10 research outputs found
Identification of Human NK17/NK1 Cells
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-c. 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-c, 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-c. 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-c and are consequently named NK17/NK1 cells. They also express CD161, NKp30, NKp44, NKp46, NKG2D, CD158, CCL22, IL-2Rb and the common c chain but not CD127 or IL-23R. Further, they possess T-bet and RORct transcription factors. Antibodies to IL-1b, IL-6, IL-21, or TGF-b1 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
Methodology for modeling of city sustainable development based on fuzzy logic: a practical case
IL-2 induces the polarization of NK17/NK1 cells.
<p>A) Supernatants were collected from different cell numbers (5×10<sup>5</sup> and 1×10<sup>6</sup>/mL) of CD56<sup>+</sup>CCR4<sup>+</sup> cells, and examined for the levels of IL-17 and IFN-γ by ELISA. Mean±SEM of three experiments. B) Enriched CD56<sup>+</sup> NK cells were incubated with IL-2 only (no treatment), or with IL-2 plus neutralizing antibodies to IL-1β, IL-6, IL-21, TGF-β1, or a combination of isotype control goat and mouse antibodies for 6 days. The cells were collected, and labeled with FITC-conjugated anti-CCR4 and intracellularly with PE-conjugated antibodies to IL-17 and IFN-γ. Mean±SEM of positive cells collected from the blood of three donors. C) CD56<sup>+</sup> NK cells were labeled with isotype control (not shown) or FITC-conjugated anti-CCR4 and PE-conjugated anti-IL-2Rβ antibodies or intracellularly with PE-conjugated anti-common γ chain (IL-2Rγ). Numbers show the percentages of positive cells. D) CD56<sup>+</sup> NK cells were incubated overnight with 100 ng/mL PMA plus ionomycin. FITC-conjugated CCR4 cells were gated and examined for the production of IL-17 and IFN-γ. Labeling with isotype control antibodies is also shown. Numbers show the percentages of positive cells.</p
NK17/NK1 cells are abundant in the CSF of MS patients.
<p>A) NK cells were isolated from the blood of MS patients. CD56<sup>+</sup> 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<sup>+</sup>CCR4<sup>+</sup> 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<sup>+</sup> 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<sup>-</sup> 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.</p
MS patients examined in this study.
<p>PB = peripheral blood. CSF = cerebrospinal fluid. RRMS = relapsing-remitting MS.</p
Phenotypes of CD56<sup>+</sup> cells.
<p>IL-2-activated CD56<sup>+</sup> cells were isolated by EasySep human CD56 positive selection kit. They were examined for the expression of CD3, CD14, CD19 and CD56. Background control using isotype antibodies are also shown. Numbers indicate the percentage of positive cells. One of 5 experiments performed.</p
Only CD56<sup>+</sup>CCR4<sup>+</sup> produce IL-17 and IFN-γ.
<p>CD56<sup>+</sup> 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.</p