Journal:

Article Title: BCA-1 is highly expressed in Helicobacter pylori -induced mucosa-associated lymphoid tissue and gastric lymphoma

doi:

Figure Lengend Snippet: In situ expression of BCA-1 in chronic Hp gastritis. Expression is observed in the whole area of lymphoid aggregates (a) and is restricted to the mantle zone in secondary lymphoid follicles (c). Intermediate distribution (b) is found occasionally. (d–f) Localization of follicular dendritic cells as detected in serial sections by immunohistochemical staining for CD21. Bottom: In situ expression of BCA-1 in a secondary follicle of a normal lymph node. Antisense and sense hybridization is shown. ×100 (original magnification).

Article Snippet: Goat anti-human BCA-1 (1:100; R&D Systems Inc., Minneapolis, Minnesota, USA) and rabbit anti-human CXCR5 (IgG purified, 1:100) were used to detect BCA-1 and its receptor.

Techniques: In Situ, Expressing, Immunohistochemical staining, Staining, Hybridization

Journal:

Article Title: BCA-1 is highly expressed in Helicobacter pylori -induced mucosa-associated lymphoid tissue and gastric lymphoma

doi:

Figure Lengend Snippet: BCA-1 and CXCR5 expression in chronic Hp gastritis. (a) In situ expression of BCA-1 is shown in the mantle zone of a secondary lymphoid follicle. The sense probe control (b) and staining for B lymphocytes (CD20+) (c) are shown in serial sections of the same follicle. ×100 (original magnification). BCA-1 (d) and CXCR5 (f) immunostaining is shown with the corresponding isotype-matched controls (e and g). Staining of a frozen section of a tonsil (h) and control (i) shows BCA-1 in association with small round and stellate cells, suggesting expression by lymphocytes and dendritic cells. ×200 (original magnification).

Article Snippet: Goat anti-human BCA-1 (1:100; R&D Systems Inc., Minneapolis, Minnesota, USA) and rabbit anti-human CXCR5 (IgG purified, 1:100) were used to detect BCA-1 and its receptor.

Techniques: Expressing, In Situ, Staining, Immunostaining

Journal:

Article Title: BCA-1 is highly expressed in Helicobacter pylori -induced mucosa-associated lymphoid tissue and gastric lymphoma

doi:

Figure Lengend Snippet: Expression of BCA-1 and CXCR5 in gastric MALT lymphomas. In situ expression of BCA-1 in (a) low-grade lymphoma with surrounding normal gastric mucosa, ×40 (original magnification); and in high-grade lymphoma (b and c), ×200 and ×400, respectively (original magnification). Immunostaining of BCA-1 and CXCR5 in high-grade lymphoma (d and e), ×400 and ×200, respectively (original magnification).

Article Snippet: Goat anti-human BCA-1 (1:100; R&D Systems Inc., Minneapolis, Minnesota, USA) and rabbit anti-human CXCR5 (IgG purified, 1:100) were used to detect BCA-1 and its receptor.

Techniques: Expressing, In Situ, Immunostaining

Journal: Cancer immunology research

Article Title: Radiofrequency ablation remodels the tumor microenvironment and promotes neutrophil-mediated abscopal immunomodulation in pancreatic cancer

