Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A ) Expression of CD27 and IgD on CD19 + B cells. Right: Percentage of IgD + CD27 − naïve B cells and IgD − CD27 − DN B cells. ( B ) Expression of CD27 and CD38 on CD19 + B cells. Right: Percentages of CD27 hi CD38 hi ASCs and CD27 + CD38 − memory B cells. ( C ) Expression of CD138 on CD27 hi CD38 hi ASCs. Right: Percentage of CD138 + ASCs and CD138 − ASCs. ( D ) Expression of CD11c and CD21 on CD19 + B cells. Right: Percentage of CD11c + CD21 − CD19 + B cells. [(A) to (D)] HC ( n = 63), irAE ( n = 33), RAC ( n = 46), and ICI ( n = 20). ( E to G ) GSEA was performed on the B cells between irAE and ICI. (E) Significantly enriched pathways in B cells from irAE and ICI. GSEA plots of IFN-α and IFN-γ response (F), and oxidative phosphorylation (G). ( H ) The volcano plots of the citrullinated or noncitrullinated relative IgG or IgM isotype autoantigen levels comparing RA versus HC, irAE versus ICI, irAE versus HC, or irAE versus RA. The autoantigens were labeled when P < 0.01. TNF-α reactivities resulted from the administration of anti–TNF-α therapy for the treatment of RA. ( I ) Immunoglobulin isotype levels in the plasma were measured by multiplex assay. HC ( n = 22), irAE ( n = 34), RAC ( n = 46), and ICI ( n = 26). ( J ) CXCL13 levels in the plasma were measured by enzyme-linked immunosorbent assay (ELISA). HC ( n = 20), irAE ( n = 34) RAC ( n = 47), and ICI ( n = 23). ( K ) B-cell activating factor (BAFF) levels in the plasma were measured by multiplex assay. HC ( n = 21), irAE ( n = 34), RAC ( n = 47), and ICI ( n = 18). Data in graphs represent mean ± SEM. Significance was tested by one-way ANOVA [(A) to (D) and (I) to (K)] and logistic regression (H). [(A) to (D) and (F) to (K)] ICI, ICI control.

Article Snippet: For CXCL13, IL-21, and CX3CL1 measurements, the following kits were used: Human CXCL13/BLC/BCA-1 Quantikine Enzyme-Linked Immunosorbent Assay (ELISA) (R&D Systems, catalog no. DCX130), Human IL-21 DuoSet ELISA (R&D Systems, catalog no. DY8879-05), and Human CX3CL1/Fractalkine DuoSet ELISA (R&D Systems, DY365); all steps were performed according to the manufacturer’s instructions.

Techniques: Expressing, Phospho-proteomics, Immunopeptidomics, Labeling, Clinical Proteomics, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: Untreated hyperacute HIV, but not ART early-treated hyperacute HIV, is associated with elevation of plasma cytokines that have distinct kinetics. a Interferon gamma-induced protein 10 (IP-10/CXCL-10). b Monokine induced by gamma interferon (MIG/CXCL-9). c Monocyte chemoattractant protein 1 (MCP-1). d Interleukin 12 (IL-12). e Soluble IL-2 receptor (IL-2R). f Interleukin 8 (IL-8). g Interferon gamma (IFN-gamma). h Interleukin-1 receptor antagonist (IL-1RA). i B cell-activating factor (BAFF/BLYS/TNFSF13B). j Chemokine (C-X-C motif) ligand 13 (CXCL13). k Soluble CD14. l Interferon alpha (IFN-alpha). N = 12 for untreated hyperacute HIV-infected participants (except CXCL13 and BAFF with N = 10). N = 8 for ART early-treated hyperacute HIV-infected individuals (except CXCL13 and BAFF with N = 6 and IFN-alpha with N = 7). Cytokine levels for one of the untreated participants were measured 434 days instead of 238–263 days after the detection of viremia. Each symbol represents an individual participant. Except for IFN-alpha, red symbols show the plasma levels in untreated participants and blue symbols show the plasma levels in ART early-treated participants. Horizontal lines and error bars in the scatter plots represent the median and interquartile range. In l (IFN-alpha), every colored line represents a participant. Statistical test used: Wilcoxon matched-pairs signed-rank test. P values < 0.05 were considered significant. * P < 0.05, ** P < 0.01, *** P < 0.001. “Pre” refers to the pre-infection time point

