Using distinct molecular signatures of human monocytes and dendritic cells to predict adjuvant activity and pyrogenicity of TLR agonists

Abstract

We present a systematic study that defines molecular profiles of adjuvanticity and pyrogenicity induced by agonists of human Toll-like receptor molecules in vitro. Using P(3)CSK(4), Lipid A and Poly I:C as model adjuvants we show that all three molecules enhance the expansion of IFNgamma(+)/CD4(+) T cells from their naïve precursors following priming with allogeneic DC in vitro. In contrast, co-culture of naive CD4(+) T cells with allogeneic monocytes and TLR2/TLR4 agonists only resulted in enhanced T cell proliferation. Distinct APC molecular signatures in response to each TLR agonist underline the dual effect observed on T cell responses. Using protein and gene expression assays, we show that TNF-alpha and CXCL10 represent DC-restricted molecular signatures of TLR2/TLR4 and TLR3 activation, respectively, in sharp contrast to IL-6 produced by monocytes upon stimulation with P(3)CSK(4) and Lipid A. Furthermore, although all TLR agonists are able to up-regulate proIL-1beta specific gene in both cell types, only monocyte activation with Lipid A results in detectable IL-1beta release. These molecular profiles, provide a simple screen to select new immune enhancers of human Th1 responses suitable for clinical application.

Fig. 1

Effect of TLR2, TLR4 and TLR3 agonists on naive T cell activation. a CFSE-labeled CB-PBMC were cultured alone (white bars), or stimulated with allogeneic CD14⁺ Mo (hatched bars) or Mo-derived DC (black bars), in the presence of CSK₄, P₃CSK₄, Lipid A, or Poly I:C, as indicated in the bottom line. T cell proliferation was assessed by measuring the frequency of CFSE^dim/CD3⁺/CD4⁺ lymphocytes by FACS analysis. b Unlabeled CB-PBMC were stimulated as in a and assed for T cell differentiation by detection of intracellular IL-4 versus IFN-γ on gated CD3⁺/CD4⁺ lymphocytes; numbers indicate the percentage of cytokine-producing T cells. c Frequency of CD3⁺/CD4⁺/IFN-γ⁺ cells upon priming with allogeneic CD14⁺ Mo (white bars) or DC (black bars) in the presence of CSK₄, P₃CSK₄, Lipid A or Poly I:C. Data represent mean results ± SD obtained from four independent experiments

Fig. 2

Mo and DC respond distinctly to TLR2, TLR4 and TLR3 agonists in vitro. Freshly purified CD14⁺ cells (d0) and Mo undergoing DC differentiation (d1–d5) upon culture in GM-CSF/IL-4 conditioned medium, were stimulated with CSK₄ (white bars), P₃CSK₄ (black bars), Lipid A (hatched bars) and Poly I:C (dotted bars). Secretion of IL-6 (a) and TNF-α (b) in culture supernatants was assessed by specific ELISA assays. Data are given as mean results ± SD obtained in four donors independently tested and are expressed in pg/ml/10⁵ cells. c Time-dependent variation in cell size (FSC-H) and surface expression of CD14 and CD1a during Mo culture in GM-CSF/IL-4. Plots show profiles obtained with cells at days 0, 1, 3 and 5 of differentiation by FACS analysis. Results are representative of four experiments

Fig. 3

Time-course analysis of cytokines release upon TLR2, TLR3 and TLR4 triggering on CD14⁺ Mo and DC in vitro. Freshly purified Mo (a, b) or Mo-derived DC (c, d) were stimulated, at 2 × 10⁵ cells/ml, with CSK₄ (open circle), P₃CSK₄ (filled circle), Lipid A (filled square) and Poly I:C (open square) and culture supernatants assessed for IL-6 (a, c) and TNF-α (b, d) secretion at different time points between 0.5 and 22 h. Representative results obtained in two out of three donors tested are shown

Fig. 4

TLR3 is specifically expressed on DC and CXCL10 represents a cell-restricted signature of activation in response to Poly I:C. Freshly isolated CD14⁺ Mo were cultured in the presence of GM-CSF/IL-4 and analyzed by FACS staining or qRT-PCR at days (d) 0, 1, 3 and 5. a TLR2 surface expression in freshly isolated Mo (d0) and terminally differentiated DC (d5). In each plot dotted lines represent the background staining with an isotype-matched control antibody. b Quantitative gene expression analysis of TLR1, TLR2, TLR3 and TLR4 specific mRNA; data are expressed as Relative Quantity (R. Q.); cells at day 5 of culture were taken as calibrator (R. Q. = 1). Time-dependent induction of CCL2 and CXCL10 chemokines in response to CSK₄ (white bars), P₃CSK₄ (black bars), Lipid A (hatched bars) and Poly I:C (dotted bars) in Mo undergoing DC differentiation in vitro is shown in c and d, respectively. Data are represented as described in Fig. 2

Fig. 5

IL1β is a molecular marker of pyrogenicity. Freshly purified CD14⁺ cells (d0) and Mo undergoing DC differentiation (d1–d5) upon culture in GM-CSF/IL-4 conditioned medium, were stimulated with CSK₄ (white bars), P₃CSK₄ (black bars), Lipid A (gray bars) and Poly I:C (dotted bars). Amounts of IL-1β a in culture supernatants, as assessed by specific ELISA assays are expressed in pg/ml/10⁵ cells. Data are representative of three independent experiments. b Dose-dependent induction of IL-1β in Mo stimulated with CSK₄ (open circle), P₃CSK₄ (filled circle) and Lipid A (filled triangle) at 10⁴–1 ng/ml in threefolds serial dilutions. Experimental data obtained from one out of three donors independently tested are expressed in pg/ml and shown as mean results of triplicate wells

Fig. 6

Prediction of adjuvant activity and safety of synthetic and natural compounds. a Correlation between amount of TNF-α released by DC and induction of Th1 responses in vitro upon stimulation with different natural and synthetic TLR-2 agonists. b Selection of putative safe adjuvants among a chemical library of 14 compounds including TLR-dependent and -independent stimuli, according to the capacity of each molecule to induce TNF-α secretion from activated DC (left panel), expansion of IFN-γ⁺/CD4⁺ T lymphocytes in MLR cultures (central panel) and IL-1β from CD14⁺ Mo in vitro (right panel). Cutoff for positive and negative selection are indicated by red and black arrows, respectively. Positively selected compounds are labeled in red

Fig. 7

Schematic representation of an applicable in vitro system for adjuvants selection. In the proposed methods chemical compounds of unknown adjuvant activity are tested for their capacity to induce IL-1β release from human Mos (Mo) and TNF-α/CXCL10 secretion by Mo-derived dendritic cells (DC). Only chemicals positive for TNF-α/CXCL10 and negative for IL-1β are selected for in vivo studies