T cell-mediated immune responses & signaling pathways activated by Toll-like Receptors
Curator: Larry H. Bernstein, MD, FCAP
Series E. 2; 6.10
Bruce A. Beutler, Jules A. Hoffmann, Ralph M. Steinman
Bruce Beutler, MD
Regental Professor; Raymond and Ellen Willie Distinguished Chair in Cancer Research, in Honor of Laverne and Raymond Willie, Sr.
Department Center for Genetics of Host Defense, Immunology
Bruce Beutler, MD, the son of the “father of red cell metabolic pathways”, Ernest Beutler, was born in Chicago and grew up in Los Angeles, where Beutler did his groundbreaking work on hemolytic red cell disorders at City of Hope Hospital. He was infected with an interest in medicine and research at an early age, somewhat analagous to a comparison with Roger and Arthur Kornberg. He attended University of California San Diego (UCSD) Medical School, where his interest in immunity was sparked by Abraham Braude, PhD, Professor Pathology and Medicine, and Chief of Microbiology.
Bruce Beutler, MD, discovered an important family of receptors that allow mammals to sense infections when they occur, triggering a powerful inflammatory response. For this work he received the 2011 Nobel Prize in Physiology or Medicine.
The period from 1950 to 1953 was critical to Abraham Braude’s career. During these years, Braude realized the importance of the microbiology laboratory not only in patient management, but also in physician education. He became convinced that infectious disease clinicians must not only be excellent microbiologists, but also exercise technical and administrative control of the diagnostic microbiology laboratory. He demonstrated and taught this principle of medical practice and education for the next 35 years as director of diagnostic microbiology and chief of infectious diseases at the Texas Southwestern Medical School of the University of Texas from 1953-57, at the University of Pittsburgh from 1957 to 1969, and at the University of California at San Diego from 1967 until his death in December, 1984. He also was my mentor as a resident in 1972, and it was remarkable how he had developed his own antibiotic diffusion plates for sensitivity studies on bacterial isolates.
He discovered that the endotoxin of cold-growing bacteria that had contaminated refrigerated blood could cause vascular collapse and death of transfused people without bacterial multiplication in the body. This observation established the importance of endotoxin as the major virulence factor of Gram negative bacteria and set the stage for his later fundamental and applied research on the pathogenesis and treatment of life-threatening septic shock. This aspect of infection, in the host defense, is perhaps important in the development of the young Beutler.
During his years in Dallas, Pittsburgh, and San Diego, Braude made outstanding research contributions in three areas, anaerobic infections, pyelonephritis, and the endotoxin shock of Gram negative bacteremia. He devised a simple, effective method for culturing fastidious anaerobes, discovered Bacteroides penicillinase, and proved that so-called “sterile” brain abscesses are caused by anaerobes. His landmark papers on brain infections and sinusitis awakened us to the major role of anaerobes in infectious disease. His work on pyelonephritis ranged from devising realistic models of infection to elegant studies that helped explain the inadequacies of the immune responses of the kidney.
Beutler received his undergraduate degree from the University of California at San Diego in 1976, and his MD degree from the University of Chicago in 1981. After two years of residency at the University of Texas Southwestern Medical Center, he became a postdoctoral fellow and then an Assistant Professor at the Rockefeller University (1983-1986), where he isolated mouse tumor necrosis factor (TNF), and was the first to recognize TNF as a key executor of the inflammatory response. Returning to Dallas in 1986 as an HHMI investigator, he designed recombinant inhibitors of TNF that are widely used in the treatment of rheumatoid arthritis and other inflammatory diseases. He also used TNF as a biological endpoint in order to identify the receptor for bacterial lipopolysaccharide (LPS). This he achieved by positionally cloning the Lps mutation of mice, known to prevent all biological responses to LPS, including TNF production. He thus concluded that Toll-like receptor 4 (TLR4) acts as the signaling core of the LPS receptor and proposed that other TLRs might also recognize conserved molecular signatures of infection.
