Journal: Frontiers in Immunology

Article Title: Age-related increase of mitochondrial content in human memory CD4+ T cells contributes to ROS-mediated increased expression of proinflammatory cytokines

doi: 10.3389/fimmu.2022.911050

Figure Lengend Snippet: Capacity of cytokine production in memory CD4 + T cell. Cells were activated with PMA/ionomycin for 5 h, adding BFA for the last 3 hours for intracellular cytokine expression. (A) Representative example of cytokine expression in memory CD4 + T cells from young and aged donors after stimulation with PMA/ionomycin. (B) Frequencies in % and geometric mean fluorescence intensity (gMFI) of IL-2, IL-4, IL-10, IL-17, IFN-γ, and TNF-α producing memory CD4 + T cells (mean ± SEM; n = 7).

Article Snippet: Incubation was conducted by dissolving 1x 10 6 cells/ml in RPMI 1640 medium (Thermo Fisher Scientific) with 5% human AB serum (Sigma Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37˚C in a 5% CO 2 atmosphere Cells were collected, washed, fixed, permeabilized (Inside staining kit, Miltenyi Biotec) and stained with a combination of monoclonal antibodies including anti-human-IFNγ (PERCP-Cy5.5, Biolegend, clone 4SB3, Cat.# 502526), anti-human-IL-2 (APC-vio770, Miltenyi Biotec, clone N7.48 A, Cat.# 130-097-011), anti-human-IL-4 (PE, Miltenyi Biotec, clone 7A3-3, Cat.# 130-091-647), anti-human-IL-17A (FITC, Miltenyi Biotec, clone CZ8-23G1, Cat.# 130-094-520), and anti-human-TNF-α (PE-Vio770 (Miltenyi Biotec, clone cA2, Cat.# 130-096-755).

Techniques: Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients

doi: 10.1371/journal.pone.0154422

Figure Lengend Snippet: SFbs from healthy donors were cultured in presence of medium (CTRL) or culture supernatants of unstimulated or anti-CD3/CD28 stimulated T helper cells of different phenotypes (classic and non-classic Th1 and Th17). A . SFbs vitality was evaluated by WST-1 assay after 24h and 48h of culture. Columns represent mean ± SE of % of viable SFbs compared to control condition (defined as 100% and shown as line in the Fig) in three experiments. Statistical analysis was performed by using the ANOVA test. B . After 48h SFbs morphology was evaluated by phase contrast microscope (magnification 200X). One representative experiment out of six is shown (one field out of five). Scale bar in all images = 100μm.

Article Snippet: Selected T cell clones with the following phenotypes: Th17 (CD161+IL-17+IFN-γ-), non-classic and classic Th1 (CD161+IL-17-IFN-γ+ and CD161-IL-17-IFN-γ+, respectively), were polyclonally stimulated with anti-CD3 plus anti-CD28 mAbs (human T Cell Activation/Expansion Kit, Miltenyi Biotec), for 72 hours to obtain culture conditioned supernatants, that were stored at -30°C.

Techniques: Cell Culture, WST-1 Assay, Control, Microscopy

Journal: PLoS ONE

Article Title: Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients

doi: 10.1371/journal.pone.0154422

Figure Lengend Snippet: SFbs from healthy donors were cultured for 48h in presence of medium alone (CTRL) or cultured supernatants of unstimulated (dark grey) or anti-CD3/CD28 stimulated (light grey) Th cells clones of different phenotypes (classic and non-classic Th1 and Th17) (A-B) or in the presence of TNF-α or IFN-γ cytokines or their combinations (D-E) in presence or absence of Etanercept (ETN) (G). Columns represent mean ± SE of % of CD106 positive SFbs (A) or of CD106 mean fluorescence intensity (MFI) (G) or both % of CD106 positive SFbs and CD106 mean fluorescence intensity (MFI) (D) of six different experiments. After 24h CD106 (B-E), TNF-αRβ (white) and IFN-γR2 (grey) (F) expression were evaluated by real time RT-PCR. Columns represents mean ± SE of mRNA expression (normalized on housekeeping gene mRNA and calculated as fold to the control, Ctrl) of three different experiments. * p < 0.05; ** p < 0.01, *** p < 0.001 stimulated condition versus ctrl or indicated by bar. IFN-γ (black), TNF-α (grey) and IL-17 (white) cytokine levels in anti-CD3/CD28 stimulated classic and non-classic Th1 and Th17-derived supernatants were evaluated by CBA flex set assay (C); columns represents mean ± SE of cytokine concentration (pg/ml) of five different supernatants for each Th phenotype.* p < 0.05 TNF-α versus IFN-γ or indicated by bar. Statistical analysis was performed by using the ANOVA test.

Article Snippet: Selected T cell clones with the following phenotypes: Th17 (CD161+IL-17+IFN-γ-), non-classic and classic Th1 (CD161+IL-17-IFN-γ+ and CD161-IL-17-IFN-γ+, respectively), were polyclonally stimulated with anti-CD3 plus anti-CD28 mAbs (human T Cell Activation/Expansion Kit, Miltenyi Biotec), for 72 hours to obtain culture conditioned supernatants, that were stored at -30°C.

Techniques: Cell Culture, Clone Assay, Fluorescence, Expressing, Quantitative RT-PCR, Control, Derivative Assay, Concentration Assay

Journal: Mediators of Inflammation

Article Title: Analysis of Th17 and Tc17 Frequencies and Antiviral Defenses in Gut-Associated Lymphoid Tissue of Chronic HIV-1 Positive Patients

doi: 10.1155/2015/395484

Figure Lengend Snippet: Activation markers, IFN- γ and IL-17A expression in CD4 + and CD8 + T cells subsets in blood and in gut. Panel (a): representative flow cytometry plots outlining CD38 and HLA-DR expression on naïve, effector, and central memory CD4 + and CD8 + lymphocytes derived from peripheral blood mononuclear cells (PBMCs) and gut-associated lymphoid tissue (GALT). Panel (b): representative flow cytometry plots outlining IFN- γ and IL-17A intracitoplasmatic expression on naïve, effector, and central memory CD4 + (Th1 and Th17) and CD8 + (Tc1 and Tc17) lymphocytes derived from PBMCs and GALT.

Article Snippet: Cultured cells were fixed, permeabilized (BD Cytofix/Cytoperm, Becton Dickinson, San Jose, CA, USA), and stained with combinations of fluorochrome-labeled monoclonal antibodies: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, CD45RO-PE-Vio770, CD27-VioBlue, IFN- γ -APC, and IL-17A-PE (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activation Assay, Expressing, Flow Cytometry, Derivative Assay

Journal: PLOS ONE

Article Title: Anti-inflammatory effect of the combined treatment of LMT-28 and kaempferol in a collagen-induced arthritis mouse model

doi: 10.1371/journal.pone.0302119

Figure Lengend Snippet: (A) Arthritis scores of mice were measured every 3 days starting at 21 days after the first immunization. (B) DBA1/J mice showed deformation and swelling of the toe and ankle joints. (C) Paraffin-embedded sections of CIA mice joint were stained with hematoxylin and eosin. (D) Histological analysis was performed based on the analysis criteria: inflammation, synovial hyperplasia, pannus formation, and erosion of cartilage and bone. (E) The protein levels of IL-6, IL-1β and IL-17 in CIA mouse serum were measured using ELISA. Data are presented as mean ± SEM. # p <0.05 vs. normal group, * p <0.05, ** p <0.01, and *** p <0.005 vs. CIA control group.

Article Snippet: The cells were incubated with anti-IL-17A-PE antibody (Miltenyi Biotec) or anti-mouse FOXP3 antibody (Invitrogen, Waltham, MA, USA) diluted in 1× permeabilization buffer (eBioscience) for 30 min at 4°C in the dark.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS ONE

Article Title: Expression of miRNAs miR-133b and miR-206 in the Il17a/f Locus Is Co-Regulated with IL-17 Production in αβ and γδ T Cells

doi: 10.1371/journal.pone.0020171

Figure Lengend Snippet: (A) CD4 + T cells from C57BL/6 mice were sorted into IL-17A + and IL-17A − populations and analyzed for expression of miR-133b and miR-206 by qRT-PCR. Dot plots show post-sort analysis of one representative experiment. (B) CD4 + T cells were sorted into GFP + (Rorγt + ) and GFP − (Rorγt − ) populations from heterozygous Rorγt reporter mice and analyzed as in (A). Dot plots show post-sort analysis of one representative experiment. Values are plotted as fold increase ( = ratio) compared to the respective negative population. The graphs show representative experiments from n = 3 independent experiments with similar results. Error bars represent SD values of triplicates from one experiment with 4–6 mice.

