Journal: bioRxiv

Article Title: Cellular and molecular events organizing the assembly of tertiary lymphoid structures in glioblastoma

doi: 10.1101/2024.07.04.601824

Figure Lengend Snippet: (A) Schematic representation of the experimental layout used to obtain data shown in panels B-K. In brief, sections from large surgical tissues of grade 4 astrocytoma cases (including GBM and IDHmut astrocytomas) were stained with H&E and pre-screened for the presence of lymphoid aggregates. Positive samples were sequentially stained using immunofluorescence (IF) to identify B cells, T cells, endothelial cells and nuclei, and confirm the presence of TLS. Finally, clinical data and staining results were correlated to perform survival analysis. (B) Representative images showing a lymphoid aggregate in an H&E-stained section (left panel) and the sequential section stained with IF antibodies for B cells (CD20), T cells (CD3), endothelial cells (CD34) and nuclei (right panel). Zoom panel shows a close-up of the lymphoid aggregate in the H&E-stained section. Scale bar in H&E section: 1 mm. Scale bar in H&E zoom and IF section: 50 µm. (C) Pie charts showing the percentage of TLS positive patients in a large GBM cohort (n=205 patients) and a small cohort of grade 4 astrocytoma patients with IDH mutation (n=24 patients). (D) Kaplan-Meier survival curve of GBM patients stratified based on TLS presence in surgical tissues. n(TLS-)=159; n(TLS+)=29. Statistics: Log-Rank test. (E) Representative IF images of TLS dominated by T cells (T cell-rich TLS, or T-TLS), TLS with a similar representation of B cells and T cells (mixed TLS, or M-TLS) and TLS with a large B cell component (B cell-rich, or B-TLS). (F) Representative IF image of a B-TLS with clear zonation of B cells and T cells. (G) Representative IF images of PNs dominated by T cells (T cell-rich PNs, or T-PNs), PNs with a similar representation of B cells and T cells (mixed PNs, or M-PNs) and PNs with a large B cell component (B cell-rich, or B-PNs). (H) Representative IF image of a B-PN with clear zonation of B cells and T cells. Yellow arrow indicates the zonated PN. Scale bars in (E-H)=100µm. (I) Pie charts showing the distribution of T cell-rich, mixed, B cell-rich and zonated structures among TLS and PNs. n(PNs)=67; n(TLS)=78. (J-K) Nuclear density in (J) TLS and (K) PNs with different T-to-B cell ratios. n=4-35 aggregates/group. Bar graphs show mean ±SEM. Statistics: one-way ANOVA with Tukey’s correction for multiple comparisons. *p<0.05; **p<0.01.

Article Snippet: Lymphoid aggregates or tumor areas were identified as distinct regions of interest (ROIs) through the use of morphology markers as guidance, such as CD3 (Origene, #UM000048BF), CD20 (Novus Biologicals, #NBP2-47840AF532) and CD31 (R&D Systems, #AF3628) antibodies as well as Syto13 (Thermo Fisher Scientific).

Techniques: Staining, Immunofluorescence, Mutagenesis

Journal: bioRxiv

Article Title: Cellular and molecular events organizing the assembly of tertiary lymphoid structures in glioblastoma

