.-H. Chen, A. M. Scipioni, + fl/fl E. Vivier, and S. L. Reiner. 2016. Cutting edge: eomesodermin is sufficient to Cre -Itga2 is only slightly decreased. Thus, the successful direct type 1 innate lymphocyte development into the conventional NK lineage. migration of mature and functional a2b1-deficient NK cells to the J. Immunol. 196: 1449–1454. dLN at 48–72 hpi and their activation in the spleen allows for 16. Fuchs, A. 2016. ILC1s in tissue inflammation and infection. Front. Immunol. 7: 104. 17. Leitinger, B., and E. Hohenester. 2007. Mammalian collagen receptors. Matrix ECTV control, even when NK numbers are altered. These findings Biol. 26: 146–155. further highlight the importance of controlling viral replication in 18. Ley, K., C. Laudanna, M. I. Cybulsky, and S. Nourshargh. 2007. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immu- the dLN during an acute virus infection. nol. 7: 678–689. + During primary infection with MCMV, Ly49H NK cells 19. Zutter, M. M., and S. A. Santoro. 1990. Widespread histologic distribution of the interacting with the viral protein m157 expand and protect mice alpha 2 beta 1 integrin cell-surface collagen receptor. Am.J.Pathol.137: 113–120. 20. Elices, M. J., and M. E. Hemler. 1989. The human integrin VLA-2 is a collagen from MCMV (5, 39, 57, 58). These MCMV-specific NK cells then receptor on some cells and a collagen/laminin receptor on others. Proc. Natl. contract and give rise to long-lived memory NK cells that con- Acad. Sci. USA 86: 9906–9910. tribute to protection upon reinfection (59–61). Our data suggest 21. Goda, S., H. Inoue, H. Umehara, M. Miyaji, Y. Nagano, N. Harakawa, H. Imai, P. Lee, J. B. Macarthy, T. Ikeo, et al. 2006. Matrix metalloproteinase-1 produced that, in addition to Ly49H interacting with m157, a2b1 acts as an by human CXCL12-stimulated natural killer cells. Am. J. Pathol. 169: 445–458. additional signal for optimal Ly49H+ NK cell proliferation. Future 22. Arase, H., T. Saito, J. H. Phillips, and L. L. Lanier. 2001. Cutting edge: the mouse NK cell-associated antigen recognized by DX5 monoclonal antibody is experiments could look into whether this affects the induction of CD49b (a 2 integrin, very late antigen-2). J. Immunol. 167: 1141–1144. memory NK cells. 23. Miyake, S., T. Sakurai, K. Okumura, and H. Yagita. 1994. Identification of In summary, a2b1 is viewed as a phenotypic hallmark of NK collagen and laminin receptor integrins on murine T lymphocytes. Eur. J. Immunol. 24: 2000–2005. cells, but its role in NK cell biology is not well understood. Our 24. Garrod, K. R., S. H. Wei, I. Parker, and M. D. Cahalan. 2007. Natural killer cells work shows that a2b1 is not required for NK cell maturation, actively patrol peripheral lymph nodes forming stable conjugates to eliminate Downloaded from migration to LNs, or acquisition of effector functions. However, MHC-mismatched targets. Proc. Natl. Acad. Sci. USA 104: 12081–12086. 25. Coombes, J. L., S. J. Han, N. van Rooijen, D. H. Raulet, and E. A. Robey. 2012. a2b1 is required for optimal virus-induced proliferation and ac- Infection-induced regulation of natural killer cells by macrophages and collagen cumulation of NK cells. Unexpectedly, despite impaired prolif- at the lymph node subcapsular sinus. Cell Rep. 2: 124–135. 26. Xu, R.-H., M. Cohen, Y. Tang, E. Lazear, J. C. Whitbeck, R. J. Eisenberg, eration, a2b1-defficient NK cells still protected from two viral G. H. Cohen, and L. J. Sigal. 