Related Experiment Videos
An engineered bifunctional recombinant molecule that regulates humoral and cellular effector functions of the immune
Maryellen C Pizzolato1, William L Fodor
1Alexion Pharmaceuticals, Inc., Cheshire, CT, USA.
Background:
Humoral and cellular defense mechanisms mediate the rejection of transplanted cells, tissues, and organs after allogeneic or xenogeneic transplantation. Inhibition of complement and T-cell costimulation are strategies aimed at increasing transplant survival.
Methods:
Engineered novel fusion proteins that contain the functional domains of human CD152 (hCTLA4) or porcine CD152 (pCD152) and human CD59 (hCD152-hCD59, pCD152-hCD59) were developed to form bifunctional chimeric proteins that retain the effector functions of both moieties. Porcine aortic endothelial cells and murine Balb/3T3 cells were transduced or transfected to express the novel fusion proteins.
Results:
Fluorescence-activated cell sorter analysis of hCD152-hCD59 transduced primary porcine aortic endothelial cells or hCD152-hCD59 and pCD152-hCD59 transfected Balb/3T3 cells determined that the molecules were expressed on the cell surface, and that they retained conformational epitopes. We demonstrate that hCD152-hCD59 and pCD152-hCD59 chimeric proteins inhibit complement-mediated cell lysis. In addition, hCD152-hCD59 or pCD152-hCD59 expression resulted in a significant reduction in T-cell activation as the result of CD152 engagement of porcine CD86 or murine CD80 in when Jurkat cells were cocultured with the hCD152-hCD59 or pCD152-hCD59 expressing cells. Antibody-blocking experiments or phosphatidylinositol phospholipase C removal of the glycosyl-phosphatidylinositol-linked molecules resulted in increased serum-mediated cytolysis and eliminated the costimulatory blockade.
Conclusions:
These data illustrate that a single molecule can confer resistance to humoral and cellular immune attack.