IntroductionThe innate and adaptive immune systems include cytotoxic lymphocytes (natural killer [NK] and cytotoxic T lymphocyte [CTL] cells) that are important for responses to intracellular pathogens and tumor cells. Although there are several mechanisms by which these cells kill (eg, Fas/Fas ligand, tumor necrosis factor [TNF]/TNF receptor, and Toll receptors), the major mechanism is that of the perforin/granzyme (granule exocytosis) pathway. [1][2][3][4] This pathway is critical for host mechanisms of defense, including viral clearance and tumor cell killing. Dysregulation of this pathway is associated with a number of human diseases, such as hemophagocytic lymphohistiocytosis, Griscelli syndrome, and X-linked lymphoproliferative disease. 1,[5][6][7] The granzymes are serine proteases that are packaged into the specialized cytotoxic granules of CTL and NK cells. It is believed that individual cells are armed with both perforin and granzymes, and that expression is limited to CD8 ϩ T lymphocytes and NK cells. 8 Although human CD4 ϩ T lymphocyte clones have been shown to express perforin and granzymes, the functional significance of the granule exocytosis pathway in these cells has not yet been clearly established. [9][10][11][12][13][14] Recent data from Kelso et al have suggested that individual cells may express different combinations of granzymes, implying an additional level of control in the process of cellular cytotoxicity. 15 Most studies of granzyme expression have relied on Northern blots, RNA protection assays, and/or reverse-transcriptionpolymerase chain reaction (RT-PCR) analysis to define cellular expression patterns. In addition, the majority of these studies have analyzed bulk cell populations. 8 There are 2 prior studies that have reported single-cell expression of either granzyme A or granzyme B in human CD8 ϩ T-cell subsets using intracellular flow cytometry. 16,17 In this report, we examine the dual expression patterns of both granzymes A and B using granzyme-specific monoclonal antibodies in an intracellular flow cytometry assay. We used this assay to characterize the expression patterns of granzymes A and B in resting and activated human peripheral blood mononuclear cells (PBMCs). Most CD56 ϩ 8 Ϫ NK cells, nearly all CD56 ϩ 8 ϩ NKT cells, and approximately half of the circulating CD8 ϩ T lymphocytes were found to coexpress both granzymes A and B. While few resting CD4 ϩ T lymphocytes expressed granzyme A or B, activation of CD4 ϩ T lymphocytes with concanavalin A (ConA)/ interleukin-2 (IL-2), or with antibodies directed against CD3/CD28 or CD3/CD46, induced high levels of granzyme B expression, but not granzyme A. Naive CD4 ϩ CD45RA ϩ T cells stimulated with antibodies to CD3/CD46 to generate adaptive Tr cells strongly expressed granzyme B, while stimulation with antibodies to CD3/CD28 was ineffective at driving granzyme B expression. In contrast, memory CD4 ϩ CD45RO ϩ T cells were found to express granzyme B with both modes of stimulation. Finally, we show that these activated granzyme ...