Combined TLR2/4-Activated Dendritic/Tumor Cell Fusions Induce Augmented Cytotoxic T Lymphocytes

Induction of antitumor immunity by dendritic cell (DC)-tumor fusion cells (DC/tumor) can be modulated by their activation status. In this study, to address optimal status of DC/tumor to induce efficient antigen-specific cytotoxic T lymphocytes (CTLs), we have created various types of DC/tumor: 1) un-activated DC/tumor; 2) penicillin-killed Streptococcus pyogenes (OK-432; TLR4 agonist)-activated DC/tumor; 3) protein-bound polysaccharides isolated from Coriolus versicolor (PSK; TLR2 agonist)-activated DC/tumor; and 4) Combined OK-432- and PSK-activated DC/tumor. Moreover, we assessed the effects of TGF-β1 derived from DC/tumor on the induction of MUC1-specific CTLs. Combined TLR2- and TLR4-activated DC/tumor overcame immune-suppressive effect of TGF-β1 in comparison to those single activated or un-activated DC/tumor as demonstrated by: 1) up-regulation of MHC class II and CD86 expression on DC/tumor; 2) increased fusion efficiency; 3) increased production of fusions derived IL-12p70; 4) activation of CD4⁺ and CD8⁺ T cells that produce high levels of IFN-γ; 5) augmented induction of CTL activity specific for MUC1; and 6) superior efficacy in inhibiting CD4⁺CD25⁺Foxp3⁺ T cell generation. However, DC/tumor-derived TGF-β1 reduced the efficacy of DC/tumor vaccine in vitro. Incorporating combined TLRs-activation and TGF-β1-blockade of DC/tumor may enhance the effectiveness of DC/tumor-based cancer vaccines and have the potential applicability to the field of adoptive immunotherapy.     

The TLR7/8 ligand resiquimod targets monocyte-derived dendritic cell differentiation via TLR8 and augments functional dendritic cell generation

Imidazoquinolone compounds, such as resiquimod are Toll-like receptor (TLR) 7/8 ligands representing novel immune response modifiers undergoing clinical testing. Resiquimod has been reported to modulate conventional human monocyte-derived DC (moDC) differentiation, but the role of TLR7 and TLR8 is unclear. We directly dissected the TLR7- and TLR8-dependency by employing selective TLR7 ligands and resiquimod-coculture experiments with inhibitory oligonucleotides (iODN) suppressing TLR7, TLR7+8 or TLR7+8+9. Selective TLR7 ligands did not affect conventional moDC differentiation as analyzed by CD14/CD1a expression. iODN experiments confirmed that resiquimod’s effects during DC differentiation were antagonized only with TLR8 iODNs. Direct comparison of resiquimod DC with TLR7- and control-DC revealed significantly higher T-cell costimulatory molecule and MHC class II expression. Resiquimod DC promoted significantly stronger allogeneic T-cell proliferation and stronger naïve CD4⁺ T-cell proliferation. These results indicate the relevance of TLR8 for human monocyte-derived DC differentiation and maturation and may be relevant for clinical trials employing resiquimod.     

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.     

Identification and Quantification of Total Coumarins from Urtica dentata Hand and Its Roles in Promoting Immune Tolerance via TLR4-Mediated Dendritic Cell Immaturation

Urtica dentata Hand (UDH) is traditionally used in the Alpine region as a herbal medicine. Immunotherapy using total coumarins (TC) of UDH has been proposed, yet the cellular and molecular mechanisms remain incompletely characterized. Additionally, there is no method available for the quantification of the main coumarins in UDH. We describe maturation-resistant, TC-conditioned dendritic cell (DC), which expressed much lower MHC class II (I-Ak) and CD86, showed reduced capacity to stimulate effector T cell responses and upregulated PD-Ll (programmed death ligand-1). TC-DC-stimulated regulatory cells (Treg) were superior alloantigen-specific suppressor of the T effector response as compared to those stimulated by control (CTR)-DC. Furthermore, TC-conditioned DC increased the levels of Foxp3 and CTLA-4 in the CD25 T cell population. TC-DC downregulated toll like receptor 4 (TLR4) protein expression in response to LPS. This indicates that down-regulation of TLR4 in response to TC on DC is a critical signaling pathway that regulates the phenotype and function of DC. We also established a sensitive and specific high-performance liquid chromatography-diodearray detection-mass spectrometry (HPLC-DAD-MS) method for simultaneous identification of its main coumarins, 6,6',7,7'-tetramethoxyl-8,8'-biscoumarin (1), 7,7'-dihydroxy-6,6'-dimethoxy-8,8'-biscoumarin (2), 7,7'-dimethoxy-6,6'-biscoumarin (3), and scoparone (4). A demonstration of this mechanism and the method for identification and quantification of TC in UDH endorsed their potential as a tolerance-promoting herbal medicine to prevent or treat transplantation rejection and autoimmune diseases.     

Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2

The ability of dendritic cells (DC) to initiate immune responses in naive T cells is dependent upon a maturation process that allows the cells to develop their potent Ag-presenting capacity. Although immature DC can be derived in vitro by treatment of peripheral blood monocytes with GM-CSF and IL-4, additional signals such as those provided by TNF-alpha, CD40 ligand, or LPS are required for complete maturation and maximum APC function. Because we recently found that microbial lipoproteins can activate monocytes and DC through Toll-like receptor (TLR) 2, we also investigated whether lipoproteins can drive DC maturation. Immature DC were cultured with or without lipoproteins and were monitored for expression of cell surface markers indicative of maturation. Stimulation with lipopeptides increased expression of CD83, MHC class II, CD80, CD86, CD54, and CD58, and decreased CD32 expression and endocytic activity; these lipopeptide-matured DC also displayed enhanced T cell stimulatory capacity in MLR, as measured by T cell proliferation and IFN-gamma secretion. The lipid moiety of the lipopeptide was found to be essential for induction of maturation. Preincubation of maturing DC with an anti-TLR2 blocking Ab before addition of lipopeptide blocked the phenotypic and functional changes associated with DC maturation. These results demonstrate that lipopeptides can stimulate DC maturation via TLR2, providing a mechanism by which products of bacteria can participate in the initiation of an immune response.     

TLR8 stimulation enhances cetuximab-mediated natural killer cell lysis of head and neck cancer cells and dendritic cell cross-priming of EGFR-specific CD8+ T cells

Background

Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that prolongs survival in the treatment for head and neck cancer (HNC), but only in 10–20 % of patients. An immunological mechanism of action such as natural killer (NK) cell–mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR-bearing cells.

Objective

To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells.

Methods

Peripheral blood mononuclear cells (PBMC), NK, DC, and CD8⁺ T cells were isolated and analyzed using ⁵¹Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR_853-861 tetramer staining.

Results

TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p < 0.01) and HNC patients (p < 0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p < 0.001). TLR8-stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8⁺ T cells in the presence of cetuximab.

Discussion

VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination and for determining biomarkers of response.     

TRIF Is Required for TLR4 Mediated Adjuvant Effects on T Cell Clonal Expansion

Toll like receptor 4 (TLR4) is an important pattern recognition receptor with the ability to drive potent innate immune responses and also to modulate adaptive immune responses needed for long term protection. Activation of TLR4 by its ligands is mediated by engagement of the adapter proteins MyD88 (myeloid differentiation factor 88) and TRIF (Toll-interleukin 1 receptor domain-containing adapter inducing interferon-beta). Previously, we showed that TRIF, but not MyD88, plays an important role in allowing TLR4 agonists to adjuvant early T cell responses. In this study, we investigated the T cell priming events that are regulated specifically by the TRIF signaling branch of TLR4. We found that TRIF deficiency prevented the TLR4 agonist lipid A from enhancing T cell proliferation and survival in an adoptive transfer model of T cell priming. TRIF deficient DC showed defective maturation as evidenced by their failure to upregulate co-stimulatory molecules in response to lipid A stimulation. Importantly, TRIF alone caused CD86 and CD40 upregulation on splenic DC, but both TRIF and MyD88 were required for CD80 upregulation. The impairment of T cell adjuvant effects and defective DC maturation in TRIF ^lps/lps mice after TLR4 stimulation was mainly due to loss of type I IFN production, indicating that type I interferons are central to TLR4''s adjuvant effects. These results are useful for the continued development of TLR4 based vaccine adjuvants that avoid inflammatory risks while retaining beneficial immune response.     

