Immunity

Phagocytes: the non-specific attack on any pathogen

Phagocytes are white blood cells that engulf pathogens and other particles and digest them, a process called phagocytosis. The two phagocytes you must know are neutrophils and macrophages, and both are produced from stem cells in the bone marrow. The attack is non-specific, which means the response is the same whatever has entered the body: a neutrophil has no receptor that fits one particular bacterium, and it attacks anything whose surface marks it out as not being part of the body. That is the essential difference from lymphocytes, whose receptors and antibodies fit one antigen and nothing else.

A neutrophil has a lobed nucleus, is the most abundant white blood cell in the blood, and is short-lived, surviving for only hours to a few days. It is highly mobile: it squeezes out between the cells of the capillary wall at the site of an infection and moves towards the bacteria. Neutrophils are the first phagocytes to arrive, and they destroy bacteria with hydrolytic enzymes and with strongly oxidising chemicals that they release. Because they are killed so quickly in the process, dead neutrophils and bacteria collect as pus at the site of an infection.

A macrophage develops from a monocyte that has left the blood and entered a tissue. Macrophages are larger and much longer lived than neutrophils, and they are found wherever pathogens are likely to arrive - in the lymph nodes, the spleen, the alveoli of the lungs and the sinusoids of the liver, where they are called Kupffer cells. Macrophages destroy pathogens in the same way as neutrophils, but they do something else that neutrophils do not: they display the antigens of the digested pathogen on their own cell surface membrane. This is antigen presentation, and it is the link between the non-specific and the specific immune response.

The sequence of events in a phagocyte is always the same, and examiners want each step and its correct name. The phagocyte is attracted to the site by chemicals released by the pathogen, by damaged cells and by other cells of the immune system; this movement along a chemical gradient is chemotaxis. Receptors on the phagocyte surface membrane then bind to antigens on the surface of the pathogen, so that the pathogen is recognised as non-self. The phagocyte engulfs the pathogen by endocytosis: the cell surface membrane folds around it and encloses it in a vacuole called a phagosome. Lysosomes fuse with the phagosome and release hydrolytic enzymes into it, which digest the pathogen; the soluble products are absorbed into the cytoplasm. In a macrophage, the antigens that survive digestion are then displayed on the surface membrane.

• Attraction (chemotaxis): the phagocyte moves towards the pathogen along a gradient of chemicals released by the pathogen, damaged cells and other immune cells.
• Recognition: receptors on the phagocyte surface membrane bind to non-self antigens on the surface of the pathogen.
• Engulfing: the cell surface membrane folds around the pathogen and encloses it in a vesicle called a phagosome.
• Digestion: lysosomes fuse with the phagosome and release hydrolytic enzymes that hydrolyse the pathogen.
• Absorption and presentation: the digested products are absorbed, and a macrophage displays pathogen antigens on its surface membrane.

Antigens: what makes a molecule self or non-self

An antigen is a molecule that is recognised by the immune system and triggers an immune response. Most antigens are proteins or glycoproteins on a cell surface membrane, because the shape of a protein gives it the variety that the immune system can recognise, but polysaccharides, glycolipids and free molecules such as the toxins released by bacteria can be antigens as well. The small region of an antigen that is actually bound by an antibody or a lymphocyte receptor is called an epitope, which is why one large protein antigen can carry several different epitopes.

Self antigens are the molecules on the surface of a person's own cells and tissues. In humans the most important self antigens are the proteins of the major histocompatibility complex (MHC), known in humans as the human leucocyte antigens. The immune system has to tolerate them, or it would destroy the body it lives in. Self-tolerance is learned while lymphocytes are maturing in the thymus and the bone marrow: any lymphocyte whose receptor binds strongly to a self antigen is destroyed before it is released, so only lymphocytes that ignore the body's own cells survive. When self-tolerance fails, the result is an autoimmune disease such as type 1 diabetes, in which the immune system destroys the beta cells of the pancreatic islets.

Non-self antigens are molecules that do not match a person's own self antigens. They are found on the surface of pathogens, on the toxins those pathogens release, on transplanted cells and tissues, and on some harmless particles such as pollen that provoke an allergic reaction. Because the lymphocytes that recognise them were never destroyed, they stimulate a full immune response. Cancer cells can also become non-self, because mutations make them produce proteins that normal body cells do not, and this is what allows the immune system, and many modern treatments, to attack a tumour.

Blood groups are the everyday example of self and non-self antigens. A person with blood group A has A antigens and makes antibodies against B, and a person with blood group B has B antigens and makes antibodies against A; the cells of the donor therefore carry non-self antigens and are agglutinated by the antibodies in the plasma of the recipient if the two are mismatched. The same principle applies to the rhesus D antigen, which is why the blood group and the rhesus type are both checked before a transfusion. In a kidney transplant the donor organ carries the donor's MHC antigens, which the recipient's T-killer cells attack, so donors are chosen with the closest match and the recipient is given drugs that suppress the immune response.

The primary immune response: macrophages, T-lymphocytes and B-lymphocytes

The primary immune response is what happens the first time a particular pathogen enters the body. The pathogen multiplies while the response is being organised, which is why the illness gets worse for several days before it gets better. Phagocytes act first and hold the infection in check without any specificity; then the macrophages that have digested the pathogen present its antigens on their surface membrane. Every lymphocyte carries receptors of a single shape, so only the small number of lymphocytes whose receptors are complementary to that antigen can respond - the antigen selects them, and this is called clonal selection.

A T-helper cell carries receptor proteins on its surface membrane that are complementary in shape to one antigen. When its receptor binds to the antigen displayed by a macrophage, the T-helper cell is activated and divides by mitosis to produce a clone of identical cells, all recognising the same antigen. The activated T-helper cells then release cytokines (interleukins), which are the chemical instructions that coordinate the rest of the response: they attract and stimulate more phagocytes, stimulate the T-killer cells, and above all stimulate the B-lymphocyte whose antibody receptor fits the same antigen.

T-killer cells (cytotoxic T cells) are the cells that deal with pathogens that have already got inside body cells, where antibodies cannot reach them. Once activated, a T-killer cell binds to the surface of a body cell that is displaying the antigen - an infected cell, or a cancer cell - and makes the cell surface membrane permeable by making pores in it. Ions and water flood in, hydrolytic enzymes inside the target cell are activated, and the cell is killed. Killing the host cell stops the pathogen from multiplying inside it, and the T-killer cell itself is not damaged. This is why a viral infection is defeated only when the infected cells are destroyed, not just the free virus particles.

A B-lymphocyte carries antibody molecules on its surface membrane, and those antibodies act as its receptors, each one with a binding site of one shape only. When the antigen binds to the antibody on the surface of the matching B-lymphocyte, and the B-lymphocyte is also stimulated by cytokines from the T-helper cells, it is activated and divides by mitosis into a clone of identical cells. Most of the clone becomes plasma cells, which are packed with rough endoplasmic reticulum and secrete antibody into the blood and lymph; some become memory cells. The antibody secreted is specific to the antigen that started the response, because the plasma cells are clones of the one B-lymphocyte whose receptors fitted it.

Memory cells and the secondary immune response

After the primary response has cleared the infection, most of the cloned lymphocytes die, but a small population survives as memory cells: memory B-lymphocytes and memory T-lymphocytes. They are not producing antibody, but they carry the same receptors and they remain in the blood, in the lymph and in the lymph nodes for years, sometimes for life. The concentration of antibody in the plasma falls once the infection is over, so it is the memory cells, not free antibody, that carry the protection forward.

If the same antigen enters the body a second time, these memory cells are already present in far greater numbers than the original naive lymphocytes. They recognise the antigen at once and divide rapidly, so plasma cells appear within a day or two instead of after several days, and the concentration of antibody in the blood rises much higher and stays high for much longer. T-killer memory cells multiply just as quickly and start destroying infected cells almost immediately. The pathogen is therefore destroyed before it can multiply enough to damage the body, so the person may have no symptoms at all, or only a mild illness.

This is the secondary immune response, and it is the reason long-term immunity exists: someone who has recovered from measles is almost never infected again, because a huge population of memory cells specific to the measles virus is waiting. The picture on a graph is unmistakable - the primary response peaks low and falls away, while the secondary response starts sooner, rises steeply to a much higher concentration of antibody and stays elevated for longer.

There are limits. Memory cells slowly decline in number over the years, which is why a booster dose of a vaccine is given to top the population back up. Memory cells are also specific to the antigens they were made against, so a pathogen that changes its surface antigens escapes them: the influenza virus mutates its surface proteins so often that last year's memory cells no longer fit, which is why a new influenza vaccine is offered each year. Antibody that crosses the placenta from mother to fetus is not a substitute for memory cells, because the fetus receives the antibody but none of the cells that make it.

Antibodies: how the molecular structure produces the function

An antibody (an immunoglobulin) is a globular protein with quaternary structure built from four polypeptide chains: two long heavy chains and two short light chains. The chains are held together by disulfide bridges, and the whole molecule is Y-shaped, with a flexible region (the hinge) between the arms and the stem. Each chain has a constant region, which is the same in all antibodies of a class, and a variable region, which differs from one antibody to the next and is found at the tips of the two arms.

The variable regions of the two arms together form the antigen-binding site. Its shape is complementary to the shape of one epitope, so an antibody binds to only one antigen - the same principle as an enzyme and its substrate, but with binding instead of catalysis. Because the molecule has two identical binding sites and a flexible hinge, one antibody can bind to two separate antigens at the same time, linking them together. The constant regions have no effect on which antigen is bound; they decide what happens after binding, because they are recognised by receptors on phagocytes and by the proteins of the complement system.

That structure gives four examinable functions. Agglutination: one antibody links two pathogens, and many antibodies together clump the pathogens into a mass, which stops them spreading through the body and makes them an easy target. Neutralisation: by binding to the active site of a toxin, or to the attachment proteins of a virus, the antibody stops the toxin working or stops the virus entering a host cell. Marking for phagocytosis: the constant region binds to receptors on the surface of a phagocyte, so the pathogen is engulfed far more readily. Lysis: antibodies bound to a bacterial surface activate the complement system, whose proteins make holes in the bacterial cell surface membrane so that the bacterium bursts.

An antibody does not itself destroy anything: it has no hydrolytic activity, and a question that says 'antibodies digest bacteria' loses marks. The destruction is carried out by phagocytes and by the complement system, which is what the constant region arranges. The enormous variety of variable regions is generated before the lymphocyte ever meets an antigen, when the genes for the antibody chains are rearranged during lymphocyte development, so the body makes many millions of lymphocytes with different binding sites and is ready for antigens it has never met.

• Agglutination: two binding sites per molecule clump pathogens together so they cannot spread.
• Neutralisation: binding covers the active site of a toxin or the attachment protein of a virus.
• Marking for phagocytosis: the constant region binds to receptors on a phagocyte, which then engulfs the pathogen.
• Lysis: antibodies on a bacterial surface activate the complement system, which makes pores in the cell surface membrane.

Monoclonal antibodies: the hybridoma method

A natural immune response is polyclonal: many different B-lymphocyte clones are stimulated, so the plasma cells secrete a mixture of antibodies that bind to different epitopes. A monoclonal antibody is a single antibody produced by one clone of cells, so every molecule is identical and binds to the same epitope of the same antigen. That uniformity is what makes monoclonal antibodies reliable as diagnostic and therapeutic tools. The problem is that a B-lymphocyte taken out of the body and grown in culture divides only a limited number of times, while the cells that divide indefinitely - tumour cells - do not make the antibody you want.

The hybridoma method solves this by joining the two. A mouse is injected with the antigen, and after a few weeks its spleen contains B-lymphocytes that are making the antibody of interest. Those spleen cells are removed and fused with myeloma cells, which are tumour cells of the bone marrow that divide indefinitely; fusion is encouraged with polyethylene glycol or a brief electric pulse. The resulting fused cells are called hybridoma cells, and each one has both useful properties: it secretes antibody like the B-lymphocyte it came from, and it divides endlessly like the myeloma cell.

Not every hybridoma makes the right antibody, and some are unstable, so the cells are screened - usually with an enzyme-linked immunosorbent assay that detects the wanted antibody, or by diluting the cells until each culture is grown from a single cell. The one clone that produces the antibody of interest is then grown on a large scale in culture vessels, and the antibody is harvested from the medium and purified. Because all the cells are descendants of one hybridoma, all the antibody molecules are identical.

The first monoclonal antibodies were made entirely in mice, and a human patient's immune system can recognise the mouse protein as foreign and make antibodies against it, which shortens the useful life of the treatment. This is avoided by genetic engineering: the variable regions from the mouse antibody are joined to constant regions from a human antibody to make a humanised monoclonal antibody, which is far less likely to be rejected. Trastuzumab, used against some breast cancers, is a humanised monoclonal antibody.

Monoclonal antibodies in the diagnosis and treatment of disease

The whole value of a monoclonal antibody comes from its specificity. One antibody binds to one antigen and nothing else, so an antibody can be used as a molecular probe that finds one particular molecule in a mixture as complicated as blood or urine. To make the binding visible, the antibody is attached to a label: a coloured particle, an enzyme that changes the colour of a substrate, a fluorescent dye or a radioisotope. Wherever the label appears, the antigen must be present, so the test identifies the molecule without any need to culture the pathogen first.

In diagnosis, monoclonal antibodies detect either the pathogen itself or the antibodies the patient has made against it. A pregnancy test uses antibodies to human chorionic gonadotrophin, a hormone produced by the early embryo, so a line of colour appears on the test strip when the hormone is present in the urine. An enzyme-linked immunosorbent assay (ELISA) is used to detect the antigens of HIV, hepatitis B and malaria parasites, or the antibodies a person has made against them, and because it detects molecules rather than whole organisms it can give a result from a blood sample within hours. Monoclonal antibodies are also used to check blood groups before a transfusion, to identify the species or strain of a pathogen in an outbreak, and to show which antigens a biopsy sample of a tumour is carrying, which helps to decide which treatment will work.

In treatment, a monoclonal antibody can be aimed at a molecule on the surface of the cells that are causing the problem. Trastuzumab (Herceptin) binds to the HER2 receptor on the surface of breast cancer cells that overproduce it: the antibody blocks the receptor so that the signals telling the cells to divide are no longer received, and its constant region also attracts the cells of the immune system to destroy the cancer cell. Rituximab binds to a protein called CD20 on B-lymphocytes and is used against some lymphomas. A third use is as a delivery system: a cytotoxic drug or a radioisotope is attached to the antibody, so the poison reaches the cancer cells that carry the antigen and the healthy tissue around them receives only a small dose.

The method has real limits, and evaluation questions want both sides. A monoclonal antibody works against one epitope, so a tumour or a pathogen that stops making that antigen, or changes it, escapes the treatment altogether. The antibody is a foreign protein if it was made in a mouse, so the patient may make antibodies against it and destroy it before it works. It is expensive to develop and produce, and because it interferes with the immune system it can increase the risk of infection. Used against an antigen that is present on healthy cells as well as on the target cells, it will damage both.

Active and passive, natural and artificial immunity, and vaccination programmes

Active immunity means the person's own lymphocytes have responded to an antigen: their B-lymphocytes have produced plasma cells and memory cells, so the antibody was made inside their own body. It takes days to develop, and it is long-lasting because memory cells remain. Passive immunity means the antibodies came from somewhere else, so the recipient does not make them and no memory cells are produced. It gives protection immediately, but the borrowed antibodies are foreign proteins that are gradually broken down, so the protection lasts only a few weeks. The test question to ask yourself is simply whether the person's own lymphocytes did the work.

The second pair of terms describes how the immunity arose. Natural immunity results from normal life processes with no medical intervention; artificial immunity results from a medical procedure such as an injection. Cross the two pairs and you have the four types. Natural active immunity follows an infection that the person recovers from, such as chickenpox. Natural passive immunity is the antibody a fetus receives across the placenta and a baby receives in the first milk (colostrum), which shelters the baby while its own immune system matures. Artificial active immunity follows a vaccination. Artificial passive immunity follows an injection of antibodies, such as tetanus antitoxin given after a wound, rabies immunoglobulin given after a bite, or anti-D immunoglobulin given to a rhesus-negative mother.

A vaccine works because it contains antigens without the danger of the disease: killed pathogens, weakened (attenuated) pathogens that cannot multiply enough to cause illness, inactivated toxins called toxoids, or purified antigen subunits, often with an adjuvant such as an aluminium salt that makes the immune response stronger. The antigens stimulate the same chain of events as a real infection - macrophages present the antigen, T-helper cells release cytokines, B-lymphocytes divide into plasma cells - so the person produces antibody and, crucially, memory cells, without becoming ill. When the real pathogen arrives later, the memory cells produce the secondary response and the pathogen is destroyed before symptoms appear. Some vaccines need repeated or booster doses, partly because memory cell numbers fall with time and partly because a pathogen such as influenza changes its surface antigens.

Vaccination programmes control the spread of a disease by making the population, not just the individual, resistant to it. If a high enough proportion of people are immune, an infected person is unlikely to meet a susceptible person, so the pathogen cannot pass on and the outbreak dies out. This is herd immunity, and it protects the people who cannot be vaccinated at all - babies, people whose immune system is suppressed by disease or treatment, and those who are allergic to a vaccine component. That is why programmes aim for coverage of almost everyone and why a fall in uptake is dangerous: measles is so infectious that coverage of around 95 per cent of the population is needed to keep it from spreading, and when uptake of the MMR vaccine fell in the United Kingdom after the publication in 1998 of a now retracted and discredited paper linking it to autism, measles outbreaks returned and the country lost its measles elimination status in 2019. The greatest success is smallpox, which was declared eradicated by the World Health Organization in 1980 after a global programme of vaccination and case finding. Poliomyelitis has been pushed back in the same way since the Global Polio Eradication Initiative began in 1988, and the wild virus is now found only in a few countries, showing that a programme depends on reaching every child, keeping vaccines cold and safe from the cold chain breaking, and maintaining public confidence.