Ly49r Activation Receptor Drives Self-MHC–Educated NK Cell Immunity Against Cytomegalovirus Infection

Ly49R activation receptor drives self-MHC–educated NK cell immunity against cytomegalovirus infection

Awndre Gamachea,b, John M. Cronka,b, William T. Nasha,b,c, Patryk Puchalskib,c, Alyssa Gillespieb,c, Hairong Weib,c,1, Laurie Graya, Marie-Louise Hammarskjolda, Wenhao Xua, and Michael G. Browna,b,c,2

aMicrobiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA 22903; bBeirne Carter Center for Immunology Research, University of Virginia, Charlottesville, VA 22903; and cDivision of Nephrology, Department of Medicine, University of Virginia, Charlottesville, VA 22903

Edited by Lewis L. Lanier, University of California San Francisco Medical Center, San Francisco, CA, and approved November 20, 2019 (received for review July 29, 2019) Natural killer (NK) cells mediate vital control of cancer and viral The H-2Dk class I molecule promotes NK cell-mediated infection. They rely on MHC class I (MHC I)-specific self-receptors to control of murine CMV (MCMV) infection in different mouse identify and lyse diseased cells without harming self-MHC strains, including MA/My and C57L.Dk (18–21). Host resistance I-bearing host cells. NK cells bearing inhibitory self-receptors for in these strains was found to correspond with the Ly49G2 in- host MHC I also undergo education, referred to as licensing, which hibitory receptor encoded in most Ly49 haplotypes so far studied causes them to become more responsive to stimulation via (18, 22, 23). Importantly, Ly49G2 self-receptor allotypes expressed activation receptor signaling. Previous work has shown that in MA/My and C57L.Dk mice can license NK cells via H-2Dk, licensed NK cells selectively expand during virus infections and whereas others (e.g., Ly49G2b6) cannot (18, 24, 25). This licensing they are associated with improved clinical response in human effect correlates with H-2Dk–dependent virus control and is abol- + patients experiencing certain chronic virus infections, including ished by specifically depleting Ly49G2 NK cells prior to MCMV HIV and hepatitis C virus. However, the importance of inhibitory infection (18, 24, 26–28). self-receptors in NK-mediated virus immunity is debated as they Although the importance of inhibitory self-receptors for MHC also limit signals in NK cells emanating from virus-specific activa- k I in virus immunity is still debated, Ly49 activation receptors have tion receptors. Using a mouse model of MHC I-dependent (H-2D ) been shown to specifically recognize and target NK cell lysis of virus immunity, we discovered that NK cells depend on the Ly49G2

virus-infected host cells. For example, Ly49H, which binds IMMUNOLOGY AND INFLAMMATION inhibitory self-receptor to mediate virus control, which coincided MCMV m157, directs virus-specific NK cell lysis of infected target with host survival during murine cytomegalovirus infection. This cells in B6 mice (29, 30). Similarly, Ly49L recognition of MCMV antiviral effect further requires active signaling in NK cells via the gp34–H-2Dk complexes was shown to mediate MHC I-dependent Ly49R activation receptor that also binds H-2Dk. In tandem, these MCMV resistance in BALB.K mice (19, 20, 31). Activation re- functionally discordant Ly49 self-receptors increase NK cell pro- + liferation and effector activity during infection, resulting in se- ceptors thus might also contribute in Ly49G2 NK cell re- lective up-regulation of CD25 and KLRG1 in virus-specific sponses during MCMV infection, as predicted (26). The Ly49R + + Ly49R Ly49G2 NK cells. Our findings establish that paired activation receptor, encoded in MA/My-related Ly49 haplotypes, self-receptors act as major determinants of NK cell-mediated virus sensing and immunity. Significance

exome sequencing | gene editing | Ly49 receptor allotypes | NK cell Natural killer (NK) cells provide essential host protection against education | viral immunity cancer and infection. Inhibitory self-receptors for ubiquitously expressed MHC class I (MHC I) molecules are needed to educate atural killer (NK) cells are innate lymphocytes that play a NK cells and help prevent NK cells from attacking autologous Nnonredundant role in sustaining host immunity to virus in- cells in the host. NK cells also utilize activation self-receptors that fections (1, 2). They respond to environmental cues by integrating can augment NK cell responsiveness to viral infections. We show signals from diverse arrays of activation and inhibitory receptors, herein that a distinct pair of functionally discordant self- receptors work in tandem to identify and eliminate infected including structurally unrelated killer Ig-like receptor (KIR) or targets, which coincides with host survival, primarily because Ly49 receptors expressed in different species. Both human KIR they effectively increase NK cell activation and proliferation and rodent Ly49 families include germline-encoded inhibitory and during infection. activation receptors that bind highly polymorphic host (self) MHC I molecules and control NK effector functions. Adaptive selection Author contributions: A. Gamache, J.M.C., W.T.N., A. Gillespie, and M.G.B. designed for binding self MHC I in the different species presumably un- research; A. Gamache, J.M.C., P.P., A. Gillespie, H.W., and M.G.B. performed research; J.M.C., L.G., M.-L.H., W.X., and M.G.B. contributed new reagents/analytic tools; A. Gamache derlies convergent diversification of clustered KIR or Ly49 re- and A. Gillespie curated data; J.M.C., W.X., and M.G.B. generated the GO mice; ceptor genes, which aids in pathogen protection and reproductive A. Gamache, J.M.C., A. Gillespie, and M.G.B. analyzed data; and A. Gamache and M.G.B. functions (3, 4). wrote the paper. Both KIR and Ly49 inhibitory self-receptors help tune NK The authors declare no competing interest. cells during interaction with host MHC I (5, 6). “Self-aware” NK This article is a PNAS Direct Submission. cells that have been tuned are said to be educated or licensed, as Published under the PNAS license. evidenced by enhanced effector function following activation re- Data deposition: The data reported in this paper have been deposited in the Gene − ceptor stimulation, and the ability to kill missing-self (MHC I ) Expression Omnibus (GEO) database (accession nos. GSE132394 and GSM3861587– GSM3861589). target cells (7–9). Licensed NK cells may improve clinical outcomes 1Present Address: Department of Microbiology, School of Medicine, University of Ala- in human patients with chronic virus infections, including hepatitis bama at Birmingham, Birmingham, AL 35205. – Cvirus(HCV)orHIV(1012). Indeed, licensed NK cells have 2To whom correspondence may be addressed. Email: [email protected]. been found to respond and accumulate during several different This article contains supporting information online at https://www.pnas.org/lookup/suppl/ human virus infections, including Hantavirus, Chikungunya virus, doi:10.1073/pnas.1913064117/-/DCSupplemental. hepatitis B virus, HCV, HIV, and cytomegalovirus (CMV) (13–17).