doi: 10.1101/2024.07.04.601824

Figure Lengend Snippet: In this figure, data in panels A-H were produced using targeted Padlock probe-based in situ sequencing, and data in panels I-M were produced using CosMx single-cell spatial analysis. (A) Representative image of a zonated TLS identified in human GBM tissue using targeted Padlock probe-based in situ sequencing. The image shows highly clustered signals from CD3D+CD3E (T cell) and MS4A1 (B cell) transcripts, segregated into clear zones. (B-H) Location of the following transcripts within the zonated TLS shown in panel A: (B) CD79A and CD79B; (C) CD40; (D) TCRB1+TCRB2; (E) CXCR4; (F) CCL5; (G) CD7 and (H) GPR183. In (A-H): spatial coordinates of transcripts are indicated by colored symbols. White = nuclei. Dotted yellow line separates B versus T cell zone of the shown TLS. (I) Schematic representation of the layout used for the CosMx single-cell spatial transcriptomics assay. In brief, FFPE sections of human GBM tissue were co-stained with fluorescent antibodies/dyes to visualize the nuclear (H3) versus cytoplasmic (18S RNA) cell fractions, as well as with in situ hybridization (ISH) probes targeted against 1030 mRNAs. Fields of view (FOVs) were selected, and probes were detected using complementary reporter probes tagged with fluorophores. Probe signal was detected within each FOV using cycles of fluorescence imaging, and probe location on the tissue was recorded using tissue coordinates. Cells were annotated based on clustering analysis and gene expression profiles. Annotated cells were plotted back onto their tissue coordinates to visualize their location. (J) UMAP of main cell clusters annotated in GBM tissue sections using CosMx single-cell spatial analysis (n=4 patients). T= T cell; B= B cell, Fb= fibroblast. (K,L) Spatial organization of T cells, B cells, endothelial cells (ECs), myeloid cells and fibroblasts in (J) T cell-rich TLS (T-TLS) and (D) B cell-rich TLS (B-TLS). Images show representative FOVs containing T-TLS and B-TLS. Bar graphs to the right of each image display the number of cells within the shown FOVs, for each plotted cell type. (M,N) Expression of IL7R in single cells within T-TLS and B-TLS visualized as (L) an expression heatmap or (B) a bar graph displaying normalized counts of IL7R in each cell within the shown FOVs.

Article Snippet: Lymphoid aggregates or tumor areas were identified as distinct regions of interest (ROIs) through the use of morphology markers as guidance, such as CD3 (Origene, #UM000048BF), CD20 (Novus Biologicals, #NBP2-47840AF532) and CD31 (R&D Systems, #AF3628) antibodies as well as Syto13 (Thermo Fisher Scientific).

Techniques: Produced, In Situ, Sequencing, Staining, In Situ Hybridization, Fluorescence, Imaging, Expressing

Journal: JCI Insight

Article Title: Type 2 innate immunity promotes the development of pulmonary fibrosis in Hermansky-Pudlak syndrome

doi: 10.1172/jci.insight.178381

Figure Lengend Snippet: ( A ) ILC2 staining in human lung tissues. Anti-GATA3, -CD90/Thy1, and a cocktail of 4 antibodies against CD3, CD56, CD20, and CD79α were employed to stain human lung tissues in normal individuals, idiopathic pulmonary fibrosis (IPF) patients, and HPS patients. ILC2s are identified as GATA3 + CD90/Thy1 + , and antibody cocktail–negative cells (purple cells). The corresponding phase-contrast image shows lung tissue architecture in normal individuals, and pathologic changes in IPF and HPS lungs. ( B ) Counting of ILC2s (normalized per area) under ×40 magnification using an immunofluorescence microscope. Values are mean ± SEM with a minimum of 4 samples in each group. Comparisons between groups were conducted by 2-way ANOVA with Bonferroni’s post hoc test. *** P ≤ 0.001. Scale bars: 20 μm.

Article Snippet: The following primary antibodies (dilution; catalog/clone number, manufacturer) were used for overnight incubation at 4°C: GATA3 (1:250; ab199428, Abcam), CD90 (1:200; AF2067, R&D Systems), CD56 (1:200; ab9272, Abcam), CD20 (1:100; IGEL/773, Novus), CD79A (1:100; NB100-64347ss, Novus), CD-3e (4:100; ab699, Abcam), and α-SMA (1:200; NB300–978, Novus).

