Thymic involution: how a chance observation saved years of work

An interview with the VIB following the recent publication of our article:

The thymus is an organ crucial for the functioning of our immune system. During aging or infection the thymus can shrink severely, a process called involution. Although the mediators that trigger involution are known, the mechanisms regulating the sensitivity to their presence remained a mystery. Now, Smaragda Papadopoulou from the Bart De Strooper Lab and James Dooley from the Adrian Liston Lab describe in Nature Immunology a microRNA network that plays a key role. A chance observation kick-started the collaboration.

What did you discover about the regulation of thymic involution?

Adrian Liston: The main finding was the tight regulation by miR-29a over sensitivity to thymic involution. miR-29a serves to suppress the involution response, in effect "saving" involution for those situations where we really need it, such as during a major infection. Knowing what drives the reaction of the thymus is important, since it is the only place where T cells can develop. No thymus, no T cells, no infection prevention.

Is there an application side to those results?

For most of us, being born with a healthy thymus, we will generate enough T cells to last a life-time. Thymus involution during an infection is generally not a problem, nor the slow progressive involution that occurs from birth. The major problem is among the very elderly and with radiation/chemotherapy patients. If we could reverse thymic involution in those populations, we could rejuvenate their T cell population, providing them with a younger, more robust, immune system.

How did you go from studying regulatory T-cells to the regulation of thymic involution?

We have been interested in both the thymic epithelium and microRNA for years, so it was natural for us to look at what microRNA does in the thymic epithelium. As for thymic involution in particular, that was observation-driven. When we knocked out microRNA in the thymic epithelium using a Cre-Lox system, the main phenotype was chronic involution. But working out which microRNA is important was an enormous task. The big breakthrough for us was serendipitous. The Bart De Strooper Lab had generated a novel knockout mouse with a defect in one particular microRNA, miR-29a, to look at the neurophenotype. A conversation, a quick look and just by chance this microRNA turned out to be the one we needed for our lead. This enabled us to start a cross-disciplinary collaboration years before anyone else even knew there was a story there.

Did you use or design any new technologies for this research?

Far from it. The most important read-out in this work was the humble cell count. There are still enormous opportunities for high-level research using basic technologies. In this particular case the edge we had was a new mouse strain (the miR-29a knockout) and a new permutation of old mouse strains (Foxn1-Cre and Dicer-flox), but the rest was simply applying old techniques to a new problem. Immunology has so many fascinating questions that remain under-investigated that we spend our time working out which ones to tackle next, rather than designing new technology.

What’s the next step in your microRNA research?

MicroRNA are such interesting molecules. So tiny, they hold only a fraction of the information of a normal gene, yet they are incredibly versatile, affecting multiple completely unrelated targets in every cell type. We pretty much cracked the role of miR-29a in the thymic epithelium, but we are sure it is doing a lot more in other cell types of the immune system.

For the full research results see:

Aikaterini S. Papadopoulou#, James Dooley#*, Michelle A. Linterman, Wim Pierson, Olga Ucar, Bruno Kyewski, Saulius Zuklys, Georg A. Hollander, Patrick Matthys, Daniel H. Gray, Bart De Strooper and Adrian Liston. #Equal first authors. *Co-corresponding authors. 'The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor.' 2012. Nature Immunology. 13 p181.  Pubmed | Direct access

Generation of a family-specific virus through repeated human passage

Generation of a family-specific virus through repeated human passage

Hayden A M Liston¹, Lydia E Makaroff¹ and Adrian Liston ^1,2*
1 Sleepytown University, Brussels 1060, Belgium
² VIB, Leuven 3000, Belgium
*send correspondance to adrian.liston@gmail.com

