Critical role for the chemokine receptor CXCR6 in NK cell–mediated antigen-specific memory of haptens and viruses

Journal name:
Nature Immunology
Volume:
11,
Pages:
1127–1135
Year published:
DOI:
doi:10.1038/ni.1953
Received
Accepted
Published online

Abstract

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell–expressed CXCR6.

At a glance

Figures

  1. Liver NK cells develop specific memory of haptens.
    Figure 1: Liver NK cells develop specific memory of haptens.

    (a) Hapten-specific CHS responses in naive Rag2−/−Il2rg−/− mice (n = 10–15 per group) that received hepatic CD45+NK1.1+Thy-1+ NK cells (1 × 105) from naive (vehicle-exposed; Acetone) or DNFB- or OXA-sensitized Rag1−/− donors (Sensitization) and were challenged 24 h or 4 months after transfer; ear swelling was calculated after 24 h by subtraction of background swelling in naive mice from that in recipients of NK cells. *P < 0.01 and **P < 0.001 (analysis of variance (ANOVA)). (b) Flow cytometry analysis of the survival and population expansion of adoptively transferred NK cells 2 weeks after the 4-month challenge in a, presented as the number of liver-resident CD45+ NK1.1+ cells. Results were similar for mice challenged with DNFB or OXA, so data were pooled according to donor sensitization. No NK1.1+ cells were detected in mock (PBS)-injected control recipient Rag2−/−Il2rg−/− mice (data not shown). (c) Hapten-specific CHS responses in naive C57BL/6 mice (n = 10–15 per group) that received sorted CD45+NK1.1+CD3Thy-1+ NK cells (1 × 105) from donors transgenic for expression of GFP-tagged actin, then were challenged 6 weeks later with OXA (left) or DNFB (right) and analyzed as in a. *P < 0.001 and **P < 0.0001 (ANOVA). (d) Recruitment of memory NK cells to challenge sites in naive Rag2−/−Il2rg−/− mice (n = 6–7 per group) that received adoptive transfer of mixtures of hepatic CD45+NK1.1+Thy-1+ NK cells from naive CD45.1+ wild-type donors (C57BL/6) and from CD45.2+ DNFB- or OXA-sensitized wild-type or donors transgenic for expression of GFP-tagged actin (1 × 105 each) and, 1 month later, were challenged in the ears with either DNFB or OXA; livers and ears collected at 24, 48 and 72 h were analyzed by flow cytometry for NK cells whose origin was distinguished by congenic or fluorescent markers (results presented as mean of all mice at 24, 48 and 72 h). No NK cells were found in acetone-challenged control ears (data not shown). *P < 10−11 (ANOVA). Data are representative of three to five independent experiments (pooled results; error bars, s.d.).

  2. Liver NK cells develop specific memory of viral antigens.
    Figure 2: Liver NK cells develop specific memory of viral antigens.

    (a) Virus-specific DTH in naive Rag2−/−Il2rg−/− mice (n = 8–10 per group) that received adoptive transfer of splenic or hepatic CD45+ NK1.1+ NK cells (8 × 104) 1 month after immunization of Rag1−/− donor mice, followed by challenge of ears of recipient mice by subcutaneous injection 2 months later and analysis after 24 h (as in Fig. 1a). P values, Student's t-test. (b) Survival of Rag2−/−Il2rg−/− mice (n = 15–19 per group) treated by the sensitization and transfer protocol in a, then infected intranasally with influenza A strain A/PR/8/34 (500 PFU) 3 months after NK cell transfer. P values, log-rank Mantel-Cox test. (c) Survival of the recipients in a (n = 19 per group) infected with influenza A strain A/PR/8/34 (500 PFU) 2 months after assessment of DTH; results are correlated to DTH. *P = 0.0001 (Spearman correlation). (d) Survival of Rag1−/− mice (n = 8–12 per group) immunized with PR8-VLPs, M1-VLPs or UV-VSV, then challenged 1 month later with live virus (2,500 PFU influenza strain A/PR/8/34 intranasally or 500 PFU VSV intravenously). P values, log-rank Mantel-Cox test. (e) Survival of Rag2−/− mice (n = 15–22 per group) immunized with VLPs containing influenza (PR8-VLP) or HIV-1 (HIV-VLP) or with UV-VSV, then challenged 1 month later by intramuscular injection of VSV at the median lethal dose (250 PFU). *P = 0.0116 (log-rank Mantel-Cox test). (f) Virus-specific DTH in Rag1−/− mice (n = 10–15 per group) immunized with HA-containing VLPs (PR8) or HA-free VLPs (M1), then challenged 1 month later and analyzed as in a. P values, unpaired Student's t-test. Data are representative of three to five independent experiments (pooled results; error bars, s.d.).

  3. Mouse liver NK cells recognize and discriminate between HIV-1 and influenza A.
    Figure 3: Mouse liver NK cells recognize and discriminate between HIV-1 and influenza A.

