Combination Of Toll-like Receptor Research Articles

Intracavitary Spraying of Nanoregulator-Encased Hydrogel Modulates Cholesterol Metabolism of Glioma-Supportive Macrophage for Postoperative Glioblastoma Immunotherapy.

Glioblastoma multiforme (GBM) is notoriously resistant to immunotherapy due to its intricate immunosuppressive tumor microenvironment (TME). Dysregulated cholesterol metabolism is implicated in the TME and promotes tumor progression. Here, it is found that cholesterol levels in GBM tissues are abnormally high, and glioma-supportive macrophages (GSMs), an essential "cholesterol factory", demonstrate aberrantly hyperactive cholesterol metabolism and efflux, providing cholesterol to fuel GBM growth and induce CD8+ T cells exhaustion. Bioinformatics analysis confirms that high 7-dehydrocholesterol reductase (DHCR7) level in GBM tissues associates with increased cholesterol biosynthesis, suppressed tumoricidal immune response, and poor patient survival, and DHCR7 expression level is significantly elevated in GSMs. Therefore, an intracavitary sprayable nanoregulator (NR)-encased hydrogel system to modulate cholesterol metabolism of GSMs is reported. The degradable NR-mediated ablation of DHCR7 in GSMs effectively suppresses cholesterol supply and activates T-cell immunity. Moreover, the combination of Toll-like receptor 7/8 (TLR7/8) agonists significantly promotes GSM polarization to antitumor phenotypes and ameliorates the TME. Treatment with the hybrid system exhibits superior antitumor effects in the orthotopic GBM model and postsurgical recurrence model. Altogether, the findings unravel the role of GSMs DHCR7/cholesterol signaling in the regulation of TME, presenting a potential treatment strategy that warrants further clinical trials.

An exploratory trial of a novel R-ISV-RO technique in the treatment of advanced solid tumors.

e14666 Background: The immunotherapy of bone and soft tissue sarcomas remains a challenge. Our pre-clinical trial confirmed that the combination of Toll-like receptor 7 (TLR7) agonist imiquimod (R837) and OX-40 agonist had prominent synergistic effects. Immunoradiotherapy, especially Stereotactic Body Radiotherapy (SBRT) could further activate specific immune response. We hypothesized that intratumoral injection of “RO” (R837+αOX40) combined with SBRT will realize tumor control and reduce adverse reactions. Meanwhile, the synergetic application of PD-1 mAb systematically will improve the effectiveness of immunotherapy. Methods: This is a single-arm, single-center, open-label, exploratory trial that explore intratumoral injection of an emulsion of “RO” in combination with SBRT as “in situ vaccination” for advanced solid tumor (Ethics number: 2021-133-02). The inclusion criteria included: 1) advanced solid tumors which progress after standard therapies, 2) presence of one or more injectable lesions. SBRT to injected lesions was at a dose of 5-8Gy per fraction at a total dose of 20-32Gy. Imiquimod (0.75mg/ml) and αOX40 (2.5mg/ml) were prepared as emulsion and intratumoral injected into the target lesion for 4 times. Patients were also systematically administrated with PD-1 mAb (serplulimab). The primary endpoint was objective response rate (ORR) of target lesions. The secondary endpoint mainly included disease control rate (DCR) of target lesions, progression free survival (PFS), overall survival (OS), and clinical benefit rate (CBR), etc. Results: From May 2021 to October 2022, 30 patients were eligible for this trial (60% were sarcomas). 46.7% and 33.3% had received radiotherapy or PD-1/PD-L1 mAb respectively before. The ORR, DCR and CBR of target lesions were 29.2%, 91.7% and 41.7%, while these of systemic efficacy were 21.7%, 82.6% and 17.4%, respectively. In sarcomas, the ORR, DCR and CBR of target lesions were 25.0%, 93.8% and 50.0%, while these of systemic efficacy were 20.0%, 93.3% and 26.7%, respectively. Systemic median PFS were 3.67 months. The median event-free survival (EFS) of target lesions and median OS were not reached. Among the evaluable target lesions, 6-month EFS was 85.7% in sarcomas (6/7) and 76.9% in all patients (10/13). The most common TRAEs were leukopenia (50%), hypoproteinemia (30%) and anemia (20%), demonstrating that treatment is tolerable. Grade≥3 treatment-related adverse events (TRAE) occurred in 3 patients, including myocarditis (6.7%) and thyroiditis (3.3%). Conclusions: In situ vaccination with “RO” emulsion, along with SBRT was tolerable and induced anti-tumor immunity, which would also trigger systemic response when synergized with PD-1 mAb. This in situ vaccination has been proved to be a promising option for advanced solid tumors especially for sarcomas. Clinical trial information: ChiCTR2100053870 .

