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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC Sep 1, 2010.
Published in final edited form as:
PMCID: PMC2745285
NIHMSID: NIHMS131925
Cutting Edge: IL-15-independent NK cell response to mouse cytomegalovirus infection1
Joseph C. Sun,* Averil Ma, and Lewis L. Lanier*2
* Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, CA 94143
Department of Medicine, University of California, San Francisco, CA 94143
2 Correspondence: Lewis.Lanier/at/ucsf.edu
Natural Killer (NK) cells respond rapidly during viral infection. The development, function, and survival of NK cells are thought to be dependent on Interleukin (IL)-15. In mice lacking IL-15, NK cells are found in severely decreased numbers. Surprisingly, following infection of IL-15- and IL-15Rα-deficient mice with mouse cytomegalovirus (MCMV), we measured a robust proliferation of Ly49H-bearing NK cells in lymphoid and non-lymphoid organs, capable of cytokine secretion and cytolytic function. Remarkably, even in Rag2−/− × Il2rg−/− mice, a widely used model of NK cell deficiency, we detected a significant number of NK cells one week after MCMV infection. In these mice, we measured a greater than 300-fold expansion of NK cells, which was dependent on recognition of the m157 viral glycoprotein ligand and IL-12. Together, these findings demonstrate a previously unrecognized independence of NK cells on IL-15 or other common-γ signaling cytokines during their response against viral infection.
IL-15 and its receptor (IL-15Rα) are important in the homeostasis of NK cells and memory CD8+ T cells (110). IL-15 bound to the receptor IL-15Rα on the surface of dendritic cells is “trans-presented” to IL-15–responsive cells bearing the shared IL-2 and IL-15 receptor common-β chain (CD122) (1117). During infection, dendritic cells respond to inflammatory cytokines, leading to the production of IL-15 and IL-15Rα (1315, 1820). Expression of IL-15 and IL-15Rα on activated myeloid cells has thus been thought to contribute to NK cell responses against pathogens. Although mice deficient in IL-15 or the IL-15 receptor severely lack peripheral NK cells, a small population of NK cells (<0.1%) is detectable in the spleen (3, 5). We sought to determine whether these NK cells that arise in the absence of IL-15 signals can mount effector responses against viral infection.
Mice and infections
C57BL/6 (B6) and Rag2−/− × Il2rg−/− B6 mice were purchased from the National Cancer Institute and Taconic, respectively. Il15−/−, Il15ra−/−, Il15−/− × Il15ra−/−, and Rag1−/− × Il2rb−/− B6 mice were bred at UCSF. Experiments were done according to the UCSF Institutional Animal Care and Use Committee guidelines. 5×104 PFU of a salivary gland stock of MCMV (Smith strain) or MCMV-Δm157 was injected intraperitoneally (21). 750 μg of neutralizing anti-IL-12 p70 (clone C17.8) was injected intraperitoneally 24 hours prior to infection.
Flow cytometry and functional assays
Cells were stained with antibodies against NK1.1, CD3, Ly49H, Ly49D, KLRG1, NKp46, NKG2D, CD27, and DX5 (CD49b) (eBioscience or BD Pharmingen). Flow cytometry was performed using a LSRII with CELLQuest software (Becton Dickinson). Splenocytes were enriched for NK cells by using a NK cell isolation kit (Miltenyi Biotec), followed by AutoMACS magnetic bead separation. NK cells were incubated in tissue culture plates treated with N-(1-(2,3-dioleoyloxyl)propyl)-N,N,N-trimethylammonium methylsulphate (Sigma) and coated with anti-NK1.1 or anti-Ly49H or PBS for 5 h at 37 °C in the presence of Golgiplug (BD Pharmingen), followed by staining for LAMP-1 and intracellular IFN-γ (BD Pharmingen) (22). NK cells were used as effector cells in a 4 hr 51Cr release assay (23) against Ba/F3 and m157-transfected Ba/F3 cells (22).
Functional NK cell responses in IL-15Rα and IL-15 deficient mice
The spleens of Il15ra−/− mice contain <0.1% CD3, NK1.1+ NK cells, compared with 2–5% in wild-type (WT) B6 mice (5). The absolute number of NK cells is decreased and the percentage of NK cells bearing the Ly49H receptor is lower in Il15ra−/− (~10%) compared to WT mice (~50%) (Fig. 1A). During the NK cell response against MCMV in WT mice, the Ly49H+ NK cells preferentially proliferate during the first several days of infection (21, 24, 25), a response specific for the m157 gene product of MCMV (22, 26). When we infected WT and Il15ra−/− mice with MCMV, both mice showed an increase in Ly49H+ NK cell numbers and comprised >80% of total NK cells at day 7 post-infection (PI) (Fig. 1A). A similar expansion was not observed in the Ly49D+ Ly49H NK cell subset (Fig. 1A). With precursor numbers of ~2×104 total Ly49H+ NK cells in the spleen, the absolute number of Ly49H+ NK cells in Il15ra−/− mice at day 7 PI expanded approximately 72-fold to become comparable to the numbers found in uninfected WT B6 mice (greater than 106) (Fig. 1B). NK cells from MCMV-infected Il15ra−/− mice expressed comparable levels of activating receptors (NK1.1, NKp46, Ly49H, and NKG2D) and activation markers (KLRG1 and CD27) as WT mice (Fig. 1C). When NK cells at day 7 PI were stimulated ex vivo with anti-NK1.1 or -Ly49H, these cells upregulated LAMP-1 and produced IFN-γ (Fig. 1D), demonstrating that NK cells do not require IL-15 signals to mediate effector functions during MCMV infection.
Figure 1
Figure 1
Expansion of NK cells in WT and Il15ra−/− mice
Il15−/− mice are also deficient in NK cells (3). On day 7 PI, we observed robust expansion of Ly49H+ NK cells in the spleen of MCMV-infected Il15−/− mice (Fig. 2A). Expression of KLRG1, a NK cell activation marker (27), was comparable in WT and Il15−/− mice (Supplemental Fig. 1). With <104 total Ly49H+ NK cells in the spleen prior to infection, the absolute number of Ly49H+ NK cells in Il15−/− mice at day 7 PI was >105, representing a 50-fold increase in absolute numbers (Fig 2A). We tested the ability of Ly49H+ NK cells from Il15−/− mice to kill m157-bearing target cells. Ly49H+ NK cells isolated at day 7 PI from MCMV-infected Il15−/− mice were able to efficiently lyse m157-bearing target cells (Fig. 2B).
Figure 2
Figure 2
NK cell expansion in WT and Il15−/− mice
To test whether specific viral ligand (and not inflammation alone) is required to drive NK cell proliferation, we infected Il15−/− mice with MCMV or a mutant strain lacking m157 (MCMV-Δm157). Unlike MCMV-infected Il15−/− mice, which contained a large percentage and absolute number of Ly49H+ NK cells at day 7 PI (45.3-fold expansion), infection of Il15−/− mice with MCMV-Δm157 did not generate many NK cells (1.7-fold expansion) compared to uninfected controls (Fig. 2, C and D). The diminished proliferation of NK cells during infection with MCMV-Δm157 is not due to defective replication as this mutant virus is equally or more virulent than WT MCMV (28). Adoptive transfer of WT NK cells into Il15−/− recipient mice results in the rapid loss of the transferred NK cells (110); however, during infection with MCMV, we measured large numbers of transferred NK cells (CD45.1+) at day 7 PI in spleen and liver of the Il15−/− recipients (Supplemental Fig. 2, A and B). At later time points after MCMV infection (day 15 and 30 PI), transferred NK cells were difficult to recover (data not shown), suggesting that following the resolution of infection, NK cells again require IL-15 for survival. Expansion and survival of adoptively transferred WT NK cells were not observed in Il15−/− mice infected with MCMV-Δm157 (Supplemental Fig. 2, A and B). Altogether, these experiments demonstrate that both viral infection and m157 are required for robust NK cell proliferation in the setting of IL-15 deficiency.