    (a) Ear swelling in naive Rag2−/−Il2rg−/− mice (n = 12–15 per group) that received adoptively transferred hepatic (left) or splenic (right) CD45+NK1.1+ NK cells (8 × 104 cells per mouse) from Rag1−/− donor mice immunized with VLPs containing influenza (PR8) or HIV-1 (HIV) 1 month before transfer; recipients were challenged by subcutaneous injection of VLPs into one ear and PBS in the other ear and were assessed 2 months after transfer. NS, not significant; *P < 0.01 and **P < 0.001 (unpaired Student's t-test). (b) Ear swelling in C57BL/6 Rag1−/− mice (left) and BALB/c Rag2−/− mice (right) immunized with VLPs and challenged 1 month later (n = 10–15 mice per group). P values, unpaired Student's t-test. Background ear swelling in nonimmunized mice was subtracted from ear swelling in the experimental groups. Data are representative of three to five independent experiments (pooled results; error bars, s.d.).

  4. NK cell-expressed CXCR6 is required for NK cell-mediated adaptive immunity to haptens.
    Figure 4: NK cell–expressed CXCR6 is required for NK cell–mediated adaptive immunity to haptens.

    (a) Frequency of CXCR6-expressing CD45+ NK1.1+ NK cells from Cxcr6+/− mice on a Rag1-sufficient (C57BL/6) or Rag1−/− background in different tissues, assessed by flow cytometry. LN, lymph node; BM, bone marrow. (b) Ear swelling in naive Rag2−/−Il2rg−/− mice (n = 10–12 per group) that received 1 × 105 NK cells from DNFB-sensitized Rag1−/−Cxcr6+/− donor spleen or liver, sorted for expression of NK1.1 and GFP; recipient mice were challenged 1 month later with DNFB on one ear and solvent on the other. (c) DNFB-induced CHS in lymphocompetent C57BL/6 mice (left) and Rag1−/− C57BL/6 mice (right; n = 10–12 mice per group). (d) DNFB-induced CHS in C57BL/6 mice (left) and Rag1−/− C57BL/6 mice (right) sensitized with hapten and given mAb to CXCR6 (100 μg per mouse) or isotype-matched control antibody intravenously 24 h before DNFB challenge (n = 10–15 mice per group). *P < 0.01, **P < 0.001 and ***P < 0.0001 (unpaired Student's t-test (a,c,d) or ANOVA (b)). Data are representative of three to five independent experiments (pooled results; error bars, s.d.).

  5. NK cell-expressed CXCR6 is required for NK cell-mediated adaptive immunity to viruses.
    Figure 5: NK cell–expressed CXCR6 is required for NK cell–mediated adaptive immunity to viruses.

    (a) Antiviral DTH responses in Rag1−/− C57BL/6 mice (left) or Rag2−/− BALB/c mice (right) immunized and challenged with various combinations of VLPs and UV-VSV (below graphs) and given mAb to CXCR6 (100 μg per mouse) or isotype-matched control antibody 24 h before challenge. P values, unpaired Student's t-test. (b) Survival of Rag1−/− and Rag2−/− mice (n = 8–12 per group) immunized with PR8-VLP or M1-VLP, challenged 1 month later by lethal infection with influenza A strain A/PR/8/34 (2,500 PFU for Rag1−/− (left) and 10,000 PFU for Rag2−/− (right)) and injected with mAb to CXCR6 (100 μg per mouse) or isotype-matched control antibody on days 1 and 5. P values, log-rank Mantel-Cox test. Data are representative of three to five independent experiments (pooled results; error bars, s.d.).

  6. CXCR6 regulates hepatic NK cell homeostasis.
    Figure 6: CXCR6 regulates hepatic NK cell homeostasis.

    (a) Frequency of GFP+ and GFP NK cell subsets (identified as CD45+NK1.1+ cells) in various organs of Cxcr6+/− and Cxcr6−/− mice. *P < 0.01 and **P < 0.001 (unpaired Student's t-test). (b) Ratio of NK cell subsets (CXCR6+/CXCR6) in liver and spleen of wild-type (Cxcr6+/+), Cxcr6+/− and Cxcr6−/− mice (all C57BL/6; n = 12–15 per group). *P < 0.00001 (unpaired Student's t-test). (c) Distribution of NK cells recovered from liver or spleen 1 month after adoptive transfer of sorted subsets (1 × 105 cells) into Rag2−/−Il2rg−/− recipients (n = 10–15 per group). *P < 0.001 and **P < 0.0001 (unpaired Student's t-test). (d) Flow cytometry of NK cells in livers and spleens of Rag2−/−Il2rg−/− recipients (n = 8–10 per group) 2 weeks after transfer of mixtures of 1 × 105 GFP+ or GFP CD45+NK1.1+ NK cells sorted from Cxcr6+/− or Cxcr6−/− donors. *P < 0.001 and **P < 0.0001 (unpaired Student's t-test). (e) Ear swelling in Rag2−/−Il2rg−/− mice (n = 8 per group) that received 8 × 104 DNFB-primed CD45+ NK1.1+ GFP+ NK cells from Cxcr6+/− or Cxcr6−/− donors and were challenged 24 h or 1 month later. *P < 0.01 and **P < 0.001 (unpaired Student's t-test). Data are representative of three to five independent experiments (pooled results; error bars, s.d.).

