Journal: Cell Reports

Article Title: Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in naive and COVID-19 recovered individuals

doi: 10.1016/j.celrep.2021.109570

Figure Lengend Snippet:

Article Snippet: APC-efluor780 anti-human CD25 (clone BC96) , Invitrogen , 47-0259-42.

Techniques: Recombinant, Software, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus

doi: 10.3389/fimmu.2024.1380481

Figure Lengend Snippet: Manual gating strategy applied to PBMCs in both HC and SLE for the identification of (A) T-cell, NK-cell, NKT-cell subsets in the first panel, and (B) B-cell and monocyte subsets in the second panel in activated state. In both panels, cell debris, doublets and dead cells were excluded. (A) In the first panel, live cells were initially gated to CD3 + T-cells, CD56 dim NK-cells, CD56 high NK-cells and CD3 + CD56 + NKT-cells. T-cells were further gated to CD4 + T-cells, CD8 + T-cells, CD4 + CD8 + double positive T-cells (DPT) and CD4 - CD8 - double negative (DNT). CD8 + T-cells were subdivided into CD8 dim and CD8 high T-cells and DPTs were categorized as CD4 high or CD8 high DPTs. NKTs were further gated to CD4 + , CD8 + and CD4 - CD8 - DN NKTs. The activation state was defined based on CD25 expression in this panel. (B) In the second panel, B-cells were identified by CD19 expression, and subsequent gating distinguished CD27 - naive B-cells, CD27 + memory B-cells, and CD27 ++ CD38 ++ plasmablasts. CD19 - cells were further categorized into CD14 ++ CD16 - classical monocytes, CD14 + CD16 - transitional monocytes, and CD14 ++ CD16 + intermediate monocytes. Non-classical monocytes were defined as CD14 + CD16 ++ HLA-DR ++ cells. Representative pseudocolor plots and histograms are provided.

Article Snippet: Dilution of antibodies: anti-human CD3 efluor450 (Cat. 48-0038-42, clone: UCHT1, Thermo Fisher Scientific, Waltham, Massachusetts, USA) 80x; anti-human CD4 Alexa Fluor 700 ® (Cat. 317426, clone: OKT4, BioLegend) 80x; anti-human CD8 PerCP (Cat. MHCD0831, clone: 3B5, Thermo Fisher Scientific) 80x; anti-human CD56 BV605 (Cat.562780, clone: NCAM16.2, Beckton Dickinson, Franklin Lakes, USA) 80x; anti-human CD25 Super BrightTM 645 (Cat. 64-0259-42, clone: BC96, Thermo Fisher Scientific) 40x.

Techniques: Activation Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus

doi: 10.3389/fimmu.2024.1380481

Figure Lengend Snippet: Frequencies of main immune subsets identified in PBMCs of heathy control (HC) individuals and SLE patients. Frequencies were measured in the (A) unstimulated resting state and (B) after 72-hour activation. T-cells, NK-cells and NKT-cells were stimulated with CytoStim™ and recombinant IL-2 (n = 13 for HC; n = 13 for SLE), while B-cells and monocytes received a cocktail of LPS and TLR9 agonist (n = 18 for HC, n = 18 for SLE). (C) For the activated cells of the first panel, the percentage of CD25 + cells within each population is shown (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). (D) Spearman correlation between the population frequencies of the SLE patients and their SLEDAI-2K scores or anti-double stranded DNA (anti-ds-DNA) antibody levels. Each point on scatter plot represents one individual subject, and the line represents the linear regression. Next to each plot, Spearman correlation coefficients (r) and associated P values.

Article Snippet: Dilution of antibodies: anti-human CD3 efluor450 (Cat. 48-0038-42, clone: UCHT1, Thermo Fisher Scientific, Waltham, Massachusetts, USA) 80x; anti-human CD4 Alexa Fluor 700 ® (Cat. 317426, clone: OKT4, BioLegend) 80x; anti-human CD8 PerCP (Cat. MHCD0831, clone: 3B5, Thermo Fisher Scientific) 80x; anti-human CD56 BV605 (Cat.562780, clone: NCAM16.2, Beckton Dickinson, Franklin Lakes, USA) 80x; anti-human CD25 Super BrightTM 645 (Cat. 64-0259-42, clone: BC96, Thermo Fisher Scientific) 40x.

