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: 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: Nature Communications

Article Title: Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis

doi: 10.1038/s41467-024-46564-6

Figure Lengend Snippet: A Axl and MerTK gene expression (regularised-log normalised reads) in synovial tissue of early arthritis treatment-naive RA patients ( n = 87) according to the histological pathotype defined as Pauci-Immune (P-I, in green), Diffuse-Myeloid (D-M, in red), and Lympho-Myeloid (L-M, in blue). Data are represented as mean ±SEM. p values indicated were calculated using the Kruskal–Wallis test with Dunn’s post hoc test. B Correlation between synovial AXL (top panels) and MERTK (bottom panels) gene expression and semi-quantitative scores (0–4) of B cells (CD20), T cells (CD3), plasma cells (CD138), and sublining macrophages (CD68SL). p values and r -coefficients were calculated using the two-tailed Pearson correlation test. C Regression model analysis with interaction term to estimate the correlation of AXL with MERTK expression in relation to clinical response to conventional synthetic Disease-Modifying-Anti-Rheumatic-Drugs. The clinical response was assessed by EULAR criteria with DAS28(CRP) after 6-months of treatment (good responders in light blue; moderate and non-responders in orange). p-interaction is not significant. The scatter plots show the regression line of the fitted negative binomial generalised mixed effects model with the error bars showing 95% confidence interval (fixed effects). D 2D polar plot of transcript modules containing AXL and MERTK in synovial tissue characterised by lympho-myeloid (L-M), diffuse-myeloid (D-M), and pauci-immune (P-I) pathotypes. Different colours show pairwise comparisons between the three pathotypes: upregulation in one group only (D-M: red, P-I: green and L-M: blue) or in two groups (D-M/P-I: yellow, L-M/P-I: light blue, L-M/D-M: purple). E , F Heatmaps showing the correlation between AXL and MERTK synovial transcript levels at baseline and cytokines and growth factors relevant to the inflammatory response ( E ) and clinical parameters ( F ). The red/blue scale represents the Spearman r coefficient, calculated using the two-tailed Spearman correlation test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CRP, C-Reactive Protein; ESR, erythrocyte sedimentation rate; DAS28 disease activity score 28; TJC/28, tender joints count (0-28); SJC/28, swollen joints count (0-28); VAS GH, Visual Analogue Scale General Health (0–100); HAQ health assessment questionnaire.

Article Snippet: The following fluorescent markers were used to determine the morphology of the tissue: CD68-AF532 (clone KP1, Cat. N. NB100-683 Novus) for macrophages, CD20-DL594 (clone IGEL/773, Cat. N. NBP2-44745, Novus) for B cells, and CD3-AF647 (clone UMAB54, Cat. N. TA807198, Origene) for T cells; nuclei were counterstained with Syto13 (Cat. N. S7575, ThermoFisher).

