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: Mediators of Inflammation

Article Title: LPA Promotes T Cell Recruitment through Synthesis of CXCL13

doi: 10.1155/2015/248492

Figure Lengend Snippet: Effect of LPA on CXCL13 secretion in the murine air pouch with or without TNF- α pretreatment. (a) Six-day-old air pouches were produced in the dorsal skin of mice and injected with TNF- α or the vehicle for 16 h prior to stimulation with LPA for 2 h. The nontreated (NT) group was injected with vehicle only (PBS-BSA). The air pouch exudates ( n = 3) were collected and pooled for qualitative analysis of cytokine/chemokine secretion using the Proteome Profiler Mouse Antibody Array Panel (a). (b) Normalized data representing CXCL13 pixel density are the mean from two independent experiments.

Article Snippet: CXCL13 ELISA dual kit, rat anti-mouse CXCL13 antibody (rat IgG2A, clone 143614), control rat IgG2A (clone 54447), and Proteome ProfilerTM Mouse Cytokine Array Panel A were purchased from R&D Systems Inc. (Minneapolis, MN, USA).

Techniques: Produced, Injection, Ab Array

Journal: Mediators of Inflammation

Article Title: LPA Promotes T Cell Recruitment through Synthesis of CXCL13

doi: 10.1155/2015/248492

Figure Lengend Snippet: Effect of LPA and TNF- α on CXCL13 secretion in the air pouch. (a), (b) Kinetics of LPA and of TNF- α -mediated CXCL13 secretion. (a) LPA (3 μ g) or (b) TNF- α (50 ng) was injected into air pouches and air pouch exudates were collected at indicated times. (c) Kinetics of LPA-induced CXCL13 secretion in air pouches pretreated with TNF- α . TNF- α was injected 16 h before LPA stimulation. Air pouch exudates were collected at indicated times. (d) Comparison of LPA-mediated CXCL13 secretion in untreated and TNF- α -primed air pouches. TNF- α or vehicle was injected 16 h prior to administration of LPA for 2 h. Exudates were collected and cytokine/chemokine secretion was measured by ELISA. The nontreated (NT) groups were injected with vehicle only (PBS-BSA). Data are the mean ± SE from three independent experiments performed with at least five mice per group. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: CXCL13 ELISA dual kit, rat anti-mouse CXCL13 antibody (rat IgG2A, clone 143614), control rat IgG2A (clone 54447), and Proteome ProfilerTM Mouse Cytokine Array Panel A were purchased from R&D Systems Inc. (Minneapolis, MN, USA).

Techniques: Injection, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: LPA Promotes T Cell Recruitment through Synthesis of CXCL13

doi: 10.1155/2015/248492

Figure Lengend Snippet: Effect of the CXCL13 neutralizing antibody on LPA-mediated lymphocyte recruitment into TNF- α -primed air pouches. Air pouch tissues were pretreated with TNF- α (50 ng) for 16 h prior to administration of LPA (3 μ g). Where indicated, the anti-CXCL13 neutralizing antibody or the isotype control antibody (IgG2A, 10 μ g) was administered into the air pouches 15 min prior to stimulation with LPA for 4 h. The absolute numbers of CD3 + T cells in air pouch lavage fluids were determined by flow cytometry as described in . Data are the mean ± SE of at least 5 mice/group. ∗∗ P < 0.01 by analysis of variance.

Article Snippet: CXCL13 ELISA dual kit, rat anti-mouse CXCL13 antibody (rat IgG2A, clone 143614), control rat IgG2A (clone 54447), and Proteome ProfilerTM Mouse Cytokine Array Panel A were purchased from R&D Systems Inc. (Minneapolis, MN, USA).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: The expression of CXCL13 in human serum, prostate tissue and cell lines. ( A ) Serum CXCL13 concentration in BPH patients and healthy controls. ( B , C ) The protein and mRNA expression of CXCL13 in BPH tissues and normal ones. ( D – F ) The mRNA and protein expression of CXCL13 in BPH-1 and WPMY-1 cells. Representative blots are shown. * p < 0.05, ** p < 0.01 and *** p < 0.001. ( G , H ) Immunofluorescence localization of CXCL13 in BPH tissues and normal ones. ( I , J ) Immunofluorescence localization of CXCL13 in BPH-1 and WPMY-1 cells. DAPI (blue) indicates nuclear staining and Cy3-immunofluorescence (red) indicates CXCL13 protein staining. Representative graphs are shown. All scale bars are 100 μm.

Article Snippet: According to the manufacturer’s instructions, human CXCL13 ELISA kit (Abcam, Cambridge, UK, Cat. ab179881) was used to detect serum CXCL13 concentration in BPH patients and healthy controls.

