Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Mar;6(1):34-46.
doi: 10.1002/iid3.190. Epub 2017 Sep 27.

IL-12 and IL-15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells

Theresa Hydes  1 , Angela Noll  2 , Gabriela Salinas-Riester  3 , Mohammed Abuhilal  4 , Thomas Armstrong  4 , Zaed Hamady  4 , John Primrose  4 , Arjun Takhar  4 , Lutz Walter  2 , Salim I Khakoo  1
Affiliations

IL-12 and IL-15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells

Theresa Hydes et al. Immun Inflamm Dis. 2018 Mar.

Abstract

Introduction: Murine hepatic NK cells exhibit adaptive features, with liver-specific adhesion molecules CXCR6 and CD49a acting as surface markers.

Methods: We investigated human liver-resident CXCR6+ and CD49a+ NK cells using RNA sequencing, flow cytometry, and functional analysis. We further assessed the role of cytokines in generating NK cells with these phenotypes from the peripheral blood.

Results: Hepatic CD49a+ NK cells could be induced using cytokines and produce high quantities of IFNγ and TNFα, in contrast to hepatic CXCR6+ NK cells. RNA sequencing of liver-resident CXCR6+ NK cells confirmed a tolerant immature phenotype with reduced expression of markers associated with maturity and cytotoxicity. Liver-resident double-positive CXCR6 + CD49a+ hepatic NK cells are immature but maintain high expression of Th1 cytokines as observed for single-positive CD49a+ NK cells. We show that stimulation with activating cytokines can readily induce upregulation of both CD49a and CXCR6 on NK cells in the peripheral blood. In particular, IL-12 and IL-15 can generate CXCR6 + CD49a+ NK cells in vitro from NK cells isolated from the peripheral blood, with comparable phenotypic and functional features to liver-resident CD49a+ NK cells, including enhanced IFNγ and NKG2C expression.

Conclusion: IL-12 and IL-15 may be key for generating NK cells with a tissue-homing phenotype and strong Th1 cytokine profile in the blood, and links peripheral activation of NK cells with tissue-homing. These findings may have important therapeutic implications for immunotherapy of chronic liver disease.

Keywords: CD49a antigen; chemokine receptor 6 protein; cytokines; human liver; natural killer cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(a) Representative flow cytometry plots showing gating strategy and individual frequencies of CD49a+ and CXCR6+ NK cell populations within the peripheral blood and hepatic perfusate. (b) A comparison of the frequency of CD49a+ (n = 20) and CXCR6+ (n = 22) NK cells within the peripheral blood and hepatic perfusate (paired samples). Dot plots display individual values. (Wilcoxon matched pairs test). (c) Distribution of frequencies of CD49a+ (n = 35) and CD49a + CXCR6+ (n = 27) NK cells within the hepatic lymphocyte population. Dot plot displays individual values and median. Individuals with high frequencies of CD49a+ NK cells are plotted with a cross. Representative flow cytometry plots gated on NK cells showing examples of individuals with average and high frequencies of CD49a + CXCR6+ NK cells. (d) Frequencies of CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, and CD49a‐CXCR6− NK cell subsets in the human liver (n = 27). Dot plots display individual values and median. (e) Comparison of frequency of CD16 (n = 12), CD57 (n = 12), CD69 (n = 11), NKG2C (n = 22), and KIR+ (n = 9) NK cells between liver‐resident subpopulations CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, and CD49a‐CXCR6− (Wilcoxon matched pairs test). Representative flow cytometry plots gated on CD49a± and CXCR6± NK cells showing expression of CD16, CD57, CD69, NKG2C, and KIR. (f) Percentage of IFNγ+ (n = 7) and TNFα+ (n = 6) NK cells within the hepatic CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, and CD49a‐CXCR6− NK cell populations following stimulation with IL‐12 10 ng/ml and IL‐15 1 ng/ml for 12 h, respectively. Dot plots display individual values and median. (Wilcoxon matched pairs test). Representative flow cytometry plots gated on CD49a± and CXCR6± NK cells showing IFNγ and TNFα production. p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001****.
Figure 2
Figure 2
RNA‐sequencing of CXCR6+ and CXCR6‐ NK cells isolated from the liver perfusate from three patients, resulting in six paired samples (s77 + s78, s102 + s107, s108 + s109). All three patients had undergone resection for colorectal metastases and had either a normal background liver or mild steatosis. Genes with a p value (adjusted for multiple comparisons according to Benjamini Hochberg) of <0.05 were analysed using: (a) Euclidian distance matrix displaying the overall similarity between samples, (b) a heat map displaying the top 75 differentially expressed genes in CXCR6+ and CXCR6− NK cells, and (c) differential expression of other selected genes of interest between CXCR6+ and CXCR6− NK cells.
Figure 3
Figure 3
(a) Representative flow cytometry plots gated on NK cells, individual frequencies shown. (b) Percentage of CD49a+ and CXCR6+ NK cells in the peripheral blood at rest (day 0) and following incubation with IL‐2, IL‐12, IL‐15, IL‐18, and the cytokine cocktail (n = 8). Dot plots display individual values. (Wilcoxon matched pairs test). (c) Percentage of CD49a + CXCR6+ NK cells in the liver at rest (day 0) and following incubation with IL‐2, IL‐12, IL‐15, IL‐18, and a cytokine cocktail at day 6. Median percentages are shown. Dot plots display individual values. (d) Day 6 CFSE MFI of hepatic CD49a+ vs. CXCR6+ NK cells following culture with IL‐2, IL‐12, IL‐15, IL‐18, and the cytokine cocktail (n = 8). Dot plots display individual values and median (Wilcoxon matched pairs test). Representative flow cytometry histograms from one individual showing CFSE MFI of CD49a+ and CXCR6+ NK cells at day 6 following culture with IL‐15. p < 0.05*, p < 0.01**.
Figure 4
Figure 4
(a) Representative flow cytometry plots gated on NK cells, individual frequencies shown. (b) Percentage of CD49a+ and CXCR6+ NK cells in the peripheral blood at rest (day 0) and following incubation with IL‐2, IL‐12, IL‐15, IL‐18, and the cytokine cocktail (n = 9). Dot plots display individual values. (Wilcoxon matched pairs test). (c) Absolute number of CD49a± and CXCR6± NK cells at rest (day 0) and following incubation with IL‐2, IL‐12, IL‐15, IL‐18, and the cytokine cocktail. (n = 9). Dot plots display individual values. Median absolute cell numbers shown in table below. (d) Percentage of CD49a + CXCR6+ NK cells in the peripheral blood at rest (day 0) and following incubation with IL‐2, IL‐12, IL‐15, IL‐18, and a cytokine cocktail at day 6. Median percentages are shown. Dot plots display individual values. (e) Day six CFSE MFI of CD49a+ vs CXCR6+ NK cells in the peripheral blood following culture with IL‐2, IL‐12, IL‐15, IL‐18, and the cytokine cocktail (n = 8). Dot plots display individual values and median (Wilcoxon matched pairs test). Representative flow cytometry histograms from one individual showing CFSE MFI of CD49a+ and CXCR6+ NK cells at day 6 following culture with IL‐15. (f) Frequency of CD49a+ and CXCR6+ NK cells at rest and following a 12 h culture with media only, IL‐12 10 ng/ml, or IL‐15 25 ng/ml using purified NK cells (n = 12). Dot plots display median. (Wilcoxon matched pairs test). p < 0.05*, p < 0.01**, p < 0.001***.
Figure 5
Figure 5
(a) A comparison of the frequencies of CD69+ (n = 11 liver, n = 6 cytokine‐induced) and NKG2C+ (n = 21 liver, n = 8 cytokine‐induced) NK cells between CD49a + CXCR6+ populations isolated from the liver and those generated in the peripheral blood following 6 days of culture with IL‐15. Dot plots display individual values and median. (Mann Whitney U test). (b) A comparison of the frequencies of CD56bright (n = 8), CD69+ (n = 6), and NKG2C+ (n = 8) NK cells within CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, CD49a‐CXCR6− NK subsets generated in the peripheral blood following 6 days of culture with IL‐15. Dot plots display individual values and median. (Wilcoxon matched pairs test). (c) A comparison of the frequency of IFNγ+ NK cells (n = 7 liver, n = 15 cytokine‐induced) between CD49a + CXCR6+ populations isolated from the liver and those generated in the peripheral blood following stimulation with IL‐12 for 12 h. Dot plots display individual values and median. (Mann Whitney U test). (d) A comparison of the frequency of IFNγ+ NK cells (n = 15) between CD49a + CXCR6+, CD49a + CXCR6−, CD49a‐CXCR6+, CD49a‐CXCR6− NK subsets generated in the peripheral blood following stimulation with IL‐12 for 12 h. Dot plots display individual values and median. (Wilcoxon matched pairs test). p < 0.05*, p < 0.001***.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Norris, S. , Collins C., Doherty D. G., Smith F., McEntee G., Traynor O., Nolan N., Hegarty J., and O'Farrelly C.. 1998. Resident human hepatic lymphocytes are phenotypically different from circulating lymphocytes. J. Hepatol. 28(1):84–90. - PubMed
    1. Doherty, D. G. , and O'Farrelly C.. 2000. Innate and adaptive lymphoid cells in the human liver. Immunol. Rev. 174:5–20. - PubMed
    1. Khakoo, S. I. , Thio C. L., Martin M. P., Brooks C. R., Gao X., Astemborski J., Cheng J., Goedert J. J., Vlahov D., Hilgartner M., et al. 2004. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 305(5685):872–874. - PubMed
    1. Knapp, S. , Warshow U., Hegazy D., Brackenbury L., Guha I. N., Fowell A., Little A. M., Alexander G. J., Rosenberg W. M., Cramp M. E., et al. 2010. Consistent beneficial effects of killer cell immunoglobulin‐like receptor 2DL3 and group 1 human leukocyte antigen‐C following exposure to hepatitis C virus. Hepatology 51(4):1168–1175. - PMC - PubMed
    1. Vidal‐Castiñeira, J. R. , López‐Vázquez A., Díaz‐Peña R., Alonso‐Arias R., Martínez‐Borra J., Pérez R., Fernández‐Suárez J., Melón S., Prieto J., Rodrigo L., et al. 2010. Effect of killer immunoglobulin‐like receptors in the response to combined treatment in patients with chronic hepatitis C virus infection. J. Virol. 84(1):475–481. - PMC - PubMed

Publication types

LinkOut - more resources