Cell adhesion plays a fundamental role in controlling normal cellular behavior. Integrins are a large family of adhesion molecules that play an important role in mediating lymphocyte interactions with other cells and with components of the extracellular matrix. The distinct array of integrins expressed on the surface of a cell at any given time dictate the spectrum of ECM and cell surface ligands with which the cell can interact. Changes in integrin expression can dramatically affect lymphocyte recirculation and activation. Unlike most cell types in the body that continuously stay in a distinct anatomical location, cells of the immune system require the ability to alternate between adhesive and nonadhesive states in order to function properly. Normal lymphocyte recirculation requires that cells be in a relatively nonadherent state so that rapid movement throughout the vascular and lymphatic systems is not impeded. Movement of cells into peripheral lymphoid organs and other tissues, where antigen recognition occurs, involves a multi-step adhesion process that allows circulating cells to slow down and extravasate through the vascular endothelium. This thesis addresses two important mechanisms that influence lymphocyte adhesion: (1) regulation of cell surface β1 integrin expression and (2) activation-dependent regulation of β1 integrin function. Results using normal Jurkat T cells and α4-deficient Jurkat cells have suggested potential mechanisms that might be used by lymphocytes to regulate cell surface expression of β1 integrins. These studies indicate that expression of individual α subunits may be regulated differently in lymphocytes. Our results suggest a role for both post-transcriptional and post-translation control of α2 subunit protein expression and a role for post-transcriptional regulation of the α5 subunit. In addition, re-expression experiments suggest that the presence of α4 protein may influence cell surface expression of other a subunits. Studies with HL60 transfectants expressing CD2/28 chimeric receptors have implicated phosphatidylinositol 3-kinase (PI 3-K) as playing an important role in the ability of CD28 to regulate integrin-mediated adhesion. However, substitution of the CD28 SH2-binding motif YMNM with the YVKM motif from CTLA-4 indicated that recruitment and activation of PI 3-K is not a sufficient signal to upregulate β1 integrin-mediated adhesion since this receptor could still mediate PI 3-K activation but not integrin upregulation. Advisors/Committee Members: Shimizu, Yoji (advisor), Koomey, J. Michael (advisor).

In chickens, B cells undergo part of their development in a unique organ, the bursa of Fabricius. The exact stages of B cell development that occur in the bursa and how these stages are regulated have only been partly defined. Diversification of chicken immunoglobulin (Ig) genes has been shown to occur by intrachromosomal gene conversion during B cell development in the bursa of Fabricius. Based on V(D)J recombination studies, B cell progenitors arise at extrabursal sites early in ontogeny and must then migrate through the vasculature to the bursa. We have determined that the carbohydrate structure sialyl Lewis ^X, previously shown to be a ligand for the selectin family of adhesion molecules, is developmentally regulated in a tissue-specific manner during chicken B cell development. Sialyl Lewis ^X expression appears to facilitate the migration of B cell progenitors to the bursa by serving as a ligand for a lectin-like adhesion receptor. Changes in glycosylation phenotype during development have also allowed us to distinguish three populations of B cells at distinct levels of maturation in the bursa. The initial B cell progenitors that migrate to the bursa and undergo proliferative expansion without undergoing gene conversion express the carbohydrate epitope sialyl Lewis ^X. During embryogenesis, bursal B cells undergo a developmental switch in surface glycosylation resulting in the loss of sialyl Lewis ^X expression and the acquisition of the related carbohydrate structure Lewis ^X. Expression of Lewis ^X identifies bursal lymphocytes that have begun to undergo gene conversion. Finally, bursal cells appear to undergo further differentiation after completing gene conversion and down-regulate Lewis ^X expression prior to emigrating from the bursa. Cells undergoing gene conversion also selectively express the recombinase activating gene RAG-2. Therefore, we examined the potential role of RAG-2 in gene conversion by gene disruption in a B cell line, DT40, that undergoes gene conversion in culture. Subclones of DT40 in which both copies of the RAG-2 coding regions are deleted undergo gene conversion at similar rates as wild type cells, demonstrating that the RAG-2 product is not absolutely required for gene conversion. Advisors/Committee Members: Thompson, Craig B. (advisor), Koomey, J. Michael (advisor).