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 gamma (IFN-gamma), as the delivered gene can continuously produce that interferon. However which immune cell type should optimally express IFN-gamma is unclear as the phenotypes of both DC and T cells are enhanced by it. Here, we used AAV to compare and contrast IFN-gamma gene delivery into DC or T cells, and versus the addition of exogenous IFN-gamma, for stimulating carcinoembryonic antigen-(CEA-) specific CTL. It was found that AAV/IFN-gamma delivery into T cells (autocrine) resulted in T cell populations with the highest CD8(+)/CD4(+) ratio, highest IFN-gamma(+)/IL-4(+) ratio, highest CD69(+),CD8(+) levels, and lowest CD4(+)/CD25(+) levels, all consistent with the strongest Th1 response. Most importantly, AAV/IFN-gamma 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-gamma autocrine gene delivery into T cells is worthy of further study towards maximizing the generation of antigen-specific anticancer CTL killers.

Figure 1

Structure of the cell treatment protocol. Shown is the temporal treatment of the Mo/DC and T cells and is self-descriptive. However, note that AAV/IFN-γ is used to infect Mo/DC at day zero, or naive T cells just prior to coincubation with AAV/antigen-loaded DC on day 5.

Figure 2

Chromosomal integration of AAV/IFN-γ vector DNA into DC and T cells. Cells were infected as described in the Materials and Methods section and then analyzed for proviral integration by, first, PCR amplification of the vector-chromosomal junctions, followed by agarose gel electrophoresis of the PCR products, Southern blotting and probing with ³²P labeled vector DNA. Each band represents a separate AAV integrant within the cell population. (a) shows the results from DC. (b) shows the results from T cells.

Figure 3

Analysis of IFN-γ expression in transduced DC and T cells by RT-PCR. Cells were infected, mRNA isolated and analyzed by RT-PCR as described in Section 2. (a) shows the results from DC. (b) shows the results from T cells. Note that both AAV/IFN-γ transduced DC and T cells demonstrate IFN-γ expression. Also note that untransduced T cells expressed some IFN-γ, but that AAV/IFN-γ transduced T cells demonstrate higher IFN-γ expression. This is confirmed in Figure 4.

Figure 4

Transduction efficiency of AAV delivery into DC and T cells by intracellular staining. Cells were infected as described in Section 2 and analyzed by intracellular staining for IFN-γ. (a) shows the results from DC infection. (b) shows the results from T cell infection. Note that both AAV/IFN-γ transduced DC and T cells demonstrate high levels of IFN-γ expression, but that T cells exhibit a signifiucant basal level of expression.

Figure 5

IL-12 and IL-10 expression in DC Analysis of IL-12 and IL-10 expression in DC under treatment with exogenous IFN-γ treatment versus AAV/IFN-γ transduction. Note that the IL-12/IL-10 ratio was slightly enhanced by AAV/IFN-γ transduction.

Figure 6

Secretion of IFN-γ from AAV-transduced cells over time. Cells were treated as indicated and secretion of IFN-γ was measured in the conditioned medium at the indicated time by ELISA. The conditions include AAV/IFN-γ delivery into DC (with concurrent AAV/CEA delivery), (a); AAV/IFN-γ delivery into T cells, (b); AAV/IFN-γ only delivery into DC, (c); cells with no treatment, (d).

Figure 7

Proliferation of T cells. Tritium incorporation by T cells is shown under various treatments.

Figure 8

Enhanced CTL killing by IFN-γ autocrine delivery. (a) shows CTL killing after the indicated treatment, of CEA-positive targets, SW480 cells, by standard ⁵¹Cr release assay. All assays were done at a effector: target ratio of 20 : 1. Note that AAV/IFN-γ autocrine delivery resulted in CTL with the highest killing ability. (b) shows a similar CTL killing assay using CEA+ LCL cells as the target. Note that, again, AAV/IFN-γ autocrine delivery resulted in CTL with the highest killing ability.