Journal: bioRxiv

Article Title: Tertiary lymphoid structures induced by CXCL13-producing CD4 + T cells increase tumor infiltrating CD8 + T cells and B cells in ovarian cancer

doi: 10.1101/2021.12.01.470493

Figure Lengend Snippet: (A) Representative TLS in tissues stained by H&E and CXCL13 (Fast RED) by RNA ISH. Scale bars indicate 100 µm. (B) TLS presence ratio based on CXCL13 gene expression. Analysis by Fisher’s exact test in 28 cases with microarray data. (C) Characterization of the immune infiltrate in tumors according to CXCL13 gene expression (n=28). Correlation was determined by Spearman’s correlation test. (D) (E) The distribution of infiltrating immune cells into the tumor site and CXCL13 gene expression using CIBERSORT (n=522). Correlation was determined by Spearman’s correlation test. (F) Overall survival of patients with HGSC by CXCL13 gene expression (TCGA n=217, KOV n=28). Patients with CXCL13 high defined if CXCL13 gene expression was above the median. Analyses were performed with Kaplan-Meier estimates, log-rank tests and Wilcoxon tests. The level of significance was set as * P <0.05, ** P <0.01, and **** P <0.0001.

Article Snippet: Mouse recombinant CXCL13 (R&D Systems) treatment was initiated one day after the tumor inoculation and administered intraperitoneally at 1 μg/mouse every other day for five times.

Techniques: Staining, Gene Expression, Microarray

Journal: bioRxiv

Article Title: Tertiary lymphoid structures induced by CXCL13-producing CD4 + T cells increase tumor infiltrating CD8 + T cells and B cells in ovarian cancer

doi: 10.1101/2021.12.01.470493

Figure Lengend Snippet: (A) Fluorescent double staining of CXCL13 (red) and CD4 (green), and CXCL13 (red) and CD8 (white) by RNA ISH in TLS. Images of four representative TLS are shown. (B) Fluorescent double staining of CXCL13 (red) and CD4 (green), and CXCL13 (red) and CD8 (white) by RNA ISH in TIL. The upper and lower pictures are representative TIL images from the same patient. Nuclei are stained with DAPI (blue). Scale bars indicate 100 µm. Co-localization of CXCL13 with CD4 or CD8 is shown in the bar graph as the relative positive ratio quantified using BZ-H4C/hybrid cell count software.

Article Snippet: Mouse recombinant CXCL13 (R&D Systems) treatment was initiated one day after the tumor inoculation and administered intraperitoneally at 1 μg/mouse every other day for five times.

Techniques: Double Staining, Staining, Cell Counting, Software

Journal: bioRxiv

Article Title: Tertiary lymphoid structures induced by CXCL13-producing CD4 + T cells increase tumor infiltrating CD8 + T cells and B cells in ovarian cancer

doi: 10.1101/2021.12.01.470493

Figure Lengend Snippet: (A) Representative images of early TLS. (B) Representative images of follicle-formed TLS. Upper panels show CXCL13 (RNA ISH, Fast RED) and lower panels show FDC (CD21 IHC, DAB). (C) Fluorescence double staining of CXCL13 (red) and CD4 (green), CXCL13 (red) and CD8 (white), and CXCL13 (red) and CD21 (light blue) in representative early TLS and follicle-formed TLS. Nuclei are stained with DAPI (blue). Scale bar indicates 100 µm. Co-localization of CXCL13 with CD4, CD8, or CD21 is shown in the bar graph as the relative positive ratio quantified using BZ-H4C/hybrid cell count software.

Article Snippet: Mouse recombinant CXCL13 (R&D Systems) treatment was initiated one day after the tumor inoculation and administered intraperitoneally at 1 μg/mouse every other day for five times.

