Peptide-Induced Antigen-Specific CD4⁺CD25⁺FoxP3⁺ T Cells Suppress Cytotoxicity T Cell Responses Directed Against the AAV Capsid.

Abstract 3769

Recent advances in adeno-associated viral (AAV) vector-mediated gene transfer continue to offer hope for the correction of monogenic disorders such as hemophilia B. However, unanticipated T cell responses directed against viral capsid epitopes may limit the efficacy of AAV gene transfer. A phase I clinical study in which an AAV2 vector expressing human factor IX (FIX) was delivered systemically provided the first evidence that AAV vector administration at high doses may trigger the expansion of memory CD8⁺ T cells directed against AAV capsid epitopes. This response was associated with the loss of FIX transgene expression and a transient increase in liver enzymes. Additional studies in human subjects undergoing AAV gene transfer suggest that the capsid antigen load is an important determinant of capsid-specific T cell activation. Thus, strategies for the modulation of capsid T cell responses could contribute to achieving sustained transgene expression following high dose delivery of AAV.

MHC class II peptide ligands identified within the human IgG Fc fragment (Tregitopes, Blood 2008;112:3303) have been shown to expand regulatory T cells (Tregs). Restimulation of human peripheral blood mononuclear cells (PBMC) in vitro with AAV capsid antigen in the presence of Tregitopes resulted in the suppression of capsid-specific CD8⁺ T cells and in the expansion of CD4⁺CD25⁺FoxP3⁺ Tregs.

To better define the nature of Tregitope-induced Tregs, we depleted CD25⁺ cells from PBMC prior to in vitro restimulation. This completely prevented capsid-specific CTL suppression and the expansion of Tregs, suggesting that Tregitopes act by expanding natural Tregs. Cytokine ELISA on conditioned media from PBMC co-cultured with AAV antigen and Tregitopes showed a 50% decrease in IL-2 levels and a >500-fold increase in IL-10 levels. These results suggest that the effect of Tregitopes may be cytokine mediated. To test this hypothesis, we used a transwell system in which the CD4⁺ T cell fraction of Tregitope-restimulated PBMC was co-cultured with the capsid-specific CD8⁺ T cells. Without cell contact, a nearly 50% suppression of anti-capsid CD8⁺ T cell responses was observed. Further evidence supporting the role of cytokine-mediated suppression came from the observation that Tregitope-treated capsid-specific CD8⁺ T cells appeared to be anergic, and depletion of CD4⁺ T cells (Tregs) followed by a 24-hour incubation of CD8⁺CD4⁻ T cells with IL-2 restored >80% of CTL activity.

Finally, antigen specificity of Tregitope-induced Tregs was tested by expanding PBMC in vitro with HLA-B*0702-restricted epitopes from either the AAV capsid or the Epstein-Barr Virus (EBV). After in vitro restimulation, negatively-isolated CD4⁺ T cells expanded in the presence of EBV antigen and Tregitopes were co-incubated with either CD8⁺ T cells expanded against the AAV capsid or against EBV. Suppression of CTL activity was observed only when EBV Tregs were co-incubated with EBV CD8⁺ T cells. Similarly, Tregs isolated from AAV and Tregitope cultures suppressed AAV-specific CD8⁺ T cells but not EBV-specific CD8⁺T cells. These results suggest that inhibition of CD8⁺ T cell responses is antigen-specific.

We conclude that Tregitopes induce the expansion of Tregs, which can mediate a potent antigen-specific inhibition of CD8⁺ T cell responses directed to the AAV capsid.