Journal: The Journal of Immunology Author Choice

Article Title: Defining T Cell Subsets in Human Tonsils Using ChipCytometry

doi: 10.4049/jimmunol.2100063

Figure Lengend Snippet: Intracellular markers for ChipCytometry cell suspension staining

Article Snippet: 1 , BCL6 , REA373 , PE , Miltenyi Biotec.

Techniques: Suspension

Journal: The Journal of Immunology Author Choice

Article Title: Defining T Cell Subsets in Human Tonsils Using ChipCytometry

doi: 10.4049/jimmunol.2100063

Figure Lengend Snippet: Phenotyping of tonsil-derived, CD3-enriched cells by cell suspension ChipCytometry. Four human tonsils were processed to single-cell suspensions, positively enriched for CD3 + T cells, barcoded, loaded onto a microfluidic chip, fixed, and subsequently analyzed by ChipCytometry with a panel of 19 fluorochrome-labeled Abs. ( A ) Density plot shows t -SNE analysis of concatenated samples, including all CD3 + CD19 − cells. ( B ) Clusters that were identified via FlowSOM were projected onto the t -SNE coordinates and color coded as indicated. ( C ) A heatmap showing median marker intensities within each cell cluster was generated to characterize cell clusters and derive cell labels. The heatmap color represents median marker expression across all four aggregated samples. ( D ) T-SNE plots show the expression of 16 markers within the aggregated samples. ( E ) Top panel, Within CD4 + T cells, the subsets TFH, pre-TFH, and non-TFH cells were identified according to expression of CXCR5 and PD1. Center panel, BCL6 expression is shown within non-TFH (red), pre-TFH (blue), and TFH (orange) cell subsets in human tonsils. Bottom panel, T-SNE plot shows non-TFH (red), pre-TFH (blue), TFH (orange), and non-CD4 T cells (black). Plots in (E) are derived from one donor and are representative of four donors.

Article Snippet: 1 , BCL6 , REA373 , PE , Miltenyi Biotec.

Techniques: Derivative Assay, Suspension, Labeling, Marker, Generated, Expressing

Journal: Frontiers in Immunology

Article Title: A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration

doi: 10.3389/fimmu.2021.675146

Figure Lengend Snippet: Detection of different TLS phenotypes by 7-color multiplex immunohistochemistry in human melanoma tissues. Examples for (A) early TLS: dense CD20 + lymphocyte aggregates with CXCL13 + cells and the presence of some interspersed CD4 + T cells; (B) primary follicular TLS: CD20 + lymphocyte aggregates interspersed with CD4 + T cells and the presence of a CD21 + but CD23 - dendritic network, surrounded by CXCL13 expressing cells; (C) secondary follicular TLS with a BCL6 - germinal center: CD21 + and CD23 + dendritic networks within CD20 + lymphocyte aggregates with interspersed CD4 + T cells, surrounded by CXCL13 expressing cells. No accessory expression of BCL6 in lymphatic cells. Germinal centers are identifiable in routine light microscopy (see Pathology View, bottom left). An example for a BCL6 + secondary follicular TLS is given in Figure 5A . Images for each of the individual markers and their composites (for clarity without DAPI staining) are shown, together with the corresponding Pathology View (respective bottom left). Scale bars represent 100 µm. (D) Spatial annotation of tumor areas. Left: Definition of the invasive tumor front (red, 2) and the respective intra- and extratumoral compartments (brown, 1 and yellow, 3, respectively) in a tissue section of a human melanoma lymph node metastasis. Extratumoral compartments include site-specific and adipose tissue (yellow). Right: Allocation of intra- (brown/black) and extratumoral perimeters (green) of increasing radiuses (1 – max. 6 mm) drawn within and around the invasive tumor front. Counts and area of TLS were referred to the tissue area (yellow) within the respective intra- and extratumoral perimeters. TLS, tertiary lymphoid structures.

Article Snippet: First, each of the following antibodies was established on four-micrometer sections from FFPE tissue of human tonsil: CD20 (mouse monoclonal IgG2a, clone L26, 1:2000, Agilent, M0755), CD4 (mouse monoclonal IgG1, clone 4B12, 1:500, Agilent, M7310), CXCL13 (rabbit polyclonal IgG, 1:1000, Proteintech, 10927-1-AP), CD21 (rabbit polyclonal IgG, 1:3600, Proteintech, 24374-1-AP), CD23 (rabbit monoclonal IgG, clone SP23, 1:900, Novus Biologicals, NB120-16702) and BCL6 (mouse monoclonal IgG1, clone 1E6B1, 1:24000, Proteintech, 66340-1-Ig).

Techniques: Multiplex Assay, Immunohistochemistry, Expressing, Light Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration

doi: 10.3389/fimmu.2021.675146

Figure Lengend Snippet: Metastatic tumor sites: TLS density and spatial distribution. (A) Densities of extratumoral secondary follicular TLS (left) and BCL6 - germinal centers (GC BCL6 - , right) in distant metastatic lymph node and skin sites. (B) Densities of secondary follicular TLS (left) and BCL6 - germinal centers (right) over intra- and extratumoral compartments in distant metastatic lymph node and skin sites. In all boxplots, lower and upper hinges correspond to the first and third quartiles, center line to the median. Upper and lower whisker extend from the hinge to the largest value no further than 1.5 times the interquartile range. Values outside this range are shown as outliers (black circles). Individual patient values are shown as black dots.

