147sm anti cd20  (fluidigm)

Journal: Immunology

Article Title: Delineation of the Human Germinal Centre Immune Landscape Using Multiplex Imaging Analysis.

doi: 10.1111/imm.13955

Figure Lengend Snippet: FIGURE 1 | TFH cells constitute a phenotypically heterogeneous cell compartment. (A) Tonsillar single cell suspensions (n = 4) were analysed using a CyTOF (N = 34 parameters) assay. The gating scheme for the identification of main cell subsets is shown. (B) Heatmap showing the relative mean expression of selected protein markers in bulk CD4hi memory, CD4hiCXCR5loPD1lo memory and TFH cell subsets (n = 8) in different tonsils (D1- D4). A colour indicator is provided. (C) UMAP analysis demonstrating the clustering of TFH clusters across four individual donors based on selected protein markers. (D) UMAP analysis showing the original clusters and annotations of CD4 tonsillar cells based on scRNA analysis (upper, left panel). The prevalence of PD1hi cells (upper, middle panel) and the subset of CD8−CD20−CD4+PD1+ TFH cells (upper, right panel) are shown. Expression of selected marker genes (middle row) and cluster membership per donor (lower row, right) are shown for UMAPs and re-clustering generated using the selected cells from five donors and the genes selected from protein analyses (lower row, left).

Article Snippet: Amount Rhodium 103Rh Cell- ID Standard Biotools 201103A 0.6 μL CD8 113ln RPA- T8 BioLegend 301018 0.6 μL CD4 115In RPA- T4 BioLegend 300515 0.6 μL CCR6 141Pr 11A9 Standard Biotools 3141014A 1 μL CD19 142Nd HIB19 Standard Biotools 3142001B 1.6 μL CCR6 144Nd G034E3 Standard Biotools 3141003A 1.88 μL CD20 147Sm 2H7 Standard Biotools 3147001B 0.7 μL ICOS 148Nd C398.4A Standard Biotools 3148019B 1 μL CCR4 149Sm L291H4 Standard Biotools 3149029A 1.5 μL CD40L 152Sm TRAP1 BD Biosciences 555698 3 μL TIGIT 153Eu MBSA43 Standard Biotools 3153019B 2 μL CD3 154Sm UCHT1 Standard Biotools 3154003B 0.5 μL CD27 155Gd L128 Standard Biotools 3155001B 1 μL CXCR3 156Gd G025H7 Standard Biotools 3156004B 0.8 μL CCR7 159 Tb G043H7 Standard Biotools 3159003A 1 μL CD30 162Dy BY88 BioLegend 333902 3 μL CXCR5 164Dy RF8B2 Standard Biotools 3164029B 1.2 μL CD45RO 165Ho UCHL1 Standard Biotools 3165011B 0.7 μL CD38 167Er HIT2 Standard Biotools 3167001B 0.6 μL CD45RA 170Er HI100 Standard Biotools 3171002B 1.6 μL HLA- DR 174Yb L243 Standard Biotools 3174001B 0.7 μL CD279/PD- 1 175Lu EH12.2H7 Standard Biotools 3175008B 1 μL CD127 176Yb A019D5 Standard Biotools 3176004B 0.7 μL CTLA4 194Pt Ipilimumab BMS ACB3065 2.5 μL CD57 198Pt NK- 1 BD Biosciences 555618 1 μL CD16 209Bi 3G8 Standard Biotools 3209002B 1.5 μL TABLE 2 | Antibodies used for the mass cytometry intracellular characterisation of tonsillar- derived cells.

Techniques: Expressing, Marker, Generated

Journal: Immunology

Article Title: Delineation of the Human Germinal Centre Immune Landscape Using Multiplex Imaging Analysis.

doi: 10.1111/imm.13955

Figure Lengend Snippet: FIGURE 2 | In situ phenotypic heterogeneity of TFH cells revealed by mIF imaging. (A) Representative fluorescent images showing the expres- sion of biomarkers CD3 (blue), CD20 (red), CD8 (yellow), CD4 (green), PD1 (cyan) and CD57 (grey) used in the mIF assay. Representative images from one tonsil are shown (scale bar: 150 μm). (B) The histocytometry gating scheme for the identification of B (CD20hi/dim), CD3hi, CD4hi and follic- ular (F) CD3hiPD1hi cells in an LN is shown. Individual follicular areas were identified based on the density of CD20hi/dim cells. All follicular areas were combined (using the FlowJo10 module in one follicle for downstream analysis. The total extrafollicular area is shown. (C) The expression (raw mean fluorescence intensities) of individual biomarkers in F-CD3hiPD1hi TFH, F-CD3hiCD4hiPD1lo non-TFH, extrafollicular (EF)-CD3hiCD8hi and F- CD20hi/dimCD3lo B cells are shown as histograms (upper two rows) or 2D contour plots (biomarker vs. PD1) (lower two rows). Imaging data from an LN were used. (D) Six Histocytometry identified follicular areas (top row) and 2D dot plots showing the associated expression of biomarkers used vs. PD1 in CD3loCD4lo (as reference cell subset-grey) and CD3hiPD1hi TFH cells (purple).

Article Snippet: Amount Rhodium 103Rh Cell- ID Standard Biotools 201103A 0.6 μL CD8 113ln RPA- T8 BioLegend 301018 0.6 μL CD4 115In RPA- T4 BioLegend 300515 0.6 μL CCR6 141Pr 11A9 Standard Biotools 3141014A 1 μL CD19 142Nd HIB19 Standard Biotools 3142001B 1.6 μL CCR6 144Nd G034E3 Standard Biotools 3141003A 1.88 μL CD20 147Sm 2H7 Standard Biotools 3147001B 0.7 μL ICOS 148Nd C398.4A Standard Biotools 3148019B 1 μL CCR4 149Sm L291H4 Standard Biotools 3149029A 1.5 μL CD40L 152Sm TRAP1 BD Biosciences 555698 3 μL TIGIT 153Eu MBSA43 Standard Biotools 3153019B 2 μL CD3 154Sm UCHT1 Standard Biotools 3154003B 0.5 μL CD27 155Gd L128 Standard Biotools 3155001B 1 μL CXCR3 156Gd G025H7 Standard Biotools 3156004B 0.8 μL CCR7 159 Tb G043H7 Standard Biotools 3159003A 1 μL CD30 162Dy BY88 BioLegend 333902 3 μL CXCR5 164Dy RF8B2 Standard Biotools 3164029B 1.2 μL CD45RO 165Ho UCHL1 Standard Biotools 3165011B 0.7 μL CD38 167Er HIT2 Standard Biotools 3167001B 0.6 μL CD45RA 170Er HI100 Standard Biotools 3171002B 1.6 μL HLA- DR 174Yb L243 Standard Biotools 3174001B 0.7 μL CD279/PD- 1 175Lu EH12.2H7 Standard Biotools 3175008B 1 μL CD127 176Yb A019D5 Standard Biotools 3176004B 0.7 μL CTLA4 194Pt Ipilimumab BMS ACB3065 2.5 μL CD57 198Pt NK- 1 BD Biosciences 555618 1 μL CD16 209Bi 3G8 Standard Biotools 3209002B 1.5 μL TABLE 2 | Antibodies used for the mass cytometry intracellular characterisation of tonsillar- derived cells.

Techniques: In Situ, Imaging, Expressing, Fluorescence, Biomarker Discovery

Journal: Immunology

Article Title: Delineation of the Human Germinal Centre Immune Landscape Using Multiplex Imaging Analysis.