Techniques: Control, Activation Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus

doi: 10.3389/fimmu.2024.1380481

Figure Lengend Snippet: Binding capacity of five lectins (AAL, Gal-1, Gal-3, SNA and Siglec-1) to T-cell subsets in PBMCs of 13 SLE patients and 13 age- and sex-matched healthy control (HC) individuals analyzed by flow cytometry. The binding of fluorochrome-conjugated lectins was quantified as median fluorescence intensity (MFI) in the (A) unstimulated resting state and (B) after 72-hour activation. In the latter case, T-cells in the whole PBMC sample were stimulated with CytoStim™ and recombinant IL-2. (C) For the activated T-cells, lectin binding ratio was calculated between CD25 + and CD25 - cells within each T-cell subset (MFI CD25+ /MFI CD25- ). The symbol “#” marks significant difference (P ≤ 0.05) between CD25 + and CD25 - fractions, and asterisks between HC and SLE, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Data represents the mean + SEM (solid bars: HC; striped: SLE). (D) Spearman correlation between the lectin binding of activated T-cell subsets of SLE patients and their SLEDAI-2K indices. (E) Spearman correlation between the lectin binding ratio of activated CD25 + per CD25 - T-cell subsets of SLE patients and their SLEDAI-2K scores. Each point on scatter plot represents one subject, and the line represents the linear regression. Next to each plot, Spearman correlation coefficients (r) and associated P values.

Article Snippet: Dilution of antibodies: anti-human CD3 efluor450 (Cat. 48-0038-42, clone: UCHT1, Thermo Fisher Scientific, Waltham, Massachusetts, USA) 80x; anti-human CD4 Alexa Fluor 700 ® (Cat. 317426, clone: OKT4, BioLegend) 80x; anti-human CD8 PerCP (Cat. MHCD0831, clone: 3B5, Thermo Fisher Scientific) 80x; anti-human CD56 BV605 (Cat.562780, clone: NCAM16.2, Beckton Dickinson, Franklin Lakes, USA) 80x; anti-human CD25 Super BrightTM 645 (Cat. 64-0259-42, clone: BC96, Thermo Fisher Scientific) 40x.

Techniques: Binding Assay, Control, Flow Cytometry, Fluorescence, Activation Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus

doi: 10.3389/fimmu.2024.1380481

Figure Lengend Snippet: Lectin binding properties and FlowSOM clustering analysis of manually gated NK-cell subsets of 13 SLE patients and 13 age- and sex-matched HCs. The binding of fluorochrome-conjugated lectins was quantified as median fluorescence intensity (MFI) in the (A) unstimulated resting state and (B) after 72-hour activation. In the latter case, NK-cells in the whole PBMC sample were stimulated with CytoStim™ and recombinant IL-2. (C) For the activated NK-cells, lectin binding ratio was calculated between CD25 + and CD25 - cells within each NK-cell subset (MFI CD25+ /MFI CD25- ). The symbol “#” marks significant difference (P ≤ 0.05) between CD25 + and CD25 - fractions. For the clustering analysis, activated (D) CD56 dim NK-cell and (F) CD56 high NK-cell populations of HCs and SLE patients were concatenated and FlowSOM was combined with t-SNE visualization. On the left t-SNE plots, varying colors correspond to relative cell density, while on the right t-SNE plots FlowSOM metaclusters (MC01-MC06) are shown with different colors. Heatmap is generated from the MFI profile of FlowSOM metaclusters with rows representing each cluster and columns the markers of interest. Bar graphs denote the percentage of metaclusters within the analyzed NK-cell subset. Data are presented as mean + SEM (solid bars: HC; striped bars: SLE). *P ≤ 0.05, **P ≤ 0.01. Spearman correlation between the metacluster frequencies of CD56 dim (E) and CD56 high (G) NK-cells of SLE patients and their SLEDAI-2K scores. Each point on scatter plot represents one subject, and the line represents the linear regression. Next to each plot, Spearman correlation coefficients (r) and associated p values.

