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Diabetes and other autoimmune endocrine diseases
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Hua Yu, Ph.D.
Yale University
Jesse Collins
Yale University
Maria Bettini, Ph.D.
Baylor College of Medicine
Xiaoli Wang, Ph.D.
Washington University in St.Louis
Daved Fremont, Ph.D.
Washington University in St.Louis
Peter Cresswell, Ph.D.
Yale University
David Spiegel, Ph.D., M.D.
Yale University
Kevan Herold, MD
Professor
Yale Univeristy
Previous clinical trials using biologics-based broad-spectrum T cell- and B cell-depleting molecules for the treatment of autoimmune diabetes have shown promising, yet mixed, results. Their varied extent of success may be due to their non-specific action and failure to permanently and completely remove the pathogenic subpopulations.
As CD8+ T cells, the most dominant cell type in human insulitis, are thought to be the primary mediator of b-cells damage, we thus designed a strategy by adapting chimeric antigen receptor engineered T (CART) technology to directly target these pathogenic T cells. The newly generated CAR construct maintains original transmembrane and intracellular components, while the extracellular scFv antigen-binding domain was replaced with HLA-A2/b2-microglobulin(B2M) complex that is linked with either diabetes-associated immunodominant antigenic peptides zinc transporter 8(ZnT8)186-194 or negative control peptide HIV Gag77-85. 70-80% of lentivirus-transduced Jurkat cells were stained positive with anti-HLA-A2 antibody, suggesting HLA-A2/B2M complexes were correctly folded and expressed. Antigenic ZnT8 and HIV peptide sequences were also verified as accurate by N-terminal sequencing. To determine whether CAR signaling is sustained in transduced Jurkat cells, we co-cultured Jurkat cells together with ZnT8186-194-reactive or non-reactive CD8+ T-cells isolated from patients with T1D. As expected, expression of CD69 was significantly elevated in transduced Jurkat cells presenting ZnT8186-194 peptide and cultured with ZnT8186-194-reactive T-cells. Since CAR-transduced Jurkat cells can selectively recognize T cells in an antigen-specific manner, we are now expressing CAR in primary T cells to test their ability to deplete autoreactive T-cells both in vitro and in vivo.