human cxcl13 protein  (Bio-Techne corporation)

Journal: iScience

Article Title: Long trimer-immunization interval and appropriate adjuvant reduce immune responses to the soluble HIV-1-envelope trimer base

doi: 10.1016/j.isci.2024.108877

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Article Snippet: CXCL13 levels in NHP plasma were measured using Human CXCL13/BLC/BCA-1 Quantikine ELISA Kit (bio-techne/R&D systems) following instruction, with Human CXCL13 protein at 2-fold dilutions start at 500pg/ml as the standard for quantification.

Techniques: Virus, Neutralization, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: PD-1 + CXCR5 − CD4 + Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma

doi: 10.1136/jitc-2020-002101

Figure Lengend Snippet: A PD-1 + CXCR5 − CD4 + Th-CXCL13 cell subpopulation in the TLSs of NPC tumors. (A) Volcano plot showing degS in tumor-derived CD4 + T cells in comparison with CD4 + T cells from normal tissue. Representative genes are labeled. (B) IHC staining of representative tumor and normal tissue sections showing CXCL13 + expression in the TLS-like structures. (C) MIHC staining of a representative tumor section showing the coexpression of CD4 + (green), CD20 + (magenta), CXCL13 + (red) and Ki67 + (yellow) in the TLS, with nuclei counterstained with DAPI (blue). (D) t-SNE plot showing different cell clusters of all single cells from 10× Genomics analysis of scRNA sequences. (E) Identification of the cell markers of the CD4 + Th-CXCL13 cell subpopulation in the tumor. (F) Cell-Talker was used to identify putative interactions between the CD4 + Th-CXCL13 cell subpopulation and other cell subsets in the tumors and normal tissue. (G) Representative dot plots showing CXCL13, IFN-γ, IL-21 and IL-10 expressions in CD4 + TILs from tumors and eight identified cell subsets, including Th0 (IFN-γ − IL-21 − CXCL13 − ), Th1 (IFN-γ + IL-21 − CXCL13 − ), Tfh (IFN-γ − IL-21 + CXCL13 − ), Th-CXCL13 (IFN-γ − IL-21 − CXCL13 + ), Th1/Tfh (IFN-γ + IL-21 + CXCL13 − ), Th1/Th-CXCL13 (IFN-γ + IL-21 − CXCL13 + ), Tfh/Th-CXCL13 (IFN-γ − IL-21 + CXCL13 + ) and Th1/Tfh/Th-CXCL13 (IFN-γ + IL-21 + CXCL13 + ). (H) Statistical analysis showing the coexpression of IFN-γ, IL-21 and/or IL-10 in CD4 + CXCL13 + T cells from TILs and PBMCs (n=12). (I) Representative dot plots and statistical analysis showing the expression of IFN-γ and IL-21 in IL-10 − CD4 + CXCL13 + T cells from TILs and PBMCs (n=12). Data were expressed as the means±SD, and paired two-tailed Student t-test. (J) IHC staining of a representative tumor section showing the coexpression of IFN-γ, CXCL13 and IL-21 in the TLS. (K) FACS analysis and heat map showing the phenotypical characteristics of the identified Th0, Th1, Tfh, Th-CXCL13, Th1/Tfh, Th1/Th-CXCL13, Tfh/Th-CXCL13, and Th1/Tfh/Th-CXCL13 subsets of TILs and PBMCs (n=5). Data were expressed as the means. ***P<0.0001. FACS, fluorescence-activated cell sorting; IFN-γ, interferon gamma; IHC, immunohistochemistry; IL, interleukin; MIHC, multiplex immunohistochemical; N, normal tissue; NPC, nasopharyngeal carcinoma; PBMC, peripheral blood mononuclear cell; T, tumor; TIL, tumor-infiltrating lymphocyte; TLS, tertiary lymphoid structure; t-SNE, T-distribution neighborhood embedding algorithm; DEGs, differentially expressed genes.

