Maturation, activation, and protection of dendritic cells induced by double-stranded RNA

Abstract

The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses.

Figure 1

Susceptibility to influenza virus infection of DC populations. (A and B) Percentage of DCs expressing viral proteins at high levels (A) or high plus low levels (B) 14 h after infection with different doses of PR8; immature DCs (▪), TNF-α–matured DCs (▵), LPS-matured DCs (⋄), IFN-α–treated DCs (○). The proportion of DCs expressing viral proteins and the level of expression were comparable when measured at 40 h (data not shown). (C and D) Level of expression of viral protein in DCs infected with PR8 at 5 HAU/ml (C) or 0.1 HAU/ml (D). The DCs were either untreated (1) or pretreated for 30 h with TNF-α (2), LPS (3), or IFN-α (4). Second antibody control (0).

Figure 2

LPS-matured DCs express MxA protein and are more resistant than immature DCs to the cytopathic effect of influenza virus at high MOI. Immature DCs were left untreated (A and H) or were infected with PR8 at 1 HAU/ml (B and I), 10 HAU/ml (C and L) or 30 HAU/ ml (D and M). Mature DCs (40 h after LPS) were left untreated (E and N) or were infected with PR8 at 10 HAU/ml (F and O) or 100 HAU/ml (G and P). After 24 h the cells were tested for the expression of MxA and influenza HA proteins (A–G). Viable, early and late apoptotic cells were evaluated in the same samples by staining with FITC-labeled Annexin V and propidium iodide (H–P).

Figure 3

MxA expression is rapidly induced in DCs by LPS, poly I:C and viral infection. Time course of MxA upregulation as detected by intracellular staining (A) or immunoblotting (B). (C) Time course of type I IFN production in culture supernatant. DCs were stimulated with the following: 50 U/ml IFN-α (○, panel A only), LPS (▵), 20 μg/ml poly I:C (▪), 1 HAU PR8 (•), TNF-α (▿), and CD40L (⋄). (D) MxA induction after 5 h of stimulation in the absence (black bar) or in the presence of two neutralizing sheep antisera to human type I IFN: Iivari, hatched bars, and Kaaleppi, empty bars.

Figure 4

dsRNA and PR8 infection induce upregulation of total protein and HLA class I synthesis in untreated as well as in IFN-α–pretreated DCs. Total protein synthesis (hatched bars) and HLA class I synthesis (black bars) were measured in DCs (A and C) or Hela cells (B and D) 5 h after stimulation with 20 μg/ml poly I:C, 5 HAU PR8, or medium alone. The cells were either untreated (A and B) or pretreated for 24 h with 500 U/ml IFN-α (C and D).

Figure 5

Increase in MHC class I biosynthesis and stability induced by PR8 infection allows efficient presentation of viral antigen to cytotoxic T cells. (A) Synthetic rate and stability of HLA class I molecules in immature DCs and in DCs that were stimulated for 5 h with 5 HAU/ml PR8 or LPS. Labeled class I molecules were precipitated after 1 h of chase (time 0) or after 12 or 24 h. (B) Half-life of labeled HLA class I molecules quantitated by PhosphorImager in immature DCs (○), LPS-treated DC (•), or PR8-infected DC (▴). (C) Proliferative response of an HLA-A2– restricted M58-66–specific T cell clone cultured with graded numbers of HLA-A2⁺ DCs. DCs were either pulsed with 1 μM M58-66 peptide (circles) or infected with 5 HAU/ml PR8 with (triangles) or without (squares) IFN-α (500 U/ml) pretreatment. DCs were tested 5 h (open symbols) or 24 h after pulsing (filled symbols).

Figure 6

PR8 infection and poly I:C increase the T cell stimulatory capacity of DCs. Proliferative response of cord blood naive T cells stimulated with graded numbers of irradiated allogeneic DCs. DCs were untreated (•) or pretreated for 24 h with 50 U/ml IFN-α (×), LPS (⋄), TNF-α (▵), or 20 μg/ml poly I:C (□), or infected with 3 HAU/ml PR8 (○).

Figure 7

Poly I:C primes for a Th1 response. IFN-γ and IL-4 production by polyclonal T cell lines generated by stimulating purified CD45RA⁺CD4⁺ T cells with allogeneic DCs. A, TNF-α–matured DCs; B, poly I:C-matured DCs; C, poly I:C-matured DCs in the presence of neutralizing antibodies to IL-12. A strong Th1 response was also obtained with LPS-matured DCs (data not shown). In some experiments both anti–IL-12 and anti–IFN-α antibodies were required to inhibit Th1 development. Anti–IL-12 antibodies did not affect polarization induced by TNF-α–matured or immature DCs.