Diabetes and other autoimmune endocrine diseases
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.