www.pnas.org/cgi/doi/10.1073/pnas.1913064117 PNAS Latest Articles | 1of11 Downloaded by guest on October 1, 2021 isinterestinginthisregardasitwasshowntomodestlybindsoluble were termed G Out1 and G Out2 (GO1 and GO2) (SI Appendix, H-2Dk tetramers (32, 33). Moreover, the Ly49DB6 activation re- Fig. S1B). ceptor, an allele variant of Ly49R, was shown to augment virus- Ly49G2 allotype-specific staining showed that NK cells specific NK responses during MCMV infection. Thus, we in- from GO founder offspring had reduced cell surface Ly49G2L terrogated Ly49R’sroleinH-2Dk-dependent resistance to MCMV expression (SI Appendix,Fig.S1C). Direct sequencing revealed infection. We discovered that the discordant Ly49G2 and Ly49R identical cytosine insertions in GO1 and GO2 Ly49g2 alleles at self-receptors enable NK cells to mediate MHC I-dependent virus the anticipated CRISPR/Cas9 target site, resulting in Ly49G2 control and overall host survival. Our findings highlight a vital role truncation within the stalk region prior to a critical dimeriza- for such paired self-receptor systems that rely on licensing to tion domain (Fig. 1 B and C). Both GO founders transmitted increase activation receptor-driven antiviral NK cell effector their mutations through the germline to establish homozy- activities. gous Ly49g2GO1-andLy49g2GO2-null mice, which can be iden- L Results tified from littermates carrying Ly49g2 alleles using HRM PCR + (Fig. 1D). Generation of Ly49g2-Deficient Mice. Ly49G2 NK cells were pre- L k k GO mice were further crossed with NKC -D to establish viously shown to mediate H-2D -dependent MCMV resistance in GO1 k GO2 k k L k NKC -D and NKC -D strains for virus resistance studies. MA/My, C57L.D ,andB6.NKC -D mice (18, 28). A specific role of L Ly49G2 in virus control, however, remained poorly defined. Thus, we We found NK cells from both strains lack Ly49G2 NK cell used CRISPR/Cas9 genome editing to initially generate B6.NKCL surface expression (Fig. 1E). Whole-genome exome sequencing con- (NKCL) mice deficient in Ly49G2 expression. The C57L allele of firmed Ly49g2 cytosine insertions in both GO strains. Moreover, only Ly49g2L exon 4 was selectively targeted in NKCB6/L heterozygous WT exome sequences (i.e., no mutations) were detected in highly re- embryos, which aided in genotypic and allotypic screening for mutant lated Ly49 genes for the regions spanning the CRISPR target founders (Fig. 1A and SI Appendix,Fig.S1). Two NKCB6/L founders site in Ly49g2 (SI Appendix,TablesS1–S3). Highly specific Ly49 carrying exon 4 indels were identified using Ly49g2-specific high- gene-editing thus selectively abolished Ly49G2 surface ex- resolution melting (HRM) PCR and the resultant mutant alleles pression on GO NK cells.

A B C

Ly49G2L

L B6.NKC Ly49G2GO1 Hybrid CRISPR B6 / L aa#86 female Embryos B6.NKC X ? B6 Offspring male (HRM PCR Screen) D E F G NKCL-Dk NKCGO1-Dk NKCGO2-Dk NKCL-Dk NKCGO1-Dk ) 6 0.15 L 10 8 - Ly49g2L / GO1 - Ly49g2 6 0.05 GO1 4 - Ly49g2 2 FSC

-0.05 0 CD27 NK cells (x10 Diﬀerence RFU k k 78 80 82 L -D -D Temperature (ºC) Ly49G2 GO1 NKC CD11b NKC

H 2×105 PFU 5×104 PFU 0 0 -1 -1 -2 -2 (CMV/β-actin) (CMV/β-actin) 10 10 -3 -3 Log Log DL DL -4 -4 k k k k L k k k L k L -D -D -D -D C L -D -D -D L -D GO1 GO2 NK /GO1 GO1 NKC L / GO2 L NKC NKC NKC NKC NKC NKC NKC NKC G2 Depleted

Fig. 1. NK cells develop normally in Ly49g2-deficient GO mice but fail to control MCMV infection. (A) Diagram of CRISPR/Cas9-mediated editing of Ly49g2L genomic DNA (gDNA) and the breeding scheme used to generate Ly49g2-mutant founders. The protospacer adjacent motif (PAM) sequence is indicated in red. (B) Sequence flanking the CRISPR sgRNA/Cas9 target site of WT Ly49g2L and mutant Ly49g2GO1 alleles. The PAM sequence is underlined and a single cytosine insertion is shown in red. (C) Schematic of putative truncation site. (D) Ly49g2 exon 4-specific HRM PCR was performed with tail gDNA from WT (Ly49g2L), heterozygous (Ly49g2L/GO1), and GO1 (Ly49g2GO1) mice. (E) Representative flow plots show Ly49G2 staining of NK cells from the spleens of uninfected NKCL-Dk, NKCGO1-Dk, and NKCGO2-Dk mice. (F) Spleen NK cell numbers in uninfected NKCL-Dk and NKCGO1-Dk mice. (G) CD27 and CD11b profiles of spleen NK cells from uninfected NKCL-Dk and NKCGO1-Dk mice. (H) Mice were infected intraperitoneally with 2 × 105 PFU (Left)or5× 104 PFU (Right) MCMV and evaluated for spleen virus levels 90 h postinfection. Each symbol represents an individual mouse and error bars indicate mean ± SD. DL, detection limit. Data in D are representative of >20 independent experiments. Data in E–G are representative of 3 independent experiments with 3 to 4 mice per group. (H, Left) Two to 4 mice per group. (Right) Combined data from 3 separate experiments with 3 to 5 mice per group. Error bars indicate mean ± SD.

2of11 | www.pnas.org/cgi/doi/10.1073/pnas.1913064117 Gamache et al. Downloaded by guest on October 1, 2021 NK Cells Develop Normally in Ly49g2-Deficient GO Mice but Fail to Ly49G2 and Ly49R Receptors Engage MHC I Dk. Prior work showed Control MCMV Infection. Homozygous GO mice breed well and that both Ly49G2 and Ly49R receptors can bind soluble develop normally, without obvious health defects. Additionally, H-2Dk tetramers (32). We hypothesized that these discordant NK cell numbers and CD27 and CD11b expression profiles are self-receptors for a shared MHC I ligand may be an important similar in NKCGO1-Dk and NKCL-Dk mice (Fig. 1 F and G). element for specific virus control. To pursue this question, we Thus, Ly49G2 deficiency did not appreciably alter NK cell de- established a reporter cell system (34) to examine whether MA/ velopment at baseline. My- or C57L-derived self-receptor allotypes bind Dk.Incompar- We next assessed Ly49G2’s effect on host resistance by com- ison to control J7 cells, J7.ZGM,J7.ZGL, and J7.ZRL reporter paring spleen virus levels several days after MCMV infection. In cells were selectively stained and stimulated with Ly49-specific comparison to WT Ly49G2 (NKCL-Dk), both GO strains displayed mAbs 4D11 or 12A8 (Fig. 3 A and B). Moreover, each of these higher MCMV levels as in NKCL or NKCL-Dk mice depleted of reporter cell lines specifically responded when cocultured with + Ly49G2 NK cells (Fig. 1H). Thus, the Ly49G2 inhibitory self- YB2/0-Dk cells, but not YB2/0 cells (Fig. 3B). Thus, Ly49G2 and receptor is required for NK cells to mediate MHC I-dependent Ly49R self-receptors both bind H-2Dk. MCMV control. Considering that Ly49 activation receptor expression on NK cells is sensitive to the presence of its cognate ligand in the host MHC I Dk-Dependent MCMV Control Is Comparable to That Mediated via (35–37), we further examined Ly49R expression on NK cells in Ly49H and Is Abolished by Ly49R Neutralization. MHC I-independent H-2Dk–disparate mice. Consistent with the results obtained us- MCMV resistance has been well characterized in B6 mice; however, ing reporter cells, we found that Ly49R expression varied in less is known about the extent of viral control conferred by MHC I- direct relation to host H-2Dk (Fig. 3 C and D), confirming that dependent mechanisms in comparison. As expected, NKCL NK cells Dk is a cognate ligand for both self-receptors. Since licensed NK that lack m157-specific Ly49H receptors failed to control MCMV in cells display greater responsiveness to activation receptor stim- + the absence of H-2Dk (Fig. 2A). In contrast, NKCL-Dk Ly49G2 NK ulation than their unlicensed counterparts (7–9), we next assessed cells controlled MCMV as effectively as virus-specific NK cells in B6 whether Ly49G2 is a primary licensing receptor in NKCL-Dk mice. mice (Fig. 2A). These data demonstrate H-2Dk–dependent MCMV Following stimulation with plate-bound mAbs specific for Nkp46 resistance is as robust as that provided by Ly49H. or Ly49R, we observed a significantly higher percentage of re- + We then interrogated a role for activation receptors in NKCL- sponsive Ly49R NK cells from mice that express the licensing Dk mice. Strikingly, the Ly49R-specific mAb 12A8 selectively receptor and its cognate ligand (Fig. 3 E and F). A small fraction abolished MCMV resistance in comparison to NKp46- or NKG2D- of responsive NK cells (2 to 4%) from each strain were also ob- IMMUNOLOGY AND INFLAMMATION blocking mAbs (Fig. 2B).TheextentofmAb12A8’s effect was served to respond during control stimulation, which suggests + comparable to selective depletion of Ly49G2 NK cells, which immobilized IgG may elicit low-level CD16 signaling. However, suggested both receptors may be important in MCMV control. In background stimulation via control Ig was inadequate to elicit + contrast to Ly49G2 NK cells depletion, however, total NK cell disparate responses in NK cells from the different strains. The + numbers were unaffected by the α-Ly49R mAb. Rather, a compa- Ly49G2 licensing self-receptor thus enhances Ly49R NK cell + rable subset of Ly49R NK cells remained after treatment, which responsiveness in NKCL-Dk mice. was readily detected using another Ly49R-reactive mAb, clone We next tested whether Ly49G2 and Ly49R self-receptors can 4E5 (SI Appendix,Fig.S2A). Moreover, prolonged 12A8 recognize MCMV-infected M2-10B4 targets bearing H-2Dk ligands. treatment in NKCL-Dk mice did not interfere with IFN-γ pro- Despite IFN-β–induced H-2Dk surface expression on M2-10B4 cells duction during NK cell stimulation via the Nkp46 activation (SI Appendix,Fig.S3), only the Ly49G2 reporters significantly + receptor, nor did it alter the ability of Ly49G2 NK cells to responded. Additionally, despite both Ly49G2 and Ly49R reporters reject non-Dk bone marrow cell targets in vivo (SI Appendix, responding to targets infected with MCMV for 24 to 72 h (Fig. 3G), Fig. S2 B–D). These data indicate mAb 12A8 treatment func- Ly49G2 signals diminished as MCMV infection progressed and tionally neutralizes Ly49R signaling without depleting or broadly H-2Dk cell surface expression declined (SI Appendix,Fig.S3), whereas impairing NK effector cells. We infer that Ly49R signaling in Ly49R signaling was maintained throughout. Intriguingly, IFN-β + Ly49G2 NK cells is required to mediate MHC I Dk-dependent treatment of MCMV-infected targets prevented Ly49R-specific rec- MCMV resistance. ognition, whereas Ly49G2 reporters were undeterred. Together, these data demonstrate that while both self-receptors recognize H-2Dk, their binding affinities and MCMV response patterns differ.