Nature Junior 8(2) 103-7 

Background. Effective control over viral infection relies on the host carrying appropriate HLA alleles for viral antigen presentation. The explosive expansion of viruses like small-pox into previously isolated human populations demonstrates the potential for certain viral strains to have a disproportionate effect on particular racial groups. As yet, however, a virus with pathogenic potential restricted to the family level has not been identified. Objective. To generate a family-specific virus in an experimental setting, in order to test the feasibility of this occurrence in nature. Methods. A common cold virus was repeatedly passaged between two related individuals for six months. Mechanisms of transmission included frequent kisses, the placement of hands and feet into the mouth and in one instance direct vomiting into the mouth. Results. A single viral strain was propagated with the capacity to chronically infect both members of this family, while having seemingly non-pathological consequences upon exposure to unrelated individuals. The pathogenic loci are predicted to be a dominant HLA carried by both family members, as the experimental inoculation of a third individual, related to one family member but not the other, did not result in pathology. Conclusions. Generation of a family-specific virus is feasible through repeated experimental transfer between family members. A natural situation analogous to the experimental set-up used here would be the transmission that can occur between parents and young children with low levels of personal hygiene. The dominant activity of the HLA cluster in this infection suggests the generation of a regulatory T cell population which inhibits effective immunity against the family-specific virus.

Key Words: virus, horizontal transfer, HLA, human genetics, regulatory T cell.

Balance of the immune system determined by newly discovered T cells

From the VIB Press release: A newly discovered cell type helps to determine the balance of the immune system. The cells are derived from FoxP3(+) regulatory T cells, which recently have been demonstrated to suppress disease during transplantation. Nature Medicine and Blood, two high-ranking scientific journals, are publishing findings by the Autoimmune Genetics Laboratory about this discovery.

 

Innumerable people suffer from immune system disorders. If the immune system is overactive, it can result in allergies and autoimmune diseases such as Systemic lupus erythematosus (SLE). If the immune system is not active enough, infections or tumors occur. It is therefore essential to maintain the right balance.

 

The Autoimmune Genetics Laboratory is investigating a new type of cell that helps to maintain this balance. These ‘follicular regulatory T cells’ (Tfrs) suppress the process by which antibodies are produced during infections and SLE. The Tfrs themselves are daughter cells of FoxP3(+) T cells, key regulators of the immune system.

 

From lupus to cancer

In order to prevent disease the immune system needs to be in balance. If the system is too active, it produces antibodies against harmless substances, as is the case with allergies. An overactive immune system can even attack the body’s own tissues, causing autoimmune diseases such as lupus, rheumatism and diabetes. However, an insufficiently active immune system opens the way for rampant infections and tumors.

 

A complex network of regulatory cells is active to maintain this balance in our bodies. This must ensure that the immune system is sufficiently active and selective so that pathogenic intruders are recognized and eliminated in good time, but at the same time that it does not get out of control. This network of regulatory and activating cells is a long way from being fully unraveled. Dozens of researchers around the world are working to map this network.

 

This Tfr research is a joint collaboration between researchers from VIB-K.U.Leuven, the Australian National University (Aus) and the University of Cambridge (U.K.).

 

Publications

Linterman et al, Foxp3(+) follicular regulatory T cells control the germinal center response, Nat Med, 2011, doi:10.1038/nm.2425

 

Tian et al, Foxp3+ regulatory T cells exert asymmetric control over murine helper responses by inducing Th2 cell apoptosis, Blood, 2011, doi:10.1182/blood-2011-04-346056

The verdict on Andrew Wakefield: Fraud

In 1998 Andrew Wakefield published a paper which has severely damaged public health in the last ten years. Based on his observations of only twelve children, nine that he claimed had autism, and without a control group, he concluded that the measles/mumps/rubella vaccine caused autism. As a hypothesis, this was fine, unlikely, but not impossible. He saw nine children with autism, reported that their parents linked this onset with the MMR vaccine, and put it in the literature. Why on earth on underpowered observation like this made it into the Lancet is beyond me, but there is nothing wrong with even outlandish hypotheses being published in the scientific literature. Was it a real observation, or just an effect of a small sample size? Was it a causative link, or just due to coincidence in timing?

As with any controversial hypothesis, after this one was published a large number of good scientists went out and tested it. It was tested over and over and over again, and the results are conclusive - there is no link between the MMR vaccine and autism.

In itself, this was of no shame to Andrew Wakefield. Every creative scientist comes up with multiple hypotheses that end up being wrong. People publish hypotheses all the time, then disprove them themselves or have them disproven by others. If you can't admit being wrong, you can't do science, and it is in fact the mark of a good scientist to be able to generate hypotheses that others seek to knock down. Ten of the thirteen authors on the study were able to see the new data and renounce the hypothesis.

The shame to Andrew Wakefield is not that his hypothesis was wrong. No, the shame he has brought upon himself was by being unscientific, unscrupulous and unethical:

1. Firstly, Wakefield did not present his paper as a hypothesis generator, to be tested by independent scientists. Instead he went straight to the media and made the outrageous claim that his paper was evidence that the MMR vaccine should be stopped. This is not the way science or medicine works and was a conclusion unsupported by the data. Worst of all it was a conclusion that many parents without scientific training were tricked into believing. Vaccination rates for MMR went down (autism rates have remained unchanged) and children started dying again of easily preventable childhood diseases. A doctor does not see half a dozen children that developed leukemia after joining a football team and then hold a press conference telling parents that playing sports causes cancer in children, which is the direct equivalent of Wakefield's actions.
2. Secondly, it has now been conclusively demonstrated that his original data was fraudulent. Interviews with the parents of the original nine children with autism show that he faked much of the data of the time of onset, taking cases where autism started before the MMR vaccine and reversing the dates to suggest that the vaccine started the autism. Analysis of the medical records of these children show that as well as the timing being incorrect, many of the symptoms were simply faked and non-existent. The evidence on this charge alone makes Wakefield guilty of professional misconduct and criminal fraud.
3. Thirdly, unknown to the coauthors of the study and the parents of the children, Wakefield had a financial conflict of interest. Before the study had begun, Wakefield had been paid £435 643 to find a link between vaccines and disease as part of a lawsuit. Every scientist must disclose their financial interests in publication so that possible conflicts are known - Wakefield did not. If he had disclosed this to the press conferences the media may have been slightly more skeptical about his outlandish claims.

These last two issues, scientific misconduct and financial conflict of interest, are the reason why the paper was formally retracted by the Lancet. Studies that are wrong don't get retracted, they just get swamped by correct data and gradually forgotten. Instead, the retraction indicates that the Wakefield paper was fradulent and should never have been published in the first place. Likewise, the British General Medical Council investigated the matter and found that Wakefield "failed in his duties as a responsible consultant" and acted "dishonestly and irresponsibly", and thus struck him off the medical registry.

The worst part about this sorry affair is that it is still dampening down vaccination rates. Literally hundreds of studies, with a combined cohort size of a million children, have found no link between the MMR vaccine and autism, yet one fraudulent and retracted study of nine children is still talked about by parents. Some parents are withholding this lifesaving medical treatment from their children, and their good intentions do nothing to mitigate the fact that cases of measles and mumps are now more than 10 times more likely than they were in 1998, and confirmed deaths have resulted. And Andrew Wakefield, the discredited and disbarred doctor who started this all? Making big money in the US by selling fear to worried parents, and deadly disease to children who have no say in it at all.



The historic quandary of antibody production

The mechanism by which antibodies were formed was once one of the oldest and most perplexing mysteries of immunology. The properties of antibody generation, with the capacity of the immune system to generate specific antibodies against any foreign challenge – even artificial compounds which had never previously existed – defied the known laws of genetics.