Article Snippet: IL-17 producing and non-producing CD4 + αβ T cells were sorted from mixed spleen and peripheral lymph node cells from C57BL/ 6 N mice after revealing IL-17 secreting cells using the mouse IL-17A Secretion Assay (Miltenyi) and subsequent FACS-sorting. γδ T cells were sorted from mixed spleen and peripheral lymph node cells from C57BL/6-Tcrd-H2BeGFP mice based on eGFP and CCR6 expression.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Expression of miRNAs miR-133b and miR-206 in the Il17a/f Locus Is Co-Regulated with IL-17 Production in αβ and γδ T Cells

doi: 10.1371/journal.pone.0020171

Figure Lengend Snippet: (A, B) Spleen and peripheral lymph node cells were isolated from DO11.10 mice, cocultered with sex-matched BALB/c irradiated feeder cells and polarized to either Th1 or treated with TGF-β, IL-6, IL-23, IL-1β, IL-21 and TNF-α in various combinations (4cytokines = TGF-β, IL-6, IL-23 and IL-1β). Values show fold increase ( = ratio) compared to cells cultured only with Ova 323–329 /antibodies. Expression levels of miR-133b (A) and miR-206 (B) were analyzed by qRT-PCR. (C) Secreted IL-17A was determined in culture supernatants of each condition from the cells in (A) and (B) by ELISA. Values show absolute amounts of IL-17A in the cell culture supernatant in ng/ml. Error bars show ±SEM of n = 3 experiments with 2–3 mice per experiment. (D), (E) Scatter plot of IL-17A protein concentration versus relative expression of miR-133b (D) and miR-206 (E) and correlation coefficient for ELISA compared to qRT-PCR.

Article Snippet: IL-17 producing and non-producing CD4 + αβ T cells were sorted from mixed spleen and peripheral lymph node cells from C57BL/ 6 N mice after revealing IL-17 secreting cells using the mouse IL-17A Secretion Assay (Miltenyi) and subsequent FACS-sorting. γδ T cells were sorted from mixed spleen and peripheral lymph node cells from C57BL/6-Tcrd-H2BeGFP mice based on eGFP and CCR6 expression.

Techniques: Isolation, Irradiation, Cell Culture, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: PLoS ONE

Article Title: Expression of miRNAs miR-133b and miR-206 in the Il17a/f Locus Is Co-Regulated with IL-17 Production in αβ and γδ T Cells

doi: 10.1371/journal.pone.0020171

Figure Lengend Snippet: CD4 + T cells were isolated from peripheral blood of 3 human healthy donors, enriched using magnetic beads and then FACS sorted into 3 different populations: IL-17A + , IL-17A − IFNγ + and IL-17A − IFN-γ − . Expression levels of miR-133b and miR-206 were analyzed by qRT-PCR. Values are plotted as fold increase compared to the respective IL-17A − IFN-γ − population. P-value for miR-206 in IL-17A + compared to IFN-γ + expressing cells was 0.0645. Error bars show ±SEM for the 3 different donors.

Article Snippet: IL-17 producing and non-producing CD4 + αβ T cells were sorted from mixed spleen and peripheral lymph node cells from C57BL/ 6 N mice after revealing IL-17 secreting cells using the mouse IL-17A Secretion Assay (Miltenyi) and subsequent FACS-sorting. γδ T cells were sorted from mixed spleen and peripheral lymph node cells from C57BL/6-Tcrd-H2BeGFP mice based on eGFP and CCR6 expression.

Techniques: Isolation, Magnetic Beads, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Expression of miRNAs miR-133b and miR-206 in the Il17a/f Locus Is Co-Regulated with IL-17 Production in αβ and γδ T Cells

doi: 10.1371/journal.pone.0020171

Figure Lengend Snippet: (A) In vitro assay. MACS-enriched CD4 + T cells from TCR-transgenic DO.11.10 mice were retrovirally transduced with miR-133b or miR-206 or with both and stimulated under Th17 polarization inducing conditions. Frequency of IL-17 producing CD4 + T cells among transduced (GFP + ) and non-transduced (GFP − ) cells of the same well was compared. One representative of two independent experiments with similar results is shown. (B, C) Frequency of IL-17 producing cells overexpressing miR-133b or miR-206 in vivo . Lineage negative bone marrow was transduced with miR-133b or miR-206 or with the empty vector MDH1-PGK-GFP2.0 and served to reconstitute lethally irradiated C57BL/6 wild type mice. After 8–10 weeks, chimeras were analyzed for the occurrence and frequency of IL-17 producing cells within the transduced GFP + and non-transduced GFP − lymphocytes from peripheral lymph nodes and spleen. (B) Representative gating strategy after excluding autofluorescent and B220 + cells. (C) Frequency of IL-17 producing CD4 + and CD4 − cells, respectively. At least 5 chimeric mice were individually analyzed for each condition.

Article Snippet: IL-17 producing and non-producing CD4 + αβ T cells were sorted from mixed spleen and peripheral lymph node cells from C57BL/ 6 N mice after revealing IL-17 secreting cells using the mouse IL-17A Secretion Assay (Miltenyi) and subsequent FACS-sorting. γδ T cells were sorted from mixed spleen and peripheral lymph node cells from C57BL/6-Tcrd-H2BeGFP mice based on eGFP and CCR6 expression.

Techniques: In Vitro, Transgenic Assay, Transduction, In Vivo, Plasmid Preparation, Irradiation