Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: Workflow for immunohistochemistry and image analysis. a Diagram of the immunohistochemical workflow. b Quantification of the efficiency of elution for three antibodies: anti-PD1, anti-FOXP3 and anti-CD4. Black bars represent antibody staining intensity (mean ± SEM) and gray bars represent staining intensity following elution. Each experiment was carried out three times. The dotted line is the background intensity produced by an unstained tissue section. c Flow diagram to show Visiopharm methodology ( www.visiopharm.com ) used to analyse tissue sections stained sequentially with anti-CD4, anti-PD1 and anti-FOXP3 antibodies. The algorithm first identified cell nuclei by haematoxylin counterstain following which CD4 expressing cells were marked green. Next CD4 expressing cells were interrogated for co-expression of FOXP3. There was then a branch point in the algorithm. If FOXP3 expression was detected but there was no co-expression of PD1 at a high level (PD1 hi ), as previously defined by comparison with tonsillar Tfh cells, then the cell was determined to be Treg and the nucleus was labelled yellow. However, if the cell co-expressed CD4, FOXP3 and was PD1 hi then it was determined to be a Tfr cell and the nucleus was labelled turquoise. Cells that were CD4 expressing and PD1 hi , but not FOXP3 expressing were determined to be Tfh cells and the nuclei were labelled orange. Cells of each subset were then counted digitally. d Identification of specific T-cell subsets by multiplex immunohistochemistry. Tissue sections were stained with anti-CD4, anti-PD1 (EuroMabNet, clone NAT105) and anti-FOXP3 (EuroMabNet, clone 236A). Example Tfh cell (CD4 + PD1 + FOXP3 − ) painted orange, Treg cell (CD4 + PD1 − FOXP3 + ) painted yellow and Tfr cell (CD4 + PD1 + FOXP3 + ) painted turquoise are presented

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques: Immunohistochemistry, Immunohistochemical staining, Staining, Produced, Expressing, Comparison, Multiplex Assay

Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: Architecture of CD4+ T-cell infiltration and T-cell gene signatures. a Tissue sections from 6 cases were stained with anti-CD20, anti-CD4 and anti-CD8. Surface plots generated in ImageJ ( https://imagej.net/Fiji ) are shown for each stain. Intensity has been normalised so that images can be compared. T-cell zones are evident and whilst these comprised both CD4 and CD8 T-cells, CD4 T-cells were found at higher density in 4 of 6 cases (#1, #2, #4 and #6). b Gene set enrichment analysis. Data from Arribas et al. (2012) 5 was employed to discover T-cell signatures. Utilising a false discovery rate (FDR < 0.25) for exploratory work. Two gene signatures were found to be positively enriched in MZL. NES is normalised enrichment score. c Support for use of PD1 hi and FOXP3 + as single surrogate markers of Tfh and Treg cells respectively. Numbers of Tfh and Treg cells, as defined (Fig. ), were compared against single marker expression of PD1 hi and FOXP3

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques: Staining, Generated, Marker, Expressing

Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: Automated quantitation of marker proteins. a Automated quantitation of PD1 hi and FOXP3 + cells in 15 cases of MZL. Numbers above bars represent PD1 hi FOXP3 + ratio. b Analysis of cell density data and PD1 hi :FOXP3 + ratio by type of MZL: nodal, extranodal or splenic. Box and whisker plots show median, 90th and 10th centile. c Analysis of cell density data and PD1 hi :FOXP3 + ratio by stage of disease, either early stage (I and II) or advanced stage (III and IV)

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques: Quantitation Assay, Marker, Whisker Assay

Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: Clustering of PD1 hi cells. a Left hand panels are density plots representing PD1 hi (top) and FOXP3 (bottom) for one case (lymph node tissue). The impression of greater clustering of PD1 hi cells is confirmed by statistical analysis using Ripleys K function (right hand panels). A measure of distance (r) on the x-axis is plotted against K(r) function on the y-axis. The curve for complete spatial randomness (CSR) (dotted red line) is compared to K function for MZL (solid black line) for PD1 (upper panel) and FOXP3 (lower panel). Greater upward deflection of the observed curve compared to CSR represents greater clustering with the difference between these curves calculated to provide an overall cluster score. b Cluster scores for MZL cases ( n = 15). PD1 hi cells are significantly more clustered than FOXP3 + cells (Mann-Whitney test, P < 0.01). c Immunofluorescence images. CD20 (green), Ki-67 (blue) and PD1 (red) staining of a lymph node from a patient with MZL. Low power (10×) views of the merged antibody stains show that proliferating cells occur in clusters. Three fields are shown as high power views (80×). Two of these fields are from regions that contain a high density of Ki-67 + cells (upper and lower rows) while the middle row is from a region of lower Ki-67 + density

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques: MANN-WHITNEY, Immunofluorescence, Staining

Journal: Diagnostic Pathology

Article Title: PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

doi: 10.1186/s13000-018-0750-8

Figure Lengend Snippet: PD1 hi cells cluster with Ki-67 + cells. a Illustrative cartoon depicting low and high interactions with the respective Pearson correlation and Morisita-Horn index for both patterns. b Spatial interaction between FOXP3 or PD1 and Ki-67 cells. Scatter plots displaying Ki67 + against PD1 hi cells (upper panel) and FOXP3 + cells (lower panel) measured in spatial quadrants (200μm 2 ). Only quadrants containing at least 5 cells of any population are displayed. Quadrants containing higher densities of both cell types (> 50 cells) were considered to represent high interaction. 11.3% of all quadrants analysed for Ki67 + and PD1 hi cells met this definition of high interaction compared to only 1.6% of Ki67 + and FOXP3 + cells. Analysis was repeated for different sized quadrants (100 and 400μm 2 ) confirming similar results. c Nearest neighbour analysis. Distance from either FOXP3 + cells (upper panel) or PD1 hi cells (lower panel) was plotted for 10 5 cells sampled from 10 cases. For PD1 hi cells the large peak occurring at short distances to the nearest Ki-67 + cell indicates that these cells are closer to Ki-67 + cells than FOXP3 + cells, which are more evenly distributed. All clustering and spatial analysis was performed using the statistical programme R

Article Snippet: Visiopharm apps were developed (available on application to the authors) to paint Tfh cells (CD4 + PD1 hi FOXP3 − ) orange, Treg cells (CD4 + PD1 − FOXP3 + ) yellow and Tfr cells (CD4 + PD1 hi FOXP3 + ) turquoise (Fig. and ) prior to extracting cell numbers and location coordinates.

Techniques: