20.5: Adaptive Immune System - Biology

20.5: Adaptive Immune System - Biology

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The Kiss of Death

The photomicrograph in Figure (PageIndex{1}) shows a group of killer T cells (green and red) surrounding a cancer cell (blue, center). When a killer T cell makes contact with the cancer cell, it attaches to and spreads over the dangerous target. The killer T cell then uses special chemicals stored in vesicles (red) to deliver the killing blow. This event has thus been nicknamed “the kiss of death.” After the target cell is killed, the killer T cells move on to find the next victim. Killer T cells like these are important players in the adaptive immune system.

The adaptive immune system is a subsystem of the overall immune system. An adaptive immune response is set in motion by antigens that the immune system recognizes as foreign. Unlike an innate immune response, an adaptive immune response is highly specific to a particular pathogen (or its antigen). An important function of the adaptive immune system that is not shared by the innate immune system is the creation of immunological memory or immunity. This occurs after the initial response to a specific pathogen. It allows a faster, stronger response on subsequent encounters with the same pathogen, usually before the pathogen can cause symptoms of illness.

Lymphocytes are the main cells of the adaptive immune system. They are leukocytes that arise and mature in organs of the lymphatic system, including the bone marrow and thymus. The human body normally has about 2 trillion lymphocytes, which constitute about a third of all leukocytes. Most of the lymphocytes are normally sequestered within tissue fluid or organs of the lymphatic system, including the tonsils, spleen, and lymph nodes. Only about 2 percent of the lymphocytes are normally circulating in the blood. There are two main types of lymphocytes involved in adaptive immune responses, called T cells and B cells. T cells destroy infected cells or release chemicals that regulate immune responses. B cells secrete antibodies that bind with antigens of pathogens so they can be removed by other immune cells or processes.

T Cells

There are multiple types of T cells or T lymphocytes. Major types are killer (or cytotoxic) T cells and helper T cells. Both types develop from immature T cells that become activated by exposure to an antigen.

T Cell Activation

T cells must be activated. After the pathogen is phagocytized and digested by macrophages, a part of the pathogen is displayed coupled with the MHC of the macrophage. Therefore macrophages are called antigen-presenting cells as shown in Figure (PageIndex{2}) and Figure (PageIndex{3}). B lymphocytes can also act as antigen-presenting cells. Helper T cells are more easily activated than killer T cells. Activation of killer T cells is strongly regulated and may require additional stimulation from helper T cells.

Helper T Cells

Activated helper T cells do not kill infected or cancerous cells. Instead, their role is to “manage” both innate and adaptive immune responses by directing other cells to perform these tasks. They control other cells by releasing cytokines. These are proteins that can influence the activity of many cell types, including cytotoxic killer T cells (sometimes referred to as only killer T cells), B cells, and macrophages. For example, some cytokines released by helper T cells help activate killer T cells.

Killer T Cells (Cytotoxic T Cells)

When infected body cells present pathogen antigen to a killer T cell, it gets activated (see lower panel of Figure (PageIndex{3})). Activated killer T cells induce the death of cells that bear a specific non-self antigen because they are infected with pathogens or are cancerous. The antigen targets the cell for destruction by killer T cells, which travel through the bloodstream searching for target cells to kill. Killer T cells may use various mechanisms to kill target cells. One way is by releasing toxins in granules that enter and kill infected or cancerous cells (Figure (PageIndex{3})).

Figure (PageIndex{3}): Naïve CD4+ (Helper) T cells engage MHC II molecules on antigen-presenting cells (APCs) and become activated. Clones of the activated helper T cell, in turn, activate B cells and CD8+ T (killer) cells, which become cytotoxic T cells. Cytotoxic T cells kill infected cells.

B Cells and B Cell Activation

B cells, or B lymphocytes, are the major cells involved in the creation of antibodies that circulate in blood plasma and lymph. Antibodies are large, Y-shaped proteins used by the immune system to identify and neutralize foreign invaders. Besides producing antibodies, B cells may also function as antigen-presenting cells or secrete cytokines that help control other immune cells and responses.

Before B cells can actively function to defend the host, they must be activated. As shown in Figure (PageIndex{4}), B cell activation begins when a B cell engulfs and digests an antigen. The antigen may be either free-floating or on top of the pathogen. B cell internalize antigen and present it on its MHC to a helper T cell. The T cell activates and secretes cytokines that help the B cell to multiply and the daughter cells to mature into plasma cells and memory B cells. Plasma B cells produce antibodies.

Plasma Cells

Plasma cells are antibody-secreting cells that form from activated B cells. Each plasma cell is like a tiny antibody factory. It may secrete millions of copies of an antibody, each of which can bind to the specific antigen that activated the original B cell. The specificity of an antibody to a specific antigen is illustrated in Figure (PageIndex{5}). When antibodies bind with antigens, it makes the cells bearing them easier targets for phagocytes to find and destroy. Antibody-antigen complexes may also trigger the complement system of the innate immune system, which destroys the cells in a cascade of protein enzymes. In addition, the complexes are likely to clump together (agglutinate). If this occurs, they are filtered out of the blood in the spleen or liver.


Most activated T cells and B cells die within a few days once a pathogen has been cleared from the body. However, a few of the cells survive and remain in the body as memory T cells or memory B cells. These memory cells are ready to activate an immediate response if they are exposed to the same antigen again in the future. This is the basis of immunity.

The earliest known reference to the concept of immunity relates to the bubonic plague (see Figure (PageIndex{6})). In 430 B.C., a Greek historian and general named Thucydides noted that people who had recovered from a previous bout of the plague could nurse people sick with the plague without contracting the illness a second time. We now know that this is true of many diseases and it occurs because of active immunity.

Active Immunity

Active immunity is the ability of the adaptive immune system to resist a specific pathogen because it has formed an immunological memory of the pathogen. Active immunity is adaptive because it occurs during the lifetime of an individual as an adaptation to infection with a specific pathogen and prepares the immune system for future challenges from that pathogen. Active immunity can come about naturally or artificially.

Naturally Acquired Active Immunity

Active immunity is acquired naturally when a pathogen invades the body and activates the adaptive immune system. When the initial infection is over, memory B cells and memory T cells remain that provide immunological memory of the pathogen. As long as the memory cells are alive, the immune system is ready to mount an immediate response if the same pathogen tries to infect the body again.

Artificially Acquired Active Immunity

Active immunity can also be acquired artificially through immunization. Immunization is the deliberate exposure of a person to a pathogen in order to provoke an adaptive immune response and the formation of memory cells specific to that pathogen. The pathogen is introduced in a vaccine — usually by injection, sometimes by nose or mouth (Figure (PageIndex{7})) — so immunization is also called vaccination.

In a vaccine, only part of a pathogen, a weakened form of the pathogen, or a dead pathogen is typically used. This causes an adaptive immune response without making the immunized person sick. This is how you most likely became immune to diseases such as measles, mumps, and chickenpox. Immunizations may last for a lifetime or require periodic booster shots to maintain immunity. While immunization generally has long-lasting effects, it usually takes several weeks to develop full immunity.

Immunization is the most effective method ever discovered in preventing infectious diseases. As many as 3 million deaths are prevented each year because of vaccinations. Widespread immunity due to vaccinations is largely responsible for the worldwide eradication of smallpox and the near elimination of several other infectious diseases from many populations, including such diseases as polio and measles. Immunization is so successful because it exploits the natural specificity and inducibility of the adaptive immune system.

Passive Immunity

Passive immunity results when pathogen-specific antibodies or activated T cells are transferred to a person who has never been exposed to the pathogen. Passive immunity provides immediate protection from a pathogen, but the adaptive immune system does not develop immunological memory to protect the host from the same pathogen in the future. Unlike active immunity, passive immunity lasts only as long as the transferred antibodies or T cells survive in the blood. This is usually between a few days and a few months. However, like active immunity, passive immunity can be acquired both naturally and artificially.

Naturally Acquired Passive Immunity

Passive immunity is acquired naturally by a fetus through its mother’s blood. Antibodies are transported from mother to fetus across the placenta, so babies have high levels of antibodies at birth. Their antibodies have the same range of antigen specificity as their mother’s. Passive immunity may also be acquired by an infant through the mother’s breast milk. This gives young infants protection from common pathogens in their environment while their own immune system matures.

Artificially Acquired Passive Immunity

Older children and adults can acquire passive immunity artificially through the injection of antibodies or activated T cells. This may be done when there is a high risk of infection and insufficient time for the body to develop active immunity through vaccination. It may also be done to reduce symptoms of ongoing disease or to compensate for immunodeficiency diseases (for the latter, see the concept Disorders of the Immune System).

Adaptive Immune Evasion

Many pathogens have been around for a long time, living with human populations for generations. To persist, some have evolved mechanisms to evade the adaptive immune system of human hosts. One way they have done this is by rapidly changing their non-essential antigens. This is called antigenic variation. An example of a pathogen that takes this approach is the human immunodeficiency virus (HIV). It mutates rapidly so the proteins on its viral envelope are constantly changing. By the time the adaptive immune system responds, the virus’s antigens have changed. Antigenic variation is the main reason that efforts to develop a vaccine against HIV have not yet been successful.

Another evasion approach some pathogens may take is to mask pathogen antigens with host molecules so the host’s immune system cannot detect the antigens. HIV takes this approach as well. The envelope that covers the virus is formed from the outermost membrane of the host cell.

Feature: My Human Body

If you think that immunizations are just for kids, think again. There are several vaccines recommended by the CDC for people over the age of 18. This link shows the vaccine schedule recommended for all adults aged 19 years and older. Additional vaccines may be recommended for certain adults based on specific medical conditions or other indications. Are you up to date with your vaccines? You can check with your doctor to be sure.


  1. What is the adaptive immune system?
  2. Describe the main cells of the adaptive immune system.
  3. How are lymphocytes activated?
  4. Identify two common types of T cells and their functions.
  5. How do activated B cells help defend against pathogens?
  6. Define immunity.
  7. What are two ways active immunity may come about?
  8. How does passive immunity differ from active immunity?
  9. How may passive immunity occur?
  10. What ways of evading the human adaptive immune system evolved in the human immunodeficiency virus (HIV)?
  11. Describe two ways in which B cells and T cells work together to generate adaptive immune responses.
  12. Which cells directly kill pathogen-infected or cancerous cells?

    A. Plasma cells

    B. Killer T cells

    C. Helper T cells

    D. All of the above

  13. Why do vaccinations involve the exposure of a person to a version of a pathogen?

  14. True or False. Immunization is a form of passive immunity.

  15. True or False. Antibodies transmitted from mother to child via breast milk cause the formation of memory B cells and long-term immunity.

Explore More

Watch this video to learn about the recent status of HIV vaccine development:

MCAT Biology : Immune System

One component of the immune system is the neutrophil, a professional phagocyte that consumes invading cells. The neutrophil is ferried to the site of infection via the blood as pre-neutrophils, or monocytes, ready to differentiate as needed to defend their host.

In order to leave the blood and migrate to the tissues, where infection is active, the monocyte undergoes a process called diapedesis. Diapedesis is a process of extravasation, where the monocyte leaves the circulation by moving in between endothelial cells, enters the tissue, and matures into a neutrophil.

Diapedesis is mediated by a class of proteins called selectins, present on the monocyte membrane and the endothelium. These selectins interact, attract the monocyte to the endothelium, and allow the monocytes to roll along the endothelium until they are able to complete diapedesis by leaving the vasculature and entering the tissues.

The image below shows monocytes moving in the blood vessel, "rolling" along the vessel wall, and eventually leaving the vessel to migrate to the site of infection.

Neutrophils are best described as being __________ .

derivatives of natural killer (NK) cells

part of immunological memory

part of the innate immune system

part of the adaptive immune system

part of the innate immune system

Neutrophils are one of the main players in innate immunity. Their response does not require having previously been exposed to the pathogen, and they are fairly nonspecific in their ability to digest foreign invaders.

As stated in the passage, neutrophils are derived from monocyctes, not B-cells or natural killer cells.

Example Question #1 : Types Of Immune System Cells

Type 1 diabetes is a well-understood autoimmune disease. Autoimmune diseases result from an immune system-mediated attack on one’s own body tissues. In normal development, an organ called the thymus introduces immune cells to the body’s normal proteins. This process is called negative selection, as those immune cells that recognize normal proteins are deleted. If cells evade this process, those that recognize normal proteins enter into circulation, where they can attack body tissues. The thymus is also important for activating T-cells that recognize foreign proteins.

As the figure below shows, immune cells typically originate in the bone marrow. Some immune cells, called T-cells, then go to the thymus for negative selection. Those that survive negative selection, enter into general circulation to fight infection. Other cells, called B-cells, directly enter general circulation from the bone marrow. It is a breakdown in this carefully orchestrated process that leads to autoimmune disease, such as type 1 diabetes.

Di George syndrome is a genetic disorder that results in failure of thymus formation during development, and thus in immune deficiency. A doctor examines the blood of a patient with Di George syndrome. What is she most likely to find?

Deficiency of both T-cells and B-cells

A deficiency of B-cells, with a relative abundance of T-cells

Excess production of both T-cells and B-cells

A deficiency of T-cells, with a relative abundance of B-cells

A normal complement of both B-cells and T-cells

A deficiency of T-cells, with a relative abundance of B-cells

The patient has Di George syndrome, which is characterized by a lack of thymus tissue. Based on the information in the passage, loss of the thymus is most likely to manifest as a deficiency of T-cells, while the presence of B-cells will be relatively unaffected.

Example Question #1 : Immune System

Which answer choice is a part of the adaptive immune response?

Adaptive immunity involves specialized cells in response to a specific antigen. When an antigen is detected, it must be presented to the T-cells and B-cells to initiate antibody production. Dendritic cells are involved in this presentation process, and serve as the link between innate and adaptive immunity.

Basophils, eosinophils, neutrophils, and mast cells are specialized granulocytes. Neutrophils help to phagocytose pathogens. Basophils help to repair damaged tissues. Eosinophils and mast cells are both involved in mediating the inflammatory response.

Example Question #1 : Immune System

When heart surgeries were initially performed on children, surgeons would sometimes discard the thymus because they did not know its function. These children would often die due to which lost function of the thymus?

Hypothyroidism would occur

T-cells could not be generated

T-cells would not be able to mature

T3 and T4 levels would decrease

B-cells would not be able to mature

T-cells would not be able to mature

The correct answer is that T-cells would not be able to mature. Both T-cells and B-cells are generated in the bone marrow, however, their sites of maturation are different. B-cells mature in the bone marrow, whereas T-cells mature in the thymus.

T3 and T4 are thyroid hormones, and are unlinked to the thymus. Hypothyroidism occurs due to a deficiency in these hormones.

Example Question #1 : Types Of Immune System Cells

Duchenne Muscular Dystrophy is an X-linked recessive genetic disorder, resulting in the loss of the dystrophin protein. In healthy muscle, dystrophin localizes to the sarcolemma and helps anchor the muscle fiber to the basal lamina. The loss of this protein results in progressive muscle weakness, and eventually death.

In the muscle fibers, the effects of the disease can be exacerbated by auto-immune interference. Weakness of the sarcolemma leads to damage and tears in the membrane. The body’s immune system recognizes the damage and attempts to repair it. However, since the damage exists as a chronic condition, leukocytes begin to present the damaged protein fragments as antigens, stimulating a targeted attack on the damaged parts of the muscle fiber. The attack causes inflammation, fibrosis, and necrosis, further weakening the muscle.

Studies have shown that despite the severe pathology of the muscle fibers, the innervation of the muscle is unaffected.

Which of the following does not play a key role in the adaptive immune response?

The adaptive immune response involves the presentation of antigens, identification of antigens, activation of immune response, and elimination of pathogens. Dendritic cells present the antigen to the helper T-cells, which identify it and activate B-cells and cytotoxic T-cells to eliminate the pathogen.

Natural killer cells play a key role in the innate immune response by eliminating pathogens without the recognition of antigens.

Example Question #374 : Mcat Biological Sciences

Sexually transmitted diseases are a common problem among young people in the United States. One of the more common diseases is caused by the bacterium Neisseria gonorrhoeae, which leads to inflammation and purulent discharge in the male and female reproductive tracts.

The bacterium has a number of systems to evade host defenses. Upon infection, it uses pili to adhere to host epithelium. The bacterium also uses an enzyme, gonococcal sialyltransferase, to transfer a sialyic acid residue to a gonococcal surface lipooligosaccharide (LOS). A depiction of this can be seen in Figure 1. The sialyic acid residue mimics the protective capsule found on other bacterial species.

Once infection is established, Neisseria preferentially infects columnar epithelial cells in the female reproductive tract, and leads to a loss of cilia on these cells. Damage to the reproductive tract can result in pelvic inflammatory disease, which can complicate pregnancies later in the life of the woman.

A key immune response to Neisseria in humans is the activity of macrophages. What is true of how macrophages combat infection?

Macrophages use isolation as their main defense, and wall off pathogens

Macrophages produce antibodies to target pathogens

Macrophages only recruit other cells that are then able to kill pathogens

Macrophages undergo apoptosis and release toxic compounds

Macrophages use reactive oxygen species after ingesting pathogens

Macrophages use reactive oxygen species after ingesting pathogens

Macrophages are professional phagocytic cells. They ingest pathogens, and often use reactive oxygen species to kill pathogens via a burst of radical activity in specialized cellular compartments.

Example Question #1 : Types Of Immune System Cells

Scientists use a process called Flourescent In-Situ Hybridization, or FISH, to study genetic disorders in humans. FISH is a technique that uses spectrographic analysis to determine the presence or absence, as well as the relative abundance, of genetic material in human cells.

To use FISH, scientists apply fluorescently-labeled bits of DNA of a known color, called probes, to samples of test DNA. These probes anneal to the sample DNA, and scientists can read the colors that result using laboratory equipment. One common use of FISH is to determine the presence of extra DNA in conditions of aneuploidy, a state in which a human cell has an abnormal number of chromosomes. Chromosomes are collections of DNA, the totality of which makes up a cell’s genome. Another typical use is in the study of cancer cells, where scientists use FISH labels to ascertain if genes have moved inappropriately in a cell’s genome.

Using red fluorescent tags, scientists label probe DNA for a gene known to be expressed more heavily in cancer cells than normal cells. They then label a probe for an immediately adjacent DNA sequence with a green fluorescent tag. Both probes are then added to three dishes, shown below. In dish 1 human bladder cells are incubated with the probes, in dish 2 human epithelial cells are incubated, and in dish 3 known non-cancerous cells are used. The relative luminescence observed in regions of interest in all dishes is shown below.

When the body recognizes that cells have become cancerous, it responds in part by mobilizing cell-mediated killing of cancer cells. What cells are most likely responsible for this killing?

CD8 + T-cells are also called cytotoxic T-cells. They are the main agents of cell-mediated immune cytotoxicity. This function is critical for the elimination of virally infected or cancerous cells. Macrophages, in contrast, are responsible for eliminating foreign pathogens and do not attack non-foreign cells.

Example Question #381 : Mcat Biological Sciences

Where do the T-cells of the immune system mature into functional T-cells?

T-cells are originally formed from stem cells in the bone marrow, however, T-cells, unlike B-cells, mature in the thymus. The thymus is a lymphoid organ located in the upper chest.

In contrast, B-cells are formed and mature in the bone marrow.

Example Question #1 : Immune System

Which of the following is NOT an antigen presenting cell?

This question asks which of the following is NOT an antigen presenting cell, therefore, any option that is an antigen presenting cell in the immune system is an INCORRECT answer.

During an immune response, the cells involved in antigen presentation are dendritic cells, macrophages, and B-cells. T-cells, then, must be the answer choice that is NOT an antigen presenting cell, and thus is the correct answer.

Example Question #3 : Immune System

Most scientists subscribe to the theory of endosymbiosis to explain the presence of mitochondria in eukaryotic cells. According to the theory of endosymbiosis, early pre-eukaryotic cells phagocytosed free living prokaryotes, but failed to digest them. As a result, these prokaryotes remained in residence in the pre-eukaryotes, and continued to generate energy. The host cells were able to use this energy to gain a selective advantage over their competitors, and eventually the energy-producing prokaryotes became mitochondria.

In many ways, mitochondria are different from other cellular organelles, and these differences puzzled scientists for many years. The theory of endosymbiosis concisely explains a number of these observations about mitochondria. Perhaps most of all, the theory explains why aerobic metabolism is entirely limited to this one organelle, while other kinds of metabolism are more distributed in the cellular cytosol.

Some version of phagocytosis was likely the initial event that introduced a free-living prokaryote into the host described in the passage. Which of the following cells is most commonly associated with phagocytosis?

Neutrophils are professional phagocytes of the immune system. They prevent infection by phagocytosing potential invaders.

Note, however, that neutrophils could not have been the initial cell type to phagocytose a free-living prokaryote. Neutrophils are eukaryotic, and evolved long after endosymbiosis first occurred. They are, however, still closely linked to phagocytosis in humans.

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Physical and Chemical Barriers

Before any immune factors are triggered, the skin functions as a continuous, impassable barrier to potentially infectious pathogens. Pathogens are killed or inactivated on the skin by desiccation (drying out) and by the skin’s acidity. In addition, beneficial microorganisms that coexist on the skin compete with invading pathogens, preventing infection. Regions of the body that are not protected by skin (such as the eyes and mucus membranes) have alternative methods of defense, such as tears and mucus secretions that trap and rinse away pathogens, and cilia in the nasal passages and respiratory tract that push the mucus with the pathogens out of the body. Throughout the body are other defenses, such as the low pH of the stomach (which inhibits the growth of pathogens), blood proteins that bind and disrupt bacterial cell membranes, and the process of urination (which flushes pathogens from the urinary tract).

Despite these barriers, pathogens may enter the body through skin abrasions or punctures, or by collecting on mucosal surfaces in large numbers that overcome the mucus or cilia. Some pathogens have evolved specific mechanisms that allow them to overcome physical and chemical barriers. When pathogens do enter the body, the innate immune system responds with inflammation, pathogen engulfment, and secretion of immune factors and proteins.

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Understanding Adaptive Immune System as Reinforcement Learning

The adaptive immune system of vertebrates can detect, respond to, and memorize diverse pathogens from past experience. While the selection of T helper (Th) clones is the simple and established mechanism to recognize and memorize new pathogens, the question that still remains unexplored is how the Th cells can acquire better ways to bias the responses of immune cells for eliminating pathogens more efficiently by translating the recognized antigen information into regulatory signals. In this work, we address this problem by associating the adaptive immune network organized by the Th cells with reinforcement learning (RL). By employing recent advancements of network-based RL, we show that the Th immune network can acquire the association between antigen patterns of and the effective responses to pathogens. Moreover, the clonal selection as well as other inter-cellular interactions are derived as a learning rule of this network. We also demonstrate that the stationary clone-size distribution after learning shares characteristic features with those observed experimentally. Our theoretical framework may contribute to revising and renewing our understanding of adaptive immunity as a learning system.

BIO 140 - Human Biology I - Textbook

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Chapter 24

Barrier Defenses and the Innate Immune Response

  • Describe the barrier defenses of the body
  • Show how the innate immune response is important and how it helps guide and prepare the body for adaptive immune responses
  • Describe various soluble factors that are part of the innate immune response
  • Explain the steps of inflammation and how they lead to destruction of a pathogen
  • Discuss early induced immune responses and their level of effectiveness

The immune system can be divided into two overlapping mechanisms to destroy pathogens: the innate immune response, which is relatively rapid but nonspecific and thus not always effective, and the adaptive immune response, which is slower in its development during an initial infection with a pathogen, but is highly specific and effective at attacking a wide variety of pathogens (Figure 1).

Figure 1: The innate immune system enhances adaptive immune responses so they can be more effective.

Any discussion of the innate immune response usually begins with the physical barriers that prevent pathogens from entering the body, destroy them after they enter, or flush them out before they can establish themselves in the hospitable environment of the body&rsquos soft tissues. Barrier defenses are part of the body&rsquos most basic defense mechanisms. The barrier defenses are not a response to infections, but they are continuously working to protect against a broad range of pathogens.

The different modes of barrier defenses are associated with the external surfaces of the body, where pathogens may try to enter (Table 1). The primary barrier to the entrance of microorganisms into the body is the skin. Not only is the skin covered with a layer of dead, keratinized epithelium that is too dry for bacteria in which to grow, but as these cells are continuously sloughed off from the skin, they carry bacteria and other pathogens with them. Additionally, sweat and other skin secretions may lower pH, contain toxic lipids, and physically wash microbes away.

Table 1: Barrier Defenses

Site Specific defense Protective aspect
Skin Epidermal surface Keratinized cells of surface, Langerhans cells
Skin (sweat/secretions) Sweat glands, sebaceous glands Low pH, washing action
Oral cavity Salivary glands Lysozyme
Stomach Gastrointestinal tract Low pH
Mucosal surfaces Mucosal epithelium Nonkeratinized epithelial cells
Normal flora (nonpathogenic bacteria) Mucosal tissues Prevent pathogens from growing on mucosal surfaces

Another barrier is the saliva in the mouth, which is rich in lysozyme&mdashan enzyme that destroys bacteria by digesting their cell walls. The acidic environment of the stomach, which is fatal to many pathogens, is also a barrier. Additionally, the mucus layer of the gastrointestinal tract, respiratory tract, reproductive tract, eyes, ears, and nose traps both microbes and debris, and facilitates their removal. In the case of the upper respiratory tract, ciliated epithelial cells move potentially contaminated mucus upwards to the mouth, where it is then swallowed into the digestive tract, ending up in the harsh acidic environment of the stomach. Considering how often you breathe compared to how often you eat or perform other activities that expose you to pathogens, it is not surprising that multiple barrier mechanisms have evolved to work in concert to protect this vital area.

Cells of the Innate Immune Response

A phagocyte is a cell that is able to surround and engulf a particle or cell, a process called phagocytosis . The phagocytes of the immune system engulf other particles or cells, either to clean an area of debris, old cells, or to kill pathogenic organisms such as bacteria. The phagocytes are the body&rsquos fast acting, first line of immunological defense against organisms that have breached barrier defenses and have entered the vulnerable tissues of the body.

Phagocytes: Macrophages and Neutrophils

Many of the cells of the immune system have a phagocytic ability, at least at some point during their life cycles. Phagocytosis is an important and effective mechanism of destroying pathogens during innate immune responses. The phagocyte takes the organism inside itself as a phagosome, which subsequently fuses with a lysosome and its digestive enzymes, effectively killing many pathogens. On the other hand, some bacteria including Mycobacteria tuberculosis, the cause of tuberculosis, may be resistant to these enzymes and are therefore much more difficult to clear from the body. Macrophages, neutrophils, and dendritic cells are the major phagocytes of the immune system.

A macrophage is an irregularly shaped phagocyte that is amoeboid in nature and is the most versatile of the phagocytes in the body. Macrophages move through tissues and squeeze through capillary walls using pseudopodia. They not only participate in innate immune responses but have also evolved to cooperate with lymphocytes as part of the adaptive immune response. Macrophages exist in many tissues of the body, either freely roaming through connective tissues or fixed to reticular fibers within specific tissues such as lymph nodes. When pathogens breach the body&rsquos barrier defenses, macrophages are the first line of defense (Table 2). They are called different names, depending on the tissue: Kupffer cells in the liver, histiocytes in connective tissue, and alveolar macrophages in the lungs.

A neutrophil is a phagocytic cell that is attracted via chemotaxis from the bloodstream to infected tissues. These spherical cells are granulocytes. A granulocyte contains cytoplasmic granules, which in turn contain a variety of vasoactive mediators such as histamine. In contrast, macrophages are agranulocytes. An agranulocyte has few or no cytoplasmic granules. Whereas macrophages act like sentries, always on guard against infection, neutrophils can be thought of as military reinforcements that are called into a battle to hasten the destruction of the enemy. Although, usually thought of as the primary pathogen-killing cell of the inflammatory process of the innate immune response, new research has suggested that neutrophils play a role in the adaptive immune response as well, just as macrophages do.

A monocyte is a circulating precursor cell that differentiates into either a macrophage or dendritic cell, which can be rapidly attracted to areas of infection by signal molecules of inflammation.

Table 2: Phagocytic Cells of the Innate Immune System

Cell Cell type Primary location Function in the innate immune response
Macrophage Agranulocyte Body cavities/organs Phagocytosis
Neutrophil Granulocyte Blood Phagocytosis
Monocyte Agranulocyte Blood Precursor of macrophage/dendritic cell

Natural Killer Cells

NK cells are a type of lymphocyte that have the ability to induce apoptosis, that is, programmed cell death, in cells infected with intracellular pathogens such as obligate intracellular bacteria and viruses. NK cells recognize these cells by mechanisms that are still not well understood, but that presumably involve their surface receptors. NK cells can induce apoptosis, in which a cascade of events inside the cell causes its own death by either of two mechanisms:

1) NK cells are able to respond to chemical signals and express the fas ligand. The fas ligand is a surface molecule that binds to the fas molecule on the surface of the infected cell, sending it apoptotic signals, thus killing the cell and the pathogen within it or

2) The granules of the NK cells release perforins and granzymes. A perforin is a protein that forms pores in the membranes of infected cells. A granzyme is a protein-digesting enzyme that enters the cell via the perforin pores and triggers apoptosis intracellularly.

Both mechanisms are especially effective against virally infected cells. If apoptosis is induced before the virus has the ability to synthesize and assemble all its components, no infectious virus will be released from the cell, thus preventing further infection.

Recognition of Pathogens

Cells of the innate immune response, the phagocytic cells, and the cytotoxic NK cells recognize patterns of pathogen-specific molecules, such as bacterial cell wall components or bacterial flagellar proteins, using pattern recognition receptors. A pattern recognition receptor (PRR) is a membrane-bound receptor that recognizes characteristic features of a pathogen and molecules released by stressed or damaged cells.

These receptors, which are thought to have evolved prior to the adaptive immune response, are present on the cell surface whether they are needed or not. Their variety, however, is limited by two factors. First, the fact that each receptor type must be encoded by a specific gene requires the cell to allocate most or all of its DNA to make receptors able to recognize all pathogens. Secondly, the variety of receptors is limited by the finite surface area of the cell membrane. Thus, the innate immune system must &ldquoget by&rdquo using only a limited number of receptors that are active against as wide a variety of pathogens as possible. This strategy is in stark contrast to the approach used by the adaptive immune system, which uses large numbers of different receptors, each highly specific to a particular pathogen.

Should the cells of the innate immune system come into contact with a species of pathogen they recognize, the cell will bind to the pathogen and initiate phagocytosis (or cellular apoptosis in the case of an intracellular pathogen) in an effort to destroy the offending microbe. Receptors vary somewhat according to cell type, but they usually include receptors for bacterial components and for complement, discussed below.

Soluble Mediators of the Innate Immune Response

The previous discussions have alluded to chemical signals that can induce cells to change various physiological characteristics, such as the expression of a particular receptor. These soluble factors are secreted during innate or early induced responses, and later during adaptive immune responses.

Cytokines and Chemokines

A cytokine is signaling molecule that allows cells to communicate with each other over short distances. Cytokines are secreted into the intercellular space, and the action of the cytokine induces the receiving cell to change its physiology. A chemokine is a soluble chemical mediator similar to cytokines except that its function is to attract cells (chemotaxis) from longer distances.

Visit the website linked to below to learn about phagocyte chemotaxis. Phagocyte chemotaxis is the movement of phagocytes according to the secretion of chemical messengers in the form of interleukins and other chemokines. By what means does a phagocyte destroy a bacterium that it has ingested?

