Migratory responses of cells to cellular guiding signals play important roles in regulating a wide range of physiological and pathological processes such as inflammation and autoimmune diseases, wound healing, neuron guidance, embryogenesis, and cancer metastasis 
, . In particular, chemoattractant gradients guide the migration of immune cells (i.e. chemotaxis), orchestrating cell trafficking and positioning in tissues 
. It has been shown that leukocytes express multiple different chemoattractant receptors in a cell subset dependent manner, and can integrate multiple co-existing chemotactic signals to direct their migration to specific targets in tissues that enable immune surveillance and immune responses 
. Such a multiple chemoattractants-based guiding mechanism relies on chemotactic signaling transduction through chemoattractant and their different specific cell surface receptors. In contrast, some chemoattractants share a common receptor for triggering chemotactic signaling such as chemokines CCL19 and CCL21 and their shared receptor CCR7 expressed in lymphocytes subsets and dendritic cells (DCs) 
. However, the mechanism of multiple chemoattractants with a common cell receptor for guiding cell migration is unclear.
Chemokine receptor CCR7 and its two ligands, chemokine CCL19 and CCL21, are important players in regulating lymphocytes and DCs trafficking in secondary lymphoid tissues (SLT) such as lymph nodes (LNs) 
. CCL19 and CCL21 are co-expressed in LNs with different expression patterns. CCL19 is only produced and presented in T cell zone (TCZ) in humans and mouse LNs 
. In contrast, CCL21 is produced in TCZ and is transcytosed to high endothelial venules (HEV) in human 
, and is produced and presented in both TCZ and HEV in mouse LNs 
. Inside TCZ, it has been shown that CCL19 and CCL21 are co-expressed by reticular cells with more CCL21-expressing cells than CCL19-expressing cells in the periphery of TCZ 
, suggesting the size of the CCL21 producing tissue in TCZ is possibly larger than the CCL19 producing tissue. Therefore, the profiles of overlapping CCL19 and CCL21 fields can be different in different sub-regions of TCZ. Furthermore, the production levels of CCL19 and CCL21 in SLT are significantly different with up to 100-fold higher of CCL21 production than CCL19 
but the exact difference is not defined in sub-regions. In addition, CCL19 only exhibits soluble patterns in SLT whereas CCL21 is found in both soluble and immobilized forms 
. At the cellular level, CCL19 and CCL21 have similar binding affinity with CCR7 and they are similar in inducing calcium immobilization and G protein activation 
. However, only CCL19 but not CCL21 robustly desensitizes and internalizes CCR7 
. Although both CCL19 and CCL21 are potent chemoattractants for T cells as shown using in-vitro
chemotaxis assays, their distinct roles in regulating T cell trafficking in SLT remain unclear. It has been shown that CCL21 but not CCL19 is required for T cells and DCs recruitment to SLT using CCL19/21 deficient mice and CCL19 deficient mice 
. This finding together with the significantly lower production level of CCL19 further complicates the role of CCL19 in lymphocytes and DCs trafficking in SLT. Altogether, the differential expression patterns of CCL19 and CCL21 in SLT and their differential ability for desensitizing CCR7 and for recruiting T cells and DCs to SLT present a complex and unclear picture of CCR7 ligands guided T cell migration and trafficking in SLT.
In the present study, we hypothesize that the different profiles of co-existing CCL19 and CCL21 fields in sub-regions of LNs together with the differential ability of CCL19 and CCL21 for desensitizing CCR7 provide a mechanism for fine tuning T cell trafficking in LNs. We employed a microfluidics-based approach to quantitatively analyze T cell migration in-vitro in different configurations of co-existing CCL19 and CCL21 fields that mimic the physiological conditions in different regions of LNs and the results are explained by mathematical modeling and computer simulations. The experimental and modeling results allow us to formulate a possible combinatorial guiding mechanism by co-existing CCL19 and CCL21 gradient fields for T cell migration and trafficking.