Itziar Echeverria

Enhanced T cell responses against hepatitis C virus by ex vivo targeting of adenoviral particles to dendritic cells

Injection of dendritic cells (DCs) presenting viral proteins constitutes a promising approach to stimulate T cell immunity against hepatitis C virus (HCV). Here we describe a strategy to enhance antigen loading and immunostimulatory functions of DCs useful in the preparation of therapeutic vaccines. Incubation of murine DCs with CFm40L, an adapter molecule containing the coxsackie‐adenovirus receptor fused to the ecto‐domain of murine CD40L‐induced DC maturation, produced high amounts of interleukin‐12 and up‐regulation of molecules associated with T helper 1 responses. Accordingly, targeting of an adenovirus encoding HCV NS3 protein (AdNS3) to DCs with CFm40L strongly enhanced NS3 presentation in vitro, activating interferon‐γ–producing T cells. Moreover, immunization of mice with these DCs promoted strong CD4 and CD8 T cell responses against HCV NS3. CFh40L, a similar adapter molecule containing human CD40L, enhanced transduction and maturation of human monocyte‐derived DCs. Comparison of DCs transduced with AdNS3 and CFh40L from patients with chronic HCV infection and healthy donors revealed similar maturation levels. More importantly, DCs from the patients induced NS3‐specific responses when transduced with AdNS3 and CFh40L but not with AdNS3 alone. Conclusion: DCs transduced with AdNS3 and the adapter molecule CFm/h40L exhibit enhanced immunostimulatory functions, induce robust anti‐HCV NS3 immunity in animals, and can induce antiviral immune responses in subjects with chronic HCV infection. This strategy may serve as therapeutic vaccination for patients with chronic hepatitis C. (HEPATOLOGY 2011;)

Oncostatin M Enhances the Antiviral Effects of Type I Interferon and Activates Immunostimulatory Functions in Liver Epithelial Cells

ABSTRACT Oncostatin M (OSM) is released together with type I interferon (IFN) by activated dendritic cells, suggesting a concerted action of these cytokines in the biological response against infection. We found that OSM increases the antiviral effect of IFN-α in Huh7 hepatoma cells infected with hepatitis A or hepatitis C virus and synergizes with IFN-α in the induction of antiviral genes. The combination of OSM and IFN-α led to upregulation of both STAT1 and STAT3 together with intense and prolonged activation of STAT1, STAT3, and Jak1. OSM with or without IFN-α also activated p38 mitogen-activated protein kinase, which is known to enhance transcription of IFN-α-inducible genes. Interestingly, OSM combined with IFN-α strongly induced immunoproteasome genes and other genes involved in antigen processing and presentation. Moreover, OSM, alone or in combination with IFN-α, upregulated relevant innate immunity molecules and increased the expression of intracellular adhesion molecule 1 and interleukin-15 receptor alpha (IL-15Rα) in liver cells. Hepatoma cells transfected with a plasmid encoding a viral antigen were able to activate effector T cells when pretreated with IFN-α plus OSM but not with each cytokine separately. Also, OSM, more than IFN-α, augmented the ability of Huh7 cells to transpresent IL-15 to responding lymphocytes and increased the immunostimulatory activity of liver epithelial cells by presenting a short viral peptide to sensitized cytotoxic T cells. In conclusion, OSM enhances the antiviral effects of type I interferon and cooperates with it in the induction of adaptive immune responses to pathogens. These findings may have therapeutic implications.

Low molecular weight hyaluronan preconditioning of tumor-pulsed dendritic cells increases their migratory ability and induces immunity against murine colorectal carcinoma.