Article Snippet: The bottom wells contained 235 µL PBS with or without 5 µg/mL recombinant human CXCL13 (R&D).

Techniques: Derivative Assay, Labeling, Immunohistochemistry, Expressing, Staining, Two Tailed Test, Fluorescence, FACS, Multiplex Assay, Immunohistochemical staining

Journal: Journal for Immunotherapy of Cancer

Article Title: PD-1 + CXCR5 − CD4 + Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma

doi: 10.1136/jitc-2020-002101

Figure Lengend Snippet: PD-1 + CXCR5 − CD4 + Th-CXCL13 might be involved in the formation of TLSs associated with NPC tumors. (A) Expression of the indicated Th cytokines and chemokines in sorted PD-1 + CXCR5 − CD4 + Th-CXCL13 in tumor of NPC (n=4) after 12 hours of resting, measured using a bead-based immunoarray. (B) Statistical analysis showing the correlation between the percentages of PD-1 + CXCR5 − CD4 + TILs and Th-CXCL13 cells analyzed by FACS analysis in tumor of NPC (n=50). (C) MIHC staining of a representative tumor section showing the coexpression of CD4 + (orange), CXCL13 + (magenta) and PD-1 + (green), with nuclei counterstained with DAPI (blue). (D) The expression of CXCR5 on CD3 + T, CD4 + T, CD8 + T and CD19 + B cells among PBMCs of patients with NPC. The same experiment was repeated five times. (E) Migration of CXCR5 + CD19 + B, CXCR5 + CD4 + T, CXCR5 + CD8 + T, CXCR5 − CD19 + B, CXCR5 − CD8 + T and CXCR5 − CD4 + T cells from PBMCs (n=5) in response to recombinant human CXCL13. Chemotaxis abilities of CXCR5 + subset, compared with CXCR5 − subset, were demonstrated, in which the chemotaxis index of CXCR5 − subsets was set as ‘1’. (F) MIHC staining of a representative tumor section showing CD4 + (green), CXCL13 + (magenta), CD20 + (cyan), CXCR5 + (red), PD-1 + (yellow) and DAPI (blue). (G) MIHC staining of a representative tumor section showing PD-1 + (Red), Bcl-6 + (Green) and CD21 + (Orange), with nuclei counterstained with DAPI (blue). (H) Survival analysis of the cohort stratified by CD4 + cells, PD-1 + cells, CXCR5 + cells and CXCL13 + cells identified by IHC staining, which the threshold used to define high and low is 8, 3, 6, and 4, respectively. (I) Survival analysis of the cohort stratified by CD4 + PD-1 + CXCR5 + , CD4 + PD-1 + CXCR5 − , CD4 + PD-1 - CXCR5 + and CD4 + PD-1 − CXCR5 − cells subsets among CD4 + TILs identified by MIHC staining and Halo analysis software, which the threshold used to define high and low is 0.997, 4.50, 32.1, and 42.5, respectively. FACS, fluorescence-activated cell sorting; IHC, immunohistochemistry; MIHC, multiplex immunohistochemical; NPC, nasopharyngeal carcinoma; PBMC, peripheral blood mononuclear cell; TIL, tumor-infiltrating lymphocyte; TLS, tertiary lymphoid structure.

Article Snippet: The bottom wells contained 235 µL PBS with or without 5 µg/mL recombinant human CXCL13 (R&D).

