HOW THE IMMUNE SYSTEM WORKS

What are the major obstacles to producing an AIDS vaccine for the general public?

A major obstacle to producing an effective AIDS vaccine is that it isn't certain which types of memory cells are needed. The results of trials with vaccines that only produce memory B cells and antibodies suggest that anti- bodies alone cannot protect against an HIV-1 infection. In contrast, individuals who are infected with HIV-1, but whose immune systems resist the virus for long periods of time, usually have inherited particular class I MHC molecules - suggesting that presentation of anti- gens to killer T cells is important for resistance. Consequently, most immunologists believe that an effective AIDS vaccine must generate memory killer T cells. Unfortunately, the production of memory CTLs requires that the agent used as a vaccine be capable of infecting antigen presenting cells - and this puts severe restrictions on the types of AIDS vaccines that might be safe to use. Most immunologists believe that to be effective, an AIDS vaccine must generate memory killer T cells. If true, non- infectious vaccines, which have been used to protect against many other pathogens, will be of little use against HIV-1. In principle, a weakened form of the AIDS virus could be used as a vaccine that would produce memory CTLs. However, because the AIDS virus has an extremely high mutation rate, there is great concern that an attenu- ated form of HIV-1 might mutate to become lethal again. Consequently, it is very unlikely that a vaccine which uses an attenuated version of the AIDS virus could ever be used to vaccinate the general population. A carrier vaccine could generate memory killer T cells without putting the vaccine recipient at risk for a real AIDS virus infection. So far, however, this strategy has not yielded a vaccine powerful enough to elicit an immune response against HIV-1 that is protective. Even if a safe vaccine could be devised which would produce HIV-1-specific CTLs, the high mutation rate of the AIDS virus makes it an elusive target. On average, each AIDS virus produced by an infected cell differs from the original infecting virus by at least one mutation. Consequently, the body of someone infected with HIV-1 contains not just "the" AIDS virus, but a huge collection of slightly different, HIV-1 strains. As a result, the memory cells produced by a vaccination might protect very well against the particular strain of HIV-1 used to prepare the vaccine, yet be totally useless against other mutant ver- sions of the virus present in a real infection. Indeed, the virus' ability to mutate rapidly may prove to be the most difficult problem of all to solve in making an effective AIDS vaccine. Despite all these difficulties, immunologists are working hard to produce an AIDS vaccine that can be used to protect the public - because such a vaccine is viewed as the current best hope for controlling the spread of the AIDS virus. Recently, antibodies have been discov- ered in rare AIDS patients which can neutralize many different HIV-1 variants. If a vaccine could be made which would elicit these "broadly neutralizing" antibodies in healthy individuals, it might be able to protect them from infection, at least by many of the common HIV-1 strains. And, as we have discussed, it should be much easier to make a safe vaccine that produces protective antibodies than one which gives rise to HIV-1-specific killer T cells. Nevertheless, it may turn out that antibodies are not enough, and that virus-specific CTLs may be required for protection.

Compare the advantages and disadvantages of killed virus vaccines and attenuated virus vaccines.

A potential drawback of all non-infectious vaccines is that although they will generate memory helper T cells and B cells (which can make protective antibod- ies), memory killer T cells will not be made - because antigen presenting cells will not be infected. Of course, many pathogens (e.g., extracellular bacteria) do not infect human cells at all. Consequently, the lack of memory CTLs (which kill infected cells) is not an issue in design- ing vaccines for these microbes. Also, antibodies pro- duced by memory B cells are sufficient to protect against many pathogens, including some pathogens which do infect human cells. For example, poliovirus and hepatitis B virus infect human cells. Nevertheless, the non- infectious Salk poliovirus vaccine and the hepatitis B virus subunit vaccine both work very well even though neither vaccine generates memory killer T cells. In con- trast, killed virus vaccines were unable to protect against either the measles or the mumps virus. So whether memory CTLs are required for protection depends on the particular microbe and its lifestyle. Another strategy for producing a vaccine is to use a weak- ened or "attenuated" form of the microbe. Virologists noticed that when a virus is grown in the laboratory in a cell type which is not its normal host, the virus sometimes accumulates mutations which weaken it. The Sabin polio vaccine, for example, was made by growing poliovirus, which normally reproduces in human nerve cells, in monkey kidney cells. This strategy resulted in poliovi- ruses which were still infectious, but which were so weak that they could not cause the disease in healthy individu- als. The vaccines for measles, rubella, and mumps, which most children in the United States now receive, are atten- uated virus vaccines. An attenuated vaccine can be tested on animals to get a general idea of whether the attenuation procedure has worked. However, to be sure a crippled microbe can stimulate the production of memory cells, yet not cause disease, it must be tested on humans - usually volunteers who expect to be at risk for contracting the disease. In this regard, it is interesting to note that by the time Dr Sabin was ready to test his vaccine, most people in our country had already received the Salk polio vaccine. So Dr Sabin took his vaccine to Russia and tested it there. This was at the height of the Cold War, but polio was such a dreaded disease that the Russians were delighted to be "guinea pigs" for Dr Sabin's vaccine. One important feature of attenuated virus vaccines is that they can produce memory killer T cells. This is because the crippled virus can infect antigen presenting cells and stimulate the production of CTLs before the immune system has had a chance to destroy the weak- ened invaders. However, because an attenuated vaccine contains a microbe that is infectious, there are safety issues. When a person has recently been vaccinated with an attenuated virus vaccine, he may produce enough virus to infect some of the people with whom he comes in contact. This can be an advantage if those people are healthy, because it "spreads the immunity around." However, a person whose immune system is weakened (e.g., because of chemotherapy for cancer) may not be able to subdue the attenuated virus. After all, the attenu- ated microbe in the vaccine isn't dead. It's just weak. So for those who are immunosuppressed, this type of gra- tuitous vaccination can have serious consequences. A second potential safety issue with an attenuated virus vaccine is that before the recipient's immune system subdues the weakened virus, the virus may mutate, and these mutations may restore the strength of the virus. Although this is not a very likely scenario, some healthy people who received the Sabin attenuated vaccine have contracted polio because the weakened virus mutated and regained its ability to cause disease.

Why do class switching and somatic hypermutation produce B cells that are better able to defend against invaders?

By using somatic hypermutation to make changes in the antigen-binding region of a BCR, and by using bind- ing and proliferation to select those mutations that have increased the BCR's ability to bind to antigen, B cell re- ceptors can be "fine-tuned." The result is a collection of B cells whose receptors have a higher average affinity for their cognate antigen. This whole process is called affinity maturation. So B cells can change their constant (Fc) region by class switching, and their antigen-binding (Fab) region by somatic hypermutation - and these two modifications produce B cells that are better adapted to deal with in- vaders. Both of these changes are controlled by cytokines that are provided mainly by helper T cells. As a result, B cells that are activated without T cell help (e.g., in re- sponse to carbohydrates on the surface of a bacterium) usually don't undergo either class switching or somatic hypermutation.

So far, we have encountered three types of dendritic cells: antigen presenting DCs, follicular DCs, and thymic dendritic cells. As a way to review, explain the function of each of these cell types.

Dendritic cells are a type of antigen-presenting cell (APC) that form an important role in the adaptive immune system. The main function of dendritic cells is to present antigens and the cells are therefore sometimes referred to as "professional" APCs. All secondary lymphoid organs have one anatomical feature in common: they all contain lymphoid follicles. These follicles are critical for the functioning of the adap- tive immune system, so we need to spend a little time getting familiar with them. Lymphoid follicles start life as "primary" lymphoid follicles: loose networks of follicular dendritic cells (FDCs) embedded in regions of the second- ary lymphoid organs that are rich in B cells. So lymphoid follicles are really islands of follicular dendritic cells within a sea of B cells. Although FDCs also have a starfish-like shape, they are very different from the antigen presenting dendritic cell (DCs) we talked about before. Those dendritic cells are white blood cells that are produced in the bone marrow, and which then migrate to their sentinel positions in the tissues. In contrast, follicular dendritic cells are regular old cells (like skin cells or liver cells) that take up their final positions in the secondary lymphoid organs as the embryo develops. In fact, follicular dendritic cells are already in place during the second trimester of gestation. Not only are the origins of follicular dendritic cells and antigen presenting dendritic cells quite different, these two types of starfish-shaped cells have very different functions. Whereas the role of dendritic APCs is to present antigen to T cells via MHC molecules, the func- tion of follicular dendritic cells is to display antigen to B cells. Here's how this works. Early in an infection, complement proteins bind to invaders, and some of this complement-opsonized antigen will be delivered by the lymph or blood to the secondary lymphoid organs. Follicular dendritic cells that reside in these organs have receptors on their surface which bind complement fragments, and as a result, fol- licular dendritic cells pick up and retain the opsonized antigen. In this way, follicular dendritic cells become "decorated" with antigens that are derived from the battle which is being waged out in the tissues. Moreover, by capturing large numbers of antigens and by holding them close together, FDCs display antigens in a way that can crosslink B cell receptors. Later during the battle, when antibodies have been produced, invaders opsonized by antibodies also can be retained on the surface of follicular dendritic cells, because FDCs have receptors that can bind to the constant regions of anti- body molecules. So follicular dendritic cells capture opsonized antigens and "advertise" these antigens to B cells in a configura- tion that can help activate them. Those B cells whose receptors are crosslinked by their cognate antigens hanging from these follicular dendritic "trees" proliferate to build up their numbers. And once this happens, the "follicle" begins to grow and become a center of B cell development. Such an active lymphoid follicle is called a "secondary" lymphoid follicle or a "germinal center." The role of complement-opsonized antigen in triggering the development of a germinal center cannot be overempha- sized: lymphoid follicles in humans who have a defective complement system never progress past the primary stage. The first type of cell that administers the exam for self tolerance is a thymic dendritic cell which has traveled to the thymus from the bone marrow. Although thymic DCs have the starfish-like shape that is characteristic of dendritic cells in general, they are different from either the antigen presenting dendritic cells or the follicular dendritic cells we have discussed previously. The exam question posed by a thymic dendritic cell is: do you recognize any of the self peptides displayed by the MHC molecules on my surface? The correct answer is, "No way!" because T cells with receptors that do recognize the combination of MHC molecules and self peptides are deleted. The reason this second test, which eliminates T cells that could react against our own anti- gens, is so important is that if such self-reactive T cells were not deleted, autoimmune disease could result. For example, Th cells that recognize self antigens could help B cells make antibodies that would tag our own mole- cules (e.g., the insulin proteins in our blood) for destruc- tion. In addition, CTLs could be produced that would attack our own cells. One important feature of negative selection is that thymic dendritic cells only survive for a about a week in the thymus. Consequently, they only present what you might call "current" self antigen. This is really smart, because if foreign antigens were to reach the thymus (as they certainly can do during an infection), dendritic cells could use them for testing, just as if they were authentic self antigens. As a result, any maturing T cells that recog- nize the invader would be deleted for as long as thymic dendritic cells continued to present the foreign antigens. The short lifetime of thymic dendritic cells protects against this possibility, and allows T cells to be examined only on "new material." Once foreign antigens associated with an infection have been eliminated from the body, freshly made thymic dendritic cells will no longer present foreign antigen as self - and T cells that can recognize the invader will again survive negative selection.

Why is it that some people seem to have a "good" immune system (i.e., they never get sick), whereas others seem to catch every bug that comes along? Asked another way: Which components of the immune system can differ between individuals?

Every human has three genes for class I MHC proteins (called HLA-A, HLA-B, and HLA-C), located on chromo- some six. Because we have two chromosome sixes (one from Mom and one from Dad), we all have a total of six class I MHC genes. Each class I HLA protein pairs with another protein called β2-microglobulin to make up the complete class I MHC molecule. In the human popula- tion, there are many, slightly different forms of the genes that encode the three class I HLA proteins. For example, there are at least 480 variants of the gene for the HLA-A protein, 800 different HLA-B genes, and 260 different HLA-C genes. The proteins encoded by these genes all have roughly the same shape, but they differ by one or a few amino acids. Immunologists call molecules that have many forms "polymorphic," and the class I HLA proteins certainly fit this description. In contrast, all of us have the same gene for the β2-microglobulin protein. Because they are polymorphic, class I MHC molecules can have different binding motifs, and therefore can present peptides that have different kinds of amino acids at their ends. For example, some class I MHC molecules bind to peptides that have hydrophobic amino acids at one end, whereas other MHC molecules prefer basic amino acids at this anchor position. Since humans have the possibility of expressing up to six dif- ferent class I molecules, collectively our class I molecules can present a wide variety of peptides. Moreover, although MHC I molecules are picky about binding to certain amino acids at the ends of the peptide, they are rather promiscuous in their selection of amino acids at the center of the protein fragment. As a result, a given class I MHC molecule can bind to and present a large number of different peptides, each of which "fits" with the par- ticular amino acids present at the ends of its binding groove. So the logic of class I MHC presentation is easy to understand, but why did Mother Nature make MHC molecules so polymorphic? After all, there are so many different forms in the human population that most of us inherit genes for six different class I molecules. Doesn't this seem a bit excessive? I mean, why not just let every- body express the same MHC I molecule? Well, suppose we all did have just one gene for class I MHC proteins, and that it was the same for everyone. Now imagine what might happen if a virus were to mutate so that none of its peptides would bind to that single MHC I molecule. Such a virus could wipe out the entire human population, because no killer T cells could be activated to destroy virus-infected cells. So polymor- phic MHC molecules give at least some people in the population a chance of surviving an attack by a clever pathogen. On a more personal level, the fact that each of us has the possibility of "owning" up to six different class I MHC molecules increases the probability that we will have at least one class I MHC molecule into which a given pathogen's protein fragments will fit. Indeed, AIDS patients who have inherited the maximum number of different class I MHC molecules (six) live significantly longer than patients who have genes for only five or fewer different class I molecules. The thinking here is that as the AIDS virus mutates, having a larger number of different class I molecules increases the probability that mutated viral proteins can be presented.

What safeguards are in place to prevent inappropriate activation of helper and killer T cells?

For a helper T cell to be activated, its receptors must rec- ognize their cognate antigen displayed by class II MHC molecules on the surface of an activated dendritic cell, and the Th cell must also receive co-stimulatory signals from that same dendritic cell. This requirement that two cells (the Th cell and the DC) agree that there has been an invasion is a powerful safeguard against the activation of "rogue" helper T cells - cells which might direct an attack against our own tissues, causing autoimmune disease. Although the events involved in the activation of helper T cells are pretty clear, the picture of how naive killer T cells are activated is still rather fuzzy. Doesn't it seem odd to you that something as important as CTL activation remains rather mysterious? It does to me. However, I think this fact helps explain why immunology is such an interesting subject: There are still many impor- tant features of the system to be discovered. Until recently, it was believed that for a naive killer T cell to be activated, three cells needed to be involved: a CTL with receptors that recognized the invader; an acti- vated dendritic cell, which was using its class I MHC molecules to present the invader's proteins to the CTL; and an activated helper T cell which was providing "help." One way this might happen would be for the dendritic cell, the Th cell, and the CTL to engage in a ménage à trois. However, early in an infection, when there are very few of any of these cells around, the probability is quite small that a helper T cell and a killer T cell would simultaneously nd a dendritic cell which happens to be presenting their cognate antigen. Moreover, the require- ment that three cells (the CTL, the dendritic cell, and the helper T cell) all agree that there is danger seems like overkill in terms of safeguarding against "unauthorized" activation. Experiments have now shown that, in response to an invasion by microbes which can infect cells (the microbes that CTLs are designed to defend against), T cell help appears not to be required during the initial activation of killer T cells. A two-cell interaction between a naive CTL and an activated dendritic cell is suf cient. During this meeting, the CTL's receptors recognize their cognate antigen displayed by class I MHC molecules on the den- dritic cell, and they receive the co-stimulation they need from that same dendritic cell. What this means is that the way a naive killer T cell is activated is analogous to the way a naive Th cell is activated: by encountering an activated dendritic cell. The fact that both helper T cells and CTLs can be acti- vated by a two-cell interaction makes perfect sense in terms of getting the adaptive immune system red up before invaders take over completely. However, this "helpless" activation of naive killer T cells raises an important question: What then is the role of the "quarter- back" Th cell in a CTL's response to an invasion? If Th cells are supposed to be orchestrating the immune response, just what is their contribution in terms of direct- ing killer T cells? The latest experiments suggest that when CTLs are activated without Th cell help, they proliferate somewhat to build up their numbers and they can kill infected cells. Nevertheless, these helpless T cells do not kill with high ef ciency, and they do not live very long. It is as if help- less activation of CTLs results in a small "burst" of killer T cells designed to deal quickly with invaders early in an infection. However, in order to ef ciently activate long- lived killer T cells and to generate memory killer T cells - cells which can defend against a subsequent invasion by the same attacker - assistance from helper T cells is required. It is possible that relatively late in an immune response, there may be enough activated dendritic cells, Th cells, and killer T cells present in lymph nodes and other sec- ondary lymphoid organs to make a three-cell interaction probable. Moreover, there is some evidence that a two- cell meeting between an activated dendritic cell and a helper T cell can generate chemokines which attract killer T cells, making a ménage à trois more likely. Recent experi- ments also suggest that when a helper T cell nds a den- dritic cell which is presenting its cognate antigen, these two cells remain "hooked up" for a period of hours - making it more probable that the rare CTL which also recognizes that invader will join the party. Another pos- sibility is that when helper T cells are activated, the den- dritic cells which activate them become "licensed" to activate CTLs - thus avoiding the need for all three cells to meet simultaneously. Although it still isn't clear how Th cell-dependent acti- vation of naive CTLs is accomplished, once CTLs have been activated and have proliferated, they travel to the battle scene. Although a single CTL is capable of killing many target cells sequentially, thousands of cells usually are infected during an attack. So to amplify their killing power, CTLs can proliferate once they reach the battle scene. However, most killer T cells depend on an external supply of the growth factor, IL-2, to proliferate out in the tissues - and helper T cells are the major suppliers of this cytokine. Consequently, when many CTLs are needed, (e.g., during a viral infection), helper T cells can supply the IL-2 required for killer T cells to proliferate. In this way, helper T cells can control the strength of a killer T cell response.

Give several reasons why antigen presentation by class I MHC molecules is important for the function of the adaptive immune system.