Moving in 2000 to the Scripps Research Institute, Beutler developed the largest mouse mutagenesis program in the world, and applied a forward genetic approach to decipher the signaling pathways activated by TLRs. He also identified many other molecules with non-redundant function in the immune response.
Beutler is currently a Regental Professor and Director of the Center for Genetics of Host Defense at the University of Texas Southwestern Medical Center. He also holds the Raymond and Ellen Willie Distinguished Chair in Cancer Research in honor of Laverne and Raymond Willie, Sr. He has authored or co-authored more than 300 papers, which have been cited more than 46,000 times. Before he received the Nobel Prize, his work was recognized by the Shaw Prize (2011), the Albany Medical Center Prize in Medicine and Biomedical Research (2009), election to the National Academy of Sciences and Institute of Medicine (2008), the Frederik B. Bang Award (2008), the Balzan Prize (2007), the Gran Prix Charles-Leopold-Mayer (2006), the William B. Coley Award (2005), the Robert-Koch-Prize (2004), and other honors.
“Monitored Saturation Mutagenesis of the Mouse Genome”
Bruce Beutler, University of Texas Southwestern Medical Center, TX USA
The availability of massively parallel sequencing platforms has accelerated the identification of induced mutations in the mouse genome. However, genetic mapping has remained essential to identify causative mutations, and for a time, became the rate-limiting step in mammalian forward genetics. We have recently described a procedure that permits real time identification of mutations responsible for phenotypes of any kind. By inducing mutations on a defined genetic background (C57BL/6J) and sequencing the whole exome of all G1 carriers of these mutations, we are able to identify all candidate aberrations that might cause phenotype in descendants of the G1. We then genotype all G3 mice to determine zygosity at each mutation site. Pre-genotyped G3 mice are distributed for extensive phenotypic screening. As quantitative phenotypic data are uploaded to the computer, an automated search for correlation between phenotype and genotype is initiated using dominant, recessive and semidominant models of transmission. The computer returns a list of statistical associations between specific mutations and phenotype, allowing immediate inference regarding causation. As allelic series develop at most loci, the strength of inference increases. CRISPR/Cas9 targeting is used to validate candidates identified by automated mapping. Presently, more than 65,000 point mutations disrupting the mouse coding region have been tested in this manner. To our best estimate, 18 percent of all mouse genes have been mutated to phenovariance and examined in 135 phenotypic screens. About 50,000 mutations can be surveyed annually. A list of robust associations between mouse genes and phenotypic anomalies will facilitate genetic investigations in humans, where a uniform genetic background is not available and a heavy mutation load confounds such analysis.
Dec 9, 2014 … Professor Bruce Beutler, a 2011 recipient of the Nobel Prize in Medicine, was the Keynote speaker. Professor Beutler discovered the …
In the last few years of his life, Dr. Ralph Steinman made himself into an extraordinary human lab experiment, testing a series of unproven therapies – including some he helped to create – as he waged a very personal battle with pancreatic cancer.
The winner of the 2011 Nobel prize in medicine died only three days before the award was announced.
Immunologist Ralph Steinman was honored today by the Nobel Committee for his discovery of dendritic cells, a class of immune cells that help rally the body’s natural defenses to fight disease. However, the prize is bittersweet for all who knew the 68-year-old scientist during his long career as a researcher and mentor at Rockefeller University in New York City.
“Dendritic cells are the guys who are training the fighters,” says Pawel Kalinski, an immunologist at the University of Pittsburgh in Pennsylvania, about their role in activating T cells, the body’s immune sentries. Over decades, with Steinman often leading the way, the work transformed cancer research. Cancer vaccines either using or targeting dendritic cells are now the subject of numerous clinical trials, and the first-ever cancer vaccine to be approved in the United States—called Provenge, to treat prostate cancer—injects a patients’ own dendritic cells back into their body. It went on the market last year.
“He felt that human clinical investigation was the highest form of research, that it was critical to engage in it,” Dr. Sarah Schlesinger, Steinman’s clinical lab director and colleague at New York’s Rockefeller University, told Reuters. “He had great criticism of how slowly the process moved … he was impatient with data and mice,” she added.
