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Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity
Abstract:
Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.
Insights
Cytotoxic T lymphocytes (CTL) use granzyme A (GA) for cell lysis. Reducing GA levels significantly impaired CTL-mediated target cell killing and DNA degradation, indicating GA
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Murine cytotoxic T lymphocytes (CTL) contain seven serine proteases called granzymes within their lytic granules.
- The precise role of these granzymes, particularly granzyme A (GA), in the CTL lytic process remains incompletely understood.
- Convincing evidence for an obligatory function of granzymes in CTL-mediated cytotoxicity has been lacking.
Purpose of the Study:
- To investigate the functional significance of granzyme A (GA) in the cytotoxic T lymphocyte (CTL) lytic pathway.
- To determine if the quantity of GA is a limiting factor in CTL-mediated cell killing.
- To elucidate the specific role of GA in target cell lysis and DNA degradation.
Main Methods:
- Construction and utilization of an antisense expression vector targeting granzyme A (GA) mRNA in murine CTL line AR1.
- Selection of stable transfectants based on hygromycin B resistance.
- Quantification of GA mRNA levels using antisense cDNA and assessment of GA enzymatic activity.
- Evaluation of CTL-mediated lysis of nucleated target cells and anti-CD3-coated SRBC.
- Analysis of DNA degradation in target cells and assessment of conjugate formation between CTL and target cells.
Main Results:
- Stable CTL transfectants with significantly reduced GA mRNA and enzymatic activity (3- to 10-fold lower) were successfully generated.
- These GA-deficient CTL exhibited markedly reduced lysis of nucleated target cells (50-70% lower) and impaired DNA degradation.
- Lytic activity against anti-CD3-coated SRBC was drastically reduced (90-95% lower) in the absence of sufficient GA.
- Crucially, conjugate formation between CTL and target cells remained unaffected, suggesting GA acts downstream of cell-cell interaction.
Conclusions:
- Granzyme A (GA) plays a critical and non-redundant role in the cytotoxic T lymphocyte (CTL) mediated lytic process.
- The amount of GA present in CTL is a significant limiting factor for efficient target cell lysis and DNA fragmentation.
- These findings provide strong evidence for the essential function of GA in CTL-induced apoptosis and cytolysis.