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Identification of human NK17/NK1 cells.

Pandya AD, Al-Jaderi Z, Høglund RA, Holmøy T, Harbo HF, Norgauer J, Maghazachi AA - PLoS ONE (2011)

Bottom Line: They also express CD161, NKp30, NKp44, NKp46, NKG2D, CD158, CCL22, IL-2Rβ and the common γ chain but not CD127 or IL-23R.Antibodies to IL-1β, IL-6, IL-21, or TGF-β1 do not inhibit IL-2-induced generation of NK17/NK1 cells, suggesting that IL-2 has the capacity to polarize these cells.NK17/NK1 cells identified here have not been previously described in healthy or MS patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.

ABSTRACT

Background: Natural killer (NK) cells have both cytolytic and immunoregulatory functions. We recently described that these cells release the inflammatory cytokines IL-17 and IFN-γ. However, the precise identity of the NK cell subset(s) that secrete these cytokines is not known.

Methodology/principal findings: To isolate the cells secreting IL-17 and IFN-γ, we took advantage of the findings that Th17/Th1 cells express chemokine receptors. Therefore, CD56(+)NK cells were stained with antibodies against various chemokine receptors and intracellularly with antibodies toward IL-17 and IFN-γ. Consequently, we identified previously unrecognized subset of NK cells generated from normal human peripheral blood after activation with IL-2 but not PMA plus ionomycin. The cells are characterized by the expression of CD56(+) and CCR4(+), produce IL-17 and IFN-γ and are consequently named NK17/NK1 cells. They also express CD161, NKp30, NKp44, NKp46, NKG2D, CD158, CCL22, IL-2Rβ and the common γ chain but not CD127 or IL-23R. Further, they possess T-bet and RORγt transcription factors. Antibodies to IL-1β, IL-6, IL-21, or TGF-β1 do not inhibit IL-2-induced generation of NK17/NK1 cells, suggesting that IL-2 has the capacity to polarize these cells. Notably, NK17/NK1 cells are abundant in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) without activation, and are generated from the peripheral blood of these patients after activation with IL-2.

Conclusions/significance: NK17/NK1 cells identified here have not been previously described in healthy or MS patients.

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Related in: MedlinePlus

Only CD56+CCR4+ produce IL-17 and IFN-γ.CD56+ NK cells were labeled with FITC-conjugated isotype control antibodies or FITC-conjugated anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR9 or anti-CXCR4 antibodies. They were also labeled intracellularly with PE-conjugated and APC-conjugated isotype control antibodies, or PE-conjugated anti- IFN-γ and APC-conjugated anti-IL-17 antibodies. FITC-conjugated cells were gated (more than 99% pure for the expression of a particular chemokine receptor) and examined for the production of IL-17 and IFN-γ. Labeling with isotype control antibodies is also shown. Numbers show the percentage of positive cells. This is a representative experiment of five different donors.
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pone-0026780-g002: Only CD56+CCR4+ produce IL-17 and IFN-γ.CD56+ NK cells were labeled with FITC-conjugated isotype control antibodies or FITC-conjugated anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR9 or anti-CXCR4 antibodies. They were also labeled intracellularly with PE-conjugated and APC-conjugated isotype control antibodies, or PE-conjugated anti- IFN-γ and APC-conjugated anti-IL-17 antibodies. FITC-conjugated cells were gated (more than 99% pure for the expression of a particular chemokine receptor) and examined for the production of IL-17 and IFN-γ. Labeling with isotype control antibodies is also shown. Numbers show the percentage of positive cells. This is a representative experiment of five different donors.

Mentions: Since NK cells have been shown to secrete these cytokines upon IL-2 activation [11], [12], we activated purified NK cells in vitro with IL-2 for 7 days and then sorted them into CD56+ and CD56− subsets. First, we ascertained that the CD56+ NK cells are pure since they were stained with anti-CD56 and not with anti-CD3 “T cells”, anti-CD14 “monocytes”, or anti-CD19 “B cells” (Figure 1). Cells of both CD56+ and CD56− subsets were labeled with surface anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR9, or anti-CXCR4 and intracellularly with antibodies toward IL-17 and IFN-γ. There was an upregulation of IL-17 and IFN-γ in CCR4+CD56+ and not CD56− cells (data not shown). Based on these preliminary findings, we examined IL-2 activated CD56+ cells labeled with antibody towards CCR4, as well as with anti-CCR6, anti-CCR7, anti-CCR9 or anti-CXCR4. As shown in Figure 2, about 25%, 14%, 5%, 4% and 11% of IL-2 activated CD56+ NK cells were stained with FITC-conjugated anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR9 and anti-CXCR4, respectively, whereas isotype control antibodies did not label these cells. FITC-labeled cells were gated (more than 99% pure), and stained intracellularly with PE-conjugated anti-IFN-γ and APC-conjugated anti-IL-17 or with isotype controls PE-conjugated and APC-conjugated antibodies. The results demonstrate that only CCR4+ and not CCR6+, CCR7+, CCR9+ or CXCR4+ NK cells were labeled intracellularly with antibodies to both cytokines (Figure 2). These results indicate that cells contained within the CD56+CCR4+ NK cell subset are primary targets for polarization into cells producing IL-17 and IFN-γ, designated here as NK17/NK1 synonymous with T cell terminology [14]. In addition to flow cytometric analysis, we measured the levels of these cytokine in the supernatants of NK17/NK1 cells. Results in Figure 3A show that high levels of both cytokines are released from 5×105/mL or 1×106/mL CD56+CCR4+ cells.


Identification of human NK17/NK1 cells.

