Lymphocyte migration is crucial for adaptive immunity. CCR7 and its ligands mediate the migration and positioning of T cells in lymph nodes but the underlying mechanism is complex. The research in this thesis investigated CCR7-mediated T cell migration using a microfluidics-based approach. A microfluidic method suitable for quantitative migration analysis of genetically modified lymphocyte transfectants was developed. Using this method, I demonstrated chemotaxis of Jurkat transfectants expressing wild-type or C-terminal mutated CCR7 to a CCL19 gradient, and characterized the difference in transfectant migration mediated by wild-type and mutant CCR7. The fluorescent tag allows identification of CCR7-expressing transfectants in cell migration analysis, and microscopy assessment of CCR7 dynamics in migrating cells. Furthermore, my results also showed interesting migratory behaviours of CCR7 Jurkat transfectants in a specific co-existing CCL19 and CCL21 fields. This developed method will be broadly useful for studying cell migration signalling.

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. Inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 115-126).

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [78]-93).

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed2009/07/02 ). Includes bibliographical references.

Thesis (M.Med.Sc.)--University of Hong Kong, 2003. / Includes bibliographical references (leaves 84-92). Also available in print.