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Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy. A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector. We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24-48 h and expression remains detectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects. DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100+ metastatic melanoma. Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives ( approximately 7-8 h) on the surface of HLA-A*0201+ cells. Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8+ T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.

These studies test whether allograft rejection can be blocked by interference with leukocyte adhesion, using a murine IgG2a mAb (R6.5) reactive with monkey ICAM-1 (CD54). In 16 Cynomolgus renal allograft recipients, R6.5 was administered prophylactically as the sole immunosuppressive agent for 12 days (0.01 to 2 mg/kg/day). Survival in 14 recipients with technically successful grafts was significantly prolonged (24.2 +/- 2.4 vs 9.2 +/- 0.6 days for controls; p less than 0.001). Intercellular adhesion molecule-1 (CD54) (ICAM-1) was expressed on vascular endothelium in the kidney and other organs in the monkey in a pattern similar to that in humans. During cellular rejection in controls, ICAM-1 expression increased on endothelial cells, infiltrating mononuclear leukocytes and tubular cells. Biopsies during R6.5 administration showed decreased T cell infiltration (CD2, CD8, CD4) compared with controls and decreased arterial endothelial inflammation. No changes occurred in circulating T cells, aside from variable coating with mIgG. In six of eight other recipients R6.5 administration (0.5 to 2 mg/kg/day for 10 days) reversed preexisting rejection that resulted from taper of Cyclosporine to subtherapeutic levels. Responding grafts showed decreased edema and hemorrhage but no consistent change in the infiltrate. At 1 h after the first dose, mouse IgG deposited primarily on the graft vascular endothelium without any change in the inflammatory infiltrate. Mouse IgG also deposited on the endothelium of normal organs without eliciting an inflammatory response and was cleared from the endothelium within 4 days. Inasmuch as the principal site of binding was the vascular endothelium, we hypothesize that the antibody blocks adhesion to graft ICAM-1 molecules on the vessels. Anti-ICAM-1 also binds to recipient cells and may interfere with Ag presentation and/or T cell interactions. Whatever the mechanism(s), these studies indicate that an anti-ICAM-1 antibody inhibits T cell mediated injury in vivo, and that ICAM-1 is a critical molecule in the pathogenesis of allograft rejection.

The phenotype of T lymphocyte subsets present in renal biopsies showing acute cellular allograft rejection in six patients on cyclosporine have been characterized in situ by immunoperoxidase staining, and after expansion in vitro in interleukin 2 (IL-2) by two-color flow cytometry, sorting, and functional analysis. After 8 to 42 days in organ culture, both Leu-3+ (CD4) and Leu-2+ (CD8) subsets were found in each culture, in a ratio that varied from 0.2 to 5.0, which was not significantly different than the results of in situ immunoperoxidase staining of the uncultured biopsy. The cultured cells were almost all Leu-4+ (CD3) T cells (89% +/- 4), which expressed the activation markers DR (82% +/- 6) and the IL 2 (CD25) receptor (15% +/- 4). The Leu-3+ cells were largely Leu-8- (90% +/- 6), whereas a minority of the Leu-2+ cells were Leu-15+ (CD11) (26% +/- 4). Only a small fraction of the Leu-2+ cells stained for Leu-7 (8% +/- 6). Functional analysis of FACS-purified Leu-2-3+ and Leu-2+3- populations indicated that both subsets proliferated in response to graft donor antigens in a mixed lymphocyte reaction (MLR) and produced IL 2. Only the Leu-2+3- population demonstrated donor-specific cytotoxic activity. A minor subpopulation in each culture were both Leu-3+ and Leu-2+ (2.0%). Leu-2+3+ cells from one biopsy were purified to homogeneity (99.8%), and were found to express the T cell antigen receptor complex Ti/CD3 (WT-31+, Leu-4+), but not the common thymocyte antigen CD1 (OKT6). The Leu-2+3+ cells neither responded in the MLR, nor showed any cytotoxic capacity. The Leu-2+3+ cells were capable of IL 2 but not interferon-gamma production. None of the purified cultures demonstrated NK activity. A subset of the purified Leu-2+3+ cells lost Leu-2+ during 1 to 3 wk in culture, and became Leu-2-3+. These studies provide evidence that the cells that infiltrate renal allografts during rejection include alloproliferative, lymphokine-producing cells of both Leu-2+ and Leu-3+ subsets. The Leu-2+3- cells are also highly cytotoxic against donor lymphocytes, indicating the presence of helper independent cytotoxic T cells. A minor population of Leu-2+3+ T cells that do not express donor specific function was also identified.