Techniques: Expressing, Two Tailed Test, Sedimentation, Activity Assay

Journal: Nature Communications

Article Title: Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis

doi: 10.1038/s41467-024-46564-6

Figure Lengend Snippet: A , C AXL ( A ) and MERTK ( C ) gene expression (vst normalised reads) in synovial tissue of anti-TNF inadequate responder RA patients (R4RA cohort, n = 133) according to the histological pathotype defined as Pauci-Immune (P-I, in green), Diffuse-Myeloid (D-M, in red), and Lympho-Myeloid (L-M, in blue). Data are represented as mean ±SEM. p values were calculated using the Kruskal–Wallis test with Dunn’s post hoc test. B , D Correlation between synovial AXL ( B ) and MERTK ( D ) gene expression and semi-quantitative scores (0–4) of B cells (CD20), T cells (CD3), plasma cells (CD138), and sublining macrophages (CD68SL). p values and r-coefficients were calculated using the two-tailed Pearson correlation test. E Expression of sAxl, sMerTK (pg/mL) or the ratio between sMerTK and sAxl in the supernatant of primary fibroblasts-like synoviocytes (FLS) conditioned with supernatant from M1-polarised THP1 (SN M1) or M2-polarised THP1 (SN M2), or in the respective medium used to condition the cells (SN CT). F Expression of sAxl, sMerTK (pg/mL) or the ratio between sMerTK and sAxl in the supernatant of THP1-derived macrophages conditioned with supernatant from unstimulated RA-FLS (SN FLS) or LPS stimulated FLS (SN FLS Infl.), or in the respective medium used to condition the cells (SN CT). E , F Data are represented as mean ±SEM. p values indicated were calculated using the unpaired two-tailed t test (left and middle panels) or the two-tailed Mann–Whitney test (right panel). Experiments were performed on n = 3 distinct patient-derived FLS. G , H Heatmaps showing the correlation between AXL and MERTK synovial transcript levels at baseline and cytokines and growth factors relevant to the inflammatory response ( G ) and clinical parameters ( H ). The red/blue scale represents the Spearman r coefficient, calculated using the two-tailed Spearman correlation test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CRP C-reactive protein, ESR erythrocyte sedimentation rate, DAS28 disease activity score 28, TJC/28 tender joints count (0–28), SJC/28 swollen joints count (0-28), HAQ health assessment questionnaire.

Article Snippet: The following fluorescent markers were used to determine the morphology of the tissue: CD68-AF532 (clone KP1, Cat. N. NB100-683 Novus) for macrophages, CD20-DL594 (clone IGEL/773, Cat. N. NBP2-44745, Novus) for B cells, and CD3-AF647 (clone UMAB54, Cat. N. TA807198, Origene) for T cells; nuclei were counterstained with Syto13 (Cat. N. S7575, ThermoFisher).

Techniques: Expressing, Two Tailed Test, Derivative Assay, MANN-WHITNEY, Sedimentation, Activity Assay

Journal: Nature Communications

Article Title: Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis

doi: 10.1038/s41467-024-46564-6

Figure Lengend Snippet: A Schematic representation of the Digital Spatial Profiling (DSP) approach, including the selection of three regions of interest (ROI): aggregate (characterised by the presence of CD3 + and CD20 + cells), deep sublining (characterised by the absence of CD3 + and CD20 + cells) and lining with superficial sublining (characterised by the presence of CD68 + cells). Scale bar = 100 μm. DSP was performed on 14 aggregates, 25 lining, and 33 sublining regions. B Three-way radial plot showing differential and overlapping genes across aggregate (green), lining (blue) and sublining (red) regions. AXL and MERTK genes have been labelled showing a significantly higher presence of AXL in both lining and sublining regions and a significant presence of MERTK in the lining region. Significance was internally estimated by the volcano3D package combining significance ( q < 0.05) from both one-way ANOVA and pairwise T test. C Double immunostaining of Axl (red) and MerTK (yellow) in the aggregate, sublining and lining synovial areas of RA patients. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. Representative images of n = 32 samples stained. D Expression of selected individual genes included in the AXL and/or MERTK networks in the aggregate ( n = 14, including 4 non-responder and 10 responder patients), sublining ( n = 33, including 12 non-responder and 21 responder patients) and lining ( n = 25, including 8 non-responder and 17 responder patients) synovial areas of responders (light blue) and non-responders (orange) RA patients to either tocilizumab or rituximab. Boxplots represent the median and first and third quartiles, and whiskers span to the minimum and maximum. A paired Wilcoxon test was undertaken to compare responders and non-responders.

Article Snippet: The following fluorescent markers were used to determine the morphology of the tissue: CD68-AF532 (clone KP1, Cat. N. NB100-683 Novus) for macrophages, CD20-DL594 (clone IGEL/773, Cat. N. NBP2-44745, Novus) for B cells, and CD3-AF647 (clone UMAB54, Cat. N. TA807198, Origene) for T cells; nuclei were counterstained with Syto13 (Cat. N. S7575, ThermoFisher).

Techniques: Selection, Double Immunostaining, Staining, Expressing