Published February 9, 2018, doi:10.4049/jimmunol.1701676 Th eJournal of Cutting Edge Immunology

Cutting Edge: Check Your Mice—A Point Mutation in the Ncr1 Locus Identified in CD45.1 Congenic Mice with Consequences in Mouse Susceptibility to Infection †,‡,x,{ Youngsoon Jang,*‖ Zachary J. Gerbec,‖ Taejoon Won,* Bongkumx { Choi,* Amy Podsiad, Bethany B. Moore,*, Subramaniam Malarkannan,†,‡, , and Yasmina Laouar* B6.SJL-Ptprca Pepcb/Boy (CD45.1) mice have been domain, resulting in epitope changes that permit specific used in hundreds of congenic competitive transplants, recognition by mAbs. Backcrossing of mice expressing the with the presumption that they differ from C57BL/6 CD45.1 allele into the B6 background has resulted in the mice only at the CD45 locus. In this study, we describe development of the mouse strain B6.SJL-Ptprca Pepcb/Boy a point mutation in the natural cytotoxicity receptor 1 (CD45.1). As CD45.1 mice have been backcrossed during (Ncr1) locus fortuitously identified in the CD45.1 many generations into the B6 background, these mice have strain. This point mutation was mapped at the 40th been termed congenic, with the presumption that they differ nucleotide of the Ncr1 locus causing a single amino from the B6 strain only at the CD45 locus. Surprisingly, acid mutation from cysteine to arginine at position despite extensive backcrossing, genotypic analysis revealed 14 from the start codon, resulting in loss of NCR1 that the congenic interval in which CD45.1 differs from expression. We found that these mice were more re- CD45.2 mice is almost 43 mbp encoding 306 and at sistant to CMV due to a hyper innate IFN-g response least 124 genetic polymorphisms (1). in the absence of NCR1. In contrast, loss of NCR1 In this study, we describe a point mutation in the natural increased susceptibility to influenza virus, a result that cytotoxicity receptor 1 (Ncr1) locus of CD45.1 strain, resulting is consistent with the role of NCR1 in the recog- in loss of expression. A point of critical consequence of this nition of influenza Ag, hemagglutinin. This work sheds mutation is the different susceptibility of this strain to viral on potential confounding experimental interpre- infection. Thus, using this “congenic” strain, under the pre- tation when this congenic strain is used as a tool for sumption that it differs from the B6 strain only at the CD45 tracking lymphocyte development. The Journal of locus, probably has been, and most likely will be, conducive to Immunology, 2018, 200: 000–000. confounding experimental interpretation. Materials and Methods he most common approach used for tracking immune Mice cell development in vivo takes advantage of poly- CD45.2 and CD45.1 mice were originally obtained from The Jackson T morphisms in the extracellular domain of the trans- Laboratory and maintained in our animal facility at the University of membrane receptor tyrosine phosphatase CD45 Michigan. For bone marrow mixed chimeras, CD45.2 recipient mice were (Ptprc), a 220-kDa protein expressed on all subsets of lethally irradiated (1200 rad) and injected (i.v) with 10 million donor bone marrow cells containing a mixture of CD45.2 and CD45.1 cells provided at a leukocytes. Two isoforms have been identified in mice: the 1:1 ratio. For the transfer of gut microbiota, gut content was harvested and common form is CD45.2, which is expressed by the C57BL/6 provided to recipient mice by oral gavage. As indicated, mice were either (B6) strain and is encoded by the Ptprcb allele; and an addi- infected with murine CMV (MCMV; 3500 PFU of Smith strain per gram of tional allelic variant Ptprca, which encodes the CD45.1 iso- body weight by i.p. injection), influenza A virus (50 PFU of H1N1 per mouse by intranasal inhalation), or Citrobacter rodentium (1010 CFU per form, was identified in the SJL mouse strain. CD45.1 and mouse by oral gavage). All experiments were performed in accordance with CD45.2 alleles differ by only 5 aa within the extracellular the University of Michigan Animal Care and Use Committee, and approval

*Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Foundation (to S.M.), the Nicholas Family Foundation (to S.M.), and American Asso- MI 48109; †Blood Center of Wisconsin, Milwaukee, WI 53226; ‡Department of Med- ciation of Immunologists Careers in Immunology Grant N023607 (to Y.L.). x icine, Medical College of Wisconsin, Milwaukee, WI 53226; Department of Microbi- Address correspondence and reprint requests to Dr. Yasmina Laouar, University of ology and Immunology, Medical College of Wisconsin, Milwaukee, WI 53226; { Michigan, 1150 West Medical Center Drive, Medical Science Building II, Room Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226; ‖ 6605E, Ann Arbor, MI 48109. E-mail address: [email protected] and Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109 The online version of this article contains supplemental material. ORCIDs: 0000-0002-6134-4291 (Z.J.G.); 0000-0001-5968-2016 (T.W.); 0000-0003- 3051-745X (B.B.M.); 0000-0002-8865-4791 (Y.L.). Abbreviations used in this article: B6, C57BL/6; Dok, downstream of kinase; ER, endoplasmic reticulum; GzmB, granzyme B; MCMV, murine CMV; NCR1, natural Received for publication December 13, 2017. Accepted for publication January 21, cytotoxicity receptor 1. 2018. This work was supported by National Institutes of Health Grants R01 AI102893 and Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 R01 CA179363 (to S.M.), R01 HL119682 (to B.B.M.), and R01 AI083642 (to Y.L.), the Midwest Athletes Against Childhood Cancer Fund (to S.M.), the Gardetto Family

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701676 2 CUTTING EDGE: A POINT MUTATION IN THE Ncr1 LOCUS IN CD45.1 STRAIN to use mice was granted by the University of Michigan in accordance with the facility. However, the transfer of gut content from CD45.2 National Institutes of Health requirements for the care and use of animals. mice failed to restore the expression of NCR1 in CD45.1 re- Sequencing, cloning, and transfection of the Ncr1 cipient NK cells (Fig. 2E). Finally, to demonstrate that the loss of NCR1 in CD45.1 strain is not driven by the environment, The Ncr1 open reading frame region was amplified from cDNA using the following primers, and amplified products were sequenced by Sanger we assessed the expression of NCR1 in mixed bone marrow method (University of Michigan): 59-GTTCAGCACTGGTCTGGC- chimeras. Results showed no expression of NCR1 on CD45.1- CACTGG-39 and 59-GCCAAACTTGGTAACACTCCTACC-39. For cloning derived NK cells, indicating a cell-autonomous defect (Fig. 2F). and transfection of the Ncr1 gene, the Ncr1 open reading frame was Because levels of the Ncr1 transcripts were comparable in amplified from cDNA using the following primers: 59-GGAATTAGA- GAGTTTCATGCTGCCAACACTCACTGC-39 and 59-GAATTGTG- both mouse strains (Fig. 2G), we sequenced the mRNA and GAAGTTTCTCACAAGGCCCCAGGAGTTG-39. Amplified Ncr1 gene identified a point mutation, TGT to CGT, in the 40th nu- was cloned into pMSCV-neo vector, transfected into Bosc cells for viral cleotide from the start codon leading to a single amino acid packaging, infected into MEF cells, and then selected with 500 mg/ml mutation from cysteine to arginine at the 14th aa (C14R), Geneticin for analysis. which is localized in the region of the signal peptide of NCR1 Results and Discussion (Fig. 2H). To evaluate the effects of this signal peptide CD45.1 and CD45.2 strains are not functionally equivalent mutation, we constructed expression vectors with mutant (NCR1C14R) or wild-type (NCR1WT) forms and assessed CD45.1 and CD45.2 mice were originally obtained from The outcomes on protein expression. Unlike wild-type controls, Jackson Laboratory and then maintained by breeding in our no protein was detected from the mutated construct, indi- animal facility. While performing experiments with CD45.2 cating that this signal peptide mutation was sufficient to and CD45.1 mice, we observed a pattern of responses that abrogate NCR1 expression (Fig. 2J, 2K). Signal peptides suggested inherent differences in their susceptibility to infection are responsible for numerous functions, including recogni- (Fig. 1). In response to MCMV infection, we found that tion and binding of the protein to the cytosolic signal rec- CD45.1 mice were better equipped to fight a lethal dose of ognition particle, its insertion into the endoplasmic reticulum MCMV. In response to influenza virus infection, we observed (ER) protein–conducting channels, and protein maturation opposite results, with CD45.1 mice being less protected than processes such as quality control and addition of N-linked CD45.2 mice. However, in response to C. rodentium infection, glycans (3). Examples from signal peptide mutations showed both CD45.1 and CD45.2 mouse strains were equally resistant that changes in the signal peptide hydrophobicity interfere to the enteric bacteria. Based on these results, we conclude that with the binding of the mutant preproteins to the signal CD45.1 and CD45.2 mouse strains—that are housed in our recognition and translocation into the ER (4). Even for the animal facility—are not functionally equivalent. fraction of the mutant preproteins that enter the ER, their Point mutation in the Ncr1 locus identified in the CD45.1 strain cleavage by the signal peptidase is inefficient; as a result, the uncleaved preproteins could not reach the glycosylation ma- Detailed analysis of the NK cell compartment landed one major chinery and therefore were retained in the ER (4). Alterna- difference between CD45.1 and CD45.2 strains: NCR1 ex- tively, the mutant preproteins are removed from the ER by a pression was lost in the CD45.1 strain (Fig. 2). Loss of NCR1 process of active degradation via the proteasome pathway (5). expression in the CD45.1 strain was identified in NK cells Although further studies are required to identify the exact from various organs (Fig. 2A, 2B) and innate lymphoid cells mechanism, our results imply a similar mechanism for the from the intestine (Fig. 2C). We confirmed that the loss of C14R signal peptide mutation resulting in degradation and NCR1 expression was not the result of intracellular seques- loss of NCR1. Accordingly, we termed this mutant strain tration, as shown by surface and intracellular staining and with Ncr1C14R mice. monoclonal and polyclonal anti-NCR1 Abs (Fig. 2D, 2I). C14R Given the increasing evidence of associations between host gene Mutant Ncr1 confers hyper IFN-g response without altering the expression and gut microbiota (2), we considered the possibility lytic activity that the loss of NCR1 in the CD45.1 strain is the result of a NCR1 contains two extracellular Ig domains, a type I trans- specific gut microbiota acquired during breeding in our animal membrane domain, and a short cytoplasmic tail lacking

