Journal: Immunity

Article Title: Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease

doi: 10.1016/j.immuni.2021.05.010

Figure Lengend Snippet:

Article Snippet: Anti-human CD25 PE (BC96) , Thermo , RRID: AB_1659682.

Techniques: Staining, Recombinant, Membrane, Enzyme-linked Immunosorbent Assay, Cell Counting, Flow Cytometry, Software, Enzyme-linked Immunospot, Cytometry, Luminex

Journal: Archivum Immunologiae et Therapiae Experimentalis

Article Title: Cyclosporine A, in Contrast to Rapamycin, Affects the Ability of Dendritic Cells to Induce Immune Tolerance Mechanisms

doi: 10.1007/s00005-021-00632-7

Figure Lengend Snippet: Effects of Rapa-DC and CsA-DC on T regulatory cells populations. Immature DCs, Rapa-DC and CsA-DC were cocultured with T cells. The percentage of CD4 + CD25 high Foxp3 + ( A ), CD8 + CD25 + CD28 + ( B ) and CD8 + CD25 – CD28 – ( C ) T cells were determined by flow cytometry. Results are the averages ± SD from ten different donors. * p ≤ 0.05; p values were calculated by Wilcoxon matched pair test

Article Snippet: After 5 days of coculture, the cells were harvested and labelled with the following fluorochrome-conjugated monoclonal antibodies: CD3-APC (clone: OKT3, eBioscience, USA), CD69-FITC (clone: FN50, eBioscience, USA) and CD25-PE (clone: BC96, BD Pharmingen, USA) to identify T cells or CD11c-APC and PD-L1-FITC (clone: MIH1, BD Pharmingen, USA) to identify DCs.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Evidence that recurrent Group A streptococcus tonsillitis is an immunosusceptibility disease involving antibody deficiency and aberrant Tfh cells

doi: 10.1101/356741

Figure Lengend Snippet: (A) Flow cytometry identification of GAS-specific CD4 + T cells (CD45RA-) and GAS-specific GC Tfh cells (CD45RA-CXCR5 high PD-1 high ) using an antigen-specific TCR-dependent activation induced marker (AIM) assay (OX40 + CD25 + , AIM25). Tonsil cells were left unstimulated or stimulated with 10μg/mL antibiotic-killed Lactococcus lactis (a non-pathogenic Gram positive bacteria which served as a negative control), 10μg/mL heat-inactivated antibiotic-killed GAS, or 1μg/mL staphylococcal enterotoxin B (SEB, positive control) for 18 hours. (B) Significantly more GAS-specific GC Tfh cells are detected by AIM 25 (CD25 + OX40 + ) compared to a negative control antigen. Cells were stimulated with heat-inactivated, antibiotickilled GAS (‘GAS’) or antibiotic-killed L. lactis (‘ Lactococcus ’). (C) GAS-specific CD4 + T cell frequencies in RT and non-RT tonsils. Total antigen-experienced cells were gated (CD45RA − ). (D) RT patients exhibit a bias against GAS-specific GC Tfh differentiation. Among total GASspecific CD4 + T cells (AIM 25 + CD45RA − ), a smaller fraction of cells are GC Tfh cells (CXCR5 high PD-1 high ) in RT donors compared to non-RT donors.

Article Snippet: Anti-human antibodies for the proliferation assay using HLA class II cell lines are listed here, by company, Thermo Fisher Scientific: OX40 FITC (clone Ber-ACT35), CD25 PE-Cyanine 7 (clone BC96), CD4 PerCP-eFluor710 (clone SK3); Biolegend: PD-1 BV785 (clone EH12.2H7), PD-L1 PE (clone 29E.2A3) ( ).

Techniques: Flow Cytometry, Activation Assay, Marker, Bacteria, Negative Control, Positive Control

Journal: Scientific Reports

Article Title: Immunophenotyping of rheumatoid arthritis reveals a linkage between HLA-DRB1 genotype, CXCR4 expression on memory CD4 + T cells, and disease activity

