Cxcl13

Manufactured by R&D Systems

Sourced in United States, Austria, United Kingdom, Germany

CXCL13 is a chemokine protein that plays a role in the migration and positioning of B cells. It is involved in the organization of lymphoid follicles and the recruitment of B cells to these structures.

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62 protocols using cxcl13

Immunophenotyping of Lymphoid Tumors

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Tissue samples were fixed in 10% formalin and embedded in paraffin. Tissue sections (5‐μm‐thick) were stained with hematoxylin and eosin. Immunoperoxidase studies were performed on formalin‐fixed paraffin‐embedded sections. The following monoclonal antibodies were used: CD3, CD8, CD10, CD30, BCL6 (DAKO), CD4, perforin (Novocastra Laboratories), PD1 (Abcam), CXCL13 (R&D systems), TIA‐1 (Coulter Immunology), granzymeB (Mososan), CD23 (Nichirei), and CD56 (eBioscience). Antibodies were applied after heating specimens in a microwave oven for antigen retrieval. Tissue samples were considered positive when more than 30% of the tumor cells were positive.

Eladl A.E., Shimada K., Suzuki Y., Takahara T., Kato S., Kohno K., Elsayed A.A., Wu C., Tokunaga T., Kinoshita T., Sakata‐Yanagimoto M., Nakamura S, & Satou A. (2019). EBV status has prognostic implication among young patients with angioimmunoblastic T‐cell lymphoma. Cancer Medicine, 9(2), 678-688.

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Multiplex cytokine and antibody quantification

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Cytokines were quantified in the supernatants using ELISA for IL-21 (BioLegend) and CXCL13 (R&D Systems) or CBA flex set for IL-3, IL-4, IL-5, IL-10, IL-13, IL-17A, TNF, and IFN-γ (BD), following the manufacturer’s protocol. Total human IgG, IgE, IgG4, and IgM were quantified using the Human IgGs Flex Sets (BD).

Pattarini L., Trichot C., Bogiatzi S., Grandclaudon M., Meller S., Keuylian Z., Durand M., Volpe E., Madonna S., Cavani A., Chiricozzi A., Romanelli M., Hori T., Hovnanian A., Homey B, & Soumelis V. (2017). TSLP-activated dendritic cells induce human T follicular helper cell differentiation through OX40-ligand. The Journal of Experimental Medicine, 214(5), 1529-1546.

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Signaling Pathways in Stimulated B Cells

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Purified B cells were rested in DMEM containing 1% charcoal stripped FCS, antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin), 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol for 30 min at 37°C/5% CO₂ before stimulation for various durations with 1μg/ml CXCL13 (R&D System or PeproTech) or 1 μM S1P (Sigma or Cayman) for 2, 5, 10, 30, or 60 min. The labeling of dead cells, fixation, and permeabilization were performed as described in the manufacturer’s protocol. Following permeabilization, the cells were immunostained with anti-B220, anti-IgD, anti-IgM, CD21, CD23, and CD93 for 30 minutes at 4° C. To detect phosphorylated signaling molecules antibodies against phospho-Akt Alexa Fluor 647 (D9E), phospho-Erk (197G) Alexa Fluor 647, and phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (pERM), all from Cell Signaling Technology, were used. Isotype control staining was performed using rabbit IgG isotype mAb Alexa Fluor 647 (DA1E; Cell Signaling Technology). Secondary F(ab’)₂ fragment of goat anti-Rabbit IgG(H+L) Alexa Fluor 647 (ThermoFisher) was used to detect the pERM antibodies. After washing the cells were resuspended in 250 μL 1% BSA/PBS and filtered prior to acquisition on a FACS Canto II flow cytometer (BD Biosciences).

Hwang I.Y., Park C., Harrison K, & Kehrl J.H. (2019). Biased S1PR1 Signaling in B cells Subverts Responses to Homeostatic Chemokines Severely Disorganizing Lymphoid Organ Architecture. Journal of immunology (Baltimore, Md. : 1950), 203(9), 2401-2414.

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In vitro Chemotaxis Assay for Immune Cells

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CCL21 and CXCL13 were purchased from R&D Systems. S1P was purchased from Sigma. In vitro migration assay was conducted using 96-well chemoTx chamber with 5-μm pore inserts (Neuroprobe) as described previously^{71 (link)}. In brief, single cells prepared from mouse spleen or lymph nodes were suspended at 8 × 10⁶/ml in RPMI 1640 containing 1 mg/ml BSA and 20 mM HEPES (pH 7.4), and applied to upper wells (25 μl/well). The same medium without or with CCL21, CXCL13, or S1P at indicated concentrations was applied to lower wells (29 μl/well). After 1 h at 37 °C, the content of each lower well was transferred to a polypropylene pointed-bottom tube. The cells were pelleted by centrifugation at 200 × g for 5 min, resuspended in 0.1% BSA in PBS, and stained with APC-Cy7-labeled anti-mouse CD4, PerCP-Cy5.5-labeled anti-mouse CD8, and FITC-labeled anti-mouse CD19. After washing, cells were analyzed on a FACSFortessa (BD Biosciences) and analyzed with FlowJo (FlowJo, LLC). All assays were done in duplicate.

Mashud R., Nomachi A., Hayakawa A., Kubouchi K., Danno S., Hirata T., Matsuo K., Nakayama T., Satoh R., Sugiura R., Abe M., Sakimura K., Wakana S., Ohsaki H., Kamoshida S, & Mukai H. (2017). Impaired lymphocyte trafficking in mice deficient in the kinase activity of PKN1. Scientific Reports, 7, 7663.

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Chemotaxis Assay for Cell Migration

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Cells were suspended in RPMI at 5×10⁶cells/mL, and 100 μl was placed in the upper chamber of a 24-transwell plate with a 5μm filter. Chambers were placed into wells containing media containing no chemokine (control), recombinant human CXCL12 (200ng/mL, Millipore) or CXCL13 (1000ng/mL, R&D systems). Migration was permitted for 3 hours, and cells in the lower chamber were collected and counted for 20 seconds on high speed on a Beckman Coulter FC500 flow cytometer. A 1/20 dilution of input cells was also determined.

Miller C.R., Ruppert A.S., Fobare S., Chen T.L., Liu C., Lehman A., Blachly J.S., Zhang X., Lucas D.M., Grever M.R., Tallman M.S., Flinn I.W., Rassenti L.Z., Kipps T.J., Sampath D., Coombes K.R, & Hertlein E.K. (2017). The long noncoding RNA, treRNA, decreases DNA damage and is associated with poor response to chemotherapy in chronic lymphocytic leukemia. Oncotarget, 8(16), 25942-25954.

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Quantifying Immunoglobulins and Biomarkers

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An ELISA quantitative kit for the detection of human IgG and IgM (Bethyl Laboratories, Montgomery, TX) was used to measure the concentrations of total IgG and IgM from culture supernatants and plasma. The ELISA kits of human fatty acid binding protein 2 (FABP2, Cat#: DY3078) and CXCL13 (Cat#: DCX130) were purchased from R&D System Inc (Minneapolis, MN, USA), and were used measure their concentration in the plasma from LC and HC subjects.

Zhao J., Shi J., Qu M., Zhao X., Wang H., Huang M., Liu Z., Li Z., He Q., Zhang S, & Zhang Z. (2019). Hyperactive Follicular Helper T Cells Contribute to Dysregulated Humoral Immunity in Patients With Liver Cirrhosis. Frontiers in Immunology, 10, 1915.

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Cytokine Profiling of Serum and Plasma

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Serum or plasma samples were immediately separated and kept at − 80 °C for subsequent cytokine analysis. Concentration of IL-6, IL-18, IL-17 (high sensitivity kit), IL-21, IL-22 (Affymetrix eBioscience, Vienna, Austria), IL-15, CCL20, CXCL13 and CX3CL1 (R&D Systems, Minneapolis, MN, USA) were measure by ELISA according to the manufacturer’s instructions.

Mangas-Losada A., García-García R., Leone P., Ballester M.P., Cabrera-Pastor A., Urios A., Gallego J.J., Martínez-Pretel J.J., Giménez-Garzó C., Revert F., Escudero-García D., Tosca J., Ríos M.P., Montón C., Durbán L., Aparicio L., Montoliu C, & Felipo V. (2019). Selective improvement by rifaximin of changes in the immunophenotype in patients who improve minimal hepatic encephalopathy. Journal of Translational Medicine, 17, 293.