A B Ly49R+Ly49G2+ NK Cells Are Specifically Activated during MCMV 0 **** 1 **** *** *** -1 0 Infection. Because NK cells differentiate in response to virus-induced -1 inflammation (38, 39), we examined the impact of Ly49G2- -2 + -2 -3 coexpression on Ly49R NK cell responses during MCMV in- -3 + + (CMV/β-actin) (CMV/β-actin) 10 -4 10 fection. First, we assessed differentiation profiles for Ly49R Ly49G2 -4 + + + – + – Log -5 DL Log (R G2 ) and Ly49R Ly49G2 (R G2 )NKcells.Sincethepropor- -5 DL + –

-6 -6 tions of NK cells in immature (CD27 CD11b ), transitional k k k + B6 L L + + – + B6/L ⎻ ⎻ ⎻ ⎻ ⎻ ⎻ ⎻ NKC C B6 -D -D L -D B6/L + + + + + + + L k (CD27 CD11b ), and more mature (CD27 CD11b )differentia- NK C NKC ⎻ NKC -D + + + – NKC C NKC NK NK tion stages (40, 41) were similar among R G2 and R G2 cells at 4 cIg -Ly49I -NK1.1 -Ly49G2-Ly49R -NKp46-NKG2D d postinfection (dpi) (SI Appendix,Fig.S4A and B), these data suggest that the different NK subsets undergo similar maturation Fig. 2. MHC I Dk-dependent MCMV control is abolished by Ly49R neutral- during infection. We also measured KLRG1 since virus-specific + ization. (A) Quantification of viral genomes in the spleens of the indicated Ly49H NK cells were shown to acquire and maintain KLRG1hi k NKC congenic ± D -transgene expression. (B) Quantification of viral ge- expression during MCMV infection (38, 42). We observed that a + + nomes in the spleens of mice that were treated with depleting or neutral- significantly higher fraction of NKCL-Dk R G2 NK cells izing antibodies against NK cell surface receptors prior to infection. Mice in × 5 exhibited KLRG1 terminal differentiation than their counterpart A and B were infected intraperitoneally with 2 10 PFU MCMV and eval- + – k uated for spleen virus levels 90 h postinfection. All data are representative of R G2 NK cells, and this difference was D -dependent as both hi L 2 to 5 independent experiments with 4 to 5 mice per group. Error bars in- subsets displayed similar KLRG1 frequencies in NKC mice (Fig. 4 dicate mean ± SD. ***P < 0.001, ****P < 0.0001. A and B).

Gamache et al. PNAS Latest Articles | 3of11 Downloaded by guest on October 1, 2021 J7 J7.ZGM J7.ZGL J7.ZRL L k GO1 k A B 100 C NKC -D NKC -D GO1 **** **** NKCL NKC 80 J7 23492 60 J7.ZGM **** L J7.ZG 28570 40 L J7.ZR ns

% of PMA/Iono 20 ns ns 23813 Events (% of Max) 0

-Ly49G2- -Ly49R- k 32330 IgG 9 Media YB2/0 Events (% of Max) Ly49G2 -Ly49R - YB2/0-D Ly49R 40000 D E NKCL-Dk NKCL NKCGO1-Dk 35000 **** **** F L k L GO1 k **** NKC -D NKC NKC -D 30000 60 ns

25000 -IgG Ly49R gMFI 3.75 3.72 3.71 NK Cells 40 20000 + k L k GO1 ** L -D -D NKC GO1 * ** NKC Ly49R *

NKC + NKC 20

-NKp46 ns 14.1 8.90 5.83 G M L 25 J7 J7.ZG J7.ZR %IFN- 0

FSC -IgG-I- **** -Ly49R **** -NKp46 PMA/Iono 20 **** **** **** -Ly49R 13.3 6.76 6.28 15 **** ***

10 %ofPMA/Iono

5 PMA/Iono 38.2 41.2 37.6

IFN- 0 IFN- MCMV

Fig. 3. Ly49G2 and Ly49R receptors specifically bind MHC I Dk.(A) Surface expression of chimeric Ly49 receptors on J7 reporter cells, as determined by anti- Ly49G2 (4D11) and anti-Ly49R (12A8) mAb staining of J7.ZGM, J7.ZGL, or J7.ZRL reporter cells. (B) Reporter cells were stimulated with plate-bound mAbs or target cells. (C) Representative histograms of Ly49R expression by splenic NK cells. (D) Quantification of Ly49R gMFI from C.(E) Representative intracellular IFN-γ staining of spleen NK cells from uninfected NKCL-Dk, NKCL, and NKCGO1-Dk mice following stimulation with the indicated plate-bound mAbs or PMA/ + ionomycin. (F) Percentages of splenic Ly49R NK cells that express IFN-γ from E.(G) Reporter cells were cocultured for 12 h with infected M2-10B4 cells pretreated with IFN-β 16 h before coculture, as indicated. Target cells were infected for the indicated times prior to coculture with reporter cells. Data in A and B are representative of 3 to 5 independent experiments with 2 to 5 samples per group. Data in C and D are representative of 3 experiments with 3 to 4 mice per group. Data in E and F are representative of 2 independent experiments with 4 mice per group. Data in G is representative of 2 experiments with 3 to 6 samples per group. Error bars indicate mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

CD25 up-regulation on NK cells also occurs during MCMV lacking either the self-receptor or its cognate ligand (Fig. 5 A and infection (SI Appendix, Fig. S4C), largely due to virus-induced IL- B). This finding is consistent with previous work showing that high 12 (43, 44). In mice lacking Ly49G2 or Dk, CD25 increased on viral burden induces splenic lymphopenia and lymphoid architec- + Ly49R NK cells to a greater extent than what was seen in NKCL- ture collapse (47). Additionally, there was a greater representation + + Dk mice (Fig. 4 C and D). This is likely due to the sustained in- R G2 NK cells within the NK cell compartment in NKCL-Dk mice flammatory environment in mice lacking Ly49G2 or Dk caused by (Fig. 5 C and D). This skewing is also seen near the peak of NK cell unfettered viral spread (Fig. 1G) (45). In NKCL-Dk mice, how- expansion 6 dpi (SI Appendix,Fig.S4D and E). These results + + + + ever, CD25 selectively increased on R G2 cells. We also ob- suggest Dk-licensed R G2 NK cells are the dominant responding + + served lower CD62L on R G2 NK cells in comparison to subset during MCMV infection. + – R G2 NK cells only in NKCL-Dk mice (Fig. 4 E and F), similar to We assessed whether cell survival differences might explain + + + – virus-specific NK cells in MCMV-infected B6 mice (46). Together, subset variation during infection. R G2 and R G2 NK cells + + these data suggest that Dk-licensed R G2 NK cells experienced from infected NKCL-Dk mice exhibited similar caspase activation, virus-specific activation, as opposed to more general, cytokine- which indicated that apoptosis does not explain differential subset mediated stimulation resulting from virus-induced inflammation. accumulation (SI Appendix, Fig. S5). We next measured NK cell incorporation of BrdU to gauge whether increased proliferation + + + Ly49G2 Promotes Ly49R NK Cell Accumulation and Proliferation accounts for selectively expanded R G2 NK cells. We observed + + during MCMV Infection. We showed in prior work that Dk-li- that a greater proportion of NKCL-Dk R G2 NK cells in- + + – censed Ly49G2 NK cells selectively accumulate in response to corporated BrdU during infection than their R G2 counterparts MCMV (27, 47). We thus examined whether Ly49G2 governs (Fig. 5 E and F). Variation in subset proliferation was not seen in + + + + this expansion. Whereas R G2 NK cells significantly increased in NKCL or GO1-Dk mice, suggesting that Dk-licensed R G2 NK NKCL-Dk spleens by 4 dpi, there was a notable decrease in mice cells selectively increased proliferation during infection.

4of11 | www.pnas.org/cgi/doi/10.1073/pnas.1913064117 Gamache et al. Downloaded by guest on October 1, 2021 Uninfected MCMV ABCL k L NKCL-Dk NKCL NKC -D NKC R+G2+ R+G2– 1.21 70.2 44.5 10.4 + 48.6 +

80 ** G2 + G2 + R

R 60 NK cells GO1 k NKCGO1-Dk Hi 40 NKC -D

20 Ly49R KLRG1 1.64 56.9 46.7 40.8

– 2.55 49.7 59.5

- 0 G2 +

G2 k L k + R %KLRG1 C R L -D -D NK GO1 NKC NKC Ly49R CD25 D E NKCL-Dk NKCL NKCGO1-Dk F R+G2+ R+G2+ R+G2– R+G2+ R+G2– R+G2– 80 R+G2null 60 80 * NK cells

Uninfected 60 + 40 NK cells 20 ** + 40 0 20

%CD25 k L k 0 L -D -D k L k

GO1 %CD62L

NKC Infected L -D -D NKC GO1 NKC NKC NKC CD62L NKC + + Fig. 4. Ly49R Ly49G2 NK cells are specifically activated during MCMV infection. (A and B) Flow plots and quantification of KLRG1hi splenic NK cell + subsets from uninfected or mice 4 dpi. (C and D) Flow plots and quantification of CD25 splenic NK cell subsets from mice 4 dpi. (E)HistogramsofCD62L + expression by splenic NK cell subsets from mice uninfected or 4 dpi. (F) Frequency of NK cells that remain CD62L at day 4 postinfection. Mice were infected with 5 × 104 PFU MCMV. All Data are representative of 2 to 3 independent experiments with 3 to 4 mice per group. Error bars indicate mean ± SD. *P < 0.05, **P < 0.01. IMMUNOLOGY AND INFLAMMATION

+ – To test whether subset skewing was due to an intrinsic defect pIC treatment, which indicated that R G2 NK cells are com- + – in R G2 proliferation, we injected mice with PolyI:C (pIC) to petent to undergo rapid proliferation (Fig. 5G). Taken together, mimic virus-induced inflammation and again measured NK cell these data demonstrate that the selective accumulation of + + uptake of BrdU. In contrast to the results obtained during R G2 NK cells resulted from enhanced proliferation in re- MCMV infection, both subsets responded equivalently following sponse to MCMV infection.

Uninfected MCMV ABNKCL-Dk NKCL NKCGO1-Dk Uninfected MCMV C D Uninfected MCMV )

) 33.1 37.9 18.9 56.6 6 ) 6 15 **** 6 * *** 6 5 **** + ×10 ×10 4 10 *** 4 ×10 ** 3 2 2 * 5 Ly49R Ly49R 1 0 0 7.98 8.59 NK cells (

NK cells ( 0

k L k NK cells ( Ly49G2 + – L -D -D GO1 + G2 + G2 MCMV NKC R NKC R Uninfected NKC

EGFHL k NKCL-Dk NKCL MITOTIC SPINDLE NKC -D + + + – R G2 R G2 MYC TARGETS V1 NKCL &NKCGO1Dk 33.2 22.7 20 50 E2F TARGETS + + + 40 * 15

G2 G2M CHECKPOINT + 30 10 R 20 IL6 J AK STAT3 SIGNALING GO1 k 5 %BrdU NKC -D %BrdU 10 0 APOPTOSIS 24.4 18.3 18.6 0 + –

Ly49R KRAS_SIGNALING_UP k L k + – + G2 L -D -D G2 INFLAMMATORY RESPONSE NKC GO1 R R G2 + NKC TNFA SIGNALING VIA NFKB R NKC IL2 STAT5 SIGNALING BrdU INTERFERON ALPHA RESPONSE INTERFERON GAMMA RESPONSE

0 10 20 30 40 50

-log10 FDR(q-value)

