Journal: Nature immunology

Article Title: A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells

doi: 10.1038/ni.3050

Figure Lengend Snippet: The OPN-i deficient T FH and T FR phenotype is cell-intrinsic. a, Flow cytometry of donor OT-II CD4 + T cells from spleens of Rag2 −/− Prf1 −/− hosts transferred with naïve OT-II, OT-II OPN-KO and OT-II OPN-i-KI CD4 + T cells along with wild-type B cells, followed by immunization with NP 13 -OVA in CFA and analysis 10 d later. Numbers indicate percent T FH (Foxp3 − PD1 + CXCR5 + CD4 + ) and GC B (Fas + GL7 + B220 + ) cells. b, Serum titers of total (NP 23 ) NP-specific IgG and IgG1 from recipient mice in a ( n = 6 per group). c, Flow cytometry of donor Treg from spleens of Tcr α −/− hosts transferred with sorted CD45.2 + Treg (CD25 hi CD44 int CXCR5 − CD4 + ) from the indicated mice and CD45.1 + wild-type naïve CD4 + T cells (CD25 − GITR − CD44 lo CD62L hi ) at a ratio of 1:2, followed by immunization with NP 26 -KLH in CFA and analysis 10 d later. Numbers indicate percent T FR (CD45.1 − CD44 + Foxp3 + PD1 + CXCR5 + CD4 + ) and GC B (Fas + GL7 + B220 + ) cells. d, Frequency of T FR and GC B cells in c ( n = 5 per group). e, Titers of total (NP 23 ) and high-affinity (NP 4 ) NP-specific IgG at d14 in immunized Rag2 −/− Prf1 −/− recipients of OPN-i-KI or OPN-KO T FH cells (5 × 10 4 ) and/or OPN-i-KI or OPN-KO T FR cells (2.5 × 10 4 ) (sorted as in ) and wild-type GL-7 − B cells (1 × 10 5 ) from KLH-immunized mice. All recipients ( n = 4 per group) were immunized with NP 26 -KLH in CFA. f, Donor GC B cells from spleens of recipients in e (in same order from left to right) at d22. g, Titers of NP-specific total IgG at d11 in immunized Rag2 −/− Prf1 −/− mice ( n = 3 per transfer) given OPN-i-KI or OPN-KO T FH cells and/or OPN-i-KI or OPN KO T FR cells (2.5 × 10 4 ) at different ratios and wild-type GL-7 − B cells (1 × 10 5 ) from KLH-immunized mice. Data are representative of three ( a–b ) and two ( c–g ) independent experiments. * P < 0.05 and ** P < 0.01 (unpaired two-tailed Student’s t-test; error bars, mean ± s.e.m).

Article Snippet: CD62L − CD4 + T cells (> 95%) from the indicated mouse strains injected with KLH in CFA were purified with MACS CD4 + CD62L + T-cell isolation kit (Miltenyi) and stimulated with anti-ICOS for the indicated times before fixation, permeabilization and immunostaining.

Techniques: Flow Cytometry, Two Tailed Test

Journal: Nature immunology

Article Title: A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells

doi: 10.1038/ni.3050

Figure Lengend Snippet: ICOS co-stimulation promotes an interaction between OPN-i and p85α. a, Quantitative RT-PCR analysis of Spp1 mRNA in naïve CD4 + T cells from B6 mice stimulated with anti-CD3 and anti-CD28 for 2 d, followed by resting overnight before 20 m incubation with the indicated Abs and cross-linking with goat anti-hamster Ab for 8 h or 24 h. Spp1 expression was normalized to the Rps18 control and results are presented relative to isotype-matched hamster IgG-treated cells at 8 h, set as 1. * P < 0.05 (unpaired two-tailed Student’s t-test; error bars, mean ± s.e.m of quadruplicates). b, Immunoblot analysis of OPN and actin of CD4 + T cells in a after 12 h cross-linking. c, Immunoassay of lysates of purified CD62L − CD4 + T cells from Icos −/− and wild-type mice 2 or 3 d after intraperitoneal injection with 100 μg KLH in CFA, probed with anti-Bcl-6, anti-OPN and anti-actin. d, Immunoblot analysis of lysates of sorted V β 5 + CD25 − CD44 hi GITR − CD4 + effector and CD25 + CD44 hi GITR + CD4 + regulatory T cells from pooled OT-II ( n = 10) or OT-II Icos −/− mice ( n = 15) 8 d post-immunization with OVA in CFA, probed as in c. e, Immunoassay of lysates of 293T cells transfected with plasmids encoding Flag-tagged p85α (Flag-p85α) and increasing concentrations of OPN-i, assessed by immunoprecipitation (IP) with anti-Flag and immunoblot analysis with anti-Flag and anti-OPN. f, Immunoassay of lysates of purified naïve CD4 + T cells from OPN-KO and wild-type mice stimulated with anti-CD3 and anti-CD28 for 2 d (left); or CD44 + CD4 + T cells from OT-II mice 4 d post-immunization with OVA in CFA (right), followed by resting and re-stimulation with anti-CD3 and/or anti-ICOS (as in a ) for 12 h, and assessed by IP with anti-p85α and immunoblot analysis with anti-p85α and anti-OPN. Data represent two ( a, c, d ) and three ( b, e, f ) independent experiments.