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics, Infection

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: CXCL13 is positively associated with delayed suppression of viremia in early-treated individuals. a Duration to viral suppression in days among early-treated participants. b Correlation between duration to viral suppression in days and viral load at the time of initiating ART in early-treated individuals. c Correlation between duration to viral suppression and plasma CXCL13 levels at 3 months. d Correlation between viral load at the time of initiating ART and plasma CXCL13 levels at 3 months. Each symbol represents an individual participant ( N = 6). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: The magnitude of plasma cytokines predicts CD4 + T cell and viral load dynamics in untreated hyperacute HIV infection. a Correlation between peak IFN-alpha and peak viremia. b Correlation between hyperacute soluble IL-2 receptor and peak viremia. c Correlation between hyperacute IL-1RA and viral load set point. d Correlation between hyperacute CXCL13 and nadir CD4 + T cell counts. e Correlation between hyperacute soluble IL-2 receptor and nadir CD4 + T cell counts. f Correlation between hyperacute IL-1RA and set point CD4 + T cell counts. Each symbol represents an individual participant ( N = 12 except CXCL13 with N = 10). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics, Infection

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: Plasma cytokines/chemokines are associated with reduced blood counts of lymphocytes, eosinophils, and basophils in untreated acutely HIV-infected patients. a Correlation between CXCL13 and total lymphocytes. b Correlation between CXCL13 and eosinophils. c Correlation between CXCL13 and basophils. d Correlation between MIG/CXCL9 and total lymphocytes. e Correlation between MIG/CXCL9 and eosinophils. f Correlation between MIG/CXCL9 and basophils. g Correlation between soluble IL-2 receptor and total lymphocytes. h Correlation between soluble IL-2 receptor and eosinophils. i Correlation between soluble IL-2 receptor and basophils. The measurements of cytokines and blood cell counts were in the hyperacute phase of HIV infection. Each symbol represents an individual participant ( N = 12 except CXCL13 ( a – c ) with N = 10). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics, Infection

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: Correlation network showing a summary of the relationships between cytokines, CD4 + T cell dynamics, viral load dynamics, Gag-driven viral replication capacity, and hematological parameters in untreated hyperacute HIV infection. Statistical test used: Spearman’s rank-order correlation. Red lines show significant positive correlations. Blue lines show significant inverse correlations. The width of the line indicates the strength of Spearman’s correlation coefficient (rho). Only correlations that have P < 0.05 are shown. Gag RC, Gag-driven viral replication capacity ( N = 12 except CXCL13 and BAFF with N = 10)

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Infection

Journal: International Journal of Molecular Sciences

Article Title: Chemokines Up-Regulated in Epithelial Cells Control Senescence-Associated T Cell Accumulation in Salivary Glands of Aged and Sjögren’s Syndrome Model Mice

doi: 10.3390/ijms22052302

Figure Lengend Snippet: Salivary gland epithelial cells from aged mice display a unique gene expression phenotype. ( A ) Heatmap of gene expression levels of selected function-associated genes in isolated epithelial cells from young or aged C57BL/6N mice. Each column shows the expression level of a single sample. The log2-transformed ratio of normalized signal intensities versus the median signal intensity of these samples is shown. ( B , C ) CXCL13 and p16 Ink4a mRNA expression levels in salivary gland ( B ) or pulmonary ( C ) epithelial cells harvested from young (Y) or aged (A) mice, as analyzed by quantitative real-time PCR. Values are shown as means ± SEM ( B : n = 6–9 mice per group, C : n = 5 mice per group). ND, not detected. ** p < 0.01. NS, not significant (the Student’s unpaired t -test). ( D ) Senescence-associated staining for SPiDER-βGal in frozen sections of salivary glands harvested from young and aged mice. Nuclei were stained with DAPI. Bars = 50 μm. ( E ) CXCL13 expression in the salivary glands of aged mice as detected by an immunofluorescence analysis using FITC-conjugated anti-mouse CD326 (EpCAM) mAb and an anti-mouse CXCL13 polyclonal antibody. Nuclei were stained with DAPI. Images show a Z-stack tile series of pictures. Bars = 20 μm.

Article Snippet: An equal volume of medium containing CXCL13 (0–4 μg/mL) (BioLegend) was added to the lothermower chamber in 350 μL of 0.1% BSA-RPIM 1640 medium, and cells were then cultured at 37 °C for 6 h. The number of migrated cells was analyzed by flow cytometry.

Techniques: Gene Expression, Isolation, Expressing, Transformation Assay, Real-time Polymerase Chain Reaction, Staining, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Chemokines Up-Regulated in Epithelial Cells Control Senescence-Associated T Cell Accumulation in Salivary Glands of Aged and Sjögren’s Syndrome Model Mice

doi: 10.3390/ijms22052302

Figure Lengend Snippet: Enhanced migratory rate of CD4 + T cells toward CXCL13 isolated from aged mice. ( A ) The cell surface expression of CXCR5 on naïve and TEM cells from the spleens of young (Y) and aged (A) female C57BL/6N mice was detected by flow cytometry ( n = 5–6 mice per group). Left histograms show a representative of those from each group of mice. Values are shown as means ± SEM. * p < 0.05 (the Student’s unpaired t -test). ( B – D ) Dose-dependent migratory response of TEM cells ( B ), naïve cells ( C ), and PD-1 + CD153 + and PD-1 + CD153 − TEM cells ( D ) to increasing concentrations of CXCL13 in aged female mice. Values are shown as the means ± SEM of tetraplicate measurements. These results are two independent experiments. * p < 0.05 and ** p < 0.01 versus the control group (the CXCL13 concentration is 0 μg/mL) (Dunnett’s multiple comparison test). ( E ) CD153 + CD4 + T cell localization in the SG of aged female mice was detected by an immunofluorescence analysis. Lower images show high magnification (×100) of the square area of the upper image. CD4 (green), EpCAM (red), CD153 (purple), CD4 and CD153 (white), and nuclei (blue). The scale bars of upper and lower images are 20 and 10 μm, respectively.

Article Snippet: An equal volume of medium containing CXCL13 (0–4 μg/mL) (BioLegend) was added to the lothermower chamber in 350 μL of 0.1% BSA-RPIM 1640 medium, and cells were then cultured at 37 °C for 6 h. The number of migrated cells was analyzed by flow cytometry.

Techniques: Isolation, Expressing, Flow Cytometry, Control, Concentration Assay, Comparison, Immunofluorescence

