Licensing delineates helper and effector NK cell subsets during viral infection
Anthony E. Zamora, Ethan G. Aguilar, Can M. Sungur, Lam T. Khuat, Cordelia Dunai, G. Raymond Lochhead, Juan Du, Claire Pomeroy, Bruce R. Blazar, Dan L. Longo, Jeffrey M. Venstrom, Nicole Baumgarth, William J. Murphy
Anthony E. Zamora, Ethan G. Aguilar, Can M. Sungur, Lam T. Khuat, Cordelia Dunai, G. Raymond Lochhead, Juan Du, Claire Pomeroy, Bruce R. Blazar, Dan L. Longo, Jeffrey M. Venstrom, Nicole Baumgarth, William J. Murphy
View: Text | PDF
Research Article Immunology Inflammation

Licensing delineates helper and effector NK cell subsets during viral infection

Abstract

Natural killer (NK) cells can be divided into phenotypic subsets based on expression of receptors that bind self-MHC-I molecules, a concept termed licensing or education. Here we show NK cell subsets with different migratory, effector, and immunoregulatory functions in dendritic cell and antigen (ag)-specific CD8+ T cell responses during influenza and murine cytomegalovirus infections. Shortly after infection, unlicensed NK cells localized in draining lymph nodes and produced GM-CSF, which correlated with the expansion and activation of dendritic cells, and resulted in greater and sustained ag-specific T cell responses. In contrast, licensed NK cells preferentially migrated to infected tissues and produced IFN-γ. Importantly, human NK cell subsets exhibited similar phenotypic characteristics. Collectively, our studies demonstrate a critical demarcation between the functions of licensed and unlicensed NK cell subsets, with the former functioning as the classical effector subset and the latter as the stimulator of adaptive immunity helping to prime immune responses.

Authors

Anthony E. Zamora, Ethan G. Aguilar, Can M. Sungur, Lam T. Khuat, Cordelia Dunai, G. Raymond Lochhead, Juan Du, Claire Pomeroy, Bruce R. Blazar, Dan L. Longo, Jeffrey M. Venstrom, Nicole Baumgarth, William J. Murphy

×

Figure 5

Cytokine profiles of licensed and unlicensed NK cell subsets.

Options: View larger image (or click on image) Download as PowerPoint
Cytokine profiles of licensed and unlicensed NK cell subsets. (A–C) Repr...
(A–C) Representative staining of NK cells that are GM-CSF+ or IFN-γ+ from mediastinal lymph nodes (mLNs) (A) or lung (B) after APR8 infection or liver (C) after MCMV infection for 5 days. (D) Frequency and (E) absolute number of either CD3–NK1.1+Ly49G2+, Ly49A+, or Ly49C/I+ among GM-CSF+ NK cells at day 5 after APR8 infection in mLNs of C57BL/6, or draining lymph nodes (DLNs) 5 days after MCMV infection (F). (G) DC expansion following in vitro NK-DC coculture with neutralizing antibody against GM-CSF. Numbers of DCs were determined through flow cytometry (CD19–CD11c+MHCII+) and compared with cultures lacking NK cells to determine the fold expansion that occurred. (H and I) Absolute number of NK cells that are IFN-γ+ and either CD3–NK1.1+Ly49G2+, Ly49A+, or Ly49C/I+ at day 5 after APR8 infection (H) or MCMV infection (I) in lung (H) or liver (I) of C57BL/6 mice. (J) Frequency and (K) absolute number of NK cells that are IFN-γ+ and either CD3–NK1.1+Ly49G2+, Ly49A+, or Ly49C/I+ at day 5 after APR8 infection in mLNs of C57BL/6. (L) Ratio of GM-CSF to IFN-γ production by NK subsets in mLNs of day 5 APR8-infected C57BL/6 mice. (M and N) Ratio of IFN-γ to GM-CSF production by NK subsets in lung of day 5 APR8-infected C57BL/6 mice (M) or liver of day 5 MCMV-infected C57BL/6 mice (N). (O) Representative staining after single-cell exclusion gating for the evaluation of intracellular GM-CSF and IFN-γ production by human NK cell subsets. NK cells were identified as CD3–CD56+ cells. Intracellular IFN-γ, and GM-CSF production by NK cells exclusively expressing a single KIR was detected by excluding cells expressing other KIRs. HLA class I genotyping and KIR ligand assignment were performed for each sample to distinguish licensed versus unlicensed single-positive KIR (spKIR) NK cells. (P) Absolute number of unlicensed and licensed NK cells expressing GM-CSF from 5 distinct individuals lacking the HLA-C2 ligand for KIR2DL1 (HLA-C1/C1, Bw4/Bw6). Unlicensed NK cells were operationally defined as NKG2A–spKIR2DL1+ or NKG2A–KIR–; licensed NK cells expressed spKIR2DL3 and/or spKIR3DL1. (Q) Aggregate percentage of NK cell populations that are GM-CSF+. n = 3 mice per group, representative of 2 to 3 experiments. One-way ANOVA with Tukey post-test used to compare groups. Human data: Experiments were performed in duplicate; 1–3 aggregated single-positive NK cell subsets per unlicensed or licensed group. Five distinct individuals were used to obtain NK cells from peripheral blood mononuclear cells. *P < 0.05, **P < 0.01, ***P < 0.001.