Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemical and Biomolecular Engineering

First Advisor

Daniel A. Hammer

Abstract

In order to perform an effective immune response, leukocytes must be able to exit the vasculature and enter the interstitial space. The leukocyte adhesion cascade has evolved to slow and stop cells to allow this access. Despite extensive molecular characterization, there are still significant questions regarding the biophysical constraints of the cascade. In this thesis, we explore the requirements for cells to physically complete the adhesion cascade. In the first aim, we confirm previously published predictions regarding the synergy between E-selectin and ICAM-1, showing that a consistent level of leukocyte adhesion can be maintained using varying ratios of the two molecules. We also show that T cells require O(10^0) sites/µm^2 to support tethering, O(10^1) sites/µm^2 to support rolling, and O(10^2) sites/µm^2 to cause arrest. In addition, we characterized the migration of T cells against the direction of flow. We discovered that cells determine their direction of migration within 30 seconds of arrest. We also found that cells migrating upstream transmigrate across a HUVEC monolayer faster than cells crawling downstream. In the second aim, we determined that cells attached to a surface through a series of linkages show a non-linear decrease in the critical detachment force as the number of linkages increases. We also showed that the intrinsic off rate of the linkages can control the critical force, while the spring constant of the linkages causes less of a change. Finally, in the third aim we used simulations to quantitatively predict the effect of depletion of kindlin-3 on cellular adhesion. We predicted that adhesion would be hypersensitive to kindlin-3 expression, requiring a reduction to below 20% of normal expression levels to see an effect. We also predicted that rolling velocity would be independent of kindlin-3 expression, while both time and distance to stop would increase with decreasing kindlin-3 expression. Experiments using the Jurkat T cell line supported these predictions, with a significant decrease in cell adhesion, no change in rolling velocity, and an increase in the time to stop. Together, these aims suggest that we now have the knowledge to improve leukocyte targeting through engineering the leukocyte adhesion cascade.

Additional Files

Supplemental_table__m6A_peaks.xls (690 kB)
TableS2_GO_terms.xlsx (107 kB)

Share

COinS