recombinant murine cxcl13  (PeproTech)

Journal: The Journal of Immunology Author Choice

Article Title: CXCL13 as a Novel Immune Checkpoint for Regulatory B Cells and Its Role in Tumor Metastasis

doi: 10.4049/jimmunol.2100341

Figure Lengend Snippet: Cxcl13 deficiency inhibits murine experimental pulmonary metastasis. (A) The expression levels of CXCR5 in B16-F10, LL/2, E.G7-OVA, and ID8 cell lines were assessed by flow cytometry (FCM). Mouse lymphocytes from the spleen were used as positive control, and the gray area represents the isotype control. (B) B16-F10, LL/2, E.G7-OVA, or ID8 cells were treated with 500 ng/ml rmCXCL13 for the indicated times. Cell viability was determined by CCK-8 assays (n = 3 biologically independent samples). (C) B16-F10, LL/2, or E.G7-OVA cells (1 × 106) were s.c. transplanted into the right-back flank of wild-type (WT) or Cxcl13−/− mice. Tumor volume was monitored at the indicated times (n = 5 mice). (D) B16-F10 cells (2 × 105) were i.v. injected into WT or Cxcl13−/− mice to establish experimental pulmonary metastasis models. Mice were sacrificed on day 14 for measuring lung metastases of the tumor. The gross appearance of the lungs (left panel) and the tumor nodules on the lungs (right panel) were examined (n = 5 mice). (E) WT or Cxcl13−/− mice were injected as in (D). Mice were sacrificed on day 21 for measuring lung weights. The gross appearance of the lungs (left panel) and the lung weights (right panel) were examined (n = 5 mice). (F) WT or Cxcl13−/− mice were injected with LL/2 cells as in (D). Mice were sacrificed on day 21 for measuring lung weights. The gross appearance of the lungs (left panel) and the lung weights (right panel) were examined (n = 5 mice). Data are presented as mean ± SD or mean ± SEM. Statistical significance was determined by two-way ANOVA or a two-sided unpaired t test. *p < 0.05, ***p < 0.001.

Article Snippet: Then, the cells were incubated with 500 ng/ml recombinant murine CXCL13 (rmCXCL13) (PeproTech) for indicated times followed by staining with 10% CCK-8 solution at 37°C for another 2 h. Then, the absorbance at 450 nm was measured by a microplate reader.

Techniques: Expressing, Flow Cytometry, Positive Control, Control, CCK-8 Assay, Injection

Journal: The Journal of Immunology Author Choice

Article Title: CXCL13 as a Novel Immune Checkpoint for Regulatory B Cells and Its Role in Tumor Metastasis

doi: 10.4049/jimmunol.2100341

Figure Lengend Snippet: CXCL13 deficiency reduced the infiltration of regulatory B cells into the tumor metastatic microenvironment. (A–F) B16-F10 cells (2 × 105) were i.v. injected into WT or Cxcl13−/− mice to establish experimental pulmonary metastasis models. Mice were sacrificed on days 7 (A), 14 (B), and 21 (C) after tumor injection, and then the single-cell suspensions of pulmonary metastatic tumors were prepared and subjected to FCM analysis. Representative scatterplots of the gated CD45+ lymphocytes are shown in (A)–(C). Numbers on the right indicate the percentage of CD19+ B cells producing IL-10 or not in the gated CD45+ lymphocyte population (n = 5 mice). Quantitative data are shown in (D)–(F), respectively. (G) Mice were treated in the same way as in (A). Mice were sacrificed on day 14, and single-cell suspensions of pulmonary metastatic tumors were subjected to FCM analysis. Representative scatterplots of the gated CD45+CD19+ B cells are shown in the left panel and are quantified in the right panel. Numbers on the upper right indicate the percentage of IL-35+ B cells (EBI3+P35+) in the gated CD45+CD19+ population. (n = 4 mice). FMO control, fluorescence minus one negative control. (H) Single-cell suspension samples were from (G) and subjected to FCM analysis. Representative scatterplots of the gated CD45+CD19+ B cells are shown in the left panel and are quantified in the right panel. Numbers on the upper right indicate the percentage of TGF-β+ B cells in the gated CD45+CD19+ population (n = 4 mice). FMO control, fluorescence minus one negative control. (I and J) CXCL13 deficiency had no clear effects on the CD19+ B cell population either in the lung (I) or spleen (J) under homeostatic conditions. B cell populations derived from lung or spleen of WT or Cxcl13−/− mice were assessed by FCM. Representative scatterplots of the gated CD45+ cells are shown. Numbers on the upper right indicate the percentages of CD19+ B cells in the gated CD45+ population (n = 5 mice). Quantitative data of FCM results are shown in the lower panel. Data are presented as mean ± SD. Statistical significance was determined by a two-sided unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Then, the cells were incubated with 500 ng/ml recombinant murine CXCL13 (rmCXCL13) (PeproTech) for indicated times followed by staining with 10% CCK-8 solution at 37°C for another 2 h. Then, the absorbance at 450 nm was measured by a microplate reader.

