Association of human TLR1 and TLR6 deficiency with altered immune responses to BCG vaccination in South African infants.

The development of effective immunoprophylaxis against tuberculosis (TB) remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG) vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCG's variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR) variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S) was associated with increased BCG-induced IFN-γ in both discovery (n = 240) and validation (n = 240) cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S) and TLR6_G1083C (synonymous) were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2). After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes) secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the development of adaptive immune responses after in vivo vaccination against a bacterial pathogen in humans. These findings could potentially guide novel adjuvant vaccine strategies as well as have implications for IFN-γ-based diagnostic testing for TB.

Role for MyD88, TLR2 and TLR9 but not TLR1, TLR4 or TLR6 in experimental autoimmune encephalomyelitis.

The potential roles of TLRs in the cause and pathogenesis of autoimmune CNS inflammation remain contentious. In this study, we examined the effects of targeted deletions of TLR1, TLR2, TLR4, TLR6, TLR9, and MyD88 on the induction of myelin oligodendrocyte glycoprotein 35-55 (MOG(35-55)) peptide/CFA/pertussis toxin-induced autoimmune encephalomyelitis. Although C57BL/6.Tlr1(-/-), C57BL/6.Tlr4(-/-) and C57BL/6.Tlr6(-/-) mice showed normal susceptibility to disease, signs were alleviated in female C57BL/6.Tlr2(-/-) and C57BL/6.Tlr9(-/-) mice and C57BL/6.Tlr2/9(-/-) mice of both sexes. C57BL/6.Myd88(-/-) mice were completely protected. Lower clinical scores were associated with reduced leukocyte infiltrates. These results were confirmed by passive adoptive transfer of disease into female C57BL/6.Tlr2(-/-) and C57BL/6.Tlr9(-/-) mice, where protection in the absence of TLR2 was associated with fewer infiltrating CD4(+) cells in the CNS, reduced prevalence of detectable circulating IL-6, and increased proportions of central (CD62L(+)) CD4(+)CD25(+)Foxp3(+) regulatory T cells. These results provide a potential molecular mechanism for the observed effects of TLR signaling on the severity of autoimmune CNS inflammation.

Adjuvant engineering for cancer immunotherapy: Development of a synthetic TLR2 ligand with increased cell adhesion.

The development of effective immunoadjuvants for tumor immunotherapy is of fundamental importance. The use of Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) in tumor immunotherapy has been examined in various clinical applications. Because BCG-CWS is a macromolecule that cannot be chemically synthesized, the development of an alternative synthetic molecule is necessary to ensure a constant supply of adjuvant. In the present study, a new adjuvant was designed based on the structure of macrophage-activating lipopeptide (MALP)-2, which is a Toll-like receptor (TLR)-2 ligand similar to BCG-CWS. Macrophage-activating lipopeptide-2, [S-(2,3-bispalmitoyloxypropyl)Cys (P2C) - GNNDESNISFKEK], originally identified in a Mycoplasma species, is a lipopeptide that can be chemically synthesized. A MALP-2 peptide was substituted with a functional motif, RGDS, creating a novel molecule named P2C-RGDS. RGDS was selected because its sequence constitutes an integrin-binding motif and various integrins are expressed in immune cells including dendritic cells (DCs). Thus, this motif adds functionality to the ligand. P2C-RGDS activated DCs and splenocytes more efficiently than MALP-2 over short incubation times in vitro, and the RGDS motif contributed to their activation. Furthermore, P2C-RGDS showed higher activity than MALP-2 in inducing migration of DCs to draining lymph node, and in inhibiting tumor growth in vivo. This process of designing and developing synthetic adjuvants has been named "adjuvant engineering," and the evaluation and improvement of P2C-RGDS constitutes a first step in the development of stronger synthetic adjuvants in the future.

Human TLRs 10 and 1 share common mechanisms of innate immune sensing but not signaling.