Three major models of antibody production were proposed before the correct model was derived. The first was the “side-chain” hypothesis put forward by Ehrlich in 1900, in which antibodies were essentially a side-product of a normal cellular process (Ehrlich 1900). Rather than a specific class of proteins, antibodies were just normal cell-surface proteins that bound their antigen merely by chance, and the elevated production in the serum after immunisation was simply due to the bound proteins being released by the cell so that a functional, non-bound, protein could take its place. In this model antibodies “represent nothing more than the side-chains reproduced in excess during regeneration and are therefore pushed off from the protoplasm”.

Figure 1. The “side-chain” hypothesis of antibody formation. Under the side-chain hypothesis, antibodies were normal cell-surface molecules that by chance bound antigens (step 1). The binding of antigen disrupted the normal function of the protein so the antigen-antibody complex was shed (step 2), and the cell responded by replacing the absent protein (step 3). Notably, this model explained the large generation of specific antibodies after immunisation, as surface proteins without specificity would stay bound to the cell surface and not require additional production. The model also allowed a single cell to generate antibodies of multiple specificities.

The “side-chain” model was replaced by the “direct template” hypothesis by Haurowitz in 1930. Under this alternative scenario, antibodies were a distinct class of proteins but with no fixed structure. The antibody-forming cell would take in antigen and use it as a mould on which to cast the structure of the antibody (Breinl and Haurowitz 1930). The resulting fixed-structure protein would then be secreted as an antigen-specific antibody, and the antigen reused to create more antibody. In preference to the “side-chain” hypothesis, the “direct template” hypothesis explained the enormous potential range of antibody specificities and the biochemical similarities between them, but it lacked any mechanism to explain immunological tolerance.

Figure 2. The “direct-template” hypothesis of antibody formation. The direct-template hypothesis postulated that antibodies were a specific class of proteins with highly malleable structure. Antibody-forming cells would take in circulating antigen (step 1) and use this antigen as a mould to modify the structure of antibody (step 2). Upon antibody “setting”, the fixed structure antibody was released into circulation and the antigen cast was reused (step 3). In this model specificity is cast by the antigen, and a single antibody-producing cell can generate multiple different specificities of antibody. 

A third alternative model was put forward by Jerne in 1955 (Jerne 1955). The “natural selection” hypothesis is, in retrospect, quite similar to the “clonal selection” hypothesis, but uses the antibody, rather than the cell, as the unit of selection. In this model the healthy serum contains minute amounts of all possible antibodies. After the exposure to antigen, those antibodies which bind the antigen are taken up phagocytes, and the antibodies are then used as templates to produce more antibodies for production (the reverse of the “direct template” model). As with the “direct template” model, this hypothesis was useful in explaining many aspects of the immune response, but strikingly fails to explain immunological tolerance.

Figure 3. The “natural selection” hypothesis of antibody formation. The theoretical basis of the natural selection hypothesis is the presence in the serum, at undetectable levels, of all possible antibodies, each with a fixed specificity. When antigen is introduced it binds only those antibodies with the correct specificity (step 1), which are then internalised by phagocytes (step 2). These antibodies then act as a template for the production of identical antibodies (step 3), which are secreted (step 4). As with the clonal selection theory, this model postulated fixed specificity antibodies, however it allowed single cells to amplify antibodies of multiple specificities.

When Talmage proposed a revision with more capacity to explain allergy and autoimmunity in 1957 (Talmage 1957), Burnet immediately saw the potential to create an alternative cohesive model, the “clonal selection model” (Burnet 1957). The elegance of the 1957 Burnet model was that by maintaining the basic premise of the Jerne model (that antibody specificity exists prior to antigen exposure) and restricting the production of antibody to at most a few specificities per cell, the unit of selection becomes the cell. Critically, each cell will have “available on its surface representative reactive sites equivalent to those of the globulin they produce” (Burnet 1957). This would then allow only those cells selected by specific antigen exposure to become activated and produce secreted antibody. The advantage of moving from the antibody to the cell as the unit of selection was that concepts of natural selection could then be applied to cells, both allowing immunological tolerance (deletion of particular cells) and specific responsiveness (proliferation of particular cells). As Burnet wrote in his seminal paper, “This is simply a recognition that the expendable cells of the body can be regarded as belonging to clones which have arisen as a result of somatic mutation or conceivably other inheritable change. Each such clone will have some individual characteristic and in a special sense will be subject to an evolutionary process of selective survival within the internal environment of the cell.” (Burnet 1957)

