The immune system plays a vital role in both health and disease. In the first of two parts, Quest looks at how autoimmune disease develops. In the second part, we’ll examine immune system hurdles to the development of gene therapy, and explore strategies being used to solve these problems.

Your immune system has an awe-inspiring and vital job: it alone can protect you from the millions of different harmful organisms which see you as their next meal. Bacteria, viruses, fungi and other microorganisms must all be recognized and destroyed quickly and completely to prevent runaway infection.

Your immune system must also be able to tell when one of your cells is infected, and to kill it if it can’t be saved. To do this, it has to know which cells belong to you and which don’t; in other words, it must distinguish between self and non-self. When it works, you stay healthy and free of infection. When it makes mistakes, when it sees some of your cells as the enemy, an autoimmune disease such as myasthenia gravis is the result.

How can it go so wrong? To understand this, we must first understand what the immune system is, and how it normally fights disease.

MUSTERING THE TROOPS

"The immune system is so incredibly complex that it’s hard to describe briefly," says Dr. Dan Drachman of Johns Hopkins University in Baltimore who has studied immunology for over 20 years. "Predominantly, though, it’s the white cells, and what they make."

If the immune system can be compared to a military defense force, the white cells make up most of the soldiers, from private to general. There are three main types of white cells: antigen-presenting cells, B cells and T cells. Their interactions control the immune response, for better or worse. We’ll focus on T cells, which immunologists think are the ranking members of the immune forces.

T cells, explains Drachman, a member of MDA’s Medical Advisory Committee, begin their lives in the bone marrow, early in development. Here, each creates hundreds of copies of its own unique receptor molecule which sit on its surface. This receptor is the key to its interactions with other cells. It has a two-part task.

"First," Drachman says, "it must be able to recognize the signature, so to speak, of that individual," allowing it to distinguish friend from foe.

The signature of the individual is a molecular one. Molecules called MHCs stud the surface of your cells, and the group of MHCs you have is what makes you immunologically unique. Your T cell receptors must be able to "read" this molecular signature during their surveillance duties. However, the MHC is more than just a personal marker. It’s designed to alert the T cells when it becomes infected, by presenting fragments of the enemy’s uniform, called peptides.

Peptides are created within the cell as part of a natural recycling process for your own worn out or defective proteins. Most of the protein fragments are broken down completely to be reassembled into new proteins. Some, though, are displayed on the cell surface by MHCs. When a cell becomes infected by a virus, some of these peptides are also displayed. In effect, your MHCs are continuously broadcasting the news about what types of proteins are inside, both foreign and domestic. Recognizing these enemy peptides within the MHC is the second part of the T cell’s task. When it does so, it can direct a battle plan that brings all the forces of the immune system into play.

REPELLING INVADERS

The most important of these for understanding myasthenia gravis are molecules called antibodies. Made by B cells and released into the blood stream upon command, antibodies latch onto invaders wherever they lie, sabotaging their cellular machinery like molecular monkey wrenches. They also act as flags for other circulating parts of the immune system, including macrophages which engulf and destroy bacteria, and complement molecules which punch holes in the cells the antibodies have marked.

For reasons not yet fully understood, antibodies are sometimes directed against an important muscle protein called the acetylcholine receptor. When this receptor is attacked and destroyed, muscles can’t respond effectively to the signals they get from nerve cells. The result is the muscle weakness of myasthenia gravis.

In describing how the MHC and T cell interact, Drachman likes to compare the MHC to a hot dog roll, holding a peptide fragment like a hot dog in a groove down its middle. A T cell encountering an MHC examines it with its T cell receptor, to see how well it fits. "A particular T cell has to see a pretty specific hot dog in a very specific roll, otherwise it won’t respond," Drachman says.

This presents an enormous challenge to the immune system. Peptides come in a huge variety of shapes and sizes; each type of invader could supply thousands of different types of peptides, and there are thousands of types of invaders. If the immune system is going to catch them all, it must have an equally huge variety of T cell receptors to match.

In fact, Drachman says, the invaders are overmatched. "There are about 10 quadrillion possible T cell receptors, so they can see, and react to, just about anything."

THE LINES OF DEFENSE

However, this dizzying variety is both the blessing and curse of the immune system. For within this rich field of receptor diversity lie the seeds of autoimmunity.

How is this extraordinary variety of T cell receptors generated? Tailoring them one by one as each new enemy peptide is discovered would be too slow; infection could easily get out of control before the right T cell was ready. Instead, during their development in the bone marrow, T cells make receptors at random, mixing and sorting pieces of the genes that code for them like ordering from the menu at a Chinese restaurant. Each T cell makes only one kind of receptor, but all together, they produce the full diversity you need for the rest of your life.

