Journal: PLoS ONE

Article Title: Reduced Level of the BCL11B Protein Is Associated with Adult T-Cell Leukemia/Lymphoma

doi: 10.1371/journal.pone.0055147

Figure Lengend Snippet: (A) Immunoblot analysis of BCL11B protein expression in a panel of HTLV-1-positive T-cell lines and HTLV-I-free T-cell lines. The cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with polyclonal antibodies against BCL11B (upper panel). Jurkat, MOLT4, PEER and SupT-11 are HTLV-I-free T -lymphoid leukemia cell lines; MT-2 and MT-4 are human T-cell lines transformed in vitro using HTLV-I; MT-1, Hut102, ATN-1, KKI, KOB, OMT RST and SO4 are ATLL patient-derived cell lines. The data were normalized to ß-tubulin (ß-TUB) and calibrated to the BCL11B/ß-tubulin ratio in the healthy volunteer samples as a relative expression value of 100 (lower panel). (B) RT-PCR analysis of BCL11B, Tax and FOXP3 mRNA in CD4+ T lymphocytes from healthy volunteers, HTLV-I-positive T-cell lines and HTLV-I-free T-cell lines (upper panel). (C) RT-qPCR analysis of BCL11B mRNA. The data were normalized to 28S ribosome RNA ( rRNA ) and calibrated to the BCL11B/28S -ribosome RNA ratio in the healthy volunteer sample as a relative expression value of 100 (lower panel). The data represent the mean values of the assays, which were performed in duplicate.

Article Snippet: The anti-human CD4 FITC-conjugated monoclonal and the goat anti-rabbit IgG DyLight 549-conjugated secondary antibodies were purchased from Abcam (Cambridge, UK).

Techniques: Western Blot, Expressing, SDS Page, Transformation Assay, In Vitro, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: A Comparative Study of N -glycolylneuraminic Acid (Neu5Gc) and Cytotoxic T Cell (CT) Carbohydrate Expression in Normal and Dystrophin-Deficient Dog and Human Skeletal Muscle

doi: 10.1371/journal.pone.0088226

Figure Lengend Snippet: GRMD muscle (cranial sartorius) was triple stained for Neu5Gc (green), Pax7 (A), CD11b (B), CD4 (C), CD8 (D) (all red), and DAPI (blue). Arrows indicated co-staining of Neu5Gc and Pax7 (in A), CD11b (in B) or CD4 (in C). Bar is 100 µm (A) and 50 µm (B–D) for all panels.

Article Snippet: Co-staining antibodies used in dog were mouse anti-chicken Pax7 (Developmental Studies Hybridoma Bank, clone P3U1), mouse anti-dog CD4 (AbD Serotec, MCA1998S), rat anti-dog CD8 (AbD Serotec, MCA1039GA), mouse anti-dog CD11b (AbD Serotec, MCA1777S), mouse anti-dog CD21 (AbD Serotec, MCA1781R), mouse anti-human β spectrin (Novus, NB300-574) or mouse anti-rat embryonic myosin (NovaCastra, NCL-MHCd).

Techniques: Staining

Journal: PLoS ONE

Article Title: A Comparative Study of N -glycolylneuraminic Acid (Neu5Gc) and Cytotoxic T Cell (CT) Carbohydrate Expression in Normal and Dystrophin-Deficient Dog and Human Skeletal Muscle

doi: 10.1371/journal.pone.0088226

Figure Lengend Snippet: (A) Cranial sartorius muscle sections from GR and GRMD dogs were stained with markers for satellite cells (Pax7), T lymphocytes (CD4 or CD8) or macrophages (CD11b) and quantified for numbers of cells stained per 40X visual field. (B) The percentage of cells co-stained for Neu5Gc and CD4, CD8, CD11b or Pax7 in GRMD muscles was quantified. Errors are SEM. ***P<0.001, for each GR vs. GRMD comparison in A.

