17.3: Adaptive Immunity - Biology

17.3: Adaptive Immunity - Biology

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The adaptive, or acquired, immune response takes days or even weeks to become established—much longer than the innate response; however, adaptive immunity is more specific to an invading pathogen. Adaptive immunity is an immunity that occurs after exposure to an antigen either from a pathogen or a vaccination. An antigen is a molecule that stimulates a response in the immune system. This part of the immune system is activated when the innate immune response is insufficient to control an infection. In fact, without information from the innate immune system, the adaptive response could not be mobilized. There are two types of adaptive responses: the cell-mediated immune response, which is controlled by activated T cells, and the humoral immune response, which is controlled by activated B cells and antibodies. Activated T and B cells whose surface binding sites are specific to the molecules on the pathogen greatly increase in numbers and attack the invading pathogen. Their attack can kill pathogens directly or they can secrete antibodies that enhance the phagocytosis of pathogens and disrupt the infection. Adaptive immunity also involves a memory to give the host long-term protection from reinfection with the same type of pathogen; on reexposure, this host memory will facilitate a rapid and powerful response.

B and T Cells

Lymphocytes, which are white blood cells, are formed with other blood cells in the red bone marrow found in many flat bones, such as the shoulder or pelvic bones. The two types of lymphocytes of the adaptive immune response are B and T cells (Figure 17.3.1). Whether an immature lymphocyte becomes a B cell or T cell depends on where in the body it matures. The B cells remain in the bone marrow to mature (hence the name “B” for “bone marrow”), while T cells migrate to the thymus, where they mature (hence the name “T” for “thymus”).

Maturation of a B or T cell involves becoming immunocompetent, meaning that it can recognize, by binding, a specific molecule or antigen (discussed below). During the maturation process, B and T cells that bind too strongly to the body’s own cells are eliminated in order to minimize an immune response against the body’s own tissues. Those cells that react weakly to the body’s own cells, but have highly specific receptors on their cell surfaces that allow them to recognize a foreign molecule, or antigen, remain. This process occurs during fetal development and continues throughout life. The specificity of this receptor is determined by the genetics of the individual and is present before a foreign molecule is introduced to the body or encountered. Thus, it is genetics and not experience that initially provides a vast array of cells, each capable of binding to a different specific foreign molecule. Once they are immunocompetent, the T and B cells will migrate to the spleen and lymph nodes where they will remain until they are called on during an infection. B cells are involved in the humoral immune response, which targets pathogens loose in blood and lymph, and T cells are involved in the cell-mediated immune response, which targets infected cells.

Humoral Immune Response

As mentioned, an antigen is a molecule that stimulates a response in the immune system. Not every molecule is antigenic. B cells participate in a chemical response to antigens present in the body by producing specific antibodies that circulate throughout the body and bind with the antigen whenever it is encountered. This is known as the humoral immune response. As discussed, during maturation of B cells, a set of highly specific B cells are produced that have many antigen receptor molecules in their membrane (Figure 17.3.2).

Each B cell has only one kind of antigen receptor, which makes every B cell different. Once the B cells mature in the bone marrow, they migrate to lymph nodes or other lymphatic organs. When a B cell encounters the antigen that binds to its receptor, the antigen molecule is brought into the cell by endocytosis and reappears on the surface of the cell bound to an MHC class II molecule. When this process is complete, the B cell is sensitized. In most cases, the sensitized B cell must then encounter a specific kind of T cell, called a helper T cell, before it is activated. The helper T cell must already have been activated through an encounter with the antigen (discussed below).

The helper T cell binds to the antigen-MHC class II complex and is induced to release cytokines that induce the B cell to divide rapidly, which makes thousands of identical (clonal) cells. These daughter cells become either plasma cells or memory B cells. The memory B cells remain inactive at this point, until another later encounter with the antigen, caused by a reinfection by the same bacteria or virus, results in them dividing into a new population of plasma cells. The plasma cells, on the other hand, produce and secrete large quantities, up to 100 million molecules per hour, of antibody molecules. An antibody, also known as an immunoglobulin (Ig), is a protein that is produced by plasma cells after stimulation by an antigen. Antibodies are the agents of humoral immunity. Antibodies occur in the blood, in gastric and mucus secretions, and in breast milk. Antibodies in these bodily fluids can bind pathogens and mark them for destruction by phagocytes before they can infect cells.

These antibodies circulate in the blood stream and lymphatic system and bind with the antigen whenever it is encountered. The binding can fight infection in several ways. Antibodies can bind to viruses or bacteria and interfere with the chemical interactions required for them to infect or bind to other cells. The antibodies may create bridges between different particles containing antigenic sites clumping them all together and preventing their proper functioning. The antigen-antibody complex stimulates the complement system described previously, destroying the cell bearing the antigen. Phagocytic cells, such as those already described, are attracted by the antigen-antibody complexes, and phagocytosis is enhanced when the complexes are present. Finally, antibodies stimulate inflammation, and their presence in mucus and on the skin prevents pathogen attack.

Antibodies coat extracellular pathogens and neutralize them by blocking key sites on the pathogen that enhance their infectivity (such as receptors that “dock” pathogens on host cells) (Figure 17.3.3). Antibody neutralization can prevent pathogens from entering and infecting host cells. The neutralized antibody-coated pathogens can then be filtered by the spleen and eliminated in urine or feces.

Antibodies also mark pathogens for destruction by phagocytic cells, such as macrophages or neutrophils, in a process called opsonization. In a process called complement fixation, some antibodies provide a place for complement proteins to bind. The combination of antibodies and complement promotes rapid clearing of pathogens.

The production of antibodies by plasma cells in response to an antigen is called active immunity and describes the host’s active response of the immune system to an infection or to a vaccination. There is also a passive immune response where antibodies come from an outside source, instead of the individual’s own plasma cells, and are introduced into the host. For example, antibodies circulating in a pregnant woman’s body move across the placenta into the developing fetus. The child benefits from the presence of these antibodies for up to several months after birth. In addition, a passive immune response is possible by injecting antibodies into an individual in the form of an antivenom to a snake-bite toxin or antibodies in blood serum to help fight a hepatitis infection. This gives immediate protection since the body does not need the time required to mount its own response.

Cell-Mediated Immunity

Unlike B cells, T lymphocytes are unable to recognize pathogens without assistance. Instead, dendritic cells and macrophages first engulf and digest pathogens into hundreds or thousands of antigens. Then, an antigen-presenting cell (APC) detects, engulfs, and informs the adaptive immune response about an infection. When a pathogen is detected, these APCs will engulf and break it down through phagocytosis. Antigen fragments will then be transported to the surface of the APC, where they will serve as an indicator to other immune cells. A dendritic cell is an immune cell that mops up antigenic materials in its surroundings and presents them on its surface. Dendritic cells are located in the skin, the linings of the nose, lungs, stomach, and intestines. These positions are ideal locations to encounter invading pathogens. Once they are activated by pathogens and mature to become APCs they migrate to the spleen or a lymph node. Macrophages also function as APCs. After phagocytosis by a macrophage, the phagocytic vesicle fuses with an intracellular lysosome. Within the resulting phagolysosome, the components are broken down into fragments; the fragments are then loaded onto MHC class II molecules and are transported to the cell surface for antigen presentation (Figure 17.3.4). Helper T cells cannot properly respond to an antigen unless it is processed and embedded in an MHC class II molecule. The APCs express MHC class II on their surfaces, and when combined with a foreign antigen, these complexes signal an invader.


View this animation from Rockefeller University to see how dendritic cells act as sentinels in the body’s immune system.

T cells have many functions. Some respond to APCs of the innate immune system and indirectly induce immune responses by releasing cytokines. Others stimulate B cells to start the humoral response as described previously. Another type of T cell detects APC signals and directly kills the infected cells, while some are involved in suppressing inappropriate immune reactions to harmless or “self” antigens.

There are two main types of T cells: helper T lymphocytes (TH) and the cytotoxic T lymphocytes (TC). The TH lymphocytes function indirectly to tell other immune cells about potential pathogens. TH lymphocytes recognize specific antigens presented by the MHC class II complexes of APCs. There are two populations of TH cells: TH1 and TH2. TH1 cells secrete cytokines to enhance the activities of macrophages and other T cells. TH2 cells stimulate naïve B cells to secrete antibodies. Whether a TH1 or a TH2 immune response develops depends on the specific types of cytokines secreted by cells of the innate immune system, which in turn depends on the nature of the invading pathogen.

Cytotoxic T cells (TC) are the key component of the cell-mediated part of the adaptive immune system and attack and destroy infected cells. TC cells are particularly important in protecting against viral infections; this is because viruses replicate within cells where they are shielded from extracellular contact with circulating antibodies. Once activated, the TC creates a large clone of cells with one specific set of cell-surface receptors, as in the case with proliferation of activated B cells. As with B cells, the clone includes active TC cells and inactive memory TCcells. The resulting active TC cells then identify infected host cells. Because of the time required to generate a population of clonal T and B cells, there is a delay in the adaptive immune response compared to the innate immune response.

TC cells attempt to identify and destroy infected cells before the pathogen can replicate and escape, thereby halting the progression of intracellular infections. TC cells also support NK lymphocytes to destroy early cancers. Cytokines secreted by the TH1 response that stimulates macrophages also stimulate TC cells and enhance their ability to identify and destroy infected cells and tumors. A summary of how the humoral and cell-mediated immune responses are activated appears in Figure 17.3.5.

B plasma cells and TC cells are collectively called effector cells because they are involved in “effecting” (bringing about) the immune response of killing pathogens and infected host cells.

Immunological Memory

The adaptive immune system has a memory component that allows for a rapid and large response upon reinvasion of the same pathogen. During the adaptive immune response to a pathogen that has not been encountered before, known as the primary immune response, plasma cells secreting antibodies and differentiated T cells increase, then plateau over time. As B and T cells mature into effector cells, a subset of the naïve populations differentiates into B and T memory cells with the same antigen specificities (Figure 17.3.6). A memory cell is an antigen-specific B or T lymphocyte that does not differentiate into an effector cell during the primary immune response, but that can immediately become an effector cell on reexposure to the same pathogen. As the infection is cleared and pathogenic stimuli subside, the effectors are no longer needed and they undergo apoptosis. In contrast, the memory cells persist in the circulation.


The Rh antigen is found on Rh-positive red blood cells. An Rh-negative female can usually carry an Rh-positive fetus to term without difficulty. However, if she has a second Rh-positive fetus, her body may launch an immune attack that causes hemolytic disease of the newborn. Why do you think hemolytic disease is only a problem during the second or subsequent pregnancies?

If the pathogen is never encountered again during the individual’s lifetime, B and T memory cells will circulate for a few years or even several decades and will gradually die off, having never functioned as effector cells. However, if the host is re-exposed to the same pathogen type, circulating memory cells will immediately differentiate into plasma cells and TC cells without input from APCs or TH cells. This is known as the secondary immune response. One reason why the adaptive immune response is delayed is because it takes time for naïve B and T cells with the appropriate antigen specificities to be identified, activated, and proliferate. On reinfection, this step is skipped, and the result is a more rapid production of immune defenses. Memory B cells that differentiate into plasma cells output tens to hundreds-fold greater antibody amounts than were secreted during the primary response (Figure 17.3.7). This rapid and dramatic antibody response may stop the infection before it can even become established, and the individual may not realize they had been exposed.

Vaccination is based on the knowledge that exposure to noninfectious antigens, derived from known pathogens, generates a mild primary immune response. The immune response to vaccination may not be perceived by the host as illness but still confers immune memory. When exposed to the corresponding pathogen to which an individual was vaccinated, the reaction is similar to a secondary exposure. Because each reinfection generates more memory cells and increased resistance to the pathogen, some vaccine courses involve one or more booster vaccinations to mimic repeat exposures.

The Lymphatic System

Lymph is the watery fluid that bathes tissues and organs and contains protective white blood cells but does not contain erythrocytes. Lymph moves about the body through the lymphatic system, which is made up of vessels, lymph ducts, lymph glands, and organs, such as tonsils, adenoids, thymus, and spleen.

Although the immune system is characterized by circulating cells throughout the body, the regulation, maturation, and intercommunication of immune factors occur at specific sites. The blood circulates immune cells, proteins, and other factors through the body. Approximately 0.1 percent of all cells in the blood are leukocytes, which include monocytes (the precursor of macrophages) and lymphocytes. Most cells in the blood are red blood cells. Cells of the immune system can travel between the distinct lymphatic and blood circulatory systems, which are separated by interstitial space, by a process called extravasation (passing through to surrounding tissue).

Recall that cells of the immune system originate from stem cells in the bone marrow. B cell maturation occurs in the bone marrow, whereas progenitor cells migrate from the bone marrow and develop and mature into naïve T cells in the organ called the thymus.

On maturation, T and B lymphocytes circulate to various destinations. Lymph nodes scattered throughout the body house large populations of T and B cells, dendritic cells, and macrophages (Figure 17.3.8). Lymph gathers antigens as it drains from tissues. These antigens then are filtered through lymph nodes before the lymph is returned to circulation. APCs in the lymph nodes capture and process antigens and inform nearby lymphocytes about potential pathogens.

The spleen houses B and T cells, macrophages, dendritic cells, and NK cells (Figure 17.3.9). The spleen is the site where APCs that have trapped foreign particles in the blood can communicate with lymphocytes. Antibodies are synthesized and secreted by activated plasma cells in the spleen, and the spleen filters foreign substances and antibody-complexed pathogens from the blood. Functionally, the spleen is to the blood as lymph nodes are to the lymph.

Mucosal Immune System

The innate and adaptive immune responses compose the systemic immune system (affecting the whole body), which is distinct from the mucosal immune system. Mucosa associated lymphoid tissue (MALT) is a crucial component of a functional immune system because mucosal surfaces, such as the nasal passages, are the first tissues onto which inhaled or ingested pathogens are deposited. The mucosal tissue includes the mouth, pharynx, and esophagus, and the gastrointestinal, respiratory, and urogenital tracts.

Mucosal immunity is formed by MALT, which functions independently of the systemic immune system, and which has its own innate and adaptive components. MALT is a collection of lymphatic tissue that combines with epithelial tissue lining the mucosa throughout the body. This tissue functions as the immune barrier and response in areas of the body with direct contact to the external environment. The systemic and mucosal immune systems use many of the same cell types. Foreign particles that make their way to MALT are taken up by absorptive epithelial cells and delivered to APCs located directly below the mucosal tissue. APCs of the mucosal immune system are primarily dendritic cells, with B cells and macrophages having minor roles. Processed antigens displayed on APCs are detected by T cells in the MALT and at the tonsils, adenoids, appendix, or the mesenteric lymph nodes of the intestine. Activated T cells then migrate through the lymphatic system and into the circulatory system to mucosal sites of infection.

Immune Tolerance

The immune system has to be regulated to prevent wasteful, unnecessary responses to harmless substances, and more importantly, so that it does not attack “self.” The acquired ability to prevent an unnecessary or harmful immune response to a detected foreign substance known not to cause disease, or self-antigens, is described as immune tolerance. The primary mechanism for developing immune tolerance to self-antigens occurs during the selection for weakly self-binding cells during T and B lymphocyte maturation. There are populations of T cells that suppress the immune response to self-antigens and that suppress the immune response after the infection has cleared to minimize host cell damage induced by inflammation and cell lysis. Immune tolerance is especially well developed in the mucosa of the upper digestive system because of the tremendous number of foreign substances (such as food proteins) that APCs of the oral cavity, pharynx, and gastrointestinal mucosa encounter. Immune tolerance is brought about by specialized APCs in the liver, lymph nodes, small intestine, and lung that present harmless antigens to a diverse population of regulatory T (Treg) cells, specialized lymphocytes that suppress local inflammation and inhibit the secretion of stimulatory immune factors. The combined result of Treg cells is to prevent immunologic activation and inflammation in undesired tissue compartments and to allow the immune system to focus on pathogens instead.

