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: 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