FIGURE 1. CD45.2 and CD45.1 strains exhibit different susceptibility to infection. (A) History of in-house breed- ing of CD45.2 and CD45.1 mouse strains. Mouse survival after (B) MCMV, (C) influenza virus, or (D) C. rodentium infection. Data are from two independent experiments with n = 10 mice per group. Statistics were analyzed using a log- Mantel–Cox test. The Journal of Immunology 3

2 FIGURE 2. CD45.1 strain bears a point mutation in the Ncr1 locus causing loss of NCR1 expression. NCR1 expression on (A) spleen NK cells (CD3 NK1.1+), (B) NK cells from the indicated organs, and (C) intestinal innate lymphoid cells is shown. (D) Surface and intracellular expression of NCR1 using mAb or polyclonal Abs (pAb). (E) NCR1 expression on NK cells from CD45.1 recipients 3 wk after gut content transfer. (F) NCR1 expression on donor-derived NK cells from CD45.2+ or CD45.1+ cells. (G) Genomic DNA and mRNA transcripts of Ncr1 gene. (H) mRNA and protein sequence of Ncr1 gene. (I) Amounts of NCR1 from sorted NK cells. (J and K) NCR1 expression in mutant (NCR1C14R) and wild-type (NCR1WT) transfected cells. Data are representative of three independent experiments with n = 4 per group. Statistics were analyzed using a t test. ****p , 0.0001.

ITAMs. Given the lack of inherent signaling motifs, NCR1 structure, NCR1 has been assumed to act solely as an acti- relies on adaptor molecules FCeRIg and CD3z to impart vating receptor delivering positive signals to NK cells (7–9). positive signals through their ITAMs (6). Based on this However, such a role has been challenged by opposite results 4 CUTTING EDGE: A POINT MUTATION IN THE Ncr1 LOCUS IN CD45.1 STRAIN