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Representative images of multiplex IHC staining of TLS in NPC tissue biopsy. Cells were coloured according to their staining with CD3 (green), CD4 (cyan), CD8 (orange), CD20 (red), and FAP-α (purple) proteins as indicated on top. Images are representative data from three independent NPC samples. Scale bars is 50 µm. b UMAP plots of cell clusters for B cells, CD4 + T cells, CD8 + T cells, and CAFs. Individual cell types were segregated from the major cell clusters identified in Supplementary Fig and subsequently clustered independently. Each dot represents a single cell, with colour coded according to the cell types or clusters as indicated in the legend to the right (comprising five, 16, 11, and five clusters for 97,360 B cells, 88,009 CD4 + T cells, 95,686 CD8 + T cells, and 1652 CAFs, respectively). c Heatmap plots of the normalised mean expression of TLS canonical marker genes (columns) for different cell clusters (rows) in B, CD4 + T, CD8 + T cells, and CAFs. Filled colours from blue to red represent scaled expression levels from low to high. d Representative images of Hematoxylin-Eosin staining (left) for TLSs and spatial feature plots of TLS-associated cell types (right) from the Stereo-seq (top; n = 3) and the Visium cohorts (bottom; n = 12). Dashed outlines indicate TLS areas on the H&E slides with scale bars of 0.1 mm and 300 μm. In spatial feature plots, the expression levels of the TLS-associated cluster signatures for each cell type (indicated on top) in each spot are filled with colours from black to yellow, representing scaled expression levels from low to high. e Box plots showing the signature scores of B lineage cells (Naïve_B, Memory_B, GC_B, and plasma cells), CD4_C8_CXCR5, CD8_C8_CXCL13, and iCAF_C2_CXCL13 in different tumour regions from NPC patients of the Stereo-seq (top; n = 9563) and the Visium cohorts (bottom; n = 21,753). Centre lines denote median values, and whiskers denote 1.5 × the interquartile range. P values are derived from two-sided student t-tests. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: Multiplex Assay, Immunohistochemistry, Staining, Expressing, Marker, Derivative Assay

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Heatmap showing the normalised mean expression of cluster markers, complement, adhesion molecules, and chemokines (rows) for each CAF cluster (columns). Filled colours from blue to red represent scaled expression levels from low to high. b Bar plot of GSVA results showing the selected up-regulated signalling pathways enriched in iCAF_C2_CXCL13 CAFs. c Box plots showing the proportions of fibroblast clusters among all fibroblasts in NPC samples at different stages ( n = 16; removing samples without detectable malignant cells in the NPC tissue and samples with fewer than 100 CAFs). Centre lines denote median values, and whiskers denote 1.5 × the interquartile range. P values are derived from two-sided student t-tests. d Representative images for multiplex IHC staining of CXCL13 + CAFs and CXCR5 + B cells in NPC tissue biopsy. Cells were coloured according to their staining with CXCR5 (green), CD20 (cyan), FAP-α (orange), and CXCL13 (red) proteins as indicated on top. The green, cyan, orange, and red arrows indicated positive cells with the expression of CXCR5, CD20, FAP-α, and CXCL13 proteins in NPC tissue, respectively. Images are representative data from three independent NPC samples. Scale bars are 30 µm or 15 µm, respectively. e Dot plot showing selected ligand-receptor interactions between iCAF_C2_CXCL13 CAFs and B cells (GC_B, Memory_B, Naïve_B, and plasma cells) in NPC (red) or NPN (blue) samples. Ligand-receptor interactions are indicated in rows. The means of the average expression levels of two interacting molecules are indicated by the colour heatmap (bottom panel), with blue to red representing low to high expression. P values are obtained by one-sided permutation tests and indicated by circle size. f Spatial distribution of ligand-receptor interactions in representative NPC sections from the Stereo-seq (top panel) and the Visium cohorts (bottom panel). Each ligand and its corresponding receptor are indicated on top, with different colours for low and high intensities, as indicated at the right panel. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: Expressing, Derivative Assay, Multiplex Assay, Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Schematic diagram showing the B cell adhesion assay (left panel). Bar plots showing the capability of B cell adhesion to CAFs (right panel; n = 3). B cells were cocultured with either VCAM1 - CAFs, VCAM1 + CAFs, or VCAM1 + CAFs in combination with a VCAM1 neutralising antibody. The relative number (OD value) of B cells bound to CAFs were quantified using CCK8 assays. b Bar plot showing the relative expression level of CXCL13 in fibroblasts (left panel; n = 3). Fibroblasts were transduced with an empty vector or CXCL13 plasmid. Bar plot illustrates the migration capabilities of B cells under various co-culture conditions with fibroblasts (right panel; n = 11). The B cells were co-cultured with fibroblasts overexpressing either an empty vector (Con_EV), CXCL13 alone, or CXCL13 in combination with a CXCL13-specific neutralising antibody. c Schematic diagram showing the B cell co-culture assay (left panel). Bar plots showing IgG production of B cells without (grey) or with coculture of VCAM1 + (orange) or VCAM1 - (blue) CAFs (right panel; n = 3). The IgG levels in the cocultured supernatant were determined using ELISA. d Bar plot showing the relative expression level of TNFSF13B in fibroblasts (left panel; n = 3). Fibroblasts were transduced with an empty vector or TNFSF13B plasmid. Bar plot showing the IgG production of B cells co-cultured with fibroblasts overexpressing either an empty vector (Con_EV), TNFSF13B alone, or TNFSF13B in combination with a TNFSF13B-specific neutralising antibody (right panel; n = 4). IgG level in supernatant was determined using ELISA. P values are derived from two-sided student t-tests. The data is presented as mean ± SD. OE overexpression, Neu neutralising antibody, Con control, EV empty vector. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: Cell Adhesion Assay, Expressing, Transduction, Plasmid Preparation, Migration, Co-Culture Assay, Cell Culture, Co-culture Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Over Expression, Control

