Cutting Edge: Stage-Specific Requirement of IL-18 for Antiviral NK Cell Expansion Sharline Madera and Joseph C. Sun This information is current as J Immunol 2015; 194:1408-1412; Prepublished online 14 of October 5, 2021. January 2015; doi: 10.4049/jimmunol.1402001 http://www.jimmunol.org/content/194/4/1408 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/01/14/jimmunol.140200 Material 1.DCSupplemental References This article cites 15 articles, 7 of which you can access for free at: http://www.jimmunol.org/content/194/4/1408.full#ref-list-1 http://www.jimmunol.org/

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology

Cutting Edge: Stage-Specific Requirement of IL-18 for Antiviral NK Cell Expansion Sharline Madera and Joseph C. Sun Although NK cells are considered part of the innate im- resting NK cells for maximum IFN-g production after ex vivo mune system, recent studies have demonstrated the ability stimulation (5), and synergize with IL-12 during NK cell acti- of Ag-experienced NK cells to become long-lived and con- vation (6). Although IL-18 is produced early during MCMV tribute to potent recall responses similar to T and B cells. infection (7), it is not known how IL-18 signals influence the + The precise signals that promote the generation of a long- virus-specific Ly49H NK cell response. In this study, we in- lived NK cell response are largely undefined. In this article, vestigate the direct effects of IL-18 signaling on primary and we demonstrate that NK cells require IL-18 signaling to recall NK cell responses to MCMV infection. generate a robust primary response during mouse CMV Downloaded from (MCMV) infection but do not require this signal for mem- Materials and Methods ory cell maintenance or recall responses. IL-12 signaling Mice and infections and STAT4 in activated NK cells increased the expres- All mice used in this study were bred and maintained at Memorial Sloan- sion of the adaptor MyD88, which mediates sig- Kettering Cancer Center in accordance with Institutional Animal Care and Use Committee guidelines. Mixed bone marrow chimeric mice were generated, naling downstream of the IL-18 and IL-1 receptors. and adoptive transfer studies and viral infections were performed as previously

During MCMV infection, NK cells required MyD88, described (8). http://www.jimmunol.org/ but not IL-1R, for optimal expansion. Thus, an IL-18– MyD88 signaling axis facilitates the prolific expansion of Flow cytometry and cell sorting NK cells in response to primary viral infection, but not FcRs were blocked with 2.4G2 mAb before staining with the indicated recall responses. The Journal of Immunology, 2015, 194: surface or intracellular Abs (BD, Biolegend, or eBioscience). Flow cytometry was performed on an LSR II (BD). Cell sorting was performed on an Aria II 1408–1412. cytometer (BD). All data were analyzed with FlowJo software (Tree Star). NK cell enrichment and adoptive transfers were performed as previously described (3). atural killer cells play a significant role in the control by guest on October 5, 2021 of infected, stressed, or transformed cells that may be Quantitative RT-PCR and chromatin immunoprecipitation detrimental to the host. Recent studies in mice and N Quantitative RT-PCR (qRT-PCR) and chromatin immunoprecipitation humans have demonstrated that NK cells possess adaptive (ChIP) were performed as previously described (4). The following qRT-PCR immune qualities (1). In mice infected with mouse CMV primers were used: Myd88, Forward [For]: 59-CACCTGTGTCTGGTC- (MCMV), Ly49H+ NK cells activated by the viral glycoprotein CATT-39, Reverse [Rev]: 59-AGGCTGAGTGCAAACTTG-39; Actb, For: 59-TGCGTGACATCAAAGAGAAG-39,Rev:59-CGGATGTCAACGTCA- m157 undergo extensive proliferation and contract, resulting in CACTT-39. The following quantitative PCR primers were used for ChIP the formation of a small pool of long-lived memory NK cells studies: Myd88 promoter,For:59-AAGTAGGAAACTCCACAGGCGAGC-39, that can be recalled, and exhibit heightened effector function (1). Rev: 59-TTCAAGAACAGCGATAGGCGGC-39; desert 50 kB upstream Proinflammatory strongly influence the NK cell of Foxp3,For:59-TAGCCAGAAGCTGGAAAGAAGCCA-39,Rev:59-TGA- TACCCTCCAGGTCCAACCATT-39; Zpf42 promoter,For:59-AGAGGGCGGTGT- response against MCMV infection (2). Although previous work GTACTGTGGTG-39,Rev:59-CTTCTTCTTGCACCCGGCTTGAG-39; has described the effect of proinflammatory cytokines on the Utf1 promoter, For: 59-AGTCGTTGAATACCGCGTTGCTG-39, Rev: 59- general activation of NK cells during MCMV infection (2), CTGTTGAGATGTCGCCCAAGTGC-39; Ifng promoter, For: 59-GCTC- their role in driving clonal-like expansion and memory in Ag- TGTGGATGAGAAAT-39, Rev: 59-GCTCTGTGGATGAGAAAT-39. specific NK cells is largely unknown. We previously implicated Ex vivo stimulation of NK cells IL-12, its signaling molecule STAT4, and the downstream transcription factor Zbtb32 as crucial signals in the generation Purified NK cells were stimulated for 4 (memory cells) or 18 h (for ChIP), as previously described (4). Negative and positive controls include NK cells of robust effector and memory NK cell responses against incubated with media only, or with PMA (50 ng/ml) and ionomycin (1 mg/ml), MCMV infection (3, 4). IL-18 has been suggested to “prime” respectively.

Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Address correspondence and reprint requests to Dr. Joseph C. Sun, Memorial Sloan- Kettering Cancer Center, 408 East 69th Street, ZRC-1402, New York, NY 10065. Received for publication August 11, 2014. Accepted for publication December 10, E-mail address: [email protected] 2014. The online version of this article contains supplemental material. This work was supported by National Institutes of Health Medical Scientist Training Program Grant T32GM007739 (to S.M.), National Institutes of Health Abbreviations used in this article: ChIP, chromatin immunoprecipitation; For, forward; Grant T32AI007621 (to S.M.), National Institutes of Health Grant AI100874 (to J.C.S.), MCMV, mouse CMV; PI, postinfection; qRT-PCR, quantitative RT-PCR; Rev, reverse; Cancer Research Institute grants (to S.M. and J.C.S.), and the Searle Scholars Program (to WT, wild-type. J.C.S.). Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402001 The Journal of Immunology 1409

Statistical methods viruses engineered to lack or express m157, respectively. After 2/2 2/2 All graphs depict mean 6 SEM. Two-tailed paired Student t test was used to adoptive transfer of WT and Il18r1 NK cells into Ly49h derive statistical differences. A p value ,0.05 was considered significant. Plots hosts, we observed that in contrast with MCMV, infection with and statistical analyses were produced in GraphPad Prism. MCMV lacking m157 (MCMV-Dm157) elicited an equivalent 2 2 (albeit modest) expansion of both WT and Il18r1 / NK cell Results and Discussion populations (Fig. 1D), indicating that IL-18 signaling is not Cell-intrinsic IL-18 signaling required for the primary expansion of required for the low-level proliferation of “bystander” NK cells virus-specific NK cells responding to viral infection. Consistent with this observation, Although IL-18 has previously been shown to activate NK cells infection with VSV-m157, but not with VSV-Ova, elicited a preferential Ag-specific expansion of WT NK cells over during viral infection, these studies involve directly infecting 2/2 IL-18– and IL-18R–deficient mice (7, 9). Therefore, to address Il18r1 NK cells (Fig. 1D). We next investigated whether IL-18 was required for NK cells to undergo homeostatic pro- whether IL-18 influences NK cell responses in a cell-intrinsic 2 2 liferation. We found that WT and Il18r1 / NK cells prolif- manner, we adoptively transferred equal numbers of wild-type 2 2 2 2 2 2 2 2 / 3 / (WT) and Il18r1 / NK cells into Ly49h / mice, which erated similarly when transferred into Rag2 Il2rg . harbor normal numbers of NK cells but are incapable of rec- mice, generating comparable long-lived populations 4wk ognizing the MCMV-derived m157 protein (3, 8). Postinfec- later (Fig. 1E), demonstrating IL-18 signaling is not required for tion (PI) with MCMV, WT NK cells preferentially expanded NK cells to proliferate in response to common g-chain cyto- kines. Altogether, these data suggest that IL-18 signaling directly

during the first week of infection and were higher in frequency Downloaded from 2 2 than Il18r1 / NK cells at day 7 PI (Supplemental Fig. 1A) regulates Ag-driven expansion of NK cells during viral infection, and at later time points (Fig. 1A). Consistent with the adoptive but not during bystander or homeostatic expansion. transfer experiment, we observed a similar expansion defect 2 2 2 2 by Il18r1 / Ly49H+ NK cells in WT:Il18r1 / mixed bone IL-18 signaling is necessary for optimal IFN-g production and marrow chimeric mice infected with MCMV (Fig. 1B and maturation of NK cells during infection

Supplemental Figure 1B). Together, these studies confirm Because IL-18 is required for optimal proliferation of NK cells http://www.jimmunol.org/ a cell-intrinsic requirement for IL-18 signaling in the antiviral during viral infection, we investigated additional NK cell ef- NK cell response. fector functions that may be compromised in the absence of IL- 2 2 IL-18 has been suggested to enhance IL-12–induced effector 18 signaling. At day 1.5 PI, we observed that Il18r1 / NK cells functions of NK cells such as IFN-g production (5, 6). To exhibited a defect in IFN-g production, but not granzyme determine whether IL-18 might also “prime” NK cells for B (Fig. 2A), consistent with a previous report (7). Although MCMV-driven expansion, we isolated resting NK cells from both NK cell populations became similarly activated at days 1.5 2 2 Il18 / mice and cotransferred them with equal numbers of and 7 PI (Supplemental Fig. 1C, 1D), WT NK cells were found 2 2 WT NK cells into Ly49h / hosts. Post MCMV infection, to be more mature, as measured by CD27 and CD11b staining,

2 2 2 2 by guest on October 5, 2021 WT and Il18 / Ly49H+ NK cells exhibited comparable compared with Il18r1 / cells (Fig. 2B), suggesting IL-18 may expansion and memory cell formation (Fig. 1C), indicating have a role in promoting the maturation of activated NK cells. that previous exposure to IL-18 during development or ho- Adoptive cotransfer and bone marrow chimeric studies 2 2 meostasis was not required for normal expansion in response revealed an expansion defect in the Il18r1 / NK cell response to viral challenge as long as IL-18 is present during infection. to MCMV infection, which could be a consequence of de- 2 2 To determine whether the effect of IL-18 signaling was limited creased proliferation or increased apoptosis of Il18r1 / NK to Ag-specific NK cell responses, we used MCMV and VSV cells relative to WT NK cells. We were not able to detect

FIGURE 1. IL-18R–deficient NK cells mount a defective response to viral infection. (A)WT 2 2 and Il18r1 / NK cells were cotransferred into 2 2 Ly49h / mice and infected with MCMV. The relative ratio of populations is shown for each time point compared with day 0. (B) Mixed WT: 2 2 Il18r1 / chimeric mice were infected with MCMV, and the relative ratio of Ly49H+ NK cells are shown for day 7 PI compared with un- infected. (C) Percentages of cotransferred WT and 2 2 Il18 / Ly49H+ NK cells are shown during MCMV infection. (D) Percentages of cotrans- 2 2 ferred WT and Il18r1 / Ly49H+ NK cells are shown postinfection with MCMV, MCMV- Dm157, VSV-m157, or VSV-Ova. (E) WT and 2 2 Il18r1 / NK cells were cotransferred into 2 2 2 2 Rag2 / 3 Il2rg / mice, and percentages of transferred Ly49H+ NK cells within the total cell population are shown. Data are mean 6 SEM representative of at least four independent experi- ments with at least n = 3 biological replicates per condition. *p , 0.05, paired Student t test. ns, not significant. 1410 CUTTING EDGE: IL-18–INDEPENDENT RECALL RESPONSE OF NK CELLS

regulate the contraction phase or maintenance of memory NK cells.

