Morphology of Interleukin-2-Stimulated Human Peripheral Blood Mononuclear Effector Cells Killing Glioma-Derived Tumor Cells In Vitro

Abstract

This is the first morphological study of interleukin-2-stimulated human peripheral blood mononuclear (PBM) cells resulting in lymphokine-activated killer (LAK) cell activity against human glioma-derived tumor cells in vitro, in which high-resolution differential interference video light microscopy, scanning electron microscopy, and transmission electron microscopy were used. A subset of cells within the LAK cell population are the effector cells and have an asymmetric cellular architecture characteristic of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Upon binding to target cells, the LAK effector cell nucleus is positioned away from the target cell, whereas the granules, Golgi apparatus, and microtubules orient toward the target cell. These LAK-glioma cell conjugates form very tight plasma membrane bonds with numerous interdigitations, and vesicles were found in the small extracellular spaces between the cells. This morphology was not observed in unstimulated PBM-glioma cell co-cultures. Glioma-derived cells react to LAK effector cells by blebbing, becoming round, and rapidly detaching from the substrate. The injured glioma-derived cells had a highly condensed cytoplasm and chromatin, lobular nucleus, and severe plasma membrane blebs, which are consistent with an apoptotic rather than an osmotic lysis mechanism of cell death. This study provides morphological evidence that supports a common cytotoxic mechanism for CTLs, NK cells, and LAK effector cells. The cytotoxic mechanism is based on the local exocytosis of vesicles by the effector cell into the small extracellular space between the effector-target cell conjugate. Granules found in CTLs, NK cells, and LAK cells contain a pore-forming protein that inserts holes in the target cell's plasma membrane through which a lethal substance(s) not yet identified is thought to enter the cell.

Glioma Cells Display Complex Cell Surface Topographies That Resist the Actions of Cytolytic Effector Lymphocytes

“…The physical interaction between cytolytic lymphocytes and glioma-associated ligands was described within one study (32). We now present evidence that another level of complexity may also contribute to the resistance of gliomas toward lymphocyte-mediated cytotoxicity in vitro.…”

“…We also thank the Southern California Institute for Research and Education (SCIRE) for support. We thank Dr. Linda Muul (NCI) for discussions on Glioma Topography from her previous work (32).…”

Molecules relevant for T cell‐target cell interaction are present in cytolytic granules of human T lymphocytes

“…We show that release occurs by exocytosis, whereby the granule membrane fuses with the CTL plasma membrane and dense cores and internal vesicles are externalized into the clefts. The presence of membraneous materials and dense material in clefts has been noticed before [20,53,551. In addition, we have observed that internal vesicles as well as dense cores can associate with the target cell membrane.…”

Analysis of interleukin 2 and various effector cell populations in adoptive immunotherapy of 9L rat gliosarcoma: allogeneic cytotoxic T lymphocytes prevent tumor take.

“…Thus, LAK cells may not recycle or may do so inefficiently. Direct contact of LAK cells with tumor cells may be necessary for a lethal hit (21). CTLs, however, can recycle and continue killing tumor with time (22).…”