DCs Pulsed with Novel HLA-A2-Restricted CTL Epitopes against Hepatitis C Virus Induced a Broadly Reactive Anti-HCV-Specific T Lymphocyte Response

OBJECTIVE: To determine the capacity of dendritic cells (DCs) loaded with single or multiple-peptide mixtures of novel hepatitis C virus (HCV) epitopes to stimulate HCV-specific cytotoxic T lymphocyte (CTL) effector functions. METHODS: A bioinformatics approach was used to predict HLA-A2-restricted HCV-specific CTL epitopes, and the predicted peptides identified from this screen were synthesized. Subsequent IFN-γ ELISPOT analysis detected the stimulating function of these peptides in peripheral blood mononuclear cells (PBMCs) from both chronic and self-limited HCV infected subjects (subjects exhibiting spontaneous HCV clearance). Mature DCs, derived in vitro from CD14(+) monocytes harvested from the study subjects by incubation with appropriate cytokine cocktails, were loaded with novel peptide or epitope peptide mixtures and co-cultured with autologous T lymphocytes. Granzyme B (GrB) and IFN-γ ELISPOT analysis was used to test for epitope-specific CTL responses. T-cell-derived cytokines contained in the co-cultured supernatant were detected by flow cytometry. RESULTS: We identified 7 novel HLA-A2-restricted HCV-specific CTL epitopes that increased the frequency of IFN-γ-producing T cells compared to other epitopes, as assayed by measuring spot forming cells (SFCs). Two epitopes had the strongest stimulating capability in the self-limited subjects, one found in the E2 and one in the NS2 region of HCV; five epitopes had a strong stimulating capacity in both chronic and self-limited HCV infection, but were stronger in the self-limited subjects. They were distributed in E2, NS2, NS3, NS4, and NS5 regions of HCV, respectively. We also found that mDCs loaded with novel peptide mixtures could significantly increase GrB and IFN-γ SFCs as compared to single peptides, especially in chronic HCV infection subjects. Additionally, we found that DCs pulsed with multiple epitope peptide mixtures induced a Th1-biased immune response. CONCLUSIONS: Seven novel and strongly stimulating HLA-A2-restricted HCV-specific CTL epitopes were identified. Furthermore, DCs loaded with multiple-epitope peptide mixtures induced epitope-specific CTLs responses

Enhanced Virus-Specific CD8+ T Cell Responses by Listeria monocytogenes-Infected Dendritic Cells in the Context of Tim-3 Blockade

In this study, we engineered Listeria monocytogens (Lm) by deleting the LmΔactA/ΔinlB virulence determinants and inserting HCV-NS5B consensus antigens to develop a therapeutic vaccine against hepatitis C virus (HCV) infection. We tested this recombinant Lm-HCV vaccine in triggering of innate and adaptive immune responses in vitro using immune cells from HCV-infected and uninfected individuals. This live-attenuated Lm-HCV vaccine could naturally infect human dendritic cells (DC), thereby driving DC maturation and antigen presentation, producing Th1 cytokines, and triggering CTL responses in uninfected individuals. However, vaccine responses were diminished when using DC and T cells derived from chronically HCV-infected individuals, who express higher levels of inhibitory molecule Tim-3 on immune cells. Notably, blocking Tim-3 signaling significantly improved the innate and adaptive immune responses in chronically HCV-infected patients, indicating that novel strategies to enhance the potential of antigen presentation and cellular responses are essential for developing an effective therapeutic vaccine against HCV infection

Enhanced cytotoxicity and decreased CD8 dependence of human cancer-specific cytotoxic T lymphocytes after vaccination with low peptide dose.

In mice, vaccination with high peptide doses generates higher frequencies of specific CD8+ T cells, but with lower avidity compared to vaccination with lower peptide doses. To investigate the impact of peptide dose on CD8+ T cell responses in humans, melanoma patients were vaccinated with 0.1 or 0.5 mg Melan-A/MART-1 peptide, mixed with CpG 7909 and Incomplete Freund's adjuvant. Neither the kinetics nor the amplitude of the Melan-A-specific CD8+ T cell responses differed between the two vaccination groups. Also, CD8+ T cell differentiation and cytokine production ex vivo were similar in the two groups. Interestingly, after low peptide dose vaccination, Melan-A-specific CD8+ T cells showed enhanced degranulation upon peptide stimulation, as assessed by CD107a upregulation and perforin release ex vivo. In accordance, CD8+ T cell clones derived from low peptide dose-vaccinated patients showed significantly increased degranulation and stronger cytotoxicity. In parallel, Melan-A-specific CD8+ T cells and clones from low peptide dose-vaccinated patients expressed lower CD8 levels, despite similar or even stronger binding to tetramers. Furthermore, CD8+ T cell clones from low peptide dose-vaccinated patients bound CD8 binding-deficient tetramers more efficiently, suggesting that they may express higher affinity TCRs. We conclude that low peptide dose vaccination generated CD8+ T cell responses with stronger cytotoxicity and lower CD8 dependence