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: 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: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Single-cell and spatial transcriptome profiling of T cells and TLSs in the tumor microenvironment of gastric cancer. ( A ) The spatial feature plot showing the scores of TLSs, T cells, B cells and macrophage in different patients’ spatial transcriptomic data sets. P3, patient 3; P10, patient 10. ( B ) Comparison of the scores of different immune cells between TLS-high and TLS-low spots in different patients’ spatial transcriptomic data sets. ( C ) UMAP plot of reclustered T cells showing 10 clusters annotated in different colors. ( D ) Dot plot showing the average expression levels and cell expression proportions of the differential genes in these 10 T-cell clusters. Dot size encodes the percentage of cells expressing the gene, color encodes the average per cell gene expression level. ( E ) CD4-C8-CXCL13 and CD8-C6-CXCL13 exhibiting dysfunctional and inhibitory characteristics. ( F ) The heatmap showing the characteristic gene expression in each T-cell cluster. ( G ) UMAP plot showing the expression of CD4, CD8B, PDCD1, CXCL13, ITGAE and CD160 in T cells. ( H ) The plot showing the abundance of immune cells as the score of TLSs increased using the ACRG data set. Analysis includes 300 processed samples. ( I ) Correlation of lineage-normalized cell-type frequencies in the ACRG cohort. ( J ) Scatterplot demonstrating the correlation between the CD4-C8-CXCL13 and the B cells (left panel). Scatterplot demonstrating the correlation between the CD8-C6-CXCL13 and the TLS (right panel). ( K ) Survival curves showing the association between factors and overall survival in patients from the ACRG cohort (log-rank test). Survival curves for TLS (upper panel), CD4-C8-CXCL13 (middle panel) and CD8-C6-CXCL13 (lower panel) and p values are shown. ACRG, Asian Cancer Research Group; TLSs, tertiary lymphoid structures; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Comparison, Expressing, Gene Expression