Article Snippet: CD62L − CD4 + T cells (> 95%) from the indicated mouse strains injected with KLH in CFA were purified with MACS CD4 + CD62L + T-cell isolation kit (Miltenyi) and stimulated with anti-ICOS for the indicated times before fixation, permeabilization and immunostaining.

Techniques: Quantitative RT-PCR, Incubation, Expressing, Control, Two Tailed Test, Western Blot, Purification, Injection, Transfection, Immunoprecipitation

Journal: Nature immunology

Article Title: A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells

doi: 10.1038/ni.3050

Figure Lengend Snippet: p85α chaperones nuclear translocation of OPN-i. a, Immunoassay of lysates of 293T cells transfected with plasmids encoding Flag-p85α and OPN-i wild-type or OPN-i Y166F (YF) mutant, assessed by immunoprecipitation with anti-Flag and immunoblot analysis with anti-Flag and anti-OPN. Input, immunoblot analysis of an aliquot of cell lysates without IP. b, Confocal microscopy of OPN and Bcl-6 expression by CD44 + CD4 + T cells from OPN-i-KI mice 3 d post-injection with KLH in CFA, followed by cross-linking with anti-ICOS Ab (as in ) in vitro . Cells were counterstained with DNA-intercalating dye DAPI to trace nuclear perimeters. About 25–30 cells stained with Bcl-6 were further analyzed for localization of OPN protein. Right, fluorescence intensity expressed as the mean ratio of nuclear (Nuc) to cytoplasmic (Cyt) fluorescence pixel intensity ( n = 25–30 cells). c, Immunoblot analysis of nuclear fractions of OPN-i-KI CD62L − CD4 + T cells treated with anti-CD3 and/or anti-ICOS Abs (as in ), probed with anti-OPN, anti-Lamin B1 and anti-tubulin (to validate the integrity of nuclear separation). d , Immunoblot analysis of nuclear and cytosolic fractions of 293T cells transfected with plasmids encoding OPN-i wild-type or OPN-i Y166F mutant and increasing concentrations of Flag-p85α, probed with Abs, as indicated. e, Confocal microscopy of OPN and Bcl-6 expression by CD62L − CD4 + T cells from p85α WT or KO mice. Cell treatment and analysis as in a . Original magnification ( b, e ), 600×. * P < 0.05 and *** P < 0.001, Mann-Whitney test (error bars, mean ± s.e.m) (b, e ). All results are representative of at least two independent experiments.

Article Snippet: CD62L − CD4 + T cells (> 95%) from the indicated mouse strains injected with KLH in CFA were purified with MACS CD4 + CD62L + T-cell isolation kit (Miltenyi) and stimulated with anti-ICOS for the indicated times before fixation, permeabilization and immunostaining.

Techniques: Translocation Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Confocal Microscopy, Expressing, Injection, In Vitro, Staining, Fluorescence, MANN-WHITNEY