Steinman spent his entire career on immunology research for which he won the Nobel Prize, an honor he shares with American Bruce Beutler and French biologist Jules Hoffmann for their contributions to explaining the immune system.
Steinman’s discovery of dendritic cells in 1973 led to the first therapeutic cancer vaccine, Dendreon’s Provenge, which treats men with advanced prostate cancer.
Jules A. Hoffmann (born 2 August 1941) is a Luxembourg-born French biologist. During his youth, growing up in Luxembourg, he developed a strong interest in insects under the influence of his father, Jos Hoffmann. This eventually resulted in the younger Hoffmann’s dedication to the field of biology using insects as model organisms. He currently holds a faculty position at the University of Strasbourg. He is a research director and member of the board of administrators of the National Center of Scientific Research (CNRS) in Strasbourg, France. He was elected to the positions of Vice-President (2005-2006) and President (2007-2008) of the French Academy of Sciences. Hoffmann and Bruce Beutler were jointly awarded a half share of the 2011 Nobel Prize in Physiology or Medicine for “their discoveries concerning the activation of innate immunity,”. [More specifically,the work showing increased Drosomycin expression following activation of Toll pathway in microbial infection.]
Hoffmann and Lemaitre discovered the function of the fruit fly Toll gene in innate immunity. Its mammalian homologs, the Toll-like receptors, were discovered by Beutler. Toll-like receptors identify constituents of other organisms like fungi and bacteria, and trigger an immune response, explaining, for example, how septic shock can be triggered by bacterial remains.
During his Ph.D. program under Pierre Joly, Hoffmann started his research in studying antimicrobial defenses in grasshoppers, inspired by the previous works done in the laboratory of Pierre Joly showing that no opportunistic infections were apparent in insects after the transplantation of certain organs from one to another. Hoffmann confirmed discovery of phagocytosis done by Eli Metchnikoff, through injection of Bacillus thuringiensis and observation of increase of phagocytes. In addition, he showed strong correlation between hematopoiesis and antimicrobial defenses by assessing the susceptibility of an insect to the microbial infection after X-ray treatment. Hoffmann shifts from using grasshopper model to using dipteran species in the 80s. By using Phormia terranovae, Hoffmann and his colleagues were able to identify 82-residues long antimicrobial polypeptide named Diptericin which was glycine-rich, along with other polypeptides in Drosophila melanogaster such as defensin, cecropin, and attacin. Further molecular genetic analysis revealed that the promoters for the genes encoding these antimicrobial peptides contained DNA sequences similar to the binding elements for NF-κB in mammalian DNA. Dorsal gene, critical in dorso-ventral patterning in the early embryo of Drosophila melanogaster was also identified to be in this NF-κB family. It was initially speculated by Hoffmann and colleagues that activity of Dorsal was directly linked to the expression of the Diptericin gene. However, it turned out that Diptericin was normally induced even in the loss-of-function Dorsal mutants. Further conducted research showed that Diptericin expression was dependent on the expression of imd gene. Identification of another antifungal peptide named Drosomycin and RNA blots demonstrated that two distinct pathways(Toll, Imd) exist, involving Drosomycin and Diptericin respectively. Similarities of structure and function between several members in the Drosophila embryo and members in mammals being noted, study “The Dorsoventral Regulatory Gene Cassette spảtzle/Toll/cactus Controls the Potent Antifungal Response in Drosophila Adults” by Lemaitre and Hoffmann in 1996 illuminated the possible existing innate immunity in Drosophila in response to fungal challenge. Later works identified that Toll transmembrane receptors are present in a wide variety of phyla and are conserved through evolution along with conservation of NF-κB activating cascades.
Hoffmann was a research assistant at CNRS from 1964 to 1968, and became a research associate in 1969. Since 1974 he has been a Research Director of CNRS. Between 1978 and 2005 he was Director of the CNRS research unit “Immune Response and Development in Insects”, and from 1994 to 2005 he was director of the Institute of Molecular and Cellular Biology of CNRS in Strasbourg.
The History of Toll-like receptors — Redefining Innate Immunity
The discovery of Toll-like receptors (TLRs) was an important event for immunology research and was recognized as such with the awarding of the 2011 Nobel Prize in Physiology or Medicine to Jules Hoffmann and Bruce Beutler, who, together with Ralph Steinman, the third winner of the 2011 Nobel Prize and the person who discovered the dendritic cell, were pioneers in the field of innate immunity. TLRs have a central role in immunity — in this Timeline article, we describe the landmark findings that gave rise to this important field of research.
Toll-like Receptors and Their Crosstalk with Other Innate Receptors in Infection and Immunity
Taro Kawai & Shizuo Akira
Immunity 27 May 2011; 34(5): 637–650, http://dx.doi.org/10.1016/j.immuni.2011.05.006
oll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play a central role in host cell recognition and responses to microbial pathogens. TLR-mediated recognition of components derived from a wide range of pathogens and their role in the subsequent initiation of innate immune responses is widely accepted; however, the recent discovery of non-TLR PRRs, such as C-type lectin receptors, NOD-like receptors, and RIG-I-like receptors, suggests that many aspects of innate immunity are more sophisticated and complex. In this review, we will focus on the role played by TLRs in mounting protective immune responses against infection and their crosstalk with other PRRs with respect to pathogen recognition.
Invivogen – Review
Toll-Like Receptors (TLRs) play a critical role in the early innate immune response to invading pathogens by sensing microorganism and are involved in sensing endogenous danger signals.
TLRs are evolutionarily conserved receptors are homologues of the Drosophila Toll protein, discovered to be important for defense against microbial infection . TLRs recognize highly conserved structural motifs known as pathogen-associated microbial patterns (PAMPs), which are exclusively expressed by microbial pathogens,
or danger-associated molecular patterns (DAMPs) that are endogenous molecules released from necrotic or dying cells.
PAMPs include various bacterial cell wall components such as lipopolysaccharide (LPS),peptidoglycan (PGN) and lipopeptides, as well as flagellin, bacterial DNA and viral double-stranded RNA. DAMPs include intracellular proteins such as heat shock proteins as well as protein fragments from the extracellular matrix.
Stimulation of TLRs by the corresponding PAMPs or DAMPs initiates signaling cascades leading to the activation of transcription factors, such as AP-1, NF-κB and interferon regulatory factors (IRFs). Signaling by TLRs result in a variety of cellular responses including the production of interferons (IFNs), pro-inflammatory cytokines and effector cytokines that direct the adaptive immune response.
The TLR Family
TLRs are type I transmembrane proteins characterized by an extracellular domain containing leucine-rich repeats (LRRs) and a cytoplasmic tail that contains a conserved region called the Toll/IL-1 receptor (TIR) domain. The structure of the extracellular domain of TLR3 was revealed by crystallography studies as a large horseshoe-shape .
TLRs are predominantly expressed in tissues involved in immune function, such as spleen and peripheral blood leukocytes, as well as those exposed to the external environment such as lung and the gastrointestinal tract.
Their expression profiles vary among tissues and cell types. TLRs are located on the plasma membrane with the exception of TLR3, TLR7, TLR9 which are localized in the endosomal compartment .
Ten human and twelve murine TLRs have been characterized, TLR1 to TLR10 in humans, and TLR1 toTLR9, TLR11, TLR12 and TLR13 in mice, the homolog of TLR10 being a pseudogene.
TLR2 is essential for the recognition of a variety of PAMPs from Gram-positive bacteria, including bacterial lipoproteins, lipomannans and lipoteichoic acids. TLR3 is implicated in virus-derived double-stranded RNA. TLR4 is predominantly activated by lipopolysaccharide. TLR5 detectsbacterial flagellin and TLR9 is required for response to unmethylated CpG DNA. Finally, TLR7 andTLR8 recognize small synthetic antiviral molecules , and single-stranded RNA was reported to be their natural ligand . TLR11(12) has been reported to recognize uropathogenic E.coli  and a profilin-like protein from Toxoplasma gondii .