NK cell responses in Rag2−/− × Il2rg−/− mice
Rag2−/− × Ilr2g−/− mice are currently the best model of NK cell deficiency. Without the common-γ chain (γC), NK cells cannot receive signals from any cytokine of the γC family, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In naïve Rag2−/− × Il2rg−/− mice, NK cells were barely detectable (0.05% in spleen and 0.2% in liver) (Fig. 3A and Supplemental Fig. 3). When we infected WT and Rag2−/− × Il2rg−/− mice with MCMV and measured NK cell responses at day 7 PI, Rag2−/− × Il2rg−/− mice showed an increase in total NK cell numbers in spleen (comprising 1.6% of splenocytes) and liver (comprising 1% of hepatic lymphocytes) (Fig. 3A and Supplemental Fig. 3). In Rag2−/− × Il2rg−/− mice, as with the other models of IL-15 deficiency, only the NK cells expressing Ly49H (and not Ly49D+ Ly49H NK cells) expanded vigorously, upregulating KLRG1 (Fig. 3A). With <1000 total Ly49H+ NK cells in the spleens of uninfected Rag2−/− × Il2rg−/− mice, the absolute number of Ly49H+ NK cells at day 7 PI became >105 (320-fold expansion) (Fig. 3B). Similar results were obtained analyzing Rag1−/− x Il2rb−/− mice (Supplemental Figure 4). Collectively, these data demonstrate that during MCMV infection NK cells do not require cytokines of the γC family for their activation and proliferation.
Figure 3
Figure 3
Ly49H+ NK cell expansion in Rag2−/− × Il2rg−/− mice
NK cell response in Il15−/− × Il15ra−/− mice dependent on IL-12
IL-12 is produced by dendritic cells and granulocytes in response to viral and bacterial infection and is required for the generation of Th1 cells, as well as inducing proliferation and IFN-γ in activated CD8+ T cells and NK cells (reviewed in Nat Rev Immunol. 2003 Feb;3(2):133–46). Additionally, IL-12 plays an important role in NK cell production of IFN-γ and NK cell blastogenesis during MCMV infection (29, 30), and NK cell proliferation in response to MCMV infection is somewhat impaired in Il12−/− mice (31, 32). To address whether IL-12 contributes to NK cell expansion in the setting of IL-15 deficiency, we injected Il15−/− × Il15ra−/− mice with a neutralizing anti-IL-12 antibody prior to infection. Uninfected Il15−/− × Il15ra−/− mice have very few peripheral Ly49H+ NK cells, but 7 days following infection, significant numbers and percentages of Ly49H+ NK cells were detected in the spleen (78%) and liver (91%) (Fig. 4A). However, absolute numbers of Ly49H+ NK cells at day 7 PI were ~30-fold less in anti-IL-12 treated mice compared to control mice (Fig. 4B). The overall expansion of Ly49H+ NK cells in Il15−/− × Il15ra−/− mice was ~70-fold, versus a 2-fold increase in anti-IL12 treated Il15−/− × Il15ra−/− mice (Fig. 4B). Thus, IL-12, not IL-15, contributes greatly to the overall NK cell response following MCMV infection in mice lacking the ability to produce or respond to IL-15. Future studies are required to determine whether the small number of NK cells that do proliferate during MCMV infection represent a unique IL-15-independent subset or new bone marrow emigrants that are rescued from death by IL-12 and inflammatory cytokine signaling. Moreover, although we have shown that IL-12 is involved in NK cell expansion in the absence of IL-15, other factors might also contribute to their proliferation and survival. In conclusion, our surprising findings contribute added insight into the cytokines (or lack thereof) that NK cells require during an immune response against viral infection.
Figure 4
Figure 4
Ly49H+ NK cell expansion in IL15−/− × Il15ra−/− mice is blocked with an antibody against IL-12
Supplementary Material
S1
Acknowledgments
We thank Drs. Jody Baron, Wayne Yokoyama, Anne Hill, and Ulrich Koszinowski for generously providing reagents.
Abbreviations
MCMVmouse cytomegalovirus
γCIL-2 receptor common-γ chain

Footnotes
1J.C.S. is an Irvington Postdoctoral Fellow of the Cancer Research Institute. This study was supported by NIH grant AI068129 and L.L.L. is an American Cancer Society Professor.
Disclosure
The authors have no financial conflicts of interest.
1. Burkett PR, Koka R, Chien M, Chai S, Chan F, Ma A, Boone DL. IL-15R alpha expression on CD8+ T cells is dispensable for T cell memory. Proc Natl Acad Sci U S A. 2003;100:4724–4729. [PubMed]
2. Huntington ND, Puthalakath H, Gunn P, Naik E, Michalak EM, Smyth MJ, Tabarias H, Degli-Esposti MA, Dewson G, Willis SN, Motoyama N, Huang DC, Nutt SL, Tarlinton DM, Strasser A. Interleukin 15-mediated survival of natural killer cells is determined by interactions among Bim, Noxa and Mcl-1. Nat Immunol. 2007;8:856–863. [PMC free article] [PubMed]
3. Kennedy MK, Glaccum M, Brown SN, Butz EA, Viney JL, Embers M, Matsuki N, Charrier K, Sedger L, Willis CR, Brasel K, Morrissey PJ, Stocking K, Schuh JC, Joyce S, Peschon JJ. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med. 2000;191:771–780. [PMC free article] [PubMed]
4. Koka R, Burkett PR, Chien M, Chai S, Chan F, Lodolce JP, Boone DL, Ma A. Interleukin (IL)-15R[alpha]-deficient natural killer cells survive in normal but not IL-15R[alpha]-deficient mice. J Exp Med. 2003;197:977–984. [PMC free article] [PubMed]
5. Lodolce JP, Boone DL, Chai S, Swain RE, Dassopoulos T, Trettin S, Ma A. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity. 1998;9:669–676. [PubMed]
6. Lodolce JP, Burkett PR, Boone DL, Chien M, Ma A. T cell-independent interleukin 15Ralpha signals are required for bystander proliferation. J Exp Med. 2001;194:1187–1194. [PMC free article] [PubMed]
7. Ma A, Koka R, Burkett P. Diverse functions of IL-2, IL-15, and IL-7 in lymphoid homeostasis. Annu Rev Immunol. 2006;24:657–679. [PubMed]
8. Prlic M, Blazar BR, Farrar MA, Jameson SC. In vivo survival and homeostatic proliferation of natural killer cells. J Exp Med. 2003;197:967–976. [PMC free article] [PubMed]
9. Schluns KS, Klonowski KD, Lefrancois L. Transregulation of memory CD8 T-cell proliferation by IL-15Ralpha+ bone marrow-derived cells. Blood. 2004;103:988–994. [PubMed]
10. Cooper MA, Bush JE, Fehniger TA, VanDeusen JB, Waite RE, Liu Y, Aguila HL, Caligiuri MA. In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells. Blood. 2002;100:3633–3638. [PubMed]
11. Burkett PR, Koka R, Chien M, Chai S, Boone DL, Ma A. Coordinate expression and trans presentation of interleukin (IL)-15Ralpha and IL-15 supports natural killer cell and memory CD8+ T cell homeostasis. J Exp Med. 2004;200:825–834. [PMC free article] [PubMed]
12. Dubois S, Mariner J, Waldmann TA, Tagaya Y. IL-15Ralpha recycles and presents IL-15 In trans to neighboring cells. Immunity. 2002;17:537–547. [PubMed]
13. Ferlazzo G, Pack M, Thomas D, Paludan C, Schmid D, Strowig T, Bougras G, Muller WA, Moretta L, Munz C. Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs. Proc Natl Acad Sci U S A. 2004;101:16606–16611. [PubMed]
14. Koka R, Burkett P, Chien M, Chai S, Boone DL, Ma A. Cutting edge: murine dendritic cells require IL-15R alpha to prime NK cells. J Immunol. 2004;173:3594–3598. [PubMed]
15. Lucas M, Schachterle W, Oberle K, Aichele P, Diefenbach A. Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity. 2007;26:503–517. [PMC free article] [PubMed]