We have recently shown that systemic administration of low molecular weight hyaluronan (LMW HA) significantly reduces colorectal carcinoma (CRC) growth in vivo. The elicited response is partially mediated by activated dendritic cells (DC). To potentiate the ability of DC loaded with whole tumor lysate (DC/TL) to induce immunity against CRC in mice, we aimed to study the effects of preconditioning DC with LMW HA for therapeutic vaccination. LMW HA improved maturation of ex vivo generated DC, increased IL-12, decreased IL-10 production, and enhanced a MLR activity in vitro. Although TNF-α showed a similar capacity to mature DC, preconditioning of DC/TL with LMW HA increased their ability to migrate in vitro toward CCL19 and CCL-21 in a CD44- and a TLR4-independent manner; this effect was superior to Poly(I:C), LPS, or TNF-α and partially associated with an increase in the expression of CCR7. Importantly, LMW HA dramatically enhanced the in vivo DC recruitment to tumor-regional lymph nodes. When these LMW HA-treated CRC tumor lysate-pulsed DC (DC/TL/LMW HA) were administered to tumor-bearing mice, a potent antitumor response was observed when compared to DC pulsed with tumor lysate alone and matured with TNF-α. Then, we showed that splenocytes isolated from animals treated with DC/TL/LMW HA presented a higher proliferative capacity, increased IFN-γ production, and secreted lower levels of the immunosuppressive IL-10. Besides, increased specific CTL response was observed in DC/TL/LMW HA-treated animals and induced long-term protection against tumor recurrence. Our data show that LMW HA is superior to other agents at inducing DC migration; therefore, LMW HA could be considered a new adjuvant candidate in the preparation of DC-based anticancer vaccines with potent immunostimulatory properties.

Improved dendritic cell‐based immunization against hepatitis C virus using peptide inhibitors of interleukin 10

The high levels of interleukin 10 (IL‐10) present in chronic hepatitis C virus (HCV) infection have been suggested as responsible for the poor antiviral cellular immune responses found in these patients. To overcome the immunosuppressive effect of IL‐10 on antigen‐presenting cells such as dendritic cells (DCs), we developed peptide inhibitors of IL‐10 to restore DC functions and concomitantly induce efficient antiviral immune responses. Two IL‐10‐binding peptides (p9 and p13) were selected using a phage‐displayed library and their capacity to inhibit IL‐10 was assessed in a bioassay and in STAT‐3 (signal transducer and activator of transcription 3) phosphorylation experiments in vitro. In cultures of human leukocytes where HCV core protein induces the production of IL‐10, p13 restored the ability of plasmacytoid DC to produce interferon alpha (IFN‐α) after Toll‐like receptor 9 (TLR9) stimulation. Similarly, when myeloid DCs were stimulated with CD40L in the presence of HCV core, p9 enhanced IL‐12 production by inhibiting HCV core‐induced as well as CD40L‐induced IL‐10. Moreover, in vitro, p13 potentiated the effect of maturation stimuli on human and murine DC, increasing their IL‐12 production and stimulatory activity, which resulted in enhanced proliferation and IFN‐γ production by responding T‐cells. Finally, immunization with p13‐treated murine DC induced stronger anti‐HCV T‐cell responses not only in wildtype mice but also in HCV transgenic mice and in mice transiently expressing HCV core in the liver. Conclusion: These results suggest that IL‐10 inhibiting peptides may have important applications to enhance anti‐HCV immune responses by restoring the immunostimulatory capabilities of DC. (HEPATOLOGY 2011.)

Clinical testing of a dendritic cell targeted therapeutic vaccine in patients with chronic hepatitis C virus infection.

The lack of antiviral cellular immune responses in patients with chronic hepatitis C virus (HCV) infection suggests that T-cell vaccines may provide therapeutic benefit. Due to the central role that dendritic cells (DC) play in the activation of T-cell responses, our aim was to carry out a therapeutic vaccination clinical trial in HCV patients using DC. Five patients with chronic HCV infection were vaccinated with three doses of 5 × 106 or 107 autologous DC transduced with a recombinant adenovirus encoding NS3 using the adapter protein CFh40L, which facilitates DC transduction and maturation. No significant adverse effects were recorded after vaccination. Treatment caused no changes in serum liver enzymes nor in viral load. Vaccination induced weak but consistent expansion of T-cell responses against NS3 and adenoviral antigens. Patients’ DC, as opposed to murine DC or DC from healthy subjects, secreted high IL-10 levels after transduction, inducing the activation of IL-10–producing T cells. IL-10 blockade during vaccine preparation restored its ability to stimulate anti-NS3 Th1 responses. Thus, vaccination with adenovirus-transduced DC is safe and induces weak antiviral immune responses. IL-10 associated with vaccine preparation may be partly responsible for these effects, suggesting that future vaccines should consider concomitant inhibition of this cytokine.