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: CXCL13 + CD160 + CD8 + T cells and CXCL13 + PDCD1 + CD4 + T cell around TLSs at different stages of maturation were assessed by multiplex immunofluorescence. ( A ) Resected gastric tissues stained with H&E or subjected to immunohistochemistry (IHC) detection for CD8, CXCL13, CD160 and CD20. The framed areas are shown adjacently at a higher magnification. Images were captured under a light microscope. Scale bar, 500 µm. ( B ) Multiplex immunofluorescence staining of human gastric cancer tissues for detection of CXCL13 (pink), CD160 (orange), CD8 (red), CD20 (green) and DAPI in gastric cancer tissues. Scale bar, 200 µm. ( C ) Tumor sections stained with H&E and IHC, including CD4, CXCL13, PDCD1 and CD20. The framed areas are shown adjacently at a higher magnification. Scale bar, 500 µm. ( D ) Multiplex immunofluorescence experiments were performed to detect the degree of CXCL13 + PDCD1 + CD4 + T-cell infiltration around TLSs at different stages of maturation. Antibody panel: CXCL13 (pink), PDCD1 (orange), CD4 (red), CD20 (green). Scale bar, 200 µm. ( E ) The numbers of CXCL13 + CD160 + CD8 + T cells in patients with low (n=17) versus high (n=17) levels of TLSs were compared. ( F ) The density of CXCL13 + CD160 + CD8 + T cells were higher around mature TLSs. ( G ) Percentage of CXCL13 + CD160 + CD8 + T cells among CD160 + CD8 + T cells around TLSs at different stages of maturation. Data are presented as the mean±SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. DAPI, 4’, 6-diamidino-2-phenylindole; TLSs, tertiary lymphoid structures.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Multiplex Assay, Immunofluorescence, Staining, Immunohistochemistry, Light Microscopy, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: CXCL13 + CD160 + CD8 + T cells were strongly associated with clinical response to checkpoint immunotherapy. ( A ) Transcriptome analysis revealed that the number of TLSs and CXCL13 + CD160 + CD8 + T cells were significantly higher in response (CR+PR) groups (n=12) compared with non-response (SD+PD) groups (n=33). ( B ) and ( C ) Enhanced CT scan of the abdomen before and after treatment with immunotherapy. The red arrows in the CT image indicate the tumor location. Multiplex immunofluorescence experiments were performed to assess the number of intratumoral CXCL13 + CD160 + CD8 + T cells in patients with a PR or SD response. Antibody panel: CXCL13 (pink), CD160 (orange), CD8 (red), CD20 (green), scale bar, 400 µm. ( D–F ) The Halo software was used to calculate the quantity of intratumoral CXCL13 + CD160 + CD8 + T cells both pre-immunotherapy and post-immunotherapy across patients exhibiting varied treatment responses. ( G ) Comparison of the number of CXCL13 + CD160 + CD8 + T cells between patients with CR or PR responses (n=9) and patients with SD or PD responses (n=6) prior to immunotherapy based on multiplex immunofluorescence staining. ( H ) Comparison of the number of infiltrating CXCL13 + CD160 + CD8 + T cells in patients with different responses following immunotherapy. Data are presented as the mean±SD. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test. CR, complete response; PD, progressive disease; PR, partial response; SD, stable disease; ssGSEA, single-sample gene set enrichment analysis; TLSs, tertiary lymphoid structures.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Computed Tomography, Multiplex Assay, Immunofluorescence, Software, Comparison, Staining, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: CXCL13 + CD160 + CD8 + T cells communicated with B cells through CXCL13-CXCR5 interactions. ( A ) Correlation heatmap between various immune subset enrichments using the bulk RNA-seq of gastric cancer samples from TCGA cohort. ( B ) Bubble heatmap showing cell–cell communication inference between T-cell clusters and B cells through ligand and receptor binding. ( C ) Analyzing the correlation between different types of immune cells based on the integrated scRNA-seq data sets. ( D ) UMAP plot displaying the expression of CXCL13 in T cells and the expression of CXCR5 among all cell types. ( E ) Multiplex immunofluorescence experiments were performed to detect the degree of CXCR5 + B-cell infiltration around TLSs in patients with different responses following immunotherapy. Antibody panel: CXCL13 (orange), CXCR5 (red), CD20 (green), Scale bar, 100 µm. The framed areas are shown below at a higher magnification. White triangles indicated CXCR5 + B cells. The number of infiltrating CXCR5 + B cells in patients with different responses was statistically analyzed. ( F ) Multiplex immunofluorescence images showing the markers for CD38 (green), CD138 (red), CD27 (white) and DAPI. Scale bar, 100 µm. The framed areas are shown below at a higher magnification. White triangle indicated CD38 + CD138 + CD27 + B cells; yellow triangle indicated CD38 + CD138 − CD27 + B cells. The number of infiltrating CD38 + CD138 + CD27 + B cells and CD38 + CD138 − CD27 + B cells in patients with different responses was statistically analyzed. Data are presented as the mean±SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. DAPI, 4’, 6-diamidino-2-phenylindole; PR, partial response; SD, stable disease; scRNA-seq, single-cell RNA sequencing; TCGA, The Cancer Genome Atlas; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: RNA Sequencing, Binding Assay, Expressing, Multiplex Assay, Immunofluorescence, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Targeting PDXK could promote the formation of TLSs and enhance the efficacy of immunotherapy in gastric cancer. ( A ) Heatmap displaying the metabolic feature for T-cell clusters using scMetabolism package. ( B ) Dot plot showing the expression of the genes