Article Snippet: Dilution of antibodies: anti-human CD3 efluor450 (Cat. 48-0038-42, clone: UCHT1, Thermo Fisher Scientific, Waltham, Massachusetts, USA) 80x; anti-human CD4 Alexa Fluor 700 ® (Cat. 317426, clone: OKT4, BioLegend) 80x; anti-human CD8 PerCP (Cat. MHCD0831, clone: 3B5, Thermo Fisher Scientific) 80x; anti-human CD56 BV605 (Cat.562780, clone: NCAM16.2, Beckton Dickinson, Franklin Lakes, USA) 80x; anti-human CD25 Super BrightTM 645 (Cat. 64-0259-42, clone: BC96, Thermo Fisher Scientific) 40x.

Techniques: Binding Assay, Fluorescence, Activation Assay, Recombinant, Generated

Journal: Frontiers in Immunology

Article Title: Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus

doi: 10.3389/fimmu.2024.1380481

Figure Lengend Snippet: Lectin binding profile and FlowSOM clustering analysis of manually gated NKT-cell subsets of 13 SLE patients and 13 age- and sex-matched HCs. The binding of five fluorochrome-conjugated lectins was measured as median fluorescence intensity (MFI) in the (A) unstimulated resting state and (B) after 72-hour activation. In the latter case, NKT-cells in the pool of PBMCs were stimulated with CytoStim™ and recombinant IL-2. (C) For the activated NKT-cells, the lectin binding ratio was calculated between CD25 + and CD25 - cells within each NKT-cell subset (MFI CD25+ /MFI CD25- ). The symbol “#” marks significant difference (P ≤ 0.05) between CD25 + and CD25 - fractions, and asterisks denote significance between HC and SLE. Spearman correlation was performed between the (D) AAL binding properties or (E) Siglec-1 CD25 + per CD25 - binding ratio of activated DN NKT-cells of SLE patients and their SLEDAI-2K or anti-ds-DNA antibody values. Each point on scatter plot represents one subject, with the line indicating linear regression. Next to each plot, Spearman correlation coefficients (r) and associated P values. For clustering analysis, activated (F) CD4 NKT-cell, (G) CD8 NKT-cell and (H) DN NKT-cell populations from both HCs and SLE patients were concatenated, and FlowSOM was combined with t-SNE visualization. On the left t-SNE plots, color gradients from dark blue to dark red indicates increasing cell density, whereas on the right t-SNE plots color-coded FlowSOM metaclusters (MC01-MC06) are displayed. The MFI profile of each FlowSOM metacluster is visualized in a heatmap. Bar charts denote the frequency of each metacluster within the analyzed NK-cell subset. (I) Spearman correlation between the MC04 metacluster frequency of DN NKT-cells of SLE patients and their anti-ds-DNA antibody values. Each point on scatter plot represents one subject, and the line represents the linear regression. Adjacent to the plot, Spearman correlation coefficient (r) and associated P value. Data are presented as mean + SEM (solid bars: HC; striped bars: SLE). *p ≤ 0.05, **p ≤ 0.01.

Article Snippet: Dilution of antibodies: anti-human CD3 efluor450 (Cat. 48-0038-42, clone: UCHT1, Thermo Fisher Scientific, Waltham, Massachusetts, USA) 80x; anti-human CD4 Alexa Fluor 700 ® (Cat. 317426, clone: OKT4, BioLegend) 80x; anti-human CD8 PerCP (Cat. MHCD0831, clone: 3B5, Thermo Fisher Scientific) 80x; anti-human CD56 BV605 (Cat.562780, clone: NCAM16.2, Beckton Dickinson, Franklin Lakes, USA) 80x; anti-human CD25 Super BrightTM 645 (Cat. 64-0259-42, clone: BC96, Thermo Fisher Scientific) 40x.

Techniques: Binding Assay, Fluorescence, Activation Assay, Recombinant