Article Snippet: First, each of the following antibodies was established on four-micrometer sections from FFPE tissue of human tonsil: CD20 (mouse monoclonal IgG2a, clone L26, 1:2000, Agilent, M0755), CD4 (mouse monoclonal IgG1, clone 4B12, 1:500, Agilent, M7310), CXCL13 (rabbit polyclonal IgG, 1:1000, Proteintech, 10927-1-AP), CD21 (rabbit polyclonal IgG, 1:3600, Proteintech, 24374-1-AP), CD23 (rabbit monoclonal IgG, clone SP23, 1:900, Novus Biologicals, NB120-16702) and BCL6 (mouse monoclonal IgG1, clone 1E6B1, 1:24000, Proteintech, 66340-1-Ig).

Techniques: Whisker Assay

Journal: Frontiers in Immunology

Article Title: A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration

doi: 10.3389/fimmu.2021.675146

Figure Lengend Snippet: Comparison of large BCL6 + secondary follicular TLS in human melanoma to tonsil tissue. (A) Melanoma: CD20 + lymphocyte aggregates with a germinal center surrounded by CXCL13 expressing cells. A CD21 + and CD23 + FDC network covers the complete germinal center area with randomly interspersed CD4 + T cells and BCL6 + cells. No detectable germinal center polarity, no identifiable light and dark zone areas in routine light microscopy (see Pathology View, bottom left). (B) Close up of BCL6 + CD20 + B cells and BCL6 + CD4 + T cells from a BCL6 + secondary follicular TLS in human melanoma. (C) Tonsil: note canonical germinal center polarity with polarized spatial distribution of the CD21 + and CD23 + FDC network together with CD4 + T cells (arrowheads) and a complementary localization of BCL6 + cells (arrow). Identifiable light (arrowhead) and dark zone (arrow) areas in routine light microscopy (see Pathology View, bottom left). (D) Tonsil (upper row): polarized distribution of BCL6 + , Ki67 + (arrows) and BCL6 + Ki67 + cells in germinal centers with canonical germinal center polarity and the presence of light (arrowhead) and dark zone (arrow) areas in routine light microscopy (see Pathology View, left). Melanoma (lower row): note decreased numbers and absence of polarized distribution of BCL6 + , Ki67 + and BCL6 + Ki67 + cells in melanoma germinal centers. Lack of canonical germinal center polarity and absence of light and dark zone areas in routine light microscopy (see Pathology View, left). Images for each of the individual markers and their composites (for clarity without DAPI staining) are shown, together with the corresponding Pathology View (respective (bottom) left). Scale bars represent 100 µm (A, C, D) and 5 µm (B) .

Article Snippet: First, each of the following antibodies was established on four-micrometer sections from FFPE tissue of human tonsil: CD20 (mouse monoclonal IgG2a, clone L26, 1:2000, Agilent, M0755), CD4 (mouse monoclonal IgG1, clone 4B12, 1:500, Agilent, M7310), CXCL13 (rabbit polyclonal IgG, 1:1000, Proteintech, 10927-1-AP), CD21 (rabbit polyclonal IgG, 1:3600, Proteintech, 24374-1-AP), CD23 (rabbit monoclonal IgG, clone SP23, 1:900, Novus Biologicals, NB120-16702) and BCL6 (mouse monoclonal IgG1, clone 1E6B1, 1:24000, Proteintech, 66340-1-Ig).

Techniques: Comparison, Expressing, Light Microscopy, Staining

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: The Combination of Jiedu Xiaoluo Decoction with Autologous Peripheral Blood Stem Cell Transplantation (APBSCT) Accelerates Disease Remission of Non-Hodgkin Lymphoma

doi: 10.1155/2021/2745705

Figure Lengend Snippet: Immunohistological detection for the expression and distribution of CD20, Bcl-2, Bcl-6, and Ki67 in tumor area of mice in each group. The expression of CD20 (a) and Bcl-2 (b) as determined by immunohistochemical staining in tumor tissue of each group. (c) The area of density (AOD) of CD20- and Bcl-2-positive cells in the three groups. The expression of Bcl-6 (d) and Ki67 (e) as determined by immunohistochemical staining in tumor tissue of each group. (f) The area of density (AOD) of Bcl-6- and Ki67-positive cells in the three groups. 100x or 400x indicates the magnification. ∗ Control group vs transplant group and treatment group. # Treatment group vs transplant group. ∗ or # P < 0.05; ∗∗ or ## P < 0.01.

Article Snippet: Next, the sections were blocked using 5% BSA for 1 h. 50 μ L diluted primary antibodies against CD20 (1 : 100, 60271–1, Proteintech Company, USA), Bcl-2 (1 : 200, AF6139, Affinity Company), Bcl-6 (1 : 200, bs-13606R, Bioss), and Ki67 (1 : 500, AF0198, Affinity Company) were added to each section and incubated overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Staining