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Chemotaxis Assay for Lymphocyte Migration

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Protocol was adapted from previously published procedures2, 16 as follows. Splenocytes were made into a single‐cell suspension by passing through a 0·7‐μm cell strainer (ThermoFisher Scientific) and red cell lysed. Adhesive cells were removed by incubating cells in RPMI‐1640 with 5% fetal calf serum for 30 min. Non‐adherent cells were collected and rested on ice in migration medium (RPMI‐1640 with 0·1% BSA) for 1 hr. After resting equal numbers of cells from three young or aged mice were pooled and 1 × 10⁶ cells were placed into the upper chamber of 3 μm Transwells in a 24‐well plate (Corning, Corning, NY). The lower chamber contained either 10, 100 or 500 nm S1P (Sigma‐Aldrich), 500 ng/ml, 1 μg/ml or 3 μg/ml CXCL13 (R&D Systems), or media alone. Three technical replicates were performed. Cells were incubated at 37° for 4 hr before the upper chamber was discarded and cells in the lower chamber were collected and stained for flow cytometric analysis. For relative cell counts, cells were resuspended in 200 μl staining buffer and acquired for 45 seconds at medium speed on an LSR Fortessa. Migration was then calculated as a percentage of input cells.

Turner V.M, & Mabbott N.A. (2017). Ageing adversely affects the migration and function of marginal zone B cells. Immunology, 151(3), 349-362.

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Quantification of BAFF, CXCL13, and sCD23

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B cell activating factor (BAFF) (Bender MedSystems, Austria), B-lymphocyte chemoattractant (BLC) also known as C-X-C motif chemokine 13 (CXCL13) and the soluble form of CD23 (sCD23) (R&D systems, United Kingdom) were quantified in serum samples from all groups by ELISA according to the manufacturer’s instructions. Samples were analyzed using plate reader Infinite M200 (Tecan, Switzerland).

Moura R.A., Quaresma C., Vieira A.R., Gonçalves M.J., Polido-Pereira J., Romão V.C., Martins N., Canhão H, & Fonseca J.E. (2017). B-cell phenotype and IgD-CD27- memory B cells are affected by TNF-inhibitors and tocilizumab treatment in rheumatoid arthritis. PLoS ONE, 12(9), e0182927.

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Transwell Migration Assay of T Cell Subsets

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Enriched CD4⁺ T cells were washed and resuspended in migration medium (1640 RPMI with 0.5% fetal bovine albumin) at 1 × 10⁶ cells/ml. 2 × 10⁵ cells were loaded in a 3 μm pore transwell (BD Falcon), with 800 μl of migration medium placed in lower chambers. CCL19, CCL21, CXCL13 (all R&D Systems) and oxysterol (Avanti® Polar Lipids INC, Alabaster, Alabama) were added at a concentration of 1 μg/ml (CCL19, CCL21, CXCL13) or 5 μM (oxysterol) individually or collectively (Fig. 6). Cells were allowed to migrate at 37°C for 3 hours, followed by their collection from the lower chamber were collected and staining to quantify numbers of CD45RA⁺, Tfh, and PSGL-1^hi PD-1^hi CXCR5^hi T cell populations. In some experiments, enriched CD4⁺ T cells were stained with either anti-CCR7 monoclonal antibody (16 μg/ml, R&D Systems) or anti-PSGL-1 biotin (16 μg/ml, Ancell) for 40 minutes either individually or collectively prior to migration. Before samples were loaded into for flow cytometry, the same numbers of beads (Spherotech^INC) were added into each tube to standardize cell numbers between tubes. Events were analyzed with FlowJo software.

Kim S.T., Choi J.Y., Lainez B., Schulz V.P., Karas D.E., Baum E.D., Setlur J., Gallagher P.G, & Craft J. (2018). Human Extrafollicular CD4+ T Helper Cells Help Memory B Cells Produce Immunoglobulins. Journal of immunology (Baltimore, Md. : 1950), 201(5), 1359-1372.

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