Techniques: Expressing, Concentration Assay, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: Knockdown of CXCL13 inhibited proliferation, EMT and promoted apoptosis of BPH-1 cells via ERK1/2 and AKT pathway. ( A – C ) Knockdown efficiency of CXCL13 at mRNA and protein levels. ( D , F ) Statistical analysis of apoptotic rate (%) and percentages (%) of cells at each stage. ( E , G ) Flow cytometry analysis of cell apoptosis and cell cycle. ( H ) CCK8 assay of BPH-1 cells. *** means p < 0.001 between si-con and si-2, ### means p < 0.001 between si-2 and si-2+rHuCXCL13. ( I , J ) Western blot assay of cell-cycle-, cell-apoptosis- and pathway-related proteins. ( K ) Relative densitometric quantification of cell-cycle-, cell-apoptosis- and pathway-related proteins. ( L ) Western blot assay of EMT-related proteins. ( M ) Relative densitometric quantification of EMT-related proteins. * p < 0.05, ** p < 0.01, *** p < 0.001 and ns means no significant difference.

Article Snippet: According to the manufacturer’s instructions, human CXCL13 ELISA kit (Abcam, Cambridge, UK, Cat. ab179881) was used to detect serum CXCL13 concentration in BPH patients and healthy controls.

Techniques: Knockdown, Flow Cytometry, CCK-8 Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: Overexpression of CXCL13 promoted inflammation and fibrosis of WPMY-1 cells through STAT3 pathway. ( A – C ) Overexpression efficiency of CXCL13 at the mRNA and protein levels. ( D – F ) The mRNA and protein level of fibrosis and inflammation markers after CXCL13 overexpression. ( G ) The protein level of STAT3 pathway after CXCL13 overexpression. ( H ) Relative densitometric quantification of fibrosis-, inflammation- and pathway-related proteins. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: According to the manufacturer’s instructions, human CXCL13 ELISA kit (Abcam, Cambridge, UK, Cat. ab179881) was used to detect serum CXCL13 concentration in BPH patients and healthy controls.

Techniques: Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: Immunohistochemical of CXCL13 on TMA of BPH. ( A ) A holistic view of CXCL13 stained TMA. The scale bar is 4 mm. ( B ) Representative graph of CXCL13 stained TMA. The scale bar is 0.5 mm.

Article Snippet: According to the manufacturer’s instructions, human CXCL13 ELISA kit (Abcam, Cambridge, UK, Cat. ab179881) was used to detect serum CXCL13 concentration in BPH patients and healthy controls.

Techniques: Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: The descriptive statistics of clinical parameters of BPH patients.

Article Snippet: According to the manufacturer’s instructions, human CXCL13 ELISA kit (Abcam, Cambridge, UK, Cat. ab179881) was used to detect serum CXCL13 concentration in BPH patients and healthy controls.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: The correlations between clinical parameters and CXCL13 expression of BPH patients.

Article Snippet: According to the manufacturer’s instructions, human CXCL13 ELISA kit (Abcam, Cambridge, UK, Cat. ab179881) was used to detect serum CXCL13 concentration in BPH patients and healthy controls.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: Primer sequence used for qPCR.

Article Snippet: According to the manufacturer’s instructions, human CXCL13 ELISA kit (Abcam, Cambridge, UK, Cat. ab179881) was used to detect serum CXCL13 concentration in BPH patients and healthy controls.

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) in Hyperplastic Prostate

doi: 10.3390/ijms24010056

Figure Lengend Snippet: List of primary antibodies (All antibodies we used are rabbit antibodies).

Article Snippet: According to the manufacturer’s instructions, human CXCL13 ELISA kit (Abcam, Cambridge, UK, Cat. ab179881) was used to detect serum CXCL13 concentration in BPH patients and healthy controls.

Techniques:

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: NPJ Vaccines

Article Title: Conjugation of HIV-1 envelope to hepatitis B surface antigen alters vaccine responses in rhesus macaques

doi: 10.1038/s41541-023-00775-y

Figure Lengend Snippet: a Gating strategy used to identify antigen specific TFH cells after 18 h stimulation with SEB, or HBsAg or HIV-1 peptide and protein. b Frequencies of HBsAg and Env responsive TFHs out of total lymph node TFH at week 98. c Frequencies of HBsAg responsive TFHs out of total blood TFH at week 60 and week 74. d Frequencies of HIV Env responsive TFHs out of total blood TFH at week 60 and week 74. e CXCL13 ELISA results from serum at indicated timepoints. Comparisons were made using Mann-Whitney U test, * p < 0.05, ** p < 0.01. Box plots represent the interquartile ranges, horizontal lines indicate the medians, and error bars extend to the minimum and maximum observed values.

Article Snippet: Human CXCL13 ELISA kit was purchased from R&D Systems (Minneapolis, MN) and performed according to manufacturer specifications and as previously described .

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY