TY - JOUR
T1 - Development of highly sensitive Bicistronic vector based non-radioactive antigen-specific cytotoxicity assay
AU - Gupta, Pranav
AU - Tayal, Ruchi
AU - Durgapal, Hemlata
AU - Rath, Satyajit
AU - Acharya, Subrat Kumar
AU - Panda, Subrat Kumar
PY - 2009/9/30
Y1 - 2009/9/30
N2 - In the absence of a better alternate, 51Cr release assay, with its several disadvantages is still the most common method for detection of MHC class I restricted T-cell mediated cytotoxicity. We describe a system in which the T-cell mediated cytotoxicity can be assessed using host-derived cells transfected with a bicistronic vector expressing the specific antigen and a quantifiable reporter as target cells. This overcomes the problems associated with use of radioactivity, pre-loading of target cells with reporter/antigen and the MHC restriction. We used HBV core antigen to prove the concept, as it is an established CTL target. Bicistronic vectors containing HBV core and reporter (EGFP/Fluc) gene were generated and further checked for antigen/reporter expression in human HepG2, mouse fibroblast BALB/c.3T3 and mouse lymphoma A20 cell lines. The effector cells to study the cytolytic activity were generated in vivo using BALB/c mice immunized with antigen expressing DNA clone or protein. The target cells (BALB/c.3T3 and A20) transiently transfected with bicistronic constructs were incubated with effector cells (splenocytes) from immunized mice at a different effector to target ratio. Following incubation the CTL activity was calculated by measuring the reporter luciferase in the remaining viable target cells that inversely correlates with the cytolysis of susceptible cells. The percent specific lysis measured in our assay was compared with conventional 51Cr release assay to validate this approach. This novel bicistronic vector based cytotoxicity assay demonstrated an easy to perform, antigen-specific and non-radioactive method of determining T-cell mediated cytotoxicity.
AB - In the absence of a better alternate, 51Cr release assay, with its several disadvantages is still the most common method for detection of MHC class I restricted T-cell mediated cytotoxicity. We describe a system in which the T-cell mediated cytotoxicity can be assessed using host-derived cells transfected with a bicistronic vector expressing the specific antigen and a quantifiable reporter as target cells. This overcomes the problems associated with use of radioactivity, pre-loading of target cells with reporter/antigen and the MHC restriction. We used HBV core antigen to prove the concept, as it is an established CTL target. Bicistronic vectors containing HBV core and reporter (EGFP/Fluc) gene were generated and further checked for antigen/reporter expression in human HepG2, mouse fibroblast BALB/c.3T3 and mouse lymphoma A20 cell lines. The effector cells to study the cytolytic activity were generated in vivo using BALB/c mice immunized with antigen expressing DNA clone or protein. The target cells (BALB/c.3T3 and A20) transiently transfected with bicistronic constructs were incubated with effector cells (splenocytes) from immunized mice at a different effector to target ratio. Following incubation the CTL activity was calculated by measuring the reporter luciferase in the remaining viable target cells that inversely correlates with the cytolysis of susceptible cells. The percent specific lysis measured in our assay was compared with conventional 51Cr release assay to validate this approach. This novel bicistronic vector based cytotoxicity assay demonstrated an easy to perform, antigen-specific and non-radioactive method of determining T-cell mediated cytotoxicity.
KW - Bicistronic vector
KW - Cytotoxic T lymphocytes
KW - Enschede
KW - Reporter gene
UR - http://www.scopus.com/inward/record.url?scp=70149096841&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2009.08.001
DO - 10.1016/j.jim.2009.08.001
M3 - Article
C2 - 19682994
AN - SCOPUS:70149096841
VL - 349
SP - 28
EP - 37
JO - Journal of immunological methods
JF - Journal of immunological methods
SN - 0022-1759
IS - 1-2
ER -