Immune cells are involved in a variety of cardiovascular diseases, including atherosclerosis, cardiac remodeling following myocardial infarction, and blood vessel inflammation in vasculitis. Although immune cells contained in blood vessel walls may be analyzed using flow cytometry, these cells must be separated in order to use them for adoptive transfer and immune reconstitution experiments. Current methodologies do not separate cells of interest with sufficient efficiency, viability, and purity. This is a barrer to further research. Immune cells of interest in the context of atherosclerosis are rare (approximately 3000 per mouse aorta) and therefore difficult to separate from the milieu of an atherosclerotic lesion. The work proposed here is aimed at designing a novel technique for identifying, selecting, and isolating high- value cell targets. Atherosclerosis is associated with n immune response to ApoB-100, the main lipoprotein in low density lipoprotein (LDL). Specifically, the targets of interest are T cells specific to self-peptides-SQEYSGSVANEANVY and TGAYSNASSTESASY, from ApoB-100- that may modulate atherosclerosis. Successful work involving the use of these peptides in an atheroprotective vaccination strategy motivates detailed study of these antigen-specific T cells. To accomplish separation of these cells, I propose to couple gas-filled lipid microbubbles to class II major histocompatibility complex (MHC II) multimers binding one of the antigenic peptides. After engagement of reactive T-cell receptors (TCR) on target cells to their cognate MHC-II/peptide on the multimer/microbubble complex, centrifuged buoyant microbubbles function as a positive selection mechanism to separate these cells. This project involves the design and validation of microbubble-based buoyant cell separation. Its intention is to solve the problems of low purity, viability, and yieldof current cell separation techniques. These problems present a critical barrier to progress in the field of atherosclerosis. The new technology developed through this project will be a useful platform for future cardiovascular disease research.
The focus of this proposal is to design a method to separate immune cells that are relevant to atherosclerosis disease progression. The project addresses unmet needs in cardiovascular disease research. It can be adapted to further biomedical science research in other fields as well.