Section Summary

The adaptive immune response is a slower-acting, longer-lasting, and more specific response than the innate response. However, the adaptive response requires information from the innate immune system to function. APCs display antigens on MHC molecules to naïve T cells. T cells with cell-surface receptors that bind a specific antigen will bind to that APC. In response, the T cells differentiate and proliferate, becoming TH cells or TC cells. TH cells stimulate B cells that have engulfed and presented pathogen-derived antigens. B cells differentiate into plasma cells that secrete antibodies, whereas TC cells destroy infected or cancerous cells. Memory cells are produced by activated and proliferating B and T cells and persist after a primary exposure to a pathogen. If re-exposure occurs, memory cells differentiate into effector cells without input from the innate immune system. The mucosal immune system is largely independent of the systemic immune system but functions in parallel to protect the extensive mucosal surfaces of the body. Immune tolerance is brought about by Treg cells to limit reactions to harmless antigens and the body’s own molecules.

Art Connections

Figure 17.3.6 The Rh antigen is found on Rh-positive red blood cells. Why do you think hemolytic disease is only a problem during the second or subsequent pregnancies?

Figure 17.3.6 If the blood of the mother and fetus mixes, memory cells that recognize the Rh antigen of the fetus can form in the mother late in the first pregnancy. During subsequent pregnancies, these memory cells launch an immune attack on the fetal blood cells of an Rh-positive fetus. Injection of anti-Rh antibody during the first pregnancy prevents the immune response from occurring.


active immunity
an immunity that occurs as a result of the activity of the body’s own cells rather than from antibodies acquired from an external source
adaptive immunity
a specific immune response that occurs after exposure to an antigen either from a pathogen or a vaccination
a protein that is produced by plasma cells after stimulation by an antigen; also known as an immunoglobulin
a macromolecule that reacts with cells of the immune system and which may or may not have a stimulatory effect
antigen-presenting cell (APC)
an immune cell that detects, engulfs, and informs the adaptive immune response about an infection by presenting the processed antigen on its cell surface
B cell
a lymphocyte that matures in the bone marrow
cell-mediated immune response
an adaptive immune response that is controlled by T cells
cytotoxic T lymphocyte (TC)
an adaptive immune cell that directly kills infected cells via enzymes, and that releases cytokines to enhance the immune response
dendritic cell
an immune cell that processes antigen material and presents it on the surface of its cell in MHC class II molecules and induces an immune response in other cells
effector cell
a lymphocyte that has differentiated, such as a B cell, plasma cell, or cytotoxic T cell
helper T lymphocyte (TH)
a cell of the adaptive immune system that binds APCs via MHC class II molecules and stimulates B cells or secretes cytokines to initiate the immune response
humoral immune response
the adaptive immune response that is controlled by activated B cells and antibodies
immune tolerance
an acquired ability to prevent an unnecessary or harmful immune response to a detected foreign body known not to cause disease
the watery fluid present in the lymphatic circulatory system that bathes tissues and organs with protective white blood cells and does not contain erythrocytes
memory cell
an antigen-specific B or T lymphocyte that does not differentiate into an effector cell during the primary immune response but that can immediately become an effector cell on reexposure to the same pathogen
major histocompatibility class (MHC) II molecule
a protein found on the surface of antigen-presenting cells that signals to immune cells whether the cell is normal or is infected or cancerous; it provides the appropriate template into which antigens can be loaded for recognition by lymphocytes
passive immunity
an immunity that does not result from the activity of the body’s own immune cells but by transfer of antibodies from one individual to another
primary immune response
the response of the adaptive immune system to the first exposure to an antigen
secondary immune response
the response of the adaptive immune system to a second or later exposure to an antigen mediated by memory cells

High School Biology : Understanding Adaptive Immunity

The spleen is an intra-abdominal organ whose function is __________ .

related to the regulation of body fat metabolism

excretion of liquid wastes

related mostly to immunological abilities

the production of gastrin, which it delivers to the stomach

related mostly to immunological abilities

The spleen is like a giant lymph node, and it is organized in a somewhat similar manner. Although it can be surgically removed if it is damaged, such patients are at life-long risk of death from fairly ordinary infectious processes. The spleen is a reservoir of immune competence. Blood passes through the spleen for exposure to white blood cells. When the white blood cells detect antigens or foreign particles in the blood, they initiate the immune response. The spleen is essentially a screening center to check the blood for contaminants.

Example Question #2 : Immune System

When a person is exposed to an organism and produces specific antibodies against it, this type of immunity is referred to as __________ .

Adaptive immunity occurs when antibodies are produced as a result of exposure to a pathogen or immunization. These antibodies are specific for the particular microorganism and memory cells are produced. Cell-mediated immunity is a direct form of defense based on the action of lymphocytes to attack foreign cells and destroy them. Congenital immunity is immunity one is born with. This may result from antibodies received from the mother's blood. Innate immunity is not pathogen-specific and includes the secretion of proteins and the activities of natural killer cells. Passive immunity involves the introduction of preformed antibodies into an unprotected individual. This may occur through infusion of immune globulin or antibodies that pass from the mother to the fetus through the placenta.

Example Question #3 : Immune System

How is VDJ recombination indispensible for adaptive immunity?

It prevents integration of viral DNA into host DNA

VDJ recombination is not involved in adaptive immunity

It promotes clotting and macrophage recruitment to wounds

It allows for the generation of diverse and variable antibodies that are able to recognize a myraid of antigens

It allows for the generation of diverse antigens to recognize many antibodies

It allows for the generation of diverse and variable antibodies that are able to recognize a myraid of antigens

VDJ recombination occurs during early B- and T-cell maturation, resulting in diverse antibodies and T-cells. This DNA recombination occurs between the V, D, and J segments of the antibody or T-cell before transcription occurs. As a result, a unique sequence is generated, transcribed, and then translated to a functional protein. This recombination is responsible for creating the unique series of antibodies that the body is capable of producing in order to detect the various antigens represented by foreign pathogens.

Example Question #4 : Immune System

Which of the following is true regarding B cell and T cell interactions?

Both B cells and T cells can activate each other

B cells and T cells do not activate each other

T cells can activate B cells but B cells cannot activate T cells

B cells can activate T cells but T cells cannot activate B cells

T cells can activate B cells but B cells cannot activate T cells

B cells and T cells are both part of the adaptive immunity. B cells secrete antibodies that bind to foreign antigens. Upon binding to a specific antigen, B cells can be activated by T cells, which facilitate the synthesis of specific antibodies for the antigen. This enhances the antibody-antigen binding and allows for a better immune response. T cells have receptors on their surface that detect antigens. Once they detect the antigen, T cells can activate B cells and other immune system cells (such as macrophages and neutrophils) to eliminate the foreign antigen. B cells do not play a role in the activation of T cells.

Example Question #1 : Understanding Adaptive Immunity

A researcher is analyzing a specific immune complex that is made up of an antibody-antigen complex. What can the researcher conclude about this immune response?

It involves B cells and a cell-mediated immune response.

It involves T cells and a cell-mediated immune response.

It involves T cells and a humoral immune response.

It involves B cells and a humoral immune response.

It involves B cells and a humoral immune response.

The question states that the immune complex has antibodies bound to antigens. Recall that B cells eliminate pathogens by secreting antibodies. These antibodies bind to antigens and release factors called cytokines. Cytokines recruit phagocytic cells like macrophages and neutrophils that kill the infected cell. They also activate a part of the innate immune system called the complement, which aids in the elimination of the pathogen. This type of immune response is called a humoral immune response. Elimination of the pathogen using T cells is called a cell-mediated immune response.

Note that both the humoral and the cell-mediated immune responses are very specific responses that are part of the adaptive immunity. Innate immunity involves non-specific immune responses via macrophages, granulocytes (neutrophils, eosinophils, and basophils), complement system, and NK cells.

Example Question #6 : Immune System

Which of the following is/are characteristic(s) of T cells?

I. T cells can differentiate into plasma cells.

II. T cells can differentiate into cells that inhibit activity of other T cells.

III. HIV attacks helper T cells.

Plasma cells are circulating cells that form part of adaptive immunity that secrete antibodies to specific antigens. These cells arise from naïve B cells. Broadly specific naïve B cells have the ability to bind to several antigens. Once bound, these naïve B cells differentiate into plasma cells that secrete antibodies that are very specific to the antigen. T cells facilitate this differentiation, but only B cells give rise to plasma cells.

A naïve T cell has the ability to differentiate into three kinds of cells. First, it can differentiate into a helper T cell. These cells facilitate the activation of other immune cells such as B cells, macrophages, and granulocytes. Second, a naïve T cell can differentiate into a cytotoxic T cell. These cells bind to infected cells and induce their death. Third, a naïve T cell can differentiate into a regulatory T cell. These T cells bind to the same antigens as the first two cells however, instead of initiating an immune response, they regulate it by suppressing the activity of T cells.

HIV is a virus that likes to reside inside helper T cells. A person infected with HIV will have a decreased helper T cell count, which makes the person more susceptible to other opportunistic infections (infections that only occur in immune-compromised individuals). A patient with very low helper T cell count develops AIDS and often passes away due to these opportunistic infections.

Example Question #7 : Immune System

CD8 is a surface glycoprotein found in many T cells. Which of the following T cells will NOT have a CD8?

All of the these T cells will have CD8.

A T cell that participates in the elimination of extracellular bacterial cells

A T cell that participates in the elimination of virus infected cells

A T cell that participates in the elimination of cancer cells

A T cell that participates in the elimination of extracellular bacterial cells

There are three kinds of T cells: helper T cells, cytotoxic T cells, and regulatory T cells. All T cells have glycoproteins on their surfaces that act as receptors. CD4 and CD8 are two glycoproteins that can be found on T cells. Helper T cells and regulatory T cells have CD4 glycoproteins, whereas cytotoxic T cells have CD8. These glycoproteins serve as markers to distinguish between T cell types.

The question is asking about CD8, or cytotoxic, T cells. Recall that cytotoxic T cells bind to infected cells and induce their death. Typically, cytotoxic T cells bind to infected cells that have the pathogen inside them (meaning intracellular pathogens). Intracellular pathogens include viruses and intracellular bacteria therefore, T cells that attack these cells will be CD8 cells. In addition, cytotoxic T cells also attack cancer cells therefore, these T cells will also be CD8 cells.

Extracellular bacterial cells do not infect host cells therefore, these bacteria are eliminated via the helper T cells. These T cells bind to the bacteria and activate other immune cells such as B cells, macrophages, and granulocytes that eliminate the bacteria.

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Antigen Presentation and Dendritic Cells

Concepts: Link between innate immunity and adaptive immunity, MHC-I & MHC-II mediated activation, function and maturation of dendritic cells, antigen cross-presentation, activation of T cells by dendritic cells, T cell tolerance, and regulatory T cells (Tregs)

Please watch original video from time 00:00 to 25:33 (Clip Link).

