TLR4 engagement during TLR3-induced proinflammatory signaling in dendritic cells promotes IL-10-mediated suppression of antitumor immunity

Abstract

Toll-like receptor (TLR) agonists are promising adjuvants for immune therapy of cancer, but their potential efficacy as single or combinatorial agents has yet to be fully evaluated. Here, we report that among all TLR agonists tested, dendritic cells (DC) stimulated with the TLR3 agonist polyI:C displayed the strongest activity in stimulating proinflammatory responses and the production of melanoma antigen-specific CD8(+) T cells. Simultaneous treatment with TLR7/8 agonists further improved these responses, but the inclusion of bacterial lipopolysaccharide (LPS), a TLR4 agonist, suppressed proinflammatory cytokine production. This inhibition was contingent upon rapid induction of the suppressive cytokine interleukin (IL)-10 by LPS, leading to dysregulated immune responses and it could be reversed by signal transducers and activators of transcription 3 knockdown, p38 blockade or antibodies to IL-10 and its receptor. Our findings show how certain TLR agonist combinations can enhance or limit DC responses associated with antitumor immunity, through their relative ability to induce IL-10 pathways that are immune suppressive.

Figure 1. LPS inhibits PolyI:C induced proinflammatory cytokines in human dendritic cells

MoDCs were exposed to 100ng/ml of LPS, 5ug/ml of PolyI:C, or their combination. IL-12p70 was measured (A, n=13) as was TNFα (B, n=9), IL-1β (C, n=7), IP-10 (D, n=3) and IL-10 (E, n=13) by cytokine bead array (CBA) and Luminex (range from 3–13 healthy donors). The combination of LPS and PolyI:C induced significantly lower amounts of IL-12p70, TNFα, IL-1β and IP-10 than PolyI:C alone, while inducing significantly higher amounts of IL-10 than LPS alone. MoDCs were exposed to a suboptimal dose of 500ng/ml of PolyI:C and given increasing doses of LPS while monitoring levels of IL-12p70 and IL-10 (F). IL-12p70 levels were measured in MoDCs that were either left untreated, exposed to blocking antibodies to IL-10 and IL-10R, IgG2/IgG1 control antibody or they were pre exposed to recombinant IL-10 (G, performed in triplicates, in 3 donors).

Figure 2. Inhibition of PolyI:C induced responses by LPS is mediated by IL-10, Stat3 and partially by P38

MoDCs were electroporated with STAT3 siRNA and the amount of total protein was measure by western blot (A) and quantified using densitometry (B). These cells were treated with LPS, PolyI:C or their combination, and IL-12p70 was measured (A). MoDCs prior to exposure to LPS, PolyI:C or their combination were pretreated with a P38 inhibitor 10ng/ml or wortmannin 100nM/ml, and IL-12p70 and IL-10 was quantified (B and C respectively). Phosphorylation of P38 was monitored over time upon ligation of TLR4, TLR3 or their combination (D). MoDCs were exposed to 100ng/ml of LPS, 5ug/ml of PolyI:C, 10uM of R848 and/or their combination as well as Mimic cytokine cocktail or left untreated with or without blocking antibodies to IL-10 and IL-10R with or without recombinant IL-10. Cells were then stained with anti CD80 and anti-CD86 (E) anti- PDL-1 antibody (F) or with the isotype control.

Figure 3. Kinetics of IL-10 secretion explains inflammatory capacity of TLR agonists

MoDCs were monitored for the secretion of IL-12p70 and IL-10 when either untreated or stimulated with LPS, PolyI:C, LPS/PolyI:C, R848, R848/LPS, R848/PolyI:C and Mimic. IL-12p70 and IL-10 was also monitored using the same conditions with or without blocking IL-10 and IL-10R, and with or without pretreatment with recombinant IL-10 (A, B respectively). The same experiment was repeated by monitoring IL-12p70 and IL-10 in supernatants collected over time (C, D respectively). Each experiment was performed in triplicates and repeated at least with 3 different donors.

Figure 4. Delayed addition of rhIL-10 and blocking antibodies to IL-10/IL-10R further elucidates the mechanism of TLR agonists

Monocyte derived dendritic cells were exposed to 100ng of LPS, 5ug of PolyI:C, 10uM of R848 and/or their combination as well as Mimic cytokine cocktail or left untreated +/− rhIL-10 (300ng/ml) at 0h and 9h and +/− anti IL-10/IL-10R (2ug/ml) at 0 or 9h, IL-12p70 was monitored over time in triplicate wells (n=2) by CBA (A–H).

Figure 5. Increased levels of inflammation in MoDCs translate into higher numbers of specific CD8 T cell responses

Healthy donor naïve A2.1+ CD8+ T cells were primed with peptide encoding for the HLA A2.1 restricted Mart-1/MelanA (ELAGIGILTV) epitope and the frequency of tetramer positive cells was monitored (n=6) (B). Tetramer positive cells from all conditions from a donor were expanded and examined functionally for secretion of IL-13, IL-5, IFNγ, TNFα and IL-2 (B, C, D, E and F, respectively). Using the same priming experimental setup 2 additional donors were used with a condition of αIL-10/IL10R Abs for each treatment, showing a representative donor (triplicate wells) (G). Differentially matured MoDCs and allogeneic naïve CD8+T cells were co-cultured for 7 days in a MLR reaction. Proliferated cells were stimulated over night with αCD3/CD28 beads and evaluated for the production of IFNγ (H).