Techniques: Injection, Control, Fluorescence, Negative Control, Suspension, Derivative Assay

Journal: The Journal of Immunology Author Choice

Article Title: CXCL13 as a Novel Immune Checkpoint for Regulatory B Cells and Its Role in Tumor Metastasis

doi: 10.4049/jimmunol.2100341

Figure Lengend Snippet: Reduction of regulatory B cells leads to boosted antitumor immunity in the metastatic microenvironment. (A and B) B16-F10 cells (2 × 105) were i.v. injected into WT or Cxcl13−/− mice to establish experimental pulmonary metastasis models. Mice were sacrificed on day 14, and the single-cell suspensions of pulmonary metastatic tumors were prepared and subjected to FCM analysis. Representative scatterplots of neutrophils (CD11b+Ly-6G+) and monocytes (CD11b+Ly-6C+Ly-6G−) in the tumor metastatic microenvironment are shown in (A) and quantified in (B). Neutrophils shown on the left panel were gated from the CD45+ subpopulation, and monocytes displayed on the right panel were gated from the CD45+Ly-6G− subpopulation (n = 3 mice). (C and D) Representative scatterplots of M2 Macs (CD11b+F4/80+arginase-1+CD206+) in the tumor metastatic microenvironment are shown in (C) and quantified in (D). Cells were gated from the CD45+CD11b+F4/80+ subpopulation (n = 4 mice). (E and F) Representative scatterplots of alveolar macrophages (CD11b−CD11c+) or dendritic cells (DCs; CD11b+CD11c+CD64−CD24+) in the tumor metastatic microenvironment are shown in (E) and quantified in (F). Cells were gated respectively from CD45+ or CD45+CD11b+CD11c+ subpopulations (n = 4 mice). (G) The percentages of activated CD4+ T cells (CD4+CD69+) in the tumor metastatic microenvironment were quantified. Cells were gated from the CD3+CD4+ subpopulation (n = 4 mice). (H) The percentages of activated CD8+ T cells (CD8+CD69+) in the tumor metastatic microenvironment were quantified. Cells were gated from the CD3+CD8+ subpopulation (n = 4 mice). (I) The percentages of Th17 cells (CD4+IL-17A+) in the tumor metastatic microenvironment were quantified. Cells were gated from the CD3+CD4+ subpopulation (n = I mice). (J) Percentages of IFN-γ–producing CD8+ CTLs (CD8+IFN-γ+) in the tumor metastatic microenvironment were quantified. Cells were gated from the CD3+CD8+ subpopulation (n = 4 mice). (K) Percentages of regulatory T cells (CD4+Foxp3+) in the tumor metastatic microenvironment were quantified. Cells were gated from the CD3+ subpopulation (n = 4 mice). (L) The mRNA levels of Tgfb, Prfn, Ifng, and Grzb in the tumor metastatic environment were assessed by quantitative real-time PCR. Gene expression level was normalized to 18S rRNA (n = 3 mice). Data are mean ± SD. Statistical significance was determined by a two-sided unpaired t test or two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Then, the cells were incubated with 500 ng/ml recombinant murine CXCL13 (rmCXCL13) (PeproTech) for indicated times followed by staining with 10% CCK-8 solution at 37°C for another 2 h. Then, the absorbance at 450 nm was measured by a microplate reader.