Early induced Proteins

Early induced proteins are those that are not constitutively present in the body, but are made as they are needed early during the innate immune response. Interferons are an example of early induced proteins. Cells infected with viruses secrete interferons that travel to adjacent cells and induce them to make antiviral proteins. Thus, even though the initial cell is sacrificed, the surrounding cells are protected. Other early induced proteins specific for bacterial cell wall components are mannose-binding protein and C-reactive protein, made in the liver, which bind specifically to polysaccharide components of the bacterial cell wall. Phagocytes such as macrophages have receptors for these proteins, and they are thus able to recognize them as they are bound to the bacteria. This brings the phagocyte and bacterium into close proximity and enhances the phagocytosis of the bacterium by the process known as opsonization. Opsonization is the tagging of a pathogen for phagocytosis by the binding of an antibody or an antimicrobial protein.

Complement System

The complement system is a series of proteins constitutively found in the blood plasma. As such, these proteins are not considered part of the early induced immune response , even though they share features with some of the antibacterial proteins of this class. Made in the liver, they have a variety of functions in the innate immune response, using what is known as the &ldquoalternate pathway&rdquo of complement activation. Additionally, complement functions in the adaptive immune response as well, in what is called the classical pathway. The complement system consists of several proteins that enzymatically alter and fragment later proteins in a series, which is why it is termed cascade. Once activated, the series of reactions is irreversible, and releases fragments that have the following actions:

  • Bind to the cell membrane of the pathogen that activates it, labeling it for phagocytosis (opsonization)
  • Diffuse away from the pathogen and act as chemotactic agents to attract phagocytic cells to the site of inflammation
  • Form damaging pores in the plasma membrane of the pathogen

Figure 2 shows the classical pathway, which requires antibodies of the adaptive immune response. The alternate pathway does not require an antibody to become activated.

Figure 2: The classical pathway, used during adaptive immune responses, occurs when C1 reacts with antibodies that have bound an antigen.

The splitting of the C3 protein is the common step to both pathways. In the alternate pathway, C3 is activated spontaneously and, after reacting with the molecules factor P, factor B, and factor D, splits apart. The larger fragment, C3b, binds to the surface of the pathogen and C3a, the smaller fragment, diffuses outward from the site of activation and attracts phagocytes to the site of infection. Surface-bound C3b then activates the rest of the cascade, with the last five proteins, C5&ndashC9, forming the membrane-attack complex (MAC). The MAC can kill certain pathogens by disrupting their osmotic balance. The MAC is especially effective against a broad range of bacteria. The classical pathway is similar, except the early stages of activation require the presence of antibody bound to antigen, and thus is dependent on the adaptive immune response. The earlier fragments of the cascade also have important functions. Phagocytic cells such as macrophages and neutrophils are attracted to an infection site by chemotactic attraction to smaller complement fragments. Additionally, once they arrive, their receptors for surface-bound C3b opsonize the pathogen for phagocytosis and destruction.

Inflammatory Response

The hallmark of the innate immune response is inflammation . Inflammation is something everyone has experienced. Stub a toe, cut a finger, or do any activity that causes tissue damage and inflammation will result, with its four characteristics: heat, redness, pain, and swelling (&ldquoloss of function&rdquo is sometimes mentioned as a fifth characteristic). It is important to note that inflammation does not have to be initiated by an infection, but can also be caused by tissue injuries. The release of damaged cellular contents into the site of injury is enough to stimulate the response, even in the absence of breaks in physical barriers that would allow pathogens to enter (by hitting your thumb with a hammer, for example). The inflammatory reaction brings in phagocytic cells to the damaged area to clear cellular debris and to set the stage for wound repair (Figure 3).

This reaction also brings in the cells of the innate immune system, allowing them to get rid of the sources of a possible infection. Inflammation is part of a very basic form of immune response. The process not only brings fluid and cells into the site to destroy the pathogen and remove it and debris from the site, but also helps to isolate the site, limiting the spread of the pathogen. Acute inflammation is a short-term inflammatory response to an insult to the body. If the cause of the inflammation is not resolved, however, it can lead to chronic inflammation, which is associated with major tissue destruction and fibrosis. Chronic inflammation is ongoing inflammation. It can be caused by foreign bodies, persistent pathogens, and autoimmune diseases such as rheumatoid arthritis.

There are four important parts to the inflammatory response:

  • Tissue Injury. The released contents of injured cells stimulate the release of mast cell granules and their potent inflammatory mediators such as histamine, leukotrienes, and prostaglandins. Histamine increases the diameter of local blood vessels (vasodilation), causing an increase in blood flow. Histamine also increases the permeability of local capillaries, causing plasma to leak out and form interstitial fluid. This causes the swelling associated with inflammation.
    Additionally, injured cells, phagocytes, and basophils are sources of inflammatory mediators, including prostaglandins and leukotrienes. Leukotrienes attract neutrophils from the blood by chemotaxis and increase vascular permeability. Prostaglandins cause vasodilation by relaxing vascular smooth muscle and are a major cause of the pain associated with inflammation. Nonsteroidal anti-inflammatory drugs such as aspirin and ibuprofen relieve pain by inhibiting prostaglandin production.
  • Vasodilation. Many inflammatory mediators such as histamine are vasodilators that increase the diameters of local capillaries. This causes increased blood flow and is responsible for the heat and redness of inflamed tissue. It allows greater access of the blood to the site of inflammation.
  • Increased Vascular Permeability. At the same time, inflammatory mediators increase the permeability of the local vasculature, causing leakage of fluid into the interstitial space, resulting in the swelling, or edema, associated with inflammation.
  • Recruitment of Phagocytes. Leukotrienes are particularly good at attracting neutrophils from the blood to the site of infection by chemotaxis. Following an early neutrophil infiltrate stimulated by macrophage cytokines, more macrophages are recruited to clean up the debris left over at the site. When local infections are severe, neutrophils are attracted to the sites of infections in large numbers, and as they phagocytose the pathogens and subsequently die, their accumulated cellular remains are visible as pus at the infection site.

Overall, inflammation is valuable for many reasons. Not only are the pathogens killed and debris removed, but the increase in vascular permeability encourages the entry of clotting factors, the first step towards wound repair. Inflammation also facilitates the transport of antigen to lymph nodes by dendritic cells for the development of the adaptive immune response.

Chapter Review

Innate immune responses are critical to the early control of infections. Whereas barrier defenses are the body&rsquos first line of physical defense against pathogens, innate immune responses are the first line of physiological defense. Innate responses occur rapidly, but with less specificity and effectiveness than the adaptive immune response. Innate responses can be caused by a variety of cells, mediators, and antibacterial proteins such as complement. Within the first few days of an infection, another series of antibacterial proteins are induced, each with activities against certain bacteria, including opsonization of certain species. Additionally, interferons are induced that protect cells from viruses in their vicinity. Finally, the innate immune response does not stop when the adaptive immune response is developed. In fact, both can cooperate and one can influence the other in their responses against pathogens.

CRISPR-Cas: biology, mechanisms and relevance

Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes—termed spacers—into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent.

This article is part of the themed issue ‘The new bacteriology’.

1. Introduction

Being the most abundant entities on our planet, bacterial and archaeal viruses (bacteriophages or phages) display a constant threat to prokaryotic life. In order to withstand phages, prokaryotes have evolved several defence strategies. In the past decade, the prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) caught increasing attention in the scientific community not only because of its unique adaptive nature, but also because of its therapeutic potential. This review seeks to summarize the major discoveries made in the field of CRISPR-Cas, and describes the biological roles of the system in antiviral defence and other biological pathways as well as its significance for medical application.

CRISPR-Cas is the only adaptive immune system in prokaryotes known so far. In this system, small guide RNAs (crRNAs) are employed for sequence-specific interference with invading nucleic acids. CRISPR-Cas comprises a genomic locus called CRISPR that harbours short repetitive elements (repeats) separated by unique sequences (spacers), which can originate from mobile genetic elements (MGEs) such as bacteriophages, transposons or plasmids. The so-called CRISPR array is preceded by an AT-rich leader sequence and is usually flanked by a set of cas genes encoding the Cas proteins [1–4]. To date, CRISPR-Cas systems can be divided into two main classes, which are further classified into six types and several sub-types [5–7]. The classification is based on the occurrence of effector Cas proteins that convey immunity by cleaving foreign nucleic acids. In class 1 CRISPR-Cas systems (types I, III and IV), the effector module consists of a multi-protein complex whereas class 2 systems (types II, V and VI) use only one effector protein [5].

2. Molecular mechanisms: adaptation, maturation and interference

The CRISPR-Cas system acts in a sequence-specific manner by recognizing and cleaving foreign DNA or RNA. The defence mechanism can be divided into three stages: (i) adaptation or spacer acquisition, (ii) crRNA biogenesis, and (iii) target interference (figure 1).

Figure 1. Simplified model of the immunity mechanisms of class 1 and class 2 CRISPR-Cas systems. The CRISPR-Cas systems are composed of a cas operon (blue arrows) and a CRISPR array that comprises identical repeat sequences (black rectangles) that are interspersed by phage-derived spacers (coloured rectangles). Upon phage infection, a sequence of the invading DNA (protospacer) is incorporated into the CRISPR array by the Cas1–Cas2 complex. The CRISPR array is then transcribed into a long precursor CRISPR RNA (pre-crRNA), which is further processed by Cas6 in type I and III systems (processing in type I-C CRISPR-Cas systems by Cas5d). In type II CRISPR-Cas systems, crRNA maturation requires tracrRNA, RNase III and Cas9, whereas in type V-A systems Cpf1 alone is sufficient for crRNA maturation. In the interference state of type I systems, Cascade is guided by crRNA to bind the foreign DNA in a sequence-specific manner and subsequently recruits Cas3 that degrades the displaced strand through its 3′–5′ exonucleolytic activity. Type III-A and type III-B CRISPR-Cas systems employ Csm and Cmr complexes, respectively, for cleavage of DNA (red triangles) and its transcripts (black triangles). A ribonucleoprotein complex consisting of Cas9 and a tracrRNA : crRNA duplex targets and cleaves invading DNA in type II CRISPR-Cas systems. The crRNA-guided effector protein Cpf1 is responsible for target degradation in type V systems. Red triangles represent the cleavage sites of the interference machinery.

(a) Adaptation

In a first phase, a distinct sequence of the invading MGE called a protospacer is incorporated into the CRISPR array yielding a new spacer. This event enables the host organism to memorize the intruder's genetic material and displays the adaptive nature of this immune system [1]. Two proteins, Cas1 and Cas2, seem to be ubiquitously involved in the spacer acquisition process as they can be found in almost all CRISPR-Cas types. Exceptions are the type III-C, III-D and IV CRISPR-Cas systems, which harbour no homologous proteins. Moreover, type V-C shows a minimal composition as it comprises only a putative effector protein termed C2C3 and a Cas1 homologue [5–7]. In past years, major advances have been made in revealing the biochemical and genetic principles of CRISPR-Cas immunity. However, the mechanism of spacer acquisition is still not fully understood [8,9]. The selection of protospacers and their processing before integration remain widely obscure in many CRISPR-Cas types. Recent findings, however, shed light on the biochemistry of the spacer integration process. It has been demonstrated that Cas1 and Cas2 of the type I-E system of Escherichia coli form a complex that promotes the integration of new spacers in a manner that is reminiscent of viral integrases and transposases [10–13]. Although both Cas1 and Cas2 are nucleases [14–16], the catalytically active site of Cas2 is dispensable for spacer acquisition [10–12]. A new spacer is usually incorporated at the leader-repeat boundary of the CRISPR array [1] while the first repeat of the array is duplicated [17,18].