The repertoire of specificities of the TLRs is apparently extended by the ability of TLRs to heterodimerize with one another. For example, dimers of TLR2 and TLR6 are required for responses to diacylated lipoproteins while TLR2 and TLR1 interact to recognize triacylated lipoproteins . Specificities of the TLRs are also influenced by various adapter and accessory molecules, such as MD-2 and CD14 that form a complex with TLR4 in response to LPS .
TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that leads to the production of inflammatory cytokines, and a MyD88-independent pathway associated with the stimulation of IFN-β and the maturation of dendritic cells.
The MyD88-dependent pathway is common to all TLRs, except TLR3 . Upon activation byPAMPs or DAMPs, TLRs hetero- or homodimerize inducing the recruitment of adaptor proteins via the cytoplasmic TIR domain.
Adaptor proteins include the TIR-domain containing proteins, MyD88, TIRAP (TIR-associated protein), Mal (MyD88 adaptor-like protein), TRIF (TIR domain-containing adaptor protein-inducing IFN-β) and TRAM (TRIF-related adaptor molecule).
Recruitment of MyD88 for instance, in turn recruits IRAK1 and IRAK4. IRAK4 subsequently activates IRAK1 by phosphorylation. Both IRAK1 and IRAK4 leave the MyD88-TLR complex and associate temporarily with TRAF6 leading to its ubiquitination. Bcl10 and MALT1 form oligomers that bind to TRAF6 promoting TRAF6 self-ubiquitination .
Recently, IRAK2 was shown to play a central role in TRAF6 ubiquitination . Following ubiquitination, TRAF6 forms a complex with TAB2/TAB3/TAK1 inducing TAK1 activation . TAK1 then couples to the IKK complex, which includes the scaffold protein NEMO, leading to the phosphorylation of IκB and the subsequent nuclear localization of NF-κB. Activation of NF-κB triggers the the production of pro-inflammatory cytokines such as TNF-α, IL-1 and IL-12.
Individual TLRs induce different signaling reponses by usage of the different adaptor molecules.
TLR4 and TLR2 signaling requires the adaptor TIRAP/Mal, which is involved in the MyD88-dependent pathway . TLR3 triggers the production of IFN-β in response to double-stranded RNA, in a MyD88-independent manner, through the adaptor TRIF/TICAM-1 . TRAM/TICAM-2 is another adaptor molecule involved in the MyD88-independent pathway  which function is restricted to the TLR4 pathway .
TLR3, TLR7, TLR8 and TLR9 recognize viral nucleic acids and induce type I IFNs. The signaling mechanisms leading to the induction of type I IFNs differ depending on the TLR activated. They involve the interferon regulatory
factors, IRFs, a family of transcription factors known to play a critical role in antiviral defense, cell growth and immune regulation. Three IRFs (IRF3, IRF5 and IRF7) function as direct transducers of virus-mediated TLR signaling. TLR3 and TLR4 activate IRF3 and IRF7 , while TLR7 and TLR8 activate IRF5 and IRF7 . Furthermore, type I IFN production stimulated by TLR9 ligand CpG-A has been shown to be mediated by PI(3)K and mTOR .
Significant progress has been made over the past years in the understanding of TLR function . TLRs are essential receptors in host defense against pathogens by activating the innate immune system, a prerequisite to the induction of adaptive immune responses.
Although TLR-mediated signaling is paramount in eradicating microbial infections and promoting tissue repair, the regulation must be tight. TLRs are implicated in a number of inflammatory and immune disorders and play a role in cancer .
Many single nucleotide polymorphisms have been identified in various TLR genes and are associated with particular diseases. Several therapeutic agents targeting the TLRs are now under pre-clinical and clinical
However, the complexity lies in that TLRs act as double-edged swords either promoting or inhibiting disease progression. Furthermore, therapeutic agents targeting the TLRs must be able to antagonize the harmful effects resulting without affecting host defense functions.
