NK receptors replace CD28 as the dominant source of signal 2 for cognate recognition of cancer cells by TAA-specific effector CD8+ T cells

CD28-driven “signal 2” is critical for naïve CD8+ T cell responses to dendritic cell (DC)-presented weak antigens, including non-mutated tumor-associated antigens (TAAs). However, it is unclear how DC-primed cytotoxic T lymphocytes (CTLs) respond to the same TAAs presented by cancer cells which lack CD28 ligands. Here, we show that NK receptors (NKRs) DNAM-1 and NKG2D replace CD28 during CTL re-activation by cancer cells presenting low levels of MHC I/TAA complexes, leading to enhanced proximal TCR signaling, immune synapse formation, CTL polyfunctionality, release of cytolytic granules and antigen-specific cancer cell killing. Double-transduction of T cells with recombinant TCR and NKR constructs or upregulation of NKR-ligand expression on cancer cells by chemotherapy enabled effective recognition and killing of poorly immunogenic tumor cells by CTLs. Operational synergy between TCR and NKRs in CTL recognition explains the ability of cancer-expressed self-antigens to serve as tumor rejection antigens, helping to develop more effective therapies. Dynabead-induced CTLs from 3 human donors were stimulated by anti-human CD3 (OKT3) with or without anti-human DNAM-1 (OKT3 + anti-DNAM1), anti-human NKG2D (OKT3 + anti-NKG2D), or anti-human CD28 (OKT3 + anti-CD28)for 4 hours. Raw RNA-sequencing read files are not currently provided to ensure confidentiality and protect participant’s privacy under New York State law. Researchers interested in accessing the data for replication or further analysis may request access through the Institutional Review Board (IRB) of Roswell Park Comprehensive Center, subject to approval and compliance with ethical guidelines.