Article Snippet: Co-staining antibodies used in dog were mouse anti-chicken Pax7 (Developmental Studies Hybridoma Bank, clone P3U1), mouse anti-dog CD4 (AbD Serotec, MCA1998S), rat anti-dog CD8 (AbD Serotec, MCA1039GA), mouse anti-dog CD11b (AbD Serotec, MCA1777S), mouse anti-dog CD21 (AbD Serotec, MCA1781R), mouse anti-human β spectrin (Novus, NB300-574) or mouse anti-rat embryonic myosin (NovaCastra, NCL-MHCd).

Techniques: Staining, Muscles, Comparison

Journal: Clinical and Experimental Immunology

Article Title: cDNA microarray analysis identifies NR4A2 as a novel molecule involved in the pathogenesis of Sjögren's syndrome

doi: 10.1111/cei.13000

Figure Lengend Snippet: Immunofluorescence staining of nuclear receptor subfamily 4, group A, member 2 (NR4A2) in labial salivary glands (LSGs). (a) Double staining of NR4A2 and CD3 in LSG sections of Sjögren's syndrome (SS) patients and patients with immunoglobulin G4‐related disease (IgG4‐RD). NR4A2 was expressed in the nuclei of T cells in SS. (b) Double staining for NR4A2 and CD4 in LSG sections of SS patients and IgG4‐RD patients. NR4A2 was expressed in the nuclei of CD4+ T cells in SS. (c) Double staining for NR4A2 and interleukin (IL)‐17 in LSG sections of SS patients and IgG4‐RD patients. NR4A2 was expressed in IL‐17‐expressing cells in SS. Areas surrounded by dashed lines were enlarged in the right images. Scale bars = 25 μm. DAPI = diamidino‐2‐phenylindole.

Article Snippet: CD4 + T cells in PBMCs of patients with SS ( n = 22) and age‐ and sex‐matched healthy control subjects ( n = 10), all of whom had not been treated with immunosuppressive agents, were isolated by positive selection using autoMACS Pro cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany) following the magnetic labelling of the targeted cells with anti‐human CD4 MicroBeads (Miltenyi Biotec; 130–045‐101), according to the manufacturer's protocol.

Techniques: Immunofluorescence, Staining, Double Staining, Expressing

Journal: Clinical and Experimental Immunology

Article Title: cDNA microarray analysis identifies NR4A2 as a novel molecule involved in the pathogenesis of Sjögren's syndrome

doi: 10.1111/cei.13000

Figure Lengend Snippet: Nuclear receptor subfamily 4, group A, member 2 (NR4A2) expression and T helper type 17 (Th17) differentiation of peripheral CD4+ T cells in patients with Sjögren's syndrome (SS). (a) Relative mRNA expression of NR4A2 in peripheral CD4+ T cells of patients with SS (n = 22) compared with those of healthy controls (HC, n = 10). NR4A2 was up‐regulated in peripheral CD4+ T cells of SS patients compared with HCs. (b) Representative experiments of intracellular staining of interleukin (IL)‐17 and interferon (IFN)‐γ in peripheral CD4+ T cells of HCs and patients with SS, cultured for 7 days under T helper type 17 (Th17)‐polarizing conditions or Th0 conditions. (c) The population (%) of IL‐17+IFN‐γ‐/CD4+ T cells on culture day 7 under Th17‐polarizing conditions or under Th0 conditions in HCs (n = 3) and SS patients (n = 5). Th17 differentiation of peripheral CD4+ T cells of SS patients was significantly higher than that of HCs. (d) Correlation between NR4A2 mRNA expression in CD4+ T cells at baseline and percentage of IL‐17+IFN‐γ– cells/CD4+ T cells under Th17‐polarizing conditions. Significant correlation was observed between them (Spearman's R = 0·87). *P < 0·05; NS = not significant. (a) Bars indicate average values. (a,c) Mann–Whitney U‐test. (d) Spearman's correlation. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: CD4 + T cells in PBMCs of patients with SS ( n = 22) and age‐ and sex‐matched healthy control subjects ( n = 10), all of whom had not been treated with immunosuppressive agents, were isolated by positive selection using autoMACS Pro cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany) following the magnetic labelling of the targeted cells with anti‐human CD4 MicroBeads (Miltenyi Biotec; 130–045‐101), according to the manufacturer's protocol.

Techniques: Expressing, Staining, Cell Culture, MANN-WHITNEY

Journal: Clinical and Experimental Immunology

Article Title: cDNA microarray analysis identifies NR4A2 as a novel molecule involved in the pathogenesis of Sjögren's syndrome

doi: 10.1111/cei.13000

Figure Lengend Snippet: Intracellular localization of nuclear receptor subfamily 4, group A, member 2 (NR4A2) in naive CD4+ T cells of patients with Sjögren's syndrome (SS) cultured under T helper type 17 (Th17)‐polarizing conditions. (a) Immunofluorescence cytochemistry images of NR4A2 in naive CD4+ T cells of a healthy subject (HS) during Th17 polarization. (b) Densitometric quantification of fluorescence intensity of NR4A2 compared to 0h in representative images of naive CD4+ T cells of HS cultured under Th0 and Th17‐polarizing conditions. Significant increase of NR4A2 expression was observed in cultures under Th0 and Th17‐polarizing conditions, while there was no difference between Th0 and Th17‐polarizing conditions at each time‐point. (c) Representative immunofluorescence cytochemistry images of NR4A2 in naive CD4+ T cells of HS on day 4 in cultures under Th17‐polarizing conditions in comparison with Th0 and Th1‐polarizing conditions. Intranuclear localization of NR4A2 was observed specifically under Th17‐polarizing conditions, compared with the other conditions. (d) Densitometric determination of nuclear fluorescence rate (%) of NR4A2 in representative images of naive CD4+ T cells of HS on day 4 during each polarization condition. The rate of NR4A2 nuclear expression was significantly higher under Th17‐polarizing conditions than under the other conditions. (e) Representative images of intracellular localization of NR4A2 in Th17‐polarized CD4+ T cells examined by immunofluorescence cytochemistry in patients with SS compared with HCs. (f) Densitometric determination of mean intranuclear fluorescence rate (%) of NR4A2 in naive CD4+ T cells cultured under Th0 and Th17‐polarizing conditions in HCs (n = 3) and patients with SS (n = 5). Intranuclear expression rate of NR4A2 in naive CD4+ T cells cultured under Th17‐polarizing conditions was significantly higher in SS patients than in HCs. DAPI = diamidino‐2‐phenylindole; NS = not significant; *P < 0·05. (a) Scale bars = 25 μm. (b) Mann–Whitney U‐test. (a,b) Images were obtained at similar exposure time (1/5 s). (c) Arrowheads: fluorescence of NR4A2. (c,e) Scale bars = 5 μm. (d) Kruskal–Wallis test. (a–d) Data are representative of three independent experiments. (f) Statistical comparison between HC and SS was performed by Mann–Whitney U‐test, while that between Th0 and Th17 conditions was examined by Wilcoxon's matched‐pairs test. (c–f) Images were obtained at similar exposure time (1/15 s). [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: CD4 + T cells in PBMCs of patients with SS ( n = 22) and age‐ and sex‐matched healthy control subjects ( n = 10), all of whom had not been treated with immunosuppressive agents, were isolated by positive selection using autoMACS Pro cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany) following the magnetic labelling of the targeted cells with anti‐human CD4 MicroBeads (Miltenyi Biotec; 130–045‐101), according to the manufacturer's protocol.

Techniques: Cell Culture, Immunofluorescence, Fluorescence, Expressing, Comparison, MANN-WHITNEY

Journal: Clinical and Experimental Immunology

Article Title: cDNA microarray analysis identifies NR4A2 as a novel molecule involved in the pathogenesis of Sjögren's syndrome

doi: 10.1111/cei.13000

Figure Lengend Snippet: Inhibition of intranuclear localization of nuclear receptor subfamily 4, group A, member 2 (NR4A2) with importazole (IPZ) in naive CD4+ T cells of healthy subjects (HS) cultured under T helper type 17 (Th17)‐polarizing conditions. (a) Representative images of intracellular localization of NR4A2 (day 4) in naive CD4+ T cells cultured under Th0 and Th17‐polarizing conditions, treated with dimethylsulphoxide (DMSO) (control) or importazole (IPZ). (b) Intranuclear fluorescence rate (%) of NR4A2 in naive CD4+ T cells cultured under Th0 and Th17‐polarizing conditions, treated with DMSO (control) or IPZ in representative images. Intranuclear localization of NR4A2 was inhibited significantly with IPZ under Th17‐polarizing conditions. (c) Representative flow cytometry data of Th17 differentiation (day 4) of naive CD4+ T cells treated with DMSO (control) or IPZ. (d) The percentages of interleukin (IL)‐17+interferon (IFN)‐γ–/CD4+ T cells on culture day 4 under Th17‐polarizing conditions or under Th0 conditions in HS (n = 5). IPZ reduced significantly the percentage of IL‐17+IFN‐γ–CD4+ T cells. (e) The mRNA expression level of IL‐21 in naive CD4+ T cells cultured under Th0 or Th17‐polarizing conditions with IPZ or control. IPZ inhibited IL‐21 expression significantly specifically under Th17‐polarizing conditions. (f) retinoic acid receptor‐related orphan receptor C (RORC) mRNA expression level in naive CD4+ T cells cultured under Th0 or Th17‐polarizing conditions with IPZ or control. IPZ did not change RORC expression significantly under Th0 and Th17‐polarizing conditions. *P < 0·05; NS = not significant. (a) Scale bar = 5 μm. Arrowheads: fluorescence of NR4A2. (b) Mann–Whitney U‐test. (a,b) Representative data of three independent experiments. Images are obtained at similar exposure time (1/15 s). (d–f) Wilcoxon's matched‐pairs test. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: CD4 + T cells in PBMCs of patients with SS ( n = 22) and age‐ and sex‐matched healthy control subjects ( n = 10), all of whom had not been treated with immunosuppressive agents, were isolated by positive selection using autoMACS Pro cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany) following the magnetic labelling of the targeted cells with anti‐human CD4 MicroBeads (Miltenyi Biotec; 130–045‐101), according to the manufacturer's protocol.

Techniques: Inhibition, Cell Culture, Control, Fluorescence, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Clinical and Experimental Immunology

Article Title: cDNA microarray analysis identifies NR4A2 as a novel molecule involved in the pathogenesis of Sjögren's syndrome

doi: 10.1111/cei.13000

Figure Lengend Snippet: Schematic diagram of salivary glands in patients with Sjögren's syndrome (SS) depicting the roles of nuclear receptor subfamily 4, group A, member 2 (NR4A2) in the pathogenesis of SS. NR4A2 is induced by T cell receptor (TCR) stimulation and localized in the nuclei of CD4+ T cells under T helper type 17 (Th17)‐polarizing cytokine environment. Intranuclear localization of NR4A2 could be involved in Th17 cell development via interleukin (IL)‐21 expression independently of Retinoic acid receptor‐related orphan receptor gamma t (RORγt). Increased expression (a) and intranuclear localization (b) of NR4A2 in CD4+ T cells seem to contribute to increased Th17 cell development in patients with SS. The increased expression and intranuclear localization could accelerate each other by enhancing Th17‐polarizing cytokine environment. MHC‐II = major histocompatibility complex class II; IPZ = importazole; NPC = nuclear pore complex. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: CD4 + T cells in PBMCs of patients with SS ( n = 22) and age‐ and sex‐matched healthy control subjects ( n = 10), all of whom had not been treated with immunosuppressive agents, were isolated by positive selection using autoMACS Pro cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany) following the magnetic labelling of the targeted cells with anti‐human CD4 MicroBeads (Miltenyi Biotec; 130–045‐101), according to the manufacturer's protocol.

Techniques: Expressing, Immunopeptidomics