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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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NK17/NK1 cells are abundant in the CSF of MS patients.A) NK cells were isolated from the blood of MS patients. CD56+ cells were isolated and labeled with FITC-conjugated anti-CCR4 and intracellularly with PE-conjugated anti-IL-17 and anti-IFN-γ. Each dot represents the percentage of CD56+CCR4+ cells producing IL-17 and IFN-γ from an individual patient before and after activation with IL-2. B) CSF from two MS patients (P1 and P2) were sorted into CD56+ NK cells and then labeled with FITC-conjugated anti-CCR4, PE-conjugated anti-IL-17, and PE-conjugated anti-IFN-γ. Numbers show percentages of cells producing IL-17 and IFN-γ. C) This is similar to panel B, except that CD56- NK cells isolated from the same MS patients were examined. D) Cells from the CSF of a third MS patient were labeled with isotype control (not shown) or FITC-conjugated anti-CCR4 antibody and intracellularly with isotype control (not shown), or PE-conjugated anti-FITC and APC-conjugated anti-IL-17 antibodies. FITC-conjugated cells were gated (more than 99% positive for CCR4 expression), and examined for the production of intracellular cytokines. Numbers show the percentage of positive cells.
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pone-0026780-g005: NK17/NK1 cells are abundant in the CSF of MS patients.A) NK cells were isolated from the blood of MS patients. CD56+ cells were isolated and labeled with FITC-conjugated anti-CCR4 and intracellularly with PE-conjugated anti-IL-17 and anti-IFN-γ. Each dot represents the percentage of CD56+CCR4+ cells producing IL-17 and IFN-γ from an individual patient before and after activation with IL-2. B) CSF from two MS patients (P1 and P2) were sorted into CD56+ NK cells and then labeled with FITC-conjugated anti-CCR4, PE-conjugated anti-IL-17, and PE-conjugated anti-IFN-γ. Numbers show percentages of cells producing IL-17 and IFN-γ. C) This is similar to panel B, except that CD56- NK cells isolated from the same MS patients were examined. D) Cells from the CSF of a third MS patient were labeled with isotype control (not shown) or FITC-conjugated anti-CCR4 antibody and intracellularly with isotype control (not shown), or PE-conjugated anti-FITC and APC-conjugated anti-IL-17 antibodies. FITC-conjugated cells were gated (more than 99% positive for CCR4 expression), and examined for the production of intracellular cytokines. Numbers show the percentage of positive cells.

Mentions: Both Th1 secreting IFN-γ and Th17 secreting IL-17 contribute to the pathogenesis of MS and EAE. Consequently, we examined the presence of NK17/NK1 cells in the blood of patients with MS. The results from five different patients show that NK17/NK1 cells were not spontaneously found in the blood, but were generated from the peripheral blood of four patients examined upon activation with IL-2, although their frequencies were less than in normal blood (Figure 5A). Of note, blood samples were collected from MS patients age 25–53 years old, which were not different from samples collected from healthy donors. We have also included CSF from MS patients due to the possibility that this may give us an idea about the role these cells might play in a diseased organ. After isolation of non-activated CD56+ cells from the CSF of two MS patients, CCR4+ cells secreting both IL-17 and IFN-γ were abundant (Figure 5B). The frequency of CD56+CCR4+ cells (NK17/NK1 cells) was about ten-fold higher than CD56−CCR4+ cells found in the CSF (Figure 5B vs. 5C), and more than twenty-fold the numbers of non-activated CD56+CCR4+ found in the peripheral blood of MS patients (Figure 5B vs. 5A). Also, we managed to obtain enough cells from the CSF of a third MS patient which were labeled with FITC-conjugated anti-CCR4 and intracellularly with PE-conjugated anti-IFN-γ and APC-conjugated anti-IL-17. The results in Figure 5D demonstrate that about 25% of CCR4+ NK cells produced both IL-17 and IFN-γ. It is highly plausible that NK17/NK1 cells may migrate from the periphery into the CSF aided by the CCL22/CCR4 axis, and are polarized in the brain due to inflamed local microenvironment that may contribute to their generation. CSF of normal individuals was not collected due to ethical considerations. However, NK cells are either not found or found in very low numbers in the brain of normal mice [24].


Identification of human NK17/NK1 cells.