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Heatmap showing normalised mean expression of markers (naïve, effector, tissue resident, exhausted, hypoxia, activation, stem-like, toll-like receptor families, and chemokines) for each CD8 + T cell cluster. Colours from blue to red represent low to high scaled expression levels. b Representative images for multiplex IHC staining of CXCL13 + CD8 + T cells in NPC tissue biopsy. Cells coloured by PD1 (green), TIM3 (yellow), CD20 (orange), CD8 (purple), and CXCL13 (red). Arrows indicate cells expressing PD1 (green), TIM3 (yellow), CD20 (orange), CD8 (purple), and CXCL13 (red) in NPC tissue. Representative images from three independent NPC samples. Scale bars = 40 µm. c Pseudotime trajectories of CD8 + T cells (CD8_C1-C11; n = 41,736) with TCR information. Each dot represents a single cell, coloured by cluster label (top) or top 10 TCR clonotypes of CD8_C8_CXCL13 cells (bottom). d Scatter plots showing exhausted (left) and stem-like (right) scores of CD8 + T cells along pseudotime scores (x-axis) among clusters. Each dot represents a single cell, coloured by cluster label as in panel ( c ). e Box plots showing the percentage of CD8 + T cells sharing TCR clonotypes with top 10 TCR clonotypes of CD8_C8_CXCL13 cells across all CD8 + T cells. Coloured dots represent patients ( n = 3). Comparison made using a two-sided Kruskal-Wallis test across all clusters. f Representative spatial feature plots showing expression levels of CD8_C8_CXCL13 and CD8_C11_HAVCR2 signatures in each data spot from Stereo-seq (left; n = 9563) and Visium (right; n = 21,753) cohorts. Colours from black to yellow represent low to high scaled expression levels. Box plots show signature scores of CD8_C8_CXCL13 and CD8_C11_HAVCR2 in TLS, TCA, and stromal regions from Stereo-seq (left) and Visium (right) cohorts. Comparisons were made using a two-sided student t-test. g Pie charts showing contributions of ligand-receptor pairs among interactions between malignant and CD8_C8_CXCL13 cells (top) or CD8_C11_HAVCR2 cells (bottom) in NPC. Ligand-receptor pairs indicated on right panel with different colours. Percentages are shown in pies. In box plots, endpoints depict minimum and maximum values; centre lines denote median values; whiskers denote 1.5 × the interquartile range. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: Expressing, Activation Assay, Multiplex Assay, Immunohistochemistry, Comparison

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Survival analysis of the signature scores for B lineage cells (Naïve_B, Memory_B, GC_B, and plasma cells), CD4_C8_CXCR5, CD8_C8_CXCL13, and iCAF_C2_CXCL13 in two NPC cohorts (Bulk-RNA-seq, n = 147; and Microarray, n = 150). P value (y-axis) and HR (x-axis) were calculated using two-sided cox tests and plotted in different colours for specific cell types annotated at the right. PFS, progression-free survival; OS, overall survival. b Heatmap showing the normalised signature scores for B lineage cells (Naïve_B, Memory_B, GC_B, and plasma cells), CD4_C8_CXCR5, CD8_C8_CXCL13, and iCAF_C2_CXCL13 (rows) in each sample (columns) from three NPC cohorts as indicated (source). Filled colours from blue to red represent scaled expression levels from low to high. Coloured bars on top of the heatmaps indicate metastasis, OS (0: dead; 1: alive) or PFS (0 or 1 represent with or without progression), and sample source. Samples were grouped using two modules with low or high TLS cell signature (TLS-CS). c Kaplan-Meier survival curves of the two NPC cohorts (Bulk-RNA-seq, n = 147; Microarray, n = 150) with patients stratified by two modules of low or high TLS-CS. Survival duration and probability are indicated at the x- and y-axis, respectively. P value and HR were calculated using a two-sided cox test. d Box plots showing the signature scores for B lineage cells (Naïve_B, Memory_B, GC_B, and plasma cells), CD4_C8_CXCR5, CD8_C8_CXCL13, and iCAF_C2_CXCL13 in the responders (R) and non-responders (NR) to ICB (toripalimab plus chemotherapy) treatment from the Visium cohort of NPC ( n = 12). Centre lines denote median values, and whiskers denote 1.5 × the interquartile range. P values are derived from two- sided student t-tests. **** P < 2.22×10 -16 . Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: RNA Sequencing Assay, Microarray, Expressing, Derivative Assay

Journal: Nature Cancer

Article Title: Reprogramming of stroma-derived chemokine networks drives the loss of tissue organization in nodal B cell lymphoma

doi: 10.1038/s43018-026-01136-z

Figure Lengend Snippet: a , Left, UMAP of LN stromal cells by entity. Right, percentages of rFRCs and rBECs (rLN n = 5, FL n = 6, DLBCL n = 8 patients). b , Differentially expressed genes between rLN-derived ( n = 2,363 cells) and DLBCL-derived ( n = 2,983 cells) FRCs (adjusted P < 0.05, log(fold change) > 0.5). c , Homeostatic (top) and inflammatory (bottom) chemokine expression in bulk data . d , Pearson correlation of CXCL13 expression and CIBERSORTx-derived FDC fractions. e , CXCL13 plasma protein levels in FL ( n = 18 patients) and DLBCL ( n = 22 patients). Vertical lines indicate the mean per entity. f , Exemplary rLN and DLBCL mIF images, representative of n = 4 patients per entity. Scale bar, 50 μm. Dashed circles: CD21+ regions. g , mIF-derived CXCL13 and CXCR5 signals averaged across four adjacent pairs of CD21 + follicular and CD21 − extrafollicular regions per sample. h , i , Spatial transcriptomics plots of FL-LN ( h ) and DLBCL-LN ( i ) cores colored by cell type and CXCL13 – CXCR5 ligand–receptor (L–R) score. j , mIF-derived enrichment of CXCL13 + cells per cell type in DLBCL versus rLN/FL samples. Asterisks indicate P < 0.01; exact P values are provided in the source data. k , CXCL13 expression in CD8 + T cells ( n = 21,268 cells) . l , Percentage of CXCR5 + cells within CD3 − fractions measured by flow cytometry (rLN n = 7, FL n = 24, DLBCL n = 18 patients). m , Migrated rLN- and DLBCL-derived B cells in the Transwell assay (mean ± s.d., n = 3 patients per condition). For c and d : tonsil n = 10, FL 1/2/3A n = 145, FL 3B n = 48, DLBCL n = 430 patients. For f , g and j : rLN n = 4, FL n = 5, DLBCL n = 4 patients. P values in a , c , e , g and l : two-sided Wilcoxon rank-sum test. P value in m : two-sided unpaired Welch’s t test. P values in j : two-sided Fisher’s exact test. P values in c , g and j were adjusted using the Benjamini–Hochberg method. Box plots: center line, median; box, interquartile range; whiskers, 1.5× the interquartile range; points, data values. FC, fold change; T tox EM, effector memory cytotoxic T cells; hr, human recombinant.

