Our November 25, 2011 article on this blog focused on Ralph Steinman, one of the three winners of The Nobel Prize in Physiology or Medicine for 2011. That article focused on dendritic cell-based vaccines for cancer, and the application of this area of science and technology to treating Dr. Steinman’s own pancreatic cancer. Dr. Steinman died on September 30, 2011 after a four-and-a-half year battle with his disease, and was awarded the Nobel Prize three days later. He is the only person to ever have been awarded a Nobel Prize posthumously.

Now comes a Nobel Prize Essay, in the December 9, 2011 issue of Cell, entitled “Bridging Innate and Adaptive Immunity”, written by William E. Paul (Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, NIH”). It is immediately followed by an obituary for Ralph Steinman, written by Antonio Lanzavecchia and Federica Sallusto (Institute for Research in Biomedicine, Bellinzona, Switzerland).

The Nobel Prize in Physiology or Medicine for 2011 was divided, one half awarded jointly to Drs. Bruce A. Beutler (Scripps Research Institute, LA Jolla, CA and University of Texas Southwestern Medical Center, Dallas, TX) and Jules A. Hoffmann [National Center of Scientific Research (CNRS), Strasbourg, France] “for their discoveries concerning the activation of innate immunity” and the other half to Dr. Ralph M. Steinman (Rockefeller University, New York, NY) “for his discovery of the dendritic cell and its role in adaptive immunity”. So the focus of this year’s Nobel Prize in Physiology or Medicine is on the two arms of the immune response–innate and adaptive immunity, and the relationship between the two.

Innate and adaptive immunity in the early to mid-20th century

Dr. Paul’s essay is a historical exposition of how researchers came to understand the basis of the innate and the adaptive immune responses, and how they work together as a coherent system. Adaptive immunity focuses on the ability of a vertebrate organism to “learn” to respond to a specific new antigen, and to “recall” and respond to an antigen that it had been exposed to in the past. Innate immunity focuses on the ability of nearly all multicellular life forms, including plants, to respond rapidly to protect themselves against pathogens, using the inflammatory system.

The essay begins with the first ever Nobel Prize given for a discovery in immunology, in 1908. This was shared by two pioneers in the field–Paul Ehrlich and Ilya (or Élie) Metchnikoff. Ehrlich pioneered the study of what is now called adaptive immunity. His work in immunology focused on the ability of humans and animals to develop specific antibodies to toxins such as tetanus toxin and diphtheria toxin. Metchnikoff pioneered the study of what is now called innate immunity. His work resulted in the discovery of phagocytosis, the process by which certain white blood cells can ingest and destroy harmful microbes.

As outlined in Dr. Paul’s article, most of the attention of immunologists between the awarding of the 1908 Nobel Prize and the modern era was on adaptive immunity, focused on the clonal selection theory of immunity and on discoveries in the the cellular (e.g., T cells) and humoral (e.g., antibodies) arms of adaptive immunity. A key practical application of the study of adaptive immunity–from Ehrlich’s day to the present–has been the development of vaccines.

Adjuvants and Charles Janeway’s pattern recognition hypothesis

However, mid-20th century immunology had a “dirty little secret”. Immunization with a pure antigen produces either a very weak immune response, or immune tolerance. In order to obtain a strong immune response, it is necessary to co-inject an adjuvant along with the antigen. The creation of adjuvants–which is involved not only in experimental immunology, but in such practical applications as vaccines–has been something of a black art. Adjuvants used in vaccines include  oil emulsions (which are thought to serve as depots for an antigen) and aluminum hydroxide (which is thought to act as an irritant). The most famous adjuvant in experimental immunology is complete Freund’s adjuvant, a strong adjuvant that consists of killed Mycobacteria tuberculosis bacteria in a water-in-oil emulsion. (Complete Freund’s adjuvant is too toxic for use in humans.)

In 1989, the late Dr. Charles Janeway (Yale University, New Haven, CT) proposed a hypothesis to explain the need for adjuvants; this hypothesis was very fruitful in stimulating further research on the immune response. Dr. Janeway hypothesized that the immune system required both an antigen/receptor interaction (as in classic adaptive immunity) and a recognition of pathogen-associated molecular patterns (PAMPs). PAMPs would be recognized by “pattern-recognition receptors” (PRRs), which would be broadly expressed by immune and inflammatory cells. Recognition of PAMPs by cells carrying PRRs would result in an innate immune response, which would be interpreted by cells of the adaptive immune system, the lymphocytes, as “permission” to mount an adaptive response when they recognized a specific antigen. In vaccination, the function of an adjuvant would be to provide the needed PAMPs.

