Journal: Frontiers in Immunology

Article Title: B cells from anti-thyroid antibody positive, infertile women show hyper-reactivity to BCR stimulation

doi: 10.3389/fimmu.2022.1039166

Figure Lengend Snippet: Different naive and memory B cell ratio in levothyroxine treated and ATA+, euthyroid, infertile patients Prevalence of naive and memory B cell subsets (A) , B cell memory subpopulations (B) and CD25 + B cell subpopulations (C) HC: healthy controls (n=12); HT: Hashimoto’s thyroiditis, H1: hypothyroid patients with HT before treatment (n=14); H2: euthyroid patients with HT, after levothyroxine-treatment, (n=12); HIE: HT, infertile, euthyroid patients (n=14); NSw: non-switched; Sw: switched, DN: double negative memory B cells. Differences between the groups and within the B cell subpopulations were assessed with two-way ANOVA and Tukey’s post hoc test except H1-H2 comparisons where a mixed-effects analysis and the Sidak’s multiple comparisons tests were used because these groups represent the same individuals in a repeated sampling before and during treatment. Bars represent the mean ± standard deviation (SD) of percentages of lymphocyte subpopulation in each group. *p < 0.05, **p < 0.01.

Article Snippet: PBMCs (8 x 10 6 cells) were stained with the appropriate combination of fluorochrome-conjugated anti-human antibodies in 100 μl of media in order to differentiate B lymphocyte subsets: CD5 PerCP (clone UCHT2), CD19 PE (clone SJ25C1), IgD APC (clone IA6-2), CD25 BV421 (clone BC96) (all from Sony Biotechnology), CD27 PE-Cy7(clone M-T271, BioLegend).

Techniques: Sampling, Standard Deviation

Journal: Frontiers in Immunology

Article Title: B cells from anti-thyroid antibody positive, infertile women show hyper-reactivity to BCR stimulation

doi: 10.3389/fimmu.2022.1039166

Figure Lengend Snippet: Basal calcium level is elevated in B cells from ATA+, euthyroid, infertile patients Baseline median fluorescence intensity of Fluo4 in B cell subpopulations (A) , CD25- (C) and CD25+ (E) B cells. Cross tabulation shows the p values when comparing patient groups without the subpopulation-based grouping of B cells (B) , CD25- (D) and CD25+ cells (F) . HC: healthy controls (n=12); HT: Hashimoto’s thyroiditis, H1: hypothyroid patients with HT before treatment (n=14); H2: euthyroid patients with HT, after levothyroxine-treatment, (n=12); HIE: HT, infertile, euthyroid patients (n=14); NSw: non-switched; Sw: switched, DN: double negative memory B cells. Differences between the groups and within the B cell subpopulations were assessed with two-way ANOVA and Tukey’s post hoc test except H1-H2 comparisons, where a mixed-effects analysis and the Sidak’s multiple comparisons test were used because these groups represent the same individuals in a repeated sampling before and during treatment. Data are presented as follows: middle line represents the median, dot represents the mean, box: interquartile range, whiskers: min-max. *p < 0.05, **p < 0.01; Significant p values are red, non-significant p values are written in blue.

Article Snippet: PBMCs (8 x 10 6 cells) were stained with the appropriate combination of fluorochrome-conjugated anti-human antibodies in 100 μl of media in order to differentiate B lymphocyte subsets: CD5 PerCP (clone UCHT2), CD19 PE (clone SJ25C1), IgD APC (clone IA6-2), CD25 BV421 (clone BC96) (all from Sony Biotechnology), CD27 PE-Cy7(clone M-T271, BioLegend).

