Abstract
A significant complication associated with treatment of inherited protein deficiencies, such as hemophilia B, by gene replacement therapy is the potential for the activation of transgene specific B and T cells to the therapeutic protein, coagulation factor IX (F.IX). In addition to the potential for inhibitor formation as a result of MHC class II antigen presentation (CD4+ T cell-dependent activation of B cells, which may also be observed in conventional protein-based therapy), gene expression may lead to MHC class I presentation of F.IX-derived peptides to CD8+ T cells. Upon in vivo gene transfer, such immune responses to may elicit a cytotoxic T lymphocyte (CTL) response capable of destroying target cells that express the F.IX transgene product. Therefore, to better understand the role of F.IX-specific CD8+ T-cell responses, it is essential that MHC I-restricted CD8 T-cell epitopes be identified. Here, we used a peptide library consisting of 82 individual 15-mer peptides overlapping by ten residues that spans the complete human F.IX (hF.IX) protein to preliminarily identify a specific immunodominate CD8+ T-cell epitope. The peptides were pooled into groups, each containing 8–11 peptides to create a matrix of 18 pools, with each peptide represented in two pools. C3H/HeJ were immunized with 5×1010 vector genomes of E1/E3-deleted adenovirus expressing hF.IX (Ad-hF.IX) via intramuscular injection into the quadriceps. Nine days later, the harvested spleen and popliteal lymph node cells were pooled and evaluated for CD8+ T-cell responses by intracellular cytokine staining for IFN-γ after being stimulated for 5h with peptides or controls. The frequency of IFN-γ producing hF.IX-specific CD8+ T-cells was determined by flow cytometry. While 16 pools from Ad-hF.IX immunized C3H/HeJ mice showed no response above the frequency of mock-stimulated cells, lymphocytes from two overlapping pools demonstrated a ~2.5-fold increase in frequency of CD8+ IFN-γ+ cells. From these results we can conclude that peptide 74 (SGGPHVTEVEGTSFL) contains a CD8+ T cell epitope for C3H/HeJ mice (H-2k haplotype). Furthermore, splenocytes from naive mice failed to respond to any of the peptide pools. The amino acid sequence corresponding to peptide 74 is located within the catalytic domain of hF.IX. This finding is of particular interest, in that, we previously reported a peptide containing the immunodominate CD4+ T-cell epitope in C3H/HeJ is also located within the catalytic domain of hF.IX (Blood 108:408). The definitive identification of hF.IX-specific CD8+ epitopes will facilitate the evaluation of experimental gene therapy strategies in murine models by providing a reagent for in vitro stimulation of F.IX specific CD8+ lymphocytes. For example, we can now determine the efficiency of CD8+ T cell activation as a function of vector, route, and dose following in vivo gene transfer.
In vivo induction of CTL responses by recombinant adenylate cyclase ofBordetella pertussiscarrying multiple copies of a viral CD8+T-cell epitope
