Delivery of allergens with bacterial adjuvants has been shown to be a successful immunotherapeutic strategy for food allergy treatment in animal models. How microbial signals, acting through the innate immune system, reshape ongoing allergic responses is poorly understood. To investigate the contribution of Toll-like receptors (TLRs) in the response to bacterial adjuvants, we designed an in vitro system to characterize the effect of heat-killed Escherichia coli vector (HKE) on peanut-induced responses of dendritic cells (DCs) and T cells. Wild-type or TLR signaling-deficient bone marrow-derived DCs were pulsed with crude peanut extract (CPE) alone (50 microg/mL) in the presence of HKE (10(6)/mL). DC maturation was analyzed by means of flow cytometry. Treated DCs were cocultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4(+) T cells from sensitized mice. Cytokine production from DCs and T cells was measured by using Bioplex assays. Peanut-pulsed DCs induced the production of IL-4, IL-5, and IL-13, as well as IL-17 and IFN-gamma, from primed T cells. Adding HKE to CPE-pulsed DCs resulted in a significant decrease in T(H)2 cytokine production associated with an increase in IFN-gamma levels and profound attenuation of T-cell proliferation. These effects were linked to HKE-induced TLR-dependent changes in DC reactivity to CPE, especially the production of polarizing cytokines, such as IL-12. TLR signals modulate peanut-induced DC maturation in vitro, leading to changes in the T-cell response to peanut. These TLR effects must be confirmed in vivo and might constitute another alternative for allergen immunotherapies.