The mechanisms of the different CRISPR-Cas types might be conserved only to a certain extent as several studies have shown variations regarding the requirements and targets of the adaptation machinery. While Cas1 and Cas2 are sufficient to promote spacer acquisition in most studied type I CRISPR-Cas systems, type I-B further requires Cas4 for adaptation [19]. The type I-F CRISPR-Cas system of Pseudomonas aeruginosa additionally requires the interference machinery to promote the uptake of new spacers [20]. Similarly, type II-A systems require Csn2, Cas9 and tracrRNA (trans activating CRISPR RNA—see further details below) for acquisition [1,21,22]. Another, so far unique, adaptation mode was revealed for a type III-B Cas1 protein that is fused to a reverse transcriptase. Here, acquisition from both DNA and RNA was reported [23].

The selection of a target sequence that is integrated into the CRISPR locus is not random. It has been demonstrated that in type I, II and V CRISPR-Cas systems, a short sequence, called the protospacer adjacent motif (PAM), is located directly next to the protospacer and is crucial for acquisition and interference [24–29]. In type II-A CRISPR-Cas systems, the PAM-recognizing domain of Cas9 is responsible for protospacer selection [21,22]. It is believed that after protospacer selection, Cas9 recruits Cas1, Cas2 and possibly Csn2 for integration of the new spacer into the CRISPR array. This feature may be conserved among all class 2 CRISPR-Cas systems although experimental evidence is missing. For type I-E, the Cas1–Cas2 complex is sufficient for spacer selection and integration although it has been reported that the presence of the interference complex increases the frequency of integrated spacers that are adjacent to a proper PAM [24,25]. Moreover, in a process called priming, the interference machinery of several type I CRISPR-Cas systems can stimulate the increased uptake of new spacers upon crRNA-guided binding to a protospacer that was selected upon a first infection [19,25,30]. This process displays a distinct adaptation mode compared to naive spacer acquisition as it strictly requires a pre-existing spacer matching the target. It usually leads to higher acquisition rates from protospacers that lie in close proximity to the target site [25]. Interestingly, primed spacer acquisition does not depend on target cleavage as it also functions for degenerated target sites that would usually result in impaired interference [31]. The exact mechanism remains obscure but it has been demonstrated that the interference complex can recruit Cas1 and Cas2 during PAM-independent binding to DNA [32].

(b) Biogenesis

To enable immunity, the CRISPR array is transcribed into a long precursor crRNA (pre-crRNA) that is further processed into mature guide crRNAs containing the memorized sequences of invaders [33,34]. In type I and III systems, members of the Cas6 family perform the processing step yielding intermediate species of crRNAs that are flanked by a short 5′ tag. One exception is given by the type I-C systems, which do not code for Cas6 proteins. Here, the protein Cas5d processes pre-crRNA resulting in intermediate crRNAs with an 11 nt 5′ tag [33,35–38]. Further trimming of the 3′ end of the intermediate crRNA by an unknown nuclease can occur and yields mature crRNA species composed of a full spacer portion (5′ end) and a repeat-portion (3′ end), which usually displays a hairpin structure in most type I systems [39–41]. The maturation of crRNAs in class 2 CRISPR-Cas systems differs significantly. In type II systems, tracrRNA is required for the processing of the pre-crRNA. The anti-repeat sequence of this RNA enables the formation of an RNA duplex with each of the repeats of the pre-crRNA, which is stabilized by Cas9. The duplex is then recognized and processed by the host RNase III yielding an intermediate form of crRNA that undergoes further maturation by a still unknown mechanism to lead to the mature small guide RNA [42]. An RNase III-independent mechanism was discovered in the type II-C CRISPR-Cas system of Neisseria meningitidis. Here, promoter sequences were identified to lie within each repeat and some were able to initiate transcription leading to intermediate crRNA species. Even though RNase III-mediated 3′ processing of the crRNA : tracrRNA duplex was observed, it was dispensable for interference [43]. In the type V-A CRISPR-Cas system, it has been shown that Cpf1 has a dual function during CRISPR-Cas immunity. Cpf1 processes premature crRNAs [28] and, following a further maturation event of unknown nature, uses the processed crRNAs that it has generated to cleave target DNA [28,29].

(c) Interference

In the last stage of immunity, mature crRNAs are used as guides to specifically interfere with the invading nucleic acids. Class 1 systems employ Cascade (CRISPR-associated complex for antiviral defence)-like complexes to achieve target degradation, while in class 2 systems, a single effector protein is sufficient for target interference [29,39,44–49]. To avoid self-targeting, type I, II and V systems specifically recognize the PAM sequence that is located upstream (types I and V) or downstream (type II) of the protospacer [26,28,29,31,45,50–52]. In type III systems, the discrimination between self and non-self is achieved via the 5′ tag of the mature crRNA, which must not base pair with the target to enable degradation by the complex [53].

In type I systems, Cascade localizes invading DNA in a crRNA-dependent manner and further recruits the nuclease Cas3 for target degradation. Cas3 induces a nick on the foreign DNA and subsequently degrades the target DNA [54,55]. In type II CRISPR-Cas systems, the tracrRNA:crRNA duplex guides the effector protein Cas9 to introduce a double-strand break in the target DNA [45]. The interference machinery of type III systems comprises Cas10-Csm (types III-A and III-D) and Cas10-Cmr (types III-B and III-C) complexes [5], which are able to target both DNA and RNA [38,39,47,49,56–63]. Intriguingly, it has been shown that interference of type III-A and type III-B systems depends on the transcription of the target DNA [57,58]. More precisely, the subunit Cas10 cleaves the DNA while Csm3 [59,60] and Cmr4 [61] cleave the transcribed mRNA in type III-A and type III-B CRISPR-Cas systems, respectively. Interference in type V CRISPR-Cas systems shows similarities to interference in type II. An RNA duplex, consisting of tracrRNA and crRNA, is strictly required for target cleavage in type V-B systems [7]. Type V-A, however, only employ crRNA for target localization and degradation [28,29].

3. Anti-CRISPR mechanisms

Prokaryotes harbour a remarkable arsenal of defence strategies in order to coexist with their viral predators (box 1). As a part of the constant arms race between bacteria and their viral counterparts, phages have evolved different strategies to overcome antiviral defence mechanisms. This paragraph summarizes the research on how phages evade the CRISPR-Cas systems.

Box 1. CRISPR-Cas – What else? (Alternative defence mechanisms in bold type)

Apart from CRISPR-Cas systems, prokaryotes have evolved a comprehensive set of defence mechanisms to protect themselves against predators. The viral infection cycle is initiated by adsorption of the phage onto the bacterial cell surface, where the phages recognize host-specific receptors on the outer membranes or cell walls of the host. Bacteria can prevent phage adsorption by producing an extracellular matrix that physically blocks the access to the specific receptor. Further counter-strategies involve mutating phage receptors and production of competitive inhibitors that occupy the receptor and thus lead to a reduced susceptibility to phage adsorption [64–66]. In the next step of infection, phages inject their genetic material into the host. In order to block the entry of viral DNA, bacteria use the so-called superinfection exclusion (Sie) systems that are often encoded by prophages. These systems comprise a set of proteins that prevent translocation of phage DNA into the cytoplasm [67,68].

Once entered, viral DNA can be degraded by restriction-modification (RM) systems that use nucleases to recognize and cleave short motifs present on the invading DNA. Non-methylated DNA is recognized by these restriction enzymes and self-cleavage is prevented by methylation of target sites on the host genome [69,70]. Another defence strategy blocks phage propagation by sacrificing an infected host cell, thus protecting the bacterial population. These abortive infection (Abi) mechanisms use proteins that sense infections and consequently induce cell death through, e.g. membrane depolarization, inhibiting the host's translational apparatus or exploiting components of toxin-antitoxin systems [71–74]. Less well-characterized antiviral systems encompass bacteriophage exclusion (BREX) and prokaryotic Argonautes. While BREX inhibits viral replication and DNA integration of lysogenic phages [75], Argonaute proteins are DNA- or RNA-guided nucleases that cleave invading DNA in a sequence-specific manner [76–78].

Phages can escape the CRISPR-Cas interference machinery through random mutations in the protospacer region or the PAM sequence [26,51]. As a counter strategy, several type I CRISPR-Cas systems show an elevated uptake of new spacers as a direct result of mismatches in the PAM or in the targeted protospacer during primed acquisition (see §2). Moreover, the efficiency of escaping CRISPR-Cas immunity by point mutations is strongly impaired in bacterial populations that show high spacer diversity. A possible explanation for this observation is that spacer diversity increases the adaptive pressure on the virus and thus leads to rapid extinction of the predator [79].

Recent studies demonstrated that Mu-like phages, which infect Pseudomonas aeruginosa, actively inhibit their host's CRISPR-Cas systems. These phages produce anti-CRISPR (Acr) proteins that interact with components of the type I-F CRISPR-Cas interference machinery: e.g. the phage proteins AcrF1 and AcrF2 bind different subunits of Cascade and thus prevent the binding of the Csy complex to the target DNA. AcrF3 was shown to bind the nuclease Cas3, inhibiting its function in target degradation. Similar proteins were found to prevent type I-E CRISPR-Cas immunity in the same organism, thus raising the question whether Acr proteins exist for other CRISPR-Cas types [20,80–82].

In a so-far unique report of immune evasion, it has been shown that Vibrio cholerae ICP1 phages encode a type I-F CRISPR-Cas system that targets a host genomic island, known to be involved in CRISPR-unrelated anti-phage defence. Attacking the host's defence mechanism was crucial for phage propagation as the efficiency of infection was greatly reduced when targeting spacers in the viral CRISPR array were deleted. Intriguingly, analysis of phages that still managed to successfully infect the host acquired new spacers that originated from the same genomic locus, thus showing that the virus harbours a fully functional CRISPR-Cas system that is also active in acquisition [83].

4. Beyond adaptive immunity

Besides their role in prokaryotic immunity, CRISPR-Cas systems have been shown to participate in cellular pathways other than immunity.