FIGURE 3. Ncr1C14R mutant mice mount a stronger innate IFN-g response. Frequency of GzmB+ cells among NK cells (A) from polyinosinic-polycytidylic acid (PolyI:C)–treated mice, or (B) after 24 h simulation with IL-15 (20 ng/ml). (C) Percentage of NK-specific lysis of YAC-1 cells in the absence or absence of 10 mg/ml anti-NCR1 Ab. Frequency of IFN-g+ cells among NK cells stimulated with (D) IL-12 (1 ng/ml) plus IL-18 (2 ng/ml), or (E) plate-bound anti-NK1.1 (1 mg/ml) Ab for 5 h. (F) Amounts of IFN-g secreted by NK cells stimulated with anti-Ly49H Ab for 6 h. (G) MCMV titers on day 3. Frequency of IFN-g+ cells among NK cells on days 1.5 (H–J) and 5 (K–M) postinfection. Results are from in vitro restimulation with 0.5 ng/ml (L) or 1 ng/ml (H, I, K, and M) IL-12 J N C14R supplemented with 2 ng/ml IL-18 or PMA (20 ng/ml) plus ionomycin (1 mg/ml) ( ). ( ) NCR1 expression in F1 and F2 progeny and parental CD45.1Ncr1 and CD45.2 mice. Mutant and wild-type mice from F2 progeny were infected with MCMV and spleen NK cells were analyzed on day 1.5 postinfection for proliferation (O) and GzmB expression (P). IFN-g expression is shown after 5 h stimulation with (Q) IL-12 (1 ng/ml) plus 2 ng/ml IL-18 (2 ng/ml) or (R)PMA/ ionomycin. (S) Dok-1 and p38 phosphorylation from NK cells stimulated with anti-Ly49H Ab for the indicated time points (minutes). Data are representative of three to four independent experiments with n = 4 per group. Data are mean 6 SEM or shown from individual mice. Statistics were analyzed using a t test or two- way ANOVA with a Sidak correction. *p , 0.05, **p , 0.01, ***p , 0.005, ****p , 0.0001. The Journal of Immunology 5 arguing instead for a regulatory role of NCR1 in NK cell activities (10). Given the controversial data reported from Ncr1gfp/gfp and Ncr1Noe/Noe mice (11, 12), we sought to re- examine the role of NCR1 using the Ncr1C14R mutant mice. Although the efficacy of lytic activity was not altered in the absence of NCR1 (Fig. 3A–C), the IFN-g response was higher in Ncr1C14R compared with wild-type NK cells (Fig. 3D–F). To examine how this gain of function translates in vivo, mutant and wild-type mice were infected with a lethal dose of MCMV (Fig. 3G–M). We found that Ncr1C14R mice were more resistant to MCMV infection than were wild-type mice, as indicated by higher survival rates (Fig. 1B) and reduced viral loads in the liver and spleen (Fig. 3G). Analysis of NK cells on 1.5 and 5 d postinfection showed higher production of IFN-g both in the liver and spleen of Ncr1C14R compared 2 with wild-type mice, and both in Ly49H+ and Ly49H NK cell subsets lacking NCR1 expression (Fig. 3H–M). Notably, neither proliferation nor production of granzyme B (GzmB) was altered in the absence of NCR1 (Supplemental Fig. 1A), indicating a selective role of NCR1 in the regulation of innate IFN-g. One could raise the concern that, in these experi- ments, the control mice are not the mutant’s littermates. To address this issue, we repeated the above experiments using C14R WT FIGURE 4. C14R A mutant (F2 Ncr1 ) and wild-type (F2 Ncr1 ) littermates Tracing the Ncr1 mutation in CD45.1 congenic mice. ( ) NCR1 expression from CD45.1 mice purchased from commercial vendors derived from F2 progeny (Fig. 3N–R, Supplemental Fig. 1B– E) and confirmed the gain of NK cell IFN-g response in the between 2015 and 2017. Data are representative of three independent ex- periments with n =4.(B) NCR1 expression from CD45.1 mice purchased absence of NCR1. Interestingly, expression of NCR1 in F1 from The Jackson Laboratory before 2009 versus after 2015. The colony heterozygotes was intermediate, indicating that normal ex- generation of Jax B6 CD45.1 strain over time. Mice from N22 colony gen- pression requires both functional alleles. Consistent with our eration could be the source of the Ncr1C14R mutation. data, stimulation of human NK cells in the presence of anti- NCR1 blocking Ab resulted in copious amounts of IFN-g Using flow cytometry data from our archives, we were able to (13). Of note, this gain of function was specific to NCR1, as trace this mutation backward and have identified the loss of blocking NCR2 or NCR3 showed no effects on IFN-g (13). NCR1 in CD45.1 mice from experiments dated from 2009 Because levels of T-bet, Eomes, and Helios were not different (Fig. 4). Conversely, analysis of recent studies performed with in both mouse groups, (Supplemental Fig. 2), we tested the CD45.1 mice purchased after 2014 showed normal expression possibility that augmented IFN-g response in the absence of of NCR1 (Fig. 4). Colony maintenance information from The NCR1 is mediated via downstream of kinase (Dok)-1 (Fig. 3S). Jackson Laboratory indicates that the CD45.1 strain (stock no. By recruiting enzymes transducing negative signals such as 002014) had been imported into The Jackson Laboratory in RasGAP and SHIP1, Dok-1 inhibits Ras/ERK and PI3K/ 1990 from Dr. Edward Boyse at generation N22. From that Akt signaling pathways, resulting in negative regulation of time until 2009, the strain was maintained by pedigrees and leukocyte activation (14). In T cells, Dok-1 is tyrosine filial matings. Between 2009 and 2010, the B6 CD45.1 phosphorylated, resulting in negative regulation of TCR- congenic mouse colony underwent three backcrosses to the induced T cell activation. Overexpression of Dok-1 decreased inbred mouse strain C57BL/6J (N25). It is possible that this IL-2 production (15), and loss of Dok-1 enhanced TCR- backcrossing may have removed the Ncr1C14R mutation mediated signaling (16). Likewise, overexpression of Dok-1 identified in mice obtained prior to 2010; however, this has in NK cells reduced IFN-g production whereas Dok-1 gene not been directly tested. Notably, as the Ncr1C14R point mu- ablation augmented the IFN-g response (17). Consistent with tation results in loss of NCR1 expression, determining the these results, we found that the loss of NCR1 in NK cells was status of your congenic mouse colony will be easily achievable sufficient to prevent Dok-1 activation downstream of Ly49H by flow cytometry. Otherwise, using these mice, as a tool engagement, resulting in increased MAPK activation and for tracking lymphocyte development under the presumption augmented IFN-g response in NK cells (Fig. 3S). Although it that they differ from the B6 strain only at the CD45 locus, is tempting to speculate that NCR1 may serve as a regulator will certainly be conducive to confounding experimental of Dok-1 activation downstream of NK cell receptor en- interpretation. gagement, further studies are required to elucidate the specific nature of the interplay between NCR1 and Dok-1 in control of NK cell IFN-g. Acknowledgments Given the frequent use of CD45.1 strain as a tool for We thank Dr. Low-Marchelli from The Jackson Laboratory for providing help- ful information on the colony generation of the CD45.1 congenic strain. tracking lymphocyte development, our finding raised one important question: did the Ncr1C14R mutation identified in our congenic mouse colony originate from the vendor or did it Disclosures occur in our animal facility as a result of genetic drifting? The authors have no financial conflicts of interest. 6 CUTTING EDGE: A POINT MUTATION IN THE Ncr1 LOCUS IN CD45.1 STRAIN

