687. Tracking Transgene Product-Specific T Cells In Vivo
Authors: Lixin Wang, E.M. Lord, Roland Herzog
Journal: Molecular Therapy (2005)

Immune responses to the transgene product are a major hurdle for successful treatment of genetic disease by gene transfer. Characterization of T helper cell activation has been largely relied on in vitro assays due to the inability to track antigen-specific T cells in vivo. We sought to develop a system to visualize in vivo activation and trafficking of T cells after gene transfer. Previously, we documented that intramuscular administration of an AAV vector expressing chicken ovalbumin (ova) resulted in a cytotoxic T lymphocyte response that destroyed ova-transduced muscle fibers within 3-4 weeks (Mol. Ther. 9:S71). Ova was transiently detectable in the systemic circulation during the first 2 weeks until the ensuing inflammatory reaction had destroyed transduced myocytes. Theoretically, immune responses to the systemically delivered antigen or the antigen introduced into skeletal muscle tissue could have triggered T cell activation, thereby limiting duration of transgene expression. Ova-specific CD4 T cells extracted from BALB/c mice transgenic for DO11.10 T cell receptor (TCR, specific for amino acids 323-339), can be identified by flow cytometry (using anti-murine CD4 and clonotypic KJ1-26 monoclonal antibody specific to the DO11.10 TCR). Twenty-four hrs after adoptive transfer of CFSE-labeled CD4 splenocytes from DO11.10 transgenic to wild-type BALB/c mice, we injected AAV2-ova vector into the muscles of one hind limb, while the contra-lateral leg received AAV2-null vector (no transgene). At day 5 after gene transfer, CFSE cells were detected by flow cytometry in the draining lymph nodes (LN) of the AAV2-null injected leg and in the spleen at a frequency of 0.2-0.4% of total cells. As expected, around 70% of CFSE cells were also TCR. However, there was no evidence for cell division at any of the vector doses tested (1|[times]|1012, 3|[times]|1012, and 1|[times]|1013 vg/leg). In contrast, in the LN draining the AAV2-ova transduced leg, vector dose-dependent in vivo proliferation of CFSE+-TCR+- cells was evident (14-68% had been proliferating, showing 1-5 cell divisions), while CFSE+-TCR- cells did not proliferate. These data illustrate local antigen-specific T cell activation in the draining LN of the ova-expressing muscle. By day 10, an inflammatory infiltrate had developed in the AAV2-ova, but not the AAV2-null transduced muscle. Inflammation was more severe at higher vector doses and contained CD8 and IFN-g expressing cells. Immuofluorescence staining showed that the infiltrate contained a large number of CD4+TCT//Q cells, indicating that ova-specific CD4 T cells activated in the draining LN had migrated into the muscle, likely providing T help for cytotoxic T lymphocytes. Isolation of dendritic cells (DCs) followed by co-culture with BDZ.OVA.26.2 hybridoma cell line showed that the CD8a subset of DCs had activated ova-specific CD4 T cells in draining LNs. We now have an experimental system that allows us to pinpoint transgene product-specific T cell activation and migration in vivo. The implications are that systemic delivery of a protein by muscle-derived expression is limited by a local immune response in the transduced muscle tissue.