Fig. 5. Ly49G2 promotes Ly49R+ NK cell accumulation, proliferation, and differential gene expression during MCMV infection. (A and B) Total NK cells and + Ly49R NK cells from the spleens of uninfected mice and mice 4 dpi. (C and D) Distribution profiles of Ly49R and Ly49G2 on NK cells in naïve mice or mice 4 dpi. + − + + NKCL-Dk mice. Representative flow plots are shown in C. Quantified numbers of R G2 and R G2 subsets are shown in D.(E and F) BrdU incorporation after 3-h pulse BrdU treatment 4 dpi. (G) BrdU incorporation after 3-h pulse BrdU treatment 3 d after pIC injection. In A–F, mice were infected with 5 × 104 PFU MCMV. (H) Selected hallmark genes and corresponding gene enrichment analysis of NKCL-Dk splenic NK cells. Data are representative of 3 to 6 independent experiments with 3 to 4 mice per group. (G) Data are representative of 2 independent experiments with 3 to 4 mice per group. Error bars indicate mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Gamache et al. PNAS Latest Articles | 5of11 Downloaded by guest on October 1, 2021 High-Dimensional Transcriptomic Profiling of Mouse Splenic NK Cells resulted in profoundly altered gene expression to enable NK cell during MCMV Infection. Single-cell RNA sequencing (scRNA-seq) expansion. was used to evaluate transcriptomic differences in NK cells To ascertain whether strain-specific differences in NK cell gene responding to MCMV. An unbiased t-distributed stochastic expression were simply due to different extrinsic signals based on host neighbor embedding (t-SNE) approach was applied to analyze environment, we performed a similar analysis using NK cells from scRNA-seq data (48). We observed NK cells from MHC I- or infected NKCL-Dk mice only. Three distinctive t-SNE clusters were Ly49G2-disparate mouse strains clustered on the basis of sample, identified for comparison (SI Appendix,Fig.S7A). GSEA of hallmark such that NKCL-Dk clusters differed from those in NKCL and genes showed that cluster 1 (C1) was highest in genes associated with NKCGO1 (SI Appendix, Fig. S6A). This suggests that NKCL and up-regulation of cell cycle control, DNA repair, and metabolic ac- NKCGO1 NK cells are transcriptionally similar during infection, in tivity (SI Appendix,Fig.S7B–D), similar to data obtained for all contrast to NK cells from infected NKCL-Dk mice (SI Appendix, NKCL-Dk NK cells in Fig. 5H. As a dominant responding subset with Fig. S6B). Indeed, examination of gene differences in NKCL and enhanced proliferative and metabolic function, C1 NK cells likely NKCGO1,asidefromH-2D or Klra7, revealed little substantive contributed significant MCMV control in NKCL-Dk mice. In con- variation (SI Appendix,Fig.S6C). trast, analysis of clusters 2 and 3 (C2 and C3) NK cells revealed Gene set enrichment analysis (GSEA) using published hall- significant up-regulation of complement and inflammatory response mark gene sets revealed that NKCL and NKCGO1 NK gene ex- pathways as was observed for NKCL and NKCGO1 NK cells (Fig. 5H pression is significantly skewed toward up-regulation of an and SI Appendix,Fig.S7D). Together, these data indicate C2- and inflammatory response, including signaling via IL-2/STAT5, C3-type NK cell responses were not limited only to a highly in- and strong TNF-α and IFN signatures (Fig. 5H). On the flammatory environment as in infected NKCL or NKCGO1 mice. + + other hand, NKCL-Dk mice with licensed R G2 NK cells up- Rather, they represented a significant NK cell gene-expression regulated genes associated with cell cycle control and prolif- signature, possibly underpinning key antiviral activities. We infer eration. A net effect of self-receptor–dependent virus control thus C2- and C3-type NK responses may be overactivated in the

R+G2+ AB R+G2– C L k + null NKC -D R G2 4 4 WT or GO1-Dk Recipient 3 **** GO1-Dk 3 2 2 GO1 k +MCMV NKC -D 1:1 Recipient 1 1 Expansion Index FC analysis Expansion Index WT 0 0 L k GO1 k CFSE CTV NKC -D NKC -D Recipient Recipient D E F 60 40 Flow-sorted Hi

+ **** L k 30 45.1 NK cells 0 NKC -D 40 Recipient 20 -1 ** 20 10

%CD25 + – + +

%KLRG1 R G2 R G2 -2 * 0 0 R+ G2+ -3 p=0.06 R+G2–

null (CMV/ β -actin) -4 R+G2 45.2 45.2 10 60 ** 40 **** Hi -5 +

k Log GO1 k 30 B6.D Recipients NKC -D 40 20 (NK depleted / Ly49H blocked) -6 Recipient 20 – + 10 + + %CD25 +MCMV G2 G2 0 %KLRG1 0 R R FC analysis No transfer

GH I 100 + – + +

) R G2 R G2 4 5 L k p=0.0068 4 ** 23.0 98.4 ×10 NKC -D 3 50 NKCGO1-Dk 2

1 % Survival Ly49R Transferred 0

NK cells ( – + 0 + G2 + G2 R R Ly49G2 2 4 6810 Days post-infection

+ Fig. 6. Ly49G2 receptor licensing enables Ly49R NK cell activation, virus control, and host survival during MCMV infection. (A) Diagram illustrating the adoptive transfer of NKCL-Dk (WT, CFSE-labeled) and NKCGO1-Dk (GO1-Dk, CTV-labeled) donor splenic NK cells (mixed 1:1) into NKCL-Dk or NKCGO1-Dk recipients 24 h prior to MCMV infection. (B) CFSE and CTV dilution profiles of enriched donor splenic NK cells from NKCL-Dk or NKCGO1-Dk mice. (C) Quanti- + fication of expansion indices of data in B.(D) Frequency of CD25 or KLRG1hi donor NK cells in B on day 4 postinfection (E) Diagram illustrating NKCL-Dk NK enrichment and flow sorting into 2 major subpopulations. Sorted cells (∼3 × 105) were adoptively transferred into NKCB6-Dk-CD45.1 mice pretreated with α-Ly49H (3D10) and α-NK1.1(PK136) mAbs. (F) Quantification of viral genomes in the spleens of recipient mice 4 dpi. (G) Accumulation of transferred NK cells in the spleen day 4 postinfection. (H) Ly49G2 positivity 4 dpi after cell sorting. (I) Host survival curves for NKCL-Dk or NKCGO1-Dk mice following infection with 1 × 106 PFU MCMV. In A–G, mice were infected with 2.5 × 104 PFU MCMV. Data are representative of 3 to 4 independent experiments with 3 to 4 mice per group. Error bars indicate mean ± SD. In F, statistical significance determined by post hoc Dunn’s test. *P < 0.05, **P < 0.01, ****P < 0.0001. In I, data are from a single experiment with 9 to 10 mice per group. Log-ranked Mantle–Cox test was used to determine statistical significance P = 0.0068.

6of11 | www.pnas.org/cgi/doi/10.1073/pnas.1913064117 Gamache et al. Downloaded by guest on October 1, 2021 absence of highly specific antiviral NK cells. More importantly, MCMV control (29, 30). Hence, licensed NK cells may be dis- these data demonstrate that the Ly49G2 self-receptor itself pensable if virus-specific recognition by NK activation receptors drives an intrinsic difference in NK cells specifically responding is adequate to overcome tolerance. Nonetheless, we found that + + to infection, whereas extrinsic factors were also involved. licensed R G2 NK cells are essential to mediate vigorous MHC I-dependent host immunity during WT MCMV infection. + Ly49G2 Receptor Licensing Enables Ly49R NK Cell Activation, Virus Moreover, licensed NK cells disarmed by exposure to MHC Control, and Host Survival during MCMV Infection. To verify Ilo host cells can regain the capacity to mediate missing-self re- + Ly49G2’s role in the activation and expansion of Ly49R NK sponses after MCMV-induced activation (57, 58). Licensed NK cells, we cotransferred differentially labeled NKCL-Dk and cells thus may be uniquely poised to overcome self-tolerance NKCGO1-Dk NK cells into NKCL-Dk or NKCGO1-Dk recipients during MCMV infection. + + prior to MCMV infection (Fig. 6A). Remarkably, R G2 NK Although seemingly counterintuitive, inhibitory receptors have cells rapidly responded and displayed enhanced proliferation been shown to augment lymphocyte effector functions. In T cells, and expansion in NKCGO1-Dk recipients (Fig. 6 B and C). the inhibitory NKG2A receptor was shown to increase control of + + + Moreover, R G2 NK cells displayed selective up-regulation of ectromelia virus infection by promoting NKG2A CD8 T cell CD25 and KLRG1 expression, especially in NKCGO1-Dk recip- survival (59). Related to this, human inhibitory KIRs enhanced + – + ients, in comparison to either R G2 or R G2null NK cells (Fig. murine CD8 T cell proliferation ex vivo in response to stimula- 6D). Together, these data demonstrate a cell-intrinsic role for tion by dendritic cells bearing transgenic cognate HLA mole- + Ly49G2 in promoting specific Ly49R NK cell responses to cules (60). Additionally, expression of self-specific inhibitory MCMV infection. KIRs was found to coincide with increased CD8 T cell survival + + To confirm that R G2 NK cells are responsible for enhanced and better overall virus control in patients infected with HIV, + – + + virus control, we enriched R G2 and R G2 NK subsets and HCV, or human T cell leukemia virus type 1 (61). separately transferred them into B6.Dk (i.e., NKCB6) recipients. Self-MHC I-specific inhibitory receptors that license de- Since NKCL-derived NK cells are resistant to PK136 (anti- veloping NK cells also increase the extent of activation receptor NK1.1) depletion (18, 21), this system allowed us to ablate en- stimulation (7–9). Licensed NK cells that are educated on self- dogenous NKCB6 NK cells in recipients prior to transfer. Thus, MHC I undergo expansion and differentiation in response to any effects on virus control stem from the transferred NK cells MCMV infection (24, 27, 62, 63), and in HCMV-infected indi- + – (Fig. 6E). While R G2 NK cells had no impact on virus control, viduals (17). Memory NK cells expressing self-specific inhibitory + + recipients of R G2 NK cells exhibited lower viral burden and Ly49 receptors in hapten-sensitized mice likewise display en- greater accumulation of NK cells in spleen than recipients of hanced recall responses (64, 65). Despite that licensed NK cells IMMUNOLOGY AND INFLAMMATION + – R G2 NK cells (Fig. 6 F and G), thus confirming that licensed expand in these varied contexts, a basis for this response is poorly + + R G2 NK cells provide essential MCMV control. understood. We envision several possibilities may account for Ly49g2129 gene activation has been shown to occur in mature selective expansion in response to viral infection. 1) Licensing NK cells in vitro in the presence of IL-2 (49). Additionally, could increase activation receptor signals in response to virus or + Ly49G2 NK cells in B6 mice expand nonspecifically following virus-induced antigens via altering activation signal transduction bone marrow transplantation and MCMV infection (50). Whether cascades. This explanation predicts that both licensing and ac- this is due to clonal expansion or de novo Ly49g2 expression in tivation receptors can specifically recognize and respond to virus- – Ly49G2 NK cells remains uncertain, but it may be up-regulated infected target cells. 2) Sustained binding of the inhibitory in activated NK cells. Whereas most adoptively transferred receptor could promote NK cell synapse formation and conjuga- + – R G2 NK cells remained so during infection, a minor fraction tion to infected target cells. 3) The licensing receptor could clearly expressed Ly49G2 receptors (Fig. 6H). This could be the modify the activation receptor ligand so that activation signals result of Ly49g2 gene activation, or possibly clonal expansion of are increased. Ongoing studies are focused on determining how + + a rare population of residual R G2 cells remaining following the Ly49G2 receptor enables NK cells to specifically recognize – flow sorting prior to adoptive transfer. Nonetheless, G2 NK and respond to MCMV infection. + cells are not a significant precursor population to G2 NK cells Although the Ly49G2 receptor was found to be essential, virus + + and R G2 NK cells undergo dramatic clonal expansion during control was abolished when the Ly49R self-receptor was neu- MCMV infection. tralized. Two additional activation receptors implicated in H-2k– Having verified the importance of the Ly49G2 receptor on dependent MCMV resistance include Ly49L and Ly49P, which + Ly49R NK cell-mediated MCMV control in the spleen, we are potential allele variants that both bind MCMV gp34– + assessed their role in host survival by administering a sublethal Dk complexes (19, 20, 66). Indeed, adult Ly49L NK cells pro- dose of MCMV to Ly49G2 WT and GO mice. All mice with WT tected BALB.K neonates upon transfer and subsequent MCMV + + Ly49G2 NK cells survived the infection, whereas >50% of GO challenge. A role for Ly49P NK cells in vivo remains elusive mice succumbed (Fig. 6I). Thus, the Ly49G2 inhibitory self- since there is no available serologic or genetic tool to selectively receptor is essential in MHC I-dependent virus immunity and ablate this subset. Still, we found that MCMV resistance in NKCL- host survival when coexpressed on NK cells with its functionally Dk mice is abolished either by serologic or genetic depletion of + discordant Ly49R self-receptor counterpart. Ly49G2 NK cells. Moreover, the Ly49R-monospecific mAb sufficed to abrogate MCMV control to a similar extent as + Discussion immunodepletion of Ly49G2 NK cells. A role for Ly49P in MHC While a widely held paradigm suggests licensed NK cells pri- I-dependent MCMV resistance thus is unclear. marily thwart NK-mediated virus control, here we demonstrate Our data instead demonstrated expression of both Ly49G2 that the inhibitory Ly49G2 NK cell receptor is required to spe- and Ly49R receptors in individual NK cells is required to elicit cifically augment host defenses, including NK cell differentiation MCMV control. Although H-2Dk tetramers were previously and proliferation, and limit virus spread during MCMV in- shown to bind Ly49R, they were folded with human β2-micro- fection. A role for an inhibitory receptor in virus control may globulin, which could have affected the interaction (32). Con- seem paradoxical since several studies show NK-mediated anti- sistent with the prior study, we found Ly49R reporter cells were viral activities are diminished in their presence (51–54). More- specifically stimulated by Dk-bearing YB20-Dk rat lymphoma over, NK cells can mediate MCMV control in MHC I-deficient cells and MCMV-infected M2-10B4 bone marrow stromal cells. animals lacking self-receptor ligands (51, 55, 56). However, Curiously, Ly49R reporters did not respond to uninfected or + MCMV m157-specific Ly49H NK cells display MHC I-independent IFN-β–stimulated M2-10B4 cells with high Dk. We speculate that

Gamache et al. PNAS Latest Articles | 7of11 Downloaded by guest on October 1, 2021 Dk conformational differences in the different cell lines may immune strategy in nature, which underscores the need for fur- underlie disparate Ly49R responses. Nonetheless, MCMV in- ther research to determine if similar receptor pairings are pre- fected M2-10B4 cells consistently triggered Ly49R signaling, sent in humans. A better understanding of such inhibitory and which was abrogated by IFN-β treatment. We additionally found activation receptor pairs will further the development of new that Ly49R expression on mouse NK cells is regulated by host strategies to augment host immunity and improve clinical out- cell Dk expression, similar to Ly49D down-regulation in the comes in the context of viral infections, tissue transplant, presence of its ligand, Dd (37). Altogether, these data suggest the and cancer. Ly49R self-receptor is sensitive to variations in Dk expression, especially during MCMV infection. Materials and Methods Since both Ly49R and Ly49G2 self-receptors bind the same Ethics Statement. Mouse experiments were performed in accordance with the ligand, a qualitative change in H-2Dk on infected target cells Animal Welfare Act and approved by the University of Virginia Institutional might result in a loss of Ly49G2-dependent self-control, in- Animal Care and Use Committee. creased Ly49R-mediated recognition, or a combination of these Mice. B6.NKCC57L-Dk (NKCL-Dk), B6.NKCC57L (NKCC57L), and B6.Dk mice (28), as effects leading to increased NK cell activity, proliferation, and GO1 GO1 k k virus control. This might occur through NK self-receptor– well as B6.NKC (GO1) and B6.NKC -D (GO1-D ) mice, were generated dependent recognition of viral peptide ligands or virus-induced and maintained at the University of Virginia under specific pathogen-free conditions. B6.SJL-PtprcaPepcb/BoyJ mice (Jackson Laboratory) were crossed modification of host MHC I. In human, select peptides can with B6.Dk mice (28) to generate B6.Dk-CD45.1 mice. nullify stimulation of KIR inhibitory receptors by their cognate MHC I ligands (67, 68). In contrast, KIR2DS2, a human NK cell- MCMV. Salivary gland passaged MCMV (Smith Strain; ATCC) was titered on activating receptor, exhibits a strong affinity for highly conserved NIH 3T3 or M2-10B4 and intraperitoneally injected at stated doses, as de- flavivirus peptide motifs presented by HLA-C*0102 (69), which scribed previously (76, 77). Mice were injected intraperitoneally with 200 μg suggests MHC I-specific NK activation receptors can specifically PK136, 4D11 or AT8 48 h before infection to deplete NK cells. Ly49R was recognize viral antigens presented by MHC molecules. Although neutralized using 200 μg mAb 12A8 (a gift from John Ortaldo, National Ly49 receptors interface with MHC I molecules beneath the Cancer Institute, National Institutes of Health, Frederick, MD) given in- peptide binding groove, they can also display peptide selectivity traperitoneally 72 and 24 h before infection (32, 78, 79). Ly49H, NKp46, or NKG2D were, respectively, neutralized using 200 μg mAbs 3D10, 29A1.4, or (70, 71). It is possible that Ly49 activation receptors might display – similar specificity for virus peptide-modified host MHC I (72). C7 given intraperitoneally 24 h before infection (80 82). Infected mouse spleen DNA was measured for MCMV genomes via quantitative PCR, as Specific proliferation is a salient feature of antigen-dependent described previously (83). effector NK cell responses during MCMV infection. Splenic NK cells generally expand and become activated in an antigen- Antibodies and Flow Cytometry. Flow cytometry (FC) was performed using BD independent manner via cytokine stimulation (73), whereas + FACS Canto II, CytoFLEX, or Aurora Northern Lights flow cytometers. Data Ly49H NK cells exhibit DAP12-dependent proliferation in B6 were respectively collected using FACSDiva, CytExpert, or Spectroflo software + + k mice (74). Selective expansion of the R G2 subset in H-2D mice and analyzed using FlowJo (versions 9.7.2 and 10.1 to 10.4). Fluorescent mAbs + is reminiscent of that seen with MCMV m157-specific Ly49H NK were purchased from BioLegend, BD Biosciences, and eBioscience. The 2.4G2, cells. In addition to increased proliferation, these NK cells also PK136, 3D10, AT8, and 4D11 mAbs were purified from spent supernatants by + become KLRG1hi CD62Llo CD25 (38, 39, 75). Our data are thus the University of Virginia Lymphocyte Culture Center. The 12A8 mAb was consistent with increased CD25 observed for NK cells responding kindly provided by John Ortaldo. to MCMV (44). We additionally observed nonselective CD25 up- Fluorescent mAbs from BioLegend, BD Biosciences, and eBioscience were regulation on NK cells in infected mice lacking Ly49G2 or Dk, titrated for optimal resolution and used to stain CD3 (145-2C11), CD19 (6D5), NK1.1 (PK136), CD49b (DX5), NKp46 (29A1.4), Ly49G2 (4D11 and Cwy-3), consistent with the hypothesis that CD25 is regulated independent Ly49R (12A8), Ly49ROV (4E5), CD27 (LG.7F9), CD11b (M1/70), DNAM1 of Ly49 activation receptors (44). It is possible that licensed virus- (10E5), KLRG1 (2F1), Ki67 (16A8), BrdU (BU20a), IFN-γ (XMG1.2), GZMB specific NK cells are more sensitive, or have better access to IL-12 + + (NGZB), or CD62L (MEL-14). LIVE/DEAD fixable dyes (Thermo Fisher Scientific) during MCMV infection. Altogether, our data suggest R G2 NK were used to assess cell viability. cells undergo antigen-specific stimulation, which promotes their differentiation and effector functions. Adoptive Transfers. B6.Dk-CD45.1 mice were pretreated with NK depleting Ly49R signals in isolation are inadequate since Ly49G2 (PK136) and Ly49H neutralizing (3D10) mAbs 48 h before and on the day of coexpression is required for optimal NK effector function. adoptive transfer. CFSE [5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester]-labeled spleen NK cells (bulk transfer), or flow-sorted (Influx, Univer- scRNA-seq analysis revealed that extrinsic and intrinsic factors + affected NK cells expressing discordant self-receptors to increase sity of Virginia FC Core Facility) Ly49 NK subsets (≥99% purity) were intravenously injected into host mice 24 h before infection. For CFSE staining, genes for cell cycle regulation and proliferation during MCMV μ μ infection. In comparison, NK cells from mice lacking the Ly49G2 500 Lof10 M CFSE (made fresh in complete RPMI) was added dropwise to enriched NK cells in 500 μL with vortexing and then incubated for 5 min at self-receptor or its cognate ligand skewed gene expression toward room temperature before quenching in 10 mL complete RPMI. Next, 3 × inflammatory response pathways. We infer that NK proliferation 105 donor NKCL-Dk NK cells were intravenously injected into host mice 24 h and differentiation is dependent on a balance of inhibitory and + + prior to infection. The expansion index is a measure of the fold-expansion of activation receptor signaling pathways in R G2 NK cells, which the original population (total divided cells/estimated original starting shifts to overcome self-tolerance upon recognition of infected population). + – targets. We further posit that R G2 NK cells are unable to overcome disarming in the absence of licensing receptor-enhanced In Vitro Stimulation and Intracellular Cytokine Staining. Mouse splenocytes recognition of target cells. cultured in complete RPMI plus IL-2 (200 U/mL; Peprotech) were used in ex In conclusion, our data uncover an underappreciated role vivo stimulations. Splenocytes (1 to 2 million) were stimulated with immobilized mAbs 12A8, NKp46, or control IgG (plates coated with 20 μg/mL mAb for inhibitory self-receptors in promoting activation and expan- μ sion of NK cells in response to viral infection. This mechanism of overnight at 4 °C) or PMA (100 ng/mL) and ionomycin (1 g/mL) for 1 h prior to brefeldin A (BFA) addition, and an additional 4 h with BFA. Stimulated NK cell detection of viral infection is reliant upon a receptor cells were fixed and permeabilized using a kit (Cytofix/Cytoperm; BD pair with discordant functions. We predict these self-receptors Biosciences) followed by staining for intracellular cytokines at 4 °C. working in tandem may be much more sensitive to subtle variations in MHC I ligands (i.e., altered-self) so as to trigger highly BrdU Incorporation Assay. Mice were intraperitoneally injected with BrdU aggressive NK cell effector activities and increased proliferation. (1 mg/ 200 μL PBS) 3 h prior to being killed. BrdU staining was performed using This intricate host–pathogen interaction may be an important a kit (BD Biosciences) per the manufacturer’s instructions.