Antiviral NK cell response depends on MyD88, but not the IL-1R Because the IL-18R requires the adapter molecule MyD88 for downstream signaling (11), we investigated the contribution of MyD88 to the NK cell response to MCMV infection. WT: 2 2 Myd88 / mixed bone marrow chimeric mice were generated and both NK cell populations reconstituted similarly (data 2 2 not shown). Post MCMV infection, Myd88 / Ly49H+ NK cells exhibited a cell-intrinsic expansion defect compared with 2 2 WT NK cells (Fig. 3A), similar to Il18r1 / NK cells 2 2 (Fig. 1C). In addition, fewer Myd88 / NK cells produced IFN-g after MCMV infection despite comparable upregula- 2 2 tion of CD69 (Fig. 3B). Similar to Il18r1 / NK cells, 2 2 Myd88 / NK cells failed to mature as efficiently as WT NK cells at day 7 PI (Fig. 3C). When equal numbers of WT and 2/2 2/2 FIGURE 2. IL-18 is necessary for optimal NK cell maturation and IFN-g Myd88 NK cells were transferred into Ly49h hosts 2 2 Downloaded from production post MCMV infection. (A) Mixed WT:Il18r1 / chimeric mice followed by infection with MCMV, we observed preferential were infected with MCMV, and amount of IFN-g and granzyme B in NK expansion and memory cell formation of the WT NK cells 2 2 cells at day 1.5 PI are shown. (B) WT and Il18r1 / NK cells were 2/2 2 2 compared with Myd88 NK cells (Fig. 3D). The IL-1R, cotransferred into Ly49h / mice and infected with MCMV. CD27 and 2/2 + which is expressed by NK cells, is also known to use MyD88 CD11b staining is shown for WT and Il18r1 Ly49H NK cells at day 7 2/2 2/2 C 2/2 for signaling (11); however, unlike Il18r1 or Myd88 PI. ( ) Equal numbers of purified effector WT and Il18r1 NK cells (at 2/2 2/2 NK cells, Il1r NK cells expanded comparably with WT

day 7 PI) were cotransferred into a naive Ly49h host. Percentages of http://www.jimmunol.org/ Ly49H+ NK cells are shown. Data are mean 6 SEM representative of at least NK cells at day 7 PI (Fig. 3E) and showed similar effector three independent experiments with at least n = 3 biological replicates per function and phenotype as WT NK cells at days 1.5 and 7 PI condition. *p , 0.05, paired Student t test. ns, not significant. (Supplemental Fig. 2A–C). These findings support a mecha- nism for IL-18 signaling on NK cells through a MyD88- dependent signaling axis. differences in BrdU incorporation, Ki67 staining, or CFSE 2 2 dilution by WT and Il18r1 / NK cells in MCMV-infected IL-12 signaling and STAT4 increase expression of MyD88 in bone marrow chimeric mice (data not shown), consistent with activated NK cells a prior study (10). In addition, FLICA incorporation studies During MCMV infection, we observed an increase in Myd88 by guest on October 5, 2021 did not reveal any differences in pan-caspase activity between expression in sorted NK cells by microarray and qRT-PCR 2 2 Il18r1 / and WT NK cells on day 7 PI (Supplemental Fig. (Fig. 4A, data not shown). Given the important early role 2 2 1E), suggesting that Il18r1 / NK cells were not more sus- of IL-12 in activating NK cells, and a report that proin- 2 2 ceptible to apoptosis. Lastly, when WT and Il18r1 / effector flammatory cytokines induce IL-18 signaling components in NK cells were sorted from MCMV-infected hosts on day 7 human NK cells (12), we investigated whether IL-12 directly PI, and equal numbers cotransferred into naive recipients, influences the expression of the IL-18 signaling machinery. the two populations contracted at similar rates (Fig. 2C), in- Overnight stimulation of sorted NK cells with IL-12 and 2 2 dicating that Il18r1 / NK cells are not undergoing increased IL-18 also showed an increase in the expression of Myd88 by cell death at later time points and that IL-18 signaling does not qRT-PCR (Fig. 4B). STAT4 is a transcription factor that

2 2 FIGURE 3. MyD88-deficient NK cells exhibit defective proliferation during MCMV infection. (A) WT:Myd88 / chimeric mice were infected with MCMV, 2 2 and percentages of splenic NK cells are shown for uninfected and day 7 PI. (B) CD69 and IFN-g are shown for WT and Myd88 / NK cells (compared with 2 2 uninfected mice) at day 1.5 PI. (C) CD27 and CD11b staining are shown for WT and Il18r1 / Ly49H+ NK cells at day 7 PI. (D) Percentages of cotransferred 2 2 2 2 WT (CD45.1) and MyD88 / (CD45.2) Ly49H+ NK cells are shown post MCMV infection. (E) Percentages of cotransferred WT and Il1r / Ly49H+ NK cells are shown during MCMV infection. Data are mean 6 SEM representative of three independent experiments with at least n = 3 biological replicates per condition. *p , 0.05, paired Student t test. ns, not significant. The Journal of Immunology 1411

tigated its role during a recall response. Equal numbers of effector 2 2 WT and Il18r1 / NK cells were cotransferred and “parked” in a naive host for 3 wk, followed by infection with MCMV. 2 2 Surprisingly, Il18r1 / memory NK cells expanded similarly to their WT counterparts during secondary challenge by MCMV (Fig. 5C), demonstrating a stage-specific requirement for IL-18 signals for proliferation. After the peak of expansion, both cell populations contributed to equal frequencies of secondary memory NK cells. Thus, IL-18 signaling is specifically required for the primary expansion of virus-specific NK cells but is dis- pensable in the subsequent recall response. NK cells possess adaptive immune qualities in response to treatment (14) or exposure to pathogen and non- pathogen Ags (15). It is poorly understood what the cytokine FIGURE 4. IL-12 signaling induces expression of Myd88 in NK cells. (A) signal requirements are in resting and memory NK cells. In qRT-PCR analysis of Myd88 mRNA abundance in NK cells sorted from the this study, we find a stage-specific requirement for IL-18, spleen of WT mice postinfection with MCMV (n = 4 biological replicates per where IL-18 promotes the Ag-specific primary expansion of B condition). Fold expression is shown relative to naive mice. ( ) qRT-PCR resting NK cells, but not the recall response of memory NK analysis of Myd88 mRNA abundance in WT NK cells stimulated with IL-12 Downloaded from and IL-18 for 18 h (n = 3 biological replicates per condition). Fold expression cells. Furthermore, we have identified a previously unknown is shown relative to medium only. (C) Vista browser image of mouse Myd88 role for IL-12 in promoting the expression of Myd88 to en- promoter showing predicted STAT4 binding site. (D) STAT4 binding at hance the IL-18 signaling cascade and possibly sensitize NK Myd88, Ifng, and control promoters as assessed through ChIP followed by cells to lower levels of IL-18 cytokine. quantitative PCR in sorted WT NK cells stimulated with IL-12 and IL-18 for Our current findings suggest that as naive NK cells differ- 18 h. STAT4 occupancy as percent of input is shown for target (Myd88) and entiate into Ag-experienced memory cells, they become “spe- control DNA (negative control: average of gene desert 50 kb upstream of cialized” and may rely more on Ag-specific signals and less on http://www.jimmunol.org/ Foxp3, Zfp42,andUtf1 promoters; positive control: Ifng promoter). Data were confirmed in two independent experiments. (E) qRT-PCR analysis of Myd88 proinflammatory cytokine signals for clonal-like expansion 2 2 mRNA abundance in Stat4 / NK cells relative to WT NK cells from mice (16). This mechanism would allow previously Ag-experienced infected with MCMV (n = 4 biological replicates per condition). Data are NK cells to respond more robustly during subsequent patho- mean 6 SEM representative of three independent experiments. *p , 0.05, gen encounter. Because memory and recall NK cell responses paired Student t test. have also been documented in humans post viral infection (1), determining whether a homologous role for IL-18 in the recall mediates signals downstream from the IL-12R (13), and response of human NK cells may impact the use of this cy- analysis of the Myd88 promoter revealed one putative STAT4 tokine in clinical settings. by guest on October 5, 2021 binding site upstream of the transcriptional start site (Fig. 4C). STAT4 ChIP followed by quantitative PCR identified a sig- nificant enrichment of STAT4 binding directly upstream of the Myd88 transcriptional start site (Fig. 4D). This increase in STAT4 binding was dependent on IL-12 stimulation, sug- gesting Myd88 is a target gene of IL-12 signaling. Finally, we 2 2 compared the expression of Myd88 in WT and Stat4 / NK 2 2 cells during MCMV infection. Activated Stat4 / NK cells exhibited lower levels of Myd88 expression compared with WT NK cells. Altogether, these data indicate that IL-12 signaling acts through STAT4 to increase the expression of Myd88, identifying a novel role for IL-12 in potentiating IL-18 sig- naling early during MCMV infection.

