Recombinant T-cell receptors from HPA-1a specific T-cells

Abstract

Fetal- and neonatal alloimmune thrombocytopenia is a condition that can cause hemorrhage and potential brain damage or death in neonates. It is most often a result of maternal alloantibodies specific for fetal Human Platelet antigen 1a (HPA-1a) that destroy fetal platelets. The recognition of the HPA-1a peptide-MHC complex by T-cells involve a peculiar form of T-cell receptor peptide-MHC interaction in which the Leu33 residue is essential for T-cell stimulation, not by being directly presented to the TCR, but by acting as an anchor residue and increasing peptide binding and stability of the peptide-MHC complex, compared to the Pro33 allovariant. Studies of immunized women have been found to involve a diverse set of T-cells with varying responsiveness. To build a solid platform in which to study the peptide-MHC interactions involved in this condition, the goal of this project was to attempt to produce soluble, recombinant T-cell receptors from an HPA-1a-specific T cell line (D7T4) characterized from an alloimmunized woman. Through cloning and recombination of D7T4-TCRα and D7T4-TCRβ plasmids and vectors used in multigene baculoviral vectors, and their fusion, baculovirus vectors for transfection and expression of D7T4-TCRαβ in insect cells were produced. D7T4-TCRαβ was found to be expressed in its expected dimeric form in insect cells, but its functionality as a T-cell receptor was not clearly demonstrated.