Intro to Immunology Part B PDF

Introduction to Immunology-Part B RHCHP School of Pharmacy Integrated Pharmacotherapy Facilitators Reading and References Required Stephanie James, Ph...

Introduction to Immunology-Part B RHCHP School of Pharmacy Integrated Pharmacotherapy Facilitators Reading and References Required Stephanie James, PhD Integrated Pharmacotherapy 3 Introduction to Immunology Parts A and B student notes [email protected] Mathias, C. et al. Pharmacology of Immunotherapeutic Drugs. ISBN978030199241 chp.1 and 3 964-6168 Supplemental Powerpoint notes and video links Optional Flaherty, DK. Immunology for Pharmacy. Elsevier. 2012. ISBN: 978-0-323-06947-2 Learning Objectives 1. Provide a definition for all terms listed under the Definitions heading on page 2. 2. Recognize important properties of the adaptive immune response. 3. Compare and contrast humoral and cell-mediated adaptive immunity, and name the cells involved in each. 4. Define antigen and antigenic determinant. 5. Compare and contrast cellular expression and function of class I and class II MHC/HLA molecules. 6. Describe the function of antigen presenting cells, and list the specific cell types that act as antigen presenting cells. 7. Define self-tolerant and immunocompetent. 8. Define clonal selection. 9. Diagram the structure of an antibody and describe its different components (i.e. heavy and light chains, variable and constant domains, Fab and Fc regions). 10. Define antibody affinity and avidity. 11. Differentiate between five classes of antibodies (AKA immunoglobulins). 12. List and describe the effector functions of antibodies. 13. Compare and contrast B cell activation in both primary and secondary immune responses. 14. Describe the role of immunological memory in both primary and secondary immune responses. 15. Compare and contrast the functions of helper T cells (CD4) and cytotoxic T cells (CD8). 16. Describe the process of CD8 and CD4 T cell recognition of antigens. 17. Describe the steps of T cell activation. 18. Define costimulation. 19. List specific roles of CD4 helper T cells in adaptive immunity. 20. Identify the four subsets of effector CD4 T cells, TH1, TH2, TH17 and Treg cells and their function. 21. Describe T cell-mediated immunological memory. 22. Outline the timecourse of the immune response to infection. 23. Define inflammation. 24. Relate immune system interactions to the time course of an infection. 25. Describe the location and function of: Interleukin-2, CD3, CD28, CD20, CD40, PD-1, glucocorticoid, Interleukin-17, CTLA4. INTRODUCTION TO IMMUNOLOGY Definitions The human immune system is a highly complex system which functions to Immunity is the ability to resist damage from foreign substances such as microorganisms, harmful chemicals, or prevent infections and to eliminate established infections. The immune system cancer cells. is highly effective and efficient because each component has a specialized role Innate immunity, or nonspecific immunity, is a branch of in defending the body. There are two defense systems, innate and adaptive the immune system that recognizes and destroys foreign immunity, that act both independently and concertedly to protect the body from substances with the same response to each exposure. diseases and to provide immunity. While the immune system typically functions Adaptive Immunity, or specific immunity, is a branch of normally, it can become dysregulated, leading to many different disorders. These the immune system that recognizes and destroys foreign substances with faster and stronger responses each time the course notes will provide an overview of the cells, tissues, and organs that make foreign substance is encountered. up the human immune system, as well as discuss how the innate and adaptive Specificity is the ability of adaptive immunity to recognize a mechanisms are responsible for normal immune responses, which a special particular substance. focus on adapative immunity. Diversity, in relation to adaptive immunity, refers to the existance of a large number of lymphocytes with different antigenic specificities. Memory is the ability of adaptive immunity to remember CELLS, TISSUES, AND ORGANS OF THE IMMUNE SYSTEM previous encounters with a particular substance, and as a result, to respond faster, stronger, and longer. Antigens are substances that stimulate adaptive immunity, Cells of the Immune System and are divided into two groups: foreign antigens and self- antigens. Pluripotent hematopoietic stem cells in the fetal liver and in the adult bone Foreign antigens are not produced by the body, but are marrow divide and differentiate into two different cell lineages: myeloid and introduced from outside. Examples include: pollen, animal lymphoid. The myeloid lineage gives rise to neutrophils, eosinophils, basophils, dander, foods, drugs, and components of bacteria, viruses, and monocytes, as well as erythrocytes and platelets. The lymphoid lineage gives and other microorganisms. rise to T and B lymphocytes, as well as natural killer (NK) cells/lymphocytes. Self-antigens are molecules the body produces that stimu- Together, these two lineages of cells (minus the erythrocytes and platelets) late an adaptive immune response. The response can be are referred to as leukocytes, or white blood