A comparative analysis of cytokine responses, cell surface marker expression and MAPKs in DCs matured with LPS compared with a panel of TLR ligands

Dendritic cells (DCs) are professional antigen-presenting cells that play a vital role in shaping adaptive immunity. DC maturation begins when exogenous danger signals bind to the appropriate toll-like receptor (TLR) and initiate expression of cell surface markers and the secretion of cytokines. This process occurs through defined mitogen-activated protein kinase (MAPK) signalling pathways. Of the 13 known mammalian TLRs, lipopolysaccharide (LPS), which activates TLR4, is the most commonly used ligand for the maturation of DCs in vitro. This comprehensive study measures cytokine secretion and cell surface marker expression in murine bone-marrow-derived DCs following maturation with LPS compared to DCs matured with a panel of other TLR-ligands (zymosan A (TLR2/6), PGN (TLR2), poly(I:C) (TLR3), flagellin (TLR5) and CpG-ODN1826 (TLR9)). The role of MAPK signalling pathways in the maturation process was also examined. Results demonstrate that zymosan A and CpG induce comparable cytokine and cell surface marker profiles to LPS. The remaining ligands differed significantly for cytokine and CD40 expression, but not for CD80 and CD86 expression. While there were differences for MAPK signalling pathways for all ligands, the effect of the inhibitors were broadly similar. These findings broaden our knowledge of TLR ligand-matured DCs.     

TLR agonists downregulate H2-O in CD8alpha- dendritic cells

Peptide loading of MHC class II (MHCII) molecules is catalyzed by the nonclassical MHCII-related molecule H2-M. H2-O, another MHCII-like molecule, associates with H2-M and modulates H2-M function. The MHCII presentation pathway is tightly regulated in dendritic cells (DCs), yet how the key modulators of MHCII presentation, H2-M and H2-O, are affected in different DC subsets in response to maturation is unknown. In this study, we show that H2-O is markedly downregulated in vivo in mouse CD8α(-) DCs in response to a broad array of TLR agonists. In contrast, CD8α(+) DCs only modestly downregulated H2-O in response to TLR agonists. H2-M levels were slightly downmodulated in both CD8α(-) and CD8α(+) DCs. As a consequence, H2-M/H2-O ratios significantly increased for CD8α(-) but not for CD8α(+) DCs. The TLR-mediated downregulation was DC specific, as B cells did not show significant H2-O and H2-M downregulation. TLR4 signaling was required to mediate DC H2-O downregulation in response to LPS. Finally, our studies showed that the mechanism of H2-O downregulation was likely due to direct protein degradation of H2-O as well as downregulation of H2-O mRNA levels. The differential H2-O and H2-M modulation after DC maturation supports the proposed roles of CD8α(-) DCs in initiating CD4-restricted immune responses by optimal MHCII presentation and of CD8α(+) DCs in promoting immune tolerance via presentation of low levels of MHCII-peptide.     

Induction of Tolerogenic Dendritic Cells by a PEGylated TLR7 Ligand for Treatment of Type 1 Diabetes

Autoimmune diabetes mellitus (DM) results from the destruction of pancreatic islet cells by activated T lymphocytes, which have been primed by activated dendritic cells (DC). Individualized therapy with ex vivo DC manipulation and reinfusion has been proposed as a treatment for DM, but this treatment is limited by cost, and requires specialized facilities. A means of in situ modulation of the DC phenotype in the host would be more accessible. Here we report a novel innate immune modulator, 1Z1, generated by conjugating a TLR7 ligand to six units of polyethylene glycol (PEG), which skews DC phenotype in vivo. 1Z1 was less potent in inducing cytokine production by DC than the parent ligand in vitro and in vivo. In addition, this drug only modestly increased DC surface expression of activation markers such as MHC class II, CD80, and CD86; however, the expression of negative regulatory molecules, such as programmed death ligand 1 (PD-L1), and interleukin-1 receptor-associated kinase M (IRAK-M) were markedly increased. In vivo transfer of 1Z1 treated DC into prediabetic NOD mice delayed pancreatic insulitis. Daily administration of 1Z1 effectively prevented the clinical onset of hyperglycemia and reduced histologic islet inflammation. Daily treatment with 1Z1 increased PD-L1 expression in the CD11c⁺ population in peri-pancreatic lymph nodes; however, it did not induce an increase in regulatory T cells. Pharmaceutical modulation of DC maturation and function in situ, thus represents an opportunity to treat autoimmune disease.     

A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4^−/− and Myd88^−/−, but not TRIF^−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.     

The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2

Toll-like receptors (TLRs) have been found to be key elements in pathogen recognition by the host immune system. Dendritic cells (DCs) are crucial for both innate immune responses and initiation of acquired immunity. Here we focus on the potential involvement of TLR ligand interaction in DC maturation. TLR2 knockout mice and mice carrying a TLR4 mutation (C3H/HeJ) were investigated for DC maturation induced by peptidoglycan (PGN), lipopolysaccharide (LPS), or lipoteichoic acids (LTAs). All stimuli induced maturation of murine bone marrow-derived DCs in control mice. TLR2(-)/- mice lacked maturation upon stimulation with PGN, as assessed by expression of major histocompatibility complex class II, CD86, cytokine, and chemokine production, fluorescein isothiocyanate-dextran uptake, and mixed lymphocyte reactions, while being completely responsive to LPS. A similar lack of maturation was observed in C3H/HeJ mice upon stimulation with LPS. DC maturation induced by LTAs from two different types of bacteria was severely impaired in TLR2(-)/-, whereas C3H/HeJ mice responded to LTAs in a manner similar to wild-type mice. We demonstrate that DC maturation is induced by stimuli from Gram-positive microorganisms, such as PGN and LTA, with similar efficiency as by LPS. Finally, we provide evidence that TLR2 and TLR4 interaction with the appropriate ligand is essential for bacteria-induced maturation of DCs.     

The Mycobacterium avium subsp. Paratuberculosis protein MAP1305 modulates dendritic cell-mediated T cell proliferation through Toll-like receptor-4

In this study, we show that Mycobacterium avium subsp. Paratuberculosis MAP1305 induces the maturation of bone marrow-derived dendritic cells (BMDCs), a representative antigen presenting cell (APC). MAP1305 protein induces DC maturation and the production of pro-inflammatory cytokines (Interleukin (IL)-6), tumor necrosis factor (TNF)-α, and IL-1β) through Toll like receptor-4 (TLR-4) signaling by directly binding with TLR4. MAP1305 activates the phosphorylation of MAPKs, such as ERK, p38MAPK, and JNK, which is essential for DC maturation. Furthermore, MAP1305-treated DCs transform naïve T cells to polarized CD4⁺ and CD8⁺ T cells, thus indicating a key role for this protein in the Th1 polarization of the resulting immune response. Taken together, M. avium subsp. Paratuberculosis MAP1305 is important for the regulation of innate immune response through DC-mediated proliferation of CD4⁺ and CD8⁺ T cells. [BMB Reports 2014; 47(2): 115-120]     

Mycobacterium paratuberculosis CobT Activates Dendritic Cells via Engagement of Toll-like Receptor 4 Resulting in Th1 Cell Expansion

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne disease in animals and MAP involvement in human Crohn disease has been recently emphasized. Evidence from M. tuberculosis studies suggests mycobacterial proteins activate dendritic cells (DCs) via Toll-like receptor (TLR) 4, eventually determining the fate of immune responses. Here, we investigated whether MAP CobT contributes to the development of T cell immunity through the activation of DCs. MAP CobT recognizes TLR4, and induces DC maturation and activation via the MyD88 and TRIF signaling cascades, which are followed by MAP kinases and NF-κB. We further found that MAP CobT-treated DCs activated naive T cells, effectively polarized CD4⁺ and CD8⁺ T cells to secrete IFN-γ and IL-2, but not IL-4 and IL-10, and induced T cell proliferation. These data indicate that MAP CobT contributes to T helper (Th) 1 polarization of the immune response. MAP CobT-treated DCs specifically induced the expansion of CD4⁺/CD8⁺CD44^highCD62L^low memory T cells in the mesenteric lymph node of MAP-infected mice in a TLR4-dependent manner. Our results indicate that MAP CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade, suggesting that MAP CobT potentially links innate and adaptive immunity against MAP.     