Figure 4. The “clonal selection” hypothesis of antibody formation. Unlike the other models described, the clonal selection model limits each antibody-forming cell to a single antibody specificity, which presents the antibody on the cell surface. Under this scenario, antibody-forming cells that never encounter antigen are simply maintained in the circulation and do not produce secreted antibody (fate 1). By contrast, those cells (or “clones”) which encounter their specific antigen are expanded and start to secrete large amounts of antibody (fate 2). Critically, the clonal selection theory provides a mechanism for immunological tolerance, based on the principle that antibody-producing cells which encounter specific antigen during ontogeny would be eliminated (fate 3).

It is important to note that while the clonal selection theory rapidly gained support as explaining the key features of antibody production, for decades it remained a working model rather than a proven theory. Key support for the model had been generated in 1958 when Nossal and Lederberg demonstrated that each antibody producing cell has a single specificity (Nossal and Lederberg 1958), however a central premise of the model remained pure speculation – the manner by which sufficient diversity in specificity could be generated such that each precursor cell would be unique. “One aspect, however, should be mentioned. The theory requires at some stage in early embryonic development a genetic process for which there is no available precedent. In some way we have to picture a “randomization” of the coding responsible for part of the specification of gamma globulin molecules” (Burnet 1957). Describing the different theories of antibody formation in 1968, ten years after the original hypothesis was put forward, Nossal was careful to add a postscript after his support of the clonal selection hypothesis: “Knowledge in this general area, particularly insights gained from structural analysis, are advancing so rapidly that any statement of view is bound to be out-of-date by the time this book is printed. As this knowledge accumulates, it will favour some theories, but also show up their rough edges. No doubt our idea will seem as primitive to twenty-first century immunologists as Ehrlich’s and Landsteiner’s do today.” (Nossal, 1969).

It was not until the research of Tonegawa, Hood and Leder that the genetic principles of antibody gene rearrangement were discovered (Barstad et al. 1974; Hozumi and Tonegawa 1976; Seidman et al. 1979), rewriting the laws of genetics that one gene encoded one protein, and a mechanism was found for the most fragile of Burnet’s original axioms. The Burnet hypothesis, more than 50 years old and still the central tenant of the adaptive immune system, remains one of the best examples in immunology of the power of a good hypothesis to drive innovative experiments.

References

Barstad et al. (1974). "Mouse immunoglobulin heavy chains are coded by multiple germ line variable region genes." Proc Natl Acad Sci U S A 71(10): 4096-100.

Breinl and Haurowitz (1930). "Chemische Untersuchung des Prazipitates aus Hamoglobin and Anti-Hamoglobin-Serum and Bemerkungen ber die Natur der Antikorper." Z Phyisiol Chem 192: 45-55.

Burnet (1957). "A modification of Jerne's theory of antibody production using the concept of clonal selection." Australian Journal of Science 20: 67-69.

Ehrlich (1900). "On immunity with special reference to cell life." Proc R Soc Lond 66: 424-448.

Hozumi and Tonegawa (1976). "Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions." Proc Natl Acad Sci U S A 73(10): 3628-32.

Jerne (1955). "The Natural-Selection Theory of Antibody Formation." Proc Natl Acad Sci U S A 41(11): 849-57.

Nossal and Lederberg (1958). "Antibody production by single cells." Nature 181(4620): 1419-20.

Nossal (1969). Antibodies and immunity.

Seidman et al. (1979). "A kappa-immunoglobulin gene is formed by site-specific recombination without further somatic mutation." Nature 280(5721): 370-5.

Talmage. (1957). "Allergy and immunology." Annu Rev Med 8: 239-56.