Many of these T cells will, by chance, make receptors perfectly suited to react to a foreign peptide. But, just as you might expect from a random process, others will not, and so they must be weeded out. This selection process occurs in a small gland in your chest, called the thymus.

"In the thymus," Drachman says, "white cells must pass muster, and they are either kept or destroyed."

First, T cells are screened for their ability to recognize your MHC signature. Any T cell carrying a receptor which can’t bind strongly to your MHCs, which can’t recognize your particular type of hot dog roll, is killed. This ensures that all the remaining T cells in your body are looking out for you, and that none of them will be more protective of somebody else’s cells, if they are ever encountered.

The second stage of T cell screening is meant to guard against autoimmunity. As we said, each T cell receptor recognizes a specific peptide sitting in the MHC groove, and your cells display domestic peptides, as well as the foreign ones they show when they become infected. Any T cell whose receptor reacts to a domestic peptide is a potential autoimmune reaction waiting to happen.

To prevent this, T cells in the thymus are exposed to the full variety of self peptide hot dogs sitting in the MHC rolls. During this round, T cells are killed if their receptors can bind strongly. If all goes well, the only T cells that remain are those which can recognize your signature, but can’t respond to self peptides.

But, of course, all doesn’t go well, at least not all the time, as those with autoimmune disease can attest. What goes wrong?

Drachman explains that, despite the rigorous screening procedure, some self-reactive cells get through. "It’s not a perfect system. In fact, all people have cells with the potential to cause autoimmune disease. If you take a perfectly normal person, one with no autoimmune disease, sure enough, you can find some autoreactive cells."

Why don’t most of us get sick? "No one really knows. We do know there are many levels of control in the immune system, so that’s likely part of the story." Drachman notes, "Given the fact that all these potentially autoimmune cells are out there, and the immune system has to keep track of millions of self peptides which it must avoid attacking, it’s pretty amazing how rarely autoimmune disease occurs."

INFILTRATING THE DEFENSES

Exactly how it occurs is still a mystery, and the subject of a good deal of research. Currently, there are four main theories, all of which may play a part in myasthenia gravis.

According to the molecular mimicry theory, autoimmunity occurs after you get infected by a virus or bacterium that has peptides similar to those of your own cells. As Drachman explains it, your immune system responds normally to these peptides (called antigens when they are the subject of immune surveillance). However, he says, "when the immune system gets pumped up to attack the foreigners, it may not be able to distinguish between foreign antigens and closely related self antigens" and, as a result, your cells may be attacked. Even after infection subsides, responding T cells may stay on high alert, continuing to stimulate antibody production against those self antigens that look like the foreigners. This has been shown to occur in at least some autoimmune diseases.

"The most interesting current example," Drachman notes, "is the Guillain-Barré syndrome, an autoimmune disease that attacks the nerves. There’s a strong suspicion that a particular, common bacterium called sampylobacter can trigger at least some forms of Guillain-Barré."

And for myasthenia gravis? "There’s not as clear a culprit. However, we’ve done some work to show that in at least some MG patients the antibodies to their acetylcholine receptors also react to the herpes simplex virus." In other words, it’s possible that a herpes infection may be the trigger for developing MG. Drachman cautions, though, that this is likely to be responsible for only some cases.

The second major theory of autoimmunity suggests that T cells in the thymus don’t get exposed to all possible self peptides. According to the "hidden antigen theory," some peptides remain hidden during the thymic selection process, and during most of later life. Since the antigen is never seen during thymic training, the T cell that can respond to it remains alive. Since the antigen is normally never exposed during later life, autoimmunity to it doesn’t usually occur. However, if the antigen does get exposed, because of infection or trauma, it could cause an autoimmune response.

"A third theory," Drachman says, "suggests that the control systems which normally suppress autoimmune attack by our T cells somehow fail to do their jobs. Unfortunately, we don’t yet understand these higher levels of control within the immune system." If such controls are weakened during an infection, they could allow the kind of autoimmune attack proposed by the molecular mimicry theory.

Finally, Drachman says, some people may be set up genetically for developing particular autoimmune diseases. "Some people may have a particular part of their MHCs which allows an antigen to be displayed very prominently, so that T cells react strongly to it. For example, people with the autoimmune disease rheumatoid spondylitis invariably have a particular MHC type."

Though there isn’t evidence yet that this happens in people with MG, Drachman has just finished an MDA-funded study demonstrating this phenomenon in mice with an experimental form of the disease. His work shows that one strain of mice, which is susceptible to myasthenia, has an MHC that holds a particular peptide from an injected acetylcholine receptor very well.

