Figure 1. Molecular design of the HLA-DR1 CAR molecule for targeting CD4+ T cells.

A. The HLA-DR1 CAR molecule consists of HLA-DR 1st domains, an antigenic peptide covalently linked to the DRB1 chain, second domains from I-E^d to enable murine CD4 interaction, and CD28 trans-membrane/costimulatory and CD3ζ activation domains to deliver signals to the CD8+ CTL. The first domains of the HLA-DR1-peptide complex serves as the ligand for the TCR expressed by CD4+ autoimmune T cell targets. Once these cells interact, the CTL is activated by signaling through the CD28/CD3ζ domain, and the attached CD4+ T cell is lysed. B. The cDNA construct of the HLA-DR1 CAR is based on the construct expressed by our DR1 Tg mouse that enables mouse CD4 interaction. In constructs 1 and 2, cDNA encoding an antigenic peptide and linker was added to the DRB1 chain. Transmembrane (TM) and cytoplasmic (Cyt) domains of the DRB1 and DRA1 chains are replaced with corresponding CD28 and CD3ζ domains enabling intracellular signaling through the DR1 molecule. The DRB1 and DRA1 chains are separated by a ribosome skipping T2A sequence allowing both chains to be expressed off the same promoter as a single mRNA that is translated as two separate peptide chains. C. For expression of DR1 CAR in T cells, purified CD8+ T cells were transduced with the MSGV vector encoding the chimeric DR1 construct or mock transduced (control). Data shown are from the DR1-CII construct, and similar results were observed with transduction of the DR1-HA and DR1 empty constructs. Seven days after transduction, cells were stimulated with either antibodies to CD3 and CD28, or with an antibody to DR (LB3.1). Cells were stained with anti-CD8-FITC and anti-DR-Alexa 647 (antibody L243) and analyzed by flow cytometry. Stimulation of the CAR T cells in culture through the CAR molecule using the anti-DR antibody significantly enriched for CAR expressing cells.