cells, and can be distinguished beneficial or harmful, such as recognition of tumor antigens to enable tumor destruction, or the stimulation of unwanted morphologically by the presence of cytoplasmic granules and/or a bi- or multi- tissue destruction in autoimmune diseases, respectively. lobed nucleus. (Refer to Figure 1 below.) The majority of leukocytes are released Antibodies are proteins that are produced by plasma cells into the blood at maturity (except T lymphocytes which mature in the thymus), (differentiated B cells) in response to an antigen. They are where they are transported throughout the body and can migrate into tissues also referred to as immunoglobulins (Ig) because they are in response to chemotactic factors, a process referred to as chemotaxis. A more globulin proteins involved in immunity. detailed description of the specific functions attributed to each leukocyte is Opsonization is the ability to render invading pathogens more susceptible to phagocytosis (i.e. through the binding of offered in the sections below pertaining to their roles in either innate and/or antibody or complement proteins to the surface membrane). Figure 1. Nomenclature of Immune System Cells Complement is a group of ~20 proteins that comprise ~10% of the plasma globulin. When activated in the complement cascade, formation of a membrane attack complex (MAC) that mediates cell lysis, opsonization of bacterial cells, and/or attraction of immune cells to site(s) of infection can occur. Major Histocompatibility Complex (MHC) molecules are glycoproteins expressed on the surface of most of the body’s cells. MHC molecules display antigens produced or pro- cessed inside the cell on the cell’s surface to be recognized by immune cells. Lymph is the clear or yellowish fluid found in lymphatic vessels that is derived from excess interstital fluid from blood vessel capillary exchange. Costimulator is a molecule on the surface of an antigen-pre- senting cell that provides a second signal required for activa- tion of naive T cells in addition to the first signal, the antigen. Integrated Pharmacotherapy 3 2 Introduction to Immunology adaptive immune reactions. Tissues and Organs of the Immune System Primary Lymphoid Organs Fetal Liver and Adult Bone Marrow Hemopoietic progenitor cells in the fetal liver and in the adult red bone marrow give rise to all blood cells. The process of blood cell formation begins during embryogenesis and continues throughout life. The majority of leukocytes also mature within the red bone marrow prior to being released into the circulation. These include neutrophils, eosinophils, basophils, monocytes, and B lymphocytes. Thymus Gland The thymus gland produces hormones, such as thymosin, which stimulate T cell maturation. Precursor T cells (thymocytes) are released from red bone marrow and circulate to the Figure 2. Thymus Location and Histology thymus where they mature. Within the thymus, thymocytes differentiate and begin to express specific receptors for antigen. The thymus itself is a bilobed gland located just above the heart (see Figure 2 below). At birth, the thymus is one of the largest organs in the body and continues to grow an expand until puberty, when sex hormones cause it to begin to atrophy. By age 30 the thymus is a vestigal organ. The majority of maturing T lymphocytes are located in the dark-staining cortex of thymic lobules. Large numbers of lymphocytes are processed by the thymus, but few survive the maturation process and degenerate. Those that survive thymic “selection” (capable of reacting to foreign substances and not self-reactive) migrate to the lobule medulla and enter the blood. Secondary Lymphoid Organs Spleen The spleen is roughly the size of a fist, and is located on the left, superior part of the abdominal cavity. Its primary function is to filter the blood in an effort to remove dying and dead erythrocytes as well as infectious agents. To support these two distinct filtration processes, the spleen is histologically composed of two tissue types--red pulp and white pulp (see Figure 3). The red pulp is composed of vascular sinusoids, reticular fiber meshworks, and a large number of macrophages that actively remove cellular debris from aged erythrocytes that rupture as they pass through the reticular fiber meshwork. Young and healthy erythrocytes are capable of bending Figure 3. Spleen Integrated Pharmacotherapy 3 3 Introduction to Immunology Figure 4. Lymphatic System and folding through the meshwork, and can survive splenic filtration. The white pulp is lymphatic tissue consisting of predominantly B-cell areas and few CD4 (helper) T-cell areas (no CD8 T-cells). Antigen-presenting cells (discussed in further detail below) can present antigen to these lymphocytes. In addition, foreign substances in the blood passing through the spleen can stimulate an immune response directly due to the presence of T cells and B cells in the white pulp. Lymph Node Lymph nodes are small and relatively round structures that are distributed along lymphatic vessels in series (see Figure 4). They filter lymph, removing bacteria and other materials. In addition, lymphocytes congregate, function, and proliferate within lymph nodes. Similar to the splenic mechanism of blood filtration, lymph nodes slowly filter lymph