doi: 10.1158/2326-6066.CIR-22-0379

Figure Lengend Snippet: Tumor size was recorded 4 days before (Initial), right before (Pre) and 4 days after (Post) Sham or RFA treatment. Proteome arrays were performed in locally ablated tumors and serum of ablated mice and compared to Sham-treated mice (Control). A, Experimental design of RFA-treated mice. B, Growth curves show Control tumors (n = 8) significantly increased in size 4 days after treatment when compared to RFA-treated (n = 8) and non-RFA treated (n = 7) tumors. C, At the time of euthanization only Sham-treated tumors (n = 8) had significantly increased in size compared with pretreatment size; no difference in size was observed in RFA-treated (n = 8) and non-RFA treated (n = 7) tumors Pre and Post RFA. D, ImageJ quantification of necrosis, which was detected by H&E staining. RFA significantly increased necrosis on the RFA- and non-RFA-treated tumors compared to control Sham-treated tumors. E, Representative composite H&E staining of control, RFA, and non-RFA treated tumors showing necrotic areas inside dashed lines. F, ImageJ quantification showing RFA increased cleaved caspase 3+ cells in the RFA-treated and non-RFA treated tumors compared to control Sham-treated control tumors, as assessed by IHC. G, Representative IHC staining for cleaved caspase 3 in control, RFA, and non-RFA treated tumors. H, ImageJ quantification revealed RFA significantly increased the number of granzyme B+ cells in the RFA-treated tumors compared to controls and non-RFA treated tumors, as assessed by IHC. I, IHC staining for granzyme B in control, RFA, and non-RFA treated tumors. J, RFA-treated tumors (n = 3) presented increased expression of C5/C5a, IL-23 and CXCL12 compared to control (n = 2) tumor content. K, CXCL10, CXCL12, CXCL13 and TIMP-1 were significantly elevated in serum from RFA-treated (n = 4) mice compared to Sham-treated (n = 3) control serum. Time x Treatment comparisons were performed using Two-way ANOVA, treatment only comparisons by One-way ANOVA and proteome arrays were analyzed by multiple t test. Bar plots showing mean with SEM were used to represent data. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., not significant. Scale bars are 50μM.

Article Snippet: CXCL13 ELISA Mouse CXCL13/BLC/BCA-1 Quantikine ELISA Kit assay was used to determine CXCL13 levels in mouse serum, splenocytes and tumors homogenates, following the manufacturer instructions (R&D Systems, cat #MCX130).

Techniques: Control, Staining, Immunohistochemistry, Expressing

Journal: Cancer immunology research

Article Title: Radiofrequency ablation remodels the tumor microenvironment and promotes neutrophil-mediated abscopal immunomodulation in pancreatic cancer

doi: 10.1158/2326-6066.CIR-22-0379

Figure Lengend Snippet: A-B, IMC analysis of tumors 4 days after Sham or RFA treatment revealed Ly6G+CD11b+CD44+ neutrophils are enriched in non-RFA treated tumors. C, Neighborhood analysis identified immune cells and markers with strong neutrophil co-localization. D, Cluster and Cell Phenotype information of Neighborhood analysis of IMC data. E, Experimental design for neutrophil depletion in vivo followed by RFA. F, RFA locally ablated tumors treated with IgG2a isotype control (VEH, n = 6) or anti-Ly6G (ND, n = 8) did not show differences in tumor size right before (Pre) and 4 days after (Post) RFA ablation. In non-RFA treated tumors, anti-Ly6G (ND, n = 8) treatment revealed an increase in tumor size Post RFA treatment when compared to IgG2a isotype control (VEH, n = 6) treated tumors. G, Neutrophil depletion (ND; anti-Ly6G treated group) did not alter αSMA staining, detected by IHC, in RFA-treated tumors when compared to RFA-treated tumors with IgG2a (VEH); on the contrary, neutrophil depletion (ND) revealed non-RFA treated tumors presented a significant increase in αSMA compared to control non-neutrophil depleted (VEH) group. H, Neutrophil depletion did not alter CD31 staining, detected by IHC, in any of the groups. I, Neutrophil-depleted RFA treated tumors presented a significant reduction in CXCL13 content compared to both VEH + RFA and non-RFA treated tumors when assayed using a cytokine array. No differences were found in non-RFA treated tumors between treatments. J, Neutrophil depletion presented a trend in reducing systemic CXCL13 levels in RFA treated mice. Tumor volume was analyzed by paired Student’s t test. Tumor chemokine levels were studied by Two-way ANOVA. IHC and serum protein expression levels were analyzed by unpaired Student’s t test. Bar plots indicate mean with SEM. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; n.s., not significant.

Article Snippet: CXCL13 ELISA Mouse CXCL13/BLC/BCA-1 Quantikine ELISA Kit assay was used to determine CXCL13 levels in mouse serum, splenocytes and tumors homogenates, following the manufacturer instructions (R&D Systems, cat #MCX130).

Techniques: In Vivo, Control, Staining, Expressing

Journal: Cell reports

Article Title: Distinct features of a peripheral T helper subset that drives the B cell response in dengue virus infection

doi: 10.1016/j.celrep.2025.115366

Figure Lengend Snippet:

Article Snippet: A human CXCL13/BLC/BCA-1 Quantikine ELISA Kit (R&D Systems) was used to measure the CXCL13 cytokine concentration in plasma samples as a circulating GC marker.

Techniques: Purification, Virus, Recombinant, Lysis, Staining, Electron Microscopy, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Amplification, Gene Expression, Software

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: PLOS Pathogens

Article Title: Lymphoid B cells upregulate HIV-1 ex vivo and are linked to its expression in vivo

doi: 10.1371/journal.ppat.1013661

Figure Lengend Snippet: (A) TFH (CD3 + CD8 - CD19 - CXCR5 hi PD-1 hi ) were spinoculated with X4-HIV GFP reporter virus (X4-HIV) and cultured with autologous uninfected, CellTrace Blue labeled TFH or GCB (CD19 + CD3 - CD38 mid IgD - ) for 3 days in R-15 and 5μM saquinavir. CXCR5 MFI of CellTrace - GFP + and GFP - TFH was determined by flow cytometry (n = 11). (B) Supernatant from cultures of TFH spinoculated with X4-HIV and cultured with autologous uninfected CellTrace Blue labeled TFH or GCB were collected after three days. Live, CellTrace Blue - TFH cell counts were determined by flow cytometry. CXCL13 concentrations were determined by ELISA and normalized to pg/TFH (n = 6). (C) CXCR5 MFI was determined by flow cytometry on GFP + CellTrace - TFH from cultures of X4-HIV spinoculated TFH cultured with autologous uninfected, CellTrace Blue labeled TFH or GCB in the presence or absence of CXCL13 blocking antibody after 3 days (n = 10). (D) TFH (CD3 + CD8 - PD-1 hi ICOS hi ) were spinoculated with X4-HIV, then recovered for 1 hour in R-15 with 5μM saquinavir. Half of the cells were cultured 3 days in R-15 with 5μM saquinavir, while the other half were washed and subjected to a CXCL13 chemotaxis assay using 5 μm transwells. After 4 hours, cells were collected from both top and bottom wells, washed, and cultured 3 days in R-15 containing 5μM saquinavir. On day 3, the chemotaxis assay was performed on the remaining TFH untouched post spinoculation. GFP expression was determined in TFH from top and bottom wells and total GFP + and GFP - cells counts were determined using absolute count beads by flow cytometry to determine chemotactic indexes (n = 5). Horizontal bars indicate medians (A). Wilcoxon matched paired tests were performed using Graphpad Prism v10 and significance indicated: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: In some experiments 5μg/ml CXCL13 blocking antibody (clone AF801, R&D systems), 10μg/ml anti-IL-2 (clone 5334, R&D systems), 10μg/ml anti-GITRL (clone 109114, R&D systems), 5μg/ml 4–1BB-Fc (R&D systems), 10μg/ml anti-MHC-II (clone IVA12, Raybiotech), 10μg/ml sICOS (Biolegend), 10μg/ml sCD40L (Biolegend), 10μg/ml anti-CD40 (clone 82102, R&D systems), 100nM AZD5582 (SelleckChem), 5μM NIK-SMI1 (Sigma), 0.1-1μM tofocitinib (SelleckChem), 0.1-1μM ruxolitinib (SelleckChem), or DMSO as an appropriate vehicle control when necessary were added.