COVID-19 Subunit Vaccine with a Combination of TLR1/2 and TLR3 Agonists Induces Robust and Protective Immunity

The development of COVID-19 vaccines is critical in controlling global health issues under the COVID-19 pandemic. The subunit vaccines are the safest and most widely used vaccine platform and highly effective against a multitude of infectious diseases. An adjuvant is essential for subunit vaccines to enhance the magnitude and durability of immune responses. In this study, we determined whether a combination of toll-like receptor (TLR)1/2 and TLR3 agonists (L-pampo) can be a potent adjuvant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine. We measured a neutralizing antibody (nAb) and an angiotensin-converting enzyme 2 (ACE2) receptor-blocking antibody against SARS-CoV-2 receptor-binding domain (RBD). We also detected interferon-gamma (IFN-γ) production by using ELISPOT and ELISA assays. By employing a ferret model, we detected nAbs and IFN-γ producing cells and measured viral load in nasal wash after the challenge of SARS-CoV-2. We found that SARS-CoV-2 antigens with L-pampo stimulated robust humoral and cellular immune responses. The efficacy of L-pampo was higher than the other adjuvants. Furthermore, in the ferret model, SARS-CoV-2 antigens with L-pampo elicited nAb response and antigen-specific cellular immune response against SARS-CoV-2, resulting in substantially decreased viral load in their nasal wash. Our study suggests that SARS-CoV-2 antigens formulated with TLR agonists, L-pampo, can be a potent subunit vaccine to promote sufficient protective immunity against SARS-CoV-2.

Inducible epithelial resistance protects against acute viral infection and subsequent CD8+Tcell dependent lethal Immunopathology

Abstract Viral pneumonia leads to significant morbidity and mortality worldwide, demanding better understanding of the host immune response to infections for development of novel strategies to prevent respiratory infections. Our group has shown that mice treated with combination of Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are protected against wide range of respiratory pathogens, including viruses. Mice challenged intra-pharyngeally with Sendai virus showed peak viral burden on day 5 and peak mortality on day 11, overlapping with increased lung CD8+Tcells. We hypothesized that mortality is associated with immunopathology caused by CD8+T cells. Pam2-ODN aerosolization one day prior to Sendai challenge reduced lung viral burden and reduced lung CD8+T cells on day 11 and enhanced mouse survival. Depletion of CD8+T cells before infection increased baseline virus susceptibility and enhanced viral replication in vivo, congruent with the known antiviral function of CD8+T cells. Pam2-ODN pre-treatment protected mice against death following viral challenge, even in the absence of CD8+T cells, reflecting antiviral responses induced directly from epithelial cells. Notably, depletion of CD8+T cells eight days after viral challenge also significantly enhanced survival of sham pre-treated mice, indicating rescue from CD8+T cell-mediated lethal immunopathology. Our findings definitively prove that CD8+T cells, although anti-viral in nature, promote lethal immunopathology that can be prevented by Pam2-ODN pre-treatment. Importantly, Pam2-ODN pre-treatment benefitted survival regardless of when CD8+T cells were depleted and may provide an opportunity to protect vulnerable populations against respiratory infections.

Toll-like receptor signalling protects against IL-33 led virus-induced asthmatic response in a Type I Interferon independent manner

Abstract Respiratory viruses are frequent causes of asthma development and exacerbations. However, there are gaps in understanding how viral infections cause immuno-pathology in the host leading to asthma progression. We have previously shown that mice treated with a combination of Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are protected against broad range of respiratory infections induced by viruses, fungi and bacteria. Pam2-ODN treatment one day prior to virus infection leads to enhanced survival with reduced lung viral burden in mice. Further, Pam2-ODN pretreatment also inhibits acute lung pathology caused by CD8+ T cells and prevents chronic asthmatic phenotype of eosinophilia, airway hyperreactivity and mucus metaplasia. This protective effect was accompanied by reduced virus-driven lung IL-33, which is necessary to induce chronic asthmatic phenotype. Interestingly, Pam2-ODN does not induce increased production of Type I or III Interferons (IFN), nor does the protective response require intact interferon signaling. Transcriptome profiling of the mouse lungs in the chronic phase of infection led to identification of genes involved in airway remodelling, epithelial cell differentiation and chemokine signalling. More importantly, we identified genes from the Pam2-ODN treatment group that are involved in reversing the viral infection effect on day 49. Taken together, our findings reveal novel type I IFN independent mechanisms to prevent virus-induced asthma, and may provide an opportunity to protect vulnerable populations.