00:00:01.00 Hi, Ira Mellman again from Genentech. I'd like to continue our discussion
00:00:05.24 of the cell biology of the immune response, this time turning to specifically
00:00:09.22 the problem of antigen presentation and the role of dendritic cells
00:00:13.17 in linking the two aspects of the immune response
00:00:16.06 that we've already discussed, innate immunity and adaptive immunity.
00:00:19.00 Innate immunity having been discovered by Metchnikoff
00:00:21.29 and adaptive immunity by Ehrlich all about 100 years ago.
00:00:25.00 Now in order for T-cells to do their job helping B-cells respond by
00:00:31.15 making antibodies and also by helping to kill virus infected cells and other types of pathogens,
00:00:37.13 they themselves require a significant amount of help, which is provided
00:00:43.03 by the so-called antigen presenting cells or APCs. Now recognition of T-cell
00:00:49.17 receptors of their targets does not necessarily lead to cell killing.
00:00:55.00 In fact this is an interaction that has to take place in order to generate a sufficient number
00:01:00.01 of T-cells to mediate the immune response in the first place. So the signal
00:01:04.01 that is sent by the T-cell receptor to the T-cell is a consequence
00:01:08.22 of seeing its ligand serves to activate the cell to not only to kill but also to differentiate
00:01:16.21 and divide, again depending on the type of T-cell you happen to be.
00:01:20.00 These types of signals together with other types of signals provided by other types of
00:01:25.07 membrane proteins referred to as co-stimulatory molecules,
00:01:28.21 are provided by a class of cells referred to as APCs or antigen-presenting cells.
00:01:35.00 Now as we reviewed in the last lecture, T-cell receptors see two different types
00:01:40.29 of peptides, peptides bound either to MHC class II molecules or peptides bound
00:01:46.09 to MHC class I molecules. CD8 T-cells recognizing the class I molecules,
00:01:51.16 CD4 T-cells recognizing the class II molecules. This is a structural
00:01:56.20 representation of how the recognition of the peptide MHC complexes by T-cell receptors actually occurs.
00:02:04.00 So here in the upper level you see the T-cell receptor itself,
00:02:07.07 the two major antigen recognition elements of it, the alpha chain and the beta chain,
00:02:12.00 which form a complex that is specifically adapted to recognize that a particular peptide
00:02:17.03 bound to a particular MHC molecule so here you can see the peptide,
00:02:21.04 which usually ranges in 9 to 10 or more of amino acids in length,
00:02:26.21 nestled within a very specific peptide binding cleft found in this case in a MHC class I molecule
00:02:34.00 that is expressed by the antigen presenting cell expressing in turn the antigen from
00:02:39.18 which this particular peptide is derived. In order for this system to work,
00:02:43.23 it has to be amplified, it has to be selected, the T-cells have to be amplified
00:02:48.11 and have to be selected and have to be affinity matured in such a way
00:02:51.28 that they can detect their peptides with higher and higher affinity
00:02:55.14 continuously again sculpting these T-cell receptors. Now producing
00:03:00.23 the peptide MHC complex, which is obviously key to this entire process
00:03:04.00 is a job of the antigen presenting cell. Now how does the antigen presenting cell do this?
00:03:10.27 We've already talked about the fact that there is both a class I and a class II
00:03:15.28 dependent pathway or restricted pathway of antigen presentation.
00:03:21.00 The class II pathway is predominantly adapted to presenting those molecules
00:03:26.27 derived from extracellular pathogens such as extracellular bacteria or proteins,
00:03:33.11 toxins, whatever are released from bacteria. So in those cases what happens
00:03:38.16 as we already discussed in the case of B-cells, the antigens bind to the surface
00:03:42.26 of the cell, the antigen presenting cells are taken up and deposited
00:03:47.16 within endocytic vesicles within the cytoplasm of the APC.
00:03:53.11 Here the antigens are unfolded, unwound, and eventually degraded into peptides
00:03:59.04 that are loaded also in endocytic vesicles onto the class II molecules themselves.
00:04:03.00 Now how the class II molecules get there is a rather remarkable story
00:04:08.01 and a remarkable piece of cell biology and membrane traffic in its own right.
00:04:11.00 Class II molecules are membrane proteins and like almost all other membrane proteins
00:04:15.27 begin their lives at the level of the rough endoplasmic reticulum,
00:04:19.05 where they are synthesized and inserted across the ER membrane
00:04:23.00 and assembled together with a chaperone called the invariant chain in the ER
00:04:28.04 that then after assembly takes place gets transported from
00:04:32.06 the endoplasmic reticulum into the cis-golgi, through the golgi stack, emerging at the
00:04:37.13 trans-side of the golgi complex but unlike proteins that are destined for
00:04:44.10 secretion or destined to be inserted into the plasma membrane,
00:04:48.02 MHC molecules are diverted from this constitutive secretory pathway and instead
00:04:52.28 are taken into the endocytic compartment where the invariant chain is removed
00:04:57.15 and we'll come back to that in a moment, and the class II molecule rendered accessible
00:05:01.11 to the peptides that can be derived from the incoming antigen.
00:05:05.17 Following binding of the peptide to the class II molecule, this complex is taken to the cell surface
00:05:11.15 where it can now be recognized by CD4+ T-cells.
00:05:16.00 Now the class I pathway as we've discussed services predominately those antigens
00:05:21.24 that are self-synthesized by a given cell. This of course can include
00:05:28.01 any membrane protein or cytosolic protein that is synthesized as a
00:05:33.01 consequence of the viral infection. So in the case of cytosolic proteins, these proteins are made,
00:05:39.06 ubiquitinated, degraded in the cytosol by the proteosome and peptides
00:05:43.25 that are produced by the proteosome were transported into the ER, where they are then bound
00:05:48.07 to class I molecules that are again, transported from the ER to the golgi
00:05:52.05 but now rather than going to the endocytic compartment,
00:05:55.00 instead the class I molecules go directly to the cell surface. Now all antigen presenting cells
00:06:00.20 are not created equal. There are amateurs and there are professionals.
00:06:04.00 Amateurs can make the class I response in most cases, because MHC class I molecules
00:06:10.23 are expressed by virtually all nucleated cells in the body so therefore virtually
00:06:15.09 all nucleated cells are protected by the immune system against infection
00:06:19.22 by viruses, which is a good thing. The MHC class II system on the other hand
00:06:24.20 is synthesized and expressed only by a relatively restricted number of cells
00:06:29.14 in the body. In most cases our cells that are specialized cells in the immune system
00:06:35.05 B-cells, macrophages, and most importantly these dendritic cells
00:06:39.23 that we mentioned earlier. Now dendritic cells are really special
00:06:43.24 and in fact are the professionals professional. They are the Tiger Woods
00:06:48.05 of the antigen presenting cell universe. Why? Because they are by far the most efficient.
00:06:54.14 They can capture almost meaningless and non-existent amounts of antigens
00:06:58.22 and turn those antigens into small peptides that can stimulate T-cell responses. They have
00:07:05.28 the rather unique capacity to be able to capture antigen from
00:07:10.17 wherever in the body antigen is first introduced,
00:07:14.12 be it in the skin, in the lung, in the intestine wherever the antigen is captured
00:07:19.07 and then its not simply left to passive transport through the lymphatics to go back to the lymph nodes,
00:07:25.05 but rather these cells are adapted to hone in on the lymphatics and make a bee line directly
00:07:30.10 into the lymph nodes where the dendritic cells can find a huge accumulation
00:07:35.29 of both T-lymphocytes and B-lymphocytes and help their stimulatory events take place.
00:07:42.12 Perhaps most importantly, dendritic cells are the only cells in the immune system,
00:07:47.13 the only antigen presenting cell that can actually initiate the antigen specific
00:07:51.10 immune response. In other words, prior to the advent of the first of an antigens kind
00:08:00.15 coming in before you had your very first infection with influenza
00:08:05.12 you may have T-cells that are capable of responding to influenza virus,
00:08:09.00 but they are naïve, they don't really know what to do. Only the dendritic cell can wake
00:08:14.16 them up. If you delete dendritic cells from a mouse using a variety
00:08:20.07 of genetic knockouts, you find those mice are almost completely incapable
00:08:24.12 of mounting antigen specific immune responses. Why? Because only
00:08:29.08 the dendritic cell can present an antigen at a sufficient level of efficiency and with
00:08:36.27 a sufficient amount of stimulation provided to the T-cell in order to wake the T-cell up
00:08:41.29 and get the T-cell response going. Now the other side of the coin here though
00:08:48.02 is the issue of tolerance in the sense that remember one of the key abilities
00:08:54.03 of the immune system is to be able to mount virulent cytotoxic responses
00:09:00.03 protective responses to invading pathogens, but somehow minimize
00:09:05.01 if not avoid entirely destructive components that are directly against our self antigens
00:09:12.00 in other words our own tissues and selves. Dendritic cells play also a key role
00:09:17.09 in ensuring that our immune system maintains tolerance to self antigens
00:09:22.21 and we'll come back at the very end to discuss some of the more recent ideas
00:09:27.21 on how we think that takes place. Now probably though the most important
00:09:33.02 key conceptual element of how the dendritic cell system works
00:09:37.16 and why dendritic cells play such a role that is so important in linking the innate and
00:09:42.27 adaptive response is that like cells of the innate response, innate immune response,
00:09:48.13 dendritic cells can respond to exactly the same types of microbial signals
00:09:52.12 as do the macrophages. Again, by virtue of the fact that they express
00:09:57.07 the same classes of Toll-like receptors that macrophages do, but instead of under
00:10:02.28 most circumstances emitting cytotoxic compounds as a consequence of that,
00:10:07.01 they turn the information and the advent of microbial pathogens
00:10:14.05 coming into the system into the peptide language that can be understood by T-cells,
00:10:19.08 thereby linking the activation of the adaptive response
00:10:25.09 to the activation of the innate response. So then as I'm mentioning in words,
00:10:30.18 as you can see here, dendritic cells do play this really important missing link
00:10:35.21 that intimately connects innate immunity with adaptive immunity responding
00:10:41.04 to the innate signals and turning those signals into the language of the adaptive immune response.
00:10:47.00 Now this is a concept that is relatively new and certainly not nearly as
00:10:54.13 old meaning 100 years, as the first discovery of the adaptive response
00:11:00.14 and the innate response. It took almost 80 to 100 years later to really figure this out,
00:11:05.26 and that was done by this gentleman with no beard, Ralph Steinman,
00:11:10.20 who works at the Rockefeller University who was really among the very very first
00:11:14.23 to appreciate that dendritic cells have this remarkable role in being able to have a critical element
00:11:23.15 in linking the innate and adaptive responses by being just so powerful and so special
00:11:29.23 and so adept at generating T-cell responses in response to antigens
00:11:37.05 and in response to adjuvants or microbial products. Now the basic logic of
00:11:42.11 the dendritic cell system is shown here, and this is terrifically rich and complex
00:11:46.23 but indeed quite understandable. The idea is as follows, dendritic cells exist
00:11:52.22 as immature sentinels in a variety of peripheral tissues, in fact all of our peripheral tissues
00:11:58.05 contain dendritic cells. Here we are looking at the skin, at the epidermis,
00:12:02.02 where dendritic cells are intercalated in various levels in the skin,
00:12:06.27 most interestingly in the epidermis itself where these long stellate cells
00:12:12.00 are intercalated among the far more numerous keratinocytes. Indeed, the fact that these dendritic cells
00:12:19.18 existed in the skin is actually a fairly old observation made by Paul Langerhans
00:12:25.15 also who was responsible for having first identified the islets of Langerhans in the pancreas,
00:12:31.07 but Langerhans didn't know what these cells did, but indeed he identified that they were there.
00:12:36.01 We now know that they are present in the skin for the purposes of immune surveillance.
00:12:42.02 They are there to capture incoming antigens, to capture incoming pathogens,
00:12:47.18 and after that capture takes place they migrate out from the skin, enter the lymphatics,
00:12:52.28 and eventually as I already mentioned find their way into lymphoid organs
00:12:58.26 where they also now start to intercalate together with lymphocytes T-cells and B-cells.
00:13:06.00 Now by the time they get to lymphoid organs under most circumstances,
00:13:10.24 these dendritic cells change in their characteristics, they become mature.
00:13:16.21 The difference between an immature cell and a mature cell turns out to be key
00:13:21.25 in understanding exactly what happens and we'll come to that in just a moment.
00:13:26.00 Immature dendritic cells and mature dendritic cells differ from one another
00:13:31.17 in some very very important ways. Immature dendritic cells are shown here
00:13:36.05 in an immunofluorescence picture. What you can see is that all the MHC molecules,
00:13:40.24 particularly the MHC class II molecules that are expressed by
00:13:43.28 immature dendritic cells are sequestered inside the cell in lysosomes and late endosomes.
00:13:50.00 They are not at the surface, so as a result these immature DCs are relatively speaking
00:13:57.08 incapable of stimulating T-cells. In addition they don't express co-stimulatory
00:14:02.27 molecules, they're very poor at secreting cytokines and they are
00:14:07.03 relatively non-motile, so as a result they are very poor at T-cell stimulation.
00:14:12.20 What they are good at though is antigen accumulation. So we view these
00:14:16.15 cells as the sentinels that are the first ones that encounter antigen in the periphery
00:14:20.18 and then as a consequence of detecting antigen and also having the ability
00:14:27.09 to detect the innate signals encompassed or encoded in those antigens
00:14:32.06 via Toll-like receptors and other inflammatory product receptors these
00:14:37.20 dendritic cells change their morphology and also change their function
00:14:41.10 dramatically and really can do so in a remarkably short period of time.
00:14:45.16 We find that only a relatively few hours is required to transform a cell
00:14:51.14 that looks like this to a cell that looks like this, one that extends out enormous
00:14:57.00 dendrites that give them their name, that now relocate all of the
00:15:01.12 class MHC molecules that were present in lysosomes to the surface of the cells
00:15:06.11 and also induce the expression of a variety of other important molecules
00:15:11.14 such as these co-stimulatory molecules that are necessary for optimal T-cell stimulation.
00:15:18.26 So the mature dendritic cell is the cell that is most adept at antigen presentation
00:15:24.00 and antigen stimulation. This flip from the immature to the mature state
00:15:28.27 is intimately linked to the fact that the immature dendritic cell expresses
00:15:34.14 these Toll-like receptors. Were it not for that fact, not for the fact that
00:15:39.07 these cells that are intimately associated with the adaptive response
00:15:42.22 also have the capacity to respond to the most fundamental and
00:15:47.24 elemental element of the innate response we would not have this link. So it's actually
00:15:54.14 maturation that does this. Now as cell biologists interested in membrane traffic
00:15:58.19 we've been very interested over the years as have been many other groups
00:16:02.16 in trying to understand what's responsible for this dramatic morphogenetic
00:16:07.00 change that dendritic cells exhibit and how does it relate to the function
00:16:11.18 of these cells and how to these functions relate to the overall control
00:16:15.18 of the immune response. So here on the left you are looking at a diagram
00:16:20.09 of the membrane traffic phenotype if you will of an immature dendritic cell.
00:16:25.07 These are cells that are highly endocytic, they take up lots of antigen
00:16:29.20 by a variety of different mechanisms of endocytosis, those antigens come in from
00:16:33.25 the outside, find their way to endosomes and finally to lysosomes
00:16:37.13 where quite remarkably they sit unlike most other cells that degrade
00:16:41.20 very rapidly proteins and nucleic acids and lipids and carbohydrates
00:16:46.04 that make it into lysosomes in immature dendritic cells the antigen that enters lysosomes
00:16:52.24 is protected and protected from degradation. At the same time,
00:16:57.24 these cells are making a large number of MHC molecules, particularly MHC class II molecules
00:17:03.17 which instead of being taken to the cell surface where they sit,
00:17:07.26 which is what happens in most other cells, now these molecules as well are targeted to the lysosomes
00:17:12.23 but basically nothing happens, the MHC molecules sit there, the antigen sits there,
00:17:17.10 a little bit of degradation, a little bit of loading of peptide onto the MHC molecules
00:17:22.12 but really not too much takes place until these cells are exposed to a TLR, a Toll-like receptor ligand.
00:17:31.26 In other words, one of the preserved molecular patterns that are conserved
00:17:36.13 from microbe to microbe, then everything starts to change and starts to change really fast.
00:17:42.07 One of the first things that occurs is that endocytosis, at least many forms
00:17:47.27 of endocytosis are shut off to a very large extent, this reflects
00:17:53.09 the fact that these Rho-family of GTPases particularly of the molecules
00:18:00.06 Cdc42 rather than being present in a constitutively active form
00:18:06.06 is now de-activated and present not as the GTP active form but rather as the GDP inactive form.
00:18:14.02 So antigen uptake is not cut-off completely but is diminished.
00:18:17.15 Next thing that happens is that newly synthesized class II molecules,
00:18:21.18 rather than being directed entirely to lysosomes are now taken from the golgi to endosomes
00:18:28.01 and like in other cells for other types of molecules are targeted to the cell surface.
00:18:33.06 There is a lot of reasons for this, we'll come back to one of the most important
00:18:37.01 in a minute but much of it has to do as well with changes in the metabolism
00:18:43.02 of this class II associated chaperone referred to as the invariant chain. As long
00:18:48.06 as the class II molecules associated with the invariant chain the class II molecule
00:18:52.17 is taken to lysosomes. If the invariant chain is removed, the class II molecule can
00:18:57.22 now proceed out to the cell surface. This happens for a variety of reasons,
00:19:02.06 not the least of which is due to down regulation of an antiprotease
00:19:06.13 called cystatin c, which turns off the proteolytic enzyme that is normally responsible
00:19:12.01 for degrading this invariant chain. In fact the lysosomes are activated
00:19:17.27 completely in this case for a variety of reasons, probably one of the most
00:19:23.06 interesting is the activation of lysosomal acidification. Normally
00:19:28.16 lysosomes are acidic vesicles, as Metchnikoff first told us, but in immature dendritic cells
00:19:34.26 they're less acidic than they need to be. The reason for that has been described
00:19:39.22 over the last two or three years as reflecting two key events.
00:19:44.04 One, the vacuolar proton pump or the ATPase that is required to move protons
00:19:50.27 from the cytosol into the lumen of the lysosome thereby dropping its pH,
00:19:56.01 is inactive in the immature dendritic cell. As a consequence of maturation,
00:20:00.24 as a consequence of TLR stimulation, this proton pump
00:20:07.00 is activated by turning on an assembly process, now allowing protons
00:20:12.14 to be translocated from the cytosol into the lysosomal lumen
00:20:16.05 in exchange for ATP hydrolysis thereby acidifying the interior of the lysosome.
00:20:22.10 Sebastian Amigorena in Paris has further found that in some ways like
00:20:26.21 macrophages, dendritic cells are indeed capable of generating active
00:20:30.27 oxygen species but one of the most important features here
00:20:34.23 is not so much to kill the incoming pathogen but rather to further regulate
00:20:39.11 the ability of lysosomes and incoming phagocytic vesicles to acidify their lumens,
00:20:47.02 again emphasizing how important it is to the dendritic cell
00:20:51.21 to ensure that the pH of the compartments involved in antigen presentation
00:20:56.14 and antigen processing are indeed carefully regulated.
00:21:00.08 So this is basically how it works, very simply the lysosomal pH of
00:21:05.19 immature dendritic cells is slightly acidic, it has a pH of 5.5.
00:21:10.07 The lysosomal pH found in organelles of mature dendritic cells
00:21:17.03 is more acidic by one whole pH unit, 4.5.
00:21:20.23 Doesn't sound like a lot but it turns out that most of the lysosomal
00:21:24.15 proteases and nucleic acid degrading enzymes and lipid degrading enzymes
00:21:30.00 that are found in lysosomes have a very sharp pH optimum