Techniques: Fluorescence, Sampling

Journal: Frontiers in Immunology

Article Title: B cells from anti-thyroid antibody positive, infertile women show hyper-reactivity to BCR stimulation

doi: 10.3389/fimmu.2022.1039166

Figure Lengend Snippet: Naive B cells show higher Ca 2+ signal upon BCR stimulation in ATA+ euthyroid, infertile patients Calcium flux kinetic data of naive B, naive CD25- and naive CD25+ B cells (A) maximum value, (B) ending value, (C) time to reach maximum, (D) time to first 50% value, (E) time from first 50% to maximum, (F) time from maximum to second 50% value, (G) slope at the first 50% value, (H) slope at second 50% value, (I) area under curve (AUC) value. HC: healthy controls (n=12); HT: Hashimoto’s thyroiditis, H1: hypothyroid patients with HT before treatment (n=14); H2: euthyroid patients with HT, after levothyroxine-treatment, (n=12); HIE: HT, infertile, euthyroid patients (n=14); NSw: non-switched; Sw: switched, DN: double negative memory B cells. The kinetic data were analyzed by the FACSKin software. Differences between the groups were assessed with two-way ANOVA and Tukey’s post hoc test except H1-H2 comparisons where a mixed-effects analysis and the Tukey’s multiple comparisons test were used because these groups represent the same individuals in a repeated sampling before and during treatment. Data are depicted as individual values, middle line represents the mean, whiskers are set to standard deviation (SD) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: PBMCs (8 x 10 6 cells) were stained with the appropriate combination of fluorochrome-conjugated anti-human antibodies in 100 μl of media in order to differentiate B lymphocyte subsets: CD5 PerCP (clone UCHT2), CD19 PE (clone SJ25C1), IgD APC (clone IA6-2), CD25 BV421 (clone BC96) (all from Sony Biotechnology), CD27 PE-Cy7(clone M-T271, BioLegend).

Techniques: Software, Sampling, Standard Deviation

Journal: Frontiers in Immunology

Article Title: B cells from anti-thyroid antibody positive, infertile women show hyper-reactivity to BCR stimulation

doi: 10.3389/fimmu.2022.1039166

Figure Lengend Snippet: Memory B cells of ATA+, euthyroid, infertile patients have increased calcium flux upon BCR activation Selected calcium flux kinetic data of memory B cell subsets: Non-switched (NSw) memory B cells, CD25- and CD25+ NSw cells: (A) maximum value, (B) ending value, (C) area under curve (AUC) value. Immunoglobulin class-switched (Sw) memory B cells, CD25- and CD25+ Sw cells: (D) maximum value, (E) ending value, (F) area under curve (AUC) value. CD27-, IgD- double-negative (DN) memory B cells, CD25- and CD25+ DN cells: (G) maximum value, (H) ending value, (I) area under curve (AUC) value. HC: healthy controls (n=12); HT: Hashimoto’s thyroiditis, H1: hypothyroid patients with HT before treatment (n=14); H2: euthyroid patients with HT, after levothyroxine-treatment, (n=12); HIE: HT, infertile, euthyroid patients (n=14). The kinetic data were analyzed by the FACSKin software. Differences between the groups were assessed with two-way ANOVA and Tukey’s post hoc test except H1-H2 comparisons where a mixed-effects analysis and the Tukey’s multiple comparisons test were used because these groups represent the same individuals in a repeated sampling before and during treatment. Data are depicted as individual values, middle line represents the mean, whiskers are set to standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: PBMCs (8 x 10 6 cells) were stained with the appropriate combination of fluorochrome-conjugated anti-human antibodies in 100 μl of media in order to differentiate B lymphocyte subsets: CD5 PerCP (clone UCHT2), CD19 PE (clone SJ25C1), IgD APC (clone IA6-2), CD25 BV421 (clone BC96) (all from Sony Biotechnology), CD27 PE-Cy7(clone M-T271, BioLegend).

Techniques: Activation Assay, Software, Sampling, Standard Deviation

Journal: Nature

Article Title: T FH -derived dopamine accelerates productive synapses in germinal centres

doi: 10.1038/nature23013

Figure Lengend Snippet: a, Fold changes of surface ICOSL expression on mouse GC B cells that were treated with anti-CD40 (10 μg/ml) and DA (0.5, 1, 5, 10μM) for 30 minutes, with medium control set as unit 1 (n=5). b, Representative histogram and quantification of surface and intracellular ICOSL on GC and non-GC B cells (n=5). **p ≤ 0.01; nonparametric Mann-Whitney test (U test). c, RNA counts per million of ICOSL, CD40, BCL6, IL21R, CD86, BAFFR and FAS mRNA in human memory B cells stimulated with or without DA (5μM) for 2h (n=3). d , Fold changes of surface ICOSL expression on mouse GC B cells that were treated with cycloheximide (CHX, 10 μg/ml) for 4h, with medium control set as unit 1. Bars represent median values and each dot represents a single mouse. e, Fold changes of surface ICOSL expression on mouse GC B cells that were stimulated with BAFF (100ng/ml), LPS (1 or 10 μg/ml), anti-CD40 (10 μg/ml) and anti-IgM (1 or 10 μg/ml) for 30 min and 4h. Unit 1 set on medium control. f, Fold changes of surface ICOSL expression on mouse GC B cells that were treated with actinomycin D (ActD, 5 μg/ml), anti-CD40 (10 μg/ml) for 4h, with medium control set as unit 1. Bars represent median and each dot represent a single mouse (n=5). d-f , ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001; two tailed student t-test. g , Representative histogram of surface ICOSL expression on human GC B cells that were stimulated with DA (10μM) or anti-CD40 (1 μg/ml) for 30 min. h , Fold changes of surface ICOSL expression on human GC B cells stimulated with several concentrations of anti-CD40 for 4 and 8 hours, with medium control set as unit 1 (n=3). i , Bar plot showing survival of GC B in the presence of anti-CD40 (1 μg/ml) after 4 or 8 hours of stimulation (n=4). *p ≤ 0.05 and ***p ≤ 0.001; nonparametric Mann-Whitney test (U test).

Article Snippet: Tonsillar lymphocytes were stained with the following anti-human antibodies – CD4 APCCy7 (RPA-T4, BD Biosciences), CXCR5 Alexa 488 or Alexa 647 (RF8B2, BD Biosciences), PD-1 PE (MIH4, eBioscience) or BV605 or BV421 (EH12.2H7, BioLegend), CD127 FITC (11-1278, eBioscience) or BV 421 (A019D5, BioLegend), CD25 biotin (BC96, eBioscience or BioLegend) or PE-Cy7 (BC96, BD Biosciences or BioLegend), BCL6 Alexa 647 or PE-Cy7 (K112-91, BD Biosciences), CD3 APC (HIT3a, BD Biosciences) or Alexa 700 (UCHT1, BD Biosciences), CD27 FITC or APC (M-T271, BD Biosciences), CD38 FITC (HIT2, BD Biosciences) or PE (HB7, BD Biosciences), ICOSL APC (2D3, BioLegend), FAS PE-CF594 (DX2, BD Bioscience), CD40 APCCy7 (5C3, BioLegend), BAFFR PECy7 (11C1, BioLegend), CD19 PECy7 or BV605 (SJ25C1, BD Bioscience), IL21R BV421 (17A12, BioLegend), CD86 BV421 (2331/FUN-1, BD Bioscience).

Techniques: Expressing, MANN-WHITNEY, Two Tailed Test

Journal: Nature

Article Title: T FH -derived dopamine accelerates productive synapses in germinal centres

doi: 10.1038/nature23013

Figure Lengend Snippet: a , Representative images of ICAM-1 ring (white) around CD40L (pseudocolor scale) in the presence or absence of CD40 and ICOSL at physiological densities on the supported lipid bilayer (SLB) containing ICAM-1 and UCHT1. Scale bar 5 µm. b , c , Plots represent CD40L MFI of individual activated human T ( b ) or T FH ( c ) cells forming synapses (n=3). d , Representative images of chromogranin B stain in the presence or absence of ICOSL at the immunological synapse. e , Plots represent CgB fluorescent intensity of individual activated T FH and non-T FH cells forming synapses (n=3). b,c,e , ns, not significant, (***p ≤ 0.001) and (****p ≤ 0.0001) nonparametric Mann-Whitney test (U test). f , Representative images of CgB + T FH cells (red) forming synapses with allogeneic B cells (green).