2008. The orthopoxvirus type I IFN binding protein infections in their natural hosts. is essential for virulence and an effective target for vaccination. J. Exp. Med. 205: 981–992. http://www.jimmunol.org/ 27. Zurbach, K. A., T. Moghbeli, and C. M. Snyder. 2014. Resolving the titer of Acknowledgments murine cytomegalovirus by plaque assay using the M2-10B4 cell line and a low We thank Lingjuan Tang for technical assistance and the Flow Cytometry viscosity overlay. Virol. J. 11: 71. 28. Wong, E., R. H. Xu, D. Rubio, A. Lev, C. Stotesbury, M. Fang, and L. J. Sigal. and Laboratory Animal at Thomas Jefferson University for services. 2018. Migratory dendritic cells, group 1 innate lymphoid cells, and inflammatory monocytes collaborate to recruit NK cells to the virus-infected lymph node. Cell Rep. 24: 142–154. Disclosures 29. Fang, M., F. Roscoe, and L. J. Sigal. 2010. Age-dependent susceptibility to a The authors have no financial conflicts of interest. viral disease due to decreased numbers and trafficking. J. Exp. Med. 207: 2369–2381. 30. Rubio, D., R. H. Xu, S. Remakus, T. E. Krouse, M. E. Truckenmiller, R. J. Thapa, S. Balachandran, A. Alcamı´, C. C. Norbury, and L. J. Sigal. 2013. by guest on September 28, 2021 References Crosstalk between the type 1 interferon and nuclear factor kappa B pathways 1. French, A. R., and W. M. Yokoyama. 2003. Natural killer cells and viral in- confers resistance to a lethal virus infection. Cell Host Microbe 13: 701–710. fections. Curr. Opin. Immunol. 15: 45–51. 31. Xu, R.-H., E. B. Wong, D. Rubio, F. Roscoe, X. Ma, S. Nair, S. Remakus, 2. Biron, C. A., K. B. Nguyen, G. C. Pien, L. P. Cousens, and T. P. Salazar-Mather. R. Schwendener, S. John, M. Shlomchik, and L. J. Sigal. 2015. Sequential acti- 1999. Natural killer cells in antiviral defense: function and regulation by innate vation of two pathogen-sensing pathways required for type I interferon expression cytokines. Annu. Rev. Immunol. 17: 189–220. and resistance to an acute DNA virus infection. Immunity 43: 1148–1159. 3. Parker, A. K., S. Parker, W. M. Yokoyama, J. A. Corbett, and R. M. L. Buller. 32. Narni-Mancinelli, E., J. Chaix, A. Fenis, Y. M. Kerdiles, N. Yessaad, 2007. Induction of natural killer cell responses by ectromelia virus controls in- A. Reynders, C. Gregoire, H. Luche, S. Ugolini, E. Tomasello, et al. 2011. Fate fection. 81: 4070–4079. mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor. 4. Delano, M. L., and D. G. Brownstein. 1995. Innate resistance to lethal mousepox Proc. Natl. Acad. Sci. USA 108: 18324–18329. is genetically linked to the NK gene complex on chromosome 6 and correlates 33. Hayakawa, Y., and M. J. Smyth. 2006. CD27 dissects mature NK cells into two with early restriction of virus replication by cells with an NK phenotype. J. Virol. subsets with distinct responsiveness and migratory capacity. J. Immunol. 176: 69: 5875–5877. 1517–1524. 5. Bukowski, J. F., B. A. Woda, and R. M. Welsh. 1984. Pathogenesis of murine 34. Nguyen, K. B., T. P. Salazar-Mather, M. Y. Dalod, J. B. Van Deusen, X. Q. Wei, cytomegalovirus infection in natural killer cell-depleted mice. J. Virol. 52: 119–128. F. Y. Liew, M. A. Caligiuri, J. E. Durbin, and C. A. Biron. 2002. Coordinated and 6. Bukowski, J. F., B. A. Woda, S. Habu, K. Okumura, and R. M. Welsh. 1983. distinct roles for IFN-ab, IL-12, and IL-15 regulation of NK cell responses to Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis viral infection. J. Immunol. 169: 4279–4287. in vivo. J. Immunol. 131: 1531–1538. 35. Dokun, A. O., S. Kim, H. R. C. Smith, H. S. P. Kang, D. T. Chu, and 7. Fang, M., L. L. Lanier, and L. J. Sigal. 2008. A role for NKG2D in NK cell- W. M. Yokoyama. 2001. Specific and nonspecific NK cell activation during virus mediated resistance to poxvirus disease. PLoS Pathog. 4: e30. infection. Nat. Immunol. 2: 951–956. 8. Biron, C. A., K. S. Byron, and J. L. Sullivan. 1989. Severe herpesvirus infections 36. Daniels, K. A., G. Devora, W. C. Lai, C. L. O’Donnell, M. Bennett, and in an adolescent without natural killer cells. N. Engl. J. Med. 320: 1731–1735. R. M. Welsh. 2001. Murine cytomegalovirus is regulated by a discrete subset of 9. Etzioni, A., C. Eidenschenk, R. Katz, R. Beck, J. L. Casanova, and S. Pollack. natural killer cells reactive with monoclonal antibody to Ly49H. J. Exp. Med. 2005. Fatal varicella associated with selective natural killer cell deficiency. J. 194: 29–44. Pediatr. 146: 423–425. 37. Tripathy, S. K., H. R. C. Smith, E. A. Holroyd, J. T. Pingel, and W. M. Yokoyama. 10. Eidenschenk, C., E. Jouanguy, A. Alcaı¨s, J.-J. Mention, B. Pasquier, 2006. Expression of m157, a murine cytomegalovirus-encoded putative major I. M. Fleckenstein, A. Puel, L. Gineau, J.-C. Carel, E. Vivier, et al. 2006. Fa- histocompatibility class I (MHC-I)-like protein, is independent of viral regulation milial NK cell deficiency associated with impaired IL-2- and IL-15-dependent of host MHC-I. J. Virol. 80: 545–550. survival of lymphocytes. J. Immunol. 177: 8835–8843. 38. Voigt, V., C. A. Forbes, J. N. Tonkin, M. A. Degli-Esposti, H. R. C. Smith, 11. Diefenbach, A., M. Colonna, and S. Koyasu. 2014. Development, differentiation, W. M. Yokoyama, and A. A. Scalzo. 2003. Murine cytomegalovirus m157 and diversity of innate lymphoid cells. Immunity 41: 354–365. mutation and variation leads to immune evasion of natural killer cells. Proc. 12.Weizman,O.E.,N.M.Adams,I.S.Schuster,C.Krishna,Y.Pritykin, Natl. Acad. Sci. USA 100: 13483–13488. C. Lau, M. A. Degli-Esposti, C. S. Leslie, J. C. Sun, and T. E. O’Sullivan. 39. Arase, H., E. S. Mocarski, A. E. Campbell, A. B. Hill, and L. L. Lanier. 2002. 2017. ILC1 confer early host protection at initial sites of viral infection. Direct recognition of cytomegalovirus by activating and inhibitory NK cell re- Cell 171: 795–808.e12. ceptors. Science 296: 1323–1326. 13. Daussy, C., F. Faure, K. Mayol, S. Viel, G. Gasteiger, E. Charrier, J. Bienvenu, 40. Adams, E. J., Z. S. Juo, R. T. Venook, M. J. Boulanger, H. Arase, L. L. Lanier, T. Henry, E. Debien, U. A. Hasan, et al. 2014. T-bet and Eomes instruct the and K. C. Garcia. 2007. Structural elucidation of the m157 mouse cytomega- development of two distinct natural killer cell lineages in the liver and in the lovirus ligand for Ly49 natural killer cell receptors. Proc. Natl. Acad. Sci. USA bone marrow. J. Exp. Med. 211: 563–577. 104: 10128–10133. 14. Gordon, S. M., J. Chaix, L. J. Rupp, J. Wu, S. Madera, J. C. Sun, T. Lindsten, and 41. Fang, M., M. T. Orr, P. Spee, T. Egebjerg, L. L. Lanier, and L. J. Sigal. 2011. S. L. Reiner. 2012. The transcription factors T-bet and Eomes control key CD94 is essential for NK cell-mediated resistance to a lethal viral disease. Im- checkpoints of natural killer cell maturation. Immunity 36: 55–67. munity 34: 579–589. 10 ROLE OF a2b1 INTEGRIN IN NK CELLS