through lymphatic sinuses. Foreign substances within the lymph are removed by macrophages lining the sinuses and/or stimulate lymphocytes throughout the lymph node to divide, proliferate, and be released into the lymph to eventually reach the bloodstream where they circulate. Mucosa-Associated Lymphoid Tissue (MALT) Aggregates of loosely associated lymphoid tissue are located in and beneath the mucous membranes along the gastrointestinal, respiratory, urinary, and reproductive tracts and are collectively referred to as mucosa-associated lymphoid tissue (MALT). Examples include the tonsils, appendix, and Peyer’s patches in the distal half of the small intestine. Lymphatic tissue within these locations is well positioned to intercept microorganisms as they enter the body. Cells of Adaptive Immunity Phagocytic Cells Phagocytic cells function by engulfing particulate matter in a similar fashion to endocytosis (Figure 5, Page 5). Inside the cell, the engulfed contents are contained in a membrane-lined vesicle which fuses with another vesicle, a lysosome, that contains lytic enzymes which digest the phagocytosed contents. The primary phagocytic cells respective to adapative immunity are monocytes/macrophages, which reside in most tissues. Monocytes are less abundant than neutrophils, and unlike neutrophils which die a few hours after ingestion of microbes, monoctyes can survive for long periods of time in tissues where they differentiate into cells called macrophages. Both neutrophils and macrophages use several membrane receptor types to recognize microbes in the blood and tissues and to initiate phagocytosis. These include, but are not limited to, Toll-like receptors, cytokine receptors, mannose receptors, as well as receptors that recognize products of complement activation and antibody- or complement-coated microbes. Neutrophils and monoctyes migrate to sites of infection through a multistep process that involves attachment and firm adhesion to endothelial cells followed by transmigration through the endothelium. This process is mediated by the production of cytokines by leukocytes at the site of infection, which stimulate the endothelium to express proteins, called selectins, that mediate leukocyte extravasation (movement of leukocytes from capillaries into surrounding tissues.) T lymphocytes of the adaptive immune response use this same mechanism to migrate to sites of infection. Dendritic Cells Dendritic cells are highly specialized immune cells that serve as a bridge between the innate immune response and the adaptive immune response. They act as antigen-presenting cells (APCs) to activate cells of adaptive immunity--CD4 T cells, CD8 T cells, and B cells. Dendritic cells are found in epithelia and in most organs and respond to microbes by: 1) producing cytokines that recruit leukocytes to the site of infection, and 2) phagocytosing the microbe and presenting a piece of the digested microbe on its surface Integrated Pharmacotherapy 3 4 Introduction to Immunology to activate and recruit B and T lymphocytes from neighboring lymph Figure 5. Process of phagocytosis nodes. lymphocytes Lymphocytes may be either B cells or T cells. B cells can function as phagocytic cells which can produce antibodies to foreign antigens. T cells can be either a CD8 T cells, which most often fights viral infections. They can also be a CD4 T cells, which is also commonly called a helper CD4 T cell because upon activation CD4 T cells will release cytokines to stimulate other immune cells to fight off disease. Specific differences in lymphocytes are discussed below. ADAPTIVE IMMUNITY Although innate immunity can effectively combat invading pathogens, many have evolved to resist innate immunity and have become Figure 6. Specificity of Innate versus Adaptive Immunity pathogenic--capable of causing disease. The adaptive immune response is the host defense that is capable of specifically recognizing and “remembering” a large variety of pathogens. The resultant immunologic memory allows the immune system to mount an intense response to a subsequent attack by the same foreign agent. Adaptive immunity provides the body’s second line of defense by employing B and T lymphocytes. These cells express receptors which specifically recognize antigens produced by pathogens. Adaptive immunity takes considerably more time to mount than innate immunity (see Figure 6,7), and often employs and augments the innate immune system and its anti-pathogenic mechanisms to effectively eliminate foreign pathogens. Important properties of the adaptive immune response include: specificity and diversity, memory, clonal expansion (hence the time delay in mounting an adaptive immune response), and nonreactivity to self. Figure 7. The innate and adaptive immune system. Humoral and Cell-Mediated Adaptive Immunity Adaptive immunity can be divided into humoral immunity and cell-mediated immunity (see Figure 8). Humoral immunity and cell-mediated immunity are designed to provide host defense against extracellular and intracellular pathogens. Humoral Immunity Humoral immunity consists of B lymphocyte activation and the resultant antibody production by differentiated B cells, called plasma cells. Antibodies, also called immunoglobulins (Igs), are proteins secreted into the circulation