CD8(+) tumor-infiltrating T cells are deficient in perforin-mediated cytolytic activity due to defective microtubule-organizing center mobilization and lytic granule exocytosis

Tumor-infiltrating lymphocytes (TIL) are well known to be functionally impa ired typified by the inability to lyse cognate tumor cells in vitro. We hav e investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8(+) TIL are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cyt okine mRNAs, and contain perforin and granzyme B proteins which are package d into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, b ut not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rap id; mitogen-activated protein/extracellular signal-related kinase kinase, e xtracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein ki nase C are activated; and IL-2 and IFN-gamma is secreted. However, on conju gate formation between nonlytic TIL and cognate tumor cells in vitro, the m icrotubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis of preformed lytic granules and acco unting for defective TIL lytic function. Recovery of TCR-mediated, activati on-dependent MTOC mobilization and lytic activity requires proteasome funct ion, implying the existence of an inhibitor of MTOC mobilization. Our findi ngs show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction.