AAV2/IL-12 gene delivery into dendritic cells (DC) enhances CTL stimulation above other IL-12 applications: Evidence for IL-12 intracrine activity in DC

Abstract

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) holds significant promise in treating cancer and Th1 response cytokines are critical for their stimulation. Recently we reported that interleukin 7-(IL-7) and interferongamma-(IFNγ) autocrine/T cell gene delivery resulted in superior ex vivo CTL stimulation over paracrine/DC delivery. IL-12 is yet another important Th1 cytokine which affects both DC and T cells. Here, using adeno-associated virus Type 2 (AAV2) gene delivery, IL-12-paracrine/DC gene delivery gave significantly superior stimulation of carcinoembryonic antigen (CEA)-specific CTL killing over that induced by autocrine gene delivery (or exogenous IL-12 addition). This is surprising as both AAV2/IL-12-treated T cells and DC secreted approximately the same level of IL-12. Paracrine IL-12 gene delivery also resulted in highest IL-12/IL-10 secretion ratio by DC and highest CD40, CD80, CD83 and CD86 expression. Moreover, AAV2/IL-12-DC stimulated the highest T-cell IFNγ production, highest T cell proliferation, highest CD69+/CD8+ levels, and lowest level of CD25+/CD4+ Treg. These data strongly suggest that the primary activity of IL-12 during CTL generation is upon the DC. These data are also consistent with there being novel activity for IL-12 within the DC itself, not involving its surface receptor; an "intracrine" activity. Given the plethora of IL-12 studies, these data also suggest that this gene delivery comparison approach could be useful for uncovering new cytokine activities and mechanism(s) of action gone unrecognized by conventional immunologic assays. Finally, these data further suggest AAV2/IL-12 intracrine gene delivery into DC may have utility in immunotherapy protocols involving antigen-specific CTL.

Figure 1. Virus structures and experimental scheme. (A) shows the structures of the AAV2/CEA and AAV2/IL12 vectors (not to scale). (B) shows the temporal experimental protocol for transducing DC and T cells, and stimulating CTL. Note that AAV/cytokine vectors either infected Mo/DC at day zero, or naive T cells just prior to co-incubation with AAV/antigen-loaded DC.

Figure 2. Provirus integration, transgene RNA and protein expression. Shown is the AAV proviral DNA chromosomal integration into DC (panel A) and T cells (panel B) by PCR amplification of vector-chromosomal (AluI element) junctions. Also shown is the transcriptional expression of the rAAV provirus and their respective transgenes by RT-PCR analysis for CEA in transduced DC (panel C), IL-12 in transduced DC (panel D), and IL-12 in transduced T cells (panel E). Panels F-H show transduction efficiency of AAV2/CEA and AAV2/IL-12 in DC and T cells. Transduction efficiency in DC was about 90% (panels F and G). The transduction efficency of CD3+ T cells was approximately 79% (panel H).

Figure 3. Transduction efficiency and effect on DC. (A) shows IL-12 and IL-10 secretion by DC under various treatments as analyzed by ELISA. Note that AAV/IL-12 treated DC secreted the highest level of IL-12 and the lowest producing IL-10. (B) shows an analysis of IL-12 and IL-10 percent expression in DC under various treatments as analyzed by intracellular staining. Note thatAAV/IL-12 treated DC had the highest percentage of cells actively producing IL-12 and the lowest producing IL-10.

Figure 4. Characterization of T cells under indicated treatments. (A) shows the amount of IL-12 secreted by T cells at 80 h after AAV/IL-12 transduction under the indicated treatment. Note that secretion levels of T cells were similar to those of DC shown in Figure 3A. (B) shows the percentage of T cells which express IFN(-γ, after indicated treatment, as determined by intracellular staining. (C) shows the Proliferation of CD3+ T cells, after the indicated treatment, as measured by the incorporation of 3H-TdR.

Figure 5. CEA-specific CTL killing resulting from indicated treatment by standard ⁵¹Cr release assay. All panels show anti-CEA CTL killing stimulated from the indicated treatments. (A) utilizes SW480 (CEA+, HLA A2+) as targets. Note that the CTL stimulated by AAV/IL-12 transduced DC cells was significantly higher than when the T cells, themselves, were AAV/IL-12 transduced, or controls. Also note killing was MHC/HLA class I-restricted. (B) utilizes genetically altered CEA+/LCL (HLA A2+) cells as targets and gives similar results to A. (C) shows CEA-specific CTL are tested against a variety of cancer cells demonstrating antigen-specific killing. K562 cells, commonly used to demonstrate NK killing, are not significant targets of anti-CEA CTL.