SUMMARY

Cells use their surface receptors to anchor to and communicate with other cells and the extracellular matrix. Such processes usually involve interactions of multiple receptor-ligand pairs occurring independently, cooperatively, or antagonistically. The study of crosstalk among different receptor species can be benefitted by techniques that are efficient, high throughput, and easy to use. Conventional approaches have difficulties to control each ligand precisely or detangle measurement of cell adhesion by each receptor-ligand pair. This thesis describes the development of two platforms to address this deficiency and their applications to several problems in cellular engineering. The first platform of an auto-alignment protein patterning approach enables a sequential ligand presentation. This allows moving cells to interact with spatially separated ligands sequentially, thus dissecting the crosstalk between different receptors by detangling temporally their ligand interactions and reporting the effect of triggering one receptor by the altered binding of another receptor. This multi-zone system is applied to provide independent evidence for the presence of an intermediate state of the major integrins on platelets and leukocytes. An intermediate state of integrin αIIbβ3 was demonstrated by triggering GPIb with VWF-A1 immobilized on one zone and measuring platelet adhesion to fibronectin or conformation reporter antibodies coated on another zone. Furthermore, the increased binding of integrin αIIbβ3 upregulated by VWF-A1 was found in diabetic platelets. The assay required only one-drop of blood, thus potentially applicable for diabetic state monitoring upon further development. The intermediate state of integrin αLβ2 (LFA-1) was similarly studied on human neutrophils and mouse T lymphocytes. The effects of “dose” were revealed through precise control of the length and ligand density of the stimulus zone and the perfusion flowrate. The second system is to sort antigen-specific T cells based on TCR triggering studied earlier using the multi-zone device. The effective interaction of TCR-pMHC can lead to an activation of LFA-1, whose differential ICAM-1 binding amplifies the difference in pMHC binding to T cells with different T cell receptor clones. The sorting chip first triggers T cells by specific pMHC and then allows activated T cells to move laterally on a patterned ICAM-1 surface to separate cells with activated LFA-1 supported rolling. Overall, this thesis developed an approach for studying cell multi-receptor crosstalk generalizable to various receptor mechanobiology and applied to mechanistic studies to potential clinical applications, revealing the advantages of microfluidics in disease studies and diagnostics. In addition, the platform for functional antigen-specific T cell sorting should be valuable in a variety of clinical settings for diagnoses and functional immunotherapies.