Techniques: Virus, Cell Culture, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Blocking Assay, Chemotaxis Assay, Expressing

Journal: Cancers

Article Title: The Possible Role of Anti- and Protumor-Infiltrating Lymphocytes in Pathologic Complete Response in Early Breast Cancer Patients Treated with Neoadjuvant Systemic Therapy.

doi: 10.3390/cancers15194794

Figure Lengend Snippet: Figure 2. Histopathological slides of patients with total tumor-infiltrating lymphocytes (TIL) (high on (A1) and low on (A2)) and TILs subtypes (CD8+ TILs, CXCL13+ TILs, PD-1+ TILs, and FOXP3+ TILs) (high on (B1,C1,D1,E1); low on (B2,C2,D2,E2)).

Article Snippet: Retrieved epitopes were detected using monoclonal antibodies directed against CD8 (DAKO Agilent Technologies Inc. Santa Clara, CA, USA, cat. No. M7103; clone C8/144B; diluted at 1:100), FOXP3 (Epitomics, Rocklin, CA, USA, cat. No. AC0304RUO; clone EP340; diluted at 1:200), PD1 (CellMarque, Rocklin, CA, USA; cat. No. 315 M; clone MRQ22; diluted at 1:800), and CXCL13 (R&D Systems, Minneapolis, MN, USA; cat. No. MAB8012; clone 53602; diluted at 1:25).

Techniques:

Journal: Cancers

Article Title: The Possible Role of Anti- and Protumor-Infiltrating Lymphocytes in Pathologic Complete Response in Early Breast Cancer Patients Treated with Neoadjuvant Systemic Therapy.

doi: 10.3390/cancers15194794

Figure Lengend Snippet: Figure 5. Percentages of CD8+, CXCL13+, PD-1+, and FOXP3+ lymphocytes in tumor-infiltrating lymphocytes (TILs), boxplots. Abbreviations: min: minimum; max: maximum; q3: 3rd quartile; q1: 1st quartile; n: number.

Article Snippet: Retrieved epitopes were detected using monoclonal antibodies directed against CD8 (DAKO Agilent Technologies Inc. Santa Clara, CA, USA, cat. No. M7103; clone C8/144B; diluted at 1:100), FOXP3 (Epitomics, Rocklin, CA, USA, cat. No. AC0304RUO; clone EP340; diluted at 1:200), PD1 (CellMarque, Rocklin, CA, USA; cat. No. 315 M; clone MRQ22; diluted at 1:800), and CXCL13 (R&D Systems, Minneapolis, MN, USA; cat. No. MAB8012; clone 53602; diluted at 1:25).

Techniques:

Journal: Cancers

Article Title: The Possible Role of Anti- and Protumor-Infiltrating Lymphocytes in Pathologic Complete Response in Early Breast Cancer Patients Treated with Neoadjuvant Systemic Therapy.

doi: 10.3390/cancers15194794

Figure Lengend Snippet: Figure 6. The distribution of CD8+ TILs (a), CXCL13+ TILs (b), PD-1+ TILs (c), and FOXP3+ TILs (d) among the breast cancer subtypes, boxplots. The difference in separate TIL subtypes between breast cancer subtypes was statistically significant for FOXP3+ TILs only (p = 0.027), but this could not be generalized to the population (adjusted p = 0.598), see Table S1. Abbreviations: min: minimum; max: maximum; q3: 3rd quartile; q1: 1st quartile; TILs: tumor-infiltrating lymphocytes; HER-2: human epidermal growth factor receptor 2; n: number.

Article Snippet: Retrieved epitopes were detected using monoclonal antibodies directed against CD8 (DAKO Agilent Technologies Inc. Santa Clara, CA, USA, cat. No. M7103; clone C8/144B; diluted at 1:100), FOXP3 (Epitomics, Rocklin, CA, USA, cat. No. AC0304RUO; clone EP340; diluted at 1:200), PD1 (CellMarque, Rocklin, CA, USA; cat. No. 315 M; clone MRQ22; diluted at 1:800), and CXCL13 (R&D Systems, Minneapolis, MN, USA; cat. No. MAB8012; clone 53602; diluted at 1:25).

Techniques:

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