Tumor-specific CD8+ T cells can potentially be activated by two distinct mechanisms of major histocompatibility complex class I–restricted antigen presentation as follows: direct presentation by tumor cells themselves or indirect presentation by professional antigen-presenting cells (APCs). However, controversy still exists as to whether indirect presentation (the cross-priming mechanism) can contribute to effective in vivo priming of tumor-specific CD8+ T cells that are capable of eradicating cancer in patients. A clinical trial of vaccination with granulocyte macrophage–colony stimulating factor–transduced pancreatic cancer lines was designed to test whether cross-presentation by locally recruited APCs can activate pancreatic tumor-specific CD8+ T cells. Previously, we reported postvaccination delayed-type hypersensitivity (DTH) responses to autologous tumor in 3 out of 14 treated patients. Mesothelin is an antigen demonstrated previously by gene expression profiling to be up-regulated in most pancreatic cancers. We report here the consistent induction of CD8+ T cell responses to multiple HLA-A2, A3, and A24-restricted mesothelin epitopes exclusively in the three patients with vaccine-induced DTH responses. Importantly, neither of the vaccinating pancreatic cancer cell lines expressed HLA-A2, A3, or A24. These results provide the first direct evidence that CD8 T cell responses can be generated via cross-presentation by an immunotherapy approach designed to recruit APCs to the vaccination site.

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In this report, we evaluated the efficiency of stable gene transfer into established CD8(+) human tumor antigen-specific cytotoxic T cell (CTL) lines and peripheral blood lymphocytes (PBL) by oncoretroviral and lentiviral vectors. In the oncoretroviral vector, the green fluorescent protein (GFP) reporter gene was regulated by the murine stem cell virus (MSCV) promoter. In three human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors, the GFP transgene was regulated by either a chimeric MSCV/HIV-1 promoter, or cellular promoters from human housekeeping genes PGK and EF1 alpha. We found that several lines of proliferating tumor-specific CTL were poorly (=2%) transduced by the oncoretroviral vector that transduced Jurkat T cell line efficiently (=80%). In contrast, three lentiviral vectors transduced 38-63% of these proliferating CTL. More interestingly, all lentiviral vectors packaged without the HIV-1 accessory proteins transduced human bulk PBL and purified CD4(+) and CD8(+) lymphocyte subsets without prior stimulation. Detailed analysis indicated that the lentiviral vectors containing the EF1 alpha or PGK ubiquitous promoter can transduce unstimulated PBL and achieve low-level transgene expression in the absence of any T-cell activation. However, T-cell activation subsequent to the transduction of unstimulated PBL is required for high-level transgene expression. Transduced PBL expressing transgene delivered by the lentiviral vectors still preserved resting and naïve cell phenotypes. Taken together, prior T cell stimulation and HIV-1 accessory proteins are dispensable for lentivirus-mediated gene transfer into resting naïve and memory T lymphocytes. These results will have significant implications for the study of T-cell biology and for the improvement of clinical gene therapies of acquired immune deficiency syndrome (AIDS) and cancer.

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Autonomic neuropathy (AN) is common in patients with chronic liver disease. For hitherto unknown reasons, in longitudinal studies, the presence of AN has been found to be an independent predictor of mortality in patients with cirrhosis. We hypothesized that patients with AN are more likely to develop hepatic encephalopathy (HE) due to prologed intestinal transit time. In this study, we examined the incidence of new-onset HE in patients with and without AN. Seventy-two patients (Child A/B/C = 35/31/6) without evidence of HE at the time of autonomic function testing (AFT) were followed for 39.5 +/- 27.3 months. The end point of the study was the development of new onset HE. Patients were followed until death or liver transplantation. Of the 72 patients, 42 (58%) patients did not develop HE (group A) while 30 (42%) developed HE (group B) during the follow-up. Both groups had similar baseline demographics. AN was more common in group B (27/30; 90%) compared to group A (28/42; 67%) (P = 0.02). Kaplan-Meier analysis showed a trend toward a higher incidence of HE in patients with AN. Mortality was higher in group B (12/30; 40%) compared to group A (8/42; 19%) (P = 0.04). Patients with AN were more likely to develop new onset HE. Although an independent effect of AN on HE was not established in this study, we speculate that delayed intestinal transit secondary to AN may explain the higher incidence of HE in patients with AN.