Immunology under the microscope: knowing what is known

The previous post in this series can be found here.

As a passionate believer in science, I have had many debates over the dinner table or at the bar with those who consider scientific research either unimportant or ineffective. The argument often comes back to “Well, science doesn't know everything!” To say this inherently misunderstands science and scientific research. Firstly, science doesn’t know anything; science doesn’t think or know - it simply is. Science is just what exists, it is everything. Everything must be explicable otherwise it couldn’t exist, even if understanding it is beyond the capability of humanity or any other intelligence. If God exists, He presumably understands Himself and so is explicable, to Him at least. I suppose that when people make the claim that “science doesn’t know everything”, they really mean “scientists don’t know everything”.  This statement is obviously true, we don’t know everything, but it never fails to surprise me how little people often assume we do know. I don’t expect people to understand science in great detail, but it is a great shame that there is so much ignorance of the sheer amount of knowledge that we’ve gathered as a race. I have, in the past, been comprehensively informed that “we still don’t even know how genes work!”, which is very surprising news considering I thought I could explain in atomic detail how a gene is transcribed, translated and expressed as a protein – that is to say: how it works. An unfortunate mistake that many people often make is to confuse the statements “I don’t know this” and “nobody knows this”.

Perhaps this is to be expected; seen from the outside, science probably appears very sporadic: there are long periods where nothing is being discovered and then suddenly there’s a big finding and everyone gets very briefly excited before everything dies down again. This, however, is simply a product of the sensationalist way that it is often portrayed in the mainstream media, in which everything deemed too complicated or insignificant is not reported and things that do make the grade are often over-hyped. Following science in this way is a bit like following literature only by watching Hollywood adaptations of major novels. In fact, science progresses in tiny steps that can seem insignificant on their own but contribute to the field as a whole. Immunology is no different.

Molecular biology, including immunology, is now at the stage where most if not all of the general principles are well studied and pretty much understood. These days there just aren’t the same groundbreaking biology-wide findings that used to happen, like the discovery of the cell, or that nucleic acid and not protein (as was believed even up to the early twentieth century) is the information-carrier of biology. This is because the molecular organisation of life only has a finite amount of complexity – one day we might understand it in absolute detail and then there will be nothing left to learn, only new ways to apply it. As we hack away at the mine of molecular biology we have to go into more and more detail about smaller and smaller things in order to keep going deeper. Fortunately, the mine is still a very rich one for the foreseeable future: there is a lot left to learn.

To return to the military analogies that I’ve used so much (and so stylishly!) in the past: research in the early days of immunology told us that the army had tanks; that they took orders from specific Generals; and that they employed specific battlefield tactics. Work today looks more into the gearing mechanisms of the tanks; the encryption algorithms used to encode orders; the psychology of the Generals; and innumerable other minutiae of the war. This might seem indulgently over-detailed but if you want to build a tank you have to reverse-engineer one, and if you want to influence military decisions then you have to understand the thought processes of those in charge.

In this post I thought I would discuss some of the important current questions in immunological research and try to give you some idea as to the work that’s going on to try and answer them. It is worth bearing in mind that I can only cover an infinitesimal fraction of the work that’s being done and even then only in superficial detail. Those who do not work in academic research are often unaware of the huge efforts being poured into answering questions such as these and so it is easy to forget that it is happening at all. I hope to expose at least a small corner of the avalanche of data that is constantly being produced and give you just some sense of the detail with which molecular biology is now being understood.

The three examples that I am going to look at cover three broad strategies of research. The first is an example of how proteins and the complexes they form are often investigated in multiple ways to try and elucidate their function and significance. The second demonstrates that our understanding of how proteins function has to be combined into models of how they interact with one another within living systems in order to answer key questions. The third is an example of the ways in which knowledge of the biological mechanisms involved allows their exploitation for therapeutic purposes.

