Liver-resident NK cells confer adaptive immunity in skin-contact inflammation - PubMed (original) (raw)

. 2013 Apr;123(4):1444-56.

doi: 10.1172/JCI66381. Epub 2013 Mar 25.

Affiliations

Liver-resident NK cells confer adaptive immunity in skin-contact inflammation

Hui Peng et al. J Clin Invest. 2013 Apr.

Abstract

Liver natural killer (NK) cells were recently reported to possess memory-like properties in contact hypersensitivity (CHS) models. However, the phenotype and origin of these "memory" NK cells cannot be distinguished from other NK cell subpopulations. Here, we define the transcriptional, phenotypic, and functional features of liver NK cell subsets and their roles in mediating CHS. Liver NK cells can be divided into two distinct subsets: CD49a+DX5- and CD49a-DX5+. Substantial transcriptional and phenotypic differences existed between liver CD49a+DX5- NK cells and other NK cell subsets. CD49a+DX5- NK cells possessed memory potential and conferred hapten-specific CHS responses upon hapten challenge. Importantly, CD49a+DX5- NK cells were liver resident and were present in the liver sinusoidal blood, but not the afferent and efferent blood of the liver. Moreover, they appeared to originate from hepatic hematopoietic progenitor/stem cells (HPCs/HSCs) but not from the bone marrow, and maintained their phenotypes in the steady state. Our findings of liver-resident NK cells shed new light on the acquisition of memory-like properties of NK cells.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Hepatic DX5– NK cells confer memory responses in CHS.

(A) Expression of DX5 versus NK1.1 was analyzed on CD122+CD3–CD19– cells from liver, spleen, peripheral blood, BM, and iLNs of WT B6 mice. Representative FACS plots are shown. (B) The percentages of DX5– NK cells among NK (CD122+NK1.1+CD3–CD19–) cells in different organs are shown (n = 7). (C) Ear swelling in naive B6 mice that received 8 × 104 DX5+ (n = 6) or DX5– (n = 4) NK (NK1.1+ CD3– CD19–) cells from OXA-sensitized _Rag1_–/– donor liver. Recipients were challenged 1 month later with OXA on 1 ear and solvent on the other. (D) Ear swelling in naive B6 mice that received 8 × 104 hepatic NK cell subsets (n = 3 per group) or splenic NK cells (n = 6) from OXA-sensitized WT mice and were challenged 1 month later. (C and D) Data are from 1 experiment representative of at least 2 independent experiments. **P < 0.01; ***P < 0.001. Unpaired Student’s t test (C) or ANOVA (D). Means ± SEM are shown in B–D.

Figure 2

Figure 2. Hepatic NK cells with memory capacity remain DX5– during hapten sensitization.

(A) DX5– or DX5+ NK (NK1.1+CD3–CD19–) cells (105) from naive GFP transgenic mice were adoptively transferred into lethally irradiated recipients, which were sensitized with 5% OXA on days 1 and 2. On day 5, liver MNCs from recipients or control irradiated mice that did not receive any cells were adoptively transferred into secondary recipients that were challenged 1 month later. (B) DX5 expression on GFP+ NK cells in the irradiated recipients that received GFP+DX5– (top panel) or GFP+DX5+ (bottom panel) NK cells was analyzed on day 5 before transfer. (C) Ear swelling of secondary recipients described in A was measured after OXA challenge (n = 5 per group). ***P < 0.001, ANOVA. Means ± SEM are shown. Hepatocytes (D) or F4/80+ Kupffer cells (E) in the liver of sensitized (blue) or control (gray shaded) WT mice were respectively isolated by a 2-step collagenase perfusion method, then gated and analyzed for FITC expression; the statistical results show FITC MFI on sensitized (n = 6) or control (n = 2) hepatocytes. *P < 0.05, unpaired Student’s t test. Means ± SEM are shown. (F) FITC+ cells in _Rag1_–/– mice 24 hours after sensitization of FITC on 2 consecutive days. _Rag1_–/– mice without sensitization were set as controls. FITC+ cells among liver MNCs from sensitized mice were analyzed for the expression of indicated markers. (B–F) Data are representative of 2 or 3 independent experiments. SSC, side scatter.

Figure 3

Figure 3. Hepatic DX5– NK cells preferentially traffic to the liver and maintain their phenotypic features after adoptive transfer.

(A) Liver MNCs (106) from CD45.1+ mice were adoptively transferred intravenously into sublethally irradiated (IR) CD45.2+ B6 mice. Twenty-four hours after transfer, CD45.1+ NK (NK1.1+CD3–CD19–) cells were analyzed for DX5 expression in recipient liver, spleen, and BM. (B) DX5– or DX5+ liver NK cells (105) were sorted from CD45.1+ mice and intravenously transferred into sublethally irradiated CD45.2+ B6 mice. DX5 expression of donor NK cells before transfer is shown pre- and post-sort. Seven days after transfer, CD45.1+ NK cells were analyzed for DX5 expression in recipient liver and spleen. All data in this figure are representative of 2 independent experiments.

Figure 4

Figure 4. Hepatic DX5– NK cells are distinct from other NK cell populations.

(A) Comparative transcriptome analysis between DX5– and DX5+ liver NK (NK1.1+CD3–) cells was performed by Affymetrix GeneChip Mouse Genome 430 2.0 arrays. Genes that were expressed greater than or equal to 2-fold higher or lower than DX5+ NK cells are highlighted in black. The number of genes upregulated or downregulated for each comparison is indicated. (B) Differentially expressed genes in DX5– and DX5+ NK cells were selected and classified into different groups. The listed genes had greater than or equal to 2-fold differences and are shown with their signal values. (C) Distribution by functional category of upregulated genes in hepatic DX5+ and DX5– NK cell subsets. Genes with greater than or equal to 2-fold differences were included. (D) Expression of CD107a and IFN-γ is shown for stimulated DX5– and DX5+ liver NK (NK1.1+CD3–CD19–) cells. Liver MNCs were stimulated with IL-2 and IL-12 (for IFN-γ analysis) or with YAC-1 cells (for CD107a analysis). Blue lines represent staining of the indicated molecules, and gray-shaded curves represent isotype controls. (E) Statistical results of percentages of CD107a+ (n = 6) or IFN-γ+ (n = 11) cells are shown. *P < 0.05 and **P < 0.01, paired Student’s t test. Means ± SEM are shown.

Figure 5

Figure 5. CD49a is a specific surface marker of liver-resident DX5– NK cells.

(A) Expression of CD49a, NKp46, CD51, CD27, CXCR6, and Thy1.2 versus DX5 was analyzed on NK1.1+CD3–CD19– cells or NK1.1+CD3– cells from the indicated organs of WT or CXCR6+/– (for CXCR6 detection) mice. Data are representative of 4 to 6 individual mice. (B) DX5– or DX5+ liver NK (NK1.1+ CD3– CD19–) cells (105) were sorted from CD45.1+ mice and intravenously transferred into sublethally irradiated CD45.2+ B6 mice. Seven days later, NK cells from recipient liver were analyzed for the expression of CD45.1 and CD49a. Data are representative of 2 independent experiments. (C) Expression of CD49a was analyzed on NK cells from unmanipulated BALB/c and _Rag1_–/– mice. Numbers indicate the percentages of cells expressing CD49a among NKp46+CD3–CD19– (BALB/c) or NK1.1+ (_Rag1_–/–) cells. Blue lines represent staining of the indicated molecules, and gray-shaded curves represent isotype controls. Data are representative of 4 mice per group. (D) As described in Methods, blood was collected from the unfractionated liver and other indicated sites from WT B6 mice, and then MNCs were isolated. Expression of CD49a versus DX5 was analyzed on NK1.1+CD3–CD19– cells. Plots are representative of at least 5 individual mice. (E) Immunofluorescence histology of frozen sections of mouse liver stained with anti-NKp46 (green), anti-CD31 (blue), anti-CD49a (red), or anti-CD49b (DX5; red). Original magnification, ×250. (F) Histological analysis of _Rag1_–/– mouse liver stained with anti-CD49a (brown) and NK1.1 (purple). Original magnification, ×320. (E and F) Data are representative of at least 2 independent experiments.

Figure 6

Figure 6. Minimal exchange of liver CD49a+DX5– NK cells in parabiotic mice.