(a) DNA repair

Early reports suggested an involvement of the E. coli Cas1 protein in DNA repair pathways since the protein was shown to interact with components of the cellular repair machinery like RecB, RecC and RuvB. Cas1 further processed intermediate DNA structures that often occur during DNA repair and recombination like Holliday junctions, replication forks and 5′-flaps. Moreover, deletions of the cas1 gene resulted in increased sensitivity towards DNA damage and affected chromosome segregation [14]. Moreover, the participation of the RecBCD recombination system in CRISPR-Cas immunity has become more evident in recent years. The RecBCD complex recognizes double-strand DNA (dsDNA) breaks that often occur at replication forks. After recognizing damaged DNA, RecBCD subsequently degrades the DNA until it reaches a Chi site [84]. A recent study suggested that the degradation products of the repair complex serve as templates for spacer acquisition as new spacers were mainly acquired from regions that lie in close proximity of stalled replication forks. With regard to antiviral immunity, RecBCD might be the first line of defence as it recognizes and degrades linear phage DNA and thus enables the adaptation machinery to collect new spacers. Acquisition from chromosomal DNA is prevented due to the frequent distribution of Chi sites within the host genome [85]. The requirement of RecB for type I-E CRISPR-Cas immunity was ultimately proven by another study demonstrating that a recB deletion abolished naive spacer acquisition in E. coli. Interestingly, the absence of RecB did not affect primed spacer acquisition. Here, the helicases RecG and PriA were essential, whereas DNA polymerase I was crucial for both, naive and primed adaptation [86]. The emerged model suggests that, during primed adaptation, RecG and PriA recognize the R-loop structure that occurs by binding of the Cascade : crRNA complex to the DNA. As a result, Cascade dissociates from the DNA leading to the exposure of temporary single-stranded DNA regions that may stimulate spacer acquisition by Cas1 and Cas2. As Cas3 is essential for primed adaptation, the nuclease activity of the protein is likely to promote the generation of DNA fragments that are captured by the acquisition machinery. During naive adaptation, DNA damage is possibly induced by Cas1 and leads to the recruitment of the RecBCD complex as described above [85,86].

(b) Gene regulation

The involvement of CRISPR-Cas components in cellular regulatory processes became more evident in the last few years. Type II CRISPR-Cas systems seem to play a significant role in regulating virulence of pathogenic bacteria. In Francisella novicida, a ribonucleoprotein complex consisting of Cas9, tracrRNA and a unique small CRISPR-associated RNA represses the expression of a bacterial lipoprotein (BLP). Transcriptional downregulation of BLP is crucial for immune evasion as the protein can be recognized by the host's immune system. It is assumed that the ribonucleoprotein complex binds the blp transcript leading to degradation of the mRNA by Cas9 or an unknown nuclease. As a consequence, BLP is underrepresented on the cell surface resulting in a reduced immune response [87]. Similarly, Neisseria meningitidis mutant strains that lack a cas9 gene showed severe survival defects in human epithelial cells [87]. In Campylobacter jejuni, cas9 deletion mutants displayed less cytotoxicity in human cell lines. Presumably, the absence of C. jejuni Cas9 affects the biochemical composition of the bacterial cell wall and thus makes the cell more prone to antibody binding [88]. A type II-B Cas2 protein of Legionella pneumophila was crucial for infection of amoebae and thus represents another virulence-related function [89]. Transcription of an abandoned CRISPR array (no cas operon) in Listeria monocytogenes leads to the stabilization of a partially matching mRNA. Interestingly, if the CRISPR array is removed from the genome, the bacteria were able to colonize a murine liver more efficiently providing evidence for a regulatory function in virulence by an antisense RNA mechanism [90].

Endogenous regulation by CRISPR-Cas can also affect group behaviour in a bacterial population as identified in the life cycle of Myxococcus xanthus. The δ-preoteobacterium is able to produce myxospores to overcome environmental stresses like nutrient deficiency. Myxospores are produced during a complicated process that involves cooperated movement and aggregation of cells within a population. As a result, cell aggregates differentiate into the so-called fruiting bodies that contain the spores. Myxococcus xanthus possesses a type I-C CRISPR-Cas system and deletions of cas7 and cas5 lead to highly decreased sporulation. The same was true for a cas8c deletion that additionally resulted in delayed cell aggregation. Moreover, Cas8c stimulates synthesis of FruA, an important protein in the sporulation pathway [91–93]. The mechanistical involvement of Cas proteins in the formation of the fruiting body remains puzzling as a recent study added yet another level of complexity by demonstrating the involvement of a type III-B CRISPR array in fruiting body development and production of exopolysaccharides [94].

(c) Genome evolution

The acquisition of foreign DNA spacers is a crucial step in CRISPR-Cas immunity and displays the unique adaptive nature of this defence system. It has been widely reported that in some cases, spacers are derived from own genomic sequences. Targeting of the chromosome, however, results in DNA damage and will inevitably kill the bacterial cell. While self-targeting of CRISPR-Cas systems can definitely be lethal for a host organism, several studies investigated its potential role in genome evolution. Besides small-scale genetic modifications like mutations in chromosomal PAM sequences, protospacers or the cas operon, large genomic rearrangements were observed in Pectobacterium atrosepticum when spacers matched sequences in the own genome. Here, a genomic island of approximately 100 kb that is involved in plant pathogenicity was remodelled or deleted [95]. A genomic study on Thermotogales revealed the association of CRISPR loci to sites of DNA inversions and other genetic rearrangements. Even though the exact involvement of the CRISPR arrays in fostering the observed genetic alterations remains unknown, CRISPR seems to promote these evolutionary events [96]. By contrast, one bioinformatic study suggested that self-targeting of CRISPR-Cas systems is a rather undesirable effect, because it conveys autoimmunity. As an outcome, CRISPR-Cas systems become degenerated due to mutations in cas genes and the target sites or by the inactivation or deletion of whole CRISPR-Cas systems which, indeed, promotes evolutionary variations but simultaneously leads to a loss of fitness regarding antiviral defence [97].

5. Significance and applications

The use of CRISPR-Cas in therapeutic approaches has become increasingly relevant in different fields of medicine. The presence of repetitive sequences interspersed with short spacers, later known as CRISPR, has been exploited for diagnostic purposes and simple typing of Mycobacterium tuberculosis strains [98]. This helped to understand ways used by pathogens for their transmission by looking at differences in the spacer content of related strains [98,99]. This so-called spoligotyping (spacer oligotyping) has also been adapted for Salmonella enterica, Yersinia pestis and Corynebacterium diphteriae [100–105].

The use of CRISPR-Cas as a direct antimicrobial tool has been studied recently. Artificial CRISPR arrays have been designed to kill pathogenic bacteria by targeting antibiotic resistance or virulence genes. This elegant way only aims for harmful strains in a bacterial population and allows non-pathogenic strains to overgrow the pathogens [106–108]. A recent study used lysogenic phages to introduce a CRISPR-Cas system in E. coli, which targets antibiotic resistance genes. The array was designed to additionally target the genomes of lytic phages leading to immunity towards phages only of antibiotic-sensitized bacteria. More precisely, bacteria that are unlikely to acquire antibiotic resistance genes due to their engineered spacer content are also resistant to lytic phages. Thus, in the case of a phage infection, only pathogenic strains would be eradicated from the population [109].

The medical potential of the CRISPR-Cas systems goes beyond antimicrobial treatment. The introduction of efficient and precise modifications into genes of an organism displays the basis for genome engineering. Programmable nucleases are used that specifically bind genomic regions and cleave the DNA at a desired position. Zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs) have been widely used to edit DNA. Both genome editing tools rely on the same principle: a sequence-specific DNA binding domain, which provides specificity, is fused to a nuclease. Because of its simplicity, effectiveness and the possibility to target multiple genomic sites simultaneously, use of the CRISPR-Cas9 system is usually favoured over ZFN and TALEN systems [110–112]. The bacterial defence protein Cas9 is used to target almost any desired DNA sequence with the help of a targeting RNA. This single-guide RNA (sgRNA) is an engineered hybrid of the naturally occurring tracrRNA:crRNA duplex and thus simplifies its application for genome editing purposes [45].

Repurposing the CRISPR-Cas9 system for genome editing exploits the DNA repair mechanisms of eukaryotic cells: after the introduction of a double-strand DNA break, the cell can repair the damage by non-homologous end joining (NHEJ). This process is error-prone and often leads to point mutations, deletions or causes frameshifts that alter the gene product and eventually abolishes its function, which is favoured for genetic knockouts. Precise genome engineering, however, relies on another pathway, termed homology-directed repair (HDR), where a piece of DNA that shows sequence homology to the target site is used to repair the DNA via homologous recombination. This short DNA sequence can harbour any sort of insertion or alteration, allowing the integration of any desirable DNA sequence at the target site [50,113–117] (figure 2a).

Figure 2. Applications of the CRISPR-Cas9 technology. (a) Cas9 is guided by a sgRNA to induce a double-strand DNA break at a desired genomic locus. The DNA damage can be repaired by NHEJ yielding short random insertions or deletions at the target site. Alternatively, a DNA sequence that shows partial complementarity to the target site can be inserted during HDR for precise genome editing purposes. (b) Mutations in the catalytical domains of Cas9 yield a dead variant (dCas9) that binds but does not cleave DNA. The approach with dCas9 is used for transcriptional repression by binding to the promoter region of a gene and thus blocking the access for the RNA polymerase. Similarly, dCas9 can be fused to a transcriptional repressor. Red crosses represent inhibition of transcription. (c) The fusion of dCas9 to a transcriptional activator stimulates transcription of an adjacent gene by recruiting the RNA polymerase.

Mutations in the nuclease motifs of Cas9 lead to a ‘dead’ variant that is unable to cleave DNA and thus can be used to regulate transcription of a desired gene. By targeting the promoter region or the open reading frame of a gene, binding of the RNA polymerase is physically blocked and mRNA elongation is inhibited. Alternatively, dCas9 can be fused to a repressor that controls gene transcription (figure 2b). Transcriptional activation can be achieved by fusing dCas9 to a transcription activator that recruits the RNA polymerase and induces gene expression (figure 2c). In some cases, gene knock-downs are desired over gene knockouts, e.g. if the targeted gene is essential [116,118–122]. The CRISPR-Cas9 method has also been exploited for epigenome editing that allows the control of gene expression by introducing modifications like DNA methylation or histone acetylation. One study showed that a fusion protein of dCas9 and the core domain of the human acetyltransferase p300 could activate gene expression at specific sites [123]. Moreover, fusion of dCas9 to the KRAB repressor was able to induce methylation at specific enhancers leading to reduced chromatin accessibility and, thus, silencing of gene expression [124]. Precise epigenome editing has great potential to reveal site-specific chromatin modification and helps to explore the regulation of gene expression that could lead to new therapeutical strategies. Other approaches—mainly in prokaryotes—exploit the endogenous type I effector complex Cascade for similar experiments if the nuclease Cas3 is absent [125–127]. In all of the aforementioned genome manipulation strategies, the existence of a PAM adjacent to the target site is a strict requirement [9].

