The mechanisms that enable cytotoxic T lymphocytes (Tc cells) to destroy target cells are only vaguely understood. However, recent studies have identified in Tc cells and natural killer cells cytoplasmic granules that contain perforin, a cytolytic protein that resembles the ninth component of complement (C9). Antigen-specific lysis of target cells, traditionally ascribed solely to Tc cells, has now also been demonstrated in some T-helper cell (Th cell) lines, referred to here as T helper-killer or Th/c cells. We recently found a novel serine esterase that is present at greatly elevated levels in cloned murine Tc cell lines and one Th/c cell line, but not in two non-cytolytic Th cell lines. These findings suggest that the serine esterase is involved in cytolytic activity and that a variety of effector cells share a common cytolytic mechanism. To explore the role of the serine esterase in this process, we have been studying additional properties of the enzyme in murine T cells. We show here that it is a membrane-associated, disulphide-linked dimer, it has trypsin-like properties but is not a general protease, in density gradient centrifugation it sediments with perforin, it is secreted by Tc cells during their cytolytic attack on target cells, and antiserum to Tc-cell serine esterase reacts with the enzyme in Th/c cells.

Lysates of many highly cytolytic murine primary CD8+ cytotoxic T lymphocytes (CTLs) have no detectable hemolytic activity and only traces of serine esterase activity, indicating a striking paucity or absence of the perforin-rich secretory granules that are abundant in the cytoplasm of murine cloned CTL cell lines. Nevertheless, the primary CTLs are almost as resistant to granule-mediated lysis as CTL cell lines. Moreover, target cells that are lysed by all CTLs so far tested, whether primary or cell lines, show similar rapid and marked increases in intracellular calcium and breakdown of DNA into nucleosome-sized fragments. A parsimonious explanation for all of these findings is that primary CTLs, like the CTL cell lines, exercise their cytolytic activity by means of perforin, but the amounts needed are extremely small and below the level of detection by the current relatively insensitive hemolytic assays.

Cytotoxic CD8+ lymphocytes (CTLs) kill virally infected target cells by releasing cytotoxic granules. The primary objective of this study was to determine whether the activity of granzyme A, a serine protease in the killing granules of CTLs is altered in HIV-infected hemophiliacs. A sensitive colorimetric assay that measures cleavage of a synthetic substrate, N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT), was used to quantitate granzyme A activity. Granzyme A activities from hemophiliacs were normalized to to granzyme A activities of healthy donors run concurrently. Granzyme A activity in CD8+ T cells from HIV-seropositive hemophiliacs was significantly lower than granzyme A activity in cells from HIV-seronegative hemophiliacs (0.48 units +/- 0.086/CD8+ T cell and 1.573 +/- 0.434 units/CD8+ T cell, respectively; p < 0.005). These results indicate that cytotoxic cells in HIV-infected hemophiliacs have reduced granzyme A activity, which may result in a defect in CTL-mediated cell killing in these patients.