Impaired pro-inflammatory cytokine production and increased Th2-biasing ability of dendritic cells exposed to Taenia excreted/secreted antigens: A critical role for carbohydrates but not for STAT6 signaling

In cysticercosis, a parasitic disease caused by cestodes, the details of early interactions between parasite antigens and innate cells from the host are not well understood. In this study, the role of cestode-conditioned dendritic cells (DCs) in priming Th1 versus Th2 responses to bystander antigen was examined by using CD11c⁺ DCs as antigen-presenting cells and naive CD4⁺ DO11.10 lymphocytes specific to ovalbumin (OVA) as responding cells. No conventional maturation was induced in DCs exposed to Taenia crassiceps excreted/secreted antigens (TcES). The ability of TcES to affect Toll-like receptor (TLR)-mediated maturation and the pro-inflammatory response was analyzed by co-pulsing DCs with TcES and TLR ligands. DCs exposed to TcES blocked TLR4, TLR9 and Toxoplasma soluble antigen-induced phenotypic maturation. TcES-exposed DCs also blocked secretion of pro-inflammatory cytokines and alloreactive T cell proliferation, while preserving IL-10 production. DCs pulsed with TcES + OVA suppressed IFN-γ, whereas they induced greater IL-4 production by CD4⁺ DO11.10 cells. TcES with chemically-altered glycans failed to modulate TLR-mediated activation of DCs and their Th1-inhibitng ability, which was STAT6-independent. Our results reflect the capacity of TcES glyco-antigens to modulate Th1-type and inflammatory responses mediated through DC activation.     

Neisserial porin-induced dendritic cell activation is MyD88 and TLR2 dependent

Neisserial porins have been shown to act as B cell mitogens and immune adjuvants. PorA and PorB are the major outer membrane porin proteins of the human pathogen Neisseria meningitidis. We have shown that the mechanism of the immunopotentiating capability of porin involves up-regulation of the T cell costimulatory ligand, CD86. Due to neisserial porin's ability to activate B cells and potentiate immune responses, we hypothesized that porin also employs the potent immune stimulatory function of dendritic cells (DC). We examined the ability of purified N. meningitidis PorB to induce maturation of murine splenic and bone marrow-derived DC. PorB treatment induced DC maturation, as demonstrated by increased expression of CD86 and class I and II MHC molecules. In addition, PorB not only enhanced the allostimulatory activity of DC, but also augmented the ability of DC to stimulate T cells in an Ag-specific manner. PorB-matured DC secreted the inflammatory cytokine IL-6, which may have implications for the adjuvant property of porin. Induction of IL-6 by PorB is also significant because IL-6 is one of a number of cytokines produced during infection with N. meningitidis and may be involved in the inflammatory process observed during infection and disease. We previously demonstrated the requirement of MyD88 and TLR2 for PorB-induced B cell activation. In the present study, MyD88 and TLR2 were also essential for PorB-induced DC activation. This work is significant for elucidating the mechanism(s) of neisserial porin's immune stimulatory activity.     

Toxoplasma gondii-derived heat shock protein 70 stimulates maturation of murine bone marrow-derived dendritic cells via Toll-like receptor 4

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) induced maturation of bone marrow-derived dendritic cells (DCs) of wild-type (WT) C57BL/6 mice as evidenced by an increase in surface expression of MHC class I and II molecules and costimulatory molecules such as CD40, CD80, and CD86. Functionally, decreased phagocytic ability and increased alloreactive T cell stimulatory ability were observed in T.g.HSP70-stimulated DCs. These phenotypic and functional changes of T.g.HSP70-stimulated DCs were demonstrated in Toll-like receptor (TLR) 2- and myeloid differentiation factor 88 (MyD88)-deficient but not TLR4-deficient C57BL/6 mice. DCs from WT and TLR2-deficient but not TLR4-deficient mice produced IL-12 after T.g.HSP70 stimulation. T.g.HSP70-stimulated DCs from WT, TLR2-deficient, and MyD88-deficient, but not TLR4-deficient mice expressed IFN-beta mRNA. Thus, T.g.HSP70 stimulates murine DC maturation via TLR4 through the MyD88-independent signal transduction cascade.