Journal: The Journal of Biological Chemistry

Article Title: Interferon-? and Granulocyte/Monocyte Colony-stimulating Factor Production by Natural Killer Cells Involves Different Signaling Pathways and the Adaptor Stimulator of Interferon Genes (STING)

doi: 10.1074/jbc.M112.435602

Figure Lengend Snippet: A, both accessory cytokines IL-15/IL-18 and CpG-ODN are necessary for NF-κB p65 nuclear translocation. Purified NK cells were primed overnight with IL-15 and IL-18 (10 ng/ml each) and then challenged with CpG-ODN (1 μm). Nuclear translocation of p65 was measured by immunofluorescence after 60-min stimulation. Hoeschst was used to stain the nucleus. B, to quantify the results shown in A, the percentage of nuclear translocation was determined by counting 100 cells. The figure is representative of three independent experiments. ***, p < 0.001 versus unstimulated cells (control) using one-way ANOVA and Fisher least significance difference test.

Article Snippet: The murine recombinant cytokines (IL-2, IL-15) were from Peprotech, and IL-18 was from Medical and Biological Laboratories.

Techniques: Translocation Assay, Purification, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Interferon-? and Granulocyte/Monocyte Colony-stimulating Factor Production by Natural Killer Cells Involves Different Signaling Pathways and the Adaptor Stimulator of Interferon Genes (STING)

doi: 10.1074/jbc.M112.435602

Figure Lengend Snippet: Purified spleen NK cells from WT and mice deficient for MyD88, TLR9, STING, TRIF, UNC93b1, IFN-α/βR, and IL-12RB2 were stimulated with accessory cytokines (IL-15 and IL-18 at 10 ng/ml each) and CpG-ODN (1 μm) for 48 h, and IFN-γ (A) and GM-CSF (B) were measured in the supernatants by ELISA. The MyD88 inhibitory peptide (MyD-Pep) was also applied on the sting−/− cells at 25 μm. Data are mean ± S.E. of five experiments. *, p < 0.05; *, p < 0.01; ***, p < 0.001 versus WT using one-way ANOVA and Fisher least significance difference test. C, purified NK cells were primed overnight with IL-15 and IL-18 (10 ng/ml each) and challenged with CpG-ODN (1 μm) in presence of GolgiStop. IL-12p40/70 was then stained intracellularly using specific antibodies and detected on the gated NKp46+ cells. The left histogram shows the IL-12p40/70 stain as percentage of positive cells, the middle graph shows the mean fluorescence intensity (MFI), and the right histograms show the expression of IL-12Rβ2 on the NK cells cultured in the same conditions. Data are the mean ± S.E. of five experiments. ***, p < 0.001 versus cells without stimulation (control) using one-way ANOVA and Fisher least significance difference test. D, purified NK cells were stimulated with IL-15/IL-18 and CpG-ODN labeled with A488. After 60 min, the cells were fixed, stained with an anti-STING antibody, and processed for confocal microscopy. The nucleus is visualized with Hoechst. Arrows indicate spots where an proximally overlapping was observed for cytosolic STING and speckled CpG-ODN stain.

Article Snippet: The murine recombinant cytokines (IL-2, IL-15) were from Peprotech, and IL-18 was from Medical and Biological Laboratories.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Expressing, Cell Culture, Labeling, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Subtype-specific atypical B cell profiles in myasthenia gravis reveal distinct immunopathological pathways

doi: 10.3389/fimmu.2025.1608160

Figure Lengend Snippet: CD11c + B cells differentiate into ASCs and produce autoantibodies in MuSK-MG. Isolated CD11c − and CD11c + B cells were stimulated with CD40L, IL-2, IL-12, and BAFF (n = 6 for each group) for 14 days. AChR-MG subjects were stratified based on disease onset (LOMG n = 2, EOMG n = 4). In LPS-treated wells, cells were stimulated with CD40L and cytokines (IL-2, IL-4, IL-21, and BAFF) for 14 days, with LPS added 48 hours prior to harvest (n = 3). (A) Representative flow cytometry plots of CD11c + B cells from a MuSK-MG subject. Antibody-secreting cells (ASCs) were identified as CD27 ++ CD38 ++ cells. (B) Quantification of CD11c − and CD11c + B cells that differentiated into + CD27 ++ CD38 ++ ASCs. (C) Total IgG production (ng/mL) in culture supernatants as measured by ELISA. (D) Radioimmunoassay measuring anti-AChR and anti-MuSK antibody levels in culture supernatants from CD11c + B cells (fmol/mL) stimulated with CD40L, IL-2, IL-4, IL-21 and BAFF for 14 days. Data are represented as mean ± SEM.

Article Snippet: 2x10 3 cells were plated with wells seeded with 3 x10 3 MS-40L lo cells (kindly provided by the Kelsoe Laboratory) per well and cultured in complete RPMI media in the presence of recombinant human cytokines IL-2 (50 ng/mL), IL-4 (10 ng/mL), IL-21(10 ng/mL) and BAFF (10 ng/mL) (all purchased from Peprotech) for 14 days (n = 6 per group).