42. Gagliani, N., C. F. Magnani, S. Huber, M. E. Gianolini, M. Pala, P. Licona- 52. Pak-Wittel, M. A., L. Yang, D. K. Sojka, J. G. Rivenbark, and W. M. Yokoyama. Limon, B. Guo, D. R. Herbert, A. Bulfone, F. Trentini, et al. 2013. Coexpression 2013. Interferon-g mediates chemokine-dependent recruitment of natural killer of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. cells during viral infection. Proc. Natl. Acad. Sci. USA 110: E50–E59. [Published erratum appears in 2014 Nat. Med. 20: 1217.] Nat. Med. 19: 739–746. 53. Xiang, R. F., S. Li, H. Ogbomo, D. Stack, and C. H. Mody. 2018. b1 integrins are 43. Yan, X., B. D. Johnson, and R. J. Orentas. 2008. Induction of a VLA-2 (CD49b)- required to mediate NK cell killing of Cryptococcus neoformans. J. Immunol. expressing effector T cell population by a cell-based neuroblastoma vaccine 201: 2369–2376. expressing CD137L. J. Immunol. 181: 4621–4631. 54. Rebustini, I. T., C. Myers, K. S. Lassiter, A. Surmak, L. Szabova, K. Holmbeck, 44. Yoshimura, K., K. F. Meckel, L. S. Laird, C. Y. Chia, J. J. Park, K. L. Olino, V. Pedchenko, B. G. Hudson, and M. P. Hoffman. 2009. MT2-MMP-dependent R. Tsunedomi, T. Harada, N. Iizuka, S. Hazama, et al. 2009. Integrin a2me- release of collagen IV NC1 domains regulates submandibular gland branching diates selective metastasis to the liver. Cancer Res. 69: 7320–7328. morphogenesis. Dev. Cell 17: 482–493. 45. Geherin, S. A., D. Go´mez, R. A. Glabman, G. Ruthel, A. Hamann, and 55. Diaferia, G. R., A. J. Jimenez-Caliani, P. Ranjitkar, W. Yang, G. Hardiman, G. F. Debes. 2016. IL-10+ innate-like B cells are part of the skin immune system C. J. Rhodes, L. Crisa, and V. Cirulli. 2013. b1 integrin is a crucial regulator of and require a4b1 integrin to migrate between the peritoneum and inflamed skin. pancreatic b-cell expansion. Development 140: 3360–3372. J. Immunol. 196: 2514–2525. 56. Kastenmu¨ller, W., P. Torabi-Parizi, N. Subramanian, T. La¨mmermann, and 46. Becker, H. M., J. Rullo, M. Chen, M. Ghazarian, S. Bak, H. Xiao, J. B. Hay, and R. N. Germain. 2012. A spatially-organized multicellular innate immune re- M. I. Cybulsky. 2013. a1b1 integrin-mediated adhesion inhibits macrophage exit sponse in lymph nodes limits systemic pathogen spread. Cell 150: 1235–1248. from a peripheral inflammatory lesion. J. Immunol. 190: 4305–4314. 57. Lee, S.-H., A. Zafer, Y. de Repentigny, R. Kothary, M. L. Tremblay, P. Gros, 47. Yokoyama, W. M., S. R. Maxfield, and E. M. Shevach. 1989. Very early (VEA) P. Duplay, J. R. Webb, and S. M. Vidal. 2003. Transgenic expression of the and very late (VLA) activation antigens have distinct functions in T lymphocyte activating natural killer receptor Ly49H confers resistance to cytomegalovirus in activation. Immunol. Rev. 109: 153–176. genetically susceptible mice. J. Exp. Med. 197: 515–526. 48. Bentley, S. A. 1982. Collagen synthesis by bone marrow stromal cells: a 58. Cheng, T. P., A. R. French, B. F. M. Plougastel, J. T. Pingel, M. M. Orihuela, quantitative study. Br. J. Haematol. 50: 491–497. M. L. Buller, and W. M. Yokoyama. 2008. Ly49h is necessary for genetic re- 49. Chen, X. D., S. Shi, T. Xu, P. G. Robey, and M. F. Young. 