Article Snippet: Sorted cells were seeded in triplicate into the top chambers of 96-well HTS Transwell plates with 8-μm pores (Corning), with the bottom chambers containing either 1 μg ml −1 human recombinant CXCL13 (cat. no. 801-CX-025, R&D Systems) or PBS as a control.

Techniques: Derivative Assay, Expressing, Clinical Proteomics, Spatial Transcriptomics, Flow Cytometry, Transwell Assay, Recombinant

Journal: Nature Cancer

Article Title: Reprogramming of stroma-derived chemokine networks drives the loss of tissue organization in nodal B cell lymphoma

doi: 10.1038/s43018-026-01136-z

Figure Lengend Snippet: ( A ) Heatmap showing estimated cell type fractions as determined using CIBERSORTx (CSx) within a large bulk transcriptomics dataset . ( B ) Box plots depicting CSx fractions of exemplary cell types across disease entities. Boxes indicate the median (center line), interquartile range (bounds) and 1.5x interquartile range (whiskers). Data points represent patient samples. P-values were calculated using two-sided unpaired Welch’s t-test and adjusted according to Benjamini-Hochberg. ( C ) Dot plot showing Spearman correlation coefficients of chemokine expression and cell type fractions. Dot sizes represent -log10 FDR-adjusted p-value of correlation tests. ( D ) Scatter plots color-coded by disease entity correlating CXCL12 (left panel) and CCL21 (right panel) expression with CSx-derived FRC factions. P-values were calculated using Pearson correlation. ( E , F ) Bulk RNA-seq datasets , stratified by FDC abundance into high and low samples using maximally selected rank statistics based on CSx fractions, represented in a Kaplan-Meier curve showing overall survival (left panels; log-rank test) and scatter plot of FDC fractions and CXCL13 expression (right panels; Pearson correlation). T PR =T prol . For panels A – D : tonsil n = 10; FL 1/2/3 A n = 145; FL 3B n = 48; DLBCL n = 430 patients. For panel E : FDC-high n = 61; FDC-low n = 44 patients. For panel F : FDC-high n = 302; FDC-low n = 68 patients.

Article Snippet: Sorted cells were seeded in triplicate into the top chambers of 96-well HTS Transwell plates with 8-μm pores (Corning), with the bottom chambers containing either 1 μg ml −1 human recombinant CXCL13 (cat. no. 801-CX-025, R&D Systems) or PBS as a control.

Techniques: Transcriptomics, Expressing, Derivative Assay, RNA Sequencing

Journal: Nature Cancer

Article Title: Reprogramming of stroma-derived chemokine networks drives the loss of tissue organization in nodal B cell lymphoma