Dual activation of Toll-like receptors 7 and 9 impairs the efficacy of antitumor vaccines in murine models of metastatic breast cancer.

Since combination of Toll-like receptor (TLR) ligands could boost antitumor immunity, we evaluated the efficacy of dendritic cell (DC) vaccines upon dual activation of TLR9 and TLR7 in breast cancer models. DCs were generated from mouse bone marrow or peripheral blood from healthy human donors and stimulated with CpG1826 (mouse TLR9 agonist), CpG2006 or IMT504 (human TLR9 agonists) and R848 (TLR7 agonist). Efficacy of antitumor vaccines was evaluated in BALB/c mice bearing metastatic mammary adenocarcinomas. CpG-DCs improved the survival of tumor-bearing mice, reduced the development of lung metastases and generated immunological memory. However, dual activation of TLRs impaired the efficacy of DC vaccines. In vitro, we found that R848 inhibited CpG-mediated maturation of murine DCs. A positive feedback loop in TLR9 mRNA expression was observed upon CpG stimulation that was inhibited in the presence of R848. Impaired activation of NF-κB was detected when TLR9 and TLR7 were simultaneously activated. Blockade of nitric oxide synthase (NOS) and indoleamine-pyrrole-2,3-dioxygenase (IDO) improved the activation of CpG-DCs. When we evaluated the effect of combined activation of TLR9 and TLR7 in human DCs, we found that R848 induced robust DC activation that was inhibited by TLR9 agonists. These observations provide insight in the biology of TLR9 and TLR7 crosstalk and suggest caution in the selection of agonists for multiple TLR stimulation. Blockade of NOS and IDO could improve the maturation of antitumor DC vaccines. R848 could prove a useful adjuvant for DC vaccines in human patients.

Chronic Lymphocytic Leukemia Cells Acquire a Regulatory B-Cell Phenotype Modified By PD1 Signaling

Introduction: Chronic lymphocytic leukemia (CLL) cells utilize several mechanisms of survival, some propagating proliferation and preventing apoptosis through intrinsic cell cycle signals, and others suppressing anti-tumor immune responses. Patients often present with a predominant population of regulatory T-cells (Tregs), and general features of T-cell exhaustion. Given the unique phenotype of CLL cells and the observed T-cell abnormalities we hypothesized that these cells function as regulatory B-cells (Bregs). Bregs, mostly explored in the autoimmune disease setting, produce interleukin-10 (IL10), which mediates attenuation of effector T-cell responses and enhances regulatory activity. These features have also been suggested to be responsible for weakening of anti-tumor immune responses. Breg activation requires stimulation of various combinations of Toll-like receptors (TLRs), the B-cell receptor (BCR) and CD40. Our previous studies have demonstrated that TLR9-stimulated CLL cells "acquire" Breg markers as well as PD1 and PDL1, which, while not being classic Breg discriminators, are established players in immune modulation. Moreover, such stimulation resulted in inhibition of proliferation of autologous T-cells. The current study aimed to further explore the regulatory characteristics of CLL cells focusing on additional suppressive mechanisms that may have a role in CLL immune evasion, particularly, the PD1/PDL1 axis.Methods: B-cells were isolated from peripheral blood mononuclear cells (PBMCs) of untreated CLL patients (Rai stages 0-IV). These B-CLL cells were stimulated with TLR-9 agonist (ODN) or CD40 ligand (CD40L) followed by their co-culture with isolated autologous CD4+ T cells. The regulatory features of B-CLL cells were studied by testing their effect on T cells. Their proliferation was evaluated using the CFSE method following stimulation with anti-CD3/CD28 antibodies and IL2; induction of Tregs (CD4+CD25highFoxp3+ population) was assessed by FACS analysis. The involvement of the PD1/PDL1 axis was examined by incubating B-cells with antiPD1 neutralizing antibodies prior to co-culture. Cell contact dependence was evaluated by plating B-cells in hanging cell culture inserts denying B and T cell contact while allowing flow of small soluble molecules.Results: CLL cells stimulated with ODN or CD40L, induced a significant increase in Tregs: 1.35±0.1-fold (p=0.03, N=12) for ODN and 1.7±0.2-fold (p=0.008, N=14) for CD40L, occurring in 68% and 80% of patients, respectively, while co-culture with unstimulated B-CLL cells did not result in the expansion of the Treg population. Treg induction was observed only under contact conditions (N=5), suggesting that this regulatory function requires cell-to-cell contact and cannot be carried out solely by secreted factors like IL10.Neutralization of PD1 on CLL B-cells affects both Treg induction and T-cell proliferation. Following CD40L stimulation, a 1.3-fold reduction in Treg percentage was observed when PD1 signaling was blunted (N=10). In contrast, PD1 blockage of ODN-stimulated CLL cells did not reduce Treg induction; however, it did adversely affect inhibition of T-cell proliferation (10%-decrease in inhibited T-cells; N=6).Conclusions: CLL cells "acquire" a Breg phenotype and function, inhibiting T-cell proliferation and inducing Tregs. These properties, while working together to promote immune regulation and cancer evasion, are elicited by different ligands in the cell environment and are likely to be mediated via separate pathways. The involvement of B-cell-associated PD1 in the induction of Tregs and inhibition of T-cell proliferation suggests a biologic role of PD1 signaling in CLL cells, strengthening the Breg phenotype. The current study has shown that CLL cells recruit several mechanisms operating cooperatively to support immune modulation and promote their survival. DisclosuresNo relevant conflicts of interest to declare.

Abstract OT2-1-02: Novel combination of toll-like receptor (TLR)-7 agonist imiquimod and local radiotherapy in the treatment of breast cancer chest wall recurrences or skin metastases

Abstract Background/Rationale: To assess the immune and systemic anti-tumor effects of the novel combination of local radiotherapy combined with imiquimod applied topically to breast cancer metastatic to skin. Breast cancer is the most common tumor, excluding melanoma, to metastasize to the skin in women. Chest wall recurrence is debilitating for patients, substantially affecting quality of life. Current treatment modalities for unresectable lesions are rarely curative and patients ultimately die of visceral metastases, indicating the need for more effective therapies. Imiquimod (IMQ), a synthetic TLR-7 agonist has immunomodulatory activity with profound effects on the tumor environment and can lead to tumor regression of cutaneous breast cancer metastases (Adams et al, Clin Ca Res, Dec 15, 2012). Accumulating evidence indicates that the potential of local radiotherapy to convert the tumor into an in-situ vaccine can be enhanced by combination with immunotherapy to achieve a therapeutic synergy. We have previously shown in a mouse model of cutaneous breast cancer that topical IMQ synergizes with local RT to induce complete tumor regression (REF). Importantly, this approach used a local treatment to generate anti-tumor immune responses with ability to control the tumor systemically (Dewan et al, Clin Ca Res Dec 15, 2012). This trial was designed to test the feasibility of translating this therapeutic synergy in the clinic (clinicaltrials.gov NCT01421017). Methods: Eligibility includes patients with biopsy-confirmed breast cancer, measurable disease and skin metastases, ECOG PS 0-2 and adequate organ/marrow function. Radiation therapy is delivered to one area of skin metastases in five fractions of 6 Gy (days 1,3,5,8,10). IMQ 5% cream is applied topically to skin metastases overnight for 5 days/week for 8 weeks, beginning the evening of the first radiotherapy. Continuous imiquimod to all skin metastases even after completion of RT is based on our preclinical evidence of an improved effector phase of the immune response. Additional treatment cycles with IMQ/RT are permitted. Following a brief phase I portion to allow dose optimization in the event of unanticipated adverse events (3-3 design), the phase II study evaluates efficacy with a planned additional 25 patients. Primary endpoint is the response rate in untreated metastases, assessed by immune-related response criteria. Furthermore, the local tumor responses and safety of the combination will be determined; tumor FNA biopsies will be obtained to investigate signatures of immune-mediated rejection as recently described with IMQ mediated rejection of basal cell carcinomas; and peripheral lymphocytes will be examined for the induction/boosting of selected tumor antigen-specific T and B cell responses. The phase I portion has been successfully completed with 6 patients without DLT. Enrollment into the phase II portion has begun. At present, a total of 11 patients have been enrolled. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT2-1-02.

