Comparison of AAV/IL-7 autocrine (T cell) versus paracrine (DC) gene delivery for enhancing CTL stimulation and function

Abstract

Adoptive transfer of antigen-specific cytotoxic T lymphocyte (CTL) into patients holds promise in treating cancer. Such anti-cancer CTL are stimulated by professional antigen-presenting dendritic cells (DC). We hypothesize the gene delivery of various Th1-response cytokines, such as interleukin 7 (IL-7), should further enhance CTL stimulation and activity. However, the issue as to which cell type, DC (paracrine) or the T cell (autocrine), should express a particular Th1 cytokine gene for optimal CTL stimulation has never been addressed. We used adeno-associated virus-2 (AAV) to compare delivery of IL-7 and IL-2 genes into DC or T cells and to exogenous commercial cytokines for generating robust carcinoembryonic antigen (CEA)-specific CTL. AAV/IL-7 transduction of T cells (autocrine delivery) generated CTL with the highest killing capability. Consistent with this, AAV/IL-7 delivery generated T cell populations with the highest proliferation, highest interferon gamma expression, highest CD8(+):CD4(+) ratio, highest CD8(+), CD69(+) levels, and lowest CD4(+), CD25(+) (Treg) levels. These data are consistent with higher killing by the AAV/IL-7-altered CTL. These data strongly suggest that IL-7 autocrine gene delivery is optimal for CTL generation. These data also suggest Th1 cytokine autocrine versus paracrine delivery is an important issue for immuno-gene therapy and uncovers new questions into cytokine mechanism of action.

Fig. 1

Structure of the cell treatment protocol. This figure shows the temporal treatment of the Mo/DC and T cells and is self-descriptive. However, note that AAV/cytokine vectors are used to infect Mo/DC at day zero, or naive T cells just prior to co-incubation with AAV/antigen-loaded DC on day 5

Fig. 2

Secretion of IL-2 and IL-7 in AAV-transduced cells over time. a Secretion of of IL-2 from transduced DC by ELISA assay. b Secretion of IL-7 from transduced DC by ELISA assay. c Secretion of of IL-2 from transduced T cells by ELISA assay. d Secretion of of IL-7 from transduced T cells by ELISA assay

Fig. 3

Analysis of IL-12 and IL-10 expression by DC and IFNγ expression by T cells under various treatments. a Analysis of IL-12 and IL-10 expression in DC under various treatments as indicated by intracellular staining. b Percentage of T cells which express IFNγ, after indicated DC (paracrine) treatment, as determined by intracellular staining. c Percentage of T cells which express IFNγ, after indicated T cell (autocrine) treatment, as determined by intracellular staining. d Proliferation of T cells resulting from indicated treatments

Fig. 4

Enhanced CTL killing, and antigen specific killing, by IL-7 autocrine delivery. a CTL killing after the indicated treatment, of CEA-positive targets, by standard ⁵¹Cr release assay. All assays were done at a effector:target ratio of 20:1. Note that AAV/IL-7 autocrine delivery resulted in CTL with the highest killing ability. b The key for section C. c CEA-specific CTL were tested for the ability to kill a variety of cellular targets by standard ⁵¹Cr release assay. All assays were done at a effector:target ratio of 20:1. Note that only the CEA-positive SW480 cell line was killed at a significant level