Differential expression of human granzymes A, B, and K in natural killer cells and during CD8+ T cell differentiation in peripheral blood.

NK cells and cytotoxic T lymphocytes can induce apoptosis in virus-infected and transformed target cells via the granule exocytosis pathway. The key components of the cytolytic granules are perforin and several serine esterases, termed granzymes. While the cellular distribution of human granzymes A (GrA) and B (GrB) has been well characterized much less is known about the expression pattern of human granzyme K (GrK). In this study GrA, GrB, and GrK expression was analyzed in human peripheral blood lymphocytes using flow cytometry. There was a distinct population of GrK expressing CD8+ T cells with a CD27+/CD28+/CCR5high/CCR7-/perforin-/low/IFN-gamma+ memory-like phenotype, while all CD56bright NK cells were also positive for GrK. In addition, GrK was also expressed in subpopulations of CD56+ T cells, CD4+ T cells, and TCRgammadelta+ T cells. In contrast, GrB was primarily expressed in CD56dim NK cells and differentiated memory CD8+ T cells with the CD27-/low/CD28-/low/CCR5-/low/CCR7-/CD11b+/perforinhigh phenotype. Only few CD8+ T cells expressed both GrB and GrK. GrA was found to be co-expressed in all GrB- and GrK-expressing T cells. Our findings suggest that granzyme expression during the differentiation process of memory CD8+ T cells might be as follows: GrA+/GrB-/GrK+ --> GrA+/GrB+/GrK+ --> GrA+/GrB+/GrK-.

Differential expression of the granzymes A, K and M and perforin in human peripheral blood lymphocytes.

Granzymes (Gzm) are a group of serine proteases which are stored in the granules of cytotoxic lymphocytes. In humans, five granzymes have been characterized to date at the molecular level. While GzmA and GzmB have been extensively studied, little is known about GzmH, GzmK and GzmM. In this study, we describe the generation of mAbs against human GzmK and GzmM by genetic immunization. The obtained anti-GzmK and anti-GzmM mAbs are not cross-reactive with GzmA, GzmB, GzmM and GzmA, GzmB, GzmK, respectively, and show a granular staining pattern in human lymphocytes. Flow cytometric analysis of peripheral blood lymphocytes revealed that GzmA, GzmM and perforin show a similar distribution. They are expressed in almost all CD16+CD56+ NK cells, CD3+CD56+ NKT cells and gammadelta T cells as well as in 20-30% of all CD3+CD8+ TC cells. Surprisingly, GzmK was not detected in the highly cytotoxic CD16+CD56+ NK cells but was preferentially expressed in lymphocytes of the T cell lineage, staining 20% of CD3+CD8+ TC cells, 50% of CD3+CD56+ NKT cells and 40% of gammadelta T cells, as well as 60% of the small sub-population of CD56bright+ NK cells. Our data suggest that human granzymes are differentially expressed in distinct sub-populations of peripheral blood lymphocytes.

Detection of soluble human granzyme K in vitro and in vivo.

Granzymes are serine proteases released from the granules of cytotoxic lymphocytes during the induction of apoptosis. To evaluate the physiologic role of human granzyme K (GzmK), we developed a sensitive ELISA which was shown to specifically detect human GzmK in its active as well as its inactive conformation. Analysis of the lysate of lymphokine-activated killer (LAK) cells by gel filtration revealed that GzmK seems to be complexed to proteoglycans within these cells. While the expression of GzmA and B by cytotoxic lymphocytes was strongly up-regulated in response to several activating stimuli, GzmK expression did not increase significantly above constitutive levels, indicating differential regulation of these granzymes. However, low levels of GzmK were detected in plasma samples of healthy volunteers, which were in the same range as levels of GzmA and B. Furthermore, circulating levels of GzmK as well as of GzmA and B were significantly elevated in patients suffering from viral infections. We conclude that GzmK protein is produced by cytotoxic cells, and just as GzmA and B it can be released in a soluble form into the extracellular space. Furthermore, our data suggest that despite a more restricted cellular expression pattern, GzmK seems to participate in immune responses against several viruses.