Nonetheless, the potential of harnessing and directing the innate immune system with drugs targeting TLRs, to prevent or treat human inflammatory and autoimmune diseases as well as cancer, appears to be promising.
|TLR||Immune Cell Expression||PAMPs||DAMPs||Signal Adaptor||Production|
|TLR1+ TLR2||Cell surface
Mo, MΦ, DC, B
|Triacylated lipoproteins (Pam3CSK4)
|(TLR2 DAMPs listed below)||TIRAP, MyD88,
|TLR2+ TLR6||Cell surface
Mo, MΦ, MC, B
|Heat Shock Proteins
(HSP 60, 70, Gp96)
High mobility group proteins (HMGB1)
(Versican, Hyaluronic Acid fragments)
B, T, NK, DC
|dsRNA (poly (I:C))
Mo, MΦ, DC, MC,
|Heat Shock Proteins
(HSP22, 60, 70,72, Gp96)
High mobility group proteins (HMGB1)
(Versican, Heparin sulfate,
Hyaluronic Acid fragments)
Mo, MΦ, DC, IE
Mo, MΦ, DC. B
Guanosine analogs (Loxoribine)
Mo, MΦ, DC, MC
Mo, MΦ, DC, B,T
|Chromatin IgG complex||MyD88||IC,
Mo, MΦ, DC
Mo: monocytes, MΦ: marcophages, DC: dendritic cells, MC: Mast cells, B: B cells, T: T cells, IE: Intestinal epithelium, IC: Inflammatory cytokines
1. Medzhitov R. et al., 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature, 388(6640):394-7.
2. Choe J. et al., 2005. Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309; 581-585.
3. Nishiya T. & DeFranco AL., 2004. Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling proper ties of the Toll-like receptors. J Biol Chem. 279(18):19008-17.
4. Jurk M. et al., 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol, 3(6):499.
5. Heil F. et al., 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 303(5663):1526-9.
6. Zhang D. et al., 2004. A toll-like receptor that prevents infection by uropathogenic bacteria. Science. 303:1522-1526.
7. Lauw FN. et al., 2005. Of mice and man: TLR11 (finally) finds profilin. Trends Immunol. 26(10):509-11.
8. Ozinsky A. et al., 2000.The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. PNAS USA, 97(25):13766-71.
9. Miyake K., 2003. Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2. Int Immunopharmacol. 3(1):119-28.
10. Adachi O. et al., 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 9(1):143-50.
11. Sun L. et al., 2004. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol Cell. 2004 14(3):289-301.
12. Keating SE. et al., 2007. IRAK-2 participates in multiple Toll-like receptor signaling pathways to NFκB via activation of TRAF6 ubiquitination. J Biol Chem. 282: 33435-33443.
13. Kanayama A. et al., 2004. TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell. 15(4):535-48.
14. Horng T. et al., 2002.The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature. 420(6913):329- 33.
15. Yamamoto M. et al., 2002. Cutting edge: a novel Toll/IL-1 receptor domain-containing adaptor that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol. 169(12):6668-72.
16. Yamamoto M. et al., 2003. TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat Immunol. 4(11):1144-50.
17. Doyle S. et al., 2002. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity. 17(3):251-63.
18. Schoenemeyer A. et al., 2005. The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling. J Biol Chem. 280(17):17005-12.
19. Costa-Mattioli M. & Sonenberg N. 2008. RAPping production of type I interferon in pDCs through mTOR. Nature Immunol. 9: 1097-1099.
20. Kawai T. & Akira S., 2011. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity Immunity 34(5):637-50.
21. Rakoff-Nahoum S. & Medzhitov R., 2009. Toll-like receptors and cancer. Nat Revs Cancer 9:57- 63.
22. Hennessy E. et al., 2010. Targeting Toll-like receptors: emerging therapeutics? Nat Rev Drug Discov 9(4) 293-307.