doi: 10.1038/s43018-026-01136-z

Figure Lengend Snippet: ( A ) Representative triangle-thresholded mIF images showing CD21 (top panels) as well as CXCL13 (bottom panels) signal in rLN (n = 4), FL (n = 5) and DLBCL (n = 4) patient samples. Scale bar indicates 50μm. ( B ) UMAP representation of spatial transcriptomics data colored by cell type (color code depicted in panel C ). ( C ) Heatmap showing scaled expression of key marker gene expression as well chemokines. ( D ) Stacked bar plot of FDC/FRC/rFRC fractions (top panel) and violin plot of CXCL13 expression in these subsets (bottom panel). ( E ) Spatial transcriptomics plots of FL (left panels) and DLBCL (right panels) tissue cores colored by cell type (left panels) alongside magnified views of chemokine expression. ( F ) Heatmap showing scaled expression of key T cell markers across CD8 + T cell populations as measured using mIF. ( G ) Box plot showing per-sample percentage of CXCL13 + cells (as determined using Otsu thresholding) among PD1 + CD8 + effector memory T cells in the mIF dataset. Boxes indicate the median (center line), interquartile range (bounds) and 1.5x interquartile range (whiskers). Data points represent patient samples. P-values were calculated using a two-sided unpaired Welch’s t-test. ( H ) Spearman correlation coefficients of key T cell markers across all CD8 + T cell populations (n = 21,268 cells). ( I ) Volcano plot showing differentially expressed genes comparing PD1 + CD8 + effector memory T cell populations (T TOX EM-II n = 2,018; T TOX EM-III n = 9,208 cells). Labels indicate cluster-identifying genes as well as CXCL13 . Abbreviations: T TOX EM = effector memory cytotoxic T cells. For panels A , F , G : rLN n = 4; FL n = 5; DLBCL n = 4 patients. For panels B – E : FL n = 1; DLBCL n = 1 patient.

Article Snippet: Sorted cells were seeded in triplicate into the top chambers of 96-well HTS Transwell plates with 8-μm pores (Corning), with the bottom chambers containing either 1 μg ml −1 human recombinant CXCL13 (cat. no. 801-CX-025, R&D Systems) or PBS as a control.

Techniques: Spatial Transcriptomics, Expressing, Marker, Gene Expression

Journal: Nature Cancer

Article Title: Reprogramming of stroma-derived chemokine networks drives the loss of tissue organization in nodal B cell lymphoma

doi: 10.1038/s43018-026-01136-z

Figure Lengend Snippet: a , UMAP representation of a microarray dataset with homeostatic ( CXCL12 , CXCL13 , CCL19 , CCL21 ) and inflammatory ( CXCL9 , CXCL10 , CXCL11 ) chemokine expression values used as features for dimensionality reduction. Left, UMAP displaying pie charts (within each dot) that represent the k = 20 nearest neighbors, colored according to disease entity. Right, the same UMAP colored according to the mean expression of homeostatic (top) and inflammatory (bottom) chemokines. b , Bulk RNA-seq dataset stratified according to homeostatic chemokine expression into high ( n = 519 patients) and low ( n = 99 patients) groups using maximally selected rank statistics, shown as a dot plot (top) and a Kaplan–Meier curve of overall survival (bottom). The P value was calculated using the log-rank test. c , Forest plot summarizing log 10 -transformed hazard ratios (center), 95% confidence intervals (error bars) and Wald-derived P values estimated from univariate Cox proportional hazards models assessing the association between homeostatic chemokine expression and overall survival across five individual DLBCL bulk datasets , – . d , Same UMAP as in a , colored by CIBERSORTx-derived FDC fractions. e , Bulk RNA-seq dataset stratified by FDC abundance into high ( n = 255 patients) and low ( n = 364 patients) groups using maximally selected rank statistics based on CIBERSORTx fractions, shown as a Kaplan–Meier curve of overall survival (left; log-rank test) and a scatter plot of log-transformed FDC fractions and CXCL13 expression (right; Pearson correlation). f , Forest plot summarizing log 10 -transformed hazard ratios (center), 95% confidence intervals (error bars) and Wald-derived P values estimated from Cox proportional hazards models assessing the association between FDC fraction and overall survival across five individual DLBCL bulk datasets , – . For panels a and d : tonsil n = 10, FL 1/2/3A n = 145, FL 3B n = 48 and DLBCL n = 430 patients. OS, overall survival; HR, hazard ratio.

Article Snippet: Sorted cells were seeded in triplicate into the top chambers of 96-well HTS Transwell plates with 8-μm pores (Corning), with the bottom chambers containing either 1 μg ml −1 human recombinant CXCL13 (cat. no. 801-CX-025, R&D Systems) or PBS as a control.

Techniques: Microarray, Expressing, RNA Sequencing, Transformation Assay, Derivative Assay