Abstract
A major obstacle in the treatment of Hemophilia A is that patients can develop an inhibitory immune response to therapeutic doses of coagulation factor VIII (fVIII). Over the last decade, we have developed a B-cell delivered gene therapy approach to prevent the development of inhibitory antibodies (“inhibitors”) in fVIII knockout mice (see Lei and Scott, Blood105: 4865, 2005). In our murine platform, activated primary spleen B cells or bone marrow cells are transduced with a retroviral vector encoding the fVIII A2 and/or C2 domain fused to an IgG heavy chain, and these cells are injected systemically into immunocompetent fVIII knockout animals. The recipients are rendered specifically tolerant to the encoded C2 and A2 domains, as evidenced by a >90% reduction of inhibitor titers, even in primed animals. To help evaluate the potential of this approach for translation, we are developing in vitro models for tolerance induction using human T-cell clones isolated from subjects with mild hemophilia A. The clones are isolated by single-cell sorting of CD4+ cells that are labeled by fluorescent HLA-DR tetramers complexed with peptides containing fVIII epitopes, followed by expansion with HLA-DR mismatched peripheral blood mononuclear cells (PBMC), phytohaemagglutinin, and interleukin-2. Our initial model utilizes a T-cell clone from an individual with mild hemophilia A due to fVIII missense genotype A2201P, which recognizes an HLA-DRA-DRB1*0101-restricted epitope within a synthetic peptide corresponding to fVIII residues 2194–2213. All of the antigen-specific T-cell clones isolated from this subject secreted interferon-gamma (IFN-γ) when stimulated by fVIII2194–2213 presented by irradiated HLA-DR-matched PBMCs or with plate-bound anti-CD3. Because of their robust response to a clinically relevant epitope in fVIII, one of these clones that expanded well in culture was chosen for initial testing of a modified gene therapy platform similar to that developed using the murine hemophilia A model. HLA-matched peripheral blood B cells were activated with antibodies to IgM or with CD40L-expressing fibroblasts and then transduced with a modified retroviral vector containing the human C2 domain sequence in-frame with the IgG sequence. These B cells were cultured with the hemophilic T-cell clone. After pre-treatment (“tolerance-induction step”), the cells were washed and then stimulated by plate-bound anti-CD3. The subsequent IFN-γ response (measured by ELIspots and ELISA) was dramatically reduced compared to the response of same T-cell clone cultured with mock-transduced B cells. The post-treatment reduction in IFN-γ secretion was equivalent to that induced after soluble anti-CD3 pre-treatment, a known method to induce T-cell anergy in vitro. Interestingly, IL-10 was produced during the tolerance induction (pre-treatment) phase, most likely from the activated B cells. Preliminary, parallel experiments with B cells transduced with a “gutless” adenovirus vector expressing C2-Ig did not result in a similar down-regulation of the T-cell response, suggesting that this non-integrating method of expressing antigens for tolerance is not effective, at least in this system. These results are the first to demonstrate in vitro modulation of cytokine responses using DR-restricted, fVIII-specific T cells from a hemophilia A subject. Further investigations using T-cell clones from hemophilic subjects with and without anti-fVIII antibodies will allow us to explore mechanisms of tolerance and may also suggest novel approaches to reduce inhibitor titers.
The interaction between CD8+ cytotoxic T cells and Leishmania-infected macrophages.