Novel Combination of Toll-Like Receptor (TLR)-7 Agonist Imiquimod and Local Radiation Therapy in the Treatment of Metastatic Breast Cancer Involving the Skin or Chest Wall

Novel Combination of Toll-Like Receptor (TLR)-7 Agonist Imiquimod and Local Radiation Therapy in the Treatment of Metastatic Breast Cancer Involving the Skin or Chest Wall

Novel combination of toll-like receptor (TLR)-7 agonist imiquimod and local radiotherapy in the treatment of breast cancer chest wall recurrences or skin metastases.

TPS3122 Background: To assess the local and systemic effects of the novel combination of local radiotherapy (RT) with imiquimod (IMQ) applied topically to breast cancer metastatic to skin, and measure immunologic correlates (clinicaltrials.gov NCT01421017). Breast cancer is the 2nd most common tumor to metastasize to the skin. Current therapies for unresectable skin lesions are rarely curative. Patients ultimately die of visceral metastases, necessitating more effective therapies. IMQ, a synthetic TLR-7 agonist has profound effects on the tumor immune microenvironment and can lead to regression of cutaneous breast cancer metastases (Adams, Clin Ca Res, 2012). The trial was designed based on accumulated data supporting the synergy of combined RT/immunotherapy (Formenti, JNCI, 2013), and pre-clinical data demonstrating the synergy of topical IMQ and local RT in a mouse model of mammary adenocarcinoma which ulcerates through the skin, and mimics a chest wall recurrence. In the mouse model, the combination was superior with complete regressions of the treated tumors, responses at untreated sites and improved survival (Dewan, Clin Ca Res, 2012). Methods: Eligibility: patients with biopsy-confirmed breast cancer, measurable disease and skin metastases, ECOG PS 0-2 and adequate organ/marrow function. RT is delivered to 1 area of skin metastases in 5 fractions of 6 Gy (days 1, 3, 5, 8, 10). IMQ cream is applied topically 5 nights/week for 8 weeks, beginning on day 1. Following a brief phase I portion to allow dose optimization in the event of unanticipated adverse events (3-3 design), the phase II study evaluates efficacy with 25 additional patients planned. Primary endpoint is the response rate in untreated distant metastases, assessed by immune-related response criteria. The local tumor responses and safety of the combination will also be determined; tumor biopsies will be studied for immune-mediated rejection signatures and peripheral lymphocytes for antigen-specific T and B cell responses. To date, 10 patients have been enrolled. The phase I portion has been successfully completed with 6 patients without DLT. Phase II enrollment has begun. Clinical trial information: NCT01421017.

Lipid-associated membrane lipopeptides of M. arginini activate NF-κB by interacting with TLR2/1, TLR2/6, and TLR2/CD14

Various strains of mycoplasmas cause the activation of transcriptional factor NF-κB as a result of interactions with different combinations of Toll-like receptors (TLRs). It is well known that MALP-2 protein of M. fermentans activates NF-κB through interactions with TLR2/6, activates lipid-associated membrane lipopeptides (LAMPs) of M. penetrans through TLR1/2, activates LAMPs of M. pneumoniae through combinations of Toll-like receptors (TLR2/6 and TLR1/2), and activates the superantigene of M. arthritidis through TLR2- and TLR4-dependent pathways. In this study, we defined specific Toll-like receptors for LAMPs of M. arginini. For carrying out the research, we used the following cell lines, which express certain combinations of TLRs and their coreceptors: 293-null, 293-hTLR2, 293-hTLR1/2, 293-hTLR2/CD14, 293-hTLR2/6, and 293-hTLR4/CD14-MD2. It was shown that LAMPs of M. arginini cause the activation of NF-κB by interacting with TLR2/1, TLR2/6 and TLR2/CD14, but not with TLR2 or TLR4 alone.

Mycoplasma infection with M. Arginini results in NF-κB constitutive activation and inhibition of apoptosis in cells expressing toll-like receptors TLR2/6

Transcription factor NF-κB is one of the main determinants of cell survival under different stress conditions. It is known that different mycoplasma species and their structural elements activate this transcription factor upon interacting with specific combinations of Toll-like receptors on the surface of the host cell. We supposed that activation of NF-κB in response to mycoplasma infection resulted in an increased resistance of the infected cell cultures to proapoptotic factors. In the present study, we demonstrated that the infection of cells that expressed Toll-like receptors 2/6 with mycoplasma M. arginini resulted in the activation of transcription factor NF-κB and inhibited apoptosis induced by different chemotherapeutical drugs such as cisplatin, 5-fluoruracyl, taxol.

Combined Stimulation with Interleukin-18 and CpG Induces Murine Natural Killer Dendritic Cells to Produce IFN-γ and Inhibit Tumor Growth

Natural killer dendritic cells (NKDC) are a novel subtype of dendritic cells with natural killer (NK) cell properties. IFN-gamma is a pleiotropic cytokine that plays an important role in the innate immune response to tumors. Based on our previous finding that the combination of Toll-like receptor 9 ligand CpG and interleukin (IL)-4 stimulates NKDC to produce IFN-gamma, we hypothesized that NKDC are the major IFN-gamma-producing dendritic cell subtype and may play a significant role in the host antitumor response. We found that under several conditions in vitro and in vivo NKDC accounted for the majority of IFN-gamma production by murine spleen CD11c(+) cells. IL-18 alone induced NKDC to secrete IFN-gamma, and the combination of IL-18 and CpG resulted in a synergistic increase in IFN-gamma production, both in vitro and in vivo. NK cells made 26-fold less IFN-gamma under the same conditions in vitro, whereas dendritic cells produced a negligible amount. The mechanism of IFN-gamma secretion by NKDC depended on IL-12. NKDC selectively proliferated in vitro and in vivo in response to the combination of IL-18 and CpG. Systemic treatment with IL-18 and CpG reduced the number of B16F10 melanoma lung metastases. The mechanism depended on NK1.1(+) cells, as their depletion abrogated the effect. IL-18 and CpG activated NKDC provided greater tumor protection than NK cells in IFN-gamma(-/-) mice. Thus, NKDC are the major dendritic cell subtype to produce IFN-gamma. The combined use of IL-18 and CpG is a viable strategy to potentiate the antitumor function of NKDC.

Genomic organization and transcript profiling of the bovine toll-like receptor gene cluster TLR6- TLR1- TLR10

Toll-like receptors (TLRs) are a family of recognition receptors playing a crucial role in the innate immune system. Different combinations of TLRs are thought to be crucial for effective immune response, thus insight into the organization and expression of TLRs is important for understanding disease resistance. Mastitis is the most frequent and costly disease in dairy production, and the innate immune system is considered to be important in the first line defence against this disease. In the present paper we have characterized the genomic organization of TLR6- TLR1- TLR10 in a ∼ 50 kb region of bovine chromosome 6, including 5′-untranslated exons not previously described. A method for gene expression analysis was developed and used for transcription profiling of the three paralogous genes in different bovine tissues. The expression analysis showed similar expression profiles for TLR1 and TLR6, which indicate a co-regulation of these two genes in cattle. TLR10 had a different expression profile, pointing toward a stronger functional diversification compared to TLR1 and TLR6. The differences in expression are in accordance with the evolutionary history of this gene cluster, where TLR10 diverged from the common ancestral gene before the duplication event that created TLR1 and TLR6.

Domain Exchange between Human Toll-like Receptors 1 and 6 Reveals a Region Required for Lipopeptide Discrimination

Among the 10 human Toll-like receptors (TLRs), TLR2 appears to be unique in its requirement for cooperation with other TLRs, namely TLR1 and TLR6, to mediate cell signaling. Through reconstitution experiments, we have defined more precisely the function of these human TLRs. Human colonic epithelial cells cotransfected with TLR1 and -2 preferentially respond to a synthetic tripalmitoylated bacterial lipopeptide analogue (Pam(3)CSK(4)). However, examination of a wide variety of lipopeptide derivatives indicates that recognition by human TLR1 and -2 does not strictly correlate with the number or position of the acyl chains on the modified cysteine residue. Conversely, human TLR2 and -6 exclusively respond to lipopeptides possessing a diacylglycerol group. Most surprisingly, we have found that an R stereoisomer of diacylated macrophage-activating lipopeptide 2 (MALP-2) exclusively activates epithelial cells through TLR6 and -2 but not through TLR1 and -2. These results suggest that the chirality of the central carbon of the diacylglycerol group of these agonists is a structural determinant for human TLR recognition. Examination of chimeric receptors, generated by domain exchange between TLR1 and -6, has revealed that leucine-rich repeats 9-12 of the extracellular domain enable these receptors to discriminate between structurally similar lipopeptides. However, additional chimeric constructs reveal that this region alone is not sufficient to generate receptors that can functionally cooperate with TLR2. Our results support the idea that TLR1 and TLR6 diverged during evolution to differentially recognize natural lipoprotein structures and that this function has been conserved with respect to the human receptors.