Molecular Therapy, Vol 19, S1, Abstract 165
The American Society of Gene and Cell Therapy Annual Conference, Seattle, 2011
A potential challenge in gene replacement therapy for recessive diseases is the generation of antigen-specific immune responses to transgene products. One strategy to mitigate this risk is to restrict participation in the trial to subjects with missense mutations where the number of likely antigenic epitopes is low. This rationale is based on the principle of central tolerance that prevents the formation of T cells reactive to self antigens by virtue of deletion in the thymus due to high affinity interactions of T cell precursors and MHC/peptide complexes from thymic epithelial cells. In preparation for a gene therapy trial for cystic fibrosis (CF) conducted by the UK CF Gene Therapy Consortium, we established baseline levels of CFTR-specific T cells in 162 patients using a validated IFN-γ ELISPOT assay with a peptide library (15aa with 10aa overlaps divided in 6 pools; A-F) that spans the wild type CFTR. Surprisingly, we found positive results in peripheral blood mononuclear cells (PBMCs) from ∼15% of CF subjects before vector treatment. The positive samples demonstrated CFTR T cell frequencies that ranged from 93 spot forming units (SFU)/106 PBMCs (low positive group) to 541 SFU/106 PBMCs (highly positive group). Positive responses were generally found to a single peptide pool for each subject; however the pools that stimulated IFNγ were spread across the CFTR protein when the whole subject group was analyzed (4/Pool A; 10/Pool C; 4/Pool D; 1/Pool E; 2/Pool F). There was no correlation between peptides that stimulated T cell responses and underlying CFTR mutations; most CF patients had at least one ΔF508 mutation and 80% of these showed T cell responses to peptides distinct from those spanning ΔF508. Analysis of PMBCs from 2 subjects harvested two years apart revealed the same ELISPOT profile indicating these self reactive T cells are indeed stable and specific to individual CF subjects. These samples were subjected to intracellular cytokine staining to characterize the nature of the T cell responses. We found both CD4 and CD8 T cells to CFTR although frequencies were low. Similar IFN-γ ELISPOT studies in PBMCs from 24 normal human subjects revealed CFTR-specific T cells in 3 subjects although the frequencies tended to be lower than what was seen in CF subjects. T cells to dystrophin protein were recently described in PBMCs of patients with Duchenne's Muscular Dystrophy which were ascribed to exon skipping in revertant fibers exposing self-reactive epitopes. Our studies demonstrate the presence of relatively high levels of self-reactive T cells to epitopes of CFTR without having to invoke somatic reversion. These T cells are not anergic since they express IFN-γ in response to CFTR peptides however, we do not know if they possess effector activities that could contribute to the underlying disease in CF or that can modulate immunotoxicity in trials of molecular therapies like gene replacement and nonsense suppression.