Techniques: Expressing, Staining, Migration, Recombinant, Chemotaxis Assay, Immunohistochemistry, Software, Fluorescence, FACS, Multiplex Assay, Immunohistochemical staining

Journal: Journal for Immunotherapy of Cancer

Article Title: PD-1 + CXCR5 − CD4 + Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma

doi: 10.1136/jitc-2020-002101

Figure Lengend Snippet: Activated APCS induced the differentiation and expansion of the PD-1 + CXCR5 − CD4 + Th-CXCL13 cell subset through TGF-β1. (A) Representative dot plots showing PD-1 + CXCR5 − CD4 + Th-CXCL13 cells in vitro (n=5). (B) Representative dot plots showing that TGF-β1 with or without other indicated cytokines gave rise to CXCL13-producing PD-1 + CD4 + Th cells in vitro (n=5). (C) Secretion of CXCL13 by sorted PD-1 + CXCR5 − D4 + TILs (n=4) after stimulation for 8 days using anti-CD3/CD28, TGF-β1 (10 ng/mL) and/or TGF-β1 receptor inhibitor SB431542 (10 µM/mL). (D, E) The mRNA expression of the indicated cytokines was determined by RT-qPCR and the cytokine protein in supernatants was determined by ELISA (n=5). (F, G) MOs, DCs and Mφ were left untreated or stimulated with LPS for 5 hours and then cultured for 8 days with autologous T cells, naïve T cells and memory T cells from healthy donors (n=3). Proliferation (CFSE − ) and expression of CXCL13 + cells were detected by FACS. (H) Representative dot plots showing that blocking of TGF-β1, IL-1β, IL-6, TNF-α and IL-12 changed the subset composition of Th-CXCL13 cells expanded by LPS-stimulated mos from healthy donors (n=3). (I) Representative dot plots and statistical analysis showing of the expression of the indicated markers on MOs from tumors and blood (n=5). (J) Representative dot plots showing that the addition of anti-TGF-β1 and/or IL-2 or TGF-β1 and/or anti-IL-2 changed the subset composition of Th-CXCL13 cells expanded by LPS-stimulated tumor-derived MOs (n=3). Data were expressed as the means±SD, and paired two-tailed Student t-test. *P<0.05, **P<0.001, ***P<0.0001. APC, antigen-presenting cell; DC, dendritic cell; FACS, fluorescence-activated cell sorting; IFN-γ, interferon gamma; Mφ, macrophage; MO, monocyte; RT-qPCR, quantitative reverse transcription PCR; TGF-β1, transforming growth factor beta 1; TIL, tumor-infiltrating lymphocyte; TLS, tertiary lymphoid structure; TNF-α, tumor necrosis factor alpha; MFI, mean fluorescence intensity.

Article Snippet: The bottom wells contained 235 µL PBS with or without 5 µg/mL recombinant human CXCL13 (R&D).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Blocking Assay, Derivative Assay, Two Tailed Test, Fluorescence, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: PD-1 + CXCR5 − CD4 + Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma

doi: 10.1136/jitc-2020-002101

Figure Lengend Snippet: Transcriptome analysis identified SOX4 as a transcription factor related to Th-CXCL13 cells in the TLSs. (A) Human naïve CD4 + T cells from healthy donors (n=3) were differentiated in the presence of the indicated chemokines for 8 days. Outline for the screening of candidate transcription factors. (B, C) Human naïve CD4 + T cells from healthy donors (n=4) were stimulated with or without anti-CD3/CD28 and/or TGF-β1 for 24 hours, and after which the expression of Sox4 , PDCD1 and CXCL13 was assessed by RT-qPCR and immunoblotting. (D–G) Representative dot plots and statistical analysis showing the proliferation (CFSE − ) and expression of CXCL13 + cells among naïve CD4 + T cells from healthy donors (n=4) and CD4 + TILs (n=4) with or without anti-CD3/CD28 beads and TGF-β1 stimulation in the presence or absence of siRNA against SOX4. (H) MIHC staining of a representative tumor section showing CD4 + (orange), CXCL13 + (red), Sox4 + (cyan) and CD20 + (green), with nuclei counterstained with DAPI (blue). (I) Survival analysis of the cohort stratified by Sox4 + cells identified by IHC staining, which the threshold used to define high and low is 2. Data were expressed as the means±SD, and paired two-tailed Student t-test. *P<0.05, **P<0.001, ***P<0.0001. IHC, immunohistochemistry; IL, interleukin; MIHC, multiplex immunohistochemical; TGF-β1, transforming growth factor beta 1; TIL, tumor-infiltrating lymphocyte; TLS, tertiary lymphoid structure; TNF-α, tumor necrosis factor alpha.