Techniques: Fluorescence, Double Staining, Staining, Cell Counting, Software

Journal: bioRxiv

Article Title: Tertiary lymphoid structures induced by CXCL13-producing CD4 + T cells increase tumor infiltrating CD8 + T cells and B cells in ovarian cancer

doi: 10.1101/2021.12.01.470493

Figure Lengend Snippet: (A) Correlation between CXCL13 and TGF-β1 expression in TCGA (n=217) and KOV (n=28). Correlation was determined by Spearman’s correlation test. (B) Human naïve CD4 + and CD8 + T cells from a healthy donor were differentiated by TCR stimulation and TGF-β1 in the presence or absence of a TGF signal inhibitor, SB431542. The proportion of CXCL13 + cells was determined by flow cytometry. Data are shown as the mean ± SEM of four samples. Statistical significance was determined by two-tailed Student’s t -test, * P <0.05, ** P <0.01, *** P <0.001, n.s.: not significant. (C) The concentration of TGF-β1 in conditioned medium obtained from three human ovarian cancer cell lines was measured by ELISA. Data are shown as the mean ± SEM of three samples. (D) Human naïve CD4 + and CD8 + T cells from a healthy donor were differentiated with TCR stimulation and conditioned medium obtained from three human ovarian cancer cell lines in the presence or absence of a TGF signal inhibitor, SB431542. The proportion of CXCL13 + cells was determined by flow cytometry. Data are shown as the mean ± SEM of triplicates. (E) (F) (G) Human naïve CD4 + and CD8 + T cells from a healthy donor were differentiated with TCR stimulation and the indicated cytokines. The proportion of CXCL13 + cells was determined by flow cytometry (E) . The concentration of CXCL13 in the culture supernatant was measured by ELISA (F) . Data are shown as the mean ± SEM of four samples in CD4 and three samples in CD8. Statistical significance was determined by two-tailed Student’s t -test, * P <0.05, ** P < 0.01, *** P < 0.001, **** P <0.0001, n.s.: not significant. Representative dot plots of PD-1 (upper row), CXCR5 (lower row), and intracellular CXCL13 in healthy human naïve CD4 + T cells are shown (G) . a IL-2 Ab indicates anti IL-2 antibody.

Article Snippet: Mouse recombinant CXCL13 (R&D Systems) treatment was initiated one day after the tumor inoculation and administered intraperitoneally at 1 μg/mouse every other day for five times.

Techniques: Expressing, Flow Cytometry, Two Tailed Test, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Tertiary lymphoid structures induced by CXCL13-producing CD4 + T cells increase tumor infiltrating CD8 + T cells and B cells in ovarian cancer

doi: 10.1101/2021.12.01.470493

Figure Lengend Snippet: (A) Mouse rCXCL13 was administered intraperitoneally to induce TLS in a mouse ovarian cancer model. Representative H&E images of TLS formed in an omental tumor. The area of TLS per tumor area was compared between the control group and the rCXCL13 treated group (n=7, each). Statistical significance was determined by two-tailed Student’s t -test, * P <0.05. (B) TLS induced by mouse rCXCL13 (H&E) and expression of mouse CXCL13 corresponding to TLS (RNA ISH, Fast RED). (C) CD8 + T cell IHC images (DAB) in the rCXCL13 treated group and control group. Scale bars indicate 100 µm. (D) (E) The effect of rCXCL13 administration on the survival of tumor-bearing mice was compared between immunocompetent mice (D) and immunodeficient mice (E). Analyses were performed using Kaplan-Meier estimates and log-rank tests.

Article Snippet: Mouse recombinant CXCL13 (R&D Systems) treatment was initiated one day after the tumor inoculation and administered intraperitoneally at 1 μg/mouse every other day for five times.

Techniques: Control, Two Tailed Test, Expressing

Journal: Journal of Virology

Article Title: A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells

doi: 10.1128/JVI.01956-16

Figure Lengend Snippet: Construction and characterization of the rRABV expressing CXCL13. (A) Schematic diagram for the construction of LBNSE, LBNSE-GM-CSF, and LBNSE-CXCL13. The mouse CXCL13 gene was cloned and inserted into the RABV genome in place of the deleted long noncoding region, and rRABVs were rescued according to the method described in Materials and Methods. (B) A multistep growth curve was generated in BSR cells. Cells were infected with LBNSE, LBNSE-GM-CSF, or LBNSE-CXCL13 at a multiplicity of infection (MOI) of 0.01 FFU and incubated at 37°C. Viruses were harvested at 1, 2, 3, 4, and 5 dpi, and viral titers were determined. All titrations were carried out in quadruplicate, and the data are presented as the means ± standard deviations (SD). (C) Production of CXCL13 in BSR cells. Cells were infected with different viruses at MOIs of 0.001, 0.01, 0.1, and 1. After incubation at 37°C for 24 h, the culture supernatants were collected, and the CXCL13 concentrations produced by the indicated rRABVs were determined with a commercial ELISA kit. (D) Chemotactic effects of cultured medium from BSR cells infected with rRABVs at an MOI of 1 on mouse splenocytes. Splenocytes (5 × 105) were applied to the upper wells of chemotaxis chambers. Two and 4 h later, the cells migrating to the bottom chamber were counted. (E and F) BALB/c mice were inoculated via i.m. injection of 1 × 106 FFU of rRABVs. The muscles from the hind legs of mice (n = 3) were harvested at 3 and 6 dpi. Total RNA was extracted, and viral genomic RNA (vRNA) (E) and CXCL13 mRNA and CXCR5 mRNA (F) were analyzed via qRT-PCR. All the data are expressed as the means ± SD. Asterisks indicate significant differences between the indicated experimental groups.

