Mechanisms of gene therapy for tolerance

Efficient methods for gene transfer into hematopoietic cells may enable the expression of antigens involved in autoimmune processes for the induction of tolerance. Our laboratory has developed a retroviral construct which displays the major T and B cell epitope, residues 12-26, of bacteriophage λ cI protein at the N-terminus of an IgG heavy chain, and expressed this in bone marrow progenitor cells and in peripheral B cells from BALB/c mice for tolerance induction. To further clarify the mechanism by which these novel fusion proteins induce tolerance, we used transgenic mice that express IgG-12-26 constitutively in B cells (L17). We found that mice given transgenic resting, LPS-stimulated B cells or CD40L stimulated B cells were specifically tolerant to 12-26. These experiments suggest that B cells are sufficient for T cell tolerance. Our laboratory has also shown that while resting B cells had no effect on an ongoing immune, response in vivo, LPS-stimulated B-cell blasts from such 12-26-IgG transgenic mice induced tolerance in already primed BALB/c mice. Since LPS and CD40L upregulate B7.2 expression, the tolerogenicity of activated B cells can not be explained by the lack of costimulatory ligands. We also found that transgenic B cells failed to present endogenous peptide to the 12-26 specific T cell clone (T32), even when activated by LPS or CD40L. Nonetheless, these cells were competent to present exogenous 12-26 to the T32 clone; therefore, there is no general defect in APC capacity of these transgenic B cells, although a processing defect can not be eliminated. Furthermore, our data suggest that transgenic B cells could not be "back-stimulated" by the T cells to upregulate B7 (in the absence of exogenous antigen) even though they produce low levels (nM) of the fusion protein and would be expected to express endogenous specific pectides (first signal). Our most recent data show also that B cells from Tg L17 mice can not induce T cells to express IL-2R nor the early activation marker, CD69, unless external antigen is added to the culture. These data suggest that tolerance induction by 12-26-expressing B cells may be due to continuous, suboptimal stimulation of specific T cells. Experiments to test this hypothesis are in process.