Article Snippet: The bottom wells contained 235 µL PBS with or without 5 µg/mL recombinant human CXCL13 (R&D).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemistry, Two Tailed Test, Multiplex Assay, Immunohistochemical staining

Journal: Journal for Immunotherapy of Cancer

Article Title: PD-1 + CXCR5 − CD4 + Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma

doi: 10.1136/jitc-2020-002101

Figure Lengend Snippet: PD-1 + CXCR5 − CD4 + Th-CXCL13 cells promoted plasma cell differentiation and immunoglobulin production via IL-21 and CD84 interactions. (A, B) Representative dot plots and statistical analysis showing the PD-1 + CXCR5 + , PD-1 + CXCR5 − , PD-1 - CXCR5 + and PD-1 − CXCR5 − subsets of CD4 + T cells among TILs and PBMCs of patients with NPC (n=8). (C) FACS analysis and heat map showing the phenotypical characteristics of the indicated four cell subsets of CD4 + TILs from NPC tumors (n=5). (D–F) Representative dot plots and statistical analysis showing the frequency of plasma cells, and the production of IgG, IgA and IgM in T-B cell cocultures with or without sterile subset cells fixed with paraformaldehyde, cocultured with autologous memory B cells from TILs of NPC tumors (n=4). (G) Statistical analysis showing the plasma cell frequency and the production of IgG, IgA and IgM in T-B cell cocultures using sorted cell subsets cocultured with autologous memory B cells from TILs of NPC tumors (n=4) with neutralizing antibodies against CXCL13 or IL-21, respectively. (H–I) Representative dot plots and statistical analysis showing the expression of indicated SLAM family members by B-cell subsets and four cell subsets of patients with NPC (n=4), respectively. (J) FACS analysis of the conjugation efficiency of blasted PD-1 + CXCR5 + and PD-1 + CXCR5 − TILs (n=5), preincubated with or without neutralizing antibodies against CD150, CD84 or CD352, with LPS-activated B cells pulsed with a tumor suspension. (K) Statistical analysis showing the frequency of plasma cells, and the production of IgG, IgA and IgM in T-B cell cocultures using sorted PD-1 + CXCR5 + and PD-1 + CXCR5 − cocultured with autologous memory B cells from TILs of NPC tumors (n=4) with or without neutralizing antibodies against CD352 or CD84, respectively. (L) MIHC staining of a representative tumor section showing CD4 + T (red), CXCL13 + (green) and CD20 + B (cyan), with nuclei counterstained with DAPI (blue). (M) MIHC staining of a representative tumor section showing CD4 + T (magenta), CD20 + B (cyan), CXCL13 + (red), Ki67 + (green) and IgG + (orange), with nuclei counterstained with DAPI (blue). (N) MIHC staining of a representative tumor section showing CD20 + B (cyan), CXCL13 + (red), IgA + (green), IgM + (magenta) and IgG + (orange), with nuclei counterstained with DAPI (blue). data are expressed as the means±SD, and paired two-tailed Student t-test. *P<0.05, **P<0.001, ***P<0.0001. FACS, fluorescence-activated cell sorting; MIHC, multiplex immunohistochemical; NPC, nasopharyngeal carcinoma; PBMC, peripheral blood mononuclear cell; TIL, tumor-infiltrating lymphocyte.

Article Snippet: The bottom wells contained 235 µL PBS with or without 5 µg/mL recombinant human CXCL13 (R&D).

Techniques: Cell Differentiation, Expressing, Conjugation Assay, Staining, Two Tailed Test, Fluorescence, FACS, Multiplex Assay, Immunohistochemical staining