Journal: Frontiers in Immunology

Article Title: GPR182 is a broadly scavenging atypical chemokine receptor influencing T-independent immunity

doi: 10.3389/fimmu.2023.1242531

Figure Lengend Snippet:

Article Snippet: Anti-mCXCL13 , 143614 , 1:1000 , RnD Systems , MAB470.

Techniques: Concentration Assay

Journal: Frontiers in Immunology

Article Title: Siglec-H-Deficient Mice Show Enhanced Type I IFN Responses, but Do Not Develop Autoimmunity After Influenza or LCMV Infections

doi: 10.3389/fimmu.2021.698420

Figure Lengend Snippet: No influence of Hck in pDCs on the IFN-α production and reduced in vitro migration of Siglec-H ko pDCs to CCL25. (A) Strategy to sort pDCs from B16Flt3L-treated wt and Siglec-H ko spleens to use in transwell-migration assays (purity more than 90%). (B) In vitro generated pDCs from wt, Siglec-H ko and HCK ko or HFL KO mice were stimulated for 24 hrs with LPS (c=1 µg/ml), R848 (c=4 µg/ml) or CpG A (c=2,5 µg/ml) (two separate experiments are shown). The amount of IFN-α was measured by ELISA. White bars: wt, black bars: Siglec-H ko, grey bars: HCK ko (left), HFL ko (right). n.d.: not detectable. N=3 mice per genotype. Error bars are mean ± SD values. (C) Sorted pDCs were used in a transwell-migration assay to evaluate migratory capacity towards CCL25. CCL25 or CXCL13 were given in the lower chamber of the transwell and sorted pDCs were given into the upper chamber. pDCs could then migrate towards CCL25 or CXCL13 for 2 hrs. Migrated cells were counted in the lower chamber and a ratio in relation to the sample with no-chemokine in the lower chamber was calculated. As a negative control CXCL13 was used. CCL25 c= 125nM, CXCL13 c= 100nM. Ratio of migrated cells to non-chemokine control is displayed. Summary of three independent experiments with N=5 mice per genotype. Mice were aged 10-16 weeks. Mann-Whitney test, *p < 0,05. Error bars are mean ± SD values. (D) Leukocytes from the small intestine and bowel were isolated and stained as shown in . pDC percentages are shown as % of living leukocytes. Mice were aged 10-16 weeks, N=3 mice per genotype.

Article Snippet: The lower chamber was filled with 600 µl of medium including the specific chemokine ligand: CCL25 (125 nM, Biolegend) or CXCL13 (100 nM, Biolegend).

Techniques: In Vitro, Migration, Generated, Enzyme-linked Immunosorbent Assay, Transwell Migration Assay, Negative Control, MANN-WHITNEY, Isolation, Staining

Journal: Nature materials

Article Title: Colony Stimulating Factor-1 Receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates

doi: 10.1038/nmat4866

Figure Lengend Snippet: CSF1R inhibition prevents the entire (innate and adaptive) immune response to implanted biomaterials. Phase contrast images showing that fibrosis, as compared to wild type (WT) vehicle-treated control, was partially eliminated by CXCL13 neutralization, and completely eliminated with continuous CSF1R inhibition (inh.; 160 mg/kg BW GW2580 SC, daily) over a 14-day implant period ( a ). Scale bar = 2000 μm. Fibrosis was reduced the same extent as that of the B cell knockout (B KO) shown in . ( b ) Flow analysis for responding macrophages, neutrophils, and B cells dissociated directly from spheres (100 μl material in all cases) 14 days post-intraperitoneal (IP) implant, showing partial loss of cell presence with CXCL13 neutralization, and complete loss following CSF1R inhibition. ( c ) Brightfield images showing that fibrosis, compared to vehicle control, was completely eliminated by both macrophage depletion (- Mϕs) and CSF1R inhibition. Scale bar = 1000 μm. ( d ) Flow analysis for responding host innate immune macrophages and neutrophils dissociated directly from spheres (100 μl of each material) 14 days post-IP implantation, showing the loss of immune adhesion with loss of fibrosis due to either macrophage depletion (- Mϕs) or CSF1R inhibition. ( e ) NanoString expression analysis for all known cytokine and cytokine receptors (see for complete data set, excerpted here), showing similar unique factors increased across all material (hydrogel alginate, ceramic glass, and polymer polystyrene (PS)) groups. ( f ) Confocal for DAPI (cellular nuclei), macrophage marker CD68 (green), B cell marker CD19 (magenta), fibrosis-associated myofibroblast marker alpha smooth muscle actin (αSMactin, myofibroblasts, red), overlay, and brightfield imaging, showing that CXCL13 neutralization resulted in loss of B cell recruitment. Scale bar = 200 μm. ( g ) qPCR expression analysis of α-SMactin directly on retrieved spheres from Vehicle-treated (WT), B KO, and CXCL13-neutralized WT mice, plotted relative to expression levels on spheres from WT mice. Statistical analysis: one-way ANOVA with Bonferroni multiple comparison correction ***: p < 0.0001, vs Vehicle. n = 5 mice/group. Experiments repeated at least 2–3 times.