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

NK17/NK1 cells are abundant in the CSF of MS patients.A) NK cells were isolated from the blood of MS patients. CD56+ cells were isolated and labeled with FITC-conjugated anti-CCR4 and intracellularly with PE-conjugated anti-IL-17 and anti-IFN-γ. Each dot represents the percentage of CD56+CCR4+ cells producing IL-17 and IFN-γ from an individual patient before and after activation with IL-2. B) CSF from two MS patients (P1 and P2) were sorted into CD56+ NK cells and then labeled with FITC-conjugated anti-CCR4, PE-conjugated anti-IL-17, and PE-conjugated anti-IFN-γ. Numbers show percentages of cells producing IL-17 and IFN-γ. C) This is similar to panel B, except that CD56- NK cells isolated from the same MS patients were examined. D) Cells from the CSF of a third MS patient were labeled with isotype control (not shown) or FITC-conjugated anti-CCR4 antibody and intracellularly with isotype control (not shown), or PE-conjugated anti-FITC and APC-conjugated anti-IL-17 antibodies. FITC-conjugated cells were gated (more than 99% positive for CCR4 expression), and examined for the production of intracellular cytokines. Numbers show the percentage of positive cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198820&req=5

pone-0026780-g005: NK17/NK1 cells are abundant in the CSF of MS patients.A) NK cells were isolated from the blood of MS patients. CD56+ cells were isolated and labeled with FITC-conjugated anti-CCR4 and intracellularly with PE-conjugated anti-IL-17 and anti-IFN-γ. Each dot represents the percentage of CD56+CCR4+ cells producing IL-17 and IFN-γ from an individual patient before and after activation with IL-2. B) CSF from two MS patients (P1 and P2) were sorted into CD56+ NK cells and then labeled with FITC-conjugated anti-CCR4, PE-conjugated anti-IL-17, and PE-conjugated anti-IFN-γ. Numbers show percentages of cells producing IL-17 and IFN-γ. C) This is similar to panel B, except that CD56- NK cells isolated from the same MS patients were examined. D) Cells from the CSF of a third MS patient were labeled with isotype control (not shown) or FITC-conjugated anti-CCR4 antibody and intracellularly with isotype control (not shown), or PE-conjugated anti-FITC and APC-conjugated anti-IL-17 antibodies. FITC-conjugated cells were gated (more than 99% positive for CCR4 expression), and examined for the production of intracellular cytokines. Numbers show the percentage of positive cells.
Mentions: Both Th1 secreting IFN-γ and Th17 secreting IL-17 contribute to the pathogenesis of MS and EAE. Consequently, we examined the presence of NK17/NK1 cells in the blood of patients with MS. The results from five different patients show that NK17/NK1 cells were not spontaneously found in the blood, but were generated from the peripheral blood of four patients examined upon activation with IL-2, although their frequencies were less than in normal blood (Figure 5A). Of note, blood samples were collected from MS patients age 25–53 years old, which were not different from samples collected from healthy donors. We have also included CSF from MS patients due to the possibility that this may give us an idea about the role these cells might play in a diseased organ. After isolation of non-activated CD56+ cells from the CSF of two MS patients, CCR4+ cells secreting both IL-17 and IFN-γ were abundant (Figure 5B). The frequency of CD56+CCR4+ cells (NK17/NK1 cells) was about ten-fold higher than CD56−CCR4+ cells found in the CSF (Figure 5B vs. 5C), and more than twenty-fold the numbers of non-activated CD56+CCR4+ found in the peripheral blood of MS patients (Figure 5B vs. 5A). Also, we managed to obtain enough cells from the CSF of a third MS patient which were labeled with FITC-conjugated anti-CCR4 and intracellularly with PE-conjugated anti-IFN-γ and APC-conjugated anti-IL-17. The results in Figure 5D demonstrate that about 25% of CCR4+ NK cells produced both IL-17 and IFN-γ. It is highly plausible that NK17/NK1 cells may migrate from the periphery into the CSF aided by the CCL22/CCR4 axis, and are polarized in the brain due to inflamed local microenvironment that may contribute to their generation. CSF of normal individuals was not collected due to ethical considerations. However, NK cells are either not found or found in very low numbers in the brain of normal mice [24].

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