TLRs are central receptors of the innate immune system that drive host inflammation and adaptive immune responses in response to invading microbes. Among human TLRs, TLR10 is the only family member without a defined agonist or function. Phylogenetic analysis reveals that TLR10 is most related to TLR1 and TLR6, both of which mediate immune responses to a variety of microbial and fungal components in cooperation with TLR2. The generation and analysis of chimeric receptors containing the extracellular recognition domain of TLR10 and the intracellular signaling domain of TLR1, revealed that TLR10 senses triacylated lipopeptides and a wide variety of other microbial-derived agonists shared by TLR1, but not TLR6. TLR10 requires TLR2 for innate immune recognition, and these receptors colocalize in the phagosome and physically interact in an agonist-dependent fashion. Computational modeling and mutational analysis of TLR10 showed preservation of the essential TLR2 dimer interface and lipopeptide-binding channel found in TLR1. Coimmunoprecipitation experiments indicate that, similar to TLR2/1, TLR2/10 complexes recruit the proximal adaptor MyD88 to the activated receptor complex. However, TLR10, alone or in cooperation with TLR2, fails to activate typical TLR-induced signaling, including NF-kappaB-, IL-8-, or IFN-beta-driven reporters. We conclude that human TLR10 cooperates with TLR2 in the sensing of microbes and fungi but possesses a signaling function distinct from that of other TLR2 subfamily members.

Role of TLR1 and TLR6 in the host defense against disseminated candidiasis.

Toll-like receptor-1 (TLR1) and TLR6 are receptors of the TLR family that form heterodimers with TLR2. The role of TLR1 and TLR6 for the recognition of the fungal pathogen Candida albicans was investigated. TLR1 is not involved in the recognition of C. albicans, and TLR1 knock-out (TLR1-/-) mice showed a normal susceptibility to disseminated candidiasis. In contrast, recognition of C. albicans by TLR6 modulated the balance between Th1 and Th2 cytokines, and TLR6 knock-out mice displayed a defective production of IL-10 and an increased IFN-gamma release. Production of the monocyte-derived cytokines tumor necrosis factor, IL-1, and IL-6 was normal in TLR6-/- mice, and this was accompanied by a normal susceptibility to disseminated candidiasis. In conclusion, TLR6 is involved in the recognition of C. albicans and modulates the Th1/Th2 cytokine balance, but this results in a mild phenotype with a normal susceptibility of TLR6-/- mice to Candida infection.

Structural and functional evidence for the role of the TLR2 DD loop in TLR1/TLR2 heterodimerization and signaling.

The Toll/Interleukin-1 receptor (TIR) domain of the Toll-like receptors (TLRs) plays an important role in innate host defense signaling. The TIR-TIR platform formed by the dimerization of two TLRs promotes homotypic protein-protein interactions with additional cytoplasmic adapter molecules to form an active signaling complex resulting in the expression of pro- and anti-inflammatory cytokine genes. To generate a better understanding of the functional domains of TLR2 we performed a random mutagenesis analysis of the human TLR2 TIR domain and screened for TLR2/1 signaling-deficient mutants. Based upon the random mutagenesis results, we performed an alanine scanning mutagenesis of the TLR2 DD loop and part of the alphaD region. This resulted in the identification of four residues crucial for TLR2/1 signaling: Arg-748, Phe-749, Leu-752, and Arg-753. Computer-assisted energy minimization and docking studies indicated three regions of interaction in the TLR2/1 TIR-docked heterodimer. In Region I, residues Arg-748 and Phe-749 in TLR2 DD loop were involved in close contacts with Gly-676 in the TLR1 BB loop. Because this model suggested that steric hindrance would significantly alter the binding interactions between DD loop of TLR2 and BB loop of TLR1, Gly-676 in TLR1 was rationally mutated to Ala and Leu. As expected, in vitro functional studies involving TLR1 G676A and TLR1 G676L resulted in reduced PAM(3)CSK(4) mediated NF-kappaB activation lending support to the computerized predictions. Additionally, mutation of an amino acid residue (TLR2 Asp-730) in Region II also resulted in decreased activity in agreement with our model, providing new insights into the structure-function relationship of TLR2/1 TIR domains.