00:21:33.18 and they really don't work very well unless the pH that they are working in
00:21:38.08 is below pH 5, so the maturation process then drives the pH down
00:21:43.24 from a pH that's too high for optimal activity of the lysosomal proteases
00:21:48.17 to a pH that now is just right, the goldilocks effect allowing these
00:21:54.03 lysosomal proteases to do their work at optimal levels increasing
00:21:58.15 the efficiency at which peptides can be generated for association
00:22:04.12 with MHC class II molecules. Now this is a diagram just quickly as to how this works.
00:22:10.08 So here you can see a class II molecule that begins its life associated
00:22:15.18 with the invariant chain as you've seen in previous diagrams,
00:22:19.03 it consists of two membrane proteins, an alpha chain and a beta chain,
00:22:23.08 here is the invariant chain in green together with its lysosomal targeting signal.
00:22:28.00 The invariant chain is degraded by a series of proteolytic cleavages
00:22:31.26 most important of which is mediated by an enzyme called cathepsin s,
00:22:36.19 which is found most prevalently in professional antigen presenting cells such as dendritic cells.
00:22:43.24 This removes the lysosomal targeting signal from the invariant chain,
00:22:49.27 leaving a small segment of the invariant chain that is rapidly removed
00:22:53.14 by virtue of the activity of another class II associated chaperone,
00:22:57.26 not invariant chain, but something called HLA-DM,
00:23:00.28 that destabilizes the affinity of this remaining invariant chain fragment
00:23:05.04 to the peptide binding cleft of the class II molecule,
00:23:09.03 allowing the peptide to bind and displace the invariant chain derived peptide
00:23:16.24 and this then peptide MHC complex can go up to the cell surface.
00:23:21.19 I mentioned cystatin c before, it works at this stage by inhibiting
00:23:26.13 the activity of cathepsin s, it slows the cleavage, renders the cleavage less efficient
00:23:32.16 of the invariant chain making these molecules less accessible to peptide loading.
00:23:38.15 In a flow diagram in terms of what this means for membrane traffic,
00:23:43.05 here emanating from the golgi complex is the invariant chain class II complex,
00:23:48.06 enters endosomes and in immature dendritic cells nothing happens
00:23:51.27 because the level of cathepsin s is low, the activity of cathepsin s is low because
00:23:58.17 the activity of cystatin c is high and also the pH of these structures is not optimal,
00:24:04.17 and instead these class II molecules go to lysosomes. Maturation reduces
00:24:09.20 cystatin c activity, increases cathepsin s activity playing a role in helping
00:24:16.09 these class II molecules proceed on to the cell surface. Now the molecules
00:24:20.11 that had made it to lysosomes are there and also as long as the dendritic cell
00:24:26.15 remains immature not much happens. Here is a video taken by Amy Chow
00:24:32.28 in our laboratory a few years ago from a MHC molecule that has been linked
00:24:40.13 to the green fluorescent protein in immature dendritic cells, and what you
00:24:45.02 are looking at are lysosomes that are just sort of bouncing around, aimlessly
00:24:48.05 in these immature cells. So as I've mentioned earlier,
00:24:53.10 the class II molecules are there, the antigen is there and nothing is happening.
00:24:56.21 But very soon after adding LPS to this system, a ligand for a Toll-like receptor,
00:25:02.29 specifically TLR-4, you get a very different effect. Now what you can see
00:25:07.20 is that these lysosomes begin shooting out tubules and the lysosomes
00:25:13.17 start accumulating in a small dot depleting the amount of class II that is
00:25:17.29 associated with them and you can begin to see class II appearing on the surface
00:25:22.25 of the cell, all these events taking place really just over a time course of
00:25:27.20 a couple of hours. This is a video in which we are just able to visualize
00:25:31.24 the movement of some of these lysosome derived tubules from their site of
00:25:35.10 formation in lysosomes out to the periphery of cells and while I'm going
00:25:39.04 to show this to you, there are various biophysical techniques that you can use
00:25:42.05 to actually show that these tubules and vesicles derived from them will
00:25:45.22 physically fuse with the plasma membrane of the cell, delivering not only
00:25:50.15 the MHC class II molecule, but obviously also the antigen that has bound
00:25:55.28 to it as a consequence of the peptide loading event that took place
00:26:01.01 in the lysosome just at the moment of dendritic cell maturation.
00:26:05.08 All of these events take place as I said in just a couple of hours
00:26:09.07 if you wait overnight you can now see live dendritic cells that look ever much like
00:26:14.29 the static images I showed you earlier. Here a cell with all of its MHC class II molecules
00:26:21.24 on the surface, lysosomes are now stained red because none of the GFP
00:26:26.14 or green fluorescent protein coupled class II molecules are present within them anymore.
00:26:31.21 So what that means then is here one more unexpected feature of the system,
00:26:38.21 which is that MHC class II molecules can escape from lysosomes
00:26:43.21 to dendritic cell surface, by a pathway which we refer to as your retrograde transport pathway.
00:26:49.26 Anterograde transport refers to what happens when molecules
00:26:54.15 such as antigens come from the outside by endocytosis into the lysosomal compartment,
00:26:59.14 retrograde refers to what happens when they go out. Again, this is probably,
00:27:04.06 or almost certainly I should say, a microtubule driven process,
00:27:07.15 but what’s most remarkable is that it happens at all. Our conventional view of
00:27:12.00 lysosomes is really kind of summarized here in this early medieval representation,
00:27:16.19 even prior to Metchnikoff, showing that there is no escape.
00:27:20.25 This is at least the conventional view. Proteins, antigens, whatever microbes are collected,
00:27:28.03 delivered into lysosomes and simply degraded. So we had not really anticipated
00:27:34.12 that there was going to be a pathway anywhere in cell biology
00:27:39.12 that was going to allow us to recover or allow a cell such as a dendritic cell
00:27:45.04 to recover molecules in a very selective and very efficient fashion
00:27:49.19 so they can move from this degradative compartment out to the cell surface.
00:27:54.00 Now when they get out to the surface, why do they stay there?
00:27:58.03 Why don't they just come back into lysosomes by endocytosis.
00:28:01.05 Well you might say endocytosis is shut off and indeed, as I already mentioned,
00:28:05.22 it is as a consequence of down regulating active forms of Rho family GTPases,
00:28:11.29 such as Cdc42 and Rac, all of which are involved in actin assembly. So the normal
00:28:20.02 ability of immature dendritic cells to capture antigen by such processes
00:28:24.11 as macropinocytosis or phagocytosis, which are both strongly actin driven
00:28:30.18 processes as we've seen in the first video, indeed are turned off. But uptake
00:28:36.18 via endocytosis by clathrin coated pits, which are much smaller vesicular carriers
00:28:43.23 that can be formed about only 0.2 microns in diameter, as opposed to the
00:28:48.17 phagosome, which can be one or two or three or even five microns in diameter,
00:28:52.24 this pathway continues. And indeed we know that MHC class II molecules
00:28:57.19 can enter cells, and indeed can enter dendritic cells by clathrin mediated
00:29:03.00 endocytosis. So why is it that class II molecules do not enter in the mature cell?
00:29:11.27 That brings us to another emerging fundamental feature of membrane traffic,
00:29:17.11 and that is the role of protein based ubiquitination in controlling the movement
00:29:22.18 of membrane proteins from one compartment to the next. Over the last several
00:29:27.05 years, a large number of investigators have demonstrated that ubiquitin plays
00:29:32.21 a critical role in signaling, either the endocytosis of endocytic receptors into
00:29:38.09 clathrin coated pits and/or the ability of those receptors at the level of endosomes
00:29:44.08 to be sequestered into these rather oddly shaped structures
00:29:48.28 called multivesicular bodies. We know from the work of Scott Ember and others
00:29:54.09 for example that when ubiquitin is modified or a membrane protein is
00:29:59.12 modified by ubiquitin, after delivery or upon delivery to the endosomal compartment,
00:30:04.19 these ubiquitinated membrane proteins are further captured
00:30:08.25 by the endosome sequestered in these little internal vesicles that then
00:30:14.07 are delivered to late endosomes and eventually to lysosomes where they can finally
00:30:20.22 be degraded. Now this happens in dendritic cells because if you look actually
00:30:26.21 by electron microscopy in a picture taken by the late Marc Pypaert is shown here
00:30:35.11 that the class II molecules are not found on the limiting membrane of lysosomes,
00:30:40.11 but rather are found associated to a first instance with these internal vesicles.
00:30:46.09 Now this was a real surprise, a double surprise because we further thought
00:30:51.15 that of course not only everything that would go into a lysosome would be
00:30:56.06 degraded, but certainly everything that was associated with the multivesicular body
00:30:59.24 would be degraded somehow dendritic cells and class II molecules have figured this out.
00:31:05.20 Now this is how the ubiquitin system works, and I'll show you the solution
00:31:10.21 to this problem at least the first solution to the problem. Ubiquitin is a small
00:31:15.15 protein that is now well known as a consequence of a series or cascade of events
00:31:22.13 to be covalently linked to a variety of different acceptors both cytosolic proteins
00:31:29.02 as well as membrane proteins by virtue of the activity of at least two large
00:31:34.19 families of so called E3 ligases that fall either into the HECT family or
00:31:40.11 the RING family, the details of this at the moment are not important,
00:31:44.10 but what is important is that both of these E3 ligases can affix ubiquitin molecules
00:31:49.16 usually the lysine acceptors on a variety of different proteins.
00:31:54.04 Now it turns out that of all the MHC class II molecules that have been
00:32:00.26 sequenced so far there’s enormous diversity in terms of the sequences
00:32:04.28 that one finds in the cytoplasmic domains of these molecules
00:32:08.13 except for a single conserved lysine residue shown here at position 4 in the cytoplasmic domain
00:32:16.13 of the beta chain of the MHC class II molecule. When Jeoung-Sook Shin
00:32:21.06 who is now at UCSF as a faculty member was in my lab, she made this
00:32:27.02 realization and further surmised that gee if there is such an important
00:32:31.21 and well conserved lysine residue present in class II molecules,
00:32:35.15 perhaps class II molecules are subject to ubiquitination. And, indeed, not only
00:32:40.25 did she find that they are subject to ubiquitination, but that ubiquitination
00:32:45.01 is exquisitely well regulated. So here you are looking
00:32:50.09 at an SDS gel which was subjected to western blot antibody labeling procedure
00:32:57.16 to detect molecules of MHC class II that are either ubiquitinated or not ubiquitinated.
00:33:03.29 So here as you can see in immature dendritic cells class II molecules
00:33:07.26 on the beta chain are nicely ubiquitinated, but again, shortly after maturation
00:33:12.25 of these cells by stimulation of the Toll-like receptor system,
00:33:16.20 you now find that those ubiquitin molecules are no longer present on class II
00:33:22.18 we believe because they are no longer added but the most important
00:33:26.14 conclusion is that they are not there, and because they are not there,
00:33:30.03 they now lack the ability to enter the cell and become sequestered
00:33:34.06 in multivesicular bodies and in lysosomes. So the bottom line is that in
00:33:39.21 short dendritic cell because ubiquitination does not occur,
00:33:43.09 those MHC class II molecule peptide complexes that are recovered by retrograde transport
00:33:49.16 from the lysosomal compartment to the plasma membrane stay put
00:33:53.19 because they lack the information, namely the ubiquitin molecule
00:33:57.02 linked to the class II that happens in immature dendritic cells.
00:34:00.23 They lack this ubiquitin molecule and as a consequence these class II peptide complexes
00:34:06.08 stay where they can best serve the interest of the immune system
00:34:10.07 and be available for recognition by Cd4 positive T-cells.
00:34:14.09 And this is what that looks like. Dendritic cells shown here in red
00:34:19.19 can complex with an enormous number of T-cells because they express
00:34:24.29 at such high levels these MHC peptide complexes in the case of class II because
00:34:31.06 those complexes cannot be internalized after maturation by endocytosis.
00:34:37.12 Class I is a different story, and I'd like to turn to that now because it illustrates
00:34:43.07 another aspect of the system as why dendritic cells are indeed so special,
00:34:48.15 not only in terms of the role they play in the immune response,
00:34:52.17 but how that role is determined by alterations, in fact some rather
00:34:57.28 unexpected alterations in membrane traffic.
00:35:01.22 Now class I as I've already mentioned is a pathway that is mostly adept
00:35:09.16 to dealing with endogenous antigens. So the best example I can give you
00:35:15.23 is if you have an influenza virus infection and epithelial cells in your airway
00:35:23.19 are infected by influenza virus, those cells are going to be making
00:35:29.12 lots of proteins that are encoded by the virus. Those proteins are going to be degraded
00:35:34.22 in the cytosol, again following a ubiquitination event they'll be degraded
00:35:38.18 by the proteosome in the cytosol, small peptides generated from
00:35:43.03 those ubiquitinated proteins and then those small peptides translocated
00:35:48.24 into the lumen of the endoplasmic reticulum by virtue of the activity of a rather
00:35:54.04 remarkable ATP-driven membrane peptide transporter called TAP, actually TAP1 and TAP2.
00:36:03.06 So the peptide that enter into the ER are loaded onto class I molecules
00:36:07.13 and then they make their way out to the cell surface by the constitutive secretory pathway.
00:36:13.02 Now there's a flaw in this logic. Remember I told you that only dendritic cells
00:36:17.08 can start an immune response. And I also just told you that the predominate cell,
00:36:22.25 in fact possibly the only cell that is infected after you become infected by
00:36:30.03 influenza are epithelial cells. How do we guard ourselves against
00:36:36.10 the possibility that the dendritic cell doesn't become infected?
00:36:39.00 The epithelial cells are incapable of generating a robust T-cell response,
00:36:43.10 only the dendritic cells are capable of doing that but the dendritic cells are not infected,
00:36:48.16 they're not making the influenza virus specific proteins. So how do
00:36:54.13 dendritic cells deal with this? They deal with it by having developed
00:36:57.26 a rather remarkable system of membrane transport,
00:37:03.00 which is classically referred to as cross presentation. It was really first described
00:37:07.08 by an immunologist, Mike Bevan. Here it was thought to be the case
00:37:12.04 that antigens coming in from the outside rather than being restricted
00:37:16.29 to the MHC class II pathway can cross over and in fact have access to the class I pathway
00:37:23.00 and do so by somehow breaking out of the endosome lysosome system,
00:37:27.03 entering into the cytosol and becoming accessible to the ubiquitination proteosome
00:37:32.06 degradation system that then is also responsible for servicing
00:37:37.16 those antigens that are endogenously synthesized by the cell. So the peptides
00:37:43.20 that form from these cross presented antigens can enter into the ER lumen
00:37:49.09 via the TAP1 TAP2 translocator, be loaded onto class I molecules
00:37:54.29 and then as I've been describing, make it out to the cell surface.
00:37:58.09 So the way this probably works in practice in the case of virus infections
00:38:02.23 is diagrammed here, immature dendritic cells will capture and take up
00:38:08.27 just as another phagocytic load a virus infected cell, which has been killed
00:38:15.08 by virtue of its virus infection. So dead cells, apoptotic cells, necrotic cells
00:38:21.06 can be nicely recognized by dendritic cells, enter into phagosomes,
00:38:25.09 these cells are then degraded and then antigens derived from the infected cell,
00:38:30.00 most importantly the virus encoded antigens now come out into the cytosol
00:38:35.04 and can be degraded by the proteosome system and be presented on the surface
00:38:39.23 of the dendritic cell on class I molecules. Now remember while all this is going on,
00:38:46.10 proteins that are also intrinsic to this self of the infected cell,
00:38:52.14 in other words our own proteins, are not going to be immune to this,
00:38:56.01 they will also be degraded in the phagosome and some portion of them
00:39:00.25 we have to imagine will also come into the cytosol and be degraded
00:39:05.18 and be loaded onto class I molecules and presented to now
00:39:09.15 CD8 positive T-cells on the cell surface. So how is it then that
00:39:13.24 the dendritic cell can distinguish between the viral antigen and the self antigen?
00:39:18.08 How does the immune system do this? How does the immune system do that?
00:39:22.29 Now it may be apparent to some of you, but I actually had to go off
00:39:25.29 and think about this for a little bit, but it turns out that to understand
00:39:29.22 how the immune system balances tolerance and immunity responses
00:39:33.18 to self versus non-self actually has a lot again to do with dendritic cells,
00:39:38.02 specifically the property of dendritic cell maturation which we now should
00:39:42.00 come back to and look at in a little bit more detail to understand just how
00:39:45.12 these cells via the process of maturation controls the progress of the immune response.
00:39:50.21 Now remember that maturation links the two major arms of the immune system,
00:39:55.24 the innate immune response to the adaptive immune response
00:39:59.13 by detecting microbial pathogens and then turning those pathogens into
00:40:04.03 the peptides that are required to be presented to T-cells to generate adaptive immunity.
00:40:08.05 Now the maturation process itself in its first instance is controlled by this family
00:40:14.15 of Toll-like receptors that I've mentioned, that essentially act as barcode readers
00:40:19.01 and I'll come back to that in a moment, that identify and deconstruct exactly
00:40:24.16 what type of pathogen happens to be present at the time the maturation event is induced.
00:40:30.28 Now there are a number of different Toll-like receptors. Some of them are
00:40:34.15 diagrammed here. About 12 or more, I've lost count since it changes periodically,
00:40:40.19 and what these Toll-like receptors are for is that they are specific for a variety of
00:40:44.13 different components that one finds in a wide variety of different pathogens.
00:40:47.19 You find some of the Toll-like receptors expressed on the plasma membranes
00:40:51.10 of cells, other Toll-like receptors are expressed in endosomes and lysosomes.
00:40:55.05 They are specific for bacterial proteins, bacterial lipids, one of the proteins
00:40:59.14 is one called flagellin, which is the major component of the bacterial flagella.
00:41:03.14 LPS is a major lipid species found in cell walls of many bacteria,
00:41:09.25 but many of the intracellular Toll-like receptors in particular actually react with specifically
00:41:15.28 and identify specifically nucleic acids, both single stranded and double stranded RNA and DNA.
00:41:22.27 In all cases, the general property that is elicited is this property of maturation,
00:41:28.17 but not all forms of maturation are created equal because not all
00:41:32.07 Toll-like receptors are created equal, and in fact they transmit different types
00:41:36.18 of signals to the dendritic cell. So as I said, these receptors work together
00:41:41.17 in the sense of a bar code. Some firing, others not firing, depending upon
00:41:46.12 what pathogen happens to be present, and the dendritic cell reacts to this information
00:41:51.22 and undergoes a path of its own maturation that adapts itself specifically
00:41:57.28 to the type of immune threat that is coming in from the environment
00:42:02.00 again and identifies the nature of the pathogen, the nature of the threat based
00:42:06.27 on the combinatorial array of Toll-like receptor ligands that happen to be present
00:42:13.06 and associated with that particular bacterium. Now as a consequence
00:42:17.27 of detecting these different arrays of Toll-like receptor ligands,
00:42:24.04 the dendritic cell matures and what it does as a consequence of that is
00:42:30.03 of course is to secrete a wide variety of different cytokines, which are again
00:42:33.16 essentially immunological hormones. Now T-cells are very smart, they are
00:42:39.27 almost infinitely capable of recognizing a wide array of different types
00:42:45.08 of antigens. But they have to essentially be told what to do by the dendritic cell
00:42:51.21 and this is not only by virtue of identifying the particular peptide MHC complex
00:42:57.01 that is presented by the dendritic cell that actually gets the T-cell responses going,
00:43:01.08 but this mixture of cytokines that is released by dendritic cell specifically
00:43:06.05 and in a customized fashion depending upon the type of microbe that was detected
00:43:12.19 is that which actually determines what the overall differentiation of the T-cell is going to be.
00:43:19.16 So its not enough simply to stimulate T-cells as a consequence
00:43:23.05 of having them detect via the T-cell receptor, the peptide MHC complexes
00:43:27.24 that are formed by dendritic cells, but the dendritic cells add to that process
00:43:32.21 by transmitting their experience of what type of pathogen had come in,
00:43:38.14 and they do this in this case by secreting the characteristic cytokines.
00:43:42.14 So one example of this is certain types of Toll-like receptors, or stimulation by
00:43:48.29 Toll-like receptors will cause dendritic cells to release the very
00:43:53.05 potent immunogenetic cytokine, IL-12, or interleukin 12.
00:43:57.27 T-cells that detect antigen exposed on the surface of
00:44:03.10 a dendritic cell that is secreting interleukin 12 undergo a type of differentiation
00:44:08.09 that allows them to become a particular sub-class of T-cell called Th1 T-cells,
00:44:14.04 which are highly inflammatory and highly immunogenetic cells.
00:44:17.16 Now the way this looks actually in situ is shown nicely here. This is a
00:44:22.06 scanning electron micrograph showing a dendritic cell in the background
00:44:25.26 with a number of T-cells that are attached very closely, as I've shown you
00:44:31.19 earlier in one of the video images, T-cells move around quite a bit across
00:44:36.07 the surface of dendritic cells, but when they finally find a good match,
00:44:40.17 a peptide MHC to a given T-cell receptor, they have a tendency to stay there
00:44:45.14 and stay there for a fairly long period of time, hours if not more,
00:44:51.08 and over this period of time they are literally bathed in the cytokine mix
00:44:56.04 that is being released by the dendritic cell, instructing them to become
00:45:00.22 a wide array of T-cells that all recognize antigen but all have a very very different
00:45:07.27 functional outcome with respect to how the immune response works.
00:45:11.09 I've lifted some of the more popular T-cell types that one can find.
00:45:15.08 We won't go through them in any detail at all, but just simply to say that
00:45:20.11 there are many many different possible T-cell outcomes that have different effects
00:45:25.03 on the so CD8 killer cells, or cytotoxic T-lymphocytes,
00:45:30.25 CTLs are ones that actually kill their target, so a cell that is infected by a virus
00:45:37.21 as we've discussed before will be killed by a cytotoxic T-cell of this particular type.
00:45:44.11 Not any T-cell will do this, but in fact only these will. CD4 helper cells help
00:45:49.19 generate antibody responses by working in collaboration with B-cells.
00:45:53.22 Inflammatory cells, central memory, effector memory cells are all T-cells that
00:45:58.14 circulate retaining the information of the immune account that had occurred,
00:46:03.16 retaining the lessons learned from the dendritic cell, and finally one
00:46:07.06 that we'll come back to in just a moment is the regulatory T-cell,
00:46:10.03 which rather than promoting immunity, actually dampens immunity and
00:46:13.13 probably assists or plays a central role in assisting the process of tolerance.
00:46:18.04 Again, often these cells are generated by dendritic cells, but not always,
00:46:24.17 and I think here I'd like to turn to just this very issue of tolerance and recognition
00:46:31.10 of self or non-self. How does it work? Basically it works in two settings,
00:46:36.10 before birth and early after birth, in the organ called the thymus where all
00:46:44.25 T-cells have their origin, prior to the exposure of the developing fetus or organism
00:46:51.07 to any exogenous antigens at least under normal circumstances,
00:46:55.08 a critical process called negative selection occurs. Now during negative selection,
00:47:00.05 either dendritic cells in the thymus or a related but nevertheless different
00:47:05.03 type of cell which has the same function or is presumed to have the same function
00:47:09.27 in the thymus called thymic epithelial cells have the remarkable function
00:47:15.06 of being able to turn on at the level of transcription, the expression of a wide array
00:47:22.00 of almost all of the proteins that we know that will be expressed in differentiated cells
00:47:27.03 later in life in the pancreas, in the liver, in the kidney, all of these are
00:47:32.22 expressed by these cells early on in the thymus, creating peptide MHC complexes
00:47:38.02 that are recognized by these thymocytes or forming T-cells that are being born
00:47:45.11 and developing in the thymus at this very very early stage of life. Now what
00:47:50.21 happens at this stage though is really quite interesting
00:47:53.21 and also quite important, and indeed quite profound.
00:47:56.14 Rather than the recognition event of the cognate peptide MHC by the T-cell receptors
00:48:04.11 on these developing T-cells, rather than causing an immune response or
00:48:09.28 an unrestricted proliferation of the T-cells, instead the T-cells
00:48:14.02 are induced to undergo apoptosis and die. In immunological parlance,
00:48:20.03 these cells are referred to as deleted. So any T-cell that recognizes
00:48:24.18 its antigen in the environment of the thymus early in development
00:48:29.06 is negatively selected and removed from the repertoire of
00:48:36.28 all of the antigens that could possibly be seen by the T-cell receptor later in life.
00:48:43.06 So this is a terrifically important first pass, whereby the immune system,
00:48:48.07 thymic and epithelial cells and dendritic cells due to the special properties of the thymus,
00:48:53.17 which are still not quite understood, are capable of removing a wide array
00:49:00.08 of T-cell specificities that would otherwise recognize host or self proteins
00:49:07.03 causing auto-reactivity and auto-immunity. As powerful and as important
00:49:13.07 as this process is, its not 100% efficient. Some self antigens are missed,
00:49:18.20 but another critically important class of antigen that's missed
00:49:23.11 is environmental antigens, since after birth we are all bombarded
00:49:28.13 and in fact bathed in a wide number of environmental allergens such as pollen in the air,
00:49:35.07 food allergens of various types, things that may penetrate through the skin,
00:49:40.17 and if we were to amount an immune response to each one of these foreign
00:49:44.08 antigens we would be hyper-allergic and not be in a very good state.
00:49:50.14 Now there is no way that the thymus can educate our T-cell responses or
00:49:54.29 the dendritic cells can educate our T-cell responses in the thymus
00:49:57.27 to delete T-cells that might be specific for a pollen or environmental antigens.
00:50:04.25 That has to occur after birth because obviously as a fetus we are not generally
00:50:09.16 speaking exposed to too many environmental antigens that are allergic in this sense.
00:50:14.05 So this is a process that's been left to the formation of this final and critically
00:50:21.21 important and yet incredibly poorly understood form of T-lymphocyte
00:50:26.10 called the T reg, or regulatory T-cell. Now a lot of these are in fact formed
00:50:31.12 in the thymus, so in addition to deletion of T-cell reactivities, one also finds
00:50:36.24 that the thymus will produce an array of regulatory T-cells that recognize
00:50:42.18 a variety of self antigens that will then have a tendency to help turn off
00:50:48.01 T-cell responses that occur inappropriately later. Okay, but many regulatory T-cells,
00:50:54.26 or T regs, are not produced in the thymus but rather are produced in the periphery
00:50:59.26 as a consequence of not so much a negative selection process but a tolerogenic
00:51:04.25 process which in this case is mediated almost exclusively by the dendritic cell
00:51:09.11 not by the thymus. Now this happens under steady-state condition.
00:51:13.27 So what I mean by that is if an antigen is encountered by dendritic cells that have not
00:51:21.09 received a stimulus via a microbial type Toll-like receptor ligand to mature,
00:51:29.22 what one finds then is that all of the same processes of antigen processing
00:51:34.12 and presentation and transport of the peptide of MHC complexes
00:51:38.10 to the surface take place, dendritic cell develops to a form that is capable of efficiently
00:51:44.09 doing this and efficiently generating T-cell recognition, but nevertheless
00:51:49.14 under these conditions in the absence of a Toll-like receptor stimulus
00:51:53.18 or another inflammatory stimulus, the type of T-cell that emerges is a
00:51:59.03 regulatory T-cell, or an induced regulatory T-cell, totally as a consequence
00:52:03.06 of peripheral recognition events. So these T regs instructed again and formed
00:52:09.26 almost uniquely by peripheral dendritic cells found in our lymph nodes,
00:52:15.05 lymphoid organs and indeed all of our peripheral tissues are our last line
00:52:21.12 and in many cases our most important line of defense against mistakes
00:52:26.21 that can be made by the immune system between self and non-self,
00:52:31.11 between foreign and endogenous antigens. Again, serving to control
00:52:36.09 this balance between tolerance and immunity. Again, it is the T-cell that does it,
00:52:42.13 although to be fair we don't really understand very much about how T regs work,
00:52:47.10 this is an emerging field, an emerging problem at the moment.
00:52:51.11 But what it does seem to be quite clear based on genetic deletion results
00:52:56.04 and antibody blocking results that have been done quite recently, it is these
00:53:01.04 dendritic cells that exist under steady state non-inflammatory conditions
00:53:05.10 that really are responsible for generating these T regs.
00:53:09.21 Now this is critically important for a variety of reasons because every time a
00:53:14.19 dendritic cell matures as a consequence of being stimulated by Toll-like receptor
00:53:19.24 ligand, those dendritic cells present not only the foreign antigen
00:53:23.18 but also present every self antigen in the body that they can come in contact
00:53:27.26 with over that period of time. So every time we respond to a foreign stimulus,
00:53:33.23 we run the risk of responding to one of our own self antigens
00:53:38.21 and run the risk of developing auto-immunity. So in order to maintain
00:53:45.06 this very very careful relationship, this very very careful balance
00:53:50.07 that must occur lest auto-immune pathologies set in between immunity and tolerance,
00:53:57.13 there is this continuing production of T regs that occurs that provide with us
00:54:03.20 this important break, this important line of defense to maintain equilibrium,
00:54:09.07 maintain homeostasis, and maintain health. So the way I like to frame this
00:54:15.16 is as a hypothesis, which I stress by saying is really no more than a hypothesis
00:54:22.06 at this point, but nevertheless I think summarizes quite well the process as many
00:54:27.20 of us really believe it occurs at this point. So as I was saying,
00:54:32.00 under conditions of no infection, under the steady-state, one finds
00:54:35.28 immature dendritic cells in peripheral tissues, as shown here in the skin,
00:54:40.02 again using the same diagram we've been looking at during the course of these two lectures.
00:54:45.11 No infection, immature in the periphery, and at some point these cells
00:54:51.09 either just due to stochastic processes or due to some inductive process
00:54:56.11 migrate from the skin via the lymphatics into the lymphoid tissues.
00:55:00.22 Along the way, they undergo a type of maturation, because now in lymphoid
00:55:06.11 organs they are capable of presenting antigens, all self-antigens in this case,
00:55:10.17 or all environmental antigens, but nevertheless the type of maturation
00:55:14.20 that they undergo is one that leads to tolerance. So they are not antigen
00:55:19.08 presenting, they are not very effective antigen presenting cells as immature cells
00:55:23.21 in the periphery, they are much better at it when they are in lymphoid organs
00:55:27.18 but they are nevertheless still tolerogenic. Again, these are conditions under
00:55:31.11 steady state, meaning no infection, no inflammation. Everything changes though
00:55:36.04 when we go to the condition of infection, or inflammation. Here now
00:55:41.10 what you find is that again the dendritic cells are still immature in the periphery,
00:55:45.23 but now they encounter a Toll-like receptor stimulus as a consequence
00:55:50.16 of the advent of one or more microbes as we've been discussing. The migration
00:55:56.29 process is the same, the delivery to lymphoid organs is more or less the same,
00:56:01.14 but now the maturation that takes place is one which is not tolerogenic but rather immunogenetic.
00:56:08.19 Okay, so the T-cells that are produced by this same progenitor population of
00:56:14.22 dendritic cells, possibly, possibly there are subsets, but possibly the same
00:56:19.09 progenitor population of dendritic cells under conditions of infection,
00:56:23.11 under conditions of inflammation, yields T-cells that undergo development
00:56:27.05 not to produce T regs, but rather to produce one of the many types of
00:56:30.28 inflammatory or immunogenetic T-cells that I listed for you just a moment ago.
00:56:36.12 Now this to me comprises probably one of the most profound of all problems that
00:56:44.16 remain in the immune system. I've stated it in what may appear to some of you
00:56:49.23 at least to be relatively reasonable terms, but the fact of the matter
00:56:53.04 is we have almost no idea how these events are interconnected.
00:56:57.18 We know small details, all of which are indeed enticing, beginning with why
00:57:03.14 is it that transcriptionally one can find so many different differentiated gene
00:57:07.28 products expressed early on in the thymus? We know something about what
00:57:12.18 the transcription factors are that do this, but how all of this is regulated,
00:57:15.26 how it actually works, very very few of the details are really known,
00:57:20.03 and it really represents a terrific area for research of basic biology and also to
00:57:25.26 come up with still solutions to one of the great problems left in immunology.
00:57:31.19 There are many, but this is certainly one that tops my list. Now another reason
00:57:37.16 why this is so important is not just because of the basic biological aspect,
00:57:42.03 but also because of the disease aspect. I think increasingly as size progresses
00:57:47.21 in our understanding and our ability to do more and more complex experiments,
00:57:52.23 particularly at the systems level, an equally valid path to take in studying basic
00:58:00.10 biology is to understand disease processes. This of course has been done
00:58:05.03 by many in the past, but I think increasingly so at earlier and earlier stages
00:58:09.00 in ones scientific career and ones scientific interests, its possible to begin
00:58:14.03 to do real basic solid experiments where your question is what happens during
00:58:20.02 a particular disease process. So how does tolerance and immunity
00:58:23.18 fit into this? I've already hinted at it several times, but if you have a situation
00:58:28.06 where there is too little tolerance in other words the dendritic cells
00:58:32.10 or the thymus were not optimally efficient at deleting auto-reactive T-cells
00:58:37.24 or turning auto-reactive T-cells into T regs, or regulatory T-cells,
00:58:43.00 one can find a wide variety of diseases that fall into the broad class of auto-immune disorders,
00:58:49.24 such as auto-immune diabetes, lupus, or Myasthenia gravis. These are mediated
00:58:55.04 either by the production of pathogenic antibodies to self proteins,
00:58:59.08 or T-cells that exert direct cytotoxic effects on normal host tissues.
00:59:05.24 Another possibility is chronic inflammation, so diseases such as arthritis,
00:59:11.19 or asthma, Crohn's disease, ulcerative colitis, Multiple Sclerosis, possibly
00:59:16.22 have to do with the fact that an inflammation starts and then can't be turned off.
00:59:20.28 These may not be strictly auto-immune in many cases because for a lot of these
00:59:26.00 diseases there may not be a single antigen against which T-cells continuously
00:59:30.21 are producing new antibody via B-cell production or new cytotoxic secretions
00:59:39.03 as a consequence of the T-cells own activities, but nevertheless these
00:59:43.17 are processes that keep going because of disregulation of the balance
00:59:48.27 between tolerance and immunity. And again, in many ways one can attribute
01:00:00.00 the brute cause of all of this to dendritic cells misbehaving,
01:00:04.25 presenting antigens in the wrong context, producing the wrong type
01:00:08.26 of T-cells under a condition that doesn't call for that type of T-cell response,
01:00:13.24 and then the do-loops that emerge simply don't get turned off.
01:00:17.27 So how do we intervene in all of these things and how can we do this not only to
01:00:24.09 understand the biology, which is of course paramount, but also to understand
01:00:28.00 how therapeutically we can begin to intervene in these diseases processes
01:00:32.16 with ever higher degrees of specificity and exactness so that we can turn off
01:00:38.04 just the disease process and not interfere with normal ongoing processes
01:00:43.04 or actually do more harm than good. So now that I've moved myself from
01:00:48.23 academia to a biotech company, these are problems that are coming to the fore
01:00:54.23 on a daily basis, and its of no small matter to try and understand and grapple
01:01:01.28 with these problems, not only as a basic scientist but also as someone
01:01:06.06 who is now committed to understand how it is that you can turn that
01:01:10.07 basic science knowledge into dealing with major major health problems such as these.
01:01:15.09 It's also possible that you have too much tolerance and two sets
01:01:21.17 of very bad things can occur under these circumstances. This is different from diseases
01:01:27.05 that lead to immunodeficiency. Here you have diseases where the immune system
01:01:32.16 is often intact, but has been educated by the pathogenic organism
01:01:38.21 or as shown here by cancer cells, to evade the immune response. Cancer is I think
01:01:47.01 a particularly challenging example. Immunotherapy in cancer is something
01:01:51.16 that is now just starting to gain steam now with the first immunotherapeutic
01:01:56.28 to prostate cancer just having been approved this year, but what we
01:02:02.00 understand about cancer and the immune system tells us, at least to a first approximation,
01:02:07.25 that many cancers in fact are capable of generating immune responses,
01:02:11.14 either due to mutation or to ectopic expression of proteins that are not normally produced by a given cell.
01:02:20.03 Cancer cells in fact can elicit T-cell responses, but they've figured out,
01:02:25.12 or if they haven't figured out at least they've been selected for cells that are
01:02:29.25 capable of subverting those T-cell responses, either by turning them off,
01:02:34.02 such that when the T-cell penetrates into a tumor bed and tries to kill its target,
01:02:39.00 the target protects itself by secreting or placing on its surface molecules that in
01:02:45.05 fact will abrogate T-cell responses rendering them as immunologically, I'll just say, anergic.
01:02:53.21 Another possibility though is that cancer cells in fact by much the same way that
01:02:59.26 dendritic cells seem to do it, will seem to generate a T-regulatory or T reg responses,
01:03:05.15 again having the same effect subverting T-cell responses to cancerous cells
01:03:12.10 that would otherwise be amenable to be controlled by those T-cells, at least in theory.
01:03:18.10 Another, and I think in many ways more clear example, occurs in the case of many
01:03:25.01 chronic viral infections, such as CMV or HIV. CMV is a particularly good example,
01:03:31.25 but many other chronic viruses are as well. What happens in these cases is that
01:03:37.00 viruses have figured out how to down regulate proteins on the surface of
01:03:43.10 the infected cell, or in some cases even on the surface of dendritic cells
01:03:47.06 in such a way as to prevent, again, T-cell recognition or in some cases even T-cell responses.
01:03:54.15 The immune system in a sense gets educated to understand that the viral proteins
01:03:59.21 are not actually foreign, but indeed are part of the own host protein repertoire,
01:04:09.04 and as a consequence by tricking the immune system in this way,
01:04:12.12 the virus can replicate and can maintain its infection with impunity without
01:04:19.19 risk of detection by the immune system. So in the case of diseases where
01:04:24.29 there is too much tolerance, therapeutic intervention that one can imagine,
01:04:30.04 is how is it that you reactivate T-cell responses by convincing the dendritic cells
01:04:36.24 to break tolerance as we say and re-introduce antigens either derived from cancer cells
01:04:43.21 or from viruses under conditions that now can generate positive
01:04:48.15 immunogenic immune responses rather than just tolerogenic immune responses.
01:04:54.05 So both of these types of disease states, again, embody I think some of the most exciting
01:05:00.02 biology, both immunology and cell biology that one can think of
01:05:05.00 in the immune system, plus also offer the opportunity to the lucky and interested
01:05:10.09 scientists, which hopefully includes me, to understand how it is one can
01:05:17.00 actually make either biological agents or even small molecule drugs
01:05:21.13 to either induce tolerance under conditions where you would like to turn off
01:05:26.20 chronic inflammation or turn off auto-immunity or overcome tolerance
01:05:31.17 under conditions where you would like to re-activate the immune system,
01:05:35.08 re-educating it to go about its job and combating what are effectively
01:05:41.24 foreign agents such as cancer or chronic viruses both for therapeutic benefit. Thank you.

