CD40 ligation restores type 1 polarizing capacity in TLR4-activated dendritic cells that have ceased interleukin-12 expression

Abstract

Inflammation triggered by microbial lipopolysaccharide (LPS) through Toll-like receptor (TLR) 4 in the presence of interferon (IFN)-gamma induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation program. This DC differentiation is characterized not only by a dynamic immune activating but also by tolerance-inducing phenotype associated with down-modulation of cytokines previously considered to be irreversible. CD40L on activated T cells further modifies DC differentiation. Using DNA micro-arrays, we showed down-regulated mRNA levels of TLR signalling molecules, whereas CD40/CD40L signalling molecules were up-regulated at a time when LPS/IFN-gamma-activated DCs had ceased cytokine expression. Accordingly, we demonstrated that CD40/CD40L but not TLR4 or TLR3 signalling mediated by LPS or poly (cytidylic-inosinic) acid (poly I:C) and dsRNA re-established the capacity for secreting interleukin (IL)-12 in primarily LPS/IFN-gamma-activated DCs, which have exhausted their potential for cytokine secretion. The resulting TH1 polarizing DC phenotype - which lacked accompanying secretion of the crucial immune suppressive factor IL-10 - maintained the potential for activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T-cell-mediated stimulus at an exhausted DC state, which prevents an immune tolerant DC phenotype. These findings impact on the rational design of TLR-activated DC-based cancer vaccines for the induction of anti-tumoural CTL responses.

Figures

Figure 1

Molecules of the CD40 signalling cascade are highly induced 48 hrs after TLR4 activation at the time of cytokine exhaustion. (A) Immature DCs were stimulated with LPS/IFN‐γ for 6 hrs and compared to un‐stimulated DCs by DNA micro‐array analysis. In addition, 12, 24 and 48 hrs matured DCs receiving a 6‐hr LPS/IFN‐γ stimulus were also compared to un‐stimulated DCs. Key molecules of the TLR and CD40 signalling cascade are analysed by heat plotting over the time. Up‐regulation is indicated by red and down‐regulation by different green colouring. (B) Detailed illustration of differentially regulated genes encoding secreted proteins or proteins involved in TLR or CD40/CD40L signalling. mRNA fold expression is depicted over 48 hrs after LPS/IFN‐γ activation at a linear scale. *Down‐regulation compared to immature DCs. (C) Quality control by analysing the activation status of LPS/IFN‐γ‐treated DCs in (A). IL‐10, IL‐12 and the IDO metabolite kynurenine were measured as depicted over 48 hrs of maturation. These data were reproducible using two different donors. (D) Expression density of TLR4, CD14 and CD40 after 48 hrs of LPS/IFN‐γ activation (black histogram) compared to un‐stimulated immature DCs (dashed histogram) and isotype control stained immature DCs (filled black histogram). One out of two independent experiments using two different donors is depicted.

Figure 2

CD40L re‐induces IL‐12 secretion in TLR‐activated DCs that have exhausted their capacity for cytokine secretion. (A) Upper panel: IL‐12 secreted from DCs after a 6‐hr exposure to the indicated stimuli and the adequate co‐factors (see main text). Accumulation of IL‐12 after 48 hrs of activation was measured. Lower panel: IL‐12 secretion after a second round of stimulation analysed from 48 hrs pre‐matured DCs in (A) re‐stimulated for 48 hrs with the indicated stimuli (colour code). One representative experiment out of five is given. (B) Immune phenotype of DCs analysed in (A) measured by flow cytometry after 48 hrs of primary maturation with the indicated maturation stimuli. LPS/IFN‐γ and poly I:C/IFN‐γ stimulated DCs (black histogram) are compared to immature DCs (dashed histogram). (C) Relative increase of the expression density (mean fluorescence intensity, MFI) of depicted surface maturation markers on 48 hrs pre‐matured DCs analysed in (A) stimulated with the indicated stimuli (top left) followed by a 48‐hr re‐stimulation with the stimuli given at the bottom of the graph in comparison to primarily stimulated DCs.

Figure 3

CD40 ligation induces strong stimulatory capacity in cytokine‐exhausted TLR‐activated DCs. Allogeneic MLR using DCs 48 hrs after receiving a primary maturation stimulus as indicated at the top of each panel (LPS/IFN‐γ; circles, poly I:C/IFN‐γ; squares) followed by a 6‐hr co‐cultivation with CD40L‐SJ‐NB‐7 cells (black symbols) or control SJ‐NB‐7 cells (white symbols), both in the presence of IFN‐γ. Proliferation was assessed by tritium thymidine incorporation at the indicated DC:lymphocyte ratios. Experiments from three different donors are combined using an internal SEA/SEB proliferation control defined as 100; unstimulated T cells were normalized to 0; the proliferation ± S.D. relative to the SEA/SEB (100) and un‐stimulated (0) controls is shown.

Figure 4

CD40L ectopically expressed on cytokine‐exhausted TLR4‐activated DCs re‐induces IL‐12 secretion. (A) CD40L or GFP expression after lenti‐viral gene transfer into 6 or 48 hrs pre‐matured DCs that received a 6‐hr LPS/IFN‐γ stimulus. Expression density was measured by flow cytometry 48 hrs after transduction with the lenti‐viral constructs lv‐CD40L or lv‐GFP (grey histograms) compared to untreated DCs (filled black histogram). (B) Immune phenotype of DCs described in (A) measured 48 hrs after viral transduction. Shown is one representative out of five experiments with three different donors. (C) IL‐12 and IL‐10 secretion from DCs described in (A) measured 48 hrs after lenti‐viral transduction. *Below the detection limit of the ELISA.

Figure 5

CD40L ectopically expressed on cytokine‐exhausted TLR4‐activated DCs triggers activation of CD8+ T lymphocytes. Allogeneic MLR using DCs that were matured for 6 or 48 hrs upon receiving a 6‐hr LPS/IFN‐γ stimulus as indicated (bottom right of each panel) followed by a 6‐hr treatment with the lenti‐virus lv‐CD40L (black circles) or lv‐GFP (white circles) in comparison to only primary stimulated DCs (squares). T‐cell activation was measured by the mean fluorescence intensity (MFI) of CD25 expressed on proliferating CFSE negative CD8+ T cells. Granzyme B expressing cells are given as the percentage in CD8+ T cells. The dot plots show CD8+ granzyme B+ T cells at a DC:T cell ratio = 1:9. Shown is one representative experiment of three using three different donors. The bar diagram combines the data of three different donors, which shows fold expression ± S.D. of granzyme B expressing CD8+ T cells stimulated with lv‐CD40L DCs normalized to lv‐GFP DCs; 6 hrs LPS/IFN‐γ pre‐matured DCs; P= 0,01; 48 hrs LPS/IFN‐γ pre‐matured DCs, P= 0,004. CD40L or GFP expression as well as the phenotype of the DCs used for the depicted alloMLRs is shown in Fig. 4.