2002. Age-related sistance to murine cytomegalovirus. Immunogenetics 60: 565–573. osteoporosis in biglycan-deficient mice is related to defects in bone marrow 59. Min-Oo, G., and L. L. Lanier. 2014. Cytomegalovirus generates long-lived stromal cells. J. Bone Miner. Res. 17: 331–340. antigen-specific NK cells with diminished bystander activation to heterologous 50. Zhang, W., G. Ou, M. Hamrick, W. Hill, J. Borke, K. Wenger, N. Chutkan, J. Yu, infection. J. Exp. Med. 211: 2669–2680. Downloaded from Q. S. Mi, C. M. Isales, and X. M. Shi. 2008. Age-related changes in the oste- 60. Sun, J. C., S. Madera, N. A. Bezman, J. N. Beilke, M. H. Kaplan, and ogenic differentiation potential of mouse bone marrow stromal cells. J. Bone L. L. Lanier. 2012. Proinflammatory cytokine signaling required for the gener- Miner. Res. 23: 1118–1128. ation of natural killer cell memory. J. Exp. Med. 209: 947–954. 51. Nair, S., M. Fang, and L. J. Sigal. 2015. The natural killer cell dysfunction 61. Sun, J. C., J. N. Beilke, and L. L. Lanier. 2009. Adaptive immune features of of aged mice is due to the bone marrow stroma and is not restored by IL-15/ natural killer cells. [Published erratum appears in 2009 Nature. 457: 1168.] IL-15Ra treatment. Aging Cell 14: 180–190. Nature 457: 557–561. http://www.jimmunol.org/ by guest on September 28, 2021 the liverandbloodduringECTV. Supplementary Figure1.α2β1isnecessaryfortheaccumulationandproliferationofNKcellsin combined withatotalnof5-7mice/group. SEM ofNK1.1 (naïve) and8(infected)mice/group. mice withmean±SEM.Datacorrespondstotwoindividual experimentscombinedwithatotalnof7 numbers withmean±SEMofNK1.1 ECTV, andtheirliverbloodwereanalyzedat120hpi. spleen ofindividualCre 0.01; ***p<0.001;****p0.0001. calculated usingtheMann-WhitneyUstatisticaltestor ANOVA whennecessary. For all,*p<0.05;**p displayed asacombinationoftwoindividualexperiments withatotalnof5-7mice/groupP-valueswere cells. BargraphsshowthefrequencyofCFSE either WT andα2β1-deficientNKcellsthedilutionofCFSEbyeitherWT andα2β1-deficientNK hpi. Representativeflowplotsshowthegatingstrategyforidentificationofadoptivelytransferredcells either naïveorinfectedwith3,000pfuWT ECTVonedayposttransferandtheirspleensanalyzed at120 at a1:1ratiolabeledwith4uMCFSEandtransferredintorecipientCD45.1mice.Recipientmicewere mice/group as indicated.Dataaredisplayedacombinationoftwoindividualexperimentswithtotaln5-7 D) % Ki67+ Nk1.1+ Cells B) %NK1.1+ Cells A) %NK1.1+ Cells 60 80 20 40 10 15 10 0 0 5 2 4 6 8 0 Cr Cre Cre e + +

+ Liver **** **

Blood Cre Cr Cre Liver

** - e -

B) - Naive Naive Naive NS Dotplotsdepictthefrequencywithmean±SEMofKi67 *** + * cellsthatwereintheliverofindividualCre

% divided cells C) (CD45.2+ NK1.1+ CFSE+) # NK1.1+ cells (105) 40 60 - 20 2 4 6 orCre 0 0 Cre

Cre +

+ ***

Liver Liver Cre **** +

Itga2 -

Cr * Naiv **** *** Cre e - e D) + fl/fl cellsthatwereintheliverofindividualCre Infected Naive Dotplotsdepictthefrequenciesandtotalnumberswith mean± miceasindicated.Datacorrespondtotwoindividualexperiments + andCre C) SplenocytesfromCre + WT orα2β1-deficientNKcellsinthespleenofindividual - Itga2 fl/fl micewereinfectedwitheither3,000pfuofWT A) Dotplotsdepictthefrequenciesandtotal - orCre + Itga2 + Itga2 + NK1.1+cellsthatwereinthe fl/fl andCre fl/fl miceasindicated.Dataare - - Itga2 orCre fl/fl weremixed + Itga2 fl/fl mice