WT B6 (CD45.2) mice were parabiosed to congenic B6-CD45.1 mice. At day 14 postsurgery, the spleen and liver were harvested and flow cytometry was performed. (A) Representative dot plot of the liver and spleen gated on live CD3–NK1.1+ cells followed by a CD45.1 gate (left panels) and CD45.2 gate (right panels) for each parabiont as indicated. The percentages of CD49a+DX5– and CD49a–DX5+ cells are shown in the dot plots. (B) The percentages of CD49a+DX5– and CD49a–DX5+ cells in the liver and spleen from A are depicted in the stacked bar graph which represents 7 parabiotic pairs. The CD49a+DX5– NK cells are indicated by the solid shading, and the CD49a–DX5+ NK cells are indicated by the open box, with each column representing the indicated parabiont, according to the CD45.1+ or CD45.2+ gate.

Figure 7

Figure 7. CD49a+ NK cells have the capacity to confer hapten-specific CHS responses.

(A) Naive B6 mice received 8 × 104 CD49a+ or CD49a– liver NK cells from OXA-sensitized WT mice and were challenged 1 month later (n = 4 per group). (B) Naive B6 mice received the indicated number of CD49a+ liver NK cells from OXA-sensitized WT mice and were challenged 1 month later (n = 3 per group). (C and D) Naive B6 mice received 8 × 104 CD49a+ or CD49a– liver NK cells from OXA–-sensitized (C), or FITC–-sensitized (D) WT mice and were challenged with OXA or FITC 1 month later as indicated (n = 4 for the FITC plus OXA group; n = 5 for the other groups). All data are from 1 experiment representative of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, unpaired Student’s t test (A, C, and D), or ANOVA (B). Means ± SEM of ear swelling are shown in all panels.

Figure 8

Figure 8. The liver retains HPCs capable of generating CD49a+DX5– NK cells.

(A) Lethally irradiated CD45.2+ B6 mice that received 2 × 106 BM cells from CD45.1+ B6 mice were analyzed for the expression of DX5 on hepatic CD45.1+ NK (NK1.1+CD3–CD19–) cells. Means ± SEM are shown (n = 3 for each time point). (B) Survival of lethally irradiated B6 mice that received 3 × 106 BM cells (BMT), liver MNCs (LT), or splenocytes (ST). Lethally irradiated mice without transplantation were set as the control group. Means ± SEM are shown (n = 5 per group). (C and D) Lethally irradiated CD45.2+ B6 mice received 106 CD45.1+CD45.2+ (DP) BM cells mixed with 106 CD45.1+CD45.2– (SP) liver MNCs. One month after transfer, DP and SP cells in the recipient livers were respectively gated to analyze the expression of DX5 on NK cells. Means ± SEM are shown (n = 3 per group). (E) BM cells (2 × 106) from Nfil3–/– mice mixed with 105 hepatic NK1.1–CD3–CD19– cells from CD45.1+ B6 mice were transferred into lethally irradiated CD45.2+ B6 mice. One month after transfer, CD45.1+ NK cells in the recipient livers were analyzed for the expression of DX5 and CD49a. All data are representative of 2 independent experiments.

Similar articles

Cited by

References

    1. Vivier E, et al. Innate or adaptive immunity? The example of natural killer cells. Science. 2011;331:44–49. doi: 10.1126/science.1198687. - DOI - PMC - PubMed
    1. Raulet DH, Vance RE. Self-tolerance of natural killer cells. Nat Rev Immunol. 2006;6(7):520–531. doi: 10.1038/nri1863. - DOI - PubMed
    1. Lanier LL. Up on the tightrope: natural killer cell activation and inhibition. Nat Immunol. 2008;9(5):495–502. doi: 10.1038/ni1581. - DOI - PMC - PubMed
    1. Seaman WE, Blackman MA, Gindhart TD, Roubinian JR, Loeb JM, Talal N. beta-Estradiol reduces natural killer cells in mice. J Immunol. 1978;121(6):2193–2198. - PubMed
    1. Kumar V, Ben-Ezra J, Bennett M, Sonnenfeld G. Natural killer cells in mice treated with 89strontium: normal target-binding cell numbers but inability to kill even after interferon administration. J Immunol. 1979;123(4):1832–1838. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources