CD86 and IL-12p70 are key players for T helper 1 polarization and natural killer cell activation by Toll-like receptor-induced dendritic cells

Abstract

Background: Dendritic cells (DCs) determine the activation and polarization of T cells via expression of costimulatory molecules and secretion of cytokines. The function of DCs derived from monocytes ex vivo strongly depends on the composition of the maturation cocktail used.

Methodology/principal findings: We analyzed the effect of costimulatory molecule expression and cytokine secretion by DCs on T and natural killer (NK) cell activation by conducting a head-to-head comparison of a Toll-like receptor (TLR) agonist-based cocktail with the standard combination of proinflammatory cytokines or IL-10 alone. We could show that TLR-induced DCs are characterized by a predominance of costimulatory over coinhibitory molecules and by high secretion of IL-12p70, but not IL-10. Functionally, these signals translated into an increase in IFN-γ secreting Th1 cells and a decrease in regulatory T cells. T cell activation and polarization were dependent on IL-12p70 and CD86, but remarkably not on CD80 signaling. By means of IL-12p70 secretion, only TLR-induced DCs activated NK cells.

Conclusions/significance: TLR-matured DCs are highly suitable for application in immunotherapeutic strategies that rely on strong type 1 polarization and NK cell activation. Their effects particularly depend on high CD86 expression and IL-12p70 secretion.

Figure 1. Costimulatory profiles of TLR-3-DCs and cc-7-DCs.

TLR-3-DCs and cc-7-DCs were generated from peripheral blood of healthy donors, and expression of various costimulatory markers was analyzed by flow cytometry. *, p<0.05; **, p<0.01. (A) Expression of the cell surface antigens on both DC populations (n = 7 for B7-H4, n = 8 for CD276, n = 10 for all other markers). (B) Comparison of the CD86/CD274 ratio for TLR-3-DCs and cc-7-DCs (n = 10).

Figure 2. Costimulatory profiles of TLR-3-DCs, cc-3-DCs and IL10-3-DCs.

TLR-3-DCs, cc-3-DCs and IL10-3-DCs were generated from peripheral blood of healthy donors, and expression of various costimulatory markers was analyzed by flow cytometry. Differences between TLR-3-DCs and cc-3-DCs were tested. *, p<0.05; **, p<0.01. (A) Expression of the cell surface antigens on all three DC populations (n = 10). (B) Comparison of the CD86/CD274 ratio for all three DC populations (n = 10).

Figure 3. Cytokine secretion patterns of TLR-3-DCs, cc-3-DCs and IL10-3-DCs.

DCs generated from peripheral blood of healthy donors were analyzed for their cytokine secretion patterns (n = 9). (A) Mature DCs were cocultured with CD40L expressing mouse fibroblasts for 24 hours, the concentration of IL-12p70 and IL-10 in the supernatants was measured by CBA, and the difference to the basal secretion of the same cell populations without CD40 ligation was calculated. (B) Comparison of the IL-12p70/IL-10 ratio for TLR-3-DCs, cc-3-DCs and IL10-3-DCs.

Figure 4. Preferential induction of activated, IFN-γ secreting T cells by TLR-3-DCs.

(A to C) TLR-3-DCs and cc-7-DCs generated from peripheral blood of healthy donors were cocultured with autologous monocyte-depleted (non-adherent) PBMCs for 24 hours, and antigen-independent stimulatory capacity of the DCs on regulatory and activated T cells was quantified using flow cytometry with the markers CD4, CD25, CD127 and FoxP3. *, p<0.05; **, p<0.01. (A) Data from one representative donor. Gating strategies used to determine different T cell subsets. (B) Capacity of TLR-3-DCs and cc-7-DCs to stimulate CD4⁺CD25⁺ T cells and their regulatory (FoxP3⁺CD127^-) and non-regulatory activated (FoxP3^-CD127⁺) subsets. Differences between stimulated and unstimulated cells are shown (n = 10). (C) Ratio of activated and regulatory T cells induced by coculture. (D) TLR-3-DCs, cc-3-DCs and IL10-3-DCs were cocultured with CD3-selected autologous T cells for 4 days. Supernatants were analyzed for secretion of IFN-γ, IL-4 and IL-17A by CBA. Differences between stimulated and unstimulated cells are shown (n = 10).

Figure 5. Effect of costimulation and IL-12p70 blockade on Th1 polarization by TLR-3-DCs.

TLR-3-DCs generated from peripheral blood of 6 healthy donors were cocultured with CD3-selected autologous T cells for 4 days either alone or with addition of various combinations of blocking antibodies. Supernatants were analyzed for secretion of IFN-γ by ELISA. T cells without DC stimulation were used as a negative control. Box-and-whisker plots for the different conditions are shown. *, p<0.05.

Figure 6. IL-12p70 dependency of NK cell activation by TLR-3-DCs.

(A/B) DCs generated from peripheral blood of 10 healthy donors were cocultured with autologous monocyte-depleted (non-adherent) PBMCs for 24 hours with addition of IL-2. Activation of NK cells was analyzed by intracellular IFN-γ staining of CD3⁻CD56⁺ cells (A; circles represent single experiments, the mean is displayed as horizontal line) and by ELISA measurement of IFN-γ in the supernatant (B; box-and-whisker plots). (C/D) In a similar set of experiments, cocultures of TLR-3-DCs and autologous monocyte-depleted PBMCs were compared with or without addition of IL-12p70 blocking antibody. Activation of NK cells was analyzed by intracellular IFN-γ staining of CD3⁻CD56⁺ cells (C; n = 13) and by ELISA measurement of IFN-γ in the supernatant (D; n = 8). *, p<0.05; **, p<0.01.