Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, fine-tuning cellular behaviours in complex tissues. Using the exemplar chemokine CXCL13, a combination of computational and experimental approaches were used to study the spatial distribution of CXCL13 in lymphoid tissues, showing CXCL13 has the capacity to form both soluble and immobilized gradients. Specifically, we show that CXCL13+ follicular stromal cells form a small-world network of guidance structures. Multi-scale computational simulations of B cell migration and behaviours was used to understand CXCL13 gradients, application of genetic evolutionary algorithms (GEAs) has predict that immobilized gradients created by the FDC network is configured to promote B-cell antigen scanning and FRC network B cell migration. Consistent with this prediction, imaging analysis shows that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable cleavage product, CXCL13, with increased chemotactic potency and reduced affinity for ECM components. Our data suggests that CXCL13 can form both soluble and immobilized gradients, providing an insight into how chemotactic landscapes are shaped by the stromal cell microenvironment. By combining multi-scale models, artificial neural networks and GEAs with experimentation has permitted new insights into immune structure and function.