Article Snippet: Recombinant mouse CXCL13 proteins were purchased from R&D Systems (USA).

Techniques: Expressing, Clone Assay, Generated, Infection, Incubation, Produced, Enzyme-linked Immunosorbent Assay, Cell Culture, Chemotaxis Assay, Injection, Muscles, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells

doi: 10.1128/JVI.01956-16

Figure Lengend Snippet: Recruitment of Tfh cells by CXCL13. BALB/c mice (n = 3) were infected via i.m. injection of 1 × 106 FFU of different rRABVs, and the spleens, draining LNs, and blood were harvested at 7 and 14 dpi. Single-cell suspensions were prepared, stained with antibodies against Tfh cells and Tfh cell activation markers, and analyzed via flow cytometry. (A and B) Representative gating strategies for the detection of Tfh cells (A) and representative flow cytometric plots of Tfh cells (B) are shown. (C to E) The results of a detailed analysis for activated Tfh cells (CD4+ CXCR5+ PD-1+) at 7 and 14 dpi are presented for the spleen (C), the draining LNs (D), and the blood (E). Data are expressed as the means ± SEM (n = 3). Asterisks indicate significant differences between the indicated experimental groups.

Article Snippet: Recombinant mouse CXCL13 proteins were purchased from R&D Systems (USA).

Techniques: Infection, Injection, Staining, Activation Assay, Flow Cytometry

Journal: Journal of Virology

Article Title: A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells

doi: 10.1128/JVI.01956-16

Figure Lengend Snippet: Recruitment of GC B cells by CXCL13. BALB/c mice (n = 3) were infected via i.m. injection of 1 × 106 FFU of different rRABVs, and the spleens and draining LNs were harvested at 7 and 14 dpi. Single-cell suspensions were prepared, stained with antibodies against GC B cells and GC B cell activation markers, and analyzed via flow cytometry. (A and B) Representative gating strategies for the detection of GC B cells (A) and representative flow cytometric plots of GC B cells (B) are shown. (C and D) The results of a detailed analysis for activated GC B cells (B220+ CD95+ GL7+) at 7 and 14 dpi are presented for the spleen (C) and the draining LNs (D). Data are presented as the means ± SEM (n = 3). Asterisks indicate significant differences between the indicated experimental groups.

Article Snippet: Recombinant mouse CXCL13 proteins were purchased from R&D Systems (USA).

Techniques: Infection, Injection, Staining, Activation Assay, Flow Cytometry

Journal: Journal of Virology

Article Title: A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells

doi: 10.1128/JVI.01956-16

Figure Lengend Snippet: Expression of CXCL13 facilitates the formation of GCs. BALB/c mice (n = 3) were infected via i.m. injection of 1 × 106 FFU of different rRABVs, and the draining LNs were collected at 7 and 14 dpi. Then, the draining LNs were excised, and tissue sections were prepared and stained for germinal centers (GL7, red; B220, blue; and IgG, green). Scale bars, 500 μm or 100 μm (rightmost column only). (A) Representative results are shown. (B) The numbers of GCs formed at 7 and 14 dpi were calculated. All the data are expressed as the means ± SEM (n = 3). Asterisks indicate significant differences between the indicated experimental groups; ns, not significant.

Article Snippet: Recombinant mouse CXCL13 proteins were purchased from R&D Systems (USA).

Techniques: Expressing, Infection, Injection, Staining

Journal: Journal of Virology

Article Title: A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells

doi: 10.1128/JVI.01956-16

Figure Lengend Snippet: Plasma CXCL13 levels correlate with GC activity and VNA titers in mice. BALB/c mice were infected via i.m. injection of 1 × 106 FFU of DMEM (n = 5), LBNSE (n = 9), LBNSE-GM-CSF (n = 9), or LBNSE-CXCL13 (n = 9), and then draining (inguinal) and nondraining (cervical) LNs were collected at 7 dpi. Single-cell suspensions were prepared, stained with antibodies against GC B cells and GC B cell activation markers, and analyzed via flow cytometry. (A and B) Representative gating strategies for the detection of GC B cells (A) and representative flow cytometric plots of plasma B cells (B) are shown. (C) The results of a detailed analysis of activated GC B cells (CD20+ Ki-67+ Bcl-6+) at 7 dpi are presented for the draining and nondraining LNs. (D) Serum samples were harvested at 7 dpi, and RABV VNA titers were measured via FAVN tests as described in Materials and Methods. (E) The concentration of plasma CXCL13 was determined using a commercial ELISA kit. (F) Correlations of GC B cell activity in the draining LNs and RABV VNA titers in mice 7 days after immunization were determined. (G) Correlations of plasma CXCL13 concentrations and RABV VNA titers in mice 7 days after immunization were determined. (H) Correlations of GC B cell activity in the draining LNs and plasma CXCL13 concentrations in mice 7 days after immunization were determined. All the data are expressed as the means ± SEM (n = 3). Asterisks indicate significant differences between the indicated experimental groups.

Article Snippet: Recombinant mouse CXCL13 proteins were purchased from R&D Systems (USA).

Techniques: Clinical Proteomics, Activity Assay, Infection, Injection, Staining, Activation Assay, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells

doi: 10.1128/JVI.01956-16

Figure Lengend Snippet: Expression of CXCL13 promotes an increase in the quantity of plasma cells. BALB/c mice (n = 3) were infected via i.m. injection of 1 × 106 FFU of different rRABVs, and the bone marrow samples were harvested at 7 and 14 dpi. Single-cell suspensions were prepared, stained with antibodies against plasma B cells and plasma B cell activation markers, and analyzed via flow cytometry. (A and B) Representative gating strategies for the detection of plasma B cells (A) and representative flow cytometric plots of plasma B cells (B) are shown. (C) The results of a detailed analysis of activated plasma B cells (B220low CD138+) at 7 and 14 dpi are presented for the bone marrow samples. All the data are expressed as the means ± SEM (n = 3). Asterisks indicate significant differences between the indicated experimental groups.

Article Snippet: Recombinant mouse CXCL13 proteins were purchased from R&D Systems (USA).

Techniques: Expressing, Clinical Proteomics, Infection, Injection, Staining, Activation Assay, Flow Cytometry

Journal: Journal of Virology

Article Title: A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells

doi: 10.1128/JVI.01956-16

Figure Lengend Snippet: qRT-PCR primers used in this study

Article Snippet: Recombinant mouse CXCL13 proteins were purchased from R&D Systems (USA).

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Syndecan-1 Plays a Role in the Pathogenesis of Sjögren’s Disease by Inducing B-Cell Chemotaxis through CXCL13–Heparan Sulfate Interaction

doi: 10.3390/ijms25179375

Figure Lengend Snippet: Identification of CXCL13 binding to syndecan-1 (SDC-1). ( A ) Co-expression of SDC-1 and CXCL13 in submandibular gland tissues of NOD/ShiLtJ mice. Each section was double-stained with immunofluorescent antibodies to determine the distribution of SDC-1 (CD138, red) and CXCL13 (green) on the epithelial cells. The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). The merged images obtained by confocal microscopy show the yellow co-localization of both molecules. Magnified views of the boxed areas are shown in the right column. Scale bars = 50 and 25 µm. ( B ) Co-expression of SDC-1 and CXCL13 on normal murine mammary gland (NMuMG) cells. NMuMG cells were double-stained with anti-CXCL13 mAb (green) and anti-SDC-1 mAb (red). The sections were counterstained with DAPI (blue). Scale bar = 25 µm. ( C ) Association of SDC-1 with CXCL13 at the cell surface of NMuMG cells. NMuMG cells were incubated with 0, 50, 100, and 150 nM recombinant mouse CXCL13 for 18 h. The cell pellets of NMuMG cells were harvested following the protocol outlined in the Pierce™ Classic Magnetic IP/Co-IP Kit. The cell lysates were incubated with mouse anti-SDC-1 antibody, and anti-SDC-1 antibody binding complexes were analyzed by Western blotting assay with antibody against CXCL13 (Novus Biologicals, Centennial, CO, USA) or rat immunoglobulin G (control, Santacruz, Dallas, TX, USA).

Article Snippet: Recombinant mouse CXCL13 was purchased from R&D Systems (Minneapolis, MN, USA), and Pierce™ Classic Magnetic IP/Co-IP Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Binding Assay, Expressing, Staining, Confocal Microscopy, Incubation, Recombinant, Co-Immunoprecipitation Assay, Western Blot, Control

Journal: Nature Communications

Article Title: Human single cell RNA-sequencing reveals a targetable CD8 + exhausted T cell population that maintains mouse low-grade glioma growth

doi: 10.1038/s41467-024-54569-4

Figure Lengend Snippet: A Schematic representation of the anti-PD1 (α-PD1), anti-TIGIT (α-TIGIT), and combined (α-PD1 + TIGIT) antibody treatments. Nf1 -OPG mice were treated (200 µg/dose/ i.p., twice per week) from 12 to 16 weeks of age, and tissues were analyzed at 16 weeks. The control group was injected with anti-IgG isotype control antibodies. Created in BioRender. Chatterjee, J. (2024) BioRender.com/r98n914. B Immunohistochemistry and quantification CD8 + T cells in the entire optic nerve (IgG, n = 10 mice; α-TIGIT, n = 10 mice; α-PD1, n = 6 mice; α-PD1 + TIGIT, n = 5 mice). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated (α-TIGIT, P = 0.0054; α-PD1, P = 0.0003; α-PD1 + TIGIT, P = 0.0032). Scale bar, 200 µm. C Ccl2 RNA expression in the optic nerves of Nf1 -OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. D Volcano plot showing fold change and P value comparing Nf1 -OPG TAM to Nf1 +/ and WT monocytes in the optic nerves of 12-week-old mice. Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). E Ccr2, Cxcr3 , and Cxcr5 expression in T cell populations from 12-week-old Nf1 -OPG mouse optic nerves, color-coded by T cell type. F Cxcl9, Ccl12 , and Cxcl13 RNA expression in the optic nerves of 12-week-old WT and Nf1 -OPG mice. Data are represented relative to the WT group ( Cxcl9 ; WT n = 3, 2 pooled optic nerves per sample; Nf1 -OPG n = 4, 2 pooled optic nerves per sample; Ccl12 ; WT n = 4, 2 pooled optic nerves per sample; Nf1 -OPG n = 4, 2 pooled optic nerves per sample; Cxcl13 ; WT n = 4, 2 pooled optic nerves per sample; Nf1 -OPG n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. To evaluate statistical differences, a two-tailed non-parametric Mann–Whitney test was performed. Exact P values are indicated. ns, not significant. G Graph showing the percentage of migrated Nf1 +/- CD8 + T cells treated with medium (Control) ( n = 5), Ccl12 ( n = 6), or Cxcl13 ( n = 5). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. Created in BioRender. Chatterjee, J. (2024) BioRender.com/r98n914. H Ccl12 RNA expression in the optic nerves of Nf1 -OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 3, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Tukey’s post-test correction. Exact P values are indicated (α-TIGIT, P = 0.0012; α-PD1, P = 0.0187; α-PD1 + TIGIT, P = 0.0013). I Cxcl13 RNA expression in the optic nerves of Nf1 -OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 4, 2 pooled optic nerves per sample; α-TIGIT n = 4, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Tukey’s post test correction. Exact P values are indicated (α-TIGIT, P = 0.0003; α-PD1, P = 0.0007; α-PD1 + TIGIT, P = 0.0001).

Article Snippet: 500 μl of chemoattractant media (Ccl12 [25 ng/ml, 428-P5-025- R&D systems ] and Cxcl13 [1 µg/ml, 470-BC-025- R&D systems] ) was added to the lower chamber, and the number of T cells in the lower chambers counted 6 h later.

Techniques: Control, Injection, Immunohistochemistry, RNA Expression, Expressing, Two Tailed Test, MANN-WHITNEY