Identification and Cytolytic Function of a Novel NK Cell Surface Receptor that Binds Haymaker on the K562 Leukemia Cell Line

Abstract

Natural killer lymphocytes (NK cells) are the first line of defense (innate immunity) against viral infections and cancer since they do not require activation to deliver a lethal hit to infected/aberrant cells. In contrast, T lymphocytes require stimulation by a foreign/neo-antigen, which may take days before they are active against the pathogen (adaptive immunity). Although numerous NK cell receptor-coreceptors (ligands) have been revealed, the exact mechanism by which unstimulated (naive) nNK cells lyse the prototypical leukemia target cell line (K562) has not been fully characterized. In Aim 1, I identified the nNK cell receptor that interacts with the Haymaker (HYMKR) ligand on K562 cells. Our laboratory previously determined that HYMKR is the translocase of the outer membrane of mitochondria (TOMM40). Pull-down experiments with HYMKR polypeptide affinity resins, peptide sequencing, flow cytometry, and anti-moesin monoclonal antibody (Mab) cytotoxicity blocking results provided evidence that moesin is the nNK cells receptor for the HYMKR ligand. Moesin is a protein that typically links the inner leaf of the plasma membrane to the cytoskeleton; additionally, in nNK cells, it localizes to the cell surface, where it binds to HYMKR on leukemia cells. Mab against moesin indicated that moesin was not on the surface of freshly isolated unstimulated T lymphocytes from healthy subjects but demonstrated that it was on the surface of nNK cells and is, therefore, a marker that distinguishes unstimulated NK cells from unstimulated T cells. The anti-moesin Mab inhibited > 97 % of the LDH release from K562 cells on incubation with purified nNK cells. This finding strongly suggests that moesin-HYMKR interaction is the major death pathway utilized by unstimulated NK cells for certain leukemia cells. In Aim 2, I investigated the nNK cell cytolytic pathway involved in delivering a lethal hit to K562 cells through moesin-HYMKR interaction. I examined the expression levels of perforin and granzyme B in the nNK cell-mediated lysis of K562 cells. Flow cytometry analysis showed no change in intracellular expression levels of the cytolytic granules in nNK cells. Alternately, I examined the H+ transporting ATP synthase in nNK cell-mediated lysis of K562 cells. Flow cytometry analysis showed a time-dependent fluorescent increase of the 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) dye in K562 target cells. This preliminary result suggests that moesin-HYMKR interaction induces an influx of ions and water into K562 cells leading to osmotic lysis of the target cells. Further experiments are warranted to substantiate this finding.