1. How does the firing mechanism of the tank work?

Nature Immunology is one of the leading journals in which immunologists publish their research. The focus of this week’s edition is the ‘inflammasome’, which is a potent weapon used by the immune system’s foot soldiers. Upon encountering an invading pathogen, the inflammasome assembles within the cell to act as a platform from which the pro-inflammatory attack is launched. The inflammasome has a complex structure made up from numerous individual proteins that come together in a wheel-like arrangement highly similar to the related ‘apoptosome’, which is commonly referred to as the ‘Wheel of Death’ with no hint of hyperbole!

The apoptosome - aka 'the Wheel of Death' - a close relative of the inflammasome.

The reason why immunologists are so interested in this, and why such an important journal has dedicated most of an edition towards it, is that the inflammasome is essential to the defence of the body against bacterial pathogens and our understanding of it may better allow us to develop sophisticated anti-bacterial treatments.

The way that researchers approach this kind of issue is to attack it from all sides: looking at its structure; how, why, and when it forms; how it does what it does; how it is regulated; how it varies from cell to cell; and countless other things. There is, as I say, still much to extract from the mine of molecular biology!

Recently, there have been a number of advances in our understanding of the role and activity of this crucial weapon. It has been discovered that the inflammasome is assembled following signals from a group of receptors known as the ‘Toll-like receptors’ that recognise common pathogen components, such as viral DNA or bacterial cell walls. Biochemical analysis of cells in which the inflammasome is firing has revealed that it achieves its effect mainly through a protein called IL-1β, which is highly pro-inflammatory. It has also been learned that the inflammasome is not uniform, but in fact varies in its composition from one type of cell to the next: the tanks come in different models! This is highly significant because it opens up the opportunity to target specific types of cell with individual therapies and so better approach treatment for different infections.

It is often the case that as understanding of one area becomes more profound it will uncover links to phenomena previously considered unrelated. In immunology this is often extremely productive as it can uncover underlying contributing factors to disease. The work being done on the inflammasome has proved highly useful in explaining some diseases and so gives hope for treatment. For example, certain autoimmune  diseases, in which the body attacks itself and causes debilitating localised swelling and tissue damage, were found to be linked to mutations in a gene called NLRP3. Independent research uncovered the fact that the protein encoded by NLRP3 is a key component of the inflammasome, whilst knowledge of its structure showed how the mutations described would lead to a permanently active state, which explains the chronic inflammation suffered by patients. This extra level of understanding has allowed a therapy to be devised whereby the IL-1β used by the inflammasome can be blocked and so relieve the symptoms, which has proved to be clinically successful. This treatment may also have broader applications because oncologists have discovered that some cancers are promoted by the untimely activation of the inflammasome, leading to inflammation around the cancerous cells, which will promote tumour growth for various reasons. Thus, translational and collaborative research is proving highly lucrative from the perspective of both improved understanding and practical applications.

2.  How does a General know when to go to war?

As I’ve discussed before, T cells are the Generals of your immune system; they make the tactical decisions that win or lose the war and transmit their orders to the B cell artillery and the numerous infantry cells that swell the ranks of the immune system. T cells know that the body is under attack when the T cell receptors (TCRs) expressed at their surface recognise an antigen that does not belong in the body. Each T cell expresses a unique TCR and so will recognise only a specific foreign antigen, allowing the body to launch a rapid and specific response against the invader. As you may recall from my first post of this series, TCRs are only able to recognise antigen that is presented to them via the MHC system, whereby other cells chop up pathogens and present bits of them at their surface in the hope that a T cell will be able to recognise it and let slip the dogs of war! It has been known for a long time that it is this recognition of MHC-antigen by TCR that activates a T cell to begin fighting, a process appropriately known as ‘triggering’. The effects of triggering are quite well understood, the TCR acts as a focal point for a signalling cascade that propagates throughout as one protein in the chain activates the next, ultimately leading to changes in the expression of certain genes that control cell movement, replication and activity. The T cell is now armed and ready!

The T cell signalling cascade -  inside the mind of the a military genius!

