Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Clément, Marie‐Véronique; Haddad, Patrick; Soulié, Annie; Legros-Maïda, S; Guillet, J G; Cesar, E; Sasportes, M

doi:10.1016/0923-2494(90)90017-s

Cited by 40 publications

(24 citation statements)

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“…(Arnaout 1990; Dustin and Springer 1988;Matsumoto et al 2000b; Thomas et al 2004). Normal lysis of the K562 cell line by NK cells is dependent on the perforin-granzyme pathway and reductions in the levels of these proteins can cause a reduction in the ability of NK cells to lyse K562 targets cell (Clement et al 1990;Montel et al 1995). Intracellular staining and analysis of the NK cell (CD56 + ) subpopulation via flow cytometry found no difference in the level of lytic granule proteins between the atrazine-treated cell population and vehicle-treated cell population (Figure 2).…”

Section: Discussionmentioning

confidence: 99%

“…The value of both treatment groups was significantly different from controls (p<0/05). Untreated PBLs (1x10 6 ) cultured with target released 2412.53 ± 168.9 pg/ml of perforin. This provides further evidence that atrazine reduced the ability of NK cell to release the lytic following target cell contact.…”

Section: Atrazine Decreases the Ability Of Nk Cells To Release Granulmentioning

confidence: 99%

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In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

Rowe

Brundage

Barnett

2007

Toxicology and Applied Pharmacology

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The herbicide atrazine is a known immunotoxicant and an inhibitor of human Natural Killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24 h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4 h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine treated cells than vehicle treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

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Section: Discussionmentioning

confidence: 99%

Section: Atrazine Decreases the Ability Of Nk Cells To Release Granulmentioning

confidence: 99%

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

Rowe

Brundage

Barnett

2007

Toxicology and Applied Pharmacology

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“…To avoid such immunogenicity, we chose human granzyme B as the toxic component of our H22-based immunotoxin. Granzyme B is an apoptosisinducing serine protease naturally expressed and secreted by cytotoxic T lymphocytes and natural killer cells (23,24). …”

Section: Discussionmentioning

confidence: 99%

“…The serine protease granzyme B is an essential component of the granules released by cytotoxic T cells and natural killer cells (23,24). The cosecreted protein perforin delivers granzyme B to the cytosol of target cells (25), where its proteolytic activity induces apoptosis.…”

Section: Introductionmentioning

confidence: 99%

Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

Stahnke

Thepen

Stöcker³

et al. 2008

Molecular Cancer Therapeutics

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Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (Fc;RI), the highaffinity receptor for IgG, which is normally expressed at high levels only on activated cells of the myeloid lineage. CD64 is therefore a prime target for the specific delivery of cytotoxic agents. A promising toxin candidate is granzyme B, a human serine protease originating from cytotoxic granules of CD8 + T lymphocytes and natural killer cells. After evaluating the sensitivity of the AML-related cell line U937 toward cytosolic granzyme B, we genetically fused granzyme B to H22, a humanized single-chain antibody fragment (scFv) specific for CD64, to obtain Gb-H22(scFv), a fusion protein lacking the immunogenic properties of nonhuman immunofusions. Gb-H22(scFv) was successfully expressed in human 293T cells, secreted, and purified from cell culture supernatants. The purified protein bound specifically to CD64 + U937 cells. Despite linkage to the binding domain, the proteolytic activity of functional Gb-H22(scFv) was identical to that of free granzyme B. Target cell-specific cytotoxicity was observed with a half-maximal inhibitory concentration (IC 50 ) between 1.7 and 17 nmol/L. In addition, the induction of apoptosis in U937 cells was confirmed by Annexin A5 staining and the detection of activated caspase-3 in the cytosol. Finally, apoptosis was observed in primary CD64 + AML cells, whereas CD64 À AML cells were unaffected. This is the first report of a completely human granzyme B-based immunotoxin directed against CD64, with activity against an AML-related cell line and primary AML cells. [Mol Cancer Ther 2008;7(9):2924 -32]

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“…[18][19][20] Granzyme B is a serine protease primarily expressed in cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. 21,22 It activates apoptosis via both the caspase-dependent and caspase-independent pathways. [23][24][25][26] However, one of the major drawbacks of granzyme B is the presence of its specific inhibitor serpin B9 (PI9).…”

mentioning

confidence: 99%

Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

Schiffer

Rosinke

Jost

et al. 2014

Intl Journal of Cancer

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CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.The clonal hematopoietic stem cell disorder CMML (chronic myelomonocytic leukemia), is a highly aggressive and resistant form of leukemia. It is characterized by persistent monocytosis in the peripheral blood, at least one dysplasia component in the bone marrow, and <20% promonocytes and blasts in peripheral blood and bone marrow. 1 CMML can be classified further according to blast numbers, that is, CMML1 (<5% blasts in peripheral blood and <10% blasts in bone marrow) and CMML2 (<20% blasts in peripheral blood and 10-19% blasts in bone marrow).2,3 The disease occurs mainly in elderly people with a median survival of 15-20 months and leukemic transformation rates of 15-30%, both factors depending on the disease subtype. 4 Other diagnostic criteria include immunophenotyping based on the overlapping antigen expression patterns in CMML and acute myelomonocytic leukemia (AMML). The expression of CD56 combined with reductions in the levels of myeloid markers such as CD15 and CD13 is a unique signature of CMML monocytes.5 Furthermore, CD14 is expressed at higher levels on bone marrow monocytes in CMML compared with reactive monocytosis and normal marrow samples. 5 High levels of CD33 have also been reported. 6 Clonal cytogenetic abnormalities are found in 20-40% of CMML patients, but these do not appear to be specific. [7][8][9] The close relationship between CMML and other myelodysplastic (MDS)/MPNs makes correct diagnosis and treatment a significant challenge. Novel molecular markers described more recently include specific alleles of TET2 and CBL, but these

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Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Cited by 40 publications

References 38 publications

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

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