doi: 10.1038/srep29338

Figure Lengend Snippet: ( A ) CXCR4 expression on synovial fluid (SF) and PBMC CD4 + T cells. Left: subset ratios of memory (CD45RA-), Th1 (CXCR3), Th17 (CCR6), Tfh (CXCR5), Treg (CD25) and CXCR4 + RA synovial fluid CD4 + T cells (n = 5). Mean ± SEM values. Right: representative plots of CXCR4 expression in PBMC and synovial fluid (SF) samples from healthy donors (HD) and RA patients. The CD45RA − CXCR4 + ratios are shown in the plots. The CXCR4 positivity of the CD45RA- subset (MemoryTh subset) is HD 7.8%, SE + RA 36%, and SE + RA 87% in the representative plots. ( B ) Comparison of CXCR4 expression on CD4 + T cell subsets among healthy donors (HD), shared epitope (SE)-negative RA patients, and SE-positive RA patients (HD; n = 82, SE-RA; n = 8, SE + RA; n = 27). ( C ) Correlations between CXCR4 expression on CD4 + T cell subsets and clinical parameters in RA patients (n = 35). A Representative scatter plot is shown on the right, as in . Tfh1, Tfh2, and Tfh17 subpopulations were eliminated from the analysis ( B,C ). ( D ) Principal component analysis of immunophenotyped subsets and CXCR4 expression on CD4 + T cell subsets to summarize the differences between HD, SE-RA, and SE + RA. PC1 explained 29% and PC2 explained 16% of the total variance (HD; n = 81, SE-RA; n = 8, SE + RA; n = 25). *p < 0.05 **p < 0.01 ***P < 0.001 Spearman’s test ( C ) or Kruskal-Wallis tests with post-hoc Wilcoxon tests with Bonferroni corrections ( B ).

Article Snippet: The following antibodies were used: Human Fc Receptor Binding Inhibitor Purified (eBioscience), CD3-PE-Cy7 (UCHT1, BioLegend), CD3-PerCP-Cy5.5 (UCHT1, BioLegend), CD4-PerCP-Cy5.5 (OKT4, BioLegend), CD4-V500 (RPA-T4, BD Biosciences), CD11c-Brilliant Violet 421 (B-ly6, BD Biosciences), CD14-FITC (M5E2, BioLegend), CD16-PerCP-Cy5.5 (3G8, BioLegend), CD19-APC-Cy7 (HIB19, BioLegend), CD19-V500 (HIB19, BD Biosciences), CD24-PE (ML5, BD Biosciences), CD25-Brilliant Violet 421 (BC96, BioLegend), CD25-PE-Cy7 (BC96, eBioscience), CD27-FITC (O323, eBioscience), CD38-PE-Cy7 (HIT2, BioLegend), CD45RA-APC-Cy7 (HI100, BioLegend), CD56 -APC-Cy7 (HCD56, BioLegend), CD123-APC (AC145, Miltenyi), CD127-PE-Cy7 (eBioRDR5, eBioscience), CXCR3-Brilliant Violet 421 (1C6, BD Biosciences), CXCR4-APC (12G5, BD Biosciences), CXCR5-Alexa Fluor 488 (RF8B2, BD Biosciences), CCR6-PE (11A9, BD Biosciences), CCR7-PerCP-Cy5.5 (G043H7, Biolegend), HLA-DR-PE (L243, eBioscience), IgD-Brilliant Violet 421 (IA6-2, BD Biosciences).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Differentially activated B cells develop regulatory phenotype and show varying immunosuppressive features: a comparative study

doi: 10.3389/fimmu.2023.1178445

Figure Lengend Snippet: Differentially activated B cells suppress the activity of effector cells and mediate CD4 + T cells differentiation into Tregs (CD4 + CD25 hi ) with simultaneous induction of IL10 secretion. (A) Tregs proliferation induced by co-cultivation of differentially activated B cells and CD4 + T cells. The percentage of divided Tregs was determined by flow cytometry using CellTrace Violet dye. (B) IL10 level was determined in the medium from CD4 + T cells co-cultivated with differentially activated B cells on day 5 of cultivation using ELISA. Mean values ± SEM of four independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001 compared to untreated control, as calculated by ANOVA. (C) Activated B cells influence the proliferation rate of T helpers, T killers, and NK cells from CD19-depleted PBMCs after 5 days of co-cultivation. (D) IFNγ, TNF, IL1β, IL6, IL10 normalized levels were determined using ELISA in the medium from B cell-depleted PBMCs after co-cultivation. The average values of four independent experiments are shown. (E) Activated B cells reduce NK-mediated killing of cancer cell line MCF7. Mean values ± SD of three independent experiments is shown.

Article Snippet: Cells were stained with antibodies to the main phenotypic molecular markers of regulatory T cells: CD4-APC (clone RPA-T4, lot No. B307926, BioLegend), CD25-PE (clone BC96, lot No. 2173867, eBioscience), and CD127-FITC (clone A019D5, lot No. E-AB-F1152L, Elabscience, USA).

Techniques: Activity Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay