Autocrine, Not Paracrine, Interferon-Gamma Gene Delivery Enhances Ex Vivo Antigen-Specific Cytotoxic T Lymphocyte Stimulation and Killing

Abstract

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) shows promise in the treatment of cancer and infectious diseases. We utilize adeno-associated virus-(AAV-) based antigen gene-loaded dendritic cells (DCs) to stimulate such antigen-specific CTL. Yet further improvements in CTL stimulation and killing may result by gene delivery of various Th1-response interferons/cytokines, such as interferon γ (IFN-γ), as the delivered gene can continuously produce that interferon. However which immune cell type should optimally express IFN-γ is unclear as the phenotypes of both DC and T cells are enhanced by it. Here, we used AAV to compare and contrast IFN-γ gene delivery into DC or T cells, and versus the addition of exogenous IFN-γ, for stimulating carcinoembryonic antigen-(CEA-) specific CTL. It was found that AAV/IFN-γ delivery into T cells (autocrine) resulted in T cell populations with the highest CD8(+)/CD4(+) ratio, highest IFN-γ(+)/IL-4(+) ratio, highest CD69(+),CD8(+) levels, and lowest CD4(+)/CD25(+) levels, all consistent with the strongest Th1 response. Most importantly, AAV/IFN-γ transduction of T cells resulted in antigen-specific T cell populations with the highest killing capabilities, 49% above other treatments. These data strongly suggest that AAV/IFN-γ autocrine gene delivery into T cells is worthy of further study towards maximizing the generation of antigen-specific anticancer CTL killers.

AAV2/IL-12 gene delivery into dendritic cells (DC) enhances CTL stimulation above other IL-12 applications

“…As IL-12 is a heterodimer, we do not know if it is the p35, p40, or if both subunits are needed for intracrine activity. Moreover, our recent studies on IL-7 and IFN-γ gene delivery “tropism” is also consistent with intracrine activity for these cytokines, but within the T cells (not DC) 14 , 15 . This suggests that cytokine tropism is a more common activity than previously recognized.…”

“…The transduction efficiency of DC by AAV2/CEA and AAV2/IL-12 (MOI of 2000), as shown in Figures 2F and G was 87–92%. AAV2 is known to transduce primary T cells and DC even at relatively low multiplicities of infection 4 - 8 , 14 , 15 , 21 . The transduction efficiency of CD3+ T cells (MOI 200), as shown in Figures 2H, was 79%.…”

“…Recently we studied the activities of interleukin (IL) 2, IL-7 and interferon gamma (IFN-γ) under both autocrine and paracrine gene delivery approaches for generating CD8+ CTL. While these cytokines are known to affect the phenotypes of both DC and T cells improved CTL stimulation was only observed when IL-7 and IFNγ were delivered by the autocrine approach 14 , 15 . In sharp contrast, IL-2 gene delivery did not improve CTL generation by either the autocrine or paracrine strategy over exogenous addition 14 …”

pH-sensitive polymer-liposome-based antigen delivery systems potentiated with interferon-γ gene lipoplex for efficient cancer immunotherapy

“…Therefore, instead of IFN-γ molecule, IFN-γ-encoding gene has been used for the local production of IFN-γ by introducing the gene into immune cells or cancerous cells of diseased tissues [28][29][30][31][32].…”

“…To date, viral vectors of various kinds based on adenovirus, adeno-associated virus, and retrovirus have been used to deliver IFN-γ-encoding gene in vivo [28][29][30][31][32]. However, non-viral vectors with low immunogenicity and no pathogenicity might be preferred for gene delivery considering that virus-based gene vectors might entail risks of unexpected immune responses derived from viral components [33,34].…”