Article Snippet: To neutralize secreted CXCL13, anti-CXCL13 antibody (mAB470, R&D Systems) was injected IP in sterile 1X PBS at a dose of 100 μg/mouse, once every 3 days, starting 3 days prior to implantation as well.

Techniques: Inhibition, Control, Neutralization, Knock-Out, Expressing, Polymer, Marker, Imaging, Comparison

Journal: Nature materials

Article Title: Colony Stimulating Factor-1 Receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates

doi: 10.1038/nmat4866

Figure Lengend Snippet: 0.5 mm-sized SLG20 hydrogel spheres of were implanted into either the intraperitoneal (IP) or subcutaneous (SC) dorsal region of cynomolgus monkeys and retrieved by laparoscopy-guided tissue excision or biopsy punch after 28 days . ( a ) Laparoscopy images taken on days 0 and 28 (initial implantation and retrieval from the IP space). ( b ) H&E and Masson’s Trichrome stained histological sections of excised IP omentum tissue at 28 days for non-fibrosed fat-laden (No material, Mock) or heavily collagen-deposited and sphere-embedded (Implanted) omental tissue. Scale bars = 400 μm. ( c ) Main figure: Confocal staining showing DAPI (cellular nuclei), innate immune macrophage marker CD68 (green), and fibrosis-associated activated myofibroblast alpha smooth muscle actin (αSMactin) staining (red), showing cellular infiltration around and fibrosis deposition on an embedded 500 μm alginate sphere; Inset: additional confocal for CSF1R (green), showing positive staining on not only more distant macrophages but also material-proximal and fused foreign body giant cells (FBGCs); both 20x magnification. White scale bars: both 200 μm for each respective image. ( d ) Flow analysis showing similar host innate immune macrophage (CD68 + CD11b + , top right quadrants) and remaining neutrophil/myeloid (CD68 − CD11b + , bottom right quadrants) cells across C57BL/6 mice and cynomolgus monkeys, dissociated directly from fibrosed spheres and adjacent fibrosed omentum tissue (as percent composition) 28 days post-IP implantation. While the prominence of CD11b seems to be inverted in C57BL/6 mice vs cynomolgus monkeys, population response percentages are similar 28 days post-IP implantation. ( e ) NanoString analysis for immune markers and cytokines, originally identified in C57BL/6 mice (Note: CD66b is used here as a neutrophil marker, as Ly6g/Gr1 does not exist in NHPs or humans). Significant increases are observed for macrophage markers, as well as CSF1R and CXCL13 in both peritoneal and subcutaneous implant sites, as compared to mock (saline-injected) controls (there was no difference between SC and IP mock controls). N = 2 for IP-implanted groups; N = 4 for subcutaneous (SC) treatment groups. These experiments were performed once for SC and twice for IP delivery.

Article Snippet: To neutralize secreted CXCL13, anti-CXCL13 antibody (mAB470, R&D Systems) was injected IP in sterile 1X PBS at a dose of 100 μg/mouse, once every 3 days, starting 3 days prior to implantation as well.