Techniques: Injection, Real-time Polymerase Chain Reaction, Gene Expression

Journal: The Journal of Immunology Author Choice

Article Title: CXCL13 as a Novel Immune Checkpoint for Regulatory B Cells and Its Role in Tumor Metastasis

doi: 10.4049/jimmunol.2100341

Figure Lengend Snippet: CXCL13 deficiency enhanced the metastasis inhibitory ability of chemotherapy. (A) CTX was dissolved in sterilized PBS. WT and Cxcl113−/− mice in treatment groups were i.p. administered with CTX (100 mg/kg body weight in 0.2 ml of saline/mouse) on days 3 and 6 following i.v. injection of B16-F10 cells (2 × 105). WT and Cxcl113−/− mice in control groups were i.p. administered with the same volume of sterilized PBS at the same time points. All mice were sacrificed on day 14, and the gross appearance of the lungs was examined (n = 5 mice). (B and C) Lungs from WT and Cxcl113−/− mice in (A) were isolated for measuring lung metastases of the tumor (B) and lungs were paraffin embedded and stained with H&E (C) (n = 5 mice). Original magnification ×100. (D) Single-cell suspensions of pulmonary metastatic tumors from mice in (A) were subjected to FCM analysis. Activated CD4+ T cells (CD4+CD69+) in the tumor metastatic microenvironment were quantified and plotted as a percentage of CD3+CD4+ T cells (n = 5 mice). (E) IFN-γ+CD8+ T cells in the tumor metastatic microenvironment were quantified and plotted as a percentage of CD3+CD8+ T cells (n = 5 mice). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Then, the cells were incubated with 500 ng/ml recombinant murine CXCL13 (rmCXCL13) (PeproTech) for indicated times followed by staining with 10% CCK-8 solution at 37°C for another 2 h. Then, the absorbance at 450 nm was measured by a microplate reader.

Techniques: Saline, Injection, Control, Isolation, Staining

Journal: The Journal of Immunology Author Choice

Article Title: CXCL13 as a Novel Immune Checkpoint for Regulatory B Cells and Its Role in Tumor Metastasis

doi: 10.4049/jimmunol.2100341

Figure Lengend Snippet: Deletion of CXCL13 enhanced the efficacy of cancer immunotherapy targeting PD-1. (A) WT and Cxcl113−/− mice in treatment groups were i.p. administered with anti–PD-1 mAbs (250 μg/mouse) on days 0 and 3 following i.v. injection of B16-F10 cells (2 × 105). WT and Cxcl113−/− mice in control groups were i.p. administered with the same dose of isotype control IgG at the same time points. All mice were sacrificed on day 14 and the gross appearance of the lungs was examined (n = 5 mice). (B and C) Lungs from WT and Cxcl113−/− mice in (A) were isolated for measuring lung metastases of the tumor (B), and lungs were paraffin embedded and stained with H&E (C) (n = 5 mice). Original magnification ×100. (D) Single-cell suspension of pulmonary metastatic tumor from mice in (A) were subjected to FCM analysis. The activated CD4+ T cells (CD4+ CD69+) in the tumor metastatic microenvironment were quantified and plotted as a percentage of CD3+CD4+ T cells (n = 4 mice). (E) IFN-γ+ CD8+ T cells in the tumor metastatic microenvironment were quantified and plotted as a percentage of CD3+CD8+ T cells (n = 4 mice). (F) Regulatory T cells (CD4+Foxp3+) in the tumor metastatic microenvironment were quantified and plotted as a percentage of CD3+ T cells (n = 4 mice). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA. *p < 0.05. **p < 0.01. ***p < 0.001.