16. Mortier E, Woo T, Advincula R, Gozalo S, Ma A. IL-15Ralpha chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation. J Exp Med. 2008;205:1213–1225. [PMC free article] [PubMed]
17. Sandau MM, Schluns KS, Lefrancois L, Jameson SC. Cutting edge: transpresentation of IL-15 by bone marrow-derived cells necessitates expression of IL-15 and IL-15R alpha by the same cells. J Immunol. 2004;173:6537–6541. [PubMed]
18. Mattei F, Schiavoni G, Belardelli F, Tough DF. IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation. J Immunol. 2001;167:1179–1187. [PubMed]
19. Nguyen KB, Salazar-Mather TP, Dalod MY, Van Deusen JB, Wei XQ, Liew FY, Caligiuri MA, Durbin JE, Biron CA. Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection. J Immunol. 2002;169:4279–4287. [PubMed]
20. Zhang X, Sun S, Hwang I, Tough DF, Sprent J. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity. 1998;8:591–599. [PubMed]
21. Sun JC, Beilke JN, Lanier LL. Adaptive immune features of natural killer cells. Nature. 2009;457:557–561. [PMC free article] [PubMed]
22. Arase H, Mocarski ES, Campbell AE, Hill AB, Lanier LL. Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science. 2002;296:1323–1326. [PubMed]
23. Sun JC, Lanier LL. Tolerance of NK cells encountering their viral ligand during development. J Exp Med. 2008;205:1819–1828. [PMC free article] [PubMed]
24. Daniels KA, Devora G, Lai WC, O’Donnell CL, Bennett M, Welsh RM. Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H. J Exp Med. 2001;194:29–44. [PMC free article] [PubMed]
25. Dokun AO, Kim S, Smith HR, Kang HS, Chu DT, Yokoyama WM. Specific and nonspecific NK cell activation during virus infection. Nat Immunol. 2001;2:951–956. [PubMed]
26. Smith HR, Heusel JW, Mehta IK, Kim S, Dorner BG, Naidenko OV, Iizuka K, Furukawa H, Beckman DL, Pingel JT, Scalzo AA, Fremont DH, Yokoyama WM. Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci U S A. 2002;99:8826–8831. [PubMed]
27. Huntington ND, Tabarias H, Fairfax K, Brady J, Hayakawa Y, Degli-Esposti MA, Smyth MJ, Tarlinton DM, Nutt SL. NK cell maturation and peripheral homeostasis is associated with KLRG1 up-regulation. J Immunol. 2007;178:4764–4770. [PubMed]
28. Bubic I, Wagner M, Krmpotic A, Saulig T, Kim S, Yokoyama WM, Jonjic S, Koszinowski UH. Gain of virulence caused by loss of a gene in murine cytomegalovirus. J Virol. 2004;78:7536–7544. [PMC free article] [PubMed]
29. Orange JS, Biron CA. Characterization of early IL-12, IFN-alphabeta, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. J Immunol. 1996;156:4746–4756. [PubMed]
30. Orange JS, Biron CA. An absolute and restricted requirement for IL-12 in natural killer cell IFN-gamma production and antiviral defense. Studies of natural killer and T cell responses in contrasting viral infections. J Immunol. 1996;156:1138–1142. [PubMed]
31. Andrews DM, Scalzo AA, Yokoyama WM, Smyth MJ, Degli-Esposti MA. Functional interactions between dendritic cells and NK cells during viral infection. Nat Immunol. 2003;4:175–181. [PubMed]
32. French AR, Sjolin H, Kim S, Koka R, Yang L, Young DA, Cerboni C, Tomasello E, Ma A, Vivier E, Karre K, Yokoyama WM. DAP12 signaling directly augments proproliferative cytokine stimulation of NK cells during viral infections. J Immunol. 2006;177:4981–4990. [PubMed]