Techniques: Staining, Marker, Saline, Injection

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: Cxcl13 - and Calca -mRNAs are present in the murine tracheal epithelium. RT-PCR experiments with cDNA obtained from tracheal epithelium of two C57BL/6RJ animals using primers for Cxcl13 (250 bp), Calca (158 bp), and β-actin (300 bp). Amplicons of Calca , Cxcl13 , and β-actin are detected in both tracheal epithelium samples (TE). Controls = RNA samples processed without reverse transcriptase (-RT) and water (H 2 O) without adding cDNA

Article Snippet: To further validate the specificity of the biotinylated secondary antibodies used in combination with the CXCL13 primary antibody, 10-μm-thick tracheal sections were air-dried for 1 h and unspecific protein binding sites were saturated with 10% normal swine serum in PBS + S (0.005 M phosphate buffer, with 0.15 M NaCl, pH 7.4) for 1 h. Sections were then incubated overnight either with goat polyclonal antibody to CXCL13 (1:400 AF470, R&D Systems) and rabbit polyclonal antibody to αCGRP (1:20,000; T-4032, Peninsula Laboratories) or with rabbit polyclonal antibody to αCGRP only.

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: CXCL13-positive and neuroendocrine cells are unevenly distributed in the trachea. a – d Tracheal whole mount immunohistochemistry, CLSM, with antibodies against CXCL13 (green) and CGRP (red), labeling single neuroendocrine cells and nerve fibers. The density of neuroendocrine (CGRP-positive) cells, CXCL13-immunoreactive cells, and nerve fibers is higher between the cartilage rings (Ca). The density of neuroendocrine cells and CXCL13-immunoreactive cells decreases from cranial (larynx) ( b ) to caudal (bifurcation) ( d ); each channel is individually shown in Supplementary Fig. 1 . Numerous CGRP-positive nerve fibers ( <) are visible throughout the whole trachea. Maximum intensity projection of z-stack of confocal optical sections. e, f Cell densities (mean ± SEM) quantified on tracheal whole mounts double-labelled for CXCL13 and PGP9.5 (neuroendocrine cell marker). Immunoreactive cells dominate in intercartilage regions ( e ), and their density continuously declines along the cranio-caudal axis ( f ). In f , counts include both the area overlying a cartilage and the next intercartilage region; colour coding along the cranio-caudal axis identifies data from the same trachea. g RT-qPCR. Cxcl13 expression is about 3 times higher in tracheal rings 1–3 compared to rings 8–10

Article Snippet: To further validate the specificity of the biotinylated secondary antibodies used in combination with the CXCL13 primary antibody, 10-μm-thick tracheal sections were air-dried for 1 h and unspecific protein binding sites were saturated with 10% normal swine serum in PBS + S (0.005 M phosphate buffer, with 0.15 M NaCl, pH 7.4) for 1 h. Sections were then incubated overnight either with goat polyclonal antibody to CXCL13 (1:400 AF470, R&D Systems) and rabbit polyclonal antibody to αCGRP (1:20,000; T-4032, Peninsula Laboratories) or with rabbit polyclonal antibody to αCGRP only.

Techniques: Immunohistochemistry, Labeling, Marker, Quantitative RT-PCR, Expressing