Techniques: Staining, Immunofluorescence, Microscopy

Journal: Nature Communications

Article Title: Spatially-resolved transcriptomics reveal macrophage heterogeneity and prognostic significance in diffuse large B-cell lymphoma

doi: 10.1038/s41467-024-46220-z

Figure Lengend Snippet: A Schematic of GeoMx® DSP WTA workflow (created with BioRender.com). B , C Immunofluorescence staining of DLBCL tissues ( n = 87) and RLTs ( n = 24). In Group 1, CD68 stained macrophages (yellow), CD3 stained T cells (cyan), CD20 stained B cells (magenta), and SYTO 13 stained nuclei (blue). In Group 2, CD68 stained macrophages (yellow), NGFR illuminated LZ (green) and SYTO 13 stains nuclei (blue). After ROI selection, each cell type was segmented based on the staining signal and their corresponding masks were generated. Representative images are shown. Scale bar: 100 μm. Source data are provided as a file. D , E Cumulative density functions showed that the signatures of macrophages ( CD68 , CD163 , FCGR1A , and CSF1R ), T cells ( CD3D , CD3E , UBASH3A , CD2 , and TRBC2 ), and B cells ( MS4A1 , CD79A , CD79B , CD19 , and PAX5 ) were highly enriched in CD68+ regions, CD3+ regions, and CD20+ regions, respectively in RLTs and DLBCL tissues (Kolmogorov-Smirnov P < 0.05). Digital spatial profiling, DSP; whole transcriptome analysis, WTA; diffuse large B-cell lymphoma, DLBCL; reactive lymphoid tissues, RLTs; regions of interest, ROIs; areas of interest, AOIs; formalin-fixed paraffin-embedded, FFPE; light zone, LZ; dark zone, DZ; nerve growth factor receptor, NGFR.

Article Snippet: For Group 1, slides of DLBCL TMA were stained with macrophage marker CD68 (sc-20060 AF594, Santa Cruz biotechnology, Texas, USA), T-cell marker CD3 (A0452, Dako, California, USA), B-cell marker CD20 (NBP2-47840 AF647, Novus Biologicals, Colorado, USA) and the nuclear stain SYTO 13.

Techniques: Immunofluorescence, Staining, Selection, Generated, Formalin-fixed Paraffin-Embedded

Journal: The Journal of investigative dermatology

Article Title: Disease Association of Anti‒Carboxyethyl Lysine Autoantibodies in Hidradenitis Suppurativa.

doi: 10.1016/j.jid.2022.08.051

Figure Lengend Snippet: Figure 4. Detection of B cells and germinal center‒like structures in HS skin. (a) Representative H&E (left) and CD20 staining (right) of HS skin. Each bar ¼ 2 mm. (b) Representative staining of CD20þ B cells (left) and CD21þ germinal center‒like structures (right) in HS skin. B cells were either diffusely distributed (top two panels) or densely organized in lymphoid follicles (bottom three panels), which often but not always accompanied CD21 expression. Each bar ¼ 100 mm. (c) Representative H&E (top; bar ¼ 3 mm), CD20 (middle; bar ¼ 2 mm), and CD21 (bottom; bar ¼ 3 mm) staining of HS skin, showing fat associated and CD21þ

Article Snippet: List of antibodies used for Chromogenic Immunohistochemistry and Nanostring (including secondary antibodies) were mouse IgG2ak anti-human CD20cy (clone L26, M0755, Dako Deutschland, Hamburg, Germany), goat anti-mouse biotinylated (polyclonal antibody, BA-9200, Vector Laboratories, Burlingame, CA), rabbit anti-human CD21 (clone EP3093, ab75985, Abcam), goat anti-rabbit biotinylated (polyclonal antibody, 111-065-144, Jackson ImmunoResearch), monoclonal rabbit isotype control (ab172730, Abcam), mouse IgG2ak isotype Control eFluor 660 (50-4724, eBioscience, San Diego, CA), mouse IgG1ak anti-human pan-cytokeratin Alexa Fluor 647 (clone AE1þAE3, NBP233200AF647, Novus Biologicals), mouse IgG2ak antihuman CD20 Alexa Fluor 594 (clone IGEL/773, number NBP2-47840DL594, Novus Biologicals), and mouse IgG1ak anti-human vimentin (Alexa Fluor 488, clone E-5, number sc373717, Santa Cruz Biotechnology).

Techniques: Staining, Expressing