Journal: Nature immunology

Article Title: A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells

doi: 10.1038/ni.3050

Figure Lengend Snippet: Intranuclear OPN-i interacts with and stabilizes Bcl-6 expression. a, Immunoassay of lysates of purified CD44 + CD4 + T cells from pooled OT-II ( n = 8), OT-II OPN-KO ( n = 10) and OT-II OPN-i-KI ( n = 8) mice 7 d post-immunization with OVA in CFA, assessed by immunoprecipitation (IP) with anti-Bcl-6 and immunoblot analysis, as indicated. b, Immunoassay of lysates of 293T cells transfected with plasmids encoding OPN-i and Flag–Bcl-6 wild-type or Flag–Bcl-6 deletion mutants (lanes 1,4,7,10 in ), assessed by IP with anti-Flag and immunoblot analysis, as indicated. Bottom, ratios of precipitated OPN to Bcl-6. c, Immunoblot analysis of lysates of purified CD62L − CD4 + T cells from the indicated OT-II mice 2.5 d post-immunization with OVA in CFA, followed by resting for 2 h, treatment with or without MG132 90 m after incubation with anti-CD3 and anti-ICOS, addition of cycloheximide (CHX) 30 m later, and analysis 3 h after treatment with or without CHX. Right, ratios of Bcl-6 to actin protein. d, Bcl-6 and OPN expression in 293T cells transfected with vectors expressing Flag–Bcl-6 and/or OPN-i, treated with CHX (100 μg/ml) for 10 h. Right, percent of residual Bcl-6 protein relative to that prior to addition of CHX. e–f , Immunoassay of lysates of 293T cells transfected with the indicated plasmids and pre-treated with MG132, assessed by denaturation of lysates, IP with anti-Bcl-6 ( e ) or anti-HA ( f ) and immunoblot analysis as indicated. Bcl-6(Ubn): polyubiquitinated Bcl-6. Increasing amounts of OPN-i plasmids in f. g, Immunoblot analysis of lysates of purified CD62L − CD4 + T cells from indicated OT-II mice 7 d post-immunization with OVA in CFA, treated with (+) or without (−) DUBi for 8 h, probed with anti-Bcl-6 and anti-actin. Bottom, ratios of Bcl-6 to actin. Data represent two ( a–d, g ) and three ( e, f ) independent experiments.

Article Snippet: CD62L − CD4 + T cells (> 95%) from the indicated mouse strains injected with KLH in CFA were purified with MACS CD4 + CD62L + T-cell isolation kit (Miltenyi) and stimulated with anti-ICOS for the indicated times before fixation, permeabilization and immunostaining.

Techniques: Expressing, Purification, Immunoprecipitation, Western Blot, Transfection, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Platelet Extracellular Vesicles Are Taken up by Canine T Lymphocytes but Do Not Play a Role in Their Proliferation, Differentiation and Cytokine Production In Vitro

doi: 10.3390/ijms23105504

Figure Lengend Snippet: List of monoclonal antibodies used for labeling peripheral blood mononuclear cells (PBMCs) for flow cytometry. Abbreviations: FITC—Fluorescein isothiocyanate, PE—Phycoerythrin, AF647—Alexa Fluor 647, PB—Pacific Blue.

Article Snippet: CD62L , FMC46 , IgG2b , mouse , PB , MCA1076PB , BioRad , 1:5.

Techniques: Bioprocessing, Labeling, Flow Cytometry

Journal: Molecular Pain

Article Title: Chronic inflammatory pain decreases the glutamate vesicles in presynaptic terminals of the nucleus accumbens

doi: 10.1177/1744806918781259

Figure Lengend Snippet: Downregulation of SNARE correlative proteins in the NAc contributed to the impairment of glutamatergic synaptic transmission. (a) Western blot showed the protein expression of STX1A, SNAP-25, VAMP2, Munc18–1, and NSF. (b) Quantitative analysis of STX1A, SNAP-25, VAMP2, Munc18–1, and NSF comparing with GAPDH, the expression of VAMP2 and Munc18–1 had no change while that of STX1A, SNAP-25, and NSF was decreased in CFA group. The data are presented as the means ± SEM ( n = 5/group; * p < 0.05, ** p < 0.01 compared to the saline group). SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; NAc: nucleus accumbens; STX1A: Syntaxin 1A; SNAP: synaptosome-associated protein; VAMP2: vesicle-associated membrane protein 2; NSF: N-ethylmaleimide-sensitive factor; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; CFA: complete Freund’s adjuvant; SEM: standard error of mean; Munc18–1: mammalian uncoordinated 18–1.

Article Snippet: The primary antibodies were as follows: rabbit IgG against Syntaxin 1A (STX1A, 1:1000, Cell Signaling Technology, USA), synaptosome-associated protein-25 (SNAP-25, 1:1000), vesicle-associated membrane protein 2 (VAMP2, 1:1000), mammalian uncoordinated 18–1 (Munc18–1, 1:1000), NSF (1:1000), VGLUT2(1:1000), mouse IgG against VGLUT1 (1:500, Millipore, USA), and mouse IgG against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000, Zhuang Zhi, China).

Techniques: Transmission Assay, Western Blot, Expressing