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: CXCL13 is restricted to neuroendocrine cells in the tracheal epithelium. a , b High-resolution of tracheal whole mount immunohistochemistry, labeling CXCL13 + cells in green and CGRP + cells and nerve fibers in red, revealed CXCL13 and CGRP colocalization within the same cell. Inset in a shows the magnified region of the labeled cell with only limited overlap of immunoreactivites (scale bar 1 µm). Images were acquired using a confocal laser scanning microscope (FLUOVIEW FV3000; Olympus), single confocal optical section. b CXCL13 + cell in contact to a CGRP + nerve fiber ( <). Maximum intensity projection of z-stack of confocal optical sections. c Immunohistochemistry of a tracheal cryosection from a ChAT-eGFP animal. In the tracheal epithelium, single cells are double-stained with antibodies against PGP9.5 and CXCL13 (arrowhead) or PGP9.5 only (arrow). An eGFP-positive cell (asterisk) is not labeled with antibodies against CXCL13 or PGP9.5. PGP9.5 + nerve fibers are in contact to PGP9.5 + epithelial cells. d Triple-immunofluorescence of tracheal cryosection shows co-labeling of single epithelial cells for CXCL13 (green), CGRP (yellow), and PGP9.5 (red) (arrowheads). A single PGP9.5-labeled cell with neither CXCL13 nor CGRP immunolabeling is also present (asterisk). e–f Transmission electron microscopy. e Ultrastructure of a tracheal neuroendocrine cell (NEC) with a pyramidal or flask-like shape and a small apical part reaching the lumen with microvilli. e′ Higher magnification of the basal part, showing the presence of numerous dense core vesicles (DCV). f Ultrastructural immunohistochemistry with antibodies against CXCL13 shows an immunoreactive cell with the diffuse DAB reaction product. f′ Higher magnification of the basal part, showing the presence of numerous DCV

Article Snippet: To further validate the specificity of the biotinylated secondary antibodies used in combination with the CXCL13 primary antibody, 10-μm-thick tracheal sections were air-dried for 1 h and unspecific protein binding sites were saturated with 10% normal swine serum in PBS + S (0.005 M phosphate buffer, with 0.15 M NaCl, pH 7.4) for 1 h. Sections were then incubated overnight either with goat polyclonal antibody to CXCL13 (1:400 AF470, R&D Systems) and rabbit polyclonal antibody to αCGRP (1:20,000; T-4032, Peninsula Laboratories) or with rabbit polyclonal antibody to αCGRP only.

Techniques: Immunohistochemistry, Labeling, Laser-Scanning Microscopy, Staining, Immunofluorescence, Immunolabeling, Transmission Assay, Electron Microscopy

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: CXCL13 is expressed in two-thirds of the murine tracheal neuroendocrine cells. a , b Immunohistochemistry of tracheal whole mounts and the corresponding quantification of their immunoreactive cells; maximum intensity projections of z-stacks of confocal optical sections. a Immunohistochemistry with antibodies against CXCL13 ( a ) (green) and PGP9.5 ( a′ ) (red), labeling single neuroendocrine cells and nerve fibers. CXCL13 + /PGP9.5 + cells are indicated by arrowheads; CXCL13 − /PGP9.5 + cells are indicated by ( <). Data points in the scatter plot ( a‴ ) represent mean values of counts in one trachea ( n = 5 tracheas); mean and SEM are indicated. The pie chart shows the percentages of CXCL13 + /PGP9.5 + and CXCL13 − /PGP9.5 + cells ( n = 2254 cells pooled from 5 tracheas). b Immunohistochemistry with antibodies against CXCL13 ( b ) (green) and CGRP ( b′ ) (red), labeling single neuroendocrine cells and nerve fibers. CXCL13 + /CGRP + cells are indicated by arrowheads; CXCL13 − /CGRP + cells are indicated by ( <); CXCL13 + /CGRP − cells are indicated by (*). Data points in the scatter plot ( b‴ ) represent mean values of counts in one trachea ( n = 5 tracheas); mean and SEM are indicated. The pie chart shows the percentages of phenotypes ( n = 2650 cells pooled from 5 tracheas)

Article Snippet: To further validate the specificity of the biotinylated secondary antibodies used in combination with the CXCL13 primary antibody, 10-μm-thick tracheal sections were air-dried for 1 h and unspecific protein binding sites were saturated with 10% normal swine serum in PBS + S (0.005 M phosphate buffer, with 0.15 M NaCl, pH 7.4) for 1 h. Sections were then incubated overnight either with goat polyclonal antibody to CXCL13 (1:400 AF470, R&D Systems) and rabbit polyclonal antibody to αCGRP (1:20,000; T-4032, Peninsula Laboratories) or with rabbit polyclonal antibody to αCGRP only.

Techniques: Immunohistochemistry, Labeling

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: In silico analysis of single-cell mRNA sequencing data revealed CXCL13 expression predominantly in neuroendocrine cells of the tracheal epithelium. a – c In silico analysis of published sequencing data (GSE102580) of murine tracheal epithelial cells (Plasschaert et al. ). a SPRING plot (Uniform Manifold Approximation and Projection, UMAP) shows eight distinct cell clusters, namely basal, secretory, Krt4/13 + , ciliated, solitary cholinergic chemosensory (brush/tuft), cycling basal and solitary neuroendocrine cells, and ionocytes. b SPRING and violin plots showing that Cxcl13 -mRNA is predominantly expressed within the neuroendocrine cell cluster. c Heat map shows the most differentially expressed genes (fold change > 1) between CXCL13 + and CXCL13 − neuroendocrine cells among the 200 highest expressed genes within the neuroendocrine cell cluster and typical neuroendocrine cell marker genes (e.g., Ascl1 , Uchl1 , Calca , Chga , Chgb )

Article Snippet: To further validate the specificity of the biotinylated secondary antibodies used in combination with the CXCL13 primary antibody, 10-μm-thick tracheal sections were air-dried for 1 h and unspecific protein binding sites were saturated with 10% normal swine serum in PBS + S (0.005 M phosphate buffer, with 0.15 M NaCl, pH 7.4) for 1 h. Sections were then incubated overnight either with goat polyclonal antibody to CXCL13 (1:400 AF470, R&D Systems) and rabbit polyclonal antibody to αCGRP (1:20,000; T-4032, Peninsula Laboratories) or with rabbit polyclonal antibody to αCGRP only.

Techniques: In Silico, Sequencing, Expressing, Marker

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: CXCL13 is less expressed in murine broncho-pulmonary solitary and clustered neuroendocrine cells. Immunohistochemistry of lung cryosections with antibodies against CXCL13 (orange) and CGRP (green) and the relative frequencies of immunoreactive phenotypes. a Solitary neuroendocrine cell co-labeled with antibodies against CXCL13 and CGRP. b Solitary neuroendocrine cell only labeled with antibodies against CGRP. c A cluster of neuroendocrine cells (neuroepithelial body) consisting of more than 7 cells, 2 of them are co-labeled with antibodies against CXCL13 and CGRP. d Pie chart shows percentages of CXCL13 + /CGRP + and CXCL13 − /CGRP + -immunolabeled cells in solitary neuroendocrine cells ( n = 73 cells pooled from 5 animals). e Pie chart shows the percentage of CXCL13 + /CGRP + and CXCL13 − /CGRP + -immunolabeled cells in neuroepithelial bodies ( n = 1475 cells pooled from 5 animals)

Article Snippet: To further validate the specificity of the biotinylated secondary antibodies used in combination with the CXCL13 primary antibody, 10-μm-thick tracheal sections were air-dried for 1 h and unspecific protein binding sites were saturated with 10% normal swine serum in PBS + S (0.005 M phosphate buffer, with 0.15 M NaCl, pH 7.4) for 1 h. Sections were then incubated overnight either with goat polyclonal antibody to CXCL13 (1:400 AF470, R&D Systems) and rabbit polyclonal antibody to αCGRP (1:20,000; T-4032, Peninsula Laboratories) or with rabbit polyclonal antibody to αCGRP only.

Techniques: Immunohistochemistry, Labeling, Immunolabeling

Journal: Frontiers in Allergy

Article Title: Asymptomatic sensitization to a cow’s milk protein induces sustained neuroinflammation and behavioral changes with chronic allergen exposure

doi: 10.3389/falgy.2022.870628

Figure Lengend Snippet: MCPT-1 and CXCL13 levels in the serum. The sera isolated from the terminal blood of sham and BLG-sensitized mice were used to quantify MCPT-1 ( A ) and CXCL13 ( B ). The concentrations of the analytes were determined from respective standard curves. Values indicate the group average ± SEM pg/ml. Significant p -values are shown. Two-way ANOVA, n = 10–12 per group. One outlier identified by the ROUT method (Q = 1%) was removed from the final analysis for MCPT-1 ( A ).