9. Glasner, A., H. Ghadially, C. Gur, N. Stanietsky, P. Tsukerman, J. Enk, and References O. Mandelboim. 2012. Recognition and prevention of tumor metastasis by the NK 1. Ryan, M. A., K. J. Nattamai, E. Xing, D. Schleimer, D. Daria, A. Sengupta, receptor NKp46/NCR1. J. Immunol. 188: 2509–2515. A. Ko¨hler, W. Liu, M. Gunzer, M. Jansen, et al. 2010. Pharmacological inhibition 10. Narni-Mancinelli, E., B. N. Jaeger, C. Bernat, A. Fenis, S. Kung, A. De Gassart, of EGFR signaling enhances G-CSF-induced hematopoietic stem cell mobilization. S. Mahmood, M. Gut, S. C. Heath, J. Estelle´, et al. 2012. Tuning of natural killer Nat. Med. 16: 1141–1146. cell reactivity by NKp46 and Helios calibrates T cell responses. Science 335: 344– 2. Morgan, X. C., B. Kabakchiev, L. Waldron, A. D. Tyler, T. L. Tickle, R. Milgrom, 348. J. M. Stempak, D. Gevers, R. J. Xavier, M. S. Silverberg, and C. Huttenhower. 11. Glasner, A., H. Simic, K. Miklic´, Z. Roth, O. Berhani, I. Khalaila, S. Jonjic, and 2015. Associations between host gene expression, the mucosal microbiome, and O. Mandelboim. 2015. Expression, function, and molecular properties of the killer clinical outcome in the pelvic pouch of patients with inflammatory bowel disease. receptor Ncr1-Noe´. J. Immunol. 195: 3959–3969. Genome Biol. 16: 67. 12. Glasner, A., B. Isaacson, and O. Mandelboim. 2017. Expression and function of 3. Martoglio, B., and B. Dobberstein. 1998. Signal sequences: more than just greasy NKp46 W32R: the human homologous protein of mouse NKp46 W32R (Noe´). peptides. Trends Cell Biol. 8: 410–415. Sci. Rep. 7: 40944. 4. Henderson, J. E., N. Amizuka, H. Warshawsky, D. Biasotto, B. M. Lanske, 13. Spallanzani, R. G., N. I. Torres, D. E. Avila, A. Ziblat, X. L. Iraolagoitia, D. Goltzman, and A. C. Karaplis. 1995. Nucleolar localization of parathyroid L. E. Rossi, C. I. Domaica, M. B. Fuertes, G. A. Rabinovich, and N. W. Zwirner. hormone-related peptide enhances survival of chondrocytes under conditions that 2015. Regulatory dendritic cells restrain NK cell IFN-g production through promote apoptotic cell death. Mol. Cell. Biol. 15: 4064–4075. mechanisms involving NKp46, IL-10, and MHC class I–specific inhibitory recep- 5. Arnold, A., S. A. Horst, T. J. Gardella, H. Baba, M. A. Levine, and tors. J. Immunol. 195: 2141–2148. H. M. Kronenberg. 1990. Mutation of the signal peptide-encoding region of the 14. Acuto, O., V. Di Bartolo, and F. Michel. 2008. Tailoring T-cell receptor signals by preproparathyroid hormone gene in familial isolated hypoparathyroidism. J. Clin. proximal negative feedback mechanisms. Nat. Rev. Immunol. 8: 699–712. Invest. 86: 1084–1087. 15. Ne´morin, J. G., P. Laporte, G. Be´rube´, and P. Duplay. 2001. p62dok Negatively 6. Foster, C. E., M. Colonna, and P. D. Sun. 2003. Crystal structure of the human natural regulates CD2 signaling in Jurkat cells. J. Immunol. 166: 4408–4415. killer (NK) cell activating receptor NKp46 reveals structural relationship to other leu- 16. Dong, S., B. Corre, E. Foulon, E. Dufour, A. Veillette, O. Acuto, and F. Michel. kocyte receptor complex immunoreceptors. J. Biol. Chem. 278: 46081–46086. 2006. T cell receptor for antigen induces linker for activation of T cell-dependent 7. Sivori, S., M. Vitale, L. Morelli, L. Sanseverino, R. Augugliaro, C. Bottino, activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2. L. Moretta, and A. Moretta. 1997. p46, A novel -specific surface J. Exp. Med. 203: 2509–2518. molecule that mediates cell activation. J. Exp. Med. 186: 1129–1136. 17. Celis-Gutierrez, J., M. Boyron, T. Walzer, P. P. Pandolfi, S. Jonjic´,D.Olive, 8. Gazit, R., R. Gruda, M. Elboim, T. I. Arnon, G. Katz, H. Achdout, J. Hanna, M. Dalod, E. Vivier, and J. A. Nune`s. 2014. Dok1 and Dok2 proteins U. Qimron, G. Landau, E. Greenbaum, et al. 2006. Lethal influenza infection in the regulate natural killer cell development and function. EMBO J. 33: 1928– absence of the natural killer cell receptor gene Ncr1. Nat. Immunol. 7: 517–523. 1940. B6 CD45.2 Liver C14R A B6 CD45.1Ncr1 C14R B F2 Ncr1WT F2 Ncr1 5 30 100 10 49 41 2 2 104

25 80 103

20 102 0 60 NCR1 6 4 52 44 cells (% )

d1.5 2 3 4 5

+ 0 10 10 10 10

cells (% ) 15

+ 40 10 Ly49H

Ki6 7 20 5 Gzm B C 0 0 Total NK cells 24 20 - + NK cells - NK cells + NK cells NK cells Spleen otal NK cells otal NK cells T T y49H y49H y49H y49H L L L L Ly49H+ 80 100 NK cells 23 20 60 80 60 cells (% ) d5 40 - cells (% ) + + 40 Ly49H NK cells 20 24 20 Ki6 7 20 Gzm B 0 0 Ki67 D

+ NK cells - NK cells + NK cells - NK cells otal NK cells otal NK cells Total T T y49H y49H y49H y49H NK cells L L L L 90 92 30 100 25 80 Ly49H+ 20 60 NK cells 94 96 cells (% )

+

cells (% ) 15 + 40 d1.5 10

Ki6 7 20 5 Gzm B Ly49H- NK cells 0 0 88 92 GzmB - + NK cells - NK cells Liver + NK cells NK cells otal NK cells otal NK cells E T T y49H y49H y49H y49H L L L L Total 80 80 NK cells 33 45 60 60 cells (% ) + + d5 cells (% ) 40 40 Ly49H + NK cells 32 46 20 20 Ki6 7 Gzm B 0 0 Ly49H- NK cells 35 46 NK1.1 + NK cells - NK cells + NK cells - NK cells otal NK cells otal NK cells T T IFNγ y49H y49H y49H y49H L L L L

Supplemental Figure 1. Proliferation and GzmB expression in NK cells in response to MCMV infections are not aletred by the loss of NCR1. (A) Splenocytes and liver-infiltrating lymphocytes from infected CD45.1Ncr1C14R and CD45.2 mice were isolated on days 1.5 and 5 post-infection. Graphs summarize the frequency of cycling (Ki67+) and GzmB-expressing cells among NK cell subsets. Data are mean ± SEM. (B) Expression of NCR1 in mutant (F2 Ncr1C14R) and wild-type (F2 Ncr1WT) mice from F2 progeny. (C-E) Liver-infiltrating lymphocytes from F2 Ncr1WT and F2 Ncr1C14R were isolated on d1.5 post-infection. NK cells were analyzed for proliferation (C) and GzmB expression. (D) IFNγ expression is shown after 5 hr stimulation with 1ng/ml of IL-12 plus 2ng/ml of IL-18 in the presence of GolgiStop. Data are from three individual experiments with n = 4 mice per group. CD27+ CD27+ CD27- CD11b- CD11b+ CD11b+

A T-bet

T-bet

B Eomes % of max

Eomes B6 CD45.2 B6 CD45.1Ncr1C14R

C 2.5 2.0 1.5 1.0 0.5 Fold fo change

(mutant /wild-type) 0

T-bet Eomes Helios Dok-1 Dok-2

Supplemental Figure 2. Loss of NCR1 did not affect the expression of the transcription factors T-bet, Eomes, or Helios. (A-B). Intracellular expression of T-bet (A) or Eomes (B) in NK cells at stage D (CD27+CD11b-), stage E (CD27+CD11b+), and stage F (CD27-CD11+). (C) Total RNA was isolated from sorted splenic NK cells of CD45.1Ncr1C14R (mutant) and CD45.2 (wild-type) mice using TRIzol (Invitrogen) and cDNA was synthesized using SuperScript II Reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative gene expression was conducted on ABI Prism 7900 instrument (Applied Bioscosystems) using SYBR Green PCR Master Mix (Applied Bioscosystems). Data were analyzed by 2-ΔΔCt method and results were expressed as a fold of changein mutant versus wild-type samples. Data show average from four independent experiment ± SEM. Primer sequences used are: T-bet Forward: 5’-CAACAACCCCTTTGCCAAAG-3’ Reverse: 5’-TCCCCCAAGCAGTTGACAGT-3’ Eomes Forward: 5’-CCTTCACCTTCTCAGAGACACAGTT-3’ Reverse: 5’-TCGATCTTTAGCTGGGTGATATCC-3’ Helio Forward: 5’- GACAGTCTCTGCAGCTGTGT-3’, Reverse: 5’-CATGCACGTGTGTGCATTAAA-3’ Dok-1 Forward: 5’- GGACCAAGGTGGAGGAAAACT-3’ Reverse: 5’-CACATTCAGCCAGGCGTATC-3 Dok-2 Forward: 5’- GCAGACCTTTGGCAAGAAGTG-3’ Reverse: 5’-TCTTCTCGGGGACATCCTGG-3’ HPRT Forward: 5’-CTGGTGAAAAGGACCTCTCG-3’ Reverse: 5’-TGAAGTACTCATTATAGTCAAGGGCA