Article Snippet: Tonsillar lymphocytes were stained with the following anti-human antibodies – CD4 APCCy7 (RPA-T4, BD Biosciences), CXCR5 Alexa 488 or Alexa 647 (RF8B2, BD Biosciences), PD-1 PE (MIH4, eBioscience) or BV605 or BV421 (EH12.2H7, BioLegend), CD127 FITC (11-1278, eBioscience) or BV 421 (A019D5, BioLegend), CD25 biotin (BC96, eBioscience or BioLegend) or PE-Cy7 (BC96, BD Biosciences or BioLegend), BCL6 Alexa 647 or PE-Cy7 (K112-91, BD Biosciences), CD3 APC (HIT3a, BD Biosciences) or Alexa 700 (UCHT1, BD Biosciences), CD27 FITC or APC (M-T271, BD Biosciences), CD38 FITC (HIT2, BD Biosciences) or PE (HB7, BD Biosciences), ICOSL APC (2D3, BioLegend), FAS PE-CF594 (DX2, BD Bioscience), CD40 APCCy7 (5C3, BioLegend), BAFFR PECy7 (11C1, BioLegend), CD19 PECy7 or BV605 (SJ25C1, BD Bioscience), IL21R BV421 (17A12, BioLegend), CD86 BV421 (2331/FUN-1, BD Bioscience).

Techniques: Staining, MANN-WHITNEY

Journal: Nature

Article Title: T FH -derived dopamine accelerates productive synapses in germinal centres

doi: 10.1038/nature23013

Figure Lengend Snippet: a , Activated human T cells that express ICOS and CD40L were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3) as a basal condition with a ring of ICAM-1 surrounding a central cluster enriched in T cell receptor enriched extracellular vesicles by 15 minutes . This condition resulted in low presentation of CD40L in punctate structures detected by anti-CD40L mAb that accumulated in the same central synapse with the TCR enriched extracellular vesicles. Addition of ICOSL the SLB resulted in strong central accumulation of fluorescent ICOSL with the TCR enriched extracellular vesicles, but no increase in CD40L presentation. Addition of CD40 the SLB resulted in a significant increase in CD40L accumulation, which we refer to as reception because its receptor dependent. When ICOSL and CD40 were added the reception of CD40L was further significantly enhanced over the level observed with CD40 alone. Thus, ICOSL ligation in the centre of the immunological synapse increases CD40L reception. All levels are shown in gray scale except CD40L panels, for which the pseudocolor scale is indicated. Scale bar 5 µm. b , Human T FH cells were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3). Addition of ICOSL resulted in increased accumulation of CgB at the synapse centre. Addition of CD40 did not further increased CgB accumulation.

Article Snippet: Tonsillar lymphocytes were stained with the following anti-human antibodies – CD4 APCCy7 (RPA-T4, BD Biosciences), CXCR5 Alexa 488 or Alexa 647 (RF8B2, BD Biosciences), PD-1 PE (MIH4, eBioscience) or BV605 or BV421 (EH12.2H7, BioLegend), CD127 FITC (11-1278, eBioscience) or BV 421 (A019D5, BioLegend), CD25 biotin (BC96, eBioscience or BioLegend) or PE-Cy7 (BC96, BD Biosciences or BioLegend), BCL6 Alexa 647 or PE-Cy7 (K112-91, BD Biosciences), CD3 APC (HIT3a, BD Biosciences) or Alexa 700 (UCHT1, BD Biosciences), CD27 FITC or APC (M-T271, BD Biosciences), CD38 FITC (HIT2, BD Biosciences) or PE (HB7, BD Biosciences), ICOSL APC (2D3, BioLegend), FAS PE-CF594 (DX2, BD Bioscience), CD40 APCCy7 (5C3, BioLegend), BAFFR PECy7 (11C1, BioLegend), CD19 PECy7 or BV605 (SJ25C1, BD Bioscience), IL21R BV421 (17A12, BioLegend), CD86 BV421 (2331/FUN-1, BD Bioscience).

Techniques: Incubation, Ligation