doi: 10.1111/imm.13955

Figure Lengend Snippet: FIGURE 3 | Differential localisation and distribution profile among TFH cell subsets. (A) Representative fluorescent images showing the expres- sion of CD20 (red), Ki67 (magenta) and Bcl6 (grey) in tonsillar follicles (scale bar: 300 μm). (B) 2D plots showing the expression of Ki67 vs. Bcl6 in total follicular (F) area from one tonsil and the corresponding localisation of F-CD20hi/dimCD3loKi67loBCl6lo (blue-MZ), F-CD20hi/dimCD3loKi67hiBCl6hi (magenta-DZ) and F-CD20hi/dimCD3loKi67loBCl6hi (grey-LZ) (left). The relevant B cell subsets and their localisation for one representative follicle are also shown (right). (C) 2D plots showing the gating for the identification of B subsets (based on the expression of Ki67 and Bcl6) and CD3hiPD1hi TFH cell subsets (based on the expression of CD57, CXCR3, TIGIT and Bcl6) as well as their localisation across the areas of individual follicles (data from one LN). (D) Dot plot graphs showing accumulated data for the analysis of the minimum distance between DZ (CD20hi/dimCD3loKi67hiBcl- 6hi) (open and blue circles) or LZ (CD20hi/dimCD3loKi67loBcl6hi) (green and red circles) B cells and TFH cell subsets (CD3hiPD1hiCD57loCXCR3hi, CD3hiPD1hiCD57hiCXCR3lo) (upper panel) and the corresponding calculated cross-G function (lower panel). Follicular areas from two tonsils and two LNs were analysed, each dot represents a follicle. Statistical analysis was performed using the Wilcoxon and Mann–Whitney tests, and p values are listed.

Article Snippet: Amount Rhodium 103Rh Cell- ID Standard Biotools 201103A 0.6 μL CD8 113ln RPA- T8 BioLegend 301018 0.6 μL CD4 115In RPA- T4 BioLegend 300515 0.6 μL CCR6 141Pr 11A9 Standard Biotools 3141014A 1 μL CD19 142Nd HIB19 Standard Biotools 3142001B 1.6 μL CCR6 144Nd G034E3 Standard Biotools 3141003A 1.88 μL CD20 147Sm 2H7 Standard Biotools 3147001B 0.7 μL ICOS 148Nd C398.4A Standard Biotools 3148019B 1 μL CCR4 149Sm L291H4 Standard Biotools 3149029A 1.5 μL CD40L 152Sm TRAP1 BD Biosciences 555698 3 μL TIGIT 153Eu MBSA43 Standard Biotools 3153019B 2 μL CD3 154Sm UCHT1 Standard Biotools 3154003B 0.5 μL CD27 155Gd L128 Standard Biotools 3155001B 1 μL CXCR3 156Gd G025H7 Standard Biotools 3156004B 0.8 μL CCR7 159 Tb G043H7 Standard Biotools 3159003A 1 μL CD30 162Dy BY88 BioLegend 333902 3 μL CXCR5 164Dy RF8B2 Standard Biotools 3164029B 1.2 μL CD45RO 165Ho UCHL1 Standard Biotools 3165011B 0.7 μL CD38 167Er HIT2 Standard Biotools 3167001B 0.6 μL CD45RA 170Er HI100 Standard Biotools 3171002B 1.6 μL HLA- DR 174Yb L243 Standard Biotools 3174001B 0.7 μL CD279/PD- 1 175Lu EH12.2H7 Standard Biotools 3175008B 1 μL CD127 176Yb A019D5 Standard Biotools 3176004B 0.7 μL CTLA4 194Pt Ipilimumab BMS ACB3065 2.5 μL CD57 198Pt NK- 1 BD Biosciences 555618 1 μL CD16 209Bi 3G8 Standard Biotools 3209002B 1.5 μL TABLE 2 | Antibodies used for the mass cytometry intracellular characterisation of tonsillar- derived cells.

Techniques: Expressing, MANN-WHITNEY

Journal: Immunology

Article Title: Delineation of the Human Germinal Centre Immune Landscape Using Multiplex Imaging Analysis.

doi: 10.1111/imm.13955

Figure Lengend Snippet: FIGURE 6 | Similar phenotypic composition of follicular and extrafollicular CD8 T cells in reactive lymph nodes. (A) Representative mIF images showing the expression of CD20 (red), CD8 (yellow) and GrzB (magenta) in an LN (scale bar: 500 μm). Inserts show the CD8, GrzB staining pattern in a zoomed area. (B) The histocytometry gating scheme for the identification of CD3hiCD4loCD8hi T cells in the EF and total F of an LN. (C) Heatmap showing the prevalence (judged by absolute cell counts) of FlowSOM identified CD8 T cell subsets (P0–P7) in EF and F area for each LN tissue in- vestigated (D) Dot plot graph showing the cell counts of the P1 and P5 subsets in EF (n = 4, open circles) and F (n = 4, grey circles) areas (right). Each circle represents a different LN donor (E) 2D histocytometry plot projecting the localisation of CD3hiCD4loCD8hi and CD3hiPD1hi TFH cells across follicular areas. Representative plots from one LN are shown. (F) Dot plot graphs showing accumulated data for the analysis of the minimum dis- tance (left panel) between follicular CD3hiCD4loCD8hi cells and CD3hiPD1hiCD57hi (open circles) or CD3hiPD1hiCD57lo (grey circles) TFH cell subsets and corresponding calculated cross-G function (right panel). Each dot represents a follicle. Statistical analysis was performed using the Wilcoxon test, and p values are listed. (G) Follicular areas from four LNs were analysed, with each dot representing a follicle. The regression analysis between concatenated follicular CD3hiCD4hiCD8loPD1hi TFH and follicular CD3hiCD4loCD8hi T cell counts, for all reactive lymph node follicles of one repre- sentative LN, is shown. The R and p values are listed.

Article Snippet: Amount Rhodium 103Rh Cell- ID Standard Biotools 201103A 0.6 μL CD8 113ln RPA- T8 BioLegend 301018 0.6 μL CD4 115In RPA- T4 BioLegend 300515 0.6 μL CCR6 141Pr 11A9 Standard Biotools 3141014A 1 μL CD19 142Nd HIB19 Standard Biotools 3142001B 1.6 μL CCR6 144Nd G034E3 Standard Biotools 3141003A 1.88 μL CD20 147Sm 2H7 Standard Biotools 3147001B 0.7 μL ICOS 148Nd C398.4A Standard Biotools 3148019B 1 μL CCR4 149Sm L291H4 Standard Biotools 3149029A 1.5 μL CD40L 152Sm TRAP1 BD Biosciences 555698 3 μL TIGIT 153Eu MBSA43 Standard Biotools 3153019B 2 μL CD3 154Sm UCHT1 Standard Biotools 3154003B 0.5 μL CD27 155Gd L128 Standard Biotools 3155001B 1 μL CXCR3 156Gd G025H7 Standard Biotools 3156004B 0.8 μL CCR7 159 Tb G043H7 Standard Biotools 3159003A 1 μL CD30 162Dy BY88 BioLegend 333902 3 μL CXCR5 164Dy RF8B2 Standard Biotools 3164029B 1.2 μL CD45RO 165Ho UCHL1 Standard Biotools 3165011B 0.7 μL CD38 167Er HIT2 Standard Biotools 3167001B 0.6 μL CD45RA 170Er HI100 Standard Biotools 3171002B 1.6 μL HLA- DR 174Yb L243 Standard Biotools 3174001B 0.7 μL CD279/PD- 1 175Lu EH12.2H7 Standard Biotools 3175008B 1 μL CD127 176Yb A019D5 Standard Biotools 3176004B 0.7 μL CTLA4 194Pt Ipilimumab BMS ACB3065 2.5 μL CD57 198Pt NK- 1 BD Biosciences 555618 1 μL CD16 209Bi 3G8 Standard Biotools 3209002B 1.5 μL TABLE 2 | Antibodies used for the mass cytometry intracellular characterisation of tonsillar- derived cells.

Techniques: Expressing, Staining

Journal: Immunology

Article Title: Delineation of the Human Germinal Centre Immune Landscape Using Multiplex Imaging Analysis.

doi: 10.1111/imm.13955

Figure Lengend Snippet: FIGURE 7 | No significant differences between tonsillar and LN tissues with respect to innate immune cell subset prevalence. (A) Frequency of immune subsets from live gated cells. Colour code represents an individual (n = 3). (B) Representative mIF images showing the expression of FDC (green), IL21 (cyan), CXCL13 (grey), CD11c (yellow), CD68 (red) and CD163 (magenta) in one tonsil (scale bar: 300 μm). Inserts show the expression of individual biomarkers in a zoomed area. (C) The histocytometry gating scheme for the identification of F, EF areas and relevant cell subsets in a tonsil is shown. Dot plots showing the normalised cell counts of follicular CD4hiIL10hi, CD4hiCXCL13hi and CD4hiIL21hi T cells in LNs (n = 4, open circles) and tonsillar (n = 4, closed circles) follicular areas. Statistical analysis was performed using the Mann–Whitney U-test. (D) Representative mIF images showing the expression of FOXP3 (green), CD25 (magenta), PD1 (yellow) and CD20 (blue) in one LNs. (E) 2D Histocytometry plots show- ing the gating scheme for the identification of follicular CD4hiCD25loFOXP3hi and CD4hiCD25hiFOXP3hi cells as well as the co-expression of FOXP3 and PD1 (left panel). The relative positioning of FOXP3hiCD25loCD4hi of CD4hiCD25hiFOXP3hi T cells across the follicular areas is shown in the right panel. (F) Dot plot showing the absolute counts of CD4hiCD25loFOXP3hi and CD4hiCD25hiFOXP3hi T cells in individual follicles from three LNs an- alysed. Data were analysed using the Wilcoxon test. (G) The regression analyses between TFH and CD4hiCD25loFOXP3hi or CD4hiCD25hiFOXP3hi T cells in individual follicles from three LNs analysed are shown. Each dot represents a follicle. The R and p values are also listed.

Article Snippet: Amount Rhodium 103Rh Cell- ID Standard Biotools 201103A 0.6 μL CD8 113ln RPA- T8 BioLegend 301018 0.6 μL CD4 115In RPA- T4 BioLegend 300515 0.6 μL CCR6 141Pr 11A9 Standard Biotools 3141014A 1 μL CD19 142Nd HIB19 Standard Biotools 3142001B 1.6 μL CCR6 144Nd G034E3 Standard Biotools 3141003A 1.88 μL CD20 147Sm 2H7 Standard Biotools 3147001B 0.7 μL ICOS 148Nd C398.4A Standard Biotools 3148019B 1 μL CCR4 149Sm L291H4 Standard Biotools 3149029A 1.5 μL CD40L 152Sm TRAP1 BD Biosciences 555698 3 μL TIGIT 153Eu MBSA43 Standard Biotools 3153019B 2 μL CD3 154Sm UCHT1 Standard Biotools 3154003B 0.5 μL CD27 155Gd L128 Standard Biotools 3155001B 1 μL CXCR3 156Gd G025H7 Standard Biotools 3156004B 0.8 μL CCR7 159 Tb G043H7 Standard Biotools 3159003A 1 μL CD30 162Dy BY88 BioLegend 333902 3 μL CXCR5 164Dy RF8B2 Standard Biotools 3164029B 1.2 μL CD45RO 165Ho UCHL1 Standard Biotools 3165011B 0.7 μL CD38 167Er HIT2 Standard Biotools 3167001B 0.6 μL CD45RA 170Er HI100 Standard Biotools 3171002B 1.6 μL HLA- DR 174Yb L243 Standard Biotools 3174001B 0.7 μL CD279/PD- 1 175Lu EH12.2H7 Standard Biotools 3175008B 1 μL CD127 176Yb A019D5 Standard Biotools 3176004B 0.7 μL CTLA4 194Pt Ipilimumab BMS ACB3065 2.5 μL CD57 198Pt NK- 1 BD Biosciences 555618 1 μL CD16 209Bi 3G8 Standard Biotools 3209002B 1.5 μL TABLE 2 | Antibodies used for the mass cytometry intracellular characterisation of tonsillar- derived cells.

Techniques: Expressing, MANN-WHITNEY