Possible Involvement of CTL Granule Proteases in Target Cell DNA Breakdown

Abstract

We have carried out experiments to test whether the granule exocytosis model for lymphocyte cytotoxicity can account for the rapid target DNA breakdown seen during CTL-induced cytotoxicity. Dense granules isolated from cloned mouse CTL and from rat NK tumor cells cause target DNA breakdown during granule cytolysin-mediated lysis of tumor cells, while the purified granule cytolysin caused lysis without DNA breakdown. When target cells are permeabilized with detergent, granule extracts have the ability to release 125I-DNA from nuclei in the absence of detectable cytolysin activity. This activity formed the basis for a nuclear DNA release (NDR) assay; this activity was a property of dense granules of cytotoxic lymphocytes but generally not of other types of lymphoid cells. NDR activity in NK tumor granules had a pH optimum of 7 and was inhibited by micromolar levels of Zn+2, and could be purified away from the granule cytolysin by column chromatography. NDR activity in CTL dense granules could be inactivated by submillimolar concentrations of the protease inhibitors PMSF and DFP (but not soybean trypsin inhibitor or TLCK). In support of the relevance to CTL cytotoxicity of these findings with the NDR assay, pretreatment of CTL with PMSF in the presence of agents raising the intragranular pH inactivated 125I-DNA release from target cells (but not the 51Cr release). These results suggest that a CTL granule component(s), probably a protease, is required for target DNA breakdown.

Expression of cytoplasmic granules with T cell‐associated serine proteinase‐1 activity in Ly‐2(CD8) T lymphocytes responding to lymphocytic choriomeningitis virus in vivo

“…In addition, they support the findings of Munger et al [43] demonstrating the presence of BLT esterase-and cytolysincontaining cytoplasmic granules in primary allo-immune peritoneal exudate T cells. However, these and the present data are contradicted by observations of Berke and Rosen [20] and Dennert et al [21].…”

The Involvement of Macrophages and Lymphocytes in the Apoptosis of Enterocytes.

“…LGLs correspond to a heterogenous cell population including NK cells, cytotoxic T lymphocytes (CTLs) and lymphokine-activated killer (LAK) cells (KANEDA, 1989). Cytoplasmic granules of the lymphocytes contain cytotoxic substances including perforin (PODACK et al, 1985), a family of serine esterases (MUNGER et al, 1988;HAYES et al, 1989;ZUNINO et al, 1990), and TIA-1 (TIAN et al, 1991) as effector molecules which kill target cells.…”

Granzymes are essential for natural killer cell-mediated and perf-facilitated tumor control

“…There is ample evidence from in vitro studies that CTL/NK cell-mediated nuclear DNA breakdown (apoptosis) and death of tumor cells is critically dependent on granule exocytosis [3] and the simultaneous activation of perforin (perf) and granzymes (gzm) [4][5][6][7][8]. In fact, by using purified native material from mouse CTL or rat NK cell lines it was found that perf alone was able to induce 51 Cr release in target cells, but that substantial DNA fragmentation of the same cells only occurred with a combination of perf and gzm [4,6,7].…”