Cell adhesion is a critical requirement for most immune functions. Immune cells don't spontaneously adhere to the vascular endothelium or to each other because there are essentially no active or simultaneously expressed receptor-ligand pairs present on resting cells. The expression of some members of the important integrin superfamily of adhesion molecules is increased under selected circumstances; however integrin-mediated adhesion is primarily regulated by the reversible adoption of an adhesive state, which in selected cases has been shown to be a high affinity state. The beta2 integrins are the leukocyte-specific integrins and the mechanisms governing the regulation of integrin avidity are not well-understood. Using Jurkat T lymphoma cells, which express LFA- 1 as their sole beta2 integrin, we have developed a method to create mutant cell clones bearing LFA- 1 that is locked in either a low avidity or a high avidity state. The LFA-1 on these cells is wild-type by several criteria; hence the mutations must be in associated molecules that participate in LFA-1 regulation. Additional preliminary experiments that employed retroviral insertional mutagenesis have resulted in several different clones with mutant phenotypes linked to mutations in particular genes. Sequence analysis of DNA fragments adjacent to the retrovirus insertion sites from some of these clones and searches of the Human Genome Project databases have allowed preliminary identification of some of these genes.
In Aim 1 of this application, we propose to generate additional mutant clones with different lymphoma cells, as there appear to be preferred mutations in a particular cell line.
In Aim 2, we will continue studies of the J-101.3 and J+hil.19 clones, which bear mutations that will lead to insights into the role of the GFFKR sequence in the alpha subunit cytoplasmic domain, a nearly invariant sequence among all integrins that has been shown to be involved in avidity regulation. We will also perform biochemical and cell biological studies to investigate the mutations in the new cell clones from Aim 1.
In Aim 3, we will continue our analysis of the existing mutant cell clones created by retroviral insertion, including the identification and characterization of the gene on chromosome 20 that is involved in LFA-1 regulation. A further understanding of how lymphocytes regulate adhesion will lead to new strategies for the development of anti-inflammatory therapies with potential applications in autoimmune disease and transplantation medicine.