Precise remodelling of the genome can be used to cure gene variants that cause genetic diseases. Scientists were able to repair mutations that cause cystic fibrosis (CF) by correcting the cftr locus in cultured intestinal stem cells of CF patients [128]. Moreover, using the CRISPR-Cas9 technique, a healthy phenotype could be restored in mice suffering from hereditary tyrosinaemia, a genetic disease that causes severe liver damage [129]. Genome editing has been further used to develop antiviral therapeutic approaches. Accordingly, the genome of HIV has been successfully eradicated from latently infected cells [130]. Indeed, a recent study demonstrated that the generation of NHEJ-induced mutations in the viral genome led to replication defects of the virus. However, it also drove the generation of replication competent mutants that harbour mutations at the target site and thus are no longer targeted by Cas9 [131]. Other studies aim to alter a specific surface protein called CCR5 that serves as a co-receptor for the HI-virus to enter a host cell. Mutations in the ccr5 gene can prevent the virus from infecting a cell leading to a highly resistant but otherwise healthy phenotype. In fact, altering the wild-type ccr5 gene leads to immunity of monocytes and macrophages against HIV infections [132–134].

The CRISPR-Cas9 technique has further simplified genome-scale screens. These screens seek to identify genes that are involved in certain metabolic or pathogenetic processes by abolishing gene function and studying the resulting phenotype. Using this approach, genes that are involved in tumour growth [135] or convey susceptibility towards bacterial toxins [136] could be identified. Previously, RNA interference (RNAi) was used to knock down gene expression in a sequence-specific manner. However, RNAi only decreases the abundance of transcripts, whereas CRISPR-Cas9 enables a full knock-out of candidate genes. Moreover, multiplexing (targeting of several genetic loci at the same time) is crucial for this approach and can be achieved by using a library of different sgRNAs that is usually delivered with Cas9 by a lentiviral vector system [135–138].

6. Perspectives

Interest in the field of CRISPR-Cas has rapidly increased in recent years. Numerous studies shed light onto the underlying genetic and biochemical processes of the adaptive prokaryotic immune system thus revealing its potential in modern medicine (box 2). Undoubtedly, the versatility of different CRISPR-Cas systems is stunning and with the recent discovery of three new types we may have just begun to fully understand the significance of CRISPR-Cas as a microbial defence system. However, many aspects of the antiviral system require further insight. The process of immunization that is accomplished by incorporation of new spacers in the CRISPR array is still the most puzzling event in CRISPR-Cas immunity. The precise biochemical basis of spacer acquisition and its degree of conservation among the different types has yet to be uncovered. For instance, the function of additional proteins like Cas4 and Csn2 that have been shown to be required for adaptation needs further investigation. Primed adaptation has only been observed in type I CRISPR-Cas systems even though this process provides a great protective advantage towards mutated phages that would escape CRISPR interference.

Box 2. Milestones in CRISPR-Cas research.

First described in 1987 as unusual repetitive sequences [139], the interest in CRISPRs and their associated genes slowly increased throughout the 1990s and early 2000s. Initially believed to participate in cellular DNA repair and replicon partitioning processes, first evidence that CRISPR-Cas systems display an adaptive prokaryotic immune system was delivered in 2005 [4]. Researchers were surprised as they found that most of the interspersed sequences interspaced between identical repeats derived from extra chromosomal DNA, more specifically from phage genomes and conjugative plasmids [4,100,140]. The hypothesis was eventually proven two years later when scientists showed the incorporation of new spacers into a CRISPR-Cas locus of Streptococcus thermophilus after challenging the bacterium with a bacteriophage. The newly acquired spacers always showed perfect complementarity to sequences on the phage genome and conveyed resistance towards that particular phage upon a subsequent infection [1]. Research interest of the CRISPR field soon accelerated, leading to new discoveries that helped to understand the basic mechanisms of the immune system. In 2008, the processing of the CRISPR transcript into mature crRNAs that guide the Cascade complex of the E. coli type I-E system was experimentally validated, also giving hints that DNA rather than RNA is targeted [54]. The latter was confirmed in the same year as a study demonstrated that indeed DNA is the targeted molecule [56]. This led scientists to think about the potential role that this prokaryotic immune system might play as a DNA manipulation tool. Today, CRISPR-Cas9 is a frequently harnessed tool for genome editing purposes and major progress in understanding the underlying biochemical processes in RNA-guided Cas9 was presented in recent years. In 2010, researchers showed that Cas9 creates a single double-stranded break at a precise position on the target DNA [63]. Further insight into the mechanism was delivered 1 year later as the involvement of another small RNA, called tracrRNA, was shown. The maturation of crRNA requires tracrRNA as well as Cas9 and RNase III [42]. Evidence that the system would function heterologously in other bacteria was demonstrated in 2011, as the S. thermophilus type II CRISPR-Cas system could provide immunity in E. coli [141]. Other research had shown certain elements of the type II system, including the involvement of a PAM sequence in interference [6,26,141] but the nature of the cleavage complex remained unknown. In 2012, tracrRNA, which was previously known to be involved in crRNA maturation [42], was shown to also form an essential part of the DNA cleavage complex, with the dual tracrRNA:crRNA directing Cas9 to introduce double-strand breaks in the target DNA [45]. Further simplification of the programmed targeting was achieved by creating a single-guide RNA fusion of tracrRNA and crRNA, that guides Cas9 for sequence-specific DNA cleavage [45]. A few months following the description of the CRISPR-Cas9 technology [45], a number of publications demonstrated its power to edit genomes in eukaryotic cells and organisms, including human and mouse cells [116,117].

Another puzzling aspect is the impact of CRISPR-Cas systems on prokaryotic diversity. It has been observed that the immune systems protect not only against phages, but also against other MGEs that might have beneficial effects for an organism. In fact, the native CRISPR-Cas system is silenced in E. coli by the histone-like nucleoid structuring protein H-NS [142], raising the idea that an inactive system may be advantageous for the bacteria. In addition, CRISPR-Cas systems can interfere with plasmid conjugation and transformation of naturally competent bacteria [43,143]. Several studies show a negative correlation between the occurrence of CRISPR-Cas systems and the amount of MGEs within the chromosome, which seems like a limitation to evolutionary processes and horizontal gene transfer (HGT) [144,145]. Contradictory results were presented by an evolutionary analysis that found no significant correlation between the activity of a CRISPR-Cas system and the number of HGT events [146]. However, these relations have to be assessed in context with further factors like predatory pressure, the occurrence of other defence systems and the fitness costs that are connected to the maintenance of adaptive defence. It has been stated that bacteria may lose or inactivate their CRISPR-Cas systems when they face a high abundance of predators. In such environments, phage resistance due to, for example, receptor mutations seems to be more affordable [147]. More precisely, high viral mutation rates render adaptive immunity obsolete as the costs of adapting to a dynamic predatory habitat exceed the immunological benefits [148]. Interestingly, another study showed that simply maintaining a CRISPR-Cas system without any predatory pressure can result in an adverse balance regarding fitness costs. Here, a wild-type strain showed reduced competitive capabilities compared with a cas gene knockout mutant. In the case of phage infection, however, no increased fitness costs were observed as described above [149] and, thus demonstrating that these dynamic phage–host interactions are highly complex and need more elaboration in future scientific work.

Further research is also required on immunity-unrelated functions of CRISPR-Cas systems. Numerous studies revealed their involvement in several regulatory processes (see §4) and deeper insight is needed, for instance, when it comes to the interaction of Cas proteins with components of cellular DNA repair and recombination pathways.

Besides their fascinating role in prokaryotes, CRISPR-Cas systems undoubtedly caught most attention for their potential in medical applications and numerous other biotechnological applications like crop editing, gene drives (the ability to stimulate biased inheritance of particular genes to alter an entire population) and synthetic biology (non-medical applications are not discussed here see [150] for details). Despite the enormous potential that lies within the CRISPR-Cas9 technology, further investigation is required to make the system an applicable and safe tool for therapeutically useful approaches. Challenging issues that remain and need to be addressed in the future include off-target cleavage by Cas9. Off-target effects are a major concern when precisely remodelling the genomic content of eukaryotic cells. In some cases, genetic alterations at off-target sites were detected at higher frequencies than the desired mutation which clearly reveals the need for higher specificity of the technique [151]. Strategies preventing off-target effects include the injection of purified Cas9 directly into a cell instead of expressing the recombinant protein in the target cell. This method is convenient for fast target cleavage but also leads to the rapid decay of Cas9, thereby reducing the possibility of off-target effects [152,153]. Moreover, using two sgRNAs that target both strands of the target sequence in combination with a DNA-nicking variant of Cas9 was shown to reduce off-target effects significantly [154]. Further strategies focus on optimizing sgRNA sequences in order to achieve more reliable editing. Truncating sgRNAs by 2–3 nt was shown to improved target specificity [155]. Also, adding two guanine nucleotides at the 5′ end, directly next to the target-complementary region of the guide RNA, could reduce off-target effects [156]. Another issue that requires further investigation is the overall delivery of the CRISPR-Cas9 system into desired cells of a multicellular organism. Promising in vivo approaches include viral and non-viral vector systems that deliver Cas9 and sgRNA to the desired cells [110,157]. Moreover, ex vivo concepts rely on isolating patient-derived cells which are transplanted back after genomic editing. A major advantage in using this approach is the assessment of the genetic alteration that was introduced. Here, only correctly edited cells without malign off-target mutations are chosen for transplantation [110,157]. Although some challenges remain, it only seems to be a matter of time until CRISPR-Cas9-based genome editing will become a safe and applicable method used in a variety of therapeutic approaches.

20.5: Adaptive Immune System - Biology

My laboratory performs a research program on the host immune response in vivo with the main goal to offer valuable new ways to combat cancer. We use various modalities, including in vivo imaging, to study where, when and how immune cells are produced, traffic, and mediate regulatory or effector functions. The studies make use of both genetic mouse models, which allow manipulations and analyses of mechanisms and causality, and human patient material, to ensure that the results are anchored in clinical correlates. This dual approach gives opportunities for discovery of novel contributions of the immune response to tumor progression, new biomarkers useful for diagnosis and prognosis, and novel targets for therapeutic intervention. Dr. Pittet directs Cancer Immunology Program at CSB and collaborates with several immunology programs at Harvard Medical School, Massachusetts General Hospital and Massachusetts Institute of Technology.

In vivo imaging of immune cells at different scales. Imaging modalities include single photon emission computed tomography – X-ray computed tomography (SPECT-CT), fluorescence mediated tomography (FMT), microscopic fiber optics, and intravital multiphoton microscopy (IVM).