What I’ve just described is fairly well understood. The question that is still contentious is: how does the TCR-MHC interaction actually trigger the whole signalling cascade? The signalling that arms the cell occurs inside it, whereas the TCR-MHC interaction takes place on the outside surface of the cell. How does the information that the TCR has recognised foreign antigen on the surface cross the cell membrane and trigger activation?

This question has been an important one for the last couple of decades as understanding it is vital for the development of properly-targetted T cell therapies that may help in the treatment of autoimmunity, immune deficiencies, and cancer. There are several key players in this scenario: the TCR and MHC molecules themselves; the first member of the signalling cascade, ZAP 70; an enzyme called Lck that can add chemical phosphate groups to the intracellular side of the TCR; and another enzyme, CD45, which takes those phosphates off again. Early work in this area identified that once the TCR has been ‘phosphorylated’ by Lck, it acts as a docking station for ZAP70, which only recognises it in this form. The recruited ZAP70 is then also subject to phosphorylation by Lck and this begins the whole process of activation. Once this was known, the question became: how does Lck know when to phosphorylate the TCR and start the process?

Several research groups have been working hard to answer this question and a number of different models have been thought up to explain it. An early explanation was that the interaction of the TCR with MHC would alter the shape of the TCR and so allow it to be activated by Lck, as happens with other cell-surface receptors. This, however, is now not believed to be the case as researchers have mutated every amino acid that makes up the TCR structure and found that none has any particular effect on its triggering ability as long as it can still bind MHC, which strongly suggests that a structural change does not occur. Instead, the most likely models now deal with the segregation of molecules at the cell surface.

If the Lck is not able to access the TCR then it cannot phosphorylate it, and likewise CD45 cannot dephosphorylate it if it can’t get to it either. So, if the TCR is hidden away from Lck until it binds MHC, or hidden from CD45 once it binds it, then the signalling cascade would know when to begin. The majority of work in this area is now looking for mechanisms by which this might occur. A number of groups have developed super-resolution microscopy techniques that allow the tracking of individual proteins at the cell surface, and they have reported that the TCR and Lck are held within separate membrane microdomains in an untriggered T cell. However, other groups have observed that the TCR is constantly being phosphorylated and dephosphorylated at the cell surface, which does not support a model of Lck-TCR segregation. Instead, these groups have suggested a mechanism by which CD45 is segregated from the TCR once the TCR has recognised MHC-antigen. This occurs because CD45 is relatively very large, whereas the TCR and Lck are quite small. As the TCR and MHC interact, the two cells that they are on have to come close together, so close that a zone is formed into which CD45 cannot fit, whereas Lck can. So, any TCR that is kept within this zone by its interaction with MHC will become phosphorylated by Lck but can’t be dephosphorylated by CD45 – hence the cascade begins and the T cell leaps to action!

The kinetic segregation model of TCR triggering - CD45 is excluded from the close contact zone between the two cells, thereby allowing TCR activation. Nature Reviews Immunology, 10, 59-71 (2010).

The way that molecular biologists test models like this is often to play around with the component molecules in such a way that the model gives a predictable outcome. In the case of TCR triggering, one prediction of this model would be that a mutant CD45 with a smaller size would be able to get into the exclusion zone and so stop triggering. When researchers did this in live T cells, it was observed that the degree of triggering was proportional to the size of CD45 – i.e. small ones stopped triggering whereas large ones didn’t, as predicted by the model. Many other experiments (far too many to list here) have drawn similar conclusions, but a number of observations have been contradictory – suggesting that there is still a level of complexity to the system that we don’t yet understand.

The issue of TCR triggering is still a divisive one within T cell biology, but as more work is done towards the understanding of the proteins involved and their behaviour we will move closer to understanding it in great detail and so potentially being able to manipulate the system for our own ends.

