The idea of enhancing the immune system's ability to cope with HIV has always been attractive. Various theoretical approaches have been proposed; some have been tried, but none have yet met with much success. Among the immune-based therapies studied to date are interleukin-2 (IL-2) and Jonas Salk's therapeutic vaccine Remune, both of which have now been in clinical trials for well over a decade. While frustrating, the slow pace of this research should not be surprising. Scientists' understanding of how the immune system fights viruses has been extraordinarily limited until very recently. Over the past few years, a quiet revolution has been occurring in immunology, the science of studying the immune system.
It has long been known that T-cells are a vital component of our defense against disease, but attempts to understand exactly what these cells do -- and how they do it -- have been limited by technical difficulties. Two breakthroughs have helped remedy this situation. Mice have proven a boon to immunologists, since the genetic factors that can influence a T-cell's structure and function can be tightly controlled in mice with a combination of genetic manipulation and careful breeding. Secondly, new lab tests are allowing a much more detailed assessment of T-cell function than was previously possible.
Before being subjected to these tests, T-cells are seen only as microscopic white blobs. They belong to a family of white blood cells called lymphocytes. Once immunologists get into their prodding and probing, T-cells can start to be broken down into different subsets. Structures on the T-cell surface called clusters of differentiation (CD for short) help identify the two major subsets of T-cells: CD4 cells, also known as helper T-cells, and CD8 cells, which include killer T-cells (known to researchers as cytotoxic T-lymphocytes or CTLs). CD4 helper T-cells appear to play a coordinating role in immune responses, fighting the infection but also sending signals to other immune system players like the CD8 killer T-cells. CD8 killer T-cells perform the vital task of identifying and destroying cells that have been invaded by infectious agents.
A typical adult has a lot of T-cells running throughout their body (see table below). A logical question is, how do they know which infection to fight? This feat is accomplished by another structure on the outside of the cell called a T-cell Receptor (TCR). The TCR acts as a sort of docking bay for pieces of infectious agents (any piece of infectious agent that can trigger an immune response is called an antigen). One way to think of it is to remember the children's game where you fit various shapes into their matching holes. Each TCR is like one of those holes, having a particular shape. Only an antigen that matches the shape of that TCR will fit snugly into it. And only that snugly fitting antigen will trigger the T-cell to respond.
The metaphor of the children's game only goes so far. If the game had as many shapes and holes as there are potential antigens and TCRs, it would be a recipe for tears and tantrums. A study published in the journal Science last October estimated the number of potential TCRs at around 250 million. This is the body's way of ensuring that its T-cells are ready for anything.
Both the TCR and CD4 or CD8 marker are acquired by T-cells in an organ called the thymus, located just behind your breastbone. Each T-cell generates one out of the potential 250 million TCRs by an essentially random shuffling of the T-cell's genetic code, or DNA. Once acquired, the TCR does not change. The T-cell stays specific for one particular antigen for its entire life. The thymus churns out billions of T-cells during childhood, but production slows in adulthood. In a typical 20 year old, the thymus is thought to produce about 6 billion or so new T-cells each day. This slowly declines to around 1.8 billion by the time we get past 50 years of age. Both CD4 and CD8 T-cells get made, but CD4 T-cells tend to outnumber CD8s by a ratio of around 2:1.
When a new T-cell leaves the thymus, equipped with a freshly generated TCR, it is called a naïve T-cell. naïve means that it hasn't encountered or fought any infection. The job of the naïve T-cell is to patrol the body, looking out for antigens that are the right shape to fit the cell's TCR. Specialized cells called dendritic cells chop up bits of potentially infectious material and hold them out to naïve T-cells for inspection. This function is called antigen presentation.
The events set into motion when a patrolling naïve T-cell encounters an antigen that fits its TCR are key to understanding immunity. Using a variety of animal models, particularly mice infected with a virus called LCMV (lymphocytic choriomeningitis virus), scientists have now studied the process in great detail. Although there are varied responses to different viruses, some common themes -- thought to be highly relevant to HIV infection -- have emerged.
Dendritic cells pick up pieces of the virus to which the body has been exposed and transport them to the lymph nodes. Lymph nodes are immune system command centers, and naïve T-cells regularly pass through them on their way to and from the blood and other body tissues. When the dendritic cells arrive in the lymph nodes, they hold out pieces of the virus -- or, to use the correct term, viral antigens -- for passing T-cells to check out. This is the process of antigen presentation mentioned earlier. The dendritic cells will embrace any naïve T-cells with TCRs that dock snugly with the viral antigens (usually this means several hundred thousand naïve T-cells, because a virus gets chopped up into many different shaped antigens). This takes these T-cells off patrol and begins the process that will kick-start the antiviral immune response.
|Approximate Numbers of CD4 and CD8 T-cells in Adulthood|
|CD4 T-cells||900 billion||1.1 trillion||2 trillion|
|CD8 T-cells||400 billion||500 billion||900 billion|
|Adapted from Haynes, B. et al. Ann. Rev. Immunology 2000, Vol. 18:529-560 and Haase, AT Ann. Rev. Immunology 1999, Vol. 17:625-656.|
The T-cell/dendritic cell embrace lasts several days, during which time signals are exchanged that cause the T-cell to prepare for battle. Eventually, the T-cell becomes activated, which means that it begins making copies of itself or, to use the technical term, proliferate. One naïve T-cell can make twenty or more copies of itself, and this process generates a fleet of T-cells that all have the same TCRs. All of these T-cells are thus specifically targeting a particular viral antigen.
These activated, virus-specific T-cells leave the lymph nodes on a search-and-destroy mission. Their task is to find and eliminate any infected cells and limit the ability of the virus to reproduce. To perform this mission, activated T-cells also develop enhanced infection-fighting skills. They release chemicals called cytokines and chemokines that can communicate with other cells and, in some cases, directly block viral replication. CD8 killer T-cells release special chemicals such as perforin that cause virus-infected cells to die.
Within a week or two, T-cells have usually gained the upper hand. Viral replication is controlled, and most of these newly made, activated T-cells automatically die off. What has been long suspected, but only recently proven, is that some of the virus-specific T-cells survive. Out of the twenty or so duplicates made by each naïve T-cell that was activated by the virus, it seems that a few don't die but live on as "memory" T-cells. These memory T-cells can be thought of as a swat team that the body retains to deal with the viral infection should it ever try and cause trouble again.
Recent studies have helped show how memory T-cells prevent infections from recurring. Remember the lingering embrace between the antigen-presenting dendritic cell and the naïve T-cell? A memory T-cell can be activated and go into battle after a much shorter period -- the immune system equivalent of a hug, perhaps. Memory T-cells also seem to be able to copy themselves more rapidly, and release cytokines, chemokines and other infection-fighting chemicals almost instantly.
The reason that memory T-cells have these enhanced skills relates to the activity of genes within the cell. Genes are short stretches of DNA that contain code for making certain proteins. The proteins then perform specific functions in the body. Most of us know that we inherit genes from our parents for things like eye color. It's often less appreciated that our genes are at work every second that we are alive. Every cell in our body (except for red blood cells) contains a complete copy of our DNA blueprint (called the genome) and all our genes are contained within it. However, cells only use the genes they need to function. A T-cell uses certain genes to make the proteins it needs to fight infection. A kidney cell will use different genes to perform the waste-eliminating functions of the kidney. One way to think of it is that each cell carries an entire library containing many volumes of instructions needed for making the body. But each cell only pulls off the shelves those volumes needed to carry out that cell's specific functions.
When a naïve T-cell begins copying itself in response to an infection, genes that make infection-fighting proteins such as cytokines and chemokines are slowly switched on. Each new copy of the cell that is made seems to get better at making these proteins. The cells that survive as the memory T-cell swat team end up with these genes set in a sort of hair-trigger position -- as soon as they reencounter the infection, the genes almost immediately start producing the relevant cytokines and chemokines.
Vaccines typically work by triggering this process. Because vaccine antigens are usually either fake copies or weakened versions of the real thing, naïve T-cells with matching TCRs get activated and leave a legacy of memory T-cells ready to rapidly respond if the real deal shows up. If this year's flu vaccine is close enough to this season's flu strain when it comes around, your flu-specific memory T-cells should be able to protect you by quickly eliminating the virus from your body.
What About Antibodies?
|In addition to assisting CD8 killer T-cells, CD4 T-cells provide help to B-cells. B-cells, or B-lymphocytes, are factories for making antibodies. Antibodies are small proteins designed to lock on to infectious agents floating in the bloodstream, disarming them and marking them for elimination from the body. Viruses like HIV, which do most of their dirty work while inside cells (scientists refer to these nasties as "intracellular pathogens"), are notorious for evading antibodies. So far, scientists have struggled to find ways of generating antibodies that effectively block HIV -- what they call "neutralizing antibodies." HIV cloaks itself in an ever-mutating envelope that seems designed to shuck off the antibody attack. This has led to the highly productive focus on killer T-cells described in the main body of this article. Researchers haven't given up on antibodies, however. Several novel antibody approaches have now shown some promise in animals and are moving slowly toward human testing.|
T-cell Teamwork: Helpers and Killers
There is another important lesson that's been learned from experiments with virus-infected animals. CD4 helper and CD8 killer T-cells rarely seem to work independently when dealing with viruses. Instead, they cooperate with each other to keep the virus under control. The process of naïve T-cell activation and the eventual generation of long-lived memory T-cells apply to both CD4 and CD8 T-cells whose TCRs match the viral antigens. The memory T-cell swat team incorporates both CD4 and CD8 T-cells specific for the same virus. Studies in mice suggest that when it comes to viruses, the CD8 members of the swat team outnumber their CD4 helpers by a ratio of about 50:1.
With so many more CD8 T-cells targeting a virus than CD4 T-cells, you might wonder if the helper cells are really needed at all. Several research teams have removed CD4 cells from mice and then exposed them to virus infections to see what happens. naïve CD8 T-cells seem to get activated and leave a legacy of virus-specific memory CD8 cells in the normal way. As a result, CD8 T-cells alone can often deal with viruses that don't stay in the body for long. But viruses that can persist in the body (chronic viral infections such as certain strains of LCMV in mice and hepatitis B & C, CMV, Epstein-Barr virus and HIV in humans) are not dealt with effectively in the absence of CD4 cells. With chronic infections, the virus-specific CD8 memory T-cells lose their ability to kill virus-infected cells if they don't have some virus-specific CD4 companions to tell them to keep it up.
Based on recent study results, researchers think the virus-specific CD4 T-cells are needed to provide the CD8 T-cells with an ongoing "license to kill." This licensing is done through a middleman, which turns out to be another job for the dendritic cell. The current thinking is that virus-specific CD4 T-cells persuade the dendritic cell to show a special molecule on its surface called CD40, along with the viral antigen. The CD4 T-cell then departs. When a CD8 killer T-cell that's specific for that same viral antigen shows up, it gets a signal from the CD40 molecule, which allows it to go off and kill virus-infected cells. Without the signal delivered via CD40, the ability of the CD8 T-cell to produce perforin -- its cell-destroying weapon -- seems to be impaired.
While the first examples of this CD4/CD8 memory T-cell teamwork were from animal studies, there is now evidence that the same cooperation occurs in human viral infections. Researchers using new technologies to track T-cell responses have found that cytomegalovirus (CMV)-specific CD4 and CD8 memory T-cells are generated in response to this virus. Studies have also tried treating active CMV with infusions of CMV-specific T-cells. Tellingly, CMV-specific CD8 T-cells given alone do not work well. When people are infused with both CMV-specific CD4 and CD8 T-cells, the treatment is usually effective. One of the key differences between people who control hepatitis C infection vs. those who don't (and may be at risk for liver damage as a result), is the strength of the hepatitis C-specific CD4 T-cell response.
Since the publication of an important study by immunologist Bruce Walker and colleagues in late 1997, the significance of the HIV-specific CD4 and CD8 response has been increasingly recognized. It has long been known that everyone with HIV has HIV-specific CD8 memory T-cells. In fact, people seem to accumulate more and more of these as disease progresses. But, now that the importance of T-cell teamwork has been revealed, what about the HIV-specific CD4 memory T-cell response?
Strong HIV-specific CD4 memory T-cell responses are almost exclusively found in one group of people: long-term non-progressors. These are the individuals who have maintained low or undetectable viral loads and normal T-cell counts despite being HIV infected for 20 years or more in some cases. Additionally, these people have CD8 killer T-cells that are particularly enthusiastic when it comes to destroying HIV-infected cells.
In the majority of people with progressive HIV infection, HIV-specific CD4 memory T-cell responses are either weak or difficult to detect. Recent studies have found that HIV-specific CD4 memory T-cells are not absent, but apparently dysfunctional. One of the primary features of memory T-cells is their ability to copy themselves (proliferate) rapidly when they reencounter their target. In people with progressive HIV infection, HIV-specific CD4 memory T-cells appear unable to proliferate when they reencounter the virus.
As was seen in animals lacking CD4 cells, this dysfunction of HIV-specific CD4 memory T-cells has a domino effect on the function of their CD8 killer compadres. In July's Journal of Experimental Medicine, a team of researchers led by immunologist Sarah Rowland-Jones showed that, in progressive infection, HIV-specific CD8 memory T-cells lack the essential cell-killing substance perforin. It is important to note that Rowland-Jones studied people infected with both HIV and the common viral infection CMV. This allowed the researchers to compare the cell-killing abilities of HIV-specific and CMV-specific CD8 memory T-cells from the same person. Unlike those targeting HIV antigens, the CMV-specific CD8 memory T-cells produced normal amounts of perforin and efficiently killed CMV-infected cells. The researchers suggest that the presence of CMV-specific CD4 memory T-cells, but the absence of functional HIV-specific CD4 memory T-cells, may explain these differences.
Because HIV -- unlike almost all other viruses -- can infect naïve CD4 T-cells as they become activated in response to viral antigens, scientists speculate that the normal process that should lead to the development of HIV-specific CD4 memory T-cells is thrown off. As a result, researchers are focusing on ways to trigger the development of HIV-specific CD4 memory T-cells without the interference of HIV replication. One strategy under study is a therapeutic vaccination given to people whose HIV is being controlled by HAART. While studies have shown that HIV-specific CD4 memory T-cell responses can be generated this way, it remains uncertain how big a response might be needed to control HIV once HAART is stopped. Clinical trials are ongoing. Another strategy is structured treatment interruptions, or STIs. (See "An Update on Structured Treatment Interruptions" in this issue for an update on this approach.)
Taken together, the mix of basic science and new clinical research described here is beginning to get even skeptical immunologists excited. Louis Picker, one of the first to study HIV-specific CD4 memory T-cell responses, has outlined at least four key questions that need to be addressed by future research:
How many HIV-specific CD4 memory T-cells need to be generated?
How many different HIV antigens might need to be targeted by these T-cells?
What functions should these cells be able to perform? For instance, is the ability to make particular cytokines and chemokines important? Is the ability of the cell to make copies of itself important?
How can these responses be maintained over time?
With scientists hotly pursuing answers to these questions, the idea of immune-based containment of HIV may finally be nearing the realm of reality.
Richard Jefferys oversees the Access Project, a national database of AIDS drug assistance programs at the AIDS Treatment Data Network.