Recent Publications

Gerhard GM, Bill R, Messemaker M, Klein AM, Pittet MJ Tumor-infiltrating dendritic cell states are conserved across solid human cancers. J Exp Med. 2021218(1):ePub - PMID: 33601412 - PMCID: PMC7754678 - DOI: 10.1084/jem.20200264

Ko J, Wang Y, Carlson JCT, Marquard A, Gungabeesoon J, Charest A, Weitz D, Pittet MJ, Weissleder R Single Extracellular Vesicle Protein Analysis Using Immuno-Droplet Digital Polymerase Chain Reaction Amplification. Adv Biosyst. 20204(12):e1900307 - PMID: 33274611 - DOI: 10.1002/adbi.201900307

Pfirschke C, Engblom C, Gungabeesoon J, Lin Y, Rickelt S, Zilionis R, Messemaker M, Siwicki M, Gerhard GM, Kohl A, Meylan E, Weissleder R, Klein AM, Pittet MJ Tumor-Promoting Ly-6G + SiglecF high Cells Are Mature and Long-Lived Neutrophils. Cell Rep. 202032(12):108164 - PMID: 32966785 - PMCID: PMC7508173 - DOI: 10.1016/j.celrep.2020.108164

Pai SI, Faquin WC, Sadow PM, Pittet MJ, Weissleder R New Technology on the Horizon: Fast Analytical Screening Technique FNA (FAST-FNA) Enables Rapid, Multiplex Biomarker Analysis in Head and Neck Cancers. Cancer Cytopathol. 2020128(11):782-791 - PMID: 32841527 - DOI: 10.1002/cncy.22305

Koch PD, Pittet MJ, Weissleder R The chemical biology of IL-12 production via the non-canonical NFkB pathway. RSC Chemical Biology. 20201:166-176 - DOI: 10.1039/d0cb00022a

Research projects

Tumor microenvironment

In vitro studies often do not reliably predict the behavior of cells in vivo. Therefore our goals are: (a) to study cellular players directly in situ by means of appropriate bioimaging technologies, (b) to quantify and model information obtained by bioimaging, (c) to develop approaches for comprehensive investigation of various cell types in defined microenvironments. In cancer, a number of immune and nonimmune cell types respond to tumor stimuli and exhibit complex regulatory or effector functions, for example through cell-cell contact and/or secretion of soluble factors. We use recent advances in in vivo imaging to yield new insights into the biology of host cells with the ultimate goal to quantify cellular responses in vivo. As detailed below, we have focused our efforts on the study of T cell responses (anti-tumor CTL and suppressor Treg cells) and monocyte responses (monocyte subsets with distinct inflammatory potential and their lineage descendants).

Adaptive immune cells

T cells are specialized to recognize cells infected with intracellular pathogens or transformed cells expressing tumor-associated antigens. Cytotoxic T cells (CTL) execute their effector functions during direct physical interactions with their targets, which include release of lytic granules and secretion of cytokines. In contrast other T cells such as T regulatory (Treg) cells mediate dominant suppressive functions and can prevent the activity of CTL and other effector cells. Our understanding of how immune functions are regulated and integrated in vivo at the cellular level is, to a large extent, still speculative. We are studying how Treg cells repress CTL responses in vivo, and whether Treg cell-mediated tolerance can be reversed in therapy.

Innate immune cells

Mononuclear phagocytes exert crucial functions as scavengers and can trigger or regulate immune responses. Recent studies indicate that monocytes - the precursors of macrophages and dendritic cells - comprise separate subsets that 'commit' to specific functions. The activity of these subsets in vivo is largely unknown. We are studying tissue tropism, cellular differentiation and role in immunity of monocyte subsets in various inflammatory conditions. We are also testing whether these cells are potential targets in therapy.

Adaptive Biotechnologies and Microsoft expand partnership to decode COVID-19 immune response and provide open data access

SEATTLE and REDMOND, Wash. — March 20, 2020 Adaptive Biotechnologies Corp. (Nasdaq: ADPT) and Microsoft Corp. (Nasdaq: MSFT) on Friday announced they will leverage their existing partnership mapping population-wide adaptive immune responses to diseases at scale to study COVID-19. Finding the relevant immune response signature may advance solutions to diagnose, treat and prevent the disease, augmenting existing research efforts that primarily focus on the biology of the virus. These data will be made freely available to any researcher, public health official or organization around the world via an open data access portal.

“We can improve our collective understanding of COVID-19 by decoding the immune system’s response to the virus and the disease patterns that can be inferred from studying these data at the population level,” said Chad Robins, CEO and co-founder of Adaptive Biotechnologies. “Immune response data may enable detection of the virus in infected people not showing symptoms and improve triaging of newly diagnosed patients, potentially solving two of the challenges we are facing in the current diagnostic paradigm.”

To generate immune response data, Adaptive will open enrollment in April to collect de-identified blood samples, using a LabCorp-enabled mobile phlebotomy service, from individuals diagnosed with or recovered from COVID-19 in a virtual clinical trial managed by Covance. Immune cell receptors from these blood samples will be sequenced using Illumina platform technology and mapped to SARS-CoV-2-specific antigens that will have been confirmed by Adaptive’s proprietary immune medicine platform to induce an immune response. The immune response signature found from the initial discovery work and the initial set of samples will be uploaded to the open data access portal. Leveraging Microsoft’s hyperscale machine learning capabilities and the Azure cloud platform, the accuracy of the immune response signature will be continuously improved and updated online in real time as more trial samples are sequenced from the study.

To expedite the development and relevance of the immune response signature across the global population, the companies are seeking additional participation from institutions and research groups around the world to contribute blood samples to this open data initiative. Providence, a large health system with 51 hospitals, including the one near Seattle that treated the first U.S. COVID-19 patient, is an initial clinical collaborator.

“The solution to COVID-19 is not likely going to come from one person, one company or one country. This is a global issue, and it will be a global effort to solve it,” said Peter Lee, corporate vice president, AI and Research, Microsoft. “Making critical information about the immune response accessible to the broader research community will help advance ongoing and new efforts to solve this global public health crisis, and we can accomplish this goal through our proven TCR-Antigen mapping partnership with Adaptive.”

Timing and enrollment details about the upcoming study and the open data access portal will be coming soon. Institutions or collaborators interested in contributing blood samples can direct inquiries to [email protected]

About the Adaptive and Microsoft partnership

Adaptive and Microsoft partnered in 2018 to create a TCR-Antigen Map, an approach to translating the genetics of the adaptive immune system to understand at scale how it works. Together we are using immunosequencing and machine learning to map T-cell receptor (TCR) sequences to diseases and disease-associated antigens. Using these data, we aim to develop a blood test for the early and accurate detection of many diseases, translating the natural diagnostic capability of the immune system into the clinic. In 2019, we confirmed clinical signals in two diseases, and established our first proof of concept in Lyme Disease. We expect to submit our first clinical application to the FDA in 2020.

About Adaptive Biotechnologies

Adaptive Biotechnologies is a commercial-stage biotechnology company focused on harnessing the inherent biology of the adaptive immune system to transform the diagnosis and treatment of disease. We believe the adaptive immune system is nature’s most finely tuned diagnostic and therapeutic for most diseases, but the inability to decode it has prevented the medical community from fully leveraging its capabilities. Our proprietary immune medicine platform reveals and translates the massive genetics of the adaptive immune system with scale, precision and speed to develop products in life sciences research, clinical diagnostics, and drug discovery. We have two commercial products, and a robust clinical pipeline to diagnose, monitor and enable the treatment of diseases such as cancer, autoimmune conditions and infectious diseases. Our goal is to develop and commercialize immune-driven clinical products tailored to each individual patient. For more information, please visit

About Illumina

Illumina is improving human health by unlocking the power of the genome. Our focus on innovation has established us as the global leader in DNA sequencing and array-based technologies, serving customers in the research, clinical, and applied markets. Our products are used for applications in the life sciences, oncology, reproductive health, agriculture, and other emerging segments. To learn more, visit and follow @illumina.

About LabCorp

LabCorp (NYSE: LH), an S&P 500 company, is a leading global life sciences company that is deeply integrated in guiding patient care, providing comprehensive clinical laboratory and end-to-end drug development services. With a mission to improve health and improve lives, LabCorp delivers world-class diagnostics solutions, brings innovative medicines to patients faster, and uses technology to improve the delivery of care. LabCorp reported revenue of more than $11.5 billion in 2019. To learn more about LabCorp, visit, and to learn more about Covance Drug Development, visit

About Providence

Providence is a national, not-for-profit Catholic health system comprising a diverse family of organizations and driven by a belief that health is a human right. With 51 hospitals, 1,085 physician clinics, senior services, supportive housing and many other health and educational services, the health system and its partners employ more than 119,000 caregivers serving communities across seven states – Alaska, California, Montana, New Mexico, Oregon, Texas, and Washington, with system offices in Renton, Wash., and Irvine, Calif.

About Microsoft

Microsoft (Nasdaq “MSFT” @microsoft) enables digital transformation for the era of an intelligent cloud and an intelligent edge. Its mission is to empower every person and every organization on the planet to achieve more.

For more information, press only:

Microsoft Media Relations, WE Communications for Microsoft, (425) 638-7777, [email protected]

Beth Keshishian, Adaptive Media, (917) 912-7195, [email protected]

Lynn Lewis or Carrie Mendivil, Adaptive Investor, (415) 937-5405, [email protected]

Immunology and Microbiology

The faculty members with an immunology research focus seek to define and understand how the immune system effectively prevents disease by microbial infection or oncogenic transformation, while at the same time avoids damaging self-tissues. Many are using this knowledge to develop novel and effective disease prevention and therapeutic measures. The students enrolled in the immunology program will gain a thorough understanding of these processes in humans as well as in comparative animal models. The training includes recommended course work and research rotations in the laboratories of associated investigators, in addition to an Immunology Journal Club and weekly Seminars in Immunology hosted by the Institute for Immunology. Student fellowship opportunities are available through several NIH-sponsored training grants.

The Biology of Infectious Disease research interest group encompasses diverse experimental systems, including parasites, bacteria, fungi, viruses and disease vectors. The faculty members present a multi-disciplinary approach to the study of infectious disease and microbial pathology. Faculty research involves the study of bacterial pathogens such asBorrelia and Chlamydia, the protozoan agents of malaria, toxoplasmosis and sleeping sickness (Plasmodium, Toxoplasma and Trypanosoma), the viral pathogens Dengue virus and HIV, and the tick (Ixodes) and mosquito (Anopheles and Aedes) insect vectors that spread human pathogens that cause malaria, Lyme disease and Dengue fever.

Watch the video: Immunology. Adaptive Immunity (July 2022).


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