 3. Can we turn our arsenal against cancer?

It’s clear to us now that our immune system is a formidable force to be reckoned with. It can seek and destroy hugely varied targets differing in orders of magnitude of size, including infected cells. This surely makes it the ideal candidate for weeding out and destroying cancerous cells before they can do any significant damage, all we need to do is harness their weapons and direct them accordingly. There is, in fact, significant support for the idea of ‘immune surveillance’, whereby the immune system is constantly searching cells that express telltale ‘cancer antigens’ that are common on the surface of cells that have partially or wholly lost the ability to correctly regulate their replication and so may become cancerous. When found, these cells are destroyed, however selective pressures mean that those most resistant to detection or destruction will be more likely to survive and so may go on to pick up further mutations. This is a form of natural selection but acting on individual cells rather than whole organisms. Eventually, a cell might finally emerge from this as completely invulnerable to detection or destruction and so is free to propagate as a tumour or malignancy. This idea is supported by numerous observations, including the fact that people with weakened immune systems, such as AIDS patients, are also more likely to develop cancer.

Immune surveillance - immune cells find and kill pre-cancerous cells until those cells develop ways to evade detection and then escape to become fully cancerous.

Some of the most effective cancer treatments available use engineered antibodies that recognise antigens common to cancerous, but not normal, cells. These help the immune system to redirect its cell-killing activities towards the cancer as the antibodies bind to the troublesome cells and recruit cells expressing antibody receptors, such as neutrophils or eosinophils, to target and destroy it. Trastuzumab (commercially known as Herceptin) is an antibody therapy used to target the Her2 antigen expressed on some forms of breast cancer, and has proved massively successful in the treatment of millions of women. However, trastuzumab is somewhat unusual in its success as many other antibody therapies have fallen by the wayside in attempts to target other cancer antigens.

Recent improvements in our understanding of the immune system are now allowing the development of more sophisticated antibody-based anti-cancer treatments so that, hopefully, fewer treatments will end in failure. One ingenious way that this is being applied is to make so-called 'bispecific' antibodies that target both a cancer antigen and a specific immune system receptor. The effect of this is to directly recruit and activate more potent immune cells to the cancerous cell. An example of this is catumaxomab (trade name, Removab), which binds the cancer antigen EpCAM but also the activatory receptor CD3 that is expressed on T cells. This has shown great promise in killing EpCAM-expressing cancer cells both in culture and in animal models and is now in clinical trials. The technology required to develop bispecific antibodies has only recently become sufficiently sophisticated to achieve the right results, but it is now allowing very exciting new therapies to be developed.

Catumaxomab - modifying the immune artillery to our own ends.

Other current research is focussing on trying to promote the anti-cancer immune activity that is already present within the body. This is achieved by combining an anti-cancer antibody with a protein that inhibits the de-activating signals that regulate normal T cell function. CTLA-4 is a receptor expressed on T cells that instructs the T cell to calm down once it's been active for a certain length of time. This is to prevent excessive immune responses that might be harmful to the body, but in the case of anti-cancer immunity it actually serves to reduce the effectiveness of the immune attack. Understanding this has allowed the development of Ipilimumab (marketed as Yervoy), which binds to and inhibits CTLA-4, thereby allowing improved immune response to melanoma.

One disadvantage of this kind of therapy is that it requires repeated administration and is extremely expensive to produces. A better strategy would be to create a 'cancer vaccine' of sorts, that primes the immune system to directly target the cancerous cells themselves. Work in this field is a very hot topic and many different strategies are being attempted, mainly trying to overcome the immune tolerance that normally prevents T and B cells from targeting some types of cancerous cells. This is a very promising field that will no doubt make the headlines over the next few years, and it's likely that we'll have a post dedicated entirely to it at some point!


The future?

So, where next for immunology? Well, as I said before, there is an absolutely massive amount of research currently ongoing within the field and its translation into practical applications and other branches of biology. This pace of work is, if anything, intensifying each year as researchers have more information to go on and so more potential avenues of research. As we get deeper into the mine that we're drilling, we not only make our tunnels smaller and more specific, but also far more numerous. I would be very surprised if we don't start to see very sophisticated therapies becoming more and more common in clinical use over the next few decades. This is all thanks to the army of researchers who hack, little by little, at the rock of immunological fact to lay the foundations for these outcomes. I'll try to keep you updated here as to the most important developments - it will be fun to watch!