encoding rate-limiting enzymes of vitamin B 6 metabolism in different types of T cells. Dot size encodes the percentage of cells expressing the gene, color encodes the average per cell gene expression level. ( C ) Quantification of CXCL13 in PDTFs in the presence of different enzyme inhibitors measured by ELISA. ( D ) Representative H&E staining and PDXK immunohistochemistry of gastric cancer tissues with different responses following immunotherapy. Scale bar, 500 µm. ( E ) The schematic diagram of the animal experiments. ( F–G ) Images of tumors and tumor volume curves of 615 mice treated with various agents (n=6, each group). ( H ) Paraffin sections of mouse subcutaneous graft tumor tissue stained with H&E and IHC detection for CD8, CD20 and CXCL13. Scale bar, 100 µm. ( I ) The number (left panel) and area (right panel) of TLS per tumor area were compared between groups (n=6, each group). Data are presented as the mean±SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. AOX1, aldehyde oxidase 1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CR, complete response; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PDXK, pyridoxal kinase; PDXP, pyridoxal phosphatase; PDTFs, patient-derived tumor fragments; PHOSPHO2, phosphatase orphan 2; PNPO, pyridoxamine 5'-phosphate oxidase; PR, partial response; PSAT1, phosphoserine aminotransferase 1; s.c, subcutaneous injections; SD, stable disease; TLS, tertiary lymphoid structures.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Two Tailed Test, Saline, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Vitamin B 6 could promote the expression and secretion of CXCL13 in CD160 + CD8 + T cells. ( A ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in PDTFs in the presence of different drugs measured by ELISA. ( B ) The schematic diagram of orthotopic transplanted tumor model with diets containing various amounts of vitamin B 6 (n=6, each group). ( C ) Mouse orthotopic stomach xenograft tumor tissues stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( D ) The schematic diagram of orthotopic transplanted tumor model fed with different drugs or diets (n=6, each group). ( E ) The representative images of mouse bioluminescence imaging at week 3 (left panel) and the corresponding quantification analysis (right panel). ( F ) Representative micrographs of xenografts stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( G–H ) Density of TLSs (left panel) and ratio of tumor area occupied by TLSs (right panel). ( I ) Gating strategy for CD160 + CD8 + T cells. ( J ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in supernatants from CD160 + CD8 + T-cell cultures in the presence or absence of PL measured by ELISA. ( K ) Flow cytometric analysis and corresponding quantification of CXCL13 + CD160 + CD8 + T cells with or without PL treatment. Data are presented as the mean±SD. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-tailed Student’s t-test. ICIs, immune checkpoint inhibitors; IFN, interferon; IHC, immunohistochemistry; IL, interleukin; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PL, pyridoxol; PD-1, programmed cell death protein 1; s.c, subcutaneous injections; TLS, tertiary lymphoid structures; TNF, tumor necrosis factor.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Imaging, Two Tailed Test, Immunohistochemistry, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Vitamin B 6 could reduce the ubiquitination modification of HIF-1α by MDM2 and promote HIF-1α-mediated CXCL13 transcriptional activation. ( A ) qRT-PCR analysis showing the mRNA levels of HIF-1α in CD160 + CD8 + T cells cultured with or without PL (0.5 mM) for 48 hours. Results were normalized with GAPDH. ( B ) Western blot analysis showing the protein levels of SP1, HIF-1α, NFκB and POU5F1 in CD160 + CD8 + T cells cultured with or without PL (0.5 mM) for 48 hours. ( C ) mRNA levels of CXCL13 in CD160 + CD8 + T cells treated with PL (0.5 mM) alone or the combination of 2-MeoE2 (10 µM) and PL (0.5 mM). CXCL13 mRNA was measured by qRT-PCR. Results were normalized with GAPDH. ( D ) HEK293T cells were cultured with or without 0.5 mM PL treatment for 48 hours and then treated with cycloheximide (20 µg/mL) for the indicated duration. Western blot analysis of intracellular Flag-HIF-1α with a Flag antibody. ( E–F ) HEK293T cells were treated with PL at the indicated concentrations for 48 hours and then treated with or without MG132 (10 µM) or NH 4 Cl (20 mM). Western blot analysis of intracellular Flag-HIF-1α with a Flag antibody. ( G ) Determination of the effect of PL (0.5 mM) treatment on HIF-1α ubiquitination. ( H–I ) Co-immunoprecipitation was performed to demonstrate HIF-1α interacted with endogenous and exogenous MDM2. ( J ) HEK293T-HIF-1α or MDM2-knockout HEK293T-HIF-1α cells were treated with different concentrations of PL for 48 hours. Western blot analysis of intracellular Flag-HIF-1α levels. ( K ) HEK293T cells were transfected with Flag-HIF-1α and increasing concentrations of His-MDM2. Western blot showing Flag-HIF-1α and MDM2 expression. ( L ) HEK293T cells were transfected with the indicated plasmids for 24 hours, treated with or without 0.5 mM PL for 48 hours, and immunoprecipitated with a Flag antibody. Western blot analysis of HIF-1α ubiquitination. Data are presented as the mean±SD. ns, not significant. **p<0.01, two-tailed Student’s t-test. mRNA, messenger RNA; PL, pyridoxol; qRT-PCR, quantitative reverse transcription polymerase chain reaction; Ub, ubiquitin.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Ubiquitin Proteomics, Modification, Activation Assay, Quantitative RT-PCR, Cell Culture, Western Blot, Immunoprecipitation, Knock-Out, Transfection, Expressing, Two Tailed Test, Reverse Transcription, Polymerase Chain Reaction