8of11 | www.pnas.org/cgi/doi/10.1073/pnas.1913064117 Gamache et al. Downloaded by guest on October 1, 2021 In Vivo Cytotoxicity Assay. In vivo cytotoxicity was performed essentially as NKCL, or NKCGO1-Dk spleen NK cells (>80% viability, 90 to 95% purity) and described previously (24, 84). Briefly, a 1:1 mix of 2 × 106 Dk and no-Dk bone sequenced by the University of Virginia Genome Analysis and Technology marrow cells (in 200 μL RPMI), respectively labeled with 5 μM CFSE or CellTrace Core using a Chromium Controller instrument (10X Genomics) and the Violet (CTV), were intravenously injected into host mice. Spleens were Chromium Single Cell 3′ Reagent Kit V3 (10X Genomics) following the harvested 20 h posttransfer and analyzed for residual donor cells by FC. manufacturer’s protocol. The indexed libraries were sized using the Agilent 4200 TapeStation and pooled into equimolar concentration. Samples were Statistical Analysis. Statistical analysis was performed using Graphpad Prism pooled and sequenced in a single run to avoid batch effects. High- (v7.04). Significance was assessed using 1- or 2-way ANOVA in conjunction throughput sequencing was performed using a NextSeq 500 Sequencer with Tukey or Holms–Sidak post hoc tests unless otherwise stated (*P < 0.05, (Illumina) and the High Output Kit V2.5 (150 cycles) using the following **P < 0.01, ***P < 0.001, ****P < 0.0001). Student’s t-test or Mann–Whitney settings for Read 1 (26 cycles), Read 2 (98 cycles), and Read Index (8 cycles). U rank test was used when comparing the means of 2 independent groups. Collected data (.bcl files) were exported for data processing and quality assessment prior to further analysis using Cell Ranger (10X Genomics) data Design and In Vitro Transcription of Single-Guide RNA. An allele-specific single- analysis pipelines. Reads were aligned to the transcriptome using the guide RNA (sgRNA) (5′-GCG UGG UGC UGC AGU UAU CG-3′) was used to edit “Count” function in Cell Ranger so that expression of selected genes were Ly49g2L exon 4 based on available 129 and C57L allele sequences using https:// assigned to single cells via attached barcodes. zlab.bio/guide-design-resources, as previously described (85). The sgRNA was Cell Ranger was used to perform t-SNE clustering of single-cell data, which selected to maximize the likelihood of specific Ly49g2L exon4 editing while was further analyzed using the Loupe Cell Browser (10X Genomics). Differ- minimizing the potential to edit highly related Ly49 genes. Notably, a 5′ Gwas entially expressed genes were identified and ranked by statistical signifi-

appended to the sgRNA to ensure efficient in vitro transcription with T7 poly- cance. Statistically significant genes were further ranked by log2 FC merase. Ly49g2L allele-specific oligonucleotides (Integrated DNA Technologies) expression differences between clusters and visualized in heatmaps gener- cloned in pX330-U6-Chimeric_BB-CBh-hSpCas9 (kindly provided by Feng Zhang, ated in Prism (Graphpad v7.05). Heatmap rank = up-regulated genes per

Broad Institute of MIT, Cambridge, MA; Addgene plasmid #42230) were used to cluster (most to least) with a log2 FC of at least 1. validate gene-editing efficiency in stem cells prior to work with mouse embryos. The in vitro transcription template was amplified using a high-fidelity DNA Chimeric CD3ζ-Ly49 Reporter Cells. Cd3ζ-Ly49g2MAMY, Cd3ζ-Ly49g2C57L,and polymerase (Phusion, New England Biolabs), pX330-Ly49g2-exon4 vector, Cd3ζ-Ly49r chimeric receptor gene cassettes were generated using PCR es- and a primer designed to append the T7 promoter to the Ly49g2-sgRNA sentially as described for Ly49daz (90), although we fused Cd3z cytoplasmic encoding oligonuceltide (86). The template was purified using a kit tail and Ly49g transmembrane domain coding sequences, followed by cod- × (Qiagen QIAQuick PCR purification) followed by dialysis against 1 TE. The ing sequences for Ly49g or Ly49r ectodomains. Sequence-verified constructs template was then transcribed and its product purified using Ambion, were separately subcloned into pMXs-IRES-PURO (kindly provided by T. MEGAshortscript, and MEGAclear T7 kits. Kitamura, University of Tokyo, Tokyo, Japan). Expression constructs were transfected into 293T cells together with pMD2.G (kindly provided by Didier

L IMMUNOLOGY AND INFLAMMATION HRM PCR Genotyping for Edited Ly49g2 Alleles. Ly49g2 exon4-specific primers Trono, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland) (For 5′-GAC TAA CTT AGT TTT TCA GC-3′ and Rev 5′-GCA GTT CAT CCT TCA (Addgene plasmid #12259; http://www.addgene.org/12259/;RRID:Addg- AGT TGA-3′) spanning the sgRNA target site were designed essentially as ene_12259) and pHIT60 (kindly provided by Alan Kingsman, Oxford University, described previously (87). Primers (Integrated DNA Technologies) were op- Oxford, United Kingdom) (91) using lipofectamine. Retroviral supernatants timized and used in HRM PCR as described previously (33, 87, 88). were used to transduce J7 cells (a gift from K. Iizuka) and Ly49 receptor- expressing reporter cells were selected in puromycyin-containing media es- B6 Generation and Validation of Ly49g2 Deficient GO Mice. B6 (NKC ) (Jackson sentially as described previously (92). CD3ζ-Ly49G2C57L (also known as J7.ZGL) C57L L Labs) males were bred to superovulated B6.NKC (NKC ) females to reporter cells were flow-sorted for high expression, comparable to CD3ζ- B6/L generate B6.NKC embryos that were microinjected with Cas9 protein Ly49G2MAMY (akaalso known as J7.ZGM)andCD3ζ-Ly49R (also known as (Integrated DNA Technologies) and Ly49g2 exon4-specific sgRNA prior to J7.ZRL). Ly49 reporter cells (2 × 105) were stimulated for 8–12 h with plate- implantation into foster mothers. Offspring tail DNA was prepared using a bound mAbs, YB20, YB20-Dk,orM2-10B4(±IFN-β or MCMV infection) target L kit (Gentraprep) and screened in HRM PCR using Ly49g2 exon4-specific primers. cells, or PMA + ionomycin. LacZ activity was determined using the substrate L Five viable offspring carried Ly49g2 exon 4 indels. Two founders transmitted chlorophenol red-D-galactoside (CPRG), as described previously (93). exon4 indels through the germline and were separately crossed back to NKCL to L k generate homozygous GO mice, before further crossing to NKC -D . Data Availability. All scRNA-seq data are accessible from the National Center Ly49g2 GO alleles were validated using whole-genome exome sequenc- for Biotechnology Information Gene Expression Omnibus depository using ing of liver DNA, which was performed by the Genomic Services Lab at GEO accession no. GSE132394. Hudson Alpha essentially as described previously (89). Briefly, GO1 and GO2 FastQ files were separately aligned to the Ly49g2L reference sequence using ACKNOWLEDGMENTS. We thank Jeff Teoh, Jessica Prince, Karolina Dziewulska, – BWA-MEM in Sequencher (Gene Codes Corporation). A Burrows Wheeler Jeremy Gatesmen, and Lucas Turner for technical assistance; and Oscar – Aligner (BWA)-MEM generated BAM file was opened in Tablet (James Aguilar, Stephen Anderson, Jon Heusel, Koho Iizuka, Kevin Kane, Lewis Hutton Institute) to visualize and identify CRISPR-modified Ly49 sequences Lanier, John Ortaldo, Andrew Makrigiannis, and Aimen Shaaban for kindly overlapping the target sequence. WT and CRISPR-modified Ly49 sequences providing reagents. We are grateful for services provided by the Fold from this alignment were exported and realigned using high-stringency Change, Genetically Engineered Murine Model, and Genome Analysis and parameters (minimum overlap 25 nucleotides, minimum match 97%) in Technology Cores at the University of Virginia. This work was supported Sequencher. Individual GO Ly49 contig alignments were reviewed for nu- by Public Health Service Grant R01 AI050072 (to M.G.B.), the Department cleotide discrepancies and consensus sequences overlapping the Ly49g2 of Medicine, Division of Nephrology and the Beirne Carter Center for Immunology Research, University of Virginia, and an American Association CRISPR target site are reported in SI Appendix, Tables S1–S3. of Immunologists Career award (to M.G.B. and A. Gamache). A. Gamache and J.M.C. received support on Public Health Service Training Grant T32 scRNA-Seq Analysis of NK Cells. Single-cell cDNA libraries were prepared from AI07496. W.T.N. received support on Public Health Service Training Grant negatively enriched (3 rounds NK isolation kit, Miltenyi Biotec) NKCL-Dk, T32 DK072922.

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