Attenuated Expression of A20 Markedly Increases the Efficacy of Double-Stranded RNA-Activated Dendritic Cells As an Anti-Cancer Vaccine

Abstract

A20 is a zinc finger protein with ubiquitin-modifying activity. A20 has been described as negatively regulating signaling induced by the TNF receptor and TLR family in a number of cell types, including mouse bone marrow-derived dendritic cells (DCs). However, the expression and effect of A20 in activated human monocyte-derived DCs have not been previously evaluated. We report that DCs activated with the TLR3 ligand poly(I:C) up-regulate A20. Down-regulating A20 demonstrated its role in the functional activation of DCs. A20 down-regulated DCs showed higher activation of the transcription factors NF-κB and activator protein-1, which resulted in increased and sustained production of IL-6, IL-10, and IL-12p70. We additionally silenced the immunosuppressive cytokine IL-10 and demonstrated that IL-10 inhibits T cell proliferation. We further demonstrated that A20 down-regulated DCs skew naive CD4+ T cells toward IFN-γ producing Th1 cells, a process which is dependent on IL-12p70 and which is unaffected by IL-10. Furthermore, A20 and/or IL-10 down-regulated DCs had an enhanced capacity to prime Melan-A/MART-1 specific CD8+ T cells. Finally, we demonstrated that potent T cell stimulatory DCs are generated by the simultaneous delivery of poly(I:C12U), A20, or A20/IL-10 small interfering RNA and Ag-encoding mRNA, introducing a one step approach to improve DC-based vaccines. Together these findings demonstrate that A20 negatively regulates NF-κB and activator protein-1 in DCs and that down-regulation of A20 results in DCs with enhanced T cell stimulatory capacity.

Mucosal and systemic anti-HIV immunity controlled by A20 in mouse dendritic cells

“…A20-deficient macrophages display prolonged NF-κB activity (27,28), and A20-silenced DCs induce robust antitumor immune responses to reject engrafted tumors in immunized mice (34). Recently, Breckpot et al reported that A20-silenced human DCs had an enhanced capacity to polarize the IFN-γ-producing CD4 + T cell response and to prime the tumor-specific CD8 + T cell response (35). These results indicate that A20 plays a critical role in controlling immunostimulatory potency of APCs.…”

Lentiviral Vectors in Immunotherapy

“…Lentivectors have successfully delivered to DCs short hairpin RNAs (shRNAs) targeting A20. The abrogation of A20 expression caused DC maturation, effective CD8 cell responses and inihibition of regulatory T cells (Tregs) [55,56].…”

Dendritic Cells and Lentiviral Vectors: Mapping the Way to Successful Immuno Gene Therapy

“…Therefore, A20 down-regulation could result in prolonged NF-κB activation, resulting in DCs with enhanced stimulatory capacity. LV delivered A20-targeted shRNA (Song et al 2008) and direct introduction of siRNA (Breckpot et al 2009) were applied to down-regulate A20 in mouse and human DCs, respectively. These approaches showed that A20 controls DC maturation, cytokine production and immunostimulatory potency.…”

“…Human DCs with down-regulated A20 expression had an increased NF-κB activity and showed enhanced and sustained IL-10 and IL-12 secretion. These DCs were more potent in stimulating Melan-A CTL responses (Breckpot et al 2009). …”