Techniques: Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, RIA Assay

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: Human effector memory (EM) CD8 + T cell subsets differentially express Kv1.3 and KCa3.1. (A–C) Peripheral blood mononuclear cells from healthy individuals were stained with antibodies (Abs) to CD8, C-C chemokine receptor type 7 (CCR7), and interleukin (IL)-7Rα and sorted into IL-7Rα high and IL-7Rα low subsets of EM CD8 + T cell using a BD FACSAria ® . (A) Freshly sorted CD8 + T cell subsets were stimulated for 24 h with anti-CD3/CD28 Abs, and their KCa3.1 (IL-7Rα high , n = 23; IL-7Rα low , n = 23) and Kv1.3 (IL-7Rα high , n = 18; IL-7Rα low , n = 19) current components underlying the total current–voltage (I–V) curve were measured as follows: (i) the maximum inactivation of Kv1.3 was induced using depolarized holding voltage (−10 mV); (ii) a reverse ramp-like pulse from 60 to −120 mV was applied; and (iii) the obtained I–V curve for KCa3.1 was subtracted from the initial I–V curve obtained using a forward ramp-like pulse in the whole-cell configuration for the Kv1.3 current. (B) KCa3.1 was activated by subsequently adding 50 µM 1-ethyl-2-benzimidazolinone (1-EBIO) to verify the presence of functional KCa3.1 in IL-7Rα high ( n = 8) and IL-7Rα low ( n = 10) EM CD8 + T cells. (C) Cells were treated with a phosphatidylinositol 3-kinase inhibitor (10 µM LY294002) in IL-7Rα high ( n = 7) and IL-7Rα low ( n = 8) EM CD8 + T cells. (D) The current components of KCa3.1 (IL-7Rα high , n = 17; IL-7Rα low , n = 16) and Kv1.3 (IL-7Rα high , n = 18; IL-7Rα low , n = 19) were measured in IL-2 reversed IL-7Rα high and IL-7Rα low EM CD8 + T cells. The results were obtained by combining data from two independent experiments using two different donors. Bars and error bars represent the mean ± SEM (A–D) , and p -values were obtained using the two-tailed Student’s t -test (A,B,D) or the analysis of variance followed by Tukey’s post hoc subgroup analysis (C) .

Article Snippet: To reverse CD8 + T cells by IL-2 stimulation, freshly sorted CD8 + T cell subsets were incubated for 5 days with anti-CD3 (HIT3a, 10 µg/mL, plate-bound)/CD28 (28.2, 5 µg/mL, soluble) Abs (both from eBioscience) in the presence of recombinant human IL-2 (20 IU/mL, R&D Systems, Minneapolis, MN, USA) and then stimulated for 10 days in the presence of IL-2 alone with media changes every 3 days with IL-2 replenishment ( ) (Sim et al., manuscript submitted).

Techniques: Staining, Functional Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: Requirement of Kv1.3 for effector memory (EM) CD8 + T cell proliferation and interleukin (IL)-2 production. (A) Freshly sorted IL-7Rα high EM CD8 + T cells were labeled with carboxyfluorescein diacetate (CFSE) and stimulated for 6 days with anti-CD3/CD28 antibodies (Abs) in the presence or absence of potassium channel inhibitors such as TRAM-34 (KCa3.1 inhibitor, 5 µM) and margatoxin (Kv1.3 inhibitor, 5 nM), and their proliferation was measured by flow cytometry. Representative histograms and a quantification graph showing proliferating cells are shown. (B) Quantification of cytokines in culture supernatants from IL-7Rα high EM CD8 + T cells that were stimulated for 24 h with anti-CD3/CD28 Abs in the presence or absence of potassium channel inhibitors using a multiplex cytokine assay. Bars indicate the mean. The results are representative data from two or three independent experiments. Bars represent the mean, and p -values were obtained using the paired two-tailed Student’s t -test.

Article Snippet: To reverse CD8 + T cells by IL-2 stimulation, freshly sorted CD8 + T cell subsets were incubated for 5 days with anti-CD3 (HIT3a, 10 µg/mL, plate-bound)/CD28 (28.2, 5 µg/mL, soluble) Abs (both from eBioscience) in the presence of recombinant human IL-2 (20 IU/mL, R&D Systems, Minneapolis, MN, USA) and then stimulated for 10 days in the presence of IL-2 alone with media changes every 3 days with IL-2 replenishment ( ) (Sim et al., manuscript submitted).

Techniques: Labeling, Flow Cytometry, Multiplex Assay, Cytokine Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: KCa3.1 mediates the motility of effector memory (EM) CD8 + T cells. The migration of EM CD8 + T cells under agarose gel confinement was recorded by time-lapse microscopy (objective 40×; Zeiss Axio Observer Z1; numerical aperture = 1.3; Plan-Neofluar) and analyzed using ImageJ and Metlab. Flat polyurethane acrylate (PUA) surfaces were coated with 10 µg/mL intercellular adhesion molecule 1 (ICAM-1) and/or 2 µg/mL stromal cell-derived factor (SDF)-1α. (A) The effect of ICAM-1 and/or SDF-1α on the mean velocity ( V mean ) of interleukin (IL)-7Rα high and IL-7Rα low EM CD8 + T cells on flat PUA surfaces. The surfaces containing >35 individual cells were analyzed. (B) IL-7Rα high EM CD8 + T cells were treated with either TRAM-34 (5 µM) or margatoxin (50 nM), and the motility of the drug-treated cells on ICAM-1 and/or SDF-1α-coated flat PUA surfaces was recorded by time-lapse microscopy. The surfaces containing >20 individual cells were analyzed. (C) The migration of IL-2-reversed IL-7Rα high and IL-7Rα low EM CD8 + T cells was analyzed on ICAM-1 and/or SDF-1α-coated flat PUA surfaces. The results are representative data from two independent experiments using two different donors. Bars represent the mean, and p -values were obtained using the unpaired two-tailed Student’s t -test.

Article Snippet: To reverse CD8 + T cells by IL-2 stimulation, freshly sorted CD8 + T cell subsets were incubated for 5 days with anti-CD3 (HIT3a, 10 µg/mL, plate-bound)/CD28 (28.2, 5 µg/mL, soluble) Abs (both from eBioscience) in the presence of recombinant human IL-2 (20 IU/mL, R&D Systems, Minneapolis, MN, USA) and then stimulated for 10 days in the presence of IL-2 alone with media changes every 3 days with IL-2 replenishment ( ) (Sim et al., manuscript submitted).

Techniques: Migration, Agarose Gel Electrophoresis, Time-lapse Microscopy, Derivative Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: Decreased transendothelial migration of interleukin (IL)-7Rα low effector memory (EM) CD8 + T cells compared to that of IL-7Rα high EM CD8 + T cells. (A) freshly isolated or (B) IL-2-reversed IL-7Rα high and IL-7Rα low EM CD8 + T cells were added on top of human umbilical vein endothelial cell monolayers on the filters in the presence or absence of stromal cell-derived factor (SDF)-1α (100 ng/mL). Cells were allowed to migrate into the lower chambers or underneath the upper transwells for 20 h. Data are expressed as the number of cells that migrated across the filter. Results are representative data from two independent experiments from five to eight different donors. Bars represent the mean, and p -values were obtained using the unpaired two-tailed Student’s t -test.

Article Snippet: To reverse CD8 + T cells by IL-2 stimulation, freshly sorted CD8 + T cell subsets were incubated for 5 days with anti-CD3 (HIT3a, 10 µg/mL, plate-bound)/CD28 (28.2, 5 µg/mL, soluble) Abs (both from eBioscience) in the presence of recombinant human IL-2 (20 IU/mL, R&D Systems, Minneapolis, MN, USA) and then stimulated for 10 days in the presence of IL-2 alone with media changes every 3 days with IL-2 replenishment ( ) (Sim et al., manuscript submitted).

Techniques: Migration, Isolation, Derivative Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: Interleukin (IL)-2 and IL-15 stimulation control the KCa3.1 activity in IL-7Rα low effector memory (EM) CD8 + T cells. (A,B) To measure the current components of cytokine-stimulated IL-7Rα high and IL-7Rα low EM CD8 + T cells, cells were stimulated for 3 days with anti-CD3/CD28 antibodies in the presence of IL-2 (20 IU/mL), IL-15 (5 ng/mL), or IL-4 (5 ng/mL), and their KCa3.1 (A) and Kv1.3 (B) current components (IL-7Rα high versus IL-7Rα low : n = 17 versus n = 15; n = 8 versus n = 7; or n = 6 versus n = 7, respectively) were measured as described in Figure . Bars and error bars represent the mean ± SEM, and p -values were obtained using the unpaired two-tailed Student’s t -test. (C) The migration of cytokine-stimulated IL-7Rα low EM CD8 + T cells was analyzed as described in Figure . The results are representative data from two independent experiments using two different donors. Bars represent the mean, and p -values were obtained using the unpaired two-tailed Student’s t -test.

Article Snippet: To reverse CD8 + T cells by IL-2 stimulation, freshly sorted CD8 + T cell subsets were incubated for 5 days with anti-CD3 (HIT3a, 10 µg/mL, plate-bound)/CD28 (28.2, 5 µg/mL, soluble) Abs (both from eBioscience) in the presence of recombinant human IL-2 (20 IU/mL, R&D Systems, Minneapolis, MN, USA) and then stimulated for 10 days in the presence of IL-2 alone with media changes every 3 days with IL-2 replenishment ( ) (Sim et al., manuscript submitted).

Techniques: Activity Assay, Two Tailed Test, Migration