MHC I molecules are billboards that display on the surface of a cell, fragments of proteins manufactured by that cell. Immunologists call these "endogenous" proteins. These include ordinary cellular proteins like enzymes and structural proteins, as well as proteins encoded by viruses and other parasites that may have infected the cell. For example, when a virus enters a cell, it uses the cellular biosynthetic machinery to produce proteins encoded by viral genes. A sample of these viral proteins is then displayed by class I MHC molecules along with samples of all the normal cellular proteins. So in effect, the MHC I billboards advertise a "sampling" of all the proteins that are being made inside a cell. Almost every cell in the human body expresses class I molecules on its surface, although the number of mol- ecules varies from cell to cell. Killer T cells (also called cytotoxic lymphocytes or CTLs) inspect the protein fragments displayed by class I MHC molecules. Consequently, almost every cell is an "open book" that can be checked by CTLs to determine whether it has been invaded by a virus or other parasite and should be destroyed. Importantly, after they have been on the surface for a short while, the MHC billboards are replaced by new ones - so the class I MHC display is kept current. The way endogenous proteins are processed and loaded onto class I MHC molecules is very interesting. When mRNA is translated into protein in the cytoplasm of a cell, mistakes are sometimes made. These mistakes can result in the production of useless proteins that don't fold up correctly. In addition, proteins suffer damage due to normal wear and tear. So to make sure our cells don't ll up with defective proteins, old or useless proteins are fed into protein-destroying "machines" in the cytoplasm that function rather like wood chippers. These protein chippers are called proteasomes, and they cut proteins up into small pieces (peptides). Most of these peptides are then broken down further into individual amino acids, which are reused to make new proteins. However, some of the peptides created by the proteasomes are carried by speci c transporter proteins (TAP1 and TAP2) across the membrane of the endoplasmic reticulum (ER) - a large, sack-like structure inside the cell from which most pro- teins destined for transport to the cell surface begin their journey. Once inside the ER, some of these peptides are chosen to be loaded into the grooves of class I MHC molecules. I say "chosen," because, as we discussed, not all peptides will t. For starters, a peptide must be the right length - about nine amino acids. In addition, the amino acids at the ends of the peptide must be compatible with the anchor amino acids that line the ends of the groove of the MHC molecule. Obviously, not all of the "chips" pre ared by the proteasome will have these characteristics, and those that don't are degraded or shipped back out of the ER into the cytoplasm. Once class I MHC molecules are loaded with peptides, they proceed to the surface of the cell for display. So there are three main steps in preparing a class I display: generation of a peptide by the proteasome, transport of the peptide into the ER by the TAP transporter, and binding of the peptide to the groove of the MHC I molecule. In "ordinary" cells like liver cells and heart cells, the major function of proteasomes is to deal with defective proteins. So as you can imagine, the chippers in these cells are not too particular about how proteins are cut up - they just hack away. As a result, some of the peptides will be appropriate for MHC presentation, but most will not be. In contrast, in cells like macrophages that specialize in presenting antigen, this chipping is not so random. For example, binding of IFN-γ to receptors on the surface of a macrophage up-regulates expression of three proteins called LMP2, LMP7, and MECL1. These proteins replace three "stock" proteins which are part of the normal pro- teasome machinery. The result of this replacement is that the "customized" proteasomes now preferentially cut proteins after hydrophobic or basic amino acids. Why, you ask? Because the TAP transporter and MHC I mole- cules both favor peptides that have either hydrophobic or basic C-termini. So in antigen presenting cells, standard proteasomes are modi ed so they will produce custom- made peptides, thereby increasing the ef ciency of class I display. Proteasomes also are not too particular about the size of peptides they make, and since the magic number for class I presentation is about nine amino acids, you might imagine that the ER would be coded with useless pep- tides that were either too long or too short. However, it turns out that the TAP transporter has the highest af nity for peptides that are eight to fteen amino acids long. Consequently, the TAP transporter screens peptides pro- duced by proteasomes, and preferentially transports those that have the right kinds of C-termini and which are approximately the correct length. Once candidate peptides have been transported into the ER, enzymes then trim off excess N-terminal amino acids to make the peptide the right size for binding to class I MHC molecules. An important feature of this "chop it up and present it" system is that the majority of the proteins that are chopped up by proteasomes are newly synthesized pro teins which are structurally defective. Consequently, most proteins are displayed on class I MHC molecules soon after they are produced. This means that you don't have to wait for proteins to wear out before they can be chopped up and presented - making it possible for the immune system to react quickly to an infection.

What events are likely to be required to initiate an autoimmune reaction?

Mother Nature evolved a multilayered system in which each layer includes mech- anisms that should weed out most self-reactive cells, with lower layers catching cells that slip through tolerance induction in the layers above. This strategy works very well, but occasionally "mistakes are made" and instead of defending us against foreign invaders, the weapons of our immune system are turned back on us. Autoimmune disease results when a breakdown in the mechanisms meant to preserve tolerance of self is severe enough to cause a pathological condition. Roughly 5% of Americans suffer from some form of autoimmune disease. Although some autoimmune disorders are due to genetic defects, the majority of autoimmune diseases occur when the layers of tolerance-inducing mecha- nisms fail to eliminate self-reactive cells in genetically normal individuals. In fact, you could argue that the potential for autoimmune disease is the price we must pay for having B and T cell receptors which are so diverse that they can recognize essentially any invader. he latest thinking is that for autoimmunity to occur, at least three conditions must be met. First, an individual must express MHC molecules that efficiently present a peptide derived from the target self antigen. This means that the MHC molecules you inherit can play a major role in determining your susceptibility to autoimmune disease. For example, only about 0.2% of the US popula- tion suffers from juvenile diabetes, yet for Caucasian Americans who inherit two particular types of class II MHC genes, the probability of contracting this autoim- mune disease is increased about twenty-fold. The second requirement for autoimmunity is that the afflicted person must produce T and, in some cases, B cells which have receptors that recognize a self antigen. Because TCRs and BCRs are made by a mix-and-match strategy, the repertoire of receptors that one individual expresses will be different from that of every other indi- vidual, and will change with time as lymphocytes die and are replaced. Even the collections of TCRs and BCRs expressed by identical twins will be different. Therefore, it is largely by chance that a person will produce lym- phocytes whose receptors recognize a particular self antigen. So for autoimmune disease to occur, a person must have MHC molecules that can present a self antigen as well as lymphocytes with receptors that can recognize the self antigen - but this is not enough. There also must be environmental factors that lead to the break- down of the tolerance mechanisms which are designed to eliminate self-reactive lymphocytes. For years, physi- cians have noticed that autoimmune diseases frequently follow bacterial or viral infections, and immunologists believe that microbial attack may be one of the key envi- ronmental factors that triggers autoimmune disease. Now clearly, a viral or bacterial infection cannot be the whole story, because for most people, these infections do not result in autoimmunity. However, in conjunction with a genetic predisposition (e.g., type of MHC molecules inherited) and lymphocytes with potentially self-reactive receptors, a microbial infection may be the "last straw" that leads to autoimmune disease. a genetically suscep- tible individual is attacked by a microbe that activates T cells whose receptors just happen to cross react with a self antigen. Simultaneously, an inflammatory reac- tion takes place in the tissues where the self antigen is expressed. This inflammation could be caused either by the mimicking microbe itself, or by another, unrelated infection or trauma. As a result of this inflammatory reaction, APCs are activated that can re-stimulate self- reactive T cells. In addition, cytokines generated by the inflammatory response can upregulate class I MHC expression on normal cells in the tissues, making these cells better targets for destruction by self-reactive CTLs

What is the underlying difculty in a T cell satisfying both the requirement for MHC restriction (positive selection) and the requirement for tolerance of self (negative selection)?

Now, if you've been paying close attention, you may be wondering how any T cells could possibly pass both exams. After all, to pass the test for MHC restriction, their TCRs must recognize MHC plus self peptide. Yet to pass the tolerance exam, their TCRs must not be able to recog- nize MHC plus self peptide. Doesn't it seem that the two exams would cancel each other out, allowing no T cells to pass? It certainly does, and this is the essence of the riddle of self tolerance: how can a T cell receptor possibly mediate both positive selection (MHC restriction) and negative selection (tolerance induction)? In fact, it is even more complicated than that, because once a T cell has been educated in the thymus, its TCRs must be able to signal activation when they encounter invader-derived peptides presented by MHC molecules. So the question that vexes immunologists is: how does the same TCR, when it engages MHC-peptide complexes, signal three, very different outcomes - positive selection, negative selection, or activation? Unfortunately, I can't answer this riddle (otherwise I'd be on my way to Sweden to pick up my Nobel Prize), but I can tell you the current thinking. Immunologists believe that the events leading to MHC restriction and tolerance induction are similar to those involved in the activation of T cells: cell-cell adhesion, TCR clustering, and co- stimulation. It is hypothesized that in the thymus, posi- tive selection (survival) of T cells results from a relatively weak interaction between TCRs and MHC- self peptide displayed on cortical thymic epithelial cells. Negative selection (death) is induced by a strong interaction between TCRs and MHC-self peptide expressed on bone marrow-derived, thymic dendritic cells or medullary thymic epithelial cells. And activa- tion of T cells after they leave the thymus results from a strong interaction between TCRs and MHC-peptide displayed by professional antigen presenting cells. he question, of course, is what makes the effect of these three interactions of MHC-peptide with a T cell receptor so different - life, death, or activation? One key element appears to be the properties of the cell that "sends" the signal. In the case of MHC restriction, this is a cortical thymic epithelial cell. For tolerance induction, the cell is a bone marrow-derived dendritic cell or a med- ullary thymic epithelial cell. And for activation, the sender is a specialized antigen presenting cell. All these cells are very different, and it is likely that they differ in the cel- lular adhesion molecules they express, and in the number or type of MHC-peptide complexes they display on their surfaces. For example, it has been discovered that the proteasomes of cortical thymic epithelial cells are subtly different from the proteasomes of the cells that are respon- sible for negative selection. This could be expected to affect which self peptides are presented by these exam- iner cells. Such differences in adhesion molecules and MHC-peptide complexes could dramatically influence the strength of the signal that is sent through the T cell receptor. In addition, different types of cells are likely to express different mixtures of co-stimulatory mol- ecules - and co-stimulatory signals could change the meaning of the signal that results from TCR-MHC- peptide engagements. Not only are the cells that send the signals different, but the "receiver" (the T cell) also may change between exams. It is known that the number of TCRs on the surface of the T cell increases as the cell is educated, and it is also possible that the "wiring" within the T cell changes as the T cell matures. These differences in TCR density and signal processing could influence the interpretation of signals generated by the three types of sender cells. Although many of the pieces of the MHC restriction/ tolerance induction puzzle have been found, immunolo- gists still have not been able to assemble them into a completely consistent picture. More work is required.

How does a helper T cell know which cytokine pro le to produce?

Once helper T cells and killer T cells have been acti- vated, they are ready to go to work - to become, as immu- nologists say, "effector cells." The primary job of an effector CTL is to kill cells that have been infected by viruses or bacteria. Effector helper T cells have two main duties. First, they can remain in the blood and lymphatic circulation and travel from node to node, providing help for B cells or for killer T cells. The other duty of an effector helper T cell is to exit blood vessels at the sites where a battle is going on to provide help for the soldiers of the innate and adaptive immune systems. Helper T cells can produce many different cytokines - protein molecules which they use to communicate with the rest of the immune system. As the "quarter- back" of the immune system team, the helper T cell uses cytokines to "call the plays." These include cytokines such as TNF, IFN-γ, IL-4, IL-5, IL-6, IL-10, IL-17, and IL- 21. However, a single Th cell doesn't secrete all these different cytokines. In fact, Th cells tend to secrete subsets of cytokines - subsets which are appropriate to orchestrate an immune defense against particular invad- ers. So far, three major subsets have been identified: Th1, Th2, and Th17 (you'll see in a moment why it is Th17 instead of Th3). You shouldn't take this to mean, however, that there are only three different combinations of cytokines that can be secreted by Th cells. In fact, immu- nologists initially had a hard time finding helper T cells that secreted exactly the Th1 or Th2 cytokine subsets in humans. Clearly, there are helper T cells which secrete mixtures of cytokines that don't conform to the Th1/Th2/ Th17 paradigm, but the concept of three major subsets turns out to be quite useful in trying to make sense of the mixture of cytokines (the cytokine "profile") that Th cells produce. Of course, all of this begs the question: How does a helper T cell know which cytokines are appropriate for a given situation? Well, as any football fan knows, behind every good quarterback there is a good coach. For a helper T cell to make an informed decision about which cytokines to make, at least two pieces of infor- mation are required. First, it's necessary to know what type of invader the immune system is dealing with. Is it a virus, a bacterium, a parasite, or a fungus? Second, it is essential to determine where in the body the invaders are located. Are they in the respiratory tract, the digestive tract, or the big toe? Virgin helper T cells don't have direct access to either type of information. After all, they are busy circulating through the blood and lymph, trying to find their cognate antigen. What is needed is an "observer" who has actually been at the battle site, who has collected the pertinent information, and who can pass it along to the helper T cell. And which of the immune system cells could qualify as such an observer? The dendritic antigen presenting cell, of course! Just as the coach of a football team collects information on the opposing team and formulates a game plan, so a dendritic cell, acting as "coach" of the immune system team, collects information on the invasion, and decides how the immune system should react. That's why den- dritic cells are so important. They don't just turn naive helper T cells and killer T cells on. They actually function as the "brains" of the immune system, processing the nformation pertaining to the invasion, and producing a plan of action. What are the inputs that dendritic cells integrate to produce the game plan? These inputs are of two types. The first input comes to the dendritic cell through its pattern recognition receptors. These are the cellular recep- tors I mentioned in Lecture 4 which recognize conserved patterns that are characteristic of various classes of invad- ers. For example, Toll-like receptor 4 (TLR4) senses the presence of LPS, which is a molecule that is a component of the cell walls of Gram-negative bacteria. TLR4 also can detect proteins made by certain viruses. TLR2 specializes in identifying proteins that are "signatures" of Gram- positive bacteria. TLR3 recognizes the double-stranded RNA produced during many viral infections. And TLR9 recognizes the unmethylated DNA di-nucleotide, CpG, which is characteristic of bacterial DNA. Although TLRs were the first pattern recognition recep- tors to be characterized, additional families of pattern recognition receptors have now been discovered. Consequently, the emerging picture is that different types of antigen presenting cells (e.g., dendritic cells or macro- phages) in different parts of the body display distinct sets of these pattern recognition receptors which are "tuned" to recognize various structural features of common micro- bial invaders. By integrating the signals from these diverse pattern recognition receptors, an APC gathers information on the type of invader to be defended against. The second "scouting report" dendritic cells employ when formulating their game plan is received through various cytokine receptors on their surface. Because dif- ferent pathogens elicit the production of different cytokines during an infection, dendritic cells can learn a lot about an invader by sensing the cytokine environ- ment. Moreover, cells in different areas of the body (e.g., skin cells or cells that underlie the intestines) produce characteristic mixtures of cytokines in response to invad- ers, and these cytokines help "imprint" dendritic cells with information about the area of the body which is under attack. As a consequence, dendritic cells which observe a battle in one part of the body will have a different character from dendritic cells that are stationed in another area of the body - a "regional identity," if you will. So dendritic cells out on the front lines receive input about the invader through pattern recognition receptors and cytokine receptors. It is then up to the dendritic cell to "decode" these inputs and decide what types of weapons need to be mobilized. In addition, the cytokine environment in which DCs are activated and the pattern recognition receptors that are triggered imprint DCs with a regional identity. This allows them to remember their "roots" and dispatch the weapons of the adaptive immune system to the region of the body where they are needed. But how is the dendritic cell's game plan conveyed to the Th cell - the cell that will direct the action? There are two ways that the coach instructs the quarterback. First, there are co-stimulatory molecules which are displayed on the surface of activated dendritic cells, and the particu- lar co-stimulatory molecules that are displayed will depend on the type of invader the dendritic cell has encountered. These surface molecules can "plug into" receptor molecules on the surface of helper T cells to pass this information along. Although B7 is the best- studied co-stimulatory molecule, other co-stimulatory molecules have been identified, and more are certain to be discovered. In addition to co-stimulatory surface molecules, acti- vated dendritic cells produce cytokines which depend on the identity of the invader, and which can convey infor- mation to the helper T cell. So the bottom line is this: co-stimulatory molecules and cytokines are used by dendritic cells to convey the "game plan" to helper T cells. And the particular combination of co-stimulatory molecules and cytokines which a dendritic cell offers to a Th cell will depend on the scouting report the den- dritic cell received at the battle scene. To get a better idea of how this all works, let's look more closely at the Th1, Th2, and Th17 subsets of cytokines.

Why is T cell-independent activation of B cells important in defending us against certain pathogens?

One advantage of T cell-independent activation is that B cells can jump right into the fray without having to wait for helper T cells to be activated. The result is a speedy antibody response to those invaders that can activate B cells independent of T cell help. But there is something else important going on here. Helper T cells only recog- nize protein antigens - the peptides presented by class II MHC molecules - so if all B cell activation required T cell help, the entire adaptive immune system would be focused on proteins. This wouldn't be so great, because many of the most common invaders have carbohydrates or fats on their surface that are not found on the surface of human cells. Consequently, these carbohydrates and fats make excellent targets for recognition by the immune system. So by allowing some antigens to activate B cells without T cell help, Mother Nature did a wonderful thing: she increased the universe of antigens that the adaptive immune system can react against to include not only proteins, but carbohydrates and fats as well.

Why do you think six different proteins are required to make up a fully functional T cell receptor?

T cell receptors (TCRs) are molecules on the surface of a T cell that function as the cell's "eyes" on the world. Without these receptors, T cells would be ying blind with no way to sense what's going on outside. T cell receptors come in two avors: αβ and γδ. Each type of receptor is composed of two proteins, either α and β or γ and δ. Like the heavy and light chains of the B cell recep- tor, the genes for α, β, γ, and δ are assembled by mixing and matching gene segments. In fact, in B and T cells, the same proteins (RAG1 and RAG2) initiate the splicing o gene segments by making double-stranded breaks in chromosomal DNA. As the gene segments are mixed and matched, a "competition" ensues from which each T cell emerges with either an αβ or a γδ receptor, but not both. Generally, all the TCRs on a mature T cell are identical - although there are exceptions to this rule. Once a TCR has recognized its cognate antigen presented by an MHC molecule, the next step is to transmit a signal from the surface of the T cell, where recognition takes place, to the nucleus of the T cell. The idea is that for the T cell to switch from a resting state to a state of activation, gene expression must be altered, and these genes are, of course, located in the cell's nucleus. Normally, this type of signaling across the cell membrane involves a trans- membrane protein that has two parts: an external region which binds to a molecule (called a ligand) that is outside the cell, plus an internal region that initiates a biochemi- cal cascade which conveys the "ligand bound" signal to the nucleus. Here the TCR runs into a bit of a problem. As is true of the BCR, the αβ TCR has a perfectly ne extracellular domain that can bind to its ligand (the com- bination of MHC molecule and peptide), but the cytoplas- mic tails of the α and β proteins are only about three amino acids long - way too short to signal. To handle the signaling part, Mother Nature had to add a few bells and whistles to the TCR: a complex of proteins collectively called CD3. In humans, this signaling complex is made up of four different proteins: γ, δ, ε, and ζ (gamma, delta, epsilon, and zeta). Please note, however, that the γ and δ proteins that are part of the CD3 complex are not the same as the γ and δ proteins that make up the γδ T cell receptor. The CD3 proteins are anchored in the cell membrane, and have cytoplasmic tails that are long enough to signal just ne. The whole complex of proteins (α, β, γ, δ, ε, ζ) is trans- ported to the cell surface as a unit. If any one of these proteins fails to be made, you don't get a TCR on the surface. So most immunologists consider the functional, mature TCR to be this whole complex of proteins. After all, the α and β proteins are great for recognition, but they can't signal. And together, the γ, δ, ε, and ζ proteins signal just ne, but they are totally blind to what's going on outside the cell. You need both parts to make it work. As with BCRs, signaling involves clustering TCRs together in one area of the T cell surface. When this happens, a threshold number of kinase enzymes is recruited by the cytoplasmic tails of the CD3 proteins, and the activation signal is dispatched to the nucleus. When the α and β chains of the TCR were rst discov- ered, it was thought that the TCR was just an on/off switch whose only function was to signal activation. But now that you have heard about the CD3 proteins, let me ask you: does this look like a simple on/off switch? No way. Mother Nature certainly wouldn't make an on/off switch with six proteins! No, this TCR is quite versatile. It can send signals that result in very different out- comes, depending on how, when, and where it is trig- gered. For example, when T cells are educated in the thymus, TCRs are used to trigger suicide (death by apop- tosis) if the TCR recognizes MHC plus self peptides. Later, if its TCRs recognize their cognate antigen pre- sented by MHC molecules, but a T cell does not receive the required co-stimulatory signals, that T cell may be neutered (anergized) so it can't function. And, of course, when a TCR is presented with its cognate antigen and co-stimulatory signals are available, the TCR signals acti- vation. So this same T cell receptor, depending on the situation, signals death, anergy, or activation. In fact, there are now documented cases in which the alteration of a single amino acid in a presented peptide can change the signal from activation to death! Clearly this is no on/ off switch, and immunologists are working very hard to understand exactly how TCR signaling is "wired," and what factors in uence the signaling outcome.

Mother Nature uses "fail-safe technology" to prevent inappropriate activation of the immune system. Can you give several examples of this strategy?

This system - which involves selectin-selectin ligand binding to make the neutrophil roll, integrin-ICAM inter- actions to stop the neutrophil, and chemoattractants and their receptors on the neutrophil to facilitate exit from the blood - may seem a little over complicated. Wouldn't it be simpler just to have one pair of adhesion molecules (say, selectin and its ligand) do all three things? Yes, it would be simpler, but it would also be very dangerous. In a human there are about 100 billion endothelial cells. Suppose one of them gets a little crazy, and begins to express a lot of selectin on its surface. If selectin binding were the only requirement, neutrophils could empty out of the blood into normal tissues where they could do terrible damage. Having three types of molecules which must be expressed before neutrophils can exit the blood and spring into action helps make the system failsafe. You remember I mentioned that to completely upregu- late expression of that rst cellular adhesion molecule, selectin, takes about six hours. Doesn't this seem a bit too leisurely? Wouldn't it be better to begin recruiting neu- trophils from the blood just as soon as a macrophage senses danger? Not really. Before you start to recruit rein- forcements, you want to be sure that the attack is serious. If a macrophage encounters only a few invaders, it can usually handle the situation without help in a short time. In contrast, a major invasion involving many macro- phages can go on for days. The sustained expression of alarm cytokines from many macrophages engaged in battle is required to upregulate selectin expression, and this insures that more troops will be summoned only when they really are needed. Neutrophils are not the only blood cells that need to exit the blood and enter tissues. For example, eosinophils and mast cells, which are involved in protection against parasites, must exit the blood at the site of a parasitic infection. Monocytes, which can mature into tissue mac- rophages, also need to leave the blood stream at appropri- ate places. In addition, B cells and T cells must exit the blood and enter lymph nodes, where they can be acti- vated. And once they are activated, these cells must then be dispatched to sites of infection. This whole business is like a mail system in which there are trillions of packages (immune system cells) that must be delivered to the correct destinations. This delivery problem is solved by using the same basic strategy that works so well for neu- trophils. The key feature of the immune system's "postal service" is that the Velcro-like molecules which cause the cells to roll and stop are different from cell type to cell type and destination to destination. As a result, these cel- lular adhesion molecules actually serve as "zip codes" to insure that cells are delivered to the appropriate locations. Indeed, the selectins and their ligands are really families of molecules, and only certain members of the selectin family will pair up with certain members of the selectin ligand family. The same is true of the integrins and their ligands. Because of this two-digit zip code (type of selectin, type of integrin), there are enough "addresses" avail- able to send the many different immune system cells to all the right places. By equipping immune system cells with different adhesion molecules, and by equipping their intended destinations with the corresponding adhesion partners, Mother Nature makes sure that the different types of immune system cells will roll, stop, and exit the blood exactly where they are needed. Antigen presentation by MHC molecules is an elegant solution to a number of problems that face the immune system. Presentation by class I MHC molecules insures that killer T cells stay focused on infected cells, that innocent bystanders are not killed by mistake, and that a clever pathogen cannot hide in an infected cell by keeping all its proteins internal. MHC presentation of protein fragments greatly increases the universe of anti- gens that are available for killer T cells and helper T cells to recognize, because epitopes hidden in a folded protein are revealed. And because MHC molecules are so polymorphic, it is likely that at least some humans will have MHC molecules which can display protein fragments from any pathogen. Finally (and perhaps most importantly), helper T cells and killer T cells must recognize their cognate antigens presented by antigen presenting cells before they can be activated. This requirement for antigen presentation during activation sets up a fail-safe system in which the decision to acti- vate the adaptive immune system always involves more than one cell. But couldn't helper T cells just recognize unpresented antigen? After all, they aren't killers, so there isn't the problem of bystander killing. That's true, of course, but there is still a safety issue here. Antigen presenting cells only present antigen ef ciently when a battle is going on, and helper T cells are educated not to react to our own proteins. Consequently, both the helper T cell and the antigen presenting cell must "agree" that there has been an invasion before a helper T cell can be activated. By requiring that helper T cells only recognize pre- sented antigen, Mother Nature guarantees that the deci- sion to deploy the deadly adaptive immune system is not made by a single cell.

Describe the different roles that activated dendritic cells, activated macrophages, and activated B cells playin the presentation of antigen during the course of an infection.

Three types of antigen presenting cells have been identi ed: activated dendritic cells, activated macro- phages, and activated B cells. It's interesting that all of these are white blood cells which start life in the bone marrow, migrate out to various sites in the body, and then must be activated before they can function as antigen presenting cells. Dendritic cells have a characteristic, star sh-like shape, and they get their name from the word "dendrite," which is commonly used to describe the projections on nerve cells. The story about dendritic cells (DCs) is intriguing, because until not long ago, these cells were considered to be only a curiosity. However, it is now appreciated that these once obscure cells are the most important of all the antigen presenting cells - because dendritic cells can ini- tiate the immune response by activating virgin T cells If there is a microbial invasion, and the tissues in which a dendritic cell resides become a battle site, the dendritic cell will become "activated." Immunologists have now identi ed two different kinds of signals which can acti- vate dendritic cells. The rst type of signal comes either from other immune system cells that are engaged in battle or from dying cells. For example, both neutrophils and macrophages give off tumor necrosis factor (TNF) when they are trying to destroy an attacker, and this battle cytokine can activate dendritic cells. In addition, cells that are being killed by invaders give off chemicals that can result in dendritic cell activation. The second type of signal involved in the activation of antigen presenting cells comes from cellular receptors which recognize molecular "patterns" that are character- istic of broad classes of invaders. The "pattern recognition receptors" about which most is known are the Toll-like receptors (TLRs), and so far, eleven human TLRs have been discovered. Some are displayed on the surface of dendritic cells where they respond to invaders that are outside the cell. Other TLRs are found inside dendritic cells, and these receptors detect invaders which have already entered these cells. Macrophages also are sentinel cells that stand guard over those areas of our body that are exposed to the outside world. They are very adaptable cells which can function as garbage collectors, antigen presenting cells, or fero- cious killers, depending on the signals they receive from the microenvironment in which they reside. In a resting state, macrophages are good at tidying up, but they are not much good at antigen presentation. This is because macrophages only express enough MHC and co- stimulatory molecules to function as antigen presenting cells after they have been activated by battle cytokines such as IFN-γ, or by having their pattern-recognition receptors (e.g., their Toll-like receptors) ligated by invad- ing pathogens. So macrophages resemble dendritic cells in that they ef ciently present antigen only when there is something dangerous to present. However, it is important to recog- nize that dendritic antigen presenting cells don't kill, and macrophages don't travel. Indeed, DCs can be pic- tured as "photojournalists" who don't carry weapons, and who take snapshots of the ghting, and who then leave the battle eld to le their stories. In contrast, mac- rophages are heavily armed soldiers who must stand and ght. After all, macrophages are one of our main weapons in the early defense against invaders. However, their lack of mobility raises an interesting question: What good is the activated macrophage's capacity to present antigen if it can't travel to lymph nodes where virgin T cells are located? Here's the answer. So mature dendritic cells acti- vate virgin T cells, and activated macrophages mainly function to re-stimulate experienced T cells. The third professional APC is the activated B cell. A virgin B cell is not much good at antigen presentation, because it expresses only low levels of class II MHC molecules and little or no B7. However, once a B cell has been acti- vated, the levels of class II MHC molecules and B7 pro- teins on its surface increase dramatically. As a result, an experienced B cell is able to act as an antigen presenting cell for Th cells. B cells are not used as APCs during the initial stages of an infection, because at that time they are still naive - they haven't been activated. However, later in the course of the infection or during subsequent infec- tions, presentation of antigen by experienced B cells plays an important role. Indeed, B cells have one great advan- tage over the other APCs: B cells can concentrate antigen for presentation. Here's how this works. In summary, when an invader is rst encountered, all the B cells which could recognize that particular invader are virgins, so the important APCs are activated den- dritic cells. Then, while the battle is raging, activated macrophages on the front lines present antigen to warring T cells to keep them pumped up. Later, if this same invader is encountered again, experienced memory B cells left over from the rst attack are the most impor- tant APCs - because they can get the adaptive immune response cranked up quickly by concentrating small amounts of antigen for presentation.

Give examples of the cooperation between players on the innate system team, and tell why this cooperation is important.

To make the innate system work efficiently, there must be cooperation between players on the innate team. For example, neutrophils are "on call" from the blood. And who does the calling? The sentinel cell, the macrophage. So here we have a defense strategy in which "garbage collectors" alert the "hired guns" when their help is needed. Indeed, cooperation between macrophages and neutrophils is essential for mounting an effective defense against invading microbes. Without macrophages to summon them to sites of attack, neutrophils would just go around and around in the blood. And without neu- trophils, macrophages would be hard pressed to deal with sizable infections. Also, during a bacterial infection, molecules like LPS bind to receptors on the surface of natural killer cells, signaling that an attack is on. NK cells then respond by producing significant amounts of IFN-γ. The IFN-γ produced by NK cells can prime macro- phages, which can then be hyperactivated when their receptors also bind to LPS. When a macrophage is hyperactivated, it produces lots of TNF. A macrophage also has receptors on its surface to which this cytokine can bind, and when TNF binds to these receptors, the macrophage begins to secrete IL-12. Together, TNF and IL-12 influence NK cells to increase the amount of IFN-γ they produce. And when there is more IFN-γ around, more macrophages can be primed. There is something else interesting going on here. IL-2 is a growth factor that is produced by NK cells. Normally, NK cells don't express the receptor for IL-2, so they don't proliferate in response to this cytokine - even though they are making it. During an infection, however, macrophages produce TNF, which upregulates the expression of IL-2 receptors on the surface of NK cells. Consequently, NK cells can now react to the IL-2 they make and begin to proliferate. As a result of this proliferation, there will soon be many more NK cells to defend against an invasion - and to help activate more macrophages. So macrophages and NK cells cooperate in several different ways to strengthen the response of the innate system to an attack. Professional phagocytes and the complement system also work together. As we discussed, complement protein fragments such as iC3b can tag invaders for phagocyte ingestion. But complement opsonization also can play a role in activating macrophages. When C3 fragments that are decorating an invader bind to receptors on the surface of a macrophage, this provides an activation signal for the macrophage which is similar to that supplied by LPS. This is a good idea, because there are invaders that can be opsonized by complement, but which do not make LPS. Cooperation between the complement system and the phagocytes is not a one-way street. Activated macro- phages actually produce several of the most important complement proteins: C3, factor B, and factor D. So in the heat of battle, when complement proteins may be depleted out in the tissues, macrophages can help resupply the complement system. In addition, during an infection, macrophages secrete chemicals that increase the perme- ability of nearby blood vessels. And when these vessels become leaky, more complement proteins are released into the tissues. All these interactions between phagocytes, NK cells, and the complement proteins are examples of the many ways in which innate system players work together. Only by cooperating with each other can the players on the innate system team respond quickly and strongly to an invasion.

During their lifetimes, dendritic antigen presenting cells can be "samplers," "travelers," and "presenters." Describe what DCs are doing during each of these three stages

When a dendritic cell is activated by battle cytokines, chemicals given off by dying cells, ligation of its pattern- recognition receptors, or a combination of these signals, the lifestyle of this "wine taster" changes dramatically. No longer does the dendritic cell "sip and spit." Now it "swallows" what it has taken in. Typically, a dendritic cell remains in the tissues for about six hours after the attack begins, collecting battle antigens. At that point, phagocy- tosis ceases, and the activated dendritic cell leaves the tissues and travels through the lymphatic system to the nearest lymph node. It is its ability to "travel when acti- vated" that makes the dendritic antigen presenting cell so special. Inside a resting dendritic cell are large numbers of class II MHC molecules. When a resting DC is activated and starts to "mature" (as immunologists like to say), these "reserve" class II MHC molecules begin to be loaded with antigens from the battle scene. And by the time a DC reaches its destination - the trip usually takes about a day - these battle antigen-loaded class II MHC molecules will be prominently displayed on the surface of the cell. Also during its journey, the DC upregulates expression of its class I MHC molecules. Consequently, if the dendritic cell had been infected by a virus out at the battle scene, by the time it reaches a lymph node, fragments of viral proteins will be on display on the dendritic cell's class I MHC billboards. Finally, while traveling, the dendritic cell increases production of B7 co-stimulatory proteins. So by the time it reaches a lymph node, the mature den- dritic cell has everything it needs to activate virgin T cells: high levels of class I and class II MHC molecules loaded with the appropriate peptides, and plenty of B7 proteins. Now, why do you think it would be a good idea to have DCs, which wildly sample antigens out in the tissues, stop sampling when they begin their journey to a lymph node? Of course. Dendritic cells take a "snapshot" of what is happening on the "front lines," and carry this image to a lymph node - the place where virgin T cells congregate. There the traveling dendritic cells activate those virgin T cells whose T cell receptors recognize the invader that is "in the picture." The fact that battle cytokines such as TNF trigger the migration of DCs to a lymph node also makes perfect sense. After all, you want DCs to mature, travel to lymph nodes, and present antigen only if a battle is on. Once a dendritic cell reaches a lymph node, it only lives for about a week. This short lifetime may seem strange at rst. After all, this doesn't give a dendritic cell very long to meet up with the "right" virgin T cell that is circulating through the lymph nodes, looking for its cognate antigen. However, this short presentation life insures that den- Now, why do you think it would be a good idea to have DCs, which wildly sample antigens out in the tissues, stop sampling when they begin their journey to a lymph node? Of course. Dendritic cells take a "snapshot" of what is happening on the "front lines," and carry this image to a lymph node - the place where virgin T cells congregate. There the traveling dendritic cells activate those virgin T cells whose T cell receptors recognize the invader that is "in the picture." The fact that battle cytokines such as TNF trigger the migration of DCs to a lymph node also makes perfect sense. After all, you want DCs to mature, travel to lymph nodes, and present antigen only if a battle is on. dendritic antigen presenting cells are sentinel cells that "sample" antigens out in the tissues. If there is an invasion, DCs become activated and travel to nearby lymph nodes. There they initiate the adaptive immune response by presenting antigen collected at the battle scene to virgin T cells. Activated DCs are short-lived, and the rapid turnover of these cells insures that the "pictures" they bring to a lymph node are continuously updated. Moreover, the number of dendritic cells dis- patched from the tissues and the number of replace- ment dendritic cells recruited will depend on the severity of the attack. Consequently, the immune system is able to mount a response that is proportional to the danger posed by the invasion. Can you imagine a more ingenious system? I don't think so! Dendritic cells are classi ed as members of the innate immune system because their receptors are "hard-wired," not adaptable like those of B and T cells. However, as I'm sure you now understand, dendritic cells actually func- tion as a "bridge" between the innate and the adaptive systems.

Why does antigen presentation by class II MHC molecules make good sense?

Whereas class I MHC molecules are designed to present protein fragments to killer T cells, class II MHC mole- cules present peptides to helper T cells. And in contrast to class I MHC molecules, which are expressed on almost every kind of cell, class II molecules are expressed exclusively on cells of the immune system. This makes sense. Class I molecules specialize in display- ing proteins that are manufactured inside the cell, so the ubiquity of class I molecules gives CTLs a chance to check most cells in the body for infection. On the other hand, class II MHC molecules function as billboards that advertise what is happening outside the cell to alert helper T cells to danger. Therefore, relatively few cells expressing class II are required for this task - just enough to sample the environment in various parts of the body. The two proteins that make up the class II MHC mol- ecules (called the α and β chains) are produced in the cytoplasm and are injected into the endoplasmic reticu- lum where they bind to a third protein called the invari- ant chain. This invariant chain protein performs several functions. First, it sits in the groove of the MHC II mol- ecule and keeps it from picking up other peptides in the ER. This is important, because the ER is full of endog- enous peptides that have been processed by proteasomes for loading onto class I MHC molecules. If these protein fragments were loaded onto class II molecules, then class I and class II MHC molecules would display the same kind of peptides: those made from proteins produced in the cell. Since the goal is to have class II MHC molecules present antigens that come from outside the cell, the invariant chain performs an important function by acting as a "chaperone" that makes sure "inappropriate suitors" (endogenous peptides) don't get picked up by MHC II molecules in the ER. The invariant chain's second function is to guide class II MHC molecules out through the Golgi stack to special vesicles in the cytoplasm called "endosomes." It is within endosomes that class II MHC molecules are loaded with peptides. I have to warn you, however, that when biologists don't understand something very well, they usually call it a "-some" - a suf x that means "body." And this is no exception, because it isn't clear yet exactly what goes on inside these endosomes. The current thinking is that while class II MHC mole- cules are making their way from the ER to the endosome, proteins that are hanging around outside the cell are enclosed in a phagosome, and brought into the cell. This phagosome then merges with the endosome, and enzymes present in the endosome chop up the exogenous proteins from the phagosome into peptides. During this time, endosomal enzymes also destroy all of the invariant chain except the piece that is actually guarding the groove of the MHC molecule. Amazingly, although the exogenous proteins and the invariant chain are hacked to pieces by enzymes in the endosome, the class II MHC molecule itself remains unscathed. This is presumably because the MHC molecule is cleverly folded so that the enzymes cannot gain access to their favorite cleavage sites. Meanwhile, a cellular protein called HLA-DM, which also has traveled to the endosome, catalyzes the release of the remaining fragment of the invariant chain (called CLIP), allowing an exogenous peptide to be loaded into the now-empty groove of the class II MHC molecule. Finally, the complex of MHC plus peptide is transported to the cell surface for display. This is probably more or less what happens, but the details are still fuzzy. The important point, however, is that Mother Nature has arranged two separate loading sites and pathways for class I and class II MHC mole- cules. It is this separation of loading sites and pathways that allows the class I billboard to advertise what's going on inside the cell (for killer T cells), and the class II billboard to advertise what's happening outside (for helper T cells). Although the separation of class I and class II pathways is the "law," under certain experimental con- ditions, antigens taken up from outside a cell can be presented by class I MHC molecules. Such an unlawful use of the class I display has been termed "cross- presentation." To date, the rules governing cross- presentation have not been clearly de ned, and it is not yet known whether, under normal circumstances, cross-presentation is an important feature of the human immune system.

There is a conflict between immune surveillance against cancer and the preservation of tolerance of self antigens. Explain

it should be clear that powerful defenses exist within the cell (e.g., tumor suppressor proteins) to deal harshly with most wannabe cancer cells. Whether or not the immune system also plays a major role in protecting us against the majority of human cancers is not nearly so clear. The majority of human cancers are spontaneous tumors that are not of blood-cell origin, and it has been proposed that killer T cells might provide surveillance against these cancers. Let's try to evaluate this possibility. Imagine that a heavy smoker finally accumulates enough mutations in the cells of his lungs to turn one of them into a cancer cell. Remember, it only takes one bad cell to make a cancer. And let's suppose that because of these mutations, this cell expresses proteins that could be recognized as foreign by CTLs. Now let me ask you a question: where are this man's naive T cells while the tumor is growing in his lung? That's right. They are cir- culating through the blood, lymph, and secondary lym- phoid organs. Do they leave this circulation pattern to enter the tissues of the lung? No, not until after they have been activated. So right away, in terms of immune surveillance, we have a "traffic problem." To make self tolerance work, Mother Nature set up the traffic system so that naive T cells don't get out into the tissues where they might encounter self antigens that were not present in the thymus during tolerance induction. As a result, it's unlikely that virgin T cells ever would "see" tumor anti- gens expressed in the lung - because they just don't go there. What we have here is a serious conflict between the need to preserve tolerance of self (and avoid autoim- mune disease) and the need to provide surveillance against tumors that arise, as most tumors do, out in the tissues. And tolerance wins. sgain we see a conflict between tolerance induction and tumor surveillance. The two-key system of specific recognition plus co-stimulation was set up so that T cells which recognize self antigens out in the tissues, but which do not receive proper co-stimulation, will be anergized or killed to prevent autoimmunity. Unfortunately, this same two-key system makes it very difficult for CTLs to be activated by tumor cells that arise in the tissues. So the bottom line is that a CTL would have to perform "unnatural acts" to be activated by a tumor out in the tissues: it would have to break the traffic laws, and somehow avoid being anergized or killed. This could happen, of course, but it would be very inefficient compared to the activation of CTLs in response to, for example, a viral infection. In addition, tumor cells can mutate so that they stop producing the particular MHC molecules that CTLs are restricted to recognize. This happens quite frequently: about 15% of the tumors that have been examined have lost expression of at least one of their MHC molecules. Also, genes that encode the TAP transporters can mutate in a tumor cell, with the result that tumor antigens will not be efficiently transported for loading onto class I MHC molecules. Indeed, a tumor cell's high mutation rate is its greatest advantage over the immune system, and usually keeps these cells one step ahead of surveil- lance by CTLs. So even when it occurs, CTL surveil- lance is usually a case of "too little, too late."

******* Make a table for each of the secondary lymphoid organs we discussed (lymph node, Peyer's patch, and spleen) which lists how antigen, B cells, and T cells enter and leave these organs.

************ Lymphocytes develop and mature in the primary lymphoid organs, but they mount immune responses from the secondary lymphoid organs. A naïve lymphocyte is one that has left the primary organ and entered a secondary lymphoid organ. Naïve lymphocytes are fully functional immunologically, but have yet to encounter an antigen to respond to. In addition to circulating in the blood and lymph, lymphocytes concentrate in secondary lymphoid organs, which include the lymph nodes, spleen, and lymphoid nodules. All of these tissues have many features in common, including the following: The presence of lymphoid follicles, the sites of the formation of lymphocytes, with specific B cell-rich and T cell-rich areaS. An internal structure of reticular fibers with associated fixed macrophages. Germinal centers, which are the sites of rapidly dividing B lymphocytes and plasma cells, with the exception of the spleen. Specialized post-capillary vessels known as high endothelial venules; the cells lining these venules are thicker and more columnar than normal endothelial cells, which allow cells from the blood to directly enter these tissues. Lymph nodes function to remove debris and pathogens from the lymph, and are thus sometimes referred to as the "filters of the lymph". Any bacteria that infect the interstitial fluid are taken up by the lymphatic capillaries and transported to a regional lymph node. Dendritic cells and macrophages within this organ internalize and kill many of the pathogens that pass through, thereby removing them from the body. The lymph node is also the site of adaptive immune responses mediated by T cells, B cells, and accessory cells of the adaptive immune system. Like the thymus, the bean-shaped lymph nodes are surrounded by a tough capsule of connective tissue and are separated into compartments by trabeculae, the extensions of the capsule. In addition to the structure provided by the capsule and trabeculae, the structural support of the lymph node is provided by a series of reticular fibers laid down by fibroblasts. Lymph enters the lymph node via the subcapsular sinus, which is occupied by dendritic cells, macrophages, and reticular fibers. Within the cortex of the lymph node are lymphoid follicles, which consist of germinal centers of rapidly dividing B cells surrounded by a layer of T cells and other accessory cells. As the lymph continues to flow through the node, it enters the medulla, which consists of medullary cords of B cells and plasma cells, and the medullary sinuses where the lymph collects before leaving the node via the efferent lymphatic vessels. In addition to the lymph nodes, the spleen is a major secondary lymphoid organ. It is about 12 cm (5 in) long and is attached to the lateral border of the stomach via the gastrosplenic ligament. The spleen is a fragile organ without a strong capsule, and is dark red due to its extensive vascularization. The spleen is sometimes called the "filter of the blood" because of its extensive vascularization and the presence of macrophages and dendritic cells that remove microbes and other materials from the blood, including dying red blood cells. The spleen also functions as the location of immune responses to blood-borne pathogens. The spleen is also divided by trabeculae of connective tissue, and within each splenic nodule is an area of red pulp, consisting of mostly red blood cells, and white pulp, which resembles the lymphoid follicles of the lymph nodes. Upon entering the spleen, the splenic artery splits into several arterioles (surrounded by white pulp) and eventually into sinusoids. Blood from the capillaries subsequently collects in the venous sinuses and leaves via the splenic vein. The red pulp consists of reticular fibers with fixed macrophages attached, free macrophages, and all of the other cells typical of the blood, including some lymphocytes. The white pulp surrounds a central arteriole and consists of germinal centers of dividing B cells surrounded by T cells and accessory cells, including macrophages and dendritic cells. Thus, the red pulp primarily functions as a filtration system of the blood, using cells of the relatively nonspecific immune response, and white pulp is where adaptive T and B cell responses are mounted. Mucosa-associated lymphoid tissue (MALT) consists of an aggregate of lymphoid follicles directly associated with the mucous membrane epithelia. MALT makes up dome-shaped structures found underlying the mucosa of the gastrointestinal tract, breast tissue, lungs, and eyes. Peyer's patches, a type of MALT in the small intestine, are especially important for immune responses against ingested substances. Peyer's patches contain specialized endothelial cells called M (or microfold) cells that sample material from the intestinal lumen and transport it to nearby follicles so that adaptive immune responses to potential pathogens can be mounted.

*****Essentially all players on the innate and adaptive immune system teams must be activated before they can "get into the game." Trace the steps in the "activation cascade" which begins when an LPS-carrying, Gram- negative bacterium enters a wound, and which ends when antibodies are produced that can recognize the bacterium.

A pathogen enters the body through break in skin barrier Nonspecific immune defenses begin - the process of inflammation Macrophages engulf and partially digest some of the invaders; Macrophages display some invader fragments along with their own MHC tag on their surgace; macrophages act as antigen presenting cells and couple the nonspecific to the specific immune defense A t helper cell that can bind the MHC and has specific receptors for the antigen fragment displayed will bind to the active macrophages The macrophages will activate the specifically bound t helper via chemical signal The activated t helper will undergo several rounds of cell division, producing a large number of identical antigen specific t helpers. These then go in search of attack to activate Resting b cells will also bind antigen on their surface receptiors. Like macrophages, they will partially digest it and display it on their surface with MHC as a sign they are specific to that antigen The activated b cell with a matched antigen specificity will bind to the t helper and activate it using chemical signals (since all self reactive t cells have been destroyed no self reactive b cells can be activated) The activated b cells will now divide producing a clone of identically reactive plasma cells The plasma cells produce and secrete large quantities of antibiodies, specifically designed to attack and neutralize the antigen and/or invader that carries it

Describe "fail-safe" systems that are involved in B cell activation.

Activation of a naive B cell requires two signals. The first is the clustering of the B cell's receptors and their associated signaling molecules. However, just having its receptors crosslinked is not enough to fully activate a B cell - a second signal is required. Immunologists call this the "co-stimulatory" signal. In T cell-dependent activation, this second signal is supplied by a helper T cell. The best studied co-stimulatory signal involves direct contact between a B cell and a helper T (Th) cell. On the surface of activated Th cells are proteins called CD40L. When CD40L plugs into (ligates) a protein called CD40 on the surface of a B cell, the co-stimulatory signal is sent, and if the B cell's receptors have been crosslinked, the B cell is activated. The interaction between these two proteins, CD40 and CD40L, is clearly very important for B cell activation. Humans who have a genetic defect in either of these proteins are unable to mount a T cell-dependent antibody defense. What is important for this discussion is that if a B cell has BCRs that can recognize a molecule with repeated epitopes like, for example, your own DNA, it may proliferate, but for- tunately, no anti-DNA antibodies will be produced. The reason is that your immune system is not engaged in a battle with your own DNA, so there will be no danger signals to provide the necessary co-stimulation. On the other hand, if the innate immune system is battling a bacterial infection, and a B cell's receptors recognize a carbohydrate antigen with repeated epitopes on the surface of the bacterial invader, that B cell will produce antibodies - because danger signals from the battle field can supply the second key needed for complete B cell activation.

B cells are produced according to the principle of clonal selection. Exactly what does this mean?

After B cells do their mix-and-match thing and paste together the modules required to form the complete recipes for their heavy and light chain antibody proteins, a relatively small number of these proteins is made - a "test batch" of antibody molecules, if you will. These tester antibodies, called B cell receptors (BCRs), are trans- ported to the surface of the B cell and are tethered there with their antigen binding regions facing out. Although each B cell has roughly 100000 BCRs anchored on its surface, all the BCRs on a given B cell recognize the same antigen. The B cell receptors on the surface of a B cell act like "bait," and what they are "fishing for" is the molecule which their Fab regions have the right shape to grasp - their "cognate" antigen. Sadly, the vast majority of B cells fish in vain. For example, most of us will never be infected with the SARS virus or the AIDS virus, so our B cells which could make antibodies that recognize these viruses never will find their match. It must be very frustrating for most B cells. They fish all their lives, and never catch anything! On occasion, however, a B cell does make a catch. When a B cell's receptors bind to its cognate antigen, that B cell is triggered to double in size and divide into two daugh- ter cells - a process immunologists call proliferation. Both daughter cells then double in size and divide to produce a total of four cells, and so forth. Each cycle of cell growth and division takes about 12 hours to complete, and this period of proliferation usually lasts about a week. At the end of this time, a "clone" of roughly 20000 identical B cells will have been produced, all of which have receptors on their surface that can recognize the same antigen. Now there are enough to mount a real defense. After the selected B cells proliferate to form this large clone, most of them begin to make antibodies in earnest. The antibodies produced by these selected B cells are slightly different from the antibody molecules displayed on their surface in that there is no "anchor" to attach them to the B cell's surface. As a result, these antibodies are transported out of the B cell and into the bloodstream. One B cell, working at full capacity, can pump out about 2000 antibody molecules per second! After making this heroic effort, most of these B cells die, having worked for only about a week as antibody factories. When you think about it, this is a marvelous strategy. First, because they employ modular design, B cells use relatively few genes to create enough different antibody molecules to recognize any possible invader. Second, B cells are made on demand. So instead of filling up our bodies with a huge number of B cells which may never be used, we begin with a relatively small number of B cells, and then select the particular B cells that will be useful against the "invader du jour." Once selected, the B cells proliferate rapidly to produce a large clone of B cells whose antibodies are guaranteed to be useful against the invader. Third, after the clone of B cells has grown suffi- ciently large, most of these cells become antibody facto- ries which manufacture huge quantities of the very antibodies that are right to defend against the invader. Finally, when the invader has been conquered, most of the B cells die. As a result, we don't fill up with B cells that are appropriate to defend against yesterday's invader, but which would be useless against the enemy that attacks us tomorrow. I love this system!

in the T cell areas of secondary lymphoid organs, activated dendritic cells and Th cells interact. What goes on during this "dance"?

All secondary lymphoid organs have one anatomical feature in common: they all contain lymphoid follicles. These follicles are critical for the functioning of the adap- tive immune system, so we need to spend a little time getting familiar with them. Lymphoid follicles start life as "primary" lymphoid follicles: loose networks of follicular dendritic cells (FDCs) embedded in regions of the second- ary lymphoid organs that are rich in B cells. So lymphoid follicles are really islands of follicular dendritic cells within a sea of B cells. Although FDCs also have a starfish-like shape, they are very different from the antigen presenting dendritic cells (DCs) we talked about before. Those dendritic cells are white blood cells that are produced in the bone marrow, and which then migrate to their sentinel positions in the tissues. In contrast, follicular dendritic cells are regular old cells (like skin cells or liver cells) that take up their final positions in the secondary lymphoid organs as the embryo develops. In fact, follicular dendritic cells are already in place during the second trimester of gestation. Not only are the origins of follicular dendritic cells and antigen presenting dendritic cells quite different, these two types of starfish-shaped cells have very different functions. Whereas the role of dendritic APCs is to present antigen to T cells via MHC molecules, the func- tion of follicular dendritic cells is to display antigen to B cells. Here's how this works. Early in an infection, complement proteins bind to invaders, and some of this complement-opsonized antigen will be delivered by the lymph or blood to the secondary lymphoid organs. Follicular dendritic cells that reside in these organs have receptors on their surface which bind complement fragments, and as a result, fol- licular dendritic cells pick up and retain the opsonized antigen. In this way, follicular dendritic cells become "decorated" with antigens that are derived from the battle which is being waged out in the tissues. Moreover, by capturing large numbers of antigens and by holding them close together, FDCs display antigens in a way that can crosslink B cell receptors. Later during the battle, when antibodies have been produced, invaders opsonized by antibodies also can be retained on the surface of follicular dendritic cells, because FDCs have receptors that can bind to the constant regions of anti- body molecules. So follicular dendritic cells capture opsonized antigens and "advertise" these antigens to B cells in a configura- tion that can help activate them. Those B cells whose receptors are crosslinked by their cognate antigens hanging from these follicular dendritic "trees" proliferate to build up their numbers. And once this happens, the "follicle" begins to grow and become a center of B cell development. Such an active lymphoid follicle is called a "secondary" lymphoid follicle or a "germinal center." The role of complement-opsonized antigen in triggering the development of a germinal center cannot be overempha- sized: lymphoid follicles in humans who have a defective complement system never progress past the primary stage. Thus, we see again that for the adaptive immune system to respond, the innate system must first react to impending danger.

why are IgA antibodies called "passive" antibodies?

All together, these qualities make IgA antibodies perfect for guarding mucosal surfaces. Indeed, it is the IgA class of antibodies that is secreted into the milk of nursing mothers. These IgA antibodies coat the baby's intestinal mucosa and provide protection against pathogens that the baby ingests. This makes sense, because many of the microbes that babies encounter are taken in through their mouths - babies like to put their mouths on everything, you know. Although IgA antibodies are very effective against mucosal invaders, they are totally useless at fixing com- plement, because C1 won't bind to an IgA antibody's Fc region. Again we see that the constant region of an anti- body determines both its class and its function. This lack of complement-fixing activity is actually a good thing. If IgA antibodies could initiate the complement reaction, our mucosal surfaces would be in a constant state of inflammation in response to the pathogenic and non-pathogenic visitors that continuously assault our mucosal surfaces. And, of course, having chronically inflamed intestines would not be all that great. So IgA antibodies mainly function as "passive" antibodies that block the attachment of invaders to cells that line our mucosal surfaces, and usher these unwanted guests out of the body.

Why doesn't the interaction between B7 proteins on APCs and CTLA-4 proteins prevent the activation of naive T cells?

Athough the removal of foreign antigen is the most important factor in turning off the system, other mecha- nisms also help decrease the level of activation as the battle winds down. In Lecture 5, we discussed the B7 proteins which are expressed on the surface of antigen presenting cells, and which provide co-stimulation to T cells by plugging into receptors called CD28 on a T cell's surface. This interaction sets off a cascade of events within a T cell that reduces the total number of T cell receptors which must be crosslinked in order to activate the T cell, making activation more efficient. However, in addition to engaging stimulatory CD28 molecules, B7 proteins also can plug into another receptor on T cells called CTLA-4. In contrast to ligation of CD28, which increases activation, engagement of CTLA-4 represses activation by antagonizing the CD28 activa- tion signal within the T cell. Moreover, because B7 binds to CTLA-4 with an affinity which is thousands of times higher than its affinity for CD28, CTLA-4 also suppresses activation by occupying B7 molecules so they cannot bind to CD28. Most human T cells display CD28 on their surface, so it is always available to assist with activation. In contrast, most of a naive T cell's CTLA-4 is stored inside the cell. Once these T cells have been activated, however, more and more CTLA-4 is moved from these intracellular res- ervoirs to the cell surface where, because of its higher affinity, CTLA-4 eventually out-competes CD28 for B7 binding. As a result, early in an infection, B7 binds to CD28 and acts as a co-stimulator. Then, after the battle has been raging for a while, B7 binds mainly to CTLA-4, making it harder, instead of easier, for these T cells to be reactivated, and helping to shut down the adaptive immune response.

What are the differences between the strategies B and T memory cells use to be sure we are "covered" against a future invasion by the pathogen they remember? Why are these differences important?

B and T cell memories are similar in that both systems center around stem-cell-like central memory cells. These central memory cells reside in the secondary lymphoid organs, where they are strategically located to intercept invaders as they enter the body. In addition, memory B and T cells are more potent weapons than are naive cells because a larger fraction of them are specific for the invader they remember - and because they are easier to activate. Other aspects of B cell and T cell memory, however, are different. In response to an invasion, B cells can fine-tune their receptors through somatic hypermutation. T cells cannot. Moreover, there is no T cell equivalent of the long-lived plasma B cell. Once we have been exposed to an invader, long-lived plasma B cells continue to produce protective antibodies, frequently for a lifetime. Consequently, the weapons made by B cells (the anti- body molecules) continue to be deployed even after an invasion has been repulsed. This works well because antibodies are very specific and rather benign. Only when they tag an invader is the rest of the immune system alerted to take action. So if the invader they recognize doesn't come again, the pre-made antibodies do nothing and cause no trouble. In contrast, activated T cells produce cytokines and other chemicals which are nonspecific and which can cause severe damage to normal tissues. As a result, it would be very dangerous to have T cells remain in action once an invasion has been repulsed. Consequently, instead of continuing to function after the enemy has been defeated, as long-lived plasma cells do, effector memory T cells go "dormant." If the attacker does not return, they cause no trouble. On the other hand, if an enemy again enters the tissues where effector memory T cells are "sleeping," these cells quickly reactivate and spring into action.

Why is it important that T cells be tested to be sure they can recognize self MHC molecules? Wouldn't it be a lot simpler just to eliminate this exam?

B cells and T cells must "learn" not to recognize our own "self" antigens as dangerous, for otherwise we would all die of autoimmune disease. In addition, T cells must be "restricted" to recognize self MHC, so that the attention f T cells will be focused on MHC-peptide complexes - and not on unpresented antigen. About this time, some of the cells start to rearrange the gene segments that encode the α and β chains of the TCR. If these rearrangements are successful, a T cell begins to express low levels of the TCR and its associated, acces- sory proteins (the CD3 protein complex). As a result, these formerly nude cells soon are "dressed" with CD4, CD8, and TCR molecules on their surface. Because these T cells express both CD4 and CD8 co-receptor molecules, they are called double positive (DP) cells. During this "reverse striptease," another important change takes place. When the T cell was naked, it was resistant to death by apoptosis, because it expressed little or no Fas antigen (which can trigger death when ligated), and because it expressed high levels of Bcl-2 (a cellular protein that protects against apoptosis). In contrast, a "fully dressed" T cell of the thymic cortex expresses high levels of Fas on its surface and produces very little Bcl-2. Consequently, it is exquisitely sensitive to signals that can trigger death by apoptosis. It is in this highly vulnerable condition that a T cell is tested for tolerance of self and MHC restriction. If it fails either test, it will die. The process of testing T cells for MHC restriction is usually referred to as "positive selection." The "examin- ers" here are epithelial cells in the cortical region of the thymus, and the question a cortical epithelial cell asks of a T cell is: do you have receptors that recognize one of the self MHC molecules which I am expressing on my surface? The correct answer is, "Yes, I do!" for if its TCRs do not recognize any of these self MHC molecules, the T cell dies. When I say "self" MHC, I simply mean those MHC molecules which are expressed by the person (or mouse) who "owns" this thymus. Yes, this does seem like a no- brainer - that my T cells would be tested in my thymus on my MHC molecules - but immunologists like to emphasize this point by saying "self MHC." The MHC molecules on the surface of the cortical epi- thelial cells are actually loaded with peptides, so what a TCR really recognizes is the combination of a self MHC molecule and its associated peptide. These peptides rep- resent a "sampling" of the proteins that are being made by the cortical epithelial cells (displayed by class I MHC molecules) plus a "sampling" of all the proteins which the cortical epithelial cells have picked up from the envi- ronment within the thymus (displayed by class II MHC molecules). Let's pause for a moment between exams to ask an impor- tant question: why do T cells need to be tested to be sure that they can recognize peptides presented by self MHC molecules? After all, most humans complete their life- times without ever seeing "foreign" MHC molecules (e.g., on a transplanted organ), so MHC restriction can't be about discriminating between your MHC molecules and mine. No, MHC restriction has nothing to do with foreign versus self - it's all about focus. As we discussed in Lecture 4, we want the system to be set up so that T cells focus on antigens that are presented by MHC mol- ecules. However, T cell receptors are made by mixing and matching gene segments, so they are incredibly diverse. As a result, it is certain that in the collection of TCRs expressed on T cells, there will be many which recognize unpresented antigens, just as a B cell's receptors do. These T cells must be eliminated. Otherwise the wonderful system of antigen presentation by MHC molecules won't work. So the reason positive selection (MHC restriction) is so important is that it sets up a system in which all mature T cells will have TCRs that recognize antigen presented by MHC molecules.

Cytokines have a limited range. Why is this a good thing?

Cytokines have a very limited range. They can travel only short distances in the body before they are captured by cellular receptors or are degraded. Consequently, when we talk about helper T cells being biased toward secreting a certain cytokine profile, we are talking about something very local. Clearly, you wouldn't want every Th cell in your body to be of the Th1 type, because then you'd have no way to defend against a respiratory infec- tion. Conversely, you wouldn't want to have only Th2 cells, because the IgA or IgE antibodies made in response to the Th2 cytokines would be useless if you get a bacte- rial infection in your big toe. In fact, it is the local nature of cytokine signaling which gives the immune system the flexibility to simultaneously mount defenses against many different invaders that threaten different parts of the body.

What is the advantage of having experienced T cells circulate through selected secondary lymphoid organs?

Experienced T cells also carry passports, but they are "restricted passports," because, during activation, expres- sion of certain adhesion molecules on the T cell surface is increased, whereas expression of others is decreased. This modulation of cellular adhesion molecule expression is not random. There's a plan here. In fact, the cellular adhesion molecules that activated T cells express depend on where these T cells were activated. For example, T cells activated in a Peyer's patch will express high levels of α4β7 (the gut-specific integrin), and low levels of L-selectin (the more general, high endothelial venule adhesion molecule). As a result, T cells activated in Peyer's patches tend to return to Peyer's patches. Thus, when activated T cells recirculate, they usually exit the blood and re-enter the same type of secondary lym- phoid organ in which they originally encountered antigen. This restricted traffic pattern is quite logical. After all, there is no use having experienced helper T cells recirculate to the lymph node behind your knee if your intestines have been invaded. Certainly not. You want those experienced helper T cells to get right back to the tissues that underlie your intestines to be restimulated and to provide help. So by equipping activated T cells with restricted passports, Mother Nature insures that these cells will go back to where they are most likely to re-encounter their cognate antigens - be it in a Peyer's patch, a lymph node, or a tonsil.

What special features of the immune system in the tissues which surround the intestines help avoid an overreaction to commensal bacteria?

Experiments with mice indicate that special macro- phages patrol the tissues which underlie the intestinal wall. Although these warriors phagocytose invading bac- teria, they usually do not give off cytokines which would signal a full-blown attack. IgA is the major antibody class produced by B cells in these tissues, and these antibodies can efficiently bind to invading bacteria and usher them back out of the tissues into the intestine. However, the Fc portion of an IgA antibody cannot bind with high affinity to receptors on immune system cells to trigger an inflam- matory response - as, for example, IgG antibodies would do. So, under normal conditions, macrophages and IgA antibodies act with restraint to deal with commensal bacteria which occasionally enter the tissues from the intestines.

How does a helper T cell "call the plays" for killer T cells?

If you have a puncture wound that results in a bacterial infection or if you are attacked by a virus that replicates in the tissues, resident dendritic cells will be alerted through their pattern recognition receptors and by receiv- ing battle cytokines produced by macrophages and other cells in the inflamed tissues. These signals activate the dendritic cell and imprint it with the special characteris- tics of a reporter cell which has observed a bacterial or viral infection in the tissues. The details of exactly how this is accomplished aren't clear yet, but the result is that when this DC leaves such a battle site and travels through the lymph to a nearby lymph node, it will produce the cytokine IL-12. And when the IL-12-producing DC presents the battle antigens it has acquired to a virgin helper T cell, that Th cell will be instructed to become a helper T cell which produces the "classical" Th1 cytokines: TNF, IFN-γ, and IL-2. Why these particular cytokines? Let's see what these cytokines do. The TNF secreted by Th1 helper T cells helps activate macrophages and natural killer cells. However, macrophages only stay activated for a limited time. They are lazy fellows which like to go back to "resting and garbage collecting." Fortunately, the IFN-γ produced by Th1 cells acts as a "prod" that keeps macro- phages fired up and engaged in the battle. IFN-γ also influences B cells during class switching to produce human IgG3 antibodies. These antibodies are especially good at opsonizing viruses and bacteria and at fixing complement NK cells can kill three or four target cells in about 16 hours, but then they "tire out." The IL-2 produced by Th1 cells can "recharge" NK cells, enabling them to kill some more. In addition, IL-2 is a growth factor which stimu- lates the proliferation of CTLs, NK cells, and Th1 cells themselves, so that more of these important weapons will be available to deal with the attack. Altogether, the Th1 cytokines are the perfect package to help defend against a viral or bacterial attack in the tissues. The Th1 cytokines instruct the innate and adap- tive systems to produce cells and antibodies that are especially effective against these invaders, and keep the warriors of the immune system fired up until the invaders have been defeated..

Why is it important that B cells also be taught tolerance of self?

Immunologists once thought that it might not be neces- sary to delete B cells with receptors that recognize self antigens. The idea was that the T cells which were needed to help activate potentially self-reactive B cells would already have been killed or anergized, so that B cell tolerance would be "covered" by T cell tolerance. However, it now is clear that mechanisms also exist for tolerizing those B cells which have the potential to be self-reactive. Most B cells are tolerized where they are born - in the bone marrow. This is the rough equivalent of thymic tolerance induction for T cells. After B cells mix and match gene segments to construct the genes for their receptors, they are "tested" to see if these receptors rec- ognize self antigens that are present in the bone marrow. If its receptors do recognize a self antigen, a B cell is given another chance to rearrange its light chain gene and come up with new receptors that don't bind to a self antigen. This process is called "receptor editing." Although the details of how receptor editing works are not yet under- stood, in mice at least 25% of all B cells take advantage of this "second chance." Nevertheless, even with this oppor- tunity to try again to produce acceptable receptors, only about 10% of all B cells pass the tolerance test. The rest die in the bone marrow. After testing, B cells with receptors that do not bind to self antigens which are abundant in the bone marrow are released to circulate with the blood and lymph. Of course, induction of B cell tolerance in the bone marrow has the same problems as T cell tolerance induction in the thymus: B cells which have receptors that recognize self antigens that are rare in the marrow can slip through the cracks.

How can B cells be activated without T cell help?

In response to certain antigens, virgin B cells can also be activated with little or no T cell help, and this mode of activation is termed T cell-independent. What these antigens have in common is that they have repeated epitopes which can crosslink a ton of B cell receptors. A good example of such an antigen is a carbohydrate of the type found on the surface of many bacterial cells. A car- bohydrate molecule is made up of many repeating units, much like beads on a string. If each "bead" is recognized by the BCR as its epitope, the string of beads can bring together many, many BCRs. The crosslinking of such a large number of BCRs can partially substitute for co-stimulation by CD40L, and can cause a B cell to proliferate. But to be fully activated and produce antibodies, a naive B cell must receive a second signal. For T cell-independent activation, this second key is an unambiguous "danger signal" - a clear indication that an attack is on. One such signal is the recognition by the B cell of molecular patterns which are characteristic of certain bacteria and parasites. We will talk more about "pattern recognition receptors" in the next lecture. What is important for this discussion is that if a B cell has BCRs that can recognize a molecule with repeated epitopes like, for example, your own DNA, it may proliferate, but for- tunately, no anti-DNA antibodies will be produced. The reason is that your immune system is not engaged in a battle with your own DNA, so there will be no danger signals to provide the necessary co-stimulation. On the other hand, if the innate immune system is battling a bacterial infection, and a B cell's receptors recognize a carbohydrate antigen with repeated epitopes on the surface of the bacterial invader, that B cell will produce antibodies - because danger signals from the battle field can supply the second key needed for complete B cell activation. Of course, as is true of T cell-dependent activa- tion, T-cell independent activation is antigen specific: only those B cells whose receptors recognize the repeated epitope will be activated.

Why are mechanisms needed that can tolerize T cells once they leave the thymus?

In summary, induction of T cell tolerance is multilayered. No single mechanism of tolerance induction is 100% efficient, but because there are multiple mecha- nisms, autoimmune diseases are relatively rare. T cells with receptors that recognize antigens which are abun- dant in the secondary lymphoid organs usually are effi- ciently deleted in the thymus. Self antigens that are rare enough in the thymus to allow self-reactive T cells to escape deletion usually are also too rare to activate virgin T cells in the secondary lymphoid organs. Thus, because of their restricted traffic pattern, virgin T cells normally remain functionally ignorant of self antigens that are rare in the thymus. In addition, natural regula- tory T cells in the secondary lymphoid organs are believed to provide additional protection, probably by interfering with the activation of potentially self- reactive T cells. In those cases where virgin T cells do venture outside the blood-lymph-secondary lymphoid organ system, they generally encounter self antigens in a context that leads to anergy or death, not activation. Finally, those rare T cells that are activated by recognizing self antigens in the tissues usually die from chronic re-stimulation.

Why are cellular adhesion molecules important during T cell activation? Don't these "sticky" molecules just slow the process down?

In the lymph nodes, helper T cells quickly scan dendritic cells to see if their cognate antigen is being displayed. Indeed, a single dendritic cell typically hosts about 1000 such "visits" each hour. If a T cell does nd a dendritic cell displaying its cognate antigen, the T cell "lingers," because complete activation of a naive helper T cell actu- ally takes between four and ten hours. During this time, a number of important events take place. First, adhesion molecules on the surface of the dendritic cell bind to their adhesion partners on the T cell, helping keep the two cells together. Next, the CD4 co-receptor molecules on the surface of the T cell clip onto the class II MHC molecules on the dendritic cell and strengthen the interaction between the two cells. In addition, the engagement of its TCRs upregulates the expression of adhesion molecules on the Th cell surface, strengthening the "glue" that holds the APC and the T cell together. This is important, because the binding between a TCR and an MHC-peptide complex is actually rather weak to allow for rapid scan- ning. Consequently, the Velcro-like adhesion molecules are extremely important for T cell activation. In fact, the ability to express the adhesion molecules required to keep APCs and T cells together long enough for a threshold level of TCR engagement to be reached is one feature that sets APCs apart from "ordinary" cells. The clustering of TCRs and adhesion molecules at the point of contact between an APC and a T cell results in the formation of what immu nologists call an "immunological synapse." Engagement of a helper T cell's receptors also upregu- lates expression of CD40L proteins on its surface, and when these proteins plug into the CD40 proteins on the surface of a dendritic cell, several remarkable things happen. Although mature dendritic cells express MHC and co-stimulatory molecules (e.g., B7) when they rst enter lymph nodes, the expression level of these proteins increases when CD40 proteins on the APC are engaged by the CD40L proteins on a Th cell. In addition, engage- ment of a dendritic cell's CD40 proteins prolongs the life of the dendritic cell. This extension of a "useful" dendritic cell's life span makes perfect sense. It insures that the particular dendritic cells which are presenting a T cell's cognate antigen will stick around long enough to activate a lot of these T cells. So the interaction between the den- dritic cell and a naive helper T cell is not just one way. These cells actually perform an activation "dance" in which they stimulate each other. The end result of this cooperation is that the dendritic cell becomes a more potent antigen presenting cell, and the Th cell is activated to express the high levels of CD40L required for helping activate B cells. After activation is complete, the helper T cell and the antigen presenting cell part. The APC then goes on to activate other T cells, while the recently activated Th cells proliferate to build up their numbers. During an infec- tion, a single activated T cell can give rise to about 10 000 daughter cells during the rst week or so of proliferation. This proliferation is driven by growth factors such as IL-2. Naive T cells can make some IL-2, but they don't have IL-2 receptors on their surface - so they can't respond to this cytokine. In contrast, activated Th cells produce large amounts of IL-2, and they also express receptors for this cytokine on their surface. As a result, newly activated helper T cells stimulate their own proliferation. This cou- pling of activation to the upregulation of growth factor receptors is the essence of clonal selection: those Th cells which are selected for activation (because their TCRs recognize an invader) upregulate their growth factor receptors, and proliferate to form a clone. So the sequence of events during the activation of a helper T cell is this. Adhesion molecules mediate weak binding between the Th and the APC while TCRs engage their cognate antigen presented by the APC. Receptor engagement strengthens the adhesion between the two cells, and upregulates CD40L expression on the Th cell. CD40L then binds to CD40 on the APC and stimulates expression of MHC and co-stimulatory molecules on the APC surface. The co-stimulation provided by the APC mpli es the "TCR engaged" signal, making activation of the Th cell more ef cient. When activation is complete, the cells disengage, and the Th cell proliferates, driven by growth factors which bind to receptors that appear on the Th cell surface as a result of activation. This proliferation produces a clone of helper T cells which can recognize the invader advertised by the antigen presenting cell.

What happens when dendritic cells and helper T cells "dance"?

In the lymph nodes, helper T cells quickly scan dendritic cells to see if their cognate antigen is being displayed. Indeed, a single dendritic cell typically hosts about 1000 such "visits" each hour. If a T cell does nd a dendritic cell displaying its cognate antigen, the T cell "lingers," because complete activation of a naive helper T cell actu- ally takes between four and ten hours. During this time, a number of important events take place. First, adhesion molecules on the surface of the dendritic cell bind to their adhesion partners on the T cell, helping keep the two cells together. Next, the CD4 co-receptor molecules on the surface of the T cell clip onto the class II MHC molecules on the dendritic cell and strengthen the interaction between the two cells. In addition, the engagement of its TCRs upregulates the expression of adhesion molecules on the Th cell surface, strengthening the "glue" that holds the APC and the T cell together. This is important, because the binding between a TCR and an MHC-peptide complex is actually rather weak to allow for rapid scan- ning. Consequently, the Velcro-like adhesion molecules are extremely important for T cell activation. In fact, the ability to express the adhesion molecules required to keep APCs and T cells together long enough for a threshold level of TCR engagement to be reached is one feature that sets APCs apart from "ordinary" cells. The clustering of TCRs and adhesion molecules at the point of contact between an APC and a T cell results in the formation of what immunologists call an "immunological synapse." Engagement of a helper T cell's receptors also upregu- lates expression of CD40L proteins on its surface, and when these proteins plug into the CD40 proteins on the surface of a dendritic cell, several remarkable things happen. Although mature dendritic cells express MHC and co-stimulatory molecules (e.g., B7) when they rst enter lymph nodes, the expression level of these proteins increases when CD40 proteins on the APC are engaged by the CD40L proteins on a Th cell. In addition, engage- ment of a dendritic cell's CD40 proteins prolongs the life of the dendritic cell. This extension of a "useful" dendritic cell's life span makes perfect sense. It insures that the particular dendritic cells which are presenting a T cell's cognate antigen will stick around long enough to activate a lot of these T cells. So the interaction between the den- dritic cell and a naive helper T cell is not just one way. These cells actually perform an activation "dance" in which they stimulate each other. The end result of this cooperation is that the dendritic cell becomes a more potent antigen presenting cell, and the Th cell is activated to express the high levels of CD40L required for helping activate B cells.

Why do some people have allergies, whereas others do not?

It is clear that IgE antibodies are the bad guys in allergic reactions, but what determines whether a person will make IgE or IgG antibodies in response to an allergen? You remember from Lecture 6 that helper T cells can be "instructed" by the environment in which they are stimu- lated to secrete various cytokine subsets (e.g., Th1, Th2, or Th17). And the cytokines given off by these T cells can then influence B cells undergoing class switching to produce IgA, IgG, or IgE antibodies. For example, a ger- minal center that is populated with Th1 cells usually will produce B cells that make IgG antibodies, because Th1 cells secrete IFN-γ, which drives the IgG class switch. In contrast, B cells tend to change to IgE production if they class switch in germinal centers that contain Th2 cells which secrete IL-4 and IL-5. Consequently, the decision to produce either IgG or IgE antibodies in response to an allergen will depend heavily on the type of helper T cells present in the secondary lymphoid organ which happens to intercept the allergen. Indeed, helper T cells from allergic individuals show a much stronger bias toward the Th2 type than do Th cells from non-atopic people. So atopic individuals produce IgE antibodies because their allergen-specific helper T cells tend to be of the Th2 type. But how do they get that way? The answer to this question is not known for certain, but many immunolo- gists believe that a bias toward Th2-type helper T cells can be established early in childhood, and in some cases, even before birth

Explain how B cell memory protects us against invaders in both the near and distant future.

It is clear that antibodies can confer life-long immunity to infection. For example, in 1781, Swedish traders brought the measles virus to the isolated Faeroe Islands. In 1846, when another ship carrying sailors infected with measles visited the islands, people who were older than 64 years did not contract the disease - because they still had anti- bodies against the measles virus. Even the longest-lived antibodies (the IgG class) have a half-life of less than a month, so antibodies would have to be made continu- ously over a period of many years to provide this long- lasting protection. When B cells are activated during the initial response to an invader, three kinds of B cells are generated. First, short-lived plasma B cells are produced in the lymphoid follicles of secondary lymphoid organs. These cells travel to the bone marrow or spleen and produce huge quantities of antibodies that are specific for the attacker. Although they only live for a few days, short-lived plasma B cells produce antibodies which are extremely important in protecting us against an enemy that the immune system has never encountered before. In addition to short-lived plasma B cells, two types of memory B cells are produced in germinal centers during an invasion. Importantly, the generation of both types of memory cells requires T cell help. The first kind of memory B cell is the long-lived plasma cell. In contrast to short-lived plasma cells, which are generated rapidly after infection and which die after a few heroic days, long-lived plasma cells take up residence in the bone marrow, and continuously produce more modest amounts of antibodies. It is the long-lived plasma cells which manufacture the IgG antibodies that can provide life-long immunity to subsequent infections. So together, short-lived and long-lived plasma B cells provide both immediate and long-term antibody protection against attacks. The second type of memory B cell is the central memory B cell. These cells reside mainly in the secondary lymphoid organs, and their job is not to produce antibod- ies. Central memory B cells function as memory "stem cells" which slowly proliferate to maintain a pool of central memory B cells, and to replace those long-lived plasma cells which have died of old age. In addition, if another attack occurs, central memory cells can quickly produce more, short-lived plasma B cells. This strategy, which involves three types of B cells, makes good sense. When an invader first attacks, anti- bodies need to be made quickly to tag invaders for destruction. That's what short-lived plasma B cells do. If, at a later time, the invader attacks again, it is important to already have invader-specific antibodies made and deployed throughout the body as an immediate defense. That's the job of long-lived plasma B cells. And between attacks, readiness is maintained by central memory B cells. These cells replenish supplies of long-lived plasma cells, and also stand ready to produce a burst of short- lived plasma B cells - cells that can rapidly manufacture large quantities of invader-specific antibodies to over- whelm the enemy.

Why did mother nature make macrophages long lived and neutrophils short lived?

It would be too dangerous. Neutrophils come out of blood vessels ready to kill, and in the course of this killing, there is always damage to normal tissues. So to limit that damage, neutrophils are programmed to be short-lived. If the battle requires addi- tional neutrophils, more can be recruited from the blood - there are plenty of them there. In contrast, macrophage ct as sentinels that watch fozr invaders and signal the attack. Consequently, it makes sense that macrophages should live a long time out in the tissues.

Why is it advantageous to have Th0 helper T cells, which don't commit to a particular cytokine pro le until they reach the battle scene?

Likewise, Th0 cells can become Th2 or Th17 cells when they reach a battle site that is rich in IL-4 or IL-6 and TGFβ, respectively. So previously uncommitted Th0 cells can be "converted" by the cytokine environment at the scene of the battle to become Th1, Th2, or Th17 cells. Once helper T cells commit to producing a particular cytokine profile, they begin to secrete growth factors which are specific for that particular type of Th cell - be it Th1, Th2, or Th17. This sets up a positive feedback loop in which the "selected" Th cell proliferates and produces even more of its own growth factor - triggering further proliferation of this type of helper T cell. In addition to positive feedback, there is also negative feedback at work. For example, IFN-γ made by Th1 cells actually decreases the rate of proliferation of Th2 cells, so that fewer Th2 cells will be produced. And one of the Th2 cytokines, IL-10, acts to decrease the rate of proliferation of Th1 cells. The result of all this positive and negative feedback is a large number of helper T cells which are strongly biased toward the production of a certain subset of cytokines.

How does a helper T cell "call the plays" for B cells?

Now suppose that you have been infected by a parasite (e.g., hookworms) or you have eaten some food that is contaminated with pathogenic bacteria. In the tissues that line your intestines, a battle will be raging. Dendritic cells. Why IL-4, IL-5, and IL-13, you ask? IL-4 is a growth factor that stimulates the proliferation of helper T cells which have committed to secrete the Th2 profile of cytokines. So, like Th1 cells, Th2 cells produce their own growth factor. IL-4 also is a growth factor for B cells, and this cytokine can influence B cells to class switch to produce IgE antibodies - powerful weapons against para- sites such as hookworms. IL-5 is a cytokine which encour- ages B cells to produce IgA antibodies, antibodies that are especially useful against bacteria which invade via the digestive tract. And IL-13 stimulates the production of mucus in the intestines, which helps prevent more intes- tinal parasites or pathogenic bacteria from breaching the intestinal wall and entering the tissues. So the Th2 cytokine profile is just the ticket if you need to defend against parasites or pathogenic bacteria that have invaded via the digestive tract. Now I want to call your attention to two interesting points. First, in the figure above, you will notice that IL-4, which causes a naive Th cell to commit to becoming a Th2 cell, does not come from the dendritic cell. In fact, the source of IL-4 which initiates the Th2 commitment is still a mystery. Of course, once the helper T cell commits to the Th2 cytokine profile, there will be plenty of IL-4 around - because this is one of the cytokines Th2 cells secrete. However, the initial source of IL-4 required for Th2 commitment has not yet been identified. The second interesting point is that Th2 cells produce cytokines (IL-4 and IL-5) which can influence B cells to make either IgE antibodies (to defend against parasites) or IgA antibodies (to defend against pathogenic bacteria). However, it would be unusual for a person to be infected with parasites and pathogenic bacteria simultaneously. Consequently, you wouldn't want Th2 cells to influence B cells to make both IgE and IgA antibodies. That would be wasteful (and probably dangerous). So how does a Th2 cell decide whether to instruct B cells to make IgE or IgA antibodies? Immunologist don't have a complete answer to this question, but a major factor in this decision is the location from which the dendritic cells are dispatched. I men- tioned earlier that DCs have a "regional identity." For example, there are dendritic cells which reside in "Peyer's patches" - special areas of the intestine that are important in defending against pathogenic bacteria which have been ingested. These particular dendritic cells are "imprinted" by the environment of the Peyer's patch to deliver signals (e.g., via co-stimulatory molecules) to Th cells which cause them to assist with IgA antibody pro- duction. On the other hand, dendritic cells which are stationed beneath areas of the intestine that are suscepti- ble to invasion by parasites are imprinted so that they convey signals to Th cells which bias them to help B cells produce IgE antibodies. That's the basic idea, but the details have yet to be worked out.

What are the functions of the various secondary lymphoid organs?

Now that you are familiar with lymphoid follicles and high endothelial venules, we are ready to take a tour of some of the secondary lymphoid organs. On our tour today, we will visit a lymph node, a Peyer's patch (an example of the MALT), and the spleen. As we explore these organs, you will want to pay special attention to the "plumbing." How an organ is connected to the blood and lymphatic systems gives important clues about how it functions.With this diagram in mind, can you see how lym- phocytes (B and T cells) enter a lymph node? That's right, they can enter from the blood by pushing their way between the cells of the high endothelial venules. There is also another way lymphocytes can enter the lymph node: with the lymph. After all, lymph nodes are like "dating bars," positioned along the route the lymph takes on its way to be reunited with the blood - and B and T cells actively engage in "bar hopping," being carried from node to node by the lymph. Although lymphocytes have two ways to gain entry to a lymph node, they only exit via the lymph - those high endothelial venules won't let them back into the blood. From this discussion, it should be clear that a lymph node is a highly organized place with specific areas for antigen presenting cells, T lymphocytes, and B lym- phocytes. lymph nodes act as "lymph filters" which intercept antigen that arrives from infected tissues either alone or as dendritic cell cargo. These nodes provide a concentrated and organized environment of antigen, APCs, T cells, and B cells in which naive B and T cells can be activated, and experienced B and T cells can be restimulated. In a lymph node, naive B and T cells can mature into effector cells that produce antibod- ies (B cells), provide cytokine help (Th cells), and kill infected cells (CTLs). In short, a lymph node can do it all.o, whereas lymph nodes sample antigens from the lymph, Peyer's patches sample antigens from the intestine - and they do it by transporting these antigens through M cells. The final secondary lymphoid organ on our tour is the spleen. This organ is located between an artery and a vein, and it functions as a blood filter. Each time your heart pumps, about 5% of its output goes through your spleen. Consequently, it only takes about half an hour for your spleen to screen all the blood in your body for pathogens. As with Peyer's patches, there are no lymphatics that bring lymph into the spleen. However, in contrast to lymph nodes and Peyer's patches, where entry of B and T cells from the blood occurs only via high endothelial venules, the spleen is like an "open-house party" in which everything in the blood is invited to enter

What is the advantage of having virgin T cells circulate through all the secondary lymphoid organs?

Now, of course, you don't want T cells to just go round and round. You also want them to exit the blood at sites of infection so that CTLs can kill pathogen-infected cells and Th cells can provide cytokines that amplify the immune response and recruit even more warriors from the blood. To make this happen, experienced T cells also carry "combat passports" (adhesion mole- cules) which direct them to exit the blood at places where invaders have started an infection. These T cells employ the same "roll, sniff, stop, exit" technique that neutrophils use to leave the blood and enter inflamed tissues. For example, T cells that gained their experience in the mucosa express an integrin molecule, αEβ7, which has as its adhesion partner an addressin molecule that is expressed on inflamed mucosal blood vessels. As a result, T cells that have the right "training" to deal with mucosal invaders will seek out mucosal tissues which have been infected. In these tissues, chemokines given off by the soldiers at the front help direct T cells to the battle by binding to the chemokine receptors that appeared on the surface of the T cells during activation. In summary, naive T cells have passports that allow them to visit all the secondary lymphoid organs, but not sites of inflammation. This traffic pattern brings the entire collection of virgin T cells into contact (in the secondary lymphoid organs) with invaders that may have entered the body at any point, and greatly increases the probability that virgin T cells will be activated. The reason that virgin T cells don't carry passports to battle sites is that they couldn't do anything there anyway - they must be activated first. In contrast to virgin T cells, experienced T cells have restricted passports that encourage them to return to the same type of secondary lymphoid organ as the one in which they gained their experience. By recirculating preferentially to the kind of organ in which they first encountered antigen, T cells are more likely to be res- timulated or to find CTLs and B cells that have encoun- tered the same invader and need their help.

Discuss why the adaptive immune system may provide some surveillance against blood-cell cancers, but not against spontaneous, non-blood-cell cancers

One of the problems that CTLs have in providing sur- veillance against tumors that arise in tissues is that these tumors simply are not on the normal traffic pattern of virgin T cells - and it's hard to imagine how a CTL could be activated by a cancer it doesn't see. In contrast, most blood-cell cancers are found in the blood, lymph, and secondary lymphoid organs, and this is ideal for viewing by CTLs, which pass through these areas all the time. Thus, in the case of blood-cell cancers, the traffic pat- terns of cancer cells and virgin T cells actually intersect. Moreover, in contrast to tumors in tissues, which usually are unable to supply the co-stimulation required for activation of virgin T cells, some cancerous blood cells actually express high levels of B7, and therefore can provide the necessary co-stimulation. These properties of blood-cell cancers suggest that CTLs may provide surveillance against some of them. Unfortunately, this surveillance must be incomplete, because people with otherwise healthy immune systems still get leukemias and lymphomas. Certain viral infections can predispose a person to par- ticular types of cancer. Because Mother Nature probably designed killer T cells to defend against viral infections, it is easy to imagine that CTLs might provide surveillance against virus-associated tumors. Unfortunately, this sur- veillance is probably quite limited. Here's why. Most viruses cause "acute" infections in which all the virus-infected cells are rather quickly destroyed by the immune system. And because a dead cell isn't going to make a tumor, viruses which only cause acute infections do not play a role in cancer. This explains why most viral infections are not associated with human cancer. There are viruses, however, which can evade the immune system, and establish long-term (sometimes life- long) infections. Importantly, all viruses which have been shown to play a role in causing cancer are able to establish long-lasting infections during which they "hide" from the immune system. Because CTLs cannot destroy virus-infected cells while they are hiding, and because these hidden cells are the very ones which even- tually become cancer cells, it can be argued that CTLs do not provide effective surveillance against virus-associated cancer. Of course, you might propose that without killer T cells, more cells would be infected during a virus attack, thereby increasing the number of cells in which the virus might be able to establish a long-term, hidden infection. And this probably is true. In fact, this may help explain why humans with deficient immune systems have higher than normal rates of virus-associated tumors. However, the bottom line is that CTLs cannot provide significant surveillance against virus-infected cells once they have become cancerous, because these cancers only result from long-term viral infections - infections which CTLs cannot detect or cannot deal with effectively.In summary, in some circumstances, macrophages and NK cells may provide surveillance against certain types of cancer cells, and the immune system probably is involved in defending against some virus-associated and blood-cell cancers. In addition, the immune system may reduce the frequency of metastases or may help slow the metastatic process once a primary tumor has formed. However, I believe it is unlikely that the immune system provides significant surveillance against most solid tumors in humans during the initial stages of their development. This is my view, but not everyone agrees with me on this point.

What properties of memory B and T cells make them better able to defend against a subsequent infection?

One of the puzzles about adaptive memory is how it can be maintained for a lifetime, even if an invader has been eliminated from the body and never returns. Antibodies have life spans measured in days or weeks, long-lived plasma cells only survive for a few months, and memory effector T cells also have relatively short lives. Consequently, memory cells and antibodies must be continuously replenished to provide lasting memory. It was first assumed that "remnants" of the initial infec- tion (e.g., pieces of a virus or a bacterium) were retained in the secondary lymphoid organs, and that these anti- gens might re-stimulate central memory cells and cause them to proliferate slowly to replace long-lived plasma cells or effector memory T cells which died of old age. However, recent experiments have shown that although this type of re-stimulation does take place, memory actu- ally can be sustained without any traces of the original infection being present. It is thought that certain cytokines trigger memory T cells to proliferate slowly, independent of their TCR specificity, and that memory B cells prolifer- ate when their pattern recognition receptors or their BCRs are ligated weakly by self antigens. Nevertheless, more research is needed to fully understand how B and T cell memory is maintained once an invader has been vanquished. The adaptive immune system remembers so well and reacts so powerfully during a subsequent infection that we usually don't even know we have been reinfected. There are a number of reasons why memory cells are better able to deal with a second attack than were the inexperienced B and T cells which responded to the original invasion. First, there are many more of them. Indeed, when we are attacked for the first time, there usually is only about one B or T cell in a million which can recognize that invader. In contrast, by the time the battle is over, the pool of pathogen-specific cells will have expanded so that roughly one in a thousand of all the B or T cells will recognize the attacker. Consequently, the adaptive immune system's response to a subsequent attack is much more robust than the initial response - because there are so many more invader-specific cells "on duty." In addition to being more numerous than their inexpe- rienced predecessors, memory B and T cells are easier to activate. The latest thinking is that during re-activation of memory cells, recognition of cognate antigen is required, but at least in some cases, co-stimulation is not essential. Now why would it be advantageous to have a system in which it is difficult to activate a B or T cell the first time, but relatively easier to reactivate it? Clearly, we want activation of virgin cells to be tightly controlled, because we only want to activate the adaptive immune system when there is a real threat. Consequently, a fail- safe activation requirement for virgin B and T cells is important. On the other hand, once these cells have been through the stringent, two-key selection for primary acti- vation, we want them to respond quickly to a subsequent attack by the same invader. So making it easier for them to be re-activated makes perfect sense. There is a third reason why memory B cells are better defenders than are naive B cells: memory B cells are "upgraded" versions of the original, virgin B cells. These upgrades are of two types. First, during the course of an attack, B cells gradually switch the class of anti- body they make from the "compromise" antibody class, IgM, to one of the other classes (IgG, IgA, or IgE) which specializes in dealing with that particular kind of invader. This class switch is imprinted on the memory of the B cells that remain after an attack. As a result, memory B cells are able to produce the antibody class which is just right to protect against the invader they remember. Also, during an attack, B cells use somatic hypermuta- tion to fine-tune both their receptors and the antibodies they manufacture. Hypermutation results in upgraded B cell receptors that can detect small amounts of foreign antigen early in an attack, allowing central memory B cells to be activated quickly during a subsequent infection. Moreover, because of hypermutation, long- lived plasma cells make upgraded antibodies that can bind more tightly to the invader.

what is the difference between a co-receptor and co-activation? Why is each important?

Over 95% of the T cells in circulation have αβ T cell recep- tors, and express either CD4 or CD8 "co-receptor" mol- ecules (more about co-receptors in a bit). The αβ receptors of these "traditional" T cells recognize a complex com- posed of a peptide and an MHC molecule on the surface of a cell, and a given T cell will have receptors that rec- ognize peptides associated either with class I MHC mol- ecules or with class II MHC molecules. Importantly, the αβ receptors of a traditional T cell recognize both the peptide and the MHC molecule. n addition to the T cell receptor, there are two more molecules which are involved in antigen recognition by T cells - the CD4 and CD8 co-receptors. Now, doesn't it seem that Mother Nature got carried away when she added on these CD4 and CD8 co-receptors? I mean, there are already two proteins, α and β, to use for antigen rec- ognition, and four more, γ, δ, ε, and ζ, to use for signaling. Wouldn't you think that would do it? Apparently not, so there must be essential features of the system that require CD4 and CD8 co-receptors. Let's see what these might be. Killer T cells and helper T cells perform two very dif- ferent functions, and they "look at" two different mole- cules, class I or class II MHC, respectively, to get their cues. But how do CTLs know to focus on peptides pre- sented by class I molecules - and how do Th cells know to scan APCs for peptides presented by class II? After all, it wouldn't be so great if a CTL got confused, recognized a class II-peptide complex on an APC, and killed the antigen presenting cell! So here's where CD4 and CD8 come in. CTLs generally express CD8 and Th cells usually express CD4. These co-receptor molecules are designed to clip onto either class I MHC (CD8) or class II MHC molecules (CD4). These "clips" strengthen the adhesion between the T cell and the APC, so CD4 and CD8 co-receptors function to focus the attention of Th cells and CTLs on the proper MHC molecule. But there is more to the story, because it turns out that CD4 and CD8 are signaling molecules just like the CD3 complex of proteins. Both CD4 and CD8 have tails that extend through the cell wall and into the interior (cytoplasm) of the cell, and both of these tails have the right characteristics to signal. In addition, because CD4 is a single protein and CD8 is composed of two different proteins, the signals that these co-receptors send are likely to be quite different - perhaps as different as "help" and "kill." In contrast to CD3 molecules, which are glued rather tightly to the αβ T cell receptor on the cell surface, the CD4 and CD8 co-receptors usually are only loosely associated with the TCR/CD3 proteins. The idea is that after a TCR has engaged its cognate antigen presented by an MHC molecule, the CD4 or CD8 co- receptors then clip on and stabilize the TCR-MHC- peptide interaction, thereby strengthening the signal sent by the TCR. In naive T cells, the "connection" between the T cell's receptors and the cell's nucleus is not very good. It's as if the T cell had an electrical system in which a large resistor were placed between the sensor (the TCR) and the piece of equipment it is designed to regulate (gene expression in the nucleus). Because of this "resistor," a lot of the signal from the TCR is lost as it travels to the nucleus. The result is that a prohibitively large number of TCRs would have to engage their cognate antigen before the signal that reaches the nucleus would be strong enough to have any effect. If, however, while the TCRs are engaged, the T cell also receives co-stimulation, the signal from the TCRs is ampli ed many times, so that fewer (probably about 100-fold fewer) TCRs must be engaged to activate a naive T cell. Although a number of different molecules have been identi ed which can co-stimulate T cells, cer- tainly the best studied examples are the B7 proteins (B7-1 and B7-2) which are expressed on the surface of antigen presenting cells. B7 molecules provide co-stimulation to T cells by plugging into receptor molecules called CD28 on the T cell's surface. So in addition to having their T cell receptors ligated by MHC-peptide, naive T cells must also receive co- stimulatory signals before they can be activated. Co- stimulation can be thought of as an "ampli er" that strengthens the "I'm engaged" signal sent by a T cell's receptors, thereby lowering the threshold number of TCRs which must be crosslinked by MHC-peptide complexes. Interestingly, once a naive T cell has been activated, the connection between the TCRs and the nucleus strengthens. It is as if an experienced T cell has been "rewired" so that the resistor present in a naive T cell is bypassed. As a result of this rewiring, in an expe- rienced T cell, ampli cation of the TCR signal is not as important as it is in virgin T cells. Consequently, experienced T cells have a reduced requirement for co-stimulation. Recent experiments have suggested a mechanism by which co-stimulation might amplify the TCR signal. Here's how this is thought to work. Although it is easy to visualize the surface of a cell as a rigid covering, the fact is that the plasma membrane that cloaks a human cell is more like a viscous uid than a rigid shell. Indeed, proteins that are on the cell surface " oat" around fairly freely in this oily gunk. Importantly, the composition of the cell membrane is not homogene- ous, and certain proteins and certain types of lipid mol- ecules form aggregates called "rafts." When immunologists examined these cholesterol-rich rafts, they found that they contain a large number of the signaling molecules that are used to carry the "TCR engaged" signal from the cell surface to the nucleus. Immunologists also discov- ered that before a naive T cell is activated, most of its T cell receptors are not associated with these rafts. However, once a T cell's receptors engage their cognate antigen, the TCRs and the rafts come together. This brings the TCRs into close contact with the downstream signaling mole- cules, and that completes the circuit to the nucleus. It turns out that before naive T cells are activated, they don't have many of these lipid rafts on their surface. Most of them are stored inside the cell as parts of other mem- branous structures. And it is this dearth of rafts on the cell surface that is one of the reasons why the connection between TCRs and the nucleus in a virgin T cell is not a good one - there just aren't enough wires (downstream signaling molecules) available to ef ciently carry the signal. However, if a virgin T cell's receptors engage their cognate antigen and appropriate co-stimulation is sup- plied by the antigen presenting cell, the lipid rafts that are stored inside the cell are rushed to the surface. Now the signal from the TCR can be carried by the additional downstream signaling molecules associated with these rafts, and a strong signal can be sent to the nucleus. According to this model, the key to signal ampli cation by co-stimulation is that co-stimulation recruits lipid rafts to the surface of the T cell. Indeed, experienced T cells have many more rafts on their surfaces than do naive cells. This would explain why reactivation of experienced T cells does not require strong co-stimulation - because the rafts in experienced T cells are already on the surface, just waiting to carry the signl

Describe the events that lead to the degranulation of mast cells during an allergic reaction.

Roughly a quarter of the US population suffers from aller- gies to common environmental antigens (allergens) that either are inhaled or ingested. Hay fever and asthma are the two most common allergic diseases of the respiratory tract.The immune systems of non-allergic people respond weakly to these allergens, and produce mainly antibodies of the IgG class. In striking contrast, allergic individuals (called "atopic" individuals) produce large quantities of IgE antibodies. Indeed, the concentration of IgE antibod- ies in the blood of those with allergies can be 1000- to 10 000-fold higher than in the blood of non-atopic people! It is the overproduction of IgE antibodies in response to otherwise innocuous environmental antigens that causes allergies. In Lecture 3, we discussed the interaction of IgE anti- bodies with white blood cells called mast cells. Because mast cell degranulation is a central event in many allergic reactions, let's take a moment to review this concept. When atopic individuals first are exposed to an allergen (e.g., pollen) they produce large amounts of IgE antibod- ies which recognize that allergen. Mast cells have recep- tors on their surface that can bind to the Fc region of IgE antibodies, so that after the initial exposure, mast cells will have large numbers of these allergen-specific IgE molecules attached to their surface. Allergens are small proteins with a repeating structure to which many IgE antibodies can bind close together. So on a second or subsequent exposure, an allergen can crosslink the IgE molecules on the mast cell surface, dragging the mast cell's Fc receptors together. This clustering of Fc receptors tells mast cells to "degranulate": to release their granules, which normally are stored safely inside the mast cells, into the tissues in which they reside. Mast cell granules contain histamine and other powerful chemicals and enzymes that can cause the symptoms with which atopic individuals are intimately familiar. Interestingly, although IgE antibodies only live for about a day in the blood, once they are attached to mast cells, they have a half-life of several weeks. This means that mast cells can stay "armed" and ready to degranulate for an extended period after exposure to an allergen. Allergic reactions generally have two phases: immedi- ate and delayed. The immediate reaction to an allergen is the work of mast cells, which are stationed out in the tissues, and basophils, another granule-containing white blood cell, which can be recruited from the blood by signals given off by mast cells responding to an allergen. Like mast cells, basophils have receptors for IgE antibod- ies, and crosslinking of these receptors can lead to basophil degranulation.

Describe what happens to a patient's immune system during the course of an HIV-1 infection

Serious disease may result when our immune system does not operate at full strength. Some of these immuno- deficiencies are caused by genetic defects that disable parts of the immune network. Others are "acquired" as the consequence of malnutrition, deliberate immunosup- pression (e.g., during organ transplantation or chemo- therapy for cancer), or disease (e.g., AIDS). The early events in a human HIV-1 infection are not well characterized because the infection typically is not diag- nosed until weeks or months after exposure to the virus. However, the emerging picture is that these infections typically begin when the virus penetrates the rectal or vaginal mucosa and infects helper T cells which lie below these protective surfaces. The virus uses these cells' bio- synthetic machinery to make many more copies of itself, and the newly made viruses then infect other cells. So in the early stages of infection, the virus multiplies relatively unchecked while the innate system gives it its best shot, and the adaptive system is being mobilized. After a week or so, the adaptive system starts to kick in, and virus- specific B cells, helper T cells, and CTLs are activated, proliferate, and begin to do their thing. Consequently, during this early, "acute" phase of a viral infection, there is a dramatic rise in the number of viruses in the body (the "viral load") as the virus multiplies in infected cells. This is followed by a marked decrease in the viral load as virus-specific CTLs go to work. The immune system destroys all the invading viruses, and memory B and T cells are produced to protect against a subsequent infection by the same virus. In contrast, a full- blown HIV-1 infection always leads to a "chronic" phase that can last for ten or more years. During this phase, a fierce struggle goes on between the immune system and the AIDS virus - a struggle which the virus almost always wins.During the chronic phase of infection, viral loads decrease to low levels compared with those reached during the height of the acute phase, but the number of virus-specific CTLs and Th cells remains high - a sign that the immune system is still trying hard to defeat the virus. However, as the chronic phase progresses, the total number of Th cells slowly decreases, because these cells are killed as a consequence of the viral infection. Eventually there are not enough Th cells left to provide the help needed by virus-specific CTLs. When this happens, the number of these CTLs also begins to decline, and the viral load increases - because there are too few CTLs left to cope with newly infected cells. In the end, the immune defenses are overwhelmed, and the resulting profound state of immunosuppression leaves the patient open to unchecked infections by patho- gens that normally would not be the slightest problem for a person with an intact immune system.

Wouldn't it be better just to activate fewer T cells, so that "excess" T cells would not have to be destroyed by activation-induced cell death? Why not?

So part of the problem of disposing of obsolete weapons is solved by making many of these weapons short-lived. T cells, however, are an important exception to this "rule." In contrast to cells like neutrophils, which are pro- grammed to self-destruct after a short time on the job, T cells are designed to live a long time. The reason for this is that naive T cells must circulate again and again through the secondary lymphoid organs, looking for their particu- lar antigen on display - so it would be extremely wasteful if T cells were short-lived. On the other hand, once T cells have been activated, have proliferated in response to an attack, and have defeated the invader, the longevity of T cells could be a major problem. Indeed, at the height of some viral attacks, more than 10% of all our T cells rec- ognize that particular virus. If most of these cells were not eliminated, our bodies would soon fill up with obsolete. T cells that could only defend us against invaders from the past. Fortunately, Mother Nature recognized this problem and invented "activation-induced cell death" (AICD) - a way of eliminating obsolete T cells after they have been restimulated many times in the course of a battle. Here's how this works. CTLs have proteins called Fas ligand that are promi- nently displayed on their surface, and one way they kill is by plugging this protein into its binding partner, Fas, which is present on the surface of target cells. When these proteins connect, the target is triggered to commit suicide by apoptosis. Virgin T cells are "wired" so that they are insensitive to ligation of their Fas proteins. However, when T cells are activated and then reactivated many times during an attack, their internal wiring changes. When this happens, they become increasingly more sensi- tive to ligation of their Fas proteins either by their own Fas ligand proteins or by Fas ligand proteins on other T cells. This feature makes these "exhausted" T cells targets for Fas-mediated killing - either through suicide or homi- cide. In fact, once an invader has been vanquished, more than 90% of the T cells which responded to the attack usually die off. By this mechanism, activation-induced cell death (AICD) eliminates T cells which have been repeatedly activated, and makes room for new T cells that can protect us from the next microbes which might try to do us in.

How does a helper T cell orchestrate the actions of innate system players like macrophages and NK cells?

Some helper T cells (the so-called "Th0" cells) remain "unbiased" when they first are activated, retaining the ability to produce a wide range of cytokines. It appears that DCs tell these helper T cells where to go, but not what to do. However, once Th0 cells reach the battle scene, the cytokine environment they encounter there causes them to commit to the cytokine profile required for the defense. For example, when Th0 cells exit the blood to fight a bacterial infection in the tissues, they encounter an envi- ronment rich in IL-12. This is because Th1 cells that are already fighting the bacteria there produce IFN-γ. This cytokine, together with danger signals like the bacterial molecule LPS, activates tissue macrophages, which secrete large amounts of IL-12. And when Th0 cells receive the IL-12 signal, they "realize" what type of battle is being fought, and commit to becoming Th1 cells - which produce the cytokines needed to defend against the bacteria. Likewise, Th0 cells can become Th2 or Th17 cells when they reach a battle site that is rich in IL-4 or IL-6 and TGFβ, respectively. So previously uncommitted Th0 cells can be "converted" by the cytokine environment at the scene of the battle to become Th1, Th2, or Th17 cells.

How does T cell memory protect us against invaders in the near future and beyond?

T cells also are able to remember a previous encounter with an invader. Indeed, it has been shown that memory T cells can persist for at least a decade. T cell memory is similar, but not identical, to B cell memory. After naive T cells have been activated in response to an initial attack, and have proliferated to build up their numbers as much as 10000-fold, many of them are given passports to travel out to the tissues to do battle with the enemy. These are the "effector" T cells. After the attack has been repulsed, most effector T cells die by apoptosis, but some of them, the "memory effector T cells," remain in the tissues. There they wait quietly for a subsequent attack. If that attack comes, they rapidly reactivate, pro- liferate a bit, and begin to destroy the invaders they remember. During an attack, some activated T cells do not travel out to the tissues to battle the invaders. They remain in the secondary lymphoid organs and the bone marrow. These are the "central memory T cells." During a sub- sequent attack, central memory T cells activate quickly, and after a brief period of proliferation, most mature into effector cells, which join the memory effector T cells at the battle scene. The rest of the central memory T cells remain in the secondary lymphoid organs and wait for another attack by the same invader.

Explain why the traffic pattern of virgin T cells plays a role in maintaining tolerance of self.

Thankfully, most T cells with receptors which could rec- ognize our own proteins are eliminated in the thymus. However, central tolerance is not foolproof. If it were, every single T cell would have to be tested on every pos- sible self antigen in the thymus - and that's a lot to ask. The probability is great that T cells with receptors which have a high affinity for self antigens that are abundant in the thymus will be deleted there. However, T cells whose receptors have a low affinity for self antigens, or which recognize self antigens that are rare in the thymus, are less likely to be negatively selected. They may just "slip through the cracks" of central tolerance induction. Fortunately, the system has been set up to deal with this possibility. Virgin T cells circulate through the secondary lym- phoid organs, but are not allowed out into the tissues. This traffic pattern takes these virgins to the areas of the body where they are most likely to encounter APCs and be activated. However, the travel restriction that keeps virgin T cells out of the tissues also is important in main- taining self tolerance. The reason is that, as a rule, those self antigens which are abundant in the secondary lym- phoid organs, where virgin lymphocytes are activated, also are abundant in the thymus, where T cells are toler- ized. Therefore, as a result of the traffic pattern followed by virgin T cells, most T cells that could be activated by an abundant self antigen in the secondary lymphoid organs already will have been eliminated by seeing that same, abundant self antigen in the thymus. Conversely, T cells whose receptors recognize self anti- gens that are relatively rare in the thymus may escape deletion there. However, these same antigens usually exist at such low concentrations in the secondary lym- phoid organs that they do not activate potentially self- reactive T cells. Thus, although rare self antigens are present in the secondary lymphoid organs, and although T cells do have receptors which can recognize them, these T cells usually remain functionally "ignorant" of their presence - because the self antigens are too rare to trigger activation. Consequently, lymphocyte traffic pat- terns play a key role not only in insuring the efficient activation of the adaptive immune system, but also in preserving tolerance of self antigens.

What are the basic differences between innate system memory and adaptive system memory?

The innate immune system has a "hard-wired" memory which is extremely important in defending us against everyday invaders. This memory is the result of millions of years of experience, during which the innate system slowly evolved pattern-recognition receptors that can detect the signatures of common invaders. These receptors (e.g., the Toll-like receptors we discussed in Lecture 4) detect molecular structures which are characteristic of broad classes of microbial pathogens, and which usually are indispensable for an invader's lifestyle. Because pattern-recognition receptors are standard equipment for cells of the innate system, this ancient memory allows an immediate and robust response to invaders that have been attacking humans for a very long time. And because innate memory evolved as humans evolved, all of us have the same innate memory. Whereas the innate immune system uses hard-wired, pattern-recognition receptors to detect pathogens which also plagued our ancestors, the adaptive immune system is set up to remember attackers we encounter during our lifetime. Although B and T cells have a diverse collection of receptors that can recognize essentially any invader, there are relatively few naive B or T cells with receptors that can recognize any particular attacker - not enough to mount an immediate defense. So in practical terms, B and T cells really begin life with a blank memory. During an initial attack, pathogen-specific B and T cells prolifer- ate to build up their numbers - a process that takes one or two weeks. Only then are memory B and T cells gener- ated that are sufficiently numerous to defend against a subsequent attack by the same invader. The way this memory is achieved is somewhat different for B cells and T cells. Although both innate and adaptive immune systems remember, it is important to understand how these mem- ories differ. The innate memory is a static memory: it is not updatable - at least not on the time scale of a human lifetime. Although there may be slight genetic differences from human to human, all humans have essentially the same innate memory, which reflects the experience of the human race with common invaders that have been plagu- ing us for millions of years. In contrast, the adaptive immune system has an expandable memory which can remember any invader to which we have been exposed, be it common or rare. Moreover, the adaptive immune system's memory is personal: each of us has a different adaptive memory, depending on the particular invaders we have encoun- tered during our lifetime. In fact, even when two people have been attacked by the same microbe, their adaptive memories of that attack will be different - because the receptors on the collection of invader-specific B and T cells will differ from person to person. Indeed, because B and T cell receptors are made by a mix-and-match mecha- nism, no two humans will have the same adaptive memory.

How do macrophages and natural killer cells tell friend from foe (i.e., how do they select their targets)?

The method NK cells employ to identify their targets is quite different from that of killer T cells. Natural killer cells have no T cell receptors, but they have two other types of receptors on their surface: "activating" receptors which, when engaged, motivate the NK cell to kill; and "inhibitory" receptors which, when engaged, encourage it not to kill. The "don't kill" signal is conveyed by receptors that recognize class I MHC molecules on the surface of a potential target cell. Class I MHC molecules are found in varying amounts on the surface of most healthy cells in our bodies. Consequently, the presence of this surface molecule is an indication that a cell is doing okay. In contrast, the "kill" signals involve interactions between the activating receptors on the surface of an NK cell and unusual carbohydrates or proteins on the surface of a target cell. These peculiar surface molecules act as flags which indicate that the target cell has been "stressed," usually because it has been infected with a virus or is becoming cancerous. It is the balance between the "kill" and the "don't kill" signals which determines whether NK cells will destroy a target cell. Although a number of different signals can prime a resting macrophage, the best studied is an intercellular communication mole- cule (cytokine) called interferon gamma (IFN-γ). This cytokine is produced mainly by helper T cells and natural killer cells. In the primed state, macrophages are good antigen presenters and reasonably good killers. However, there is an even higher state of readiness, "hyperactivation," which they can attain if they receive a direct signal from an invader. For example, such a signal can be conveyed by a molecule called lipopolysaccharide (LPS for short). LPS, a component of the outer cell wall of Gram-negative bac- teria like Escherichia coli, can be shed by these bacteria, and can bind to receptors on the surface of primed macrophages. Macrophages also have receptors for mannose - the carbohydrate that is an ingredient of the cell walls of many common pathogens and which, as we discussed earlier, is a "danger signal" that can activate the complement system. When receptors on the surface of the macrophage bind to either LPS or mannose, the macrophage knows for sure that there has been an invasion. Faced with this realization, the macrophage stops proliferating, and focuses its attention on killing. In the hyperactive state, macrophages grow larger and increase their rate of phagocytosis. In fact, they become so large and phagocytic that they can ingest invaders that are as big as unicellular parasites

Some peptides are presented more ef ciently than others. What factors in uence the ef ciency of presentation by class I and class II MHC molecules?

The structures of both class I and class II MHC molecules have now been carefully analyzed, so we have a good idea of what both kinds of molecules look like. Class I molecules have a binding groove that is closed at both ends, so the small protein fragments (peptides) they present must t within the con nes of the groove (the "bun," if you will). Indeed, when immunologists pried peptides from the grasp of class I molecules and sequenced them, they found that most of them are eight to eleven amino acids in length. These peptides are anchored at the ends, and the slight variation in length is accommodated by letting the peptide bulge out a bit in the center. Every human has three genes for class I MHC proteins (called HLA-A, HLA-B, and HLA-C), located on chromo- some six. Because we have two chromosome sixes (one from Mom and one from Dad), we all have a total of six class I MHC genes. Each class I HLA protein pairs with another protein called β2-microglobulin to make up the complete class I MHC molecule. In the human popula- tion, there are many, slightly different forms of the genes that encode the three class I HLA proteins. For example, there are at least 480 variants of the gene for the HLA-A protein, 800 different HLA-B genes, and 260 different HLA-C genes. The proteins encoded by these genes all have roughly the same shape, but they differ by one or a few amino acids. Immunologists call molecules that have many forms "polymorphic," and the class I HLA proteins certainly t this description. In contrast, all of us have the same gene for the β2-microglobulin protein. Because they are polymorphic, class I MHC molecules can have different binding motifs, and therefore can present peptides that have different kinds of amino acids at their ends. For example, some class I MHC molecules bind to peptides that have hydrophobic amino acids at one end, whereas other MHC molecules prefer basic amino acids at this anchor position. Since humans have the possibility of expressing up to six dif- ferent class I molecules, collectively our class I molecules can present a wide variety of peptides. Moreover, although MHC I molecules are picky about binding to certain amino acids at the ends of the peptide, they are rather promiscuous in their selection of amino acids at the center of the protein fragment. As a result, a given class I MHC molecule can bind to and present a large number of different peptides, each of which " ts" with the par- ticular amino acids present at the ends of its binding groove.

Why are inducible T regulatory cells (iTregs) important, and how do they function?

We generally think of Th cells as providing help to turn the immune system on, but recently, another type of CD4+ T cell has been discovered which actually helps turn the system off: the inducible regulatory T cell (iTreg). These cells are called "regulatory" because, instead of producing cytokines such as TNF and IFN-γ which activate the immune system, they produce cytokines such as IL-10 and TGFβ that help restrain the system. When TGFβ binds to receptors on T cells, it reduces the proliferation rate of these cells and makes killer T cells less vicious killers. IL-10 works by binding to its receptors on T cells and blocking co-stimulatory signals (e.g., signals transmitted through CD28). This makes it more difficult to activate these cells, and decreases their rate of proliferation. These regulatory T cells are termed "inducible" because, just as naive helper T cells can be encouraged to become Th1, Th2, or Th17 cells, naive Th cells can also be "induced" to become iTregs. Here's the way this is thought to work. Under non-battle conditions, when the integrity of the intestinal barrier has not been compromised, the epithe- lial cells that line the intestine produce the cytokine TGFβ. And when naive Th cells are activated in the Peyer's patches that underlie the intestine, they are encouraged by this cytokine to become iTregs. These T cells then give off cytokines which help keep the mucosal immune system "calmed down." Now, you may remember that TGFβ is one of the cytokines that causes naive Th cells to become Th17 cells - cells which are skilled at orchestrating an inflam- matory response to a bacterial or fungal invasion. So how does the immune system decide whether Th cells that are guarding against intestinal invaders should become iTregs and restrain the immune response, or become Th17 cells and "let the dogs out"? The answer to this question is not known for certain, but it appears, as you'd expect, that it is the dendritic cell which makes this decision. If there is an invasion of pathogenic bacteria, dendritic cells begin to produce IL-6, which influences helper T cells to commit to becoming Th17 cells. If there is no real danger, and things just need to be kept calm, den- dritic cells don't produce IL-6 - and naive Th cells, under the influence of tissue-produced TGFβ become iTregs. And how do dendritic cells know the difference between pathogenic and commensal bacteria? Nobody knows for sure, but it may have to do with receptors on dendritic cells which recognize pathogenic vs. commen- sal bacteria.

In the lymphoid follicles of secondary lymphoid organs, B cells and Th cells interact. What goes on during this "dance"?

When B cells bind their cognate antigen displayed on follicular dendritic cells, the protein is taken into the B cell, cut into fragments, and presented by class II MHC molecules on the B cell surface. And once a B cell has been activated, it also expresses B7 proteins on its surface. Consequently, an activated B cell has all the goodies required to function as an APC and to restimulate helper T cells that have run out of gas. So, in a lymphoid follicle, Th cells and B cells do a "dance" in which the Th cell provides the co-stimulation (CD40L) required to activate the B cell, and the B cell provides the presented antigen and co-stimulation (B7) required to "recharge" the T cell.

For T cells being educated in the thymus, what is the functional de nition of self (i.e., what do these T cells consider to be self peptides)?

When I say "self" MHC, I simply mean those MHC molecules which are expressed by the person (or mouse) who "owns" this thymus. Yes, this does seem like a no- brainer - that my T cells would be tested in my thymus on my MHC molecules - but immunologists like to emphasize this point by saying "self MHC." The MHC molecules on the surface of the cortical epi- thelial cells are actually loaded with peptides, so what a TCR really recognizes is the combination of a self MHC molecule and its associated peptide. These peptides rep- resent a "sampling" of the proteins that are being made by the cortical epithelial cells (displayed by class I MHC molecules) plus a "sampling" of all the proteins which the cortical epithelial cells have picked up from the envi- ronment within the thymus (displayed by class II MHC molecules).

Why do most mast cells wait until their second exposure to an allergen before they degranulate? Hint: Think about the timing.

When a mast cell encounters a parasite, it dumps the contents of these granules (i.e., it "degranulates") onto the parasite to kill it. Unfortunately, in addition to killing parasites, mast cell degranulation also can cause an aller- gic reaction, and in extreme cases, anaphylactic shock. Here's how this works. An antigen (e.g., the man-of-war toxin) that can cause an allergic reaction is called an allergen. On the rst expo- sure to an allergen, some people, for reasons that are far from clear, make lots of IgE antibodies directed against the allergen. Mast cells have receptors on their surface that can bind to the Fc region of these IgE antibodies, and when this happens, the mast cells are like bombs waiting to explode. On a second exposure to the allergen, IgE antibodies that are already bound to the surfaces of mast cells can bind to the allergen. Because allergens usually are pro- teins with a repeating sequence, the allergen can crosslink many IgE molecules on the mast cell surface, dragging the Fc receptors together. This clustering of Fc receptors is similar to the crosslinking of B cell receptors in that bringing many of these receptors together results in a signal being sent. In this case, however, the signal says "degranulate," and the mast cell responds by dumping its granules into your tissues.

Discuss how the innate system deals with a virus attack.

When viruses enter (infect) human cells, they take over the cells' machinery and use it to produce many more copies of the virus. Eventually, these newly made viruses burst out of the infected cells, and go on to infect other cells in the neighborhood. We have already discussed some of the weapons the innate system can use to defend against viruses when they are outside of cells. For example, proteins of the complement system can opsonize viruses for phagocytosis by macrophages and neutrophils. In addition, complement proteins can poke holes in enveloped viruses (e.g., HIV-1) by constructing mem- brane attack complexes on the virus's surface. Although the innate system is quite effective against viruses when they are outside of cells, once viruses enter cells, the weapons the innate immune system can bring to bear are rather limited. NK cells and activated macro- phages secrete cytokines like IFN-γ and TNF that in some cases can reduce the amount of virus that infected cells produce. Secreted TNF also can kill some virus-infected cells, and cells infected by certain viruses can be killed directly by NK cells or by activated macrophages. However, each virus-infected cell can produce thousands of new viruses, and many viruses mutate rapidly, ena- bling them to evolve defenses that can protect them from the weapons of the innate immune system. Consequently, although complement proteins, professional phago- cytes, and NK cells can help contain a viral infection, especially in the early stages, more potent weapons fre- quently are required to deal with virus-infected cells. This is probably the reason Mother Nature invented the adaptive immune system - the subject of our next few lectures.

Discuss the features of an HIV-1 infection that make it difficult for the immune system to deal with.

Why is HIV-1 able to defeat an immune system that is so successful in protecting us from most other pathogens? There are two parts to this answer. The first has to do with the nature of the virus itself. All viruses are basically pieces of genetic information (either DNA or RNA) with a protective coat. For the AIDS virus, this genetic informa- tion is in the form of RNA which, after the virus enters its target cell, is copied by a viral enzyme (reverse tran- scriptase) to make a piece of "copy" DNA (cDNA). Next, the DNA of the cell is cut by another enzyme carried by the virus, and the viral cDNA is inserted into the gap in the cellular DNA. Now comes the nasty part. Once the viral DNA has been inserted into a cell's DNA, it can just sit there, and while the virus is in this "latent" state, the infected cell cannot be detected by CTLs. Recent data suggests that it only takes five to ten days for HIV-1 to initiate a latent infection and establish a reservoir of stealth virus in these "sanctuary" cells. Sometime later, in response to signals that are not fully understood, the latent virus can be "reactivated," additional copies of the virus can be produced, and more cells can be infected by these newly minted viruses. o the ability to establish a latent infection which cannot be detected by CTLs is one property of HIV-1 that makes this virus such a problem for the immune system. But it gets worse. The reverse transcriptase enzyme used to copy the HIV-1 RNA is very error-prone: it makes a "mistake" almost every time it copies a piece of viral RNA. This means that most of the new viruses produced by an infected cell are mutated versions of the virus which originally infected that cell. And some of these mutations may enable the newly made viruses to evade the immune system. For example, the virus can mutate so that a viral peptide that formerly was targeted by a CTL can no longer be recognized, or can no longer be presented by the MHC molecule that the CTL was trained to focus on. In fact, it has been shown that it only takes about ten days for these "escape mutants" to arise. When such mutations occur, that CTL will be useless against cells infected with the mutant virus, and new CTLs which recognize another viral peptide will need to be activated. Meanwhile, the virus that has escaped from surveillance by the obsolete CTLs is replicating like crazy, and every time it infects a new cell, it mutates again. Consequently, the mutation rate of the AIDS virus is so high that it can usually stay one step ahead of CTLs or antibodies directed against it. So two of the properties of HIV-1 that make it especially deadly are its ability to establish an undetectable, latent infection, and its high mutation rate. But that's only half the story. The other part has to do with the cells HIV-1 infects. This virus specifically targets cells of the immune system: helper T cells, macrophages, and dendritic cells. The "docking" protein that HIV-1 binds to when it infects a cell is CD4, the co-receptor protein found in large numbers on the surface of helper T cells. This protein also is expressed on macrophages and dendritic cells, although they have fewer CD4 molecules on their surface. By attacking these cells, the AIDS virus either disrupts their function, kills the cells, or makes them targets for killing by CTLs that recognize them as being virus-infected. So the very cells that are needed to activate CTLs and to provide them with help are damaged or destroyed by the virus. Even more insidiously, HIV-1 can turn the immune system against itself by using processes which are essential for immune function to spread and maintain the viral infection. For example, HIV-1 can attach to the surface of dendritic cells and be transported by these cells from the tissues, where there are relatively few CD4+ cells, into the lymph nodes, where huge numbers of CD4+ T cells are located. Not only are there helper T cells within easy reach in the lymph nodes, many of these cells are proliferating, making them ideal candidates to be infected and become HIV-1 "factories." Also, AIDS viruses that have been opsonized either by antibodies or by complement are retained in lymph nodes by follicular dendritic cells. This display is intended to help activate B cells. However, CD4+ T cells also pass through these forests of follicular dendritic cells, and as they do, they can be infected by the opsonized AIDS viruses. And because virus particles typically remain bound to follicular dendritic cells for months, lymph nodes actually become reservoirs of HIV-1. Indeed, HIV-1 takes advantage of the normal trafficking of immune system cells through lymph nodes, and turns these sec- ondary lymphoid organs into its own playground. In summary, the pathological consequences of an HIV-1 infection are the result of the virus' ability to slowly destroy the immune system of the patient, leading to a state of profound immunosuppression which makes the individual an inviting host for life- threatening infections. The virus is able to do this because it can establish a latent, "stealth" infection, because it has a high mutation rate, because it preferen- tially infects and disables the immune system cells that normally would defend against it, and because it uses the immune system itself to facilitate its spread through- out the body.

Describe what happens during T cell-dependent activation of B cells.

antigen, and these cells are called "naive" or "virgin" B cells. An example would be a B cell that can recognize the smallpox virus, but which happens to reside in a human who has never been exposed to smallpox. In contrast, B cells that have encountered their cognate antigen and have been activated are called "experienced." There are two ways that naive B cells can be activated to defend us against invaders. One is completely dependent on the assistance of helper T cells (T cell-dependent activation), and the second is more or less independent of T cell help (T cell-independent activation). Activation of a naive B cell requires two signals. The first is the clustering of the B cell's receptors and their associated signaling molecules. However, just having its receptors crosslinked is not enough to fully activate a B cell - a second signal is required. mmunologists call this the "co-stimulatory" signal. In T cell-dependent activation, this second signal is supplied by a helper T cell. The best studied co-stimulatory signal involves direct contact between a B cell and a helper T (Th) cell. On the surface of activated Th cells are proteins called CD40L. When CD40L plugs into (ligates) a protein called CD40 on the surface of a B cell, the co-stimulatory signal is sent, and if the B cell's receptors have been crosslinked, the B cell is activated. The interaction between these two proteins, CD40 and CD40L, is clearly very important for B cell activation. Humans who have a genetic defect in either of these proteins are unable to mount a T cell-dependent antibody defense.

What is the fundamental difference in the way the complement system is activated by the alternative pathway and by the lectin activation pathway?

complement system must be activated before it can function, and there are three ways this can happen. The first, the so- called "classical" pathway, depends on antibodies for activation. So, whereas the alternative activation pathway is spontaneous, and can be visualized as "grenades" going off randomly here and there to destroy any unprotected surface, lectin activation can be thought of as "smart bombs" that are targeted by mannose- binding lectins. This protein is called mannose-binding lectin (MBL for short). A lectin is a protein that is able to bind to a carbohydrate molecule, and mannose is a carbohydrate molecule found on the surface of many common pathogens. This is an example of an important strategy employed by the innate system: the innate system mainly focuses on pat- terns of carbohydrates and fats that are found on the surfaces of common pathogens.

Describe the main attributes of IgM, IgG, IgA, and IgE antibodies.

the constant region of an anti- body determines both its class and its function. early in an infection, when antibodies are just beginning to be made, IgM antibodies have a great advantage over IgG antibodies because they fix complement so efficiently. In addition, IgM antibod- ies are very good at "neutralizing" viruses by binding to them and preventing them from infecting cells. Because of these properties, IgM is the perfect "first antibody" to defend against viral or bacterial infections. IGG: This process is called "antibody-dependent cellular cytotoxicity" (ADCC). In ADCC, the NK cell does the killing, but the antibody identifies the target. IGA: IgA is the main antibody class that guards the mucosal surfaces of the body IGE: On the first expo- sure to an allergen, some people, for reasons that are far from clear, make lots of IgE antibodies directed against the allergen. Mast cells have receptors on their surface that can bind to the Fc region of these IgE antibodies, and when this happens, the mast cells are like bombs waiting to explode.

Imagine a splinter has punctured your big toe, and that Gram-negative bacteria have invaded the tissues surrounding the splinter. Sketch the likely sequence of events in which the various players of the innate system team deal with this invasion.

this macrophage actually has sensed the presence of the bacterium, and is reaching out a "foot" to grab it macrophages have antennae (recep- tors) on their surfaces which are tuned to recognize "danger molecules" characteristic of common microbial invaders. when macrophages eat the bacteria on that splinter in your foot, they give off chemicals which increase the flow of blood to the vicinity of the wound. The build-up of blood in this area is what makes your toe red. Some of these chemicals also cause the cells that line the blood vessels to contract, leaving spaces between them so that fluid from the capillaries can leak out into the tissues. It is this fluid which causes the swelling. In addition, chemicals released by macrophages can stimulate nerves in the tissues that surround the splinter, sending pain signals to your brain to alert you that something isn't quite right in the area of your big toe. During their battle with bacteria, macrophages also produce and give off (secrete) proteins called cytokines. These are hormone-like messengers which facilitate com- munication between cells of the immune system. Some of the cytokines alert monocytes and other immune system cells traveling in nearby capillaries that the battle is on, and influence these cells to exit the blood to help fight the rapidly multiplying bacteria. And pretty soon, you have a vigorous "inflammatory" response going on in your toe, as the innate immune system battles to eliminate the invaders. You have a large perimeter to defend, so you station sentinels (macrophages) to check for invaders. When these senti- nels encounter the enemy, they send out signals (cytokines) that recruit more defenders to the site of the battle. The macrophages then do their best to hold off the invaders until reinforcements arrive. There are other players on the innate team. For example, in addition to the "professional phagocytes" like macro- phages, which make it their business to eat invaders, the innate system also includes the complement proteins that can punch holes in bacteria, and natural killer (NK) cells which are able to destroy bacteria, parasites, virus- infected cells, and some cancer cells.