“The Antimicrobial Defence of Drosophila: a paradigm of innate immunity”, by Jules Hoffmann

Today, immunologists consider the innate arm of immunity to be at least as equally important as the adaptive for the overall host defence. The innate immunity comprises a heritable, multifaceted and highly conserved defence system which its molecular basis only now has started to be elucidated. The fundamental questions on how the microbes interact with the host during the first minutes to hours following inoculation, what genes are induced and what molecular effectors are expressed are investigated extensively both in insects and in mammals.

Addressing these issues in the antimicrobial defence of Drosophila, a highly efficient innate defence system, has provided great insight and possibilities in immunology research. The results accumulated so far converge to a theatre where two major pathways act as the major actors of these mechanisms. The first is the Spatzle-Toll cascade, triggered by infection with fungi or gram-positive bacteria, while the second is the Imd (Immune deficiency) cascade, triggered by Gram-negative bacterial invasion. These pathways signal to NF-kB response elements, orchestrating the expression of several hundreds of immune-response genes. As to which protein family serves the infection discrimination function during the microbe invasion, several classes of the Peptidoglycan Recognition Proteins (PGRP) seem to be the possible culprit.

Although the knowledge about the innate immunity emerging from the Drosophila paradigm is still very elementary, several lines of investigation imply that the aforementioned complex signalling cascades are builded and act in a similar fashion in mammals also; every element of the Toll and Imd paths are represented in mammals by the TLR4 and TNF cascades respectively.

“Malaria and Immune Responses in Mosquitoes”, by Fotis Kafatos

Humanity still pays a significant toll for malaria. Approximately 500 million new cases occur worldwide each year, and over one million people die with the disease; mostly children in the sub-Saharan area of Africa. Malaria is transmitted to humans by Anopheles mosquitoes (Anopheles gambiae) and is caused by protozoan parasites of the genus Plasmodium with Plasmodium falciparum and Plasmodium vivax being the most dangerous. Since the Anopheles gambiae is the obligatory vector of human malaria, in order for transmission to occur, the malaria parasite has to complete a complex developmental cycle in the mosquito. In his complex journey inside its vector, the Plasmodium encounters the mosquito’s immune system mechanisms which are implicated in killing it. These multifaceted interactions between the vector and the parasite, offer many novel potential targets for interventions against the disease, even before is transmitted to the human.

In essence, this novel approach is elegant in its simplicity: curing the mosquito to stop malaria. However, what are the mechanisms of the mosquito’s immune system implicated in killing parasites and moreover what are the survival strategies of the parasites? The mechanisms can be explored by analysing tissue-specific transcriptional responses of mosquito’s genes (up- or down-regulated genes) to Plasmodium infection. This particular area of research has been revolutionized by the recent completion of the Anopheles gambiae and Plasmodium falciparum genome sequencing mapping and the application of high throughput methods of functional genomics.

It is now well appreciated that the mosquito midgut epithelium comprises the most crucial barrier during the malaria transmission cycle (time bomb theory of crossing). The ability to knock-out individual genes expressed during the mosquitoparasite interactions has generated a wealth of information. Three of the mosquito immune genes that have been recently identified with these techniques and can affect the parasites life cycle within the mosquito species are the TEP1, CTL1 and LRIM1.

The proteins encoded by these genes, were shown to be true defenders of the mosquito - killing the parasite in the insect's gut. Mosquitoes lacking these genes produced more parasites than their normal counterparts did. Two other genes, CTL4 and CTLMA2, encode proteins which have the opposite effect, safeguarding the developing parasites in the mosquito gut. When these genes are silenced, the mosquito’s immune system destroyed up to 97 percent of the maturing parasites.

While many more genes still remain to be characterized and many more interactions wait to be unmasked, the aforementioned discoveries have raised new possibilities for stopping mosquitoes from spreading the parasite. The most novel approach would be to make genetically engineered mosquitoes populations (with extra genes to attack the parasites, or lacking the genes that protect them) that are refractory to the parasite and release those mosquitoes into the wild to displace existing wildtype.

