Production and Characterization of Respiratory Syncytial Virus Specific T Cell Hybridomas Using FG, a Novel Chimeric Glycoprotein
Pomaranski, Mark R.
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Respiratory syncytial virus has expressed on its surface two glycoproteins, F and G, which are considered to be primary targets for host immune responses. Through the use of a baculovirus expression vector system, a novel chimeric glycoprotein, FG, which contains the extracellular regions of both F and G glycoproteins, has been expressed in an insect derived cell line. To gain a more comprehensive understanding of the role of glycoprotein presentation in T cell stimulation, multiple T cell hybridomas specific for the chimeric FG glycoprotein were produced. Initially popliteal lymph node cells (PLNC) from BALB/c mice that had been immunized with FG were restimulated in vitro to determine optimal time and concentration of FG for maximum responsiveness to the glycoprotein. PLNC were then fused with BW5147 thymoma cells using polyethylene glycol (PEG). Resulting T cell hyridomas were then screened for specific reactivity to FG by measuring the release of IL-2 after stimulation with FG and appropriate antigen presenting cells (APC). Approximately 4% of the cell fusion hybridomas reacted specifically with FG, one of which (106) was chosen for a detailed characterization owing to its stability. Using monoclonal antibody (MAb) blocking and mouse L cells transfected with class II Major Histocompatibility Complex (MHC) genes, the hybridoma 106 was tested to determine which class II MHC molecules were presenting FG. These characterization studies have shown that the lAd determinant is the class II MHC molecule responsible for FG presentation to this particular hybridoma. The continued testing of this hybridoma will allow more complete characterization and mapping of immunological sites on the two major glycoproteins of RS virus.