Next biotinylated anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG were used at 2?g/ml and applied for 30?min at room temperature. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients. and (Supplementary Table?1). The small-world configuration is characterized by an overabundance of highly connected nodes, common connections mediating the short mean-path lengths. This property is associated with rapid information transfer and is also observed in airline routes and social networks33,34. In the context of the follicle, this property is likely to promote complement-mediated trafficking of antigen by non-cognate B cells from the subcapsular sinus to the FDC network, and also Silvestrol aglycone the migration of cognate B cells as they search for antigen within the follicle, and then present it to T cells at the interfollicular border before seeding a GC reaction5,35,36. Open in a separate window Fig. 1 The topological network properties of CXCL13+ follicular stromal cells.a Mapping confocal images of Rabbit Polyclonal to PLD2 lymph node follicles taken from Cxcl13-cre/EYFP reporter mice using the Imaris image analysis software. The FDC subnetwork is highlighted in yellow and the RC subnetwork in cyan. Distributions of Silvestrol aglycone degree centrality, edge length and local clustering coefficient are indicated for the FDC and RC subnetworks (b?d). e Distribution of shortest path lengths is indicated for the global follicular network and are compared to that of an equivalent random network with the same number of nodes and edges (f). Data represent mean??SD for value? ?0.001) and so significance was assessed using a Mann?Whitney test (value? ?0.001; ***). Data shown are from a single experiment (from a total of two independent experiments) with each data point representing a distinct follicle obtained from a single patient. c Quantification of CXCL13AF647 mobility in CD19+-positive regions of human tonsil sections. Diffusion measured in untreated tissue sections is indicated in red with values obtained for heparinase II-treated sections indicated in blue. All tissue sections were obtained from the same patient. The median [IQR] diffusion rate of CXCL13AF647 in untreated sections was calculated as 0.19 [0.001?0.79]?m2?s?1, while treatment with heparinase-II led to a significantly different (assessed using the Mann?Whitney test) diffusion coefficient of 1.6 [0.47?3.9]?m2?s?1 (test (value?=?0.06 for model 1 and infection42, is upregulated in many cancers43, and can be produced in extracellular form in cytokine-stimulated fibroblasts taken from rheumatoid arthritis patients44. Incubation of CXCL13 with Cath-B yielded two cleavage products with masses of 9.03 and 8.68?kDa, respectively (Fig.?5a). The smaller product is stable and forms across a range of enzyme substrate ratios in both humans and mice (Supplementary Fig.?4a) and is detected at pH values between 4.0 and 7.2 with an optimal turnover rate between pH 5.0 and 6.5 (Supplementary Fig.?4b). Consistent with these data, single-molecule imaging of CXCL13[1C72] diffusion in 15% Ficoll showed a higher mobility rate for the Cath-B-treated form of the molecule as compared to untreated (1.0 [0.04?3.6]?m2?s?1 and 0.61 [0.08?2.2]?m2?s?1 respectively, -dependent fluorescence changes in fura-2 loaded CXCR5-transfected Pre-B 300-19 cells induced by 30?nM CXCL13 or CXCL13[1C72]. e Dose response of calcium mobilization elicited by CXCL13 and CXCL13[1C72]. Relative units (mean??SD) were calculated as described in Methods. f CXCR5 surface expression after incubation of CXCR5-transfected Pre-B 300-19 cells with CXCL13 and CXCL13[1C72]. CXCR5 expression levels were quantified by flow cytometry analysis. Data (mean??SD) from at least four independent experiments show the percentage of surface CXCR5 Silvestrol aglycone compared to control. g Primary human B-cell migration in response to intact and truncated CXCL13 was evaluated using 5?m pore size Transwell filters. Data represent the percentage of migrated cells relative to the number of cells added to the Transwell filters. Values (mean??SD) represent at least.