Article Snippet: CXCL13 and chemokine ligand 12 (CCL12) in hippocampal brain lysates were quantified using the CXCL13 (R&D System Inc.; Cat# DY470) and CCL12 (R&D Systems, Cat# DY428) DuoSet® ELISA Kits.

Techniques: Isolation

Journal: Frontiers in Allergy

Article Title: Asymptomatic sensitization to a cow’s milk protein induces sustained neuroinflammation and behavioral changes with chronic allergen exposure

doi: 10.3389/falgy.2022.870628

Figure Lengend Snippet: Chemokine levels in the hippocampal region. The hippocampus dissected from the right brain hemisphere of each mouse was lysed to extract total proteins. Ten µg of the hippocampal lysates were used to quantify CXCL13 ( A ) and CCL12 ( B ) using ELISA. The concentrations of the analytes were determined from respective standard curves. Values indicate the group average ± SEM. Significant p -values are shown. Two-way ANOVA, n = 9–12 per group.

Article Snippet: CXCL13 and chemokine ligand 12 (CCL12) in hippocampal brain lysates were quantified using the CXCL13 (R&D System Inc.; Cat# DY470) and CCL12 (R&D Systems, Cat# DY428) DuoSet® ELISA Kits.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cancer discovery

Article Title: Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids

doi: 10.1158/2159-8290.CD-17-0833

Figure Lengend Snippet: Cytokine heatmaps Day 3 ± anti-PD-1 (a, n=28), anti-CTLA-4 (c, n=24), or anti-PD-1 + anti-CTLA-4 (e, n=24) expressed as log2 fold-change (L2FC) relative to untreated control. Absolute CCL19/CXCL13 levels (pg/mL) observed at Day 3 ± anti-PD-1 (b, n=28), anti-CTLA-4 (d, n=24), or anti-PD-1 + anti-CTLA-4 (f, n=24) (2-sided, paired, t-test, α= 0.05).

Article Snippet: Mouse CCL19 (Abcam, ab100729) and mouse CXCL13 (R&D Systems, MCX130) ELISAs were used according to manufacturer instructions.

Techniques: Control

Journal: Cancer discovery

Article Title: Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids

doi: 10.1158/2159-8290.CD-17-0833

Figure Lengend Snippet: a–b, CCL19/CXCL13 mRNA levels from melanoma biopsy samples on anti-PD1 treatment relative to pre-PD-1 (L2FC) by (a) qRT-PCR (n=12) and (b) RNA-seq (n=17 from 10 patients). c, Immune signatures (ssGSEA) in melanoma biopsy specimens (pre- and on-treatment) define immune-infiltrated (Group A, n=10 samples from 4 patients) and immune-poor tumor samples (Group B, n=17 samples from 6 patients). d–e, absolute expression (RPKM) for (d) CCL19 and CXCL13 and (e) their respective receptors, CCR7 and CXCR5 in melanoma biopsy specimens (pre- and on-treatment) in indicated sets of patient samples (group A - immune infiltrated; group B – immune poor by ssGSEA, Fig. 7c). f, Kaplan-Meier survival curve by four-way sorting of CCL19/CXCL13 expression using cutaneous melanoma (SKCM) TCGA data (38) (log-rank Mantel-Cox test). g, immune signatures (ssGSEA) in melanoma biopsy specimens (pre- and on-treatment) in clusters of patients with varying expression of CCL19 and CXCL13 in cutaneous melanoma (SKCM) TCGA. h, Heatmap of Day 3 PDOTS anti-PD1 induced cytokines (L2FC; n=14), ranked by progression-free survival (PFS) and annotated by response to anti-PD-1 therapy (CB, NCB/LTS, or NCB) and timing of sample collection.

Article Snippet: Mouse CCL19 (Abcam, ab100729) and mouse CXCL13 (R&D Systems, MCX130) ELISAs were used according to manufacturer instructions.

Techniques: Quantitative RT-PCR, RNA Sequencing, 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