Pandya AD, Al-Jaderi Z, Høglund RA, Holmøy T, Harbo HF, Norgauer J, Maghazachi AA - PLoS ONE (2011)

Only CD56+CCR4+ produce IL-17 and IFN-γ.CD56+ NK cells were labeled with FITC-conjugated isotype control antibodies or FITC-conjugated anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR9 or anti-CXCR4 antibodies. They were also labeled intracellularly with PE-conjugated and APC-conjugated isotype control antibodies, or PE-conjugated anti- IFN-γ and APC-conjugated anti-IL-17 antibodies. FITC-conjugated cells were gated (more than 99% pure for the expression of a particular chemokine receptor) and examined for the production of IL-17 and IFN-γ. Labeling with isotype control antibodies is also shown. Numbers show the percentage of positive cells. This is a representative experiment of five different donors.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198820&req=5

pone-0026780-g002: Only CD56+CCR4+ produce IL-17 and IFN-γ.CD56+ NK cells were labeled with FITC-conjugated isotype control antibodies or FITC-conjugated anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR9 or anti-CXCR4 antibodies. They were also labeled intracellularly with PE-conjugated and APC-conjugated isotype control antibodies, or PE-conjugated anti- IFN-γ and APC-conjugated anti-IL-17 antibodies. FITC-conjugated cells were gated (more than 99% pure for the expression of a particular chemokine receptor) and examined for the production of IL-17 and IFN-γ. Labeling with isotype control antibodies is also shown. Numbers show the percentage of positive cells. This is a representative experiment of five different donors.
Mentions: Since NK cells have been shown to secrete these cytokines upon IL-2 activation [11], [12], we activated purified NK cells in vitro with IL-2 for 7 days and then sorted them into CD56+ and CD56− subsets. First, we ascertained that the CD56+ NK cells are pure since they were stained with anti-CD56 and not with anti-CD3 “T cells”, anti-CD14 “monocytes”, or anti-CD19 “B cells” (Figure 1). Cells of both CD56+ and CD56− subsets were labeled with surface anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR9, or anti-CXCR4 and intracellularly with antibodies toward IL-17 and IFN-γ. There was an upregulation of IL-17 and IFN-γ in CCR4+CD56+ and not CD56− cells (data not shown). Based on these preliminary findings, we examined IL-2 activated CD56+ cells labeled with antibody towards CCR4, as well as with anti-CCR6, anti-CCR7, anti-CCR9 or anti-CXCR4. As shown in Figure 2, about 25%, 14%, 5%, 4% and 11% of IL-2 activated CD56+ NK cells were stained with FITC-conjugated anti-CCR4, anti-CCR6, anti-CCR7, anti-CCR9 and anti-CXCR4, respectively, whereas isotype control antibodies did not label these cells. FITC-labeled cells were gated (more than 99% pure), and stained intracellularly with PE-conjugated anti-IFN-γ and APC-conjugated anti-IL-17 or with isotype controls PE-conjugated and APC-conjugated antibodies. The results demonstrate that only CCR4+ and not CCR6+, CCR7+, CCR9+ or CXCR4+ NK cells were labeled intracellularly with antibodies to both cytokines (Figure 2). These results indicate that cells contained within the CD56+CCR4+ NK cell subset are primary targets for polarization into cells producing IL-17 and IFN-γ, designated here as NK17/NK1 synonymous with T cell terminology [14]. In addition to flow cytometric analysis, we measured the levels of these cytokine in the supernatants of NK17/NK1 cells. Results in Figure 3A show that high levels of both cytokines are released from 5×105/mL or 1×106/mL CD56+CCR4+ cells.

Bottom Line: They also express CD161, NKp30, NKp44, NKp46, NKG2D, CD158, CCL22, IL-2Rβ and the common γ chain but not CD127 or IL-23R.Antibodies to IL-1β, IL-6, IL-21, or TGF-β1 do not inhibit IL-2-induced generation of NK17/NK1 cells, suggesting that IL-2 has the capacity to polarize these cells.NK17/NK1 cells identified here have not been previously described in healthy or MS patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.

ABSTRACT

Background: Natural killer (NK) cells have both cytolytic and immunoregulatory functions. We recently described that these cells release the inflammatory cytokines IL-17 and IFN-γ. However, the precise identity of the NK cell subset(s) that secrete these cytokines is not known.

Methodology/principal findings: To isolate the cells secreting IL-17 and IFN-γ, we took advantage of the findings that Th17/Th1 cells express chemokine receptors. Therefore, CD56(+)NK cells were stained with antibodies against various chemokine receptors and intracellularly with antibodies toward IL-17 and IFN-γ. Consequently, we identified previously unrecognized subset of NK cells generated from normal human peripheral blood after activation with IL-2 but not PMA plus ionomycin. The cells are characterized by the expression of CD56(+) and CCR4(+), produce IL-17 and IFN-γ and are consequently named NK17/NK1 cells. They also express CD161, NKp30, NKp44, NKp46, NKG2D, CD158, CCL22, IL-2Rβ and the common γ chain but not CD127 or IL-23R. Further, they possess T-bet and RORγt transcription factors. Antibodies to IL-1β, IL-6, IL-21, or TGF-β1 do not inhibit IL-2-induced generation of NK17/NK1 cells, suggesting that IL-2 has the capacity to polarize these cells. Notably, NK17/NK1 cells are abundant in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) without activation, and are generated from the peripheral blood of these patients after activation with IL-2.

Conclusions/significance: NK17/NK1 cells identified here have not been previously described in healthy or MS patients.

Show MeSH
Related in: MedlinePlus