  7. Hepatic memory NK cells mediate hapten-specific killing in vitro.
    Figure 7: Hepatic memory NK cells mediate hapten-specific killing in vitro.

    (a) Hapten-specific killing of target B cells by naive and hapten-sensitized CD45+ NK1.1+ NK cells cultured for 12 h at various target cell/effector cell ratios (horizontal axis) with a mixture of two populations of B cells labeled with a large (CFSEhi) or small (CFSElo) amount of the cytosolic dye CFSE (n = 10–20 donor mice per group); alternatively, target and control B cells were distinguished by use of the congenic markers CD45.1 and CD45.2. CFSElo or CD45.1+ B cells served as a control; CFSEhi or CD45.2+ B cells were from wild-type donors and were haptenated with DNBS (left and middle) or were from MHC class I–deficient (MHC-KO) donors (right). Hapten-specific killing was assessed as the ratio of CFSElo to CFSEhi cells, corrected for input. *P < 10−8 and **P < 10−12, DNFB- or OXA-sensitized versus acetone (left and middle) or MHC-KO versus DNBS-labeled (right; unpaired Student's t-test). (b) Killing capacity of DNFB-primed hepatic CD45+ NK1.1+ NK cells from Cxcr6+/− or Cxcr6−/− donor mice (n = 12 donor mice per group), assessed as in a in the presence of mAb to CXCR6 or isotype-matched control mAb. *P < 0.01, **P < 0.001 and ***P < 0.00001, compared with Cxcr6−/− (unpaired Student's t-test). (c) Killing capacity of acetone- or DNFB-primed hepatic CD45+ NK1.1+ NK cells from Rag1−/− donors (n = 15 per group) at a target cell/effector cell ratio of 1:25, assessed in the presence of mAb to CXCR6 (10 μg/ml), mAb to CXCL16 (10 μg/ml) or CXCL16 (500 ng/ml); results are presented relative to those of cultures treated with isotype-matched control antibody (10 μg/ml). *P < 0.01 and **P < 0.001, compared with isotype-matched control antibody (unpaired Students t-test). (d) Flow cytometry analysis of the incorporation of anti-LAMP-1 by NK1.1+ NK cells sorted from the livers or spleens of Rag1−/− donor mice sensitized with acetone, DNFB or OXA on days 0 and 1 and injected with 100 μg mAb to CXCR6 or isotype-matched control mAb 12 h before NK cell isolation; NK cells were cultured together with DNBS-labeled B cells in the presence of fluorescein isothiocyanate–conjugated anti-LAMP-1 (10 μg/ml) with or without mAb to CXCR6 or isotype-matched control mAb (10 μg/ml) and assessed after 3 h (n = 10–18 donor mice total with 12–20 wells per group). (e) Frequency of LAMP-1+ NK cells among the cells in d. *P < 10−9 (unpaired Student's t-test). Data are representative of three to five independent experiments (pooled results; error bars, s.d.).

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Affiliations

  1. Harvard Medical School, Department of Pathology, Boston, Massachusetts, USA.

    • Silke Paust,
    • Michael P Flynn,
    • E Ashley Moseman,
    • Balimkiz Senman &
    • Ulrich H von Andrian
  2. The Ragon Institute of MIT, MGH and Harvard, Charlestown, Massachusetts, USA.

    • Silke Paust &
    • Amalio Telenti
  3. Department of Microbiology & Immunology and Emory Vaccine Center, Emory University, Atlanta, Georgia, USA.

    • Harvinder S Gill,
    • Bao-Zhong Wang &
    • Richard W Compans
  4. Texas Tech University, Department of Chemical Engineering, Lubbock, Texas, USA.

    • Harvinder S Gill
  5. Department of Human Developmental Biology, Jagiellonian University College of Medicine, Kraków, Poland.

    • Marian Szczepanik
  6. Institute of Microbiology, University Hospital, University of Lausanne, Lausanne, Switzerland.

    • Amalio Telenti
  7. Yale Medical School, Department of Medicine, New Haven, Connecticut, USA.

    • Marian Szczepanik &
    • Philip W Askenase
  8. Immune Disease Institute, Boston, Massachusetts, USA.

    • Ulrich H von Andrian

Contributions

S.P. and U.H.v.A. designed the study; S.P., H.S.G., B.Z.W. and M.F. did experiments; S.P., A.T. and B.S. collected and analyzed data; E.A.M., H.S.G., B.Z.W. and R.H.C. provided reagents; E.A.M., M.S. and P.W.A. provided technical support and conceptual advice; and S.P. and U.H.v.A. wrote the manuscript.

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The authors declare no competing financial interests.

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