Some of the earliest concepts of immunity arose over 3000 years ago with the discovery that inoculation with powdered smallpox scabs could protect people against the disease. We now understand that this protection is mediated by our adaptive immune system (AIS) which, driven by lymphocytes and DNA rearrangements, creates a myriad of receptor specificities towards many antigens. Unlike the innate immune system (IIS), the AIS requires several days to weeks to mount a full response upon first encounter with antigen. Nevertheless, once activated it responds with specificity and memory, and further encounters with the same antigen result in an accelerated and enhanced response. Since Edward Jenner’s first demonstration of vaccination in 1796, an understanding of the AIS has allowed us to treat and prevent many deadly infectious diseases. Yet, to our current knowledge, more than 96% of living organisms do not have an AIS and suffice with germ-line encoded receptors to live just as complex lifestyles as we do while maintaining sufficient immunity to pathogens. As such, how and why the AIS arose in vertebrates 400 million years ago remains an unsolved mystery.
Emergence of the Adaptive Immune System
Examples of a conventional AIS are only found in jawed vertebrates (gnathostomes), which came into existence about 400 million years ago. In order to trace the origins of the AIS, scientists have focused on the genes encoding the essential elements for generating diversity: B cell receptor (BCR), T cell receptor (TCR), Recombinase Activating Enzymes (RAG1/2) and the major histocompatibility complex (MHC). Until quite recently, very few remnants, if any, of the above molecules have been found in non-jawed vertebrates and invertebrates. This led scientists to formulate the “Big Bang Theory of the AIS”, which proposed that a large number of genes involved in adaptive immunity were acquired over a very short period of time in evolution.
To describe the Big Bang Theory, one can think of the BCR, which is generated through V(D)J recombination and can be secreted as immunoglobulins (Igs). A wide diversity of Igs exists within the gnathostomes superclass, and amongst this superclass, tetrapods have some that are specialized. Humans have several subtypes of Igs – IgG, IgE, IgA, IgD and IgM – each associated with a different effector function. IgG, IgE and IgA represent specialized Igs involved in high-affinity memory formation, inflammatory responses at epithelial surfaces, and mucosal functions respectively. Birds, amphibians and reptiles have an IgY isotype that functions similarly to IgG and is thought to be the ancestor of both IgG and IgE. IgA is also found in birds and non-avian reptiles and is believed to derive from amphibian IgX. More ancient gnathostomes lack many of the specialized Ig subtypes but possess IgM, the most evolutionarily conserved immunoglobulin. IgD is also fairly well conserved but has been lost in several species and its function remains unclear. New studies have shown the presence of other Igs such as New Antigen Receptors (IgNARs), which are homologous to IgD in cartilaginous fish, and a new isotype called IgT/IgZ that is involved in bony fish mucosal immunity. However beyond jawed-vertebrates, no orthologs of immunoglobulins have been found. Neither have TCRs nor MHC, which unlike the Igs are much more evolutionarily conserved in all gnathostomes.
Particularly puzzling was the search for orthologs of BCR, TCR, MHC and RAG genes in non-jawed vertebrate (agnathan) Transcriptome analysis had shown that lymphocyte-like cells in agnathans had similar genes as those that jawed vertebrate lymphocytes use for cellular migration, proliferation, differentiation, and intracellular signaling, yet they did not contain functional BCR or TCR. An eureka moment came when Pancer et al. in 2004 showed that the cDNA of large activated lymphoblastoid cells from lampreys, a member of the agnathans, contained an abundance of unique, highly diverse Leucine Rich Repeat (LRR) proteins. These were named Variable Lymphocyte Receptors (VLRs), and in contrast with the BCR and TCR of the conventional AIS, are made of LRR rather than Ig domains. Importantly, agnathans do not use a recombinase-based approach for DNA rearrangement. Rather, variety in lymphocyte receptors is thought to arise by a gene conversion mechanism based on sequence similarity, where LRR segments flanking the VLR gene are inserted between the constant N and C terminus for a final VLR to be produced. So instead of finding genes similar to those involved in conventional immunity, the authors stumbled upon a completely new AIS shared by non-jawed vertebrates: the alternative AIS.
Are we just not looking hard enough?
Technological advances and the sequencing of additional genomes are now indicating that the Big Bang theory is not as simple as initially thought. Ig and LRR domains are widely used by invertebrates in immune and non-immune functions, implying that the raw materials of the AIS were present even before the appearance of vertebrates. For example, the Down syndrome cell adhesion molecule (Dscam) found in insects is a member of the Immunoglobulin Superfamily (IgSF). Alternative splicing of Dscam gene exons is thought to lead to more than 18,000 different extracellular domains and, although initially described as having a role in neuronal wiring, there is evidence to suggest that Dscam may also be involved in phagocytosis. In 2006, the labs of Dr. Jonathan Rast and Dr. Sebastian Fugmann found homologues of RAG1 and RAG2 in the purple sea urchin, suggesting that RAG genes were acquired millions of years earlier than previously thought. Further sequencing projects have identified the presence of large diversified gene families of innate receptors in many invertebrate species, suggesting possible selective pressure to recognize variants of rapidly evolving pathogens.
How all of these genes came together to form a cohesive AIS that functions in association with the IIS is hard to pin down. It is thought that a RAG-mediated transposon invaded the genome and, followed by whole genome duplication, locus duplication events, and differentiation based on selective pressures, shaped the basis for the AIS.
Why do we need an adaptive immune system?
Every living organism must maintain some defensive barrier with the outside, and essential to this barrier is the ability to combat pathogens. Even bacteria have defense mechanisms such as antiviral enzymes that allow them to evade bacteriophages. Multicellular organisms have specialized cells for defense, and examples of this can be seen even in the most ancient organisms such as dictyostelium. Importantly, the defense mechanisms employed must be flexible to keep pace with the ever-changing strategies that pathogens use to evade the immune system. So at least in principle, would it not seem ideal to have a system that makes boundless amounts of receptors with specificity and memory towards any antigen that it might encounter? If so, why are there so many organisms that lack it?“One can think of warm blooded animals, size, life span etc., but nothing completely correlates with the appearance of the adaptive immune system.”
Initial speculations stated that vertebrate organisms live longer, are bigger and have more complex lifestyles than invertebrates, and so there is a greater need for the AIS. However, there are plenty of invertebrate species, such as squids and octopuses, that are larger and have longer lifespans than many vertebrates yet lack an AIS. So this school of thought has been heavily criticized and rebuked. Recently, Margaret McFall-Ngai suggested that the AIS has allowed vertebrates to have more complex interactions with their commensal bacteria, whereas David Usharauli proposed that the AIS arose to reduces the collateral damage to the tissue during chronic infection. According to Dr. Jonathan Rast, “one can think of warm blooded animals, size, life span etc., but nothing completely correlates with the appearance of the adaptive immune system.”
In addition, the presence of the AIS has its costs. Besides the maintenance of many different genes required for diversity, it also runs the risk of creating autoimmunity in the individual. In a Darwinian sense, specific traits are selected based on a cost-benefit ratio. So at least initially there must have been an advantage to maintain a complex system like the AIS, but the advantage, Rast explains, could be “a double edge sword, something that locks itself.” Along the same lines, Stephen M. Hedrick proposed in 2004 that there is no optimal solution to the problem of parasitism because pathogens will eventually find a way to evade the system. He points out that “evolution does not have foresight” and the AIS, once created, became an “appendage that generates its own necessity.”
The recent discovery of the VLR system suggested that the two different adaptive immune systems with equal potential for diversity likely arose by convergent evolution. These observations suggest that a strong selective pressure must have existed for both systems to arise independently. Rather than asking why the AIS is present in vertebrates, Rast asks why it is not present in invertebrates, and proposes that perhaps we just haven’t found it yet. In fact, some evidence of primitive AIS-like systems in invertebrates exists. Rast and his lab have already characterized many immune genes in the sea urchin by sequence similarity to genes in mammals, but they are likely missing many others. “The problem”, Rast says, “is that most immunological assays, such as graft rejection, have been perfected in mammals so it is hard to look for things that are completely different. If you look at…VLRs, they [were almost missed] because it wasn’t exactly what the scientists were looking for. But once they were discovered, progress on understanding their function went really quickly. Partly because the labs working on it were very good, but also because they were very analogous to Igs, so you could…take all those ideas that had formed over decades about V(D)J and apply them to VLRs, and it worked well. But I think there’s probably going to be other systems that aren’t going to be so analogous and that’s going to be trickier.”
The challenge then, lies in defining what exactly an immune system is and what genes to look for amidst the huge amount of sequencing data. One approach might be to look for differential expression of genes in response to disturbances and changes in the animal. Some genes, such as transcription factors are fairly straightforward, whereas genes that elicit a function might be harder. Rast explains that “there could be genes that even promote bacterial growth of some types and…maintain the relationship with the microbial world…and that could be the larger part of what immune systems do. It might just be a fairly rare event that immune systems elicit killing responses, [rather] it might be more important to just jiggle the microbial environment around the animal [to a level that is beneficial].” Certainly, as more genome sequences emerge and more experiments are done in invertebrate and vertebrate species, this quest will become easier.
Just as hundreds of years of research on the conventional AIS have improved medicine, research on other AIS could allow for great biomedical advances. The lab of Dr. Götz Ehrhardt, for example, is using the VLR system for biomarker discovery. “VLR antibodies may detect structures that conventional antibodies simply cannot see [due to structural or tolerogenic constrains],” explains Ehrhardt. In fact, they’ve already made a VLR antibody that recognizes all plasma cells more specifically than any conventional antibody. “Of course the question is,” says Ehrhardt, “what is [the antigen] that is recognized on plasma cells, and why is it on plasma cells? [Could it be] an essential component that helps a plasma cell function?” Like VLRs, there are likely many yet to be discovered molecules that could have practical applications for human health. Finding new mechanisms of immunity, for example, might hold answers to current antimicrobial problems such as antibiotic resistance. Rather than reinventing the wheel, scientists can take what evolution has developed for millions of years and employ it in our fight against disease.
Thanks to Dr. Jonathan Rast, Dr. Götz Ehrhardt, Catherine Schrankel and Nichole Escalante for helpful insights, comments and edits.
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Mayra Cruz Tleugabulova
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