Novel method for the identification and visualization of CD8+ T Cell Mediated IFN-γ Signaling in Target Cells during an Antiviral Immune Response in the Brain: Implications for Neurological Gene Therapy

PUNTEL; BARRET; SANDERSON; KROEGER; BONDALE; KENNEDY; LIU; CASTRO; LOWENSTEIN

Filadelfia

Congreso; American Society for Gene and Cell Therapy Annual Meeting; 2012

American Society for Gene and Cell Therapy

      The immune response to viral vectors is an important challenge to gene therapy, as killing of transduced cells could hamper therapeutic benefits. Long term gene transfer in humans and pre-clinical animal models can be limited by preexisting anti-adenoviral (Ad) immunity, as the adaptive immune response reduces transgene expression. In the brain, however, good transduction, even in the presence of anti-adenoviral immunity has been detected reliably. We have previously shown that during an anti-Ad immune response, CD8+ T cells infiltrate the brain, recognize cells expressing adenoviral antigens, become activated, and secrete IFNγ through the formation of specific immunological synapses. As part of this process IFN-γ becomes polarized at intercellular junctions between T cells and adenovirus infected target astrocytes.       Currently, there are no methods available to recognize individual target cells that have been exposed to IFNγ secreted by antigen specific T cells, in vivo. Thus, we designed a model to identify those brain cells that become targeted by interferon-gamma during a systemic anti-Ad immune response. This method can identify individual cells that respond to IFNγ secreted by specific anti-adenoviral cytotoxic t cells. To this end, we constructed adenoviral vectors encoding a transcriptional response element that is selectively activated by IFNγ signaling, i.e., the gamma-activated site (GAS) promoter element. In our final vector the GAS element drives expression of a transgene, Cre recombinase (Ad-GAS-Cre). Upon binding of IFNγ to its receptor, the intracellular signaling cascade activates the GAS promoter, which drives expression of the transgene Cre recombinase. In ROSA26 mice bearing a STOP sequence flanked by LoxP sites which is located upstream of a β-galactosidase (β-gal) encoding sequence within the ROSA locus, Cre recombinase activity induces β-gal expression.  We demonstrate that upon activation of a systemic immune response against adenovirus, CD8+ T cells infiltrate the brain, interact with target cells, and cause an increase in the number of cells expressing Cre recombinase. This method can be used to identify, study, and eventually determine the long term fate of infected brain cells that are specifically targeted by IFNγ secreted from cytotoxic T cells during an antiadenoviral immune response. This method will allow the characterization of the fate of Ad infected brain cells that are targets for anti-viral CD8+ T cells which infiltrate the brain. This will allow novel insights into the cellular and molecular mechanisms underlying brain immune responses and their impact on long term neurological gene therapy using Ad vectors.