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:

Journal: Frontiers in Immunology

Article Title: New Paradigm in the Role of Regulatory T Cells During Pregnancy

doi: 10.3389/fimmu.2019.00573

Figure Lengend Snippet: Immunological balance at the feto-maternal interface during early pregnancy. EVTs did not express polymorphic HLA-A, B whereas HLA-C and non-polymorphic HLA-E, G, and F were expressed. Maternal CD8 + T cells and NK cells can directly recognize paternal HLA-C and CD4 + T cells can indirectly recognize it. HLA- E and G protect EVTs from NK-cell mediated cytotoxicity. Treg cells can recognize fetal antigens via maternal antigen presenting cells (APCs) and induce tolerance in an antigen-specific manner. EVT, Extravillous trophoblast; NK, natural killer cell; Treg; regulatory T cell; APC, antigen-presenting cell.

Article Snippet: Semi-allogenic fetuses are not rejected by the maternal immune system because feto-maternal tolerance induced by CD4 + CD25 + FoxP3 + regulatory T (Treg) cells is established during pregnancy.

Techniques:

Journal: Frontiers in Immunology

Article Title: New Paradigm in the Role of Regulatory T Cells During Pregnancy

doi: 10.3389/fimmu.2019.00573

Figure Lengend Snippet: Paternal antigen-specific Treg cells in mouse models.

Article Snippet: Semi-allogenic fetuses are not rejected by the maternal immune system because feto-maternal tolerance induced by CD4 + CD25 + FoxP3 + regulatory T (Treg) cells is established during pregnancy.

Techniques: Derivative Assay

Journal: Frontiers in Immunology

Article Title: New Paradigm in the Role of Regulatory T Cells During Pregnancy

doi: 10.3389/fimmu.2019.00573

Figure Lengend Snippet: Single-cell based TCR repertoire analysis method. To study the clonality of effector Treg cells, a single-cell based T cell receptor (TCR) repertoire analysis method was used. Paired samples of maternal peripheral blood mononuclear cells and decidual lymphocytes were obtained. CD4+CD25+CD45RA-CD127low/- effector Treg cells were single-cell sorted. The cDNAs of complementarity determining lesion 3 (CDR3) in TCRβ chain and FoxP3 were amplified by RT-PCR. The nucleotides and amino acid sequences of CDR3 were analyzed.

Article Snippet: Semi-allogenic fetuses are not rejected by the maternal immune system because feto-maternal tolerance induced by CD4 + CD25 + FoxP3 + regulatory T (Treg) cells is established during pregnancy.

Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: Advanced functional materials

Article Title: Surface engineered polymersomes for enhanced modulation of dendritic cells during cardiovascular immunotherapy

doi: 10.1002/adfm.201904399

Figure Lengend Snippet: (A) Representative flow cytometric plots of mature DCs as CD80+CD86+CD11c+ gated on CD45+ live cells in both spleen and DLNs of ApoE−/− mice with and without PS-aVD or P-D2-PEG5-PS-aVD treatment. The percentages of mature DCs (CD80+CD86+CD11c+) within the total DC population (CD11c+) were quantified in both spleen (B) and DLNs (C). (D) The expression of CD80 and CD86 genes in aortas of control or P-D2-PEG5-PS-aVD treated groups, as determined by quantitative RT-PCR. (E) Representative flow cytometric plots of Tregs (Foxp3+CD25+CD4+) gated on CD45+CD3+ live cells in spleen of ApoE−/− mice. (F) Immunofluorescence staining of Foxp3 (red) in aortic sinus of ApoE−/− mice was (G) quantified in cross sections to assess Treg content in aorta. Scale bar=200μm. N = 5 mice per group in A, B, C, E; N = 5–6 mice per group in D, F, G. All results are representative of two independent experiments. The unpaired Mann-Whitney test (D, G) and two-tailed t-tests (A, B, C, E) were used for statistical significance: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Specific antibodies were used on another consecutive cross-section for macrophages (anti-CD68, 1:500, Abcam) and Treg cells (anti-Foxp3, 1:500, Abcam).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: Methylation-based reclassification and risk stratification of skull-base chordomas

doi: 10.3389/fonc.2022.960005

Figure Lengend Snippet: Immune cell infiltration and tumor microenvironment analyses between clusters. (A) Immune cell infiltration scores in cluster 1 and cluster 2 chordomas. (B) Immune cell infiltration scores in high-risk and low-risk chordomas. (C) Adaptive immune cells in cluster 1 and cluster 2 chordomas. (D) Innate immune cells in cluster 1 and cluster 2 chordomas. (E) Representative images (×20) showing more frequent presence of CD8, Treg, CD56, and fibroblasts in cluster 1 (upper panels) than cluster 2 (lower panels). (F) Adaptive immune cells in high-risk and low-risk chordomas. (G) Innate immune cells in high-risk and low-risk chordomas. (H) The association between known clinical and molecular features (sex, extent of resection [EOR], radiotherapy, recurrence status, cluster status, and risk status) and mDNAsi values of our cohort. Columns represent the sorted chordoma samples by mDNAsi values (low to high). Rows represent clinical information. (I) Violin plots of mDNAsi values in cluster 1 and cluster 2 chordoma patients. (J) Violin plots of mDNAsi values in the high-risk and low-risk chordoma patients. (K) Distribution of stromal scores, immune scores, ESTIMATE scores, and tumor purity of cluster 1 and cluster 2 chordoma patients. (L) Distribution of stromal scores, immune scores, ESTIMATE scores, and tumor purity of high-risk and low-risk chordoma patients ( *** p < 0.001, ** p < 0.01, * p < 0.05).

Article Snippet: Immunostaining analysis was used to assess the CD8 (ab32620; Abcam, Cambridge, UK), CD56 (ab133345; Abcam), Treg (72338; Cell Signaling Technology, Danvers, MA, USA), and fibroblast (ab207178; Abcam) expression levels following the manufacturers’ protocols using formalin-fixed, paraffin-embedded tissue slides.

Techniques:

Journal: OncoImmunology

Article Title: Highly clonal regulatory T-cell population in follicular lymphoma – inverse correlation with the diversity of CD8+T cells

doi: 10.1080/2162402x.2014.1002728

Figure Lengend Snippet: Figure 2. Treg localization in FL tissues. Immunohistochemical staining for Foxp3 (dark brown) was done on formalin-fixed, paraffin-embedded lymph nodes of 6 FL patients before treatment. Original magnification £200. IF, intrafollicular; PE, perifollicular. *denotes 3 cases exhibiting prominent intrafol- licular localization.

Article Snippet: Tzankov A, Meier C, Hirschmann P, Went P, Pileri SA, Dirnhofer S. Correlation of high numbers of intratumoral FOXP3+ regulatory T cells with improved survival in germinal center-like diffuse large B-cell lymphoma, follicular lymphoma and classical Hodgkin’s lymphoma.

Techniques: Immunohistochemical staining, Staining