IL-18 signaling is dispensable for recall responses by memory NK cells To assess whether IL-18 signaling regulates the functional capa- 2 2 bilities of memory NK cells, we stimulated WT and Il18r1 / memory NK cells with Ly49D, Ly49H, or IL-12 and IL-18 and measured their ability to degranulate and to make IFN-g.Similar 2 2 to resting NK cells, Il18r1 / memory NK cells produced less IFN-g than their WT counterparts only when stimulated with proinflammatory cytokines, but not with Ly49D, Ly49H, or FIGURE 5. IL-18 signaling is dispensable for recall response of NK cells. 2 2 2/2 PMA and ionomycin (Fig. 5A and data not shown). Il18r1 / Memory WT and Il18r1 NK cells were stimulated with Ly49D, IL-12 and IL-18, or PMA and ionomycin, and percent IFN-g (A) or CD107a (B) memory NK cells degranulated similarly to WT (Fig. 5B). Thus, 2 2 expression is shown. (C) Percentage of recalled WT and Il18r1 / NK cells like resting NK cells (5), memory NK cells continue to depend (at day 28 PI) are shown after secondary challenge with MCMV. Data are on IL-18 for IL-12–mediated IFN-g production. mean 6 SEM representative of five independent experiments with at least Given the importance of IL-18 signaling on the optimal ex- n = 4 biological replicates per condition. *p , 0.05, paired Student t test. ns, pansion of naive NK cells during MCMV infection, we inves- not significant. 1412 CUTTING EDGE: IL-18–INDEPENDENT RECALL RESPONSE OF NK CELLS

8. Sun, J. C., J. N. Beilke, and L. L. Lanier. 2009. Adaptive immune features of natural Disclosures killer cells. Nature 457: 557–561. The authors have no financial conflicts of interest. 9. Andrews, D. M., A. A. Scalzo, W. M. Yokoyama, M. J. Smyth, and M. A. Degli- Esposti. 2003. Functional interactions between dendritic cells and NK cells during viral infection. Nat. Immunol. 4: 175–181. References 10. French, A. R., H. Sjo¨lin, S. Kim, R. Koka, L. Yang, D. A. Young, C. Cerboni, 1. Sun, J. C., and L. L. Lanier. 2011. NK cell development, homeostasis and function: E. Tomasello, A. Ma, E. Vivier, et al. 2006. DAP12 signaling directly augments parallels with CD8(+) T cells. Nat. Rev. Immunol. 11: 645–657. proproliferative cytokine stimulation of NK cells during viral infections. J. Immunol. 2. Biron, C. A., K. B. Nguyen, G. C. Pien, L. P. Cousens, and T. P. Salazar-Mather. 177: 4981–4990. 1999. Natural killer cells in antiviral defense: function and regulation by innate 11. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami, cytokines. Annu. Rev. Immunol. 17: 189–220. K. Nakanishi, and S. Akira. 1998. Targeted disruption of the MyD88 gene results in 3. Sun, J. C., S. Madera, N. A. Bezman, J. N. Beilke, M. H. Kaplan, and L. L. Lanier. loss of IL-1- and IL-18-mediated function. Immunity 9: 143–150. 2012. Proinflammatory cytokine signaling required for the generation of natural 12. Sareneva, T., I. Julkunen, and S. Matikainen. 2000. IFN-alpha and IL-12 induce IL- killer cell memory. J. Exp. Med. 209: 947–954. 18 receptor in human NK and T cells. J. Immunol. 165: 1933–1938. 4. Beaulieu, A. M., C. L. Zawislak, T. Nakayama, and J. C. Sun. 2014. The tran- 13. Thierfelder, W. E., J. M. van Deursen, K. Yamamoto, R. A. Tripp, S. R. Sarawar, scription factor Zbtb32 controls the proliferative burst of virus-specific natural killer R. T. Carson, M. Y. Sangster, D. A. Vignali, P. C. Doherty, G. C. Grosveld, and cells responding to infection. Nat. Immunol. 15: 546–553. J. N. Ihle. 1996. Requirement for Stat4 in interleukin-12-mediated responses of 5. Chaix, J., M. S. Tessmer, K. Hoebe, N. Fuse´ri, B. Ryffel, M. Dalod, natural killer and T cells. Nature 382: 171–174. L. Alexopoulou, B. Beutler, L. Brossay, E. Vivier, and T. Walzer. 2008. Cutting 14. Cooper, M. A., J. M. Elliott, P. A. Keyel, L. Yang, J. A. Carrero, and edge: priming of NK cells by IL-18. J. Immunol. 181: 1627–1631. W. M. Yokoyama. 2009. Cytokine-induced memory-like natural killer cells. Proc. 6. Takeda, K., H. Tsutsui, T. Yoshimoto, O. Adachi, N. Yoshida, T. Kishimoto, Natl. Acad. Sci. USA 106: 1915–1919. H. Okamura, K. Nakanishi, and S. Akira. 1998. Defective NK cell activity and Th1 15. Paust, S., and U. H. von Andrian. 2011. memory. Nat. Immunol. response in IL-18-deficient mice. Immunity 8: 383–390. 12: 500–508. 7. Pien, G. C., A. R. Satoskar, K. Takeda, S. Akira, and C. A. Biron. 2000. Cutting 16. Min-Oo, G., and L. L. Lanier. 2014. Cytomegalovirus generates long-lived antigen- edge: selective IL-18 requirements for induction of compartmental IFN-gamma specific NK cells with diminished bystander activation to heterologous infection.

responses during viral infection. J. Immunol. 165: 4787–4791. J. Exp. Med. 211: 2669–2680. Downloaded from http://www.jimmunol.org/ by guest on October 5, 2021 Supplementary Figure 1.

A B Day 7 Day 14 Day 21 Day 28 Uninfected Day 7 105 25 11 105 11 5 105 8 2 105 4 2 34 30 55 35 104 104 104 104 y49H 103 103 103 103 y49H L L

102 102 102 102 0 0 0 0 19 18 4 6 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD45.2 CD45.2 s

C E ll Wildtype Wildtype ce 50 -/- Il18r1-/- Il18r1 NK

40

Uninfected + H

Number 30 Relative Cell y49

L 20

CD69 CD25 + A 10 C I

FL 0

D % Naive Day 7 PI Number Relative Cell CD11b KLRG1 CD90.2

Supplementary Figure 1. IL-18R-deficient NK exhibit expansion defect following MCMV infection. A. 106 WT (CD45.1) and Il18r1-/- (CD45.2) NK cells were co-transferred into Ly49H-deficient mice (CD45.2) and infected with MCMV. Plots are gated on total NK cells and the percentages of Ly49H+ NK cells are shown for each time point PI. B. WT (CD45.1) and Il18r1-/- (CD45.2) mixed bone marrow chimeric mice were infected with MCMV and percentages of splenic NK cells are shown (gated on TCR- - NK1.1+) for uninfected and day 7 PI. Expression of (C) CD69, CD25 at day 1.5 PI and (D) CD11b, KLRG1, and CD90.2 at day 7 PI are shown for wildtype and Il18r1-/- Ly49H+ NK cells (compared to uninfected mice) in mixed bone marrow chimeric mice. All data are representative of three experiments with 2-3 mice per time point. E. Day 7 splenocytes were incubated with FLICA® FAM-VAD-FMK and percentages of Ly49H+ FLICA+ NK cells are shown.

Supplementary Figure2.

B Wildtype A 30 Il1r -/- Uninfected Day 7 s ll ce

19 36 27 41 20 NK

+ y49H g L

N 10 F 15 30 9 23 I % 0 CD45.2 Day 0 PI Day 1.5 PI

C Wildtype Il1r -/- Uninfected Number ZĞůĂƟǀĞĞůů

CD27 KLRG1 CD90.2

Supplementary Figure 2. NK cell expansion and memory formation are independent of IL-1 during MCMV infection. A. Wildtype (CD45.1+) and Il1r-/- (CD45.2+) mixed bone marrow chimeric mice were infected with MCMV and percentages of splenic NK cells are shown (gated on TCR- - NK1.1+) for uninfected mice and at day 7 post-infection. B. Percentages of IFN- + wildtype and Il1r-/- NK cells are shown at day 0 and 1.5 PI. C. Expression of CD27, KLRG1, and CD90.2 are shown for wildtype and Il1r-/- Ly49H+ NK cells (compared to uninfected mice) at day 7 PI. All data are representative of two independent experiments with 3-4 mice per time point.