Differentiation, priming, tolerance and training

Adaptations in innate immune compartments are exceptionally diverse, as innate immune cells demonstrate substantial plasticity and adapt to various insults such as trauma, infections and vaccination, and they continue to adapt as they leave the local microenvironment of the bone marrow and travel to the blood and tissues. It is important to note that the magnitude (low versus high dose) and duration (short versus long) of stimulation induces specific adaptations in innate immune cells that reflect their requirement to either enhance immune responses or prevent immunity and excessive immunopathology. Several such adaptive programs have been described, including cell differentiation, priming, tolerance and trained immunity (Fig. 1). Because innate immune cells can undergo any of these functional adaptive programs, it is essential to precisely define the similarities and differences between these cellular adaptations to ensure the field’s focus and avoid confusion in the literature.

a, Differentiation. b, Priming. c, Trained immunity (innate immune memory). d, Tolerance.

The main difference between innate immune cells undergoing these different adaptive programs is their functional status prior to secondary challenges. Innate immune cell ‘differentiation’ (Fig. 1a) is often the change of an immature cell into its mature counterpart, which is defined by a long-term change in the functional program of the cell and is often accompanied by altered morphological characteristics caused by alterations of the tissue environment or chronic exposure to stimuli 22 . During ‘priming’ (Fig. 1b), the first stimulus changes the functional state of these cells, and their immune status (as defined by active gene transcription) does not return to basal levels before the secondary stimulation or infection. Thus, the impact of a second challenge in primed cells is often additive or synergistic with the original stimuli. In ‘trained immunity’ (Fig. 1c), in sharp contrast to priming, while the first stimulus leads to changes in the functional immune status, the immune activation status returns to the basal level following removal of the stimulus, while the epigenetic alterations persist. However, in response to homologous or heterologous challenges, both gene transcription and cell function are enhanced at much higher levels than those observed during the primary challenge. The opposite of trained immunity is innate immune ‘tolerance,’ wherein the cell is unable to activate gene transcription and does not perform its functions following restimulation (Fig. 1d). For instance, repeated or persistent exposure of macrophages to a high dose of lipopolysaccharide epigenetically enforces tolerance to prevent the expression of inflammatory genes 23 .

Therefore, studies aiming to investigate trained immunity need to clearly identify the activation state during initial stimulation (represented by effector functions such as cytokine and reactive oxygen species production, phagocytosis, killing, and so on) as well as after the removal of the initial insult. This is challenging when dealing with monocytes and macrophages, as their spectrum of states is far less defined than that of adaptive immune cells. However, improved nomenclature has been proposed for both in vitro and tissue-resident monocytes and macrophages 24 . To investigate the central effects of trained immunity via HSPCs in the BM, experiments have been conducted predominantly in vivo or ex vivo 8,10 . However, many in vitro experiments have been performed to study the peripheral impacts of trained immunity. Thus, experimental standards are necessary for expanding our knowledge in this exciting field of immunology.

Defining the adaptive programs of innate immune cells according to their functional state is important because the molecular mechanisms underlying these processes can often overlap. In this respect, the epigenetic and metabolic rewiring that specifically program cell differentiation, trained immunity, immune priming and tolerance can define these processes. Thus, there are unique signatures that define different cellular adaptations. For example, long-term changes in DNA methylation and stable changes in chromatin accessibility can accompany cell differentiation, whereas specific histone marks characterizing ‘latent enhancers,’ such as monomethylated histone H3 K4 (but not solely that), are often ‘tagged’ in trained immunity 1 . However, while specific pathways and markers differ between the various adaptive programs in innate immunity, they all use the same basic mechanisms (epigenetic, transcriptional and metabolic), but with different flavors.


We are grateful for the input on this work provided by members of the Doudna laboratory. We thank S. Floor, A.S. Lee, H.Y. Lee, R. Wilson, R. Wu and K. Zhou for technical assistance, the 8.3.1 beamline staff at the Advanced Light Source and A. Iavarone (University of California, Berkeley) for MS. We thank D. King (Howard Hughes Medical Institute, University of California, Berkeley) for Flag and HA peptides. This project was funded by a US National Science Foundation grant to J.A.D. (no. 1244557). J.K.N. and A.V.W. are supported by US National Science Foundation Graduate Research Fellowships and J.K.N. by a University of California, Berkeley Chancellor's Fellowship. P.J.K. is supported as a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. J.N. is supported by a Long-Term Postdoctoral Fellowship from the Human Frontier Science Program Organization. J.A.D. is supported as an Investigator of the Howard Hughes Medical Institute.

Adaptive Immune Response

The adaptive, or acquired, immune response takes days or even weeks to become established—much longer than the innate response however, adaptive immunity is more specific to pathogens and has memory. Adaptive immunity is an immunity that occurs after exposure to an antigen either from a pathogen or a vaccination. This part of the immune system is activated when the innate immune response is insufficient to control an infection. In fact, without information from the innate immune system, the adaptive response could not be mobilized. There are two types of adaptive responses: the cell-mediated immune response , which is carried out by T cells, and the humoral immune response , which is controlled by activated B cells and antibodies. Activated T cells and B cells that are specific to molecular structures on the pathogen proliferate and attack the invading pathogen. Their attack can kill pathogens directly or secrete antibodies that enhance the phagocytosis of pathogens and disrupt the infection. Adaptive immunity also involves a memory to provide the host with long-term protection from reinfection with the same type of pathogen on reexposure, this memory will facilitate an efficient and quick response.

V(D)J Recombination and the Evolution of the Adaptive Immune System

Copyright: © 2003 Public Library of Science. This is an open-access article distributed under the terms of the Public Library of Science Open-Access License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abbreviations: BCR, B-cell receptor C, constant CJ, coding joint D, diversity DNA-PKcs[RRK1], DNA protein kinase H, heavy J, joining L, light RAG, recombination-activating gene RSS, recombination signal sequence SCID, severe combined immunodeficiency syndrome SJ, signal joint TCR, T-cell receptor V, variable.

The immune system needs to be able to identify and ultimately destroy foreign invaders. To do so, it utilizes two major types of immune cells, T cells and B cells (or, collectively, lymphocytes). Lymphocytes display a large variety of cell surface receptors that can recognize and respond to an unlimited number of pathogens, a feature that is the hallmark of the “adaptive” immune system. To react to such a variety of invaders, the immune system needs to generate vast numbers of receptors. If the number of different types of receptors present on lymphocytes were encoded by individual genes, the entire human genome would have to be devoted to lymphocyte receptors. To establish the necessary level of diversity, B- and T-cell receptor (BCR and TCR, respectively) genes are created by recombining preexisting gene segments. Thus, different combinations of a finite set of gene segments give rise to receptors that can recognize unlimited numbers of foreign invaders. This is accomplished by a supremely well-coordinated set of reactions, starting with cleaving DNA within specific, well-conserved recombination signal sequences (RSSs). This highly regulated step is carried out by the lymphocyte-specific recombinationactivating genes (RAG1 and RAG2). The segments are then reassembled using a common cellular repair mechanism.