Drs. Hoffman and Beutler and innate immunity

Beginning in 1996, Jules Hoffmann and his colleagues elucidated the innate immune response pathway in the fruit fly Drosophila, which enables the fly to produce the antifungal peptide drosomycin, and thus to become resistant to fungal infection. This pathway is initiated by the cell surface receptor Toll, and is homologous to the interleukin 1 (IL-1)/NF-κB signaling pathway, which is a key pathway in vertebrate immune and inflammatory responses.

Dr. Janeway and his colleagues then followed up on this study, in order to identify the corresponding microbial sensors in humans. They first scanned a molecular biology database, and identified a transcript that encoded a human homologue of Drosophila Toll, which they named a “Toll-like receptor” (TLR). Since Dr. Janeway and his colleagues did not know the ligand for their TLR, they constructed a chimeric molecule in which the extracellular domain of CD4 was linked to the cytoplasmic domain of the TLR. They expressed this chimera in a human monocyte cell line. When the chimera was crosslinked with an anti-CD4 antibody, NF-κB was activated, resulting in the production of the proinflammatory cytokines IL-1, IL-6, and IL-8. This showed that humans had at least one Toll homolog (Dr. Janeway’s TLR turned out to be TLR4) and that it controlled a signaling pathway similar to those controlled by Drosophila Toll or human IL-1. The ligands for human TLRs remained unknown, as did whether TLRs were the microbial sensors/PRRs postulated by Dr. Janeway had postulated.

It was Bruce Beutler who first determined the nature of TLR recognition specificity. In the 1990s, he worked to identify the genetic defect that rendered some mice unresponsive to lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, which acts as an endotoxin in humans and other mammals. He used two closely related mouse strains, one of which was responsive to LPS (the “wild type” strain), and the other that was unresponsive (the “mutant” strain). Upon stimulation with LPS, macrophages from the wild type mouse produced tumor necrosis factor alpha (TNFα), while macrophages from mutant mice did not. Dr. Beutler used positional cloning to determine the gene that was mutant in the LPS unresponsive mice. In 1998, he and his colleagues reported that that gene was Tlr4, which codes for the very same TLR identified by Dr. Janeway and his colleagues a year earlier. Dr. Beutler’s study indicated that LPS was a direct or indirect ligand for TLR4. It also showed that one type of molecule that would fulfill the criteria for a “PAMP”, namely LPS, working via TLR4 as a “PRR”, could activate the NF-κB-IL-1 pathway.

Since the initial identification of TLR4 by Dr. Beutler and his colleagues, other researchers have identified numerous other TLRs, which are activated by a variety of bacterial and viral molecules. These include such types of molecules as single- and double-stranded RNAs, CpG oligodeoxynucleotides, bacterial flagellin, lipopeptides, and zymosan, all of which fit with Dr. Janeway’s PAMP hypothesis. Different TLRs occupy different subcelluar locations–some are on the cell surface, others in intracellular vesicles. In addition to TLRs, other types of molecules may also act as PRRs.

Dr. Steinman, dendritic cells, and the unification of innate and adaptive immunity

Now we come to the work of Ralph Steinman and his colleagues on the role of dendritic cells in adaptive immune responses, and their relationship to innate immunity.

Antibodies (whether free antibodies or antibodies on the surface of B cells) can recognize molecules on the surface of pathogens. T cell receptors, however, recognize small antigenic peptides carried by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs). This recognition, together with the activity of other signaling molecules on APCs, results in the activation of the T cell.

The requirement for an APC in T-cell activation was first recognized in the late 1960s and early 1970s. At that time, immunologists generally believed that macrophages and perhaps B cells were the major APCs. In 1973, Ralph Steinman and Zanvil Cohn identified mouse dendritic cells, which are rare cells in the spleen and lymph nodes that have a stellate morphology. In 1978, Dr. Steinman and his colleagues published evidence that dendritic cells had potent immunostimulatory activity, and were over 100 times as effective in immunostimulation as macrophages and B or T cells.

Researchers were initially skeptical about Dr. Steinman’s studies, largely based on the widely held view that the far more numerous macrophages were the major APCs. However, a series of studies by Dr. Steinman and his colleagues showed that dendritic cells are the key APCs for nearly all aspects of T cell activation, and that the potency of dendritic cells as APCs far exceeds that of macrophages and B cells.  Indeed, modern techniques that led to the deletion of dendritic cells result in a profound inability to mount adaptive immune responses.

Dendritic cells are found in perhaps every type of tissue, where they exist in an immature state. For example, the population of immature dendritic cells in the skin are known as Langerhans cells–these cells are illustrated in the figure at the top of our November 25, 2011 article. Immature dendritic cells in tissues act as sentinels of microbial infection, and function to capture antigens (e.g., antigens from pathogenic microbes, or from cells infected by viruses or bacteria). They also express TLRs.

When tissue dendritic cells are stimulated via their TLRs (e.g., by TLR4 binding to bacterial LPS), the dendritic cells change to a mature phenotype, which is specialized in antigen presentation. These mature dendritic cells migrate from the tissue into the draining lymph node. The stimulated dendritic cells in the lymphoid system upregulate class II MHC molecules and other cell surface molecules involved in antigen presentation, and they also produce cytokines involved in T cell activation. The dendritic cells thus activate T cells, and the antigens presented on their surface, as well as the pattern of cytokines they produce, determine the specificity and the type of activated T cells that will result from their actions.

Thus, the work of Dr. Steinman and his colleagues serves to integrate studies of innate and adaptive immunity, and to elucidate how these two branches of the immune system work together to enable humans and other vertebrates to mount immune responses against pathogens and other insults such as tumors.

Despite the major advances in the relationship between innate and adaptive immunity that have been made in recent years, their are still many unknowns. For example, there are minority types of T cells such as natural killer T (NKT) cells and gamma-delta (γδ) T cells, which are conventionally thought to be involved in bridging innate and adaptive immunity. However, their functions are not well understood. Moreover, there are also numerous subsets of dendritic cells, and the functions of these subsets is also not well understood. These cell types, and other unknowns in the relationship between innate and adaptive immunity might, for example, be involved in the pathogenesis of steroid-resistant asthma, the most serious type of asthma.

Implications for drug discovery and development

Our previous article on Ralph Steinman and dendritic cells emphasized the development of dendritic cell vaccines, especially those for cancer. However the broad area of the relationship between innate and adaptive immunity has been and is expected to be a major factor in discovery and development of many types of drugs, vaccines, and immunotherapies.

• Numerous cytokine-based therapies (e.g., interferons, interleukins, and TNF-related therapeutics) have already been developed and marketed. Dr. Beutler himself was the co-discoverer of TNFα in 1985,  and now there are several types of TNF inhibitors on the market.

• In the vaccine area, Dr. Steniman’s work may allow researchers to design more effective adjuvants, a key need in the design of novel anti-viral and anti-cancer vaccines.

• Several companies are developing TLR modulators as drugs or vaccine adjuvants. These include TLR agonists and antagonists. For example, Pfizer is developing the oligonucleotide TLR9 agonist vaccine adjuvant CpG7909 (in Phase 3 trials with GlaxoSmithKline’s MAGE-A3 melanoma vaccine), and another oligonucleotide TLR9 agonist product agatolimod, in combination with trastuzumab (Genentech/Roche’s Herceptin) in treatment of breast cancer (Phase 2). [Pfizer’s TLR agonists were originally developed by Coley Pharmaceuticals (Cambridge, MA), which Pfizer acquired in 2008.] TLR antagonists in development include Eisai’s eritoran tetrasodium, a TLR4 antagonist in Phase 3 trials for the treatment of sepsis and septic shock.

• Research on the role of various immune cell populations that are thought to link innate and adaptive immunity (e.g. Th17 cells, NKT cells, and γδ T cells) in steroid-resistant asthma may lead to the design of new medicines to treat this serious condition.

There are likely to be numerous other drug discovery and development applications of research on the relationship between innate and adaptive immunity that will emerge as work in this very complex area continues.
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