Expression and characterization of recombinant murine integrin β3 modified to express the human platelet antigen-1a epitope

Abstract

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition where the mother produces alloantibodies against fetal platelets during pregnancy most commonly due to an incompatibility in the human platelet antigen (HPA)-1 system; a single amino acid polymorphism located on integrin β3 with two common variants, HPA-1a and HPA-1b. Antibody responses against HPA-1a can be formed in women who are HPA-1b homozygous. No effective treatment or prevention to the FNAIT condition are available today, and much is unknown about the immunization process. Antibody production by HPA-1a-specific B cells is most likely dependent on helper T cells specific for the same antigen. To model these cellular and molecular interactions in mice, we aim to introduce the HPA-1 system in murine integrin β3 by altering four key amino acids, and to use recombinant protein to induce HPA-1a-specific B and T cell responses. Predictably, this will more closely mimic FNAIT associated immune responses compared to using human integrin β3 as an immunogen since the latter has many antigenic differences that can induce additional T cell responses. The aim of the current study was to produce recombinant HPA-1a and HPA-1b variants of murine integrin β3 and to characterize these biochemically and functionally in comparison to human integrin β3. Thus, in this study, these recombinant murine integrin β3 proteins were designed and expressed using the baculovirus system. Concurrently, recombinant human integrin β3 was expressed using an already prepared virus stock. Produced proteins containing 6xHis were captured from the supernatants on beads coated with anti-His-tag antibody. The protein production was confirmed using SDS-PAGE and western blot, and the presence of the HPA-1a epitope was verified by flow cytometry using HPA-1a-specific antibodies. HPA-1a-specific T cells isolated from an HPA-1bb mother with a child affected by FNAIT, were incubated with HLA-DRB3*01:01 positive monocytes co-cultured with antigen. HPA-1a platelets, integrin β3-derived peptide comprising the Leu33 residue and recombinant murine and human integrin β3 protein expressing the HPA-1a epitope stimulated T cell proliferation and tumor necrosis factor (TNF) production. Thus, the produced proteins can potentially be used to further study the interaction between HPA-1a specific B cells and T cells in a future murine model.