Schematic of the P815 exocytosis assay. The P815 assay is performed by coculturing patient-derived PBMNCs with P815 cells and anti-CD3 or anti-CD16 antibodies. The Fc portion of the antibodies binds the FcγRII on the P815 cells. The epitope-specific portion of the anti-CD3 antibody then binds to the T-cell receptor on CD8 T cells, and the anti-CD16 antibody binds to CD16 on NK cells. This serves to “redirect” the CTL toward the P815 target cell. In PBMNCs from healthy donors with intact lymphocyte exocytosis (A), this process causes the CD8 T and NK cells to degranulate, resulting in lytic granule fusion with the lymphocyte plasma cell membrane and translocation of CD107a protein from the inner membrane of the lytic granule to the surface of the lymphocyte, where it can be detected via flow cytometry. In contrast, PBMNCs from patients with pHLH (B) harbor loss-of-function mutations in genes required for lymphocyte exocytosis (denoted with an asterisk). Although these pHLH CD8 T and NK cells are activated by the anti-CD3 and -16 antibodies, respectively, they are unable to effectively degranulate and CD107a is not detected on the cell surface.