For foreign invaders and their proteins (antigens) that are not part of the host to elicit an immune response, the immune system must be able to recognize countless numbers of antigens. For obvious reasons, an unlimited number of unique antigen receptors cannot be genetically encoded. Rather, the necessary diversity in receptors is achieved by creating variations in the antigen-recognition regions of the receptors of both B cells and T cells. These regions are created by the pairing of two different protein segments, called polypeptide chains (heavy [H] and light [L] chains in the case of the BCR and α and β chains in the case of the TCR), which form a cleft that provides a binding site for the antigen. The mechanism that generates variation in the antigen-binding pockets of these receptors involves mixing and matching variable (V), diversity (D), and joining (J) gene segments in a process called V(D)J recombination. To assemble a single functional receptor, preexisting V, D, and J gene segments are rearranged to yield a contiguous V(D)J region, just upstream of another element of the receptor, the constant (C) region (Figure 1A).

Adaptive immunity to viruses

Innate recognition of viruses allows activation of adaptive immune responses. Dendritic cells (DCs) are potent inducers of T cell responses. However, how various populations of DCs sense virus infection and induce immune responses during a natural virus infection is unclear. OUr study demonstrated that submucosal DCs (beneath the epithelial layer), but not Langerhans cells (within the epithelial layer), are the primary inducers of Th1 immunity following genital herpes infection. Antigen presentation following mucosal viral infection is handled by the tissue-migrant submucosal DCs, while needle-introduced virus antigens are presented by lymphoid resident DCs.

In addition to the direct activation of DCs by TLRs, we showed that DCs require TLR-dependent instructive signals from the infected cells in order to induce differentiation of effector T cells. We further demonstrated the requirement for TLR-dependent signal in enabling maximum screening of cognate lymphocytes during initiation of adaptive immunity through remodeling of the lymph node arteriole. Once initiated within the lymph nodes, effector Th1 cells travel to the site of infection and eliminate virus infection. Our recent study showed that the local mucosal DCs and B cells cooperate to restimulate Th1 cells to execute protective antiviral immunity.

These studies collectively demonstrated the importance of tissue-DC interaction in the initiation of antiviral immunity. While the role of TLRs and RLRs in the initiation of adaptive immunity has been studied extensively, the role of NOD-like receptors (NLRs) in innate viral recognition and initiation of adaptive immune responses is unknown. Our recent study demonstrated that influenza virus infection triggers NLRs and it is required to elicit protective T cell and B cell immunity. We are currently using this information to design and develop novel vaccine strategies to better fight viral infections including HSV-2, influenza and human papillomavirus.

Materials and methods

We developed a mathematical model for simulating the circadian clock in the lung of a rat, the immune system under acute inflammation, and the interactions between the two systems. A schematic diagram that depicts the regulatory network is shown in Fig 1. Model equations and parameters can be found in Tables A-J in S1 File.

Circadian clock in the lung

The mammalian clock consists of interlocked transcriptional-translational feedback loops that drive the circadian oscillations of core clock components [23]. Both the master and peripheral clocks share essentially the same molecular architecture [24]. The activators CLOCK and BMAL1 dimerize to induce the transcription of target genes, including the Period genes (Per1, 2, 3), Cryptochrome genes (Cry1, 2), retinoic acid-related orphan receptor (Rora, Rorb, Rorc) and Rev-Erb nuclear orphan receptor (Rev-Erbα, Rev-Erbβ) to activate their transcription [1]. PERs and CRYs then heterodimerize and enter the nucleus to inhibit their own transcription by acting on the CLOCK-BMAL1 protein complex, and thus form the main feedback loop [1, 25, 26]. In the secondary loop, the nuclear receptors REV-ERBα, β and RORa, b, c compete for ROR regulatory element (RRE) binding sites in the promoter region of Bmal1 and respectively repress and activate its transcription. The REV-ERBs, which also repress Cry1 transcription [27], are essential for robust oscillations [28–30]. See the clock network in Fig 1, and refer to Refs. [3, 26, 28] for a detailed overview of the molecular clock architecture.

The present lung circadian clock model, inspired by Ref. [31], describes the time evolution of mRNA and corresponding protein concentrations of Per, Cry, Rev-Erb, Ror, and Bmal1, and their modulation of proximal tubule epithelial transport [32]. Following the model assumptions in Ref. [31], we grouped the three Period homologs (Per1-3) as a single Per gene and the two cryptochromes (Cry1,2) as a single Cry gene. Similarly, the two isoforms Rev-Erbα and Rev-Erbβ and three isoforms Rora, Rorb and Rorc are represented by single variables Rev-Erb and Ror, respectively. It was assumed that the CLOCK protein is constitutively expressed. We did not include post-translational protein modifications, considering that transport between the cytoplasm and the nucleus is rapid on a circadian timescale [33]. The time evolution of the core clock genes and proteins is described by Eqs. 1–12 in Table B in S1 File.

Acute immune response

The acute immune response model, inspired by Refs. [34] and [35] with some modifications, consists of eight variables: endotoxin concentration (P) the total number of activated phagocytic cells (N, which includes activated immune response cells such as neutrophils and monocytes) a non-accessible tissue damage marker (D) concentrations of pro- and anti-inflammatory cytokines, namely IL-6, TNF-α and IL-10 a tissue driven non-accessible IL-10 promoter (YIL10) and a state representing the level of slow acting anti-inflammatory mediators (CA), which comprises slow-acting anti-inflammatory agents such as cortisol and TGF-β1. Model assumptions can be found in Refs. [34] and [35].

The introduction of bacterial insult in the system activates the phagocytic cells, N, and inflicts direct tissue damage, D [36]. This is different from the work of Roy et al. [34] in which endotoxin only activates phagocytic cells. The activated cells up-regulate the production of inflammatory agents (TNF-α, IL-6, IL-10, and CA) [37]. The pro-inflammatory cytokines TNF-α and IL-6 exert a positive feedback on the system by further activating N, as well as up-regulating other cytokines [37, 38]. The anti-inflammatory cytokines IL-10 and CA, on the other hand, have a negative feedback on the system. They inhibit the activation of N and other cytokines [39, 40]. The model also incorporates tissue damage, represented by a non-accessible damage marker, D. Tissue damage further up-regulates activation of N [41] and also contributes to up-regulation of IL-10 [42, 43]. In our model, D is up-regulated by IL-6 because it has been shown that IL-6 is associated with the development of sepsis [44–46]. This differs from Ref. [34] in which damage is up-regulated by N. Note also that D should not be interpreted directly as a cell type in the model. The acute inflammatory response is described by Eqs. 13–20 in Table B in S1 File see also Fig 1.

Coupling between the circadian clock and the immune system

Most studies on circadian-immune interactions have focused on Bmal1, since inactivation of this gene is a convenient way to abrogate clock function [13, 47, 48]. Thus, care should be taken in distinguishing Bmal1-specific effects from downstream effects because other clock components act as intermediaries. The inhibitory effects that the circadian clock and the inflammatory response have on each other are shown in Fig 1. Specifically:

  • CRY proteins.Cry1 −/− Cry2 −/− mice exhibit an elevated number of T cells in the spleen with increased TNF-α levels [22, 49]. Other studies showed that Cry1 and Cry2 double KO in fibroblasts and bone-marrow-derived macrophages (BMDMs) leads to increased Il6 and Tnf-α mRNA and an hypersensitivity to lipopolysaccharide (LPS) infection [15, 50]. Furthermore, the NF-κB signaling pathway was shown to be constitutively activated in Cry1 −/− Cry2 −/− BMDMs [50]. Due to the ensuing higher constitutive inflammatory state, Cry1 −/− Cry2 −/− mice exhibit increased infiltration of leukocytes in lungs and kidneys [51]. Therefore, CRYs play an important anti-inflammatory role by downregulating inflammatory cytokines. Because IL-6 is inducible with TNF-α, effects on IL-6 in CRY double KO experiments are primarily mediated by TNF-α [50]. In our model, CRY directly inhibits the production of TNF-α, hence indirectly inhibits the TNF-α-induced IL-6 production (Eq. 17 in Table B in S1 File).
  • ROR proteins. Similarly to the CRY proteins, experiments have shown that Rora −/− mice, also known as the staggerer mutant, exhibit higher levels of IL-6 in bronchoalveolar lavage fluid, which renders them more susceptible to LPS lethality [52]. Interestingly, staggerer mutant mice have an increased production of IL-6 and TNF-α in mast cells and macrophages after LPS stimulation [53, 54]. Furthermore, overexpression of RORa in human primary smooth muscle cells inhibits TNF-α-induced expression of IL-6 [55]. The present model assumes that ROR downregulates TNF-α, thus indirectly downregulates IL-6 (Eq. 17, Table B in S1 File). Recall that the model does not distinguish between the three isoforms Rora, Rorb and Rorc.
  • REV-ERB proteins. There is compelling evidence for a role for REV-ERBα in the control of the immune system. REV-ERBα is encoded by Nr1d1 and in vivo challenge of Nr1d1 −/− mice with LPS leads to IL-6 upregulation in serum in comparison to wildtype animals [56]. REV-ERBα represses Il6 expression not only indirectly through an NF-κB binding motif but also directly through a REV-ERBα binding motif in the murine Il6 promoter region [57]. A more recent study showed that the dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelial cells further augmented inflammatory responses and chemokine activation [58]. REV-ERBα also negatively affects the expression of anti-inflammatory cytokine IL-10. Rev-ErbαmRNA binds to the IL-10 proximal promoter and represses expression in human macrophages [59]. Together, these studies reveal the role of REV-ERBα as an equilibrist. In our model, REV-ERB directly inhibits the production of IL-6 and IL-10 (Eqs. 16 and 18 in Table B in S1 File, respectively). We note that the two isoforms Rev-Erbα and Rev-Erbβ are represented by a single model variable Rev-Erb.
  • Inflammation. In a reciprocal manner, inflammation induced by agents such as LPS, TNF-α, and IFN-γ [60–64] or acute bacterial infection [65] can affect the circadian clock. In particular, rodent studies indicate that LPS transiently suppresses clock gene expression and oscillations in the SCN and peripheral tissues [33, 64, 66, 67] notably, a number studies show significant suppression of Bmal1 [15, 66, 68]. The inhibition of the circadian mechanism during endotoxemia lasts for at least 24 h [64, 68]. To represent the sustained effect of a bacterial infection on the circadian clock, we introduced a filter function for LPS (Eq. 21 in Table B in S1 File), which acts on the clock through its inhibition of Bmal1 (Eq. 5 in Table B in S1 File). The filter function decays linearly over 24h and causes circadian disruption for at least this amount of time. We assume that the effects of cytokines such as TNF-α are incorporated in the net effect of LPS on clock genes, and so we do not include direct links from cytokines to clock genes and proteins.

Model parameters

Most of the model parameters are not well characterized, and were estimated by fitting model dynamics to experimental data. Due to the transient nature of acute inflammation, this is done in a two-step process: we first fit the circadian clock model in isolation, in an infection-free state. In the absence of infection, the acute inflammation model is idle and therefore has no influence on the expression of clock genes. Likewise, clock genes have no effect on inflammation variables whose initial conditions are zero. The fitting of the clock model is done using data on the expression of circadian genes in the mouse lung (CircaDB: Animals were entrained to a 12h light:12h dark schedule for one week, then released into constant darkness. Clock gene expression was recorded starting at CT18 postrelease [69]. It is noteworthy that the present model is based on the rat, whereas the parameters for the circadian clock were based on mouse data. However, while species differences exist, core clock gene expressions of the mouse and rat lungs exhibit substantial similarities [70]. Our model inherits the period of the data which is 24 h.

In a second step, we fit the acute inflammation model together with the clock-inflammation coupling, without changing the circadian clock parameters. This is done by simultaneously fitting experimental measurements of the cytokines IL-6, TNF-α, and IL-10 in rat following the administration of endotoxin at 3 mg/kg and also at 12 mg/kg [34, 35]. In other words, we fit the time profiles for all variables (P, D, N, CA, IL-6, IL-10, TNF-α) using measured data on IL-6, IL-10 and TNF-α only. This fitting was conducted with the coupling with the clock model taken into account. Some parameters in the model were specified. The clearance rate of endotoxin, P, captured by the parameter dP in Eq. 13 was obtained from the literature [71, 72]. Parameters sIL10 and sCA from Eqs. 18 and 20 were extracted directly from the experimental data, respectively [34]. Model parameters are shown in Tables C–J in S1 File.

Parameter estimation technique.

Parameter identification for the coupled system was carried out with a nonlinear least-squares method with a normalized residual, which minimizes the error between the computed model output and the experimental data. To this end, we defined: (1) Here, yi, is the measured data at time ti. The model prediction is given by y(ti, θ), where θ represent model parameters. Q is the total number of data points. Experimental error bars were not taken into account. The cost function that we minimize is given by: (2)

The subscripts 3 and 12 refer to the injected dose of endotoxin (mg/kg). As proposed in Ref. [73], the cost function has been minimized by using an optimization function in MATLAB known as fminsearch to search for the parameter estimated values which give the best fit of the model to the experimental data. Still, obtaining our final result required a series of educated guesses, manually correcting the most obvious difference before restarting the optimization. We have found that the parameter values are not uniquely determined, as different sets of parameters provide almost the same goodness of fit. In order to assess the relative influence of each parameter on the outcomes, we performed a sensitivity analysis using the Sobol’ method (S1 File).

Sexual dimorphism in clock gene alterations under circadian disruption

In their 2012 study, Hadden et al. reported sexual dimorphism in clock gene expression in the lungs of mice exposed to chronic jet lag [12]. Male and female mice were assigned to either remain in a LD12:12 regimen or to undergo experimental chronic jet lag (CJL). Under the CJL regimen, mice were subjected to serial 8-h advances of the light/dark cycle every 2 days for 4 weeks. Then using quantitative Polymerase chain reaction (PCR) to measure the relative amount of clock gene mRNAs, Hadden et al. observed that Rev-Erbα gene expression is upregulated in CJL males and downregulated in CJL females by 98% and 70% on average, respectively. Bmal1 is downregulated in CJL females only by 43% on average, while Clock, which forms a heterodimer with Bmal1 (CLOCK:BMAL1) [69], is downregulated in males only by 26%. The repressors Per2 and Cry2 are both upregulated when compared with same-sex control animals, although Cry2 upregulation was not significant for CJL males. In particular, Per2 and Cry2 increased by 497% and 69%, respectively in CJL female mice, while Per2 increased by 230% in CJL male mice. The authors did not test the effects of chronic jet lag on Ror gene expression. This could be explained by the fact that Ror is not directly related to the shift work phenotype. Indeed, the association between Ror and shift work disorder has been shown to be weak at best [74].

We used this information to create separate mathematical models of the lung circadian clock for males and females undergoing CJL. The decrease in Clock mRNA for CJL males is not taken into account because we do not model this gene explicitly and only constitutively represent the associated CLOCK protein. In addition, NPAS2, a paralog of CLOCK, has been shown to compensate for the loss of CLOCK in peripheral circadian oscillators [75, 76]. We note also that the baseline immune system likely differs between the sexes, but due to insufficient quantitative data, we were only able to construct one baseline model. Fig 2 shows the male-to-female relative abundance of mRNAs for controls and shifters, as reported by Hadden et al., normalized by control male-to-female ratios.

