Tolerance induction via a B-cell delivered gene therapy-based protocol: Optimization and role of the Ig scaffold

Our previous studies indicated that antigen-specific tolerance could be achieved by the injection of LPS-activated B-cell blasts that were retrovirally gene-transferred with an IgG-antigen fusion construct. This system was shown to be effective for tolerance induction with a variety of inserted antigens ranging in size from a single peptide to a large multi-epitope protein in a variety of mouse strains. Moreover, it was shown to be effective in four animal models for human disease. To optimize the existing protocol, establish the role of the IgG H chain scaffold, and provide baseline for potential clinical applications, we examined the effects of different B-cell activators, including lipopolysaccharide (LPS), anti-CD40, CpG oligodeoxynucleotide (CpG-ODN), and anti-IgM plus IL-4, on B-cell proliferation, GFP transduction efficiency, and tolerance induction in vivo. The results show that all activators except CpG-ODN have similar effects on retroviral gene transfer and peptide-IgG-induced tolerance. Furthermore, dose-response analyses showed that T-cell tolerance could be induced with 10⁵ peptide-IgG LPS B-cell blasts, but that 10⁶ transduced B-cells were needed for humoral unresponsiveness. Transduced anti-IgM-induced blasts were tolerogenic at 10⁶ cells, but no dose of transduced CpG blasts was tolerogenic. Finally, to examine the role of IgG scaffold, a retroviral construct encoding λ repressor p1-102 and signal peptide of murine IgG heavy chain was engineered to allow secretion of the p1-102 domain in the same manner as that of p1-102-IgG fusion protein. The results demonstrate that not only is IgG scaffold important in tolerance induction and maintenance of the long-lasting immune hyporesponsiveness, but assembly of the IgG heterodimer may be required for the efficacy of this system.