Article Snippet: Then, the cells were incubated with 500 ng/ml recombinant murine CXCL13 (rmCXCL13) (PeproTech) for indicated times followed by staining with 10% CCK-8 solution at 37°C for another 2 h. Then, the absorbance at 450 nm was measured by a microplate reader.

Techniques: Injection, Control, Isolation, Staining, Suspension

Journal: bioRxiv

Article Title: B cell-intrinsic requirement for WNK1 kinase in T cell-dependent antibody responses

doi: 10.1101/2021.09.09.459588

Figure Lengend Snippet: (A) Irradiated RAG1-deficient mice were reconstituted with bone marrow from either Wnk1 fl/+ RCE or Wnk1 fl/fl RCE mice, or from Wnk1 fl/+ RCE or Wnk1 fl/D368A RCE mice. At least 56 d later, mice were treated with tamoxifen on 3 consecutive days and analyzed 7 d after start of tamoxifen treatment. (B) Mean±SEM levels of Wnk1 mRNA measured across the junction of exons 1 and 2 in control or WNK1-deficient splenic mature B cells, normalized to Hprt expression and to Wnk1 mRNA levels in control B cells (set to 1). (C, D) Mean±SEM numbers of mature B cells in the spleen (B220 + CD19 + CD93 - ) and lymph nodes (TCRβ - B220 + IgM + IgD + ) of RAG1-deficient mice reconstituted with Wnk1 fl/+ RCE or Wnk1 fl/fl RCE marrow (C) or with Wnk1 fl/+ RCE or Wnk1 fl/D368A RCE marrow (D), as described in A. (E) Mean±SEM binding of soluble VCAM-1 complexes to control or WNK1-deficient B cells in response to treatment with anti-IgM or CXCL13 for the indicated times. (F) Mean±SEM surface levels of CD11a, CXCR5, IgM and CD40 on control or WNK1-deficient B cells normalized to expression on control B cells (set to 100). (G) Irradiated RAG1-deficient mice were reconstituted with bone marrow from either Oxsr1 +/+ RCE or Oxsr1 fl/fl RCE mice, or from Oxsr1 +/+ Stk39 +/+ RCE or Oxsr1 fl/fl Stk39 T243A/T243A RCE mice. At least 56 d later, mice were treated with tamoxifen on 5 consecutive days and analyzed 21 d after start of tamoxifen. (H, J) Immunoblot analysis (top) of total cell lysates from splenic B cells from RAG1-deficient mice reconstituted with Oxsr1 +/+ RCE or Oxsr1 fl/fl RCE marrow (H) or with Oxsr1 +/+ Stk39 +/+ RCE or Oxsr1 fl/fl Stk39 T243A/T243A RCE marrow (J), probed with antibodies to OXSR1 and α-TUBULIN. Graph (bottom) shows mean±SEM amount of OXSR1 in the lanes above, normalized to the abundance of α-TUBULIN in each lane. (I, K) Mean±SEM numbers of mature B cells in the spleen (B220 + CD19 + CD93 - ) of RAG1-deficient mice reconstituted with Oxsr1 +/+ RCE or Oxsr1 fl/fl RCE marrow (I), or with Oxsr1 +/+ Stk39 +/+ RCE or Oxsr1 fl/fl Stk39 T243A/T243A RCE marrow (K), as described in G. Mann-Whitney test (B, C, F, H-K); Two-way ANOVA (E), Mann-Whitney test (F, H-K); * 0.01 < P < 0.05, ** 0.001 < P < 0.01; *** 0.0001 < P < 0.001, **** P < 0.0001. Sample sizes: 6 (B, D, E), 5 (C, H), 7 (F, K), 3-4 (I), 12 (J). Data are pooled from 2 independent experiments.

