Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia.

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5'-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.

A highly conserved sequence of the viral TAP inhibitor ICP47 is required for freezing of the peptide transport cycle.

The transporter associated with antigen processing (TAP) translocates antigenic peptides into the endoplasmic reticulum (ER) lumen for loading onto MHC class I molecules. This is a key step in the control of viral infections through CD8+ T-cells. The herpes simplex virus type-1 encodes an 88 amino acid long species-specific TAP inhibitor, ICP47, that functions as a high affinity competitor for the peptide binding site on TAP. It has previously been suggested that the inhibitory function of ICP47 resides within the N-terminal region (residues 1-35). Here we show that mutation of the highly conserved 50PLL52 motif within the central region of ICP47 attenuates its inhibitory capacity. Taking advantage of the human cytomegalovirus-encoded TAP inhibitor US6 as a luminal sensor for conformational changes of TAP, we demonstrated that the 50PLL52 motif is essential for freezing of the TAP conformation. Moreover, hierarchical functional interaction sites on TAP dependent on 50PLL52 could be defined using a comprehensive set of human-rat TAP chimeras. This data broadens our understanding of the molecular mechanism underpinning TAP inhibition by ICP47, to include the 50PLL52 sequence as a stabilizer that tethers the TAP-ICP47 complex in an inward-facing conformation.