Journal: EBioMedicine

Article Title: Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer.

doi: 10.1016/j.ebiom.2024.105003

Figure Lengend Snippet: Fig. 5: hMENA/hMENAΔv6 influences the expression and signaling of LTβR and the secretion of CXCL13 in CAFs. The ‘epithelial’ hMENA11a isoform in tumor cells affects CXCL13 production by TRM cells. a. qRT-PCR analysis of LTβR mRNA expression in the CAFs obtained from four different patients with NSCLC (#359, #358, #391, #405), transfected with control (Si-CNTR), and hMENA(t) pool siRNAs (Si- hMENA(t)). Data reported are the mean of technical triplicates. P value of paired 2-tailed Student’s t test is reported. b. Representative WB analysis with the indicated Abs of protein extracts from CAF #302 transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA, untreated or treated with 50 ng/mL of LIGHT for 24 h. Fold change of P-p65/p65 and of p52/p100 staining intensity (right). Data reported are the mean of 4 different experiments ± SD. Adjusted P values of One-way ANOVA followed by Tukey’s multiple comparisons procedures are reported when significant. c. CXCL13 production evaluated by ELISA assay of CAFs derived from three different patients (#302; #358; #571) transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA. Data reported are the mean of technical triplicates of pg/mL normalized for total protein content in three biological replicates. P value of paired two tailed t test, is reported d. Percentage of CXCL13 chemokine, evaluated by multiparametric flow cytometry, in ex-vivo TILs isolated from eight patients with NSCLC, after culture (24 h) with CM from H1650 tumor cells silenced for hMENA11a (Si-hMENA11a), or control (Si-CNTR). Results within CD8+ TRM (CD103+CD69+, left panel) or CD4+ TRM (CD103+CD69+, right panel) are shown. Statistical significance was determined using non-parametric Wilcoxon rank test.

Article Snippet: Levels of CXCL13 in conditioned media were determined by Enzyme-linked immunosorbent assay (ELISA) (EA 100469, Origene) after 48 h from silencing or transfection of CAFs and IMR-90 as described above.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Staining, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test, Cytometry, Ex Vivo, Isolation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: DEX-Induced SREBF1 Promotes BMSCs Differentiation into Adipocytes to Attract and Protect Residual T-Cell Acute Lymphoblastic Leukemia Cells After Chemotherapy.

doi: 10.1002/advs.202205854

Figure Lengend Snippet: Figure 2. BMSC-derived adipocytes attract T-ALL cells via CXCL13. A) Direct coculture systems of T-ALL cell lines (Jurkat or SupT1 marked by mScarlet fluorescent protein) with induced BMSC. The BODIPY marking adipocytes in green. The circles label the aggregation of T-ALL cell around adipocytes. Bar: 150 μm. B) The results of the cell migration assay. The upper row shows the GFP-labeled T-ALL cells that migrated into the bottom chamber through the Transwell. The lower row shows the coexisting system in the bottom chamber of the Transwell. Bar: 150 μm. C) Relative expression analysis

Article Snippet: The mouse plasma was collected from peripheral blood and CXCL13 was examined by the commercial Human CXCL13 ELISA kit (Elabscience, E-EL-M0190c).

Techniques: Derivative Assay, Cell Migration Assay, Labeling, Expressing