Article Snippet: Where indicated, the cells were stimulated with either 1 µg/ml recombinant murine CXCL13 (CXCL13, R&D Systems; Biotechne), 10 µg/ml AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 10 µg/ml biotin-SP AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (biotinylated anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml recombinant murine CD40L (CD40L, R&D Systems; Biotechne) or 10 µg/ml LPS from Salmonella minnesota R595 (LPS, Enzo Life Sciences, Inc.).

Techniques: Irradiation, Expressing, Binding Assay, Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: B cell-intrinsic requirement for WNK1 kinase in T cell-dependent antibody responses

doi: 10.1101/2021.09.09.459588

Figure Lengend Snippet: (A-H) Top; immunoblots of cell lysates from mouse B cells stimulated for the indicated times with anti-IgM (A-C, G) or CXCL13 (D-F, H) using WNK1-deficient or control B cells (A, D), B cells expressing kinase-inactive WNK1-D368A or control B cells (B, E), or wild-type B cells treated with vehicle (DMSO), an inhibitor of WNK-family kinases (WNK463) (C, F), a PI3K inhibitor (PI-103) or an AKT inhibitor (MK2206) (G, H), probed with antibodies to phosphorylated OXSR1 (p-OXSR1), α-TUBULIN, phosphorylated AKT (p-AKT), phosphorylated PRAS40 (p-PRAS40) or ERK2. Below; mean±SEM abundance of p-OXSR1, p-AKT and p-PRAS40 in the lanes above, normalized to the α-TUBULIN or ERK2. Mann-Whitney test; * 0.01 < P < 0.05, ** 0.001 < P < 0.01, *** 0.0001 < P < 0.001. Sample sizes: 4 (A, H), 5 (B, D, E, G), 7 (C, F). Data are pooled from two (A, B, D, E, G, H) or three (C, F) independent experiments.

Article Snippet: Where indicated, the cells were stimulated with either 1 µg/ml recombinant murine CXCL13 (CXCL13, R&D Systems; Biotechne), 10 µg/ml AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 10 µg/ml biotin-SP AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (biotinylated anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml recombinant murine CD40L (CD40L, R&D Systems; Biotechne) or 10 µg/ml LPS from Salmonella minnesota R595 (LPS, Enzo Life Sciences, Inc.).

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: B cell-intrinsic requirement for WNK1 kinase in T cell-dependent antibody responses

doi: 10.1101/2021.09.09.459588

Figure Lengend Snippet: (A-D) Mean±SEM binding of soluble ICAM-1 complexes to mouse B cells from control B cells and either WNK1-deficient B cells (A), B cells expressing kinase-inactive WNK1-D368A (B), OXSR1-deficient B cells (C) or OXSR1-deficient B cells expressing a non-phosphorylatable mutant of STK39-T243A (D), stimulated with anti-IgM or CXCL13 or treated with MnCl 2 for the indicated times. (E) Migration of control or WNK1-deficient mouse B cells from the top to the bottom chamber of a Transwell plate in response to CXCL13. (F, G) Violin plots showing mean speed (F) and displacement (G) of control and WNK1-deficient mouse B cells migrating in response to CXCL13. Dashed lines indicate median (red) and 25 th and 75 th percentiles (grey). Two-Way ANOVA (A-D), Mann-Whitney test (E-G); * 0.01 < P < 0.05, ** 0.001 < P < 0.01, *** 0.0001 < P < 0.001, **** P < 0.0001. Sample sizes: 11 (A), 6 (B), 5 (C), 9 (D), 4 mutant and 5 control (E), 5245 mutant cells and 7289 control cells (F, G), Data pooled from two independent experiments.

Article Snippet: Where indicated, the cells were stimulated with either 1 µg/ml recombinant murine CXCL13 (CXCL13, R&D Systems; Biotechne), 10 µg/ml AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 10 µg/ml biotin-SP AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (biotinylated anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml recombinant murine CD40L (CD40L, R&D Systems; Biotechne) or 10 µg/ml LPS from Salmonella minnesota R595 (LPS, Enzo Life Sciences, Inc.).

Techniques: Binding Assay, Expressing, Mutagenesis, Migration, MANN-WHITNEY