Toll-like receptor agonist primed dendritic cells overcome immunosuppressive tumor stroma associated molecules Free

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Purpose: Dendritic cells (DC) that secrete high levels of IL-12 have the capacity to prime anti-tumor immune responses. We have been able to induce high level IL-12 secretion by DC with toll-like receptor (TLR) agonists and IFN-γ. We hypothesized that DC primed in this way are refractory to the powerful immuno-inhibitory influence of the tumor micro-environment. Specifically we evaluated the immunosuppressive effects of tumor derived molecules (TDMs: IL-10, TGF-β, PGE₂ and VEGF) on TLR agonist primed, IL-12 secreting DC (DC1). Methods: The monocyte-fraction of elutriation samples from normal donors was used to generate DC. Monocytes were treated with GM-CSF and IL-4 for 24 hours. DC1 were prepared with combinations of IFN-γ and a TLR agonist (TLR4 agonist LPS or TLR8 agonist R848) or combinations of two TLR agonists. DC matured with a cocktail of cytokines that do not secrete IL-12 were used as controls for all experiments. CD40 ligand (CD40L) was added to mature all groups of DC. TDMs were added to parallel cultures either 4 hours prior to the addition of priming reagents or 1 hour prior to the addition of CD40L (i.e., after the addition of priming reagents). Culture supernatants were assayed for DC IL-12 secretion by ELISA. DC were further phenotypically assayedby flow cytometric analysis (CD80, CD86, CD83, CD14, MHCI, MHCII, and CD40). Allogeneic responses were tested by co-culturing DC1, or TDM treated DC1 with naïve CD4⁺ T cells from MHC mismatched normal donors. After 5 days, CD4⁺ T cells were harvested and re-stimulated with plate-bound anti-CD3 and anti-CD28 for 24 hours and culture supernatants were assayed for IFN-γ, IL-4 and IL-5 by ELISA. Results: TLR agonist primed DC1 secreted high levels of IL-12 (15-114ng/ml) and highly expressed CD80, CD86, CD83, MHCI, MHCII, and CD40. DC1 strongly polarized naïve CD4⁺ T cells toward a Th1 type response. IL-10 and PGE₂ added prior to the addition of priming reagents inhibited DC1 IL-12 production by 80-95%(1-10ng/ml) and 50-90%(2-34ng/ml) respectively. However, when IL-10 and PGE₂ were added after the addition of priming reagents, the inhibition rates decreased significantly to 30-50%(5-20ng/ml) and 22-30%(17-90ng/ml) respectively. TGF-β and VEGF had moderate effects on DC1 IL-12 production (10-20% decrease, 18-90ng/ml) at all time points tested. Of all the TDMs tested, only IL-10 inhibited DC1 maturation by suppressing the expression of CD80, CD86 and CD83. Allogeneic responses, as measured by IFN-γ secretion, were inhibited by 50-60% and 20-30% when IL-10 and PGE₂ were added prior to and after the addition of primingreagents respectively. TGF-β and VEGF only inhibited allogeneic responses by 20-30% and 10-30% at these time points. Conclusion: TLR agonist primed DC1 are resistant to TDM mediated immunosuppression. If administered intra-tumorally, these DC may have the capacity to modulate anti-tumor T cell responses.