“Immunoprotections vs. Immunopathology”, by Rolf Zinkernagel

Infection of a cell by a virus can have several outcomes, which depend on the replication cycle of the virus as well as the way the immune system of the organism deals with the specific infection. They can be broadly divided into three categories, with regard to the degree of cell damage they cause and their natural history.

Cytopathic acute infections (such as epidemic childhood infections) are characterized by skin and/or mucosal invasion, viraemia and possibly CNS invasion, which can be prevented by neutralizing antibodies (nAbs). Noncytopathic persistent infections (e.g. HCV, HIV-2) on the other hand are mainly dealt with via T-cell-mediated mechanisms leading to significant cellular damage (immunopathology). Finally, intermittently cytopathic infections (such as HSV and measles) represent an intermediate of the first two categories. In general, noncytopathic infections lead to immunopathology, while cytopathic infections cause less immunopathology and more immunoprotection.

Antigen (Ag) dose and timing of infection (or administration) regulates the immune responses. If the antigen remains peripherally (e.g. skin) where there are no lymph nodes, it is ignored and there is no immune response. The same result ensues when the Ag is present in all the lymphoid tissue of the body, as in vertical infections; however the mechanism involved is tolerance by deletion of reactive clones. On the contrary, if the Ag reaches a few lymph nodes within the first days of infection a strong immune response develops. Antibodies are required to control the infection.

Subsequent extralymphatic persistence of the Ag can lead to either immunologic memory, if the Ag dose is small and localized, or immunopathology, if the Ag dose is widely distributed. All these observations are employed in the design of vaccines, since manipulation of dose, time and place/mode of administration can be utilized to improve responses to vaccines.

Two different scenarios of immunopathology can be discerned. In the first, the offending agent (e.g. a virus) is known and the mechanisms involved are obvious. In the second, either there is no recognized virus or there is an endogenous factor driving the immune response. The result of this second scenario is autoimmunity. It has been observed that for antigens that when brought to lymph nodes lead to a proper immune response, autoimmunity results if the lymphoid tissue is brought to the Ag/organ; that is the case Hashimoto thyroiditis, where the thyroid turns into a large lymph node.

“Anti-viral Antibody and Immunological Memory Responses”, by Rolf Zinkernagel

In order to be able to cope with the multitude of infections, the immune system has evolved in such a way so that it develops immunological memory, after an effective immune response. B cells, responsible for the production of antibody, play a central role in this procedure. They differentiate non-self from self by the presence of polymeric structures in the former. If a microbe has a cell-surface pattern very similar to a self component, then it can evade the immune response. A minimum of 20-30 monomeric units connected into a multimer are required in order to react with B cells and trigger an immune response.

Secondary lymphoid organs are also essential for mounting an effective immune response and developing immunological memory, as are T helper cells. Pathogens like LCMV and HIV lead to destruction of follicular structures, resulting in minimal production of IgA and ineffective immune response. Only neutralizing antibodies, which are highly specific for each viral serotype, are protective; T cells, on the other hand, are less specific and largely shared between serotypes. Neutralizing Abs are of the IgG class and thus require a few weeks to develop and mature.

In order to measure immunity and assess the existence or absence of protection against a pathogen, the neutralizing antibody responses need to be measured. Some methods, however, overestimate their levels. ELISA, in particular, is not specific enough and may quantify other molecules as protective neutralizing Abs.

Eventually, immunity is the result of a delicate equilibrium between the host and the pathogen: neutralizing antibodies and T cells are necessary to eradicate an infection and develop memory, but a continuous low-grade persistence of the pathogen (or part of it) is required in order to maintain this protection and prevent immunological memory from fading out. Moreover, balance between the speed at which the virus replicates versus the speed of Ab production is of key importance, since slow Ab production can lead to the escaping of the virus.

“T lymphocyte differentiation, migration and immune regulation”, by Antonio Lanzavecchia

The adaptive immune response is initiated in organized lymphoid tissues where antigen-loaded dendritic cells (DCs) encounter antigen-specific T cells. Several special features of DCs, such as pathogen recognition, antigen capturing and processing, migratory capacity and co-stimulatory molecules and cytokines, allow them to act as the professional antigen presenting cells.

The stochastic and highly dynamic interaction of DC with T cells at the level of the immunologic synapse, results not only in the generation of terminally differentiated effector cells and intermediates but furthermore play distinctive roles in immunoregulation, and immunological memory. This is accomplished through the integration by DCs of multiple stimuli (from pathogens, inflammatory cytokines, T cells etc.) and the differential translation of peptide–MHC complexes concentration, cytokine and co-stimulatory molecule composition, and DC density into distinct Tcell fates and classes of T-cell responses (e.g. the Th1/Th2 paradigm).

In this context, novel subsets of T cells have been recently indentified and include the Th17 and Th22 cells, which their precise role is still unclear. Furthermore the regulatory role of chemokine receptors in DC and T lymphocytes are being investigated in these interactions and several studies provide great insights into the mechanisms that control T-cell priming as well as memory and effector immune responses.

“Dissecting the human antibody response to pathogens”, by Antonio Lanzavecchia

The human antibody response to pathogens constitutes one of the most efficient and advanced immune strategies, vital for survival. Antibody characteristics like multispecificity, crossreactivity, promiscuity and degeneracy along with memory B cells, which are maintained for a lifetime, offer an enormous immune advantage.

The induction of the human antibody response is through the activation of naïve B cells. In terms of naive B cells activation in vitro (ABCB1 expression precisely identifies human naiÅNve B cells), three signals are required. Unexpectedly, this in vitro approach revealed a distinct role served by TLRs in the activation of naive versus memory B cells (naive B cells do not express TLRs, but upregulate TLR-2/6/7/9/10 after BCR stimulation). In this progressive differentiation process following activation, memory B cells acquire the capacity to proliferate and differentiate in response to polyclonal stimuli such as cytokines, microbial products, TLR agonists or bystander T cell help.

Experimental evidence from several studies on the dynamics of memory B cells and the kinetics of serum antibodies during secondary immune responses and in steady state conditions, paved the way to the build up of a comprehensive model of immunologic memory: the “stem cell model”. According to this model memory B cells posses a stem cell-like behaviour, being capable of generating plasma cells and antibodies in an antigen-dependent as well as in an antigen-independent fashion. This model also puts the principles of T- and B-cell immunologic memory under the same perspective: responses are mediated in secondary lymphoid organs by central memory T cells and memory B cells, while immediate protection to secondary infection is mediated in peripheral tissues by effector memory T cells and by antibodies produced by long-lived plasma cells in the bone marrow niche. While self renewing, central memory T cells and memory B cells continuously spill out effector T cells and plasma cells, thus replenishing those that turn over.

The implications of these findings are striking in the field of vaccine design and monoclonal antibodies. In combination with a novel method to harness human memory B cells and to isolate human monoclonal antibodies (through immortalization with EBV + CpG) the aforementioned principles can be exploited for the production of neutralizing antibodies for serotherapy and for “analytic vaccinology” (antibodydriven target discovery: neutralizing antibodies are used to discover target antigens that can be appropriately formulated as vaccine).

“The impact of environmental stimuli on innate and adaptive immune responses”, by Gitta Stockinger

CD4 effector T cells are considered to be “The Orchestrators” of the immune symphony (although dendritic cells may be even more important). It is know well appreciated that many CD4 effector T cell subsets may exist, but yet their exact number, characteristics, role and plasticity is still unclear. Since the discovery of Th1 and Th2 CD4 effector T cell subsets 20 years ago, inducible regulatory T cells (iTreg) and Th17 cells (IL-17-producing T cells) were added to the armamentarium of helper T cells. In a rather simplistic way the distinct job of Treg cells is to limit immune pathology by exuberant Th1, Th2 or Th17 cells, while Th17 cells are responsible for coordinating the innate and adaptive immune responses against pathogens with the side effect of autoimmunity.

Several lines of evidence shows that for the differentiation of Th17 cells from naive CD4 T cells, the TGF-β and the proinflammatory cytokine IL-6 are the essential and sufficient factors (amplified by IL-1β and TNFα). For this programming process of Th17 cells IL-23 is not needed; nevertheless, is essential for their survival, expansion and function in vivo. Importantly, in the presence of IL-6, TGF-β subverts Th1 and Th2 differentiation for the generation of Th17 cells. Th17 cells secret a characteristic set of cytokines in particular IL-17 and IL-22. Significantly, IL-17 stimulates the innate system and amplifies the innate response in a very potent manner. What is more, nearly every cell in the body expresses receptors for IL-17.

Mouse studies assessing the transcription factors in different CD4 T cell subsets, revealed a transcriptional factor which its expression is restricted to the Th17 subset: the aryl hydrocarbon receptor (AhR). AhR is an evolutionary very conserved ligand dependent transcription factor, best known for mediating the toxicity of dioxin. AhR is also expressed in human Th17 cells. Its ligation during Th17 development markedly increases the proportion of Th17 T cells and their production of cytokines, in particular that of IL-22. CD4 T cells from AhR-deficient mice can develop Th17 cell responses, but when confronted with AhR ligand fail to produce IL-22 and to enhance Th17 cell development. Therefore AhR is an essential co-factor in the optimal differentiation of Th17 effector cells and natural AhR ligands seem to shape this differentiation in vitro.

Moreover, AhR activation during induction of experimental autoimmune encephalomyelitis (EAE - a mouse model of multiple sclerosis in which Th17 play a major pathogenic role) exacerbates its onset and severity in wild-type mice, but not in AhR-deficient mice. Therefore these AhR ligands may represent also co-factors in the development of autoimmunity.

The activation of the AhR by a potentially diverse range of endogenous and exogenous ligands and the link of the AhR with the Th17 programme, opens intriguing possibilities regarding the potential of environmental factors to initiate or augment autoimmune conditions.

“Common Regulatory Mechanisms in enteric lymphoid and neuronal organogenesis”, by Dimitrios Kioussis

Normal organogenesis requires the highly coordinated development and interaction of multiple cell types. During embryonic life, lymphoid organogenesis is dependent on tightly regulated (in terms of temporal expression) molecules, which dictate the cell fate. The process of lymphoid organogenesis entails cell migration, aggregation and colonisation. During this process, lymphoid tissue “inducer” cells (LTi) interact with stromal lymphoid tissue “organizer” cells (LTo), building the lymphoid organ primordial.

The lymphoid tissue resides in strategic sites of the body in order to safeguard it. In the gut, the major components of the gut-associated lymphoid tissue are the Peyer's patches. So, how do the haematopoietic cells colonize the gut and generate the primordia of Payer’s patches in a specific number and location? With the use of the fluorescent and Cre transgenic mice it was shown that the haematopoietic cells in the gut exhibit a random pattern of motility and interactions before their aggregation into the Payer’s patches primordia. What is more, interestingly it was found and demonstrated that the receptor tyrosine kinase RET and it’s signalling pathway, which is essential for mammalian enteric nervous system formation, is also crucial for Payer’s patch formation. Ret -/- animals never create Payer’s patches, while functional genetic analysis revealed that Gfra3-deficiency results in impairment of Payer’s patch development, suggesting that the signalling axis RET/GFR 3/ARTN is involved in this process. Moreover, the RET ligand ARTN was shown to be a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patchlike structures.

Consequently, the RET signalling pathway, is involved in the regulation of the development of both the nervous and lymphoid system in the gut, and thus has a key role in the molecular mechanisms that orchestrate intestine organogenesis. This is only one commonality between the lymphoid and the nervous system; concepts like the synapse, the memory, the plasticity, the migration and networks unifies this two universes.

“The Autoimmune Diseases: Diversity and Mechanisms”, by Argyrios Theofilopoulos

In autoimmune diseases the immune system looses in essence one of its most basic, yet still enigmatic, ability: the “Γνῶθι σαυτόν” i.e. ‘Know thyself’, inscribed at the pronaos of Apollo's temple at Delphi); the distinction of self from non-self and thus the maintenance of self-tolerance (nonaggression against native tissues). Since Paul Ehrlich’s concept of “horror autotoxicus” approximately 100 years ago, the circumvention of the normal tolerance for self has been the dominant subject of immunologic research providing recently a firm footing for the understanding of autoimmunity.

In a mechanistic way, the most straightforward autoimmunity theories to emerge can incorporated into two main concepts: a) autoimmunity due to defects in the induction of central or peripheral tolerance, and b) autoimmunity as a result of an inappropriate, yet conventional, immune response against self-antigens for which tolerance has never been established. Nevertheless, other abnormalities such as defects in the apoptosis machinery and induction of self-reactive cells by molecular mimicry may also be invoked. Only in a few individuals, however, the genetic soil is conducive for the disease to ensue.

“The Genetics of Systemic Autoimmunity”, by Argyrios Theofilopoulos

Population, family and twin studies have clearly shown that the propensity for autoimmunity is highly dependent on genetic factors, with the environmental factors precipitating or accelerating disease development. Nevertheless, autoimmune diseases are considered to be highly genetically complex entities since multiple genes contribute to the induction of pathogenic autoimmunity, with specific combinations of genes additively contributing to phenotypic expression.

These very genes that contribute to the development of autoimmune syndromes can be classified into two distinct categories: a) susceptibility genes, that play in essence an etiologic role and consist of mutations or allelic variants and b) effector genes, which are normal genes involved in disease pathogenesis but do not confer predilection.

Significant insights into the pathogenesis and mediators (e.g. immune cell types, autoantigens, MHC, co-stimulatory molecules, signal transduction molecules, chemokines, cytokines, apoptosis-related molecules) of autoimmune disorders has provided by building transgenic and gene knockout mouse models. The development of microsatellite-based dense chromosomal maps has advanced significantly the actual identification of these susceptibility genes.

“Autoimmunity and inflammation: From bench to bedside”, by Dimitrios Boumpas

It is now clear that many of the human disease entities are the consequence of dysfunctional innate and/or adaptive immune responses not only to external pathogens, but importantly to non-infectious endogenous molecules derived from a stressed host. This implies that the innate immune system has developed sensors (TLRs and NLRs) that recognise not only pathogens (Pathogen Associated Molecular Patterns/PAMPs) but also other stressors derived from the host (Stress Associated Molecular Patterns/SAMPs including among other by-products of apoptotic or necrotic cells, metabolic products and even nutricients). The realisation of this concept has encouraged researchers to take a closer look on the mechanisms of innate immunity and their contribution to the pathogenesis of diseases (either rheumatic, inflammatory or other common maladies like atherosclerosis-the list it keeps expanding).

Several studies have now shown that NLRs (nucleotide-binding and oligomerization domain (NOD) - like receptors) sense PAMPs as well as SAMPs and are involved in the regulation of innate immune responses in terms of promoting the activation of large cytoplasmic multiprotein complexes, called inflammasomes. These “guardians of the body and inflammation engines” then proteolytically activate the proinflammatory cytokines IL-1β and IL-18. Moreover they may on occasion even direct an adaptive immune response. Thus the inflammasomes could potentially be involved in the initiation, amplification and progression of the inflammatory response in several disease classes, especially in autoinflammatory as well as autoimmune diseases.