A method similar to that of the baseline system was used for the calibration of the CJL male and CJL female models. Hadden et al. did not measure clock gene expression over a 24-hour period, which would have allowed identification of rhythm differences, i.e. mesor, amplitude and phase expression of these genes, but rather reported the average expression level of clock genes. We have therefore modified the cost function, Eqs 1 and 2, to minimize the error between the average gene concentration in our models and the experimental data. A comparison between the change in mean gene expression between our models and experimental data is available in Table L in S1 File.

Only a few parameters changed drastically from the nominal values in the baseline model. Thus, we fixed the parameters that had not changed much, and repeated the calibration with a reduced set of free parameters for the CJL models: 6 parameters for the male CJL model and 10 parameters for the female CJL model. CJL model parameters can be found in Table K in S1 File. This process resulted in a change in the average concentration of clock genes in CJL models to the levels specified by the data. Consequently, the amplitude of the oscillations increased or decreased depending on the direction of the change. A previous study using the same experimental protocol has recorded sustained alterations to the average gene concentration as well as the amplitude of clock genes in the SCN and peripheral tissues [77].

At the time of this study, [12] is the only work known to us that reports quantitative data on the sexual dimorphism of clock gene expression in the lungs of mice exposed to CJL. However, other studies involving only male rats or mice have recorded changes similar to CJL males [77, 78].

A new perspective for mitigation of SARS-CoV-2 infection: priming the innate immune system for viral attack

The course of infection by SARS-CoV-2 frequently includes a long asymptomatic period, followed in some individuals by an immune dysregulation period that may lead to complications and immunopathology-induced death. This course of disease suggests that the virus often evades detection by the innate immune system. We suggest a novel therapeutic approach to mitigate the infection's severity, probability of complications and duration. We propose that priming an individual's innate immune system for viral attack shortly before it is expected to occur may allow pre-activation of the preferable trajectory of immune response, leading to early detection of the virus. Priming can be carried out, for example, by administering a standard vaccine or another reagent that elicits a broad anti-viral innate immune response. By the time that the expected SARS-CoV-2 infection occurs, activation cascades will have been put in motion and levels of immune factors needed to combat the infection will have been elevated. The infection would thus be cleared faster and with less complication than otherwise, alleviating adverse clinical outcomes at the individual level. Moreover, priming may also mitigate population-level risk by reducing need for hospitalizations and decreasing the infectious period of individuals, thus slowing the spread and reducing the impact of the epidemic. In view of the latter consideration, our proposal may have a significant epidemiological impact even if applied primarily to low-risk individuals, such as young adults, who often show mild symptoms or none, by shortening the period during which they unknowingly infect others. The proposed view is, at this time, an unproven hypothesis. Although supported by robust bio-medical reasoning and multiple lines of evidence, carefully designed clinical trials are necessary.

1. Introduction

Widespread vaccination capable of specifically neutralizing the SARS-CoV-2 virus is expected to provide the ultimate solution to the COVID-19 epidemic. However, a vaccine is still unavailable, and preventative medication is currently lacking [1,2]. We propose a novel therapeutic approach that to date has been under-explored in the COVID-19 epidemic: we suggest that priming an individual's immune system during active epidemic, by inducing a short-term anti-viral systemic activation of the innate immune system, may reduce the infection's severity, length and probability of complications.

Following viral infection, the innate immune system is activated when pattern-recognition receptors (PRRs) are engaged by microbe-associated molecular patterns (MAMPs) in viral proteins and nucleic acids [3,4]. Specifically, the endosomal toll-like receptors (TLRs) 3, 7 and 8, and the intracellular cytosolic PRRs, such as MDA5 and RIG-I, have been shown to respond to respiratory infection by RNA viruses such as coronaviruses [5–7]. These sensors recognize viral RNA, such as 5′ triphosphate single-strand RNA and double-stranded RNA, and trigger a downstream signalling cascade to ultimately induce the secretion of types I and III interferons (IFNs) and proinflammatory cytokines [4]. In turn, the IFNs stimulate their cognate receptors and induce the activation of thousands of interferon stimulated genes (ISGs) that establish an anti-viral state in the infected cells and in surrounding cells [8,9]. This state efficiently inhibits further spread of the infection, while allowing time for the activation of adaptive responses that in most cases will clear the virus from the infected individual. These cascading dynamics are also critical to guarantee sufficiently strong, but not excessive, innate and inflammatory immune responses, and a timely downregulation of these responses to protect the individual from harmful immunopathology [7].

Evidence so far suggests that in the course of COVID-19, the SARS-CoV-2 virus has an average incubation period of approximately 5 days, and up to 14 days and longer [10,11]. This long period, alongside recently published direct evidence [12], suggests that SARS-CoV-2 initially manages to evade the innate immune system during early stages of infection. Studies of related coronaviruses SARS-CoV and MERS-CoV have demonstrated that these viruses encode a large number of factors that delay or suppress anti-viral interferon responses and may be involved in the evasion of immune detection [5,12]. At later stages of the disease, uncontrolled viral replication triggers hyper-inflammatory conditions in some individuals, which can lead to induction of lung injury by a cytokine storm [12,13].

Therefore, we suggest that priming the anti-viral innate immune response prior to SARS-CoV-2 infection may trigger an enhanced anti-viral interferon response ahead of time, thus preventing immune evasion by the virus. This may direct the immune response towards the preferable route for overcoming COVID-19 and prevent the immune pathology seen in the more severe cases. We anticipate that the ensuing infection would be attenuated relative to the infection of a naïve unprimed individual, as has been shown in analogous murine model systems [14–18]. A primed infection would still allow the adaptive immune system to develop adaptive immunity to SARS-CoV-2. This adaptive immunity is required at a population scale to halt the epidemic.

Our proposal does not intend to prevent a primed individual from being infected, but—by readying the immune system ahead of time—to lessen the severity of the infection and risk of complications, and to shorten the duration of infection. At the population level, the shortened duration of infection could change the epidemic dynamics, helping to ‘flatten’ the epidemic curve and to reduce the maximal number of infected and hospitalized individuals at any time point [19,20]. To alter the population-level dynamics in this way, the infection-shortening aspect of our proposal may be important even in subclinical and asymptomatic individuals, as they are likely to be infectious and play a major role in the spread of the epidemic [21].

The ‘gold standard’ of the immune response to a pathogen is often perceived solely as the presence (or absence) of specific antibodies and T cells that allow the adaptive immune system to identify and neutralize the pathogens, and, for viruses, to kill infected cells. This traditional focus creates a misleading impression regarding the immune system's ‘bread and butter’ function: alongside components of the adaptive immune system that provide a response which is specific to a particular pathogen, there are thousands of genes that are involved in anti-viral defence and that are not pathogen-specific [22–25]. This is reflected in the extensive overlap between the sets of proteins whose production is upregulated in response to different viral infections [24,26–32].

Defence priming—upregulation of immune function in response to environmental cues, social cues or physiological cues emitted by conspecifics—is well known in plants and invertebrates [33–40]. For example, termites increase their production of immune-related proteins following interaction with nest mates that had been exposed to a pathogen [40]. Defence priming has also been shown in vertebrates, in particular via activation of components of the innate immune system [35,37,41–49]. Specifically, it has been demonstrated experimentally that activation of the mammalian immune system by various triggers, from social cues to exposure to microbes or microbially derived compounds, provides protection upon exposure to an unrelated pathogen [14–18,50–57]. For example, mice that were administered aerosolized bacterial lysate exhibited an innate immune response—increased cytokine levels—and survived an otherwise lethal exposure to Influenza A [17]. Priming of the immune system by exposure to agents other than the pathogen itself is common: priming and upregulation of the immune system by the mammal's commensal bacteria have been frequently suggested and its importance has been repeatedly demonstrated [58–62].

Most encouraging are recent experiments on priming the immune system for intermediate time scales in humans: Arts et al. [63] have recently shown that BCG vaccination against tuberculosis activates factors of the innate immune system for extended periods of time, on the order of weeks, and increases resistance to an experimental infection by an attenuated yellow fever virus. This phenomenon, dubbed ‘trained immunity’ or ‘innate immune memory’ [44,64–66], relates to the longer-lasting effects of priming the immune system, but supports the feasibility of the short-term priming that we propose here. Similarly, a decreased rate of non-targeted infections has been reported in children in the period following vaccination for measles, mumps and rubella, as well as following vaccination with live-attenuated polio virus [67–70]. A number of studies are currently exploring the potential attenuation or prevention of COVID-19 via vaccination with BCG or MMR vaccines [71–74].

Different triggers may serve to prime the immune system, readying it for attack by stimulating it to mount a short-term broad anti-viral response. Priming with bacteria and bacterially derived factors, particularly administered nasally, has been shown experimentally to significantly alleviate the severity of viral challenges that attack the respiratory system [14–18,50–52,56,57]. An even more promising category of priming agents are attenuated viruses used in vaccines, various virus-derived elements, virus-like particles and other components [63,64,71,75–80]. Such agents have been studied and tested extensively, and candidates have been highlighted specifically for their ability to trigger a broad anti-viral immune response, acting as adjuvants in anti-viral vaccines. The systemic priming can be carried out using various therapeutic agents, including many off-the-shelf products and common vaccines that are prescribed prophylactically such as influenza, polio or varicella-zoster vaccines [81–85]. Use of vaccines as triggers in such a context would aim to capitalize on the broad innate immune response that they trigger and which is transient, lasting for a number of days to a few weeks and possibly longer after administration [63,81–86]. The longer-lasting effect of gaining adaptive immunity to the specific virus or viral strain that the vaccine is designed for would be a potentially beneficial unrelated side effect and would not be expected to play a role in countering SARS-CoV-2.

Decreased incidence and rate of complications of COVID-19 have been reported in children. In the light of our proposal, this might be partially attributed to immune system priming, caused by the frequent rate of vaccination during childhood in many countries. Similarly, negative correlations have been reported between COVID-19 prevalence and severity of outcomes, and region-level prevalence of malaria, helminths and schistosomiasis [87–90]. These infectious agents, whose prevalence correlates positively with the prevalence of other infectious diseases, may have an immunomodulatory effect which primes the immune system for viral attack. This possibility warrants careful exploration. Finally, it has recently been suggested that there is a negative correlation between coverage of influenza vaccination and deaths from COVID-19 in the elderly [91], presenting another promising observation that may support our proposed perspective.

To demonstrate the potential population-level impact of our proposal, we have incorporated large-scale population priming in an SEIR model that has been used to analyse and forecast the COVID-19 epidemic trajectory in China and the continental US [21,92], using parameters previously estimated from US county-level data between 21 February 2020 and 13 March 2020 [92]. Figure 1a shows the fraction of infected and hospitalized individuals with and without priming. If priming reduces the infectious period and chance of complications by 33%, the priming agent is administered to the whole population slightly before infection rates peak, and priming is effective for a week, the maximum number of hospitalized individuals is reduced by 25%. Figure 1b explores such reductions in hospitalizations for different parameter combinations: the fraction of the population receiving the priming agent, and the factor by which priming reduces the infectious period and chance of complications that require hospitalization. Although this is a simplified model (e.g. only a single population is examined rather than a metapopulation as in [21,92]), it demonstrates the potential population-level effect that priming might have on the epidemic trajectory and its impact in a city, region or state.

Figure 1. Effect of priming on epidemic dynamics. (a) Fraction of infected (blue) and hospitalized (red) individuals in the population over time without priming (solid lines) or with priming (dashed) if priming were administered on 5 May (day 72) to the entire population (fraction of primed individuals α = 1), assuming the effect of priming ρ lasts for one week and that it reduces the infectious period and chance of hospitalization by ρ = 1.5 (i.e. by 33%). (b) Reduction in maximum daily hospitalizations due to priming for various fractions of priming α (on the x-axis) and effects of priming ρ (on the y-axis). Dynamics are based on an SEIR model where infected individuals are primed with probability α and otherwise not primed. Model parameters estimated by Pei & Shaman from US county-level incidence data between 21 February and 13 March 2020 ([92], table 3): transmission rate β = 0.635 (weighted average of documented and undocumented cases) expected latency period δ −1 = 3.59 days expected infectious period r −1 = 3.56 days, or if primed in the past week. An additional model compartment for hospitalized individuals was added: infected individuals are hospitalized at rate h = 0.014 per day [93], or h/ρ if primed in the past week, for an expected duration of γ −1 = 21 days [93]. See for Python source code.

A number of caveats are associated with our proposal. First, it is crucial that the priming does not evoke an autoimmune response. In this respect, authorized therapeutic agents such as broadly used vaccines are preferable as a first set of candidates. Second, it is necessary to test and choose priming agents that do not trigger an adverse effect (i.e. to ensure that they do not burden the immune system and make it less effective in countering the ensuing attack by SARS-CoV-2). Finally, many of the cases of severe symptoms and mortality of COVID-19, especially in the elderly, seem to involve a cytokine storm of hyper-inflammation, in which much of the damage is caused by the immune system itself [13,94,95]. It is important to ascertain whether the proposed activation of the immune system prior to infection would reduce the likelihood of immune system dysregulation and hyper-inflammation. Evidence from murine models is encouraging: the viral challenges used were characterized by a tendency to stimulate a hyper-inflammatory condition often accountable for the major damage to the host the primed individuals in these experiments suffered significantly less from such complications than the control groups [14,15,17,52,56,57]. A particular risk–benefit exploration needs to be carried out for the elderly, who are at the greatest risk for severe and lethal complications of COVID-19 [96–98]. The reduced efficacy of immune functions involved with aging [99–101] raises the concern that priming would burden their system further and reduce its ability to respond to the SAR-CoV-2 infection. However, for the same reason, early preparation of the immune system to the expected attack may be crucial and beneficial for the elderly. This may be specifically true with respect to early activation of toll-like receptors such as TLR7 that may detect SARS-COV-2 infection and trigger an appropriate immune response [5–7]. Carefully designed clinical trials will be necessary to determine the risks and opportunities of the approach we propose for the elderly.

The COVID-19 epidemic is a rare case of a rapidly spreading epidemic that can reach high infection levels in affected populations. Although this poses a major challenge, it also constitutes an Achilles' heel that can be used to attenuate the epidemic's devastating effects: once the virus has spread in a population (e.g. a specific town or city), the timing of infection for many individuals is highly predictable. Similarly, large-scale infection may be expected shortly following removal of a population lockdown, or specifically among individuals that return to the work-force when a general lockdown is gradually lifted. Our approach capitalizes on the predictability of the infection and suggests a way to prepare susceptible individuals to counter the expected attack. Even vaccines that are often prescribed without particular medical indication, such as MMR, the polio vaccine or the seasonal flu vaccine, might serve this purpose.

In light of the imminent threat posed by SARS-CoV-2 to millions around the world and the current lack of preventative therapeutic measures, our proposal could be highly beneficial. It can potentially be implemented using extant authorized therapeutic agents such as broadly used vaccines for viral diseases, and thus may involve relatively low risk and can be readily tested. Our proposal combines direct individual-level effects—reducing complication rates, hospitalization events and mortality—and effects that play out at the population level—reduction of the infectious period, including of asymptomatic yet infectious individuals, and reduction of peak hospitalization load. Given the scale of the challenge that humanity is facing, even a moderate attenuation of the duration, severity, and complication risk of COVID-19 infections may, via these direct and indirect effects, would save many lives.

Watch the video: Überblick über das unspezifische und spezifische Immunsystem -- Immunologie -- AMBOSS Video (November 2022).