Another strain, with a slightly different MHC, can’t hold the peptide well. "The MHC can’t fit the peptide, so the T cells don’t see it, and so the B cells don’t get turned on to make antibody." As a result, these mice are much less susceptible to myasthenia.

Drachman also points out that the mice aren’t originally responding to their own acetylcholine receptors; that comes later. The immunogenic peptide is from the electric ray fish, a particularly rich source of receptors. "So the mice must have both the right genetic background and be exposed to an unusual peptide. It takes a combination of factors to trigger the disease in these mice."

FIGHTING BACK

Research is progressing not just into the cause of MG, but also into new treatments. Most people with myasthenia begin with mestinon. Mestinon makes up for the loss of acetylcholine receptors on myasthenic muscle by helping the acetylcholine molecules last a little longer, so they can stimulate the remaining receptors more strongly.

When mestinon doesn’t work, current therapy relies on globally suppressing the immune system, damping down all kinds of immune responses. Prednisone, a corticosteroid hormone, is usually given first. "Prednisone has about 20 different actions, turning down just about every part of the immune system, including antibody production," Drachman says. It has some serious side effects, and a person with MG may need to be on it for life. Imuran predominantly affects T cells, and lowers antibody levels that way. Some years ago, Drachman pioneered the use of cyclosporin for MG. It also works on T cells, though in a different way.

Antibodies can be removed from the bloodstream by plasmapheresis (a blood cleansing system), but it’s only a short-term fix, since the B cells will continue to pump out more antibodies as long as they are stimulated by the T cells. Plasmapheresis is used mainly in a crisis, when muscle weakness is so severe that breathing becomes difficult.

IVIG, or intravenous immune globulin, can also be used. "IVIG is purified from blood. It’s basically antibodies against all kinds of things: polio, colds, measles, anything. We inject massive doses — more than 100 grams over a five-day period — and it’s very costly. But it seems to work for some people, though nobody knows how."

Removing the thymus gland can also help some people, though again, the reason isn’t clear.

NEW STRATEGIES

While your immune system continues to function during all these treatments, despite the immune suppression, the side effects of each can be serious. Furthermore, none of these strategies targets the autoimmune cells specifically.

"What you’d like is a treatment that gets only those cells which are causing the problem," Drachman says. "Unfortunately, you can’t go after the B cells which are making the antibody, for two reasons. The first is that to target a specific B cell, you need to go after the thing that makes it specific, namely its antibody. Unfortunately, any antibody-seeking poison would also find the circulating antibody. When they react, the resulting complexes could precipitate in the kidneys and elsewhere, and really cause a lot of damage. The second thing is that B cells, unlike T cells, are capable of mutating, so that even if you did eliminate all the MG-causing B cells, you may get new ones being stimulated by T cells before long."

So the logical target has to be T cells. Drachman is currently at work on some strategies that will specifically target the T cells involved in a response. He points out that only responding T cells will put receptors on their surfaces for a molecule called IL-2. "If you take a piece of the IL-2 molecule and attach it to a poisonous fragment of the diphtheria toxin, the active, but not the resting, T cells will take it in, and it kills them." This experimental strategy is currently being refined.

Another strategy being used in a number of immune disorders mimics the process of thymic training. In order to be eliminated, a self-reactive T cell must encounter its target in the thymus. However, it doesn’t respond there, even though its target is right in its sights.

Why not? It appears that T cells need not one, but two signals in order to respond. The first comes from the MHC with its peptide. The second signal, passed through a different receptor, is apparently not presented in the thymus. T cells that get the first, but not the second, signal become unresponsive to further stimulation, and many of them die. Once outside the thymus, T cells will normally get both signals from the cell which presents the foreign antigens, and so they respond. But an experimental drug called CTLA4-Ig blocks this second signal. It has been used successfully to prevent rejection of organ transplants, and is currently under investigation for MG. Drachman notes that if you use both the IL-2 toxin and CTLA4-Ig, you get even better results. "The effects are at least additive, maybe better. It’s very potent."

A different strategy is being investigated by MDA researcher Dr. Premkumar Christadoss of the University of Texas Medical Branch at Galveston. Christadoss uses interferon-alpha, an immune system regulator that is produced by some T cells to control others. He has found that interferon-alpha can improve the muscle weakness of mice with experimental myasthenia. He’s currently planning a clinical trial.

Drachman points out that this kind of research into new treatments for myasthenia gravis brings results even outside of the field of neuromuscular disease. "These are really the wave of the future. We think we’re going to be able to develop these strategies using myasthenia as a model, but they should be useful for hay fever, thyroiditis, rheumatoid arthritis and many other immune system diseases."