and mucosal fluids that bind to and neutralize extracellular pathogens and their toxins. The antigen-antibody complex does not directly destroy the antigens; instead, it prepares them for destruction by innate immunity through the process of opsonization or other mechanisms discussed in further detail below. Because antibodies cannot gain access to pathogens present inside infected cells, antibodies function in the extracellular environment which Integrated Pharmacotherapy 3 5 Introduction to Immunology includes bodily secretions and tissue fluids. Figure 8. Divisions of Adaptive Immunity Cell-Mediated Immunity Infectious microorganisms such as viruses are pathogens that multiply inside cells. Cell-mediated immunity can destroy infected cells either by activating cytotoxic T lymphocytes (CD8 cells) that kill infected cells directly, or by employing helper T lymphocytes (CD4 cells), which in turn, direct the activity of macrophages, cytotoxic T lymphocytes, NK cells, and other cells of innate immunity (eosinophils, neutrophils, macrophages, and dendritic cells). As you will learn below, infected cells notify the immune system of their infection by presenting processed protein fragments (antigens) on their cell surface which enables recognition by T cells. Cell-mediated immunity acts on virus-infected (or bacterially infected cells) or parasite-infected cells, cancer cells, and cells of foreign grafts (transplanted cells). Figure 9. Antigenic determinants. Antigens, MHCs, and APCs Prior to discussing humoral and cell-mediated immunity in further detail, it is necessary to introduce a few key concepts that are common to the activation of both: antigens, major histocompatibility complex proteins(also called human leukocyte antigens), and antigen-presenting cells (APCs). Taken from Seeley’s Anatomy & Physiology, 9th Ed. Antigens Antigens are molecules that activate the immune system and induce an immune response. They are foreign to the body and can vary in size from small chemical structures to larger complex molecules. Antigens consist of any of the following: proteins, polysaccharides, lipids, or nucleic acids. Proteins are the most common antigen and are the most immunogenic (the ability of an antigen to stimulate an immune response). It is hypothesized that the human immune system can recognize between 107-109 unique antigens. The part of the antigen that is recognized by the immune system is called the antigenic epitope, or determinant (Figure 9). Major Histocompatibility Complex (MHC) Proteins A normal part of every nucleated cell’s day-to-day metabolism includes the presentation of protein fragments on its surface. Hundreds of proteins are expressed in this way on the external surface of our cells. Since normally these proteins are endogenous and considered “self,” the immune system does not recognize them to be foreign or antigenic. This is a normal process by which healthy cells present parts of proteins that are being “turned-over” (proteins that have reached their lifetime and are being replaced by newer models). Why do our cells do this? It is a necessary expenditure of energy. If a cell happens to become infected by a pathogen (such as a virus), that pathogen must make proteins to carry out its work and survive. These pathogenic proteins will undergo the same fate as our own proteins--when they are being turned over, they will also be presented on the cell surface. Since they constitute foreign proteins, they will be recognized by our immune system, and that infected cell will be targeted for death. Presentation of protein fragments on the cell surface requires a class of cell surface glycoproteins called major histocompatibility complex proteins, or MHC proteins. These proteins are also known as human leukocyte antigens (HLA) in humans, and the two terms are often used interchangeably. Each individual has a unique combination of HLA proteins, and it is unlikely that any two people except identical twins would have the same HLA proteins. There are two groups of MHC/HLA proteins called Class I proteins and Class II proteins. Class I proteins are located on cell membranes of all nucleated cells (i.e. cells competent for infection), and Class II proteins are found only on specific cells of the immune Integrated Pharmacotherapy 3 6 Introduction to Immunology system referred to as antigen presenting cells Figure 10. Intracellular antigen processing and presentation on class I and class II MHC. (APCs). APCs, as discussed in further detail below, act as recruiters of other immune cells by “presenting” processed foreign antigen to them. Figure 10 displays the pathways of how proteins become presented on the cell surface by MHC (HLA) Class I and Class II pathways. MHC (HLA) proteins function to display a protein fragment, or peptide, on the cell surface to either be recognized (if foreign) or ignored (if self) by immune cells. Class I MHC/HLA display peptides generated from the breakdown of cellular proteins during normal protein recycling. Basically, small portions of all cellular proteins are displayed by class I MHC/HLA so that the immune system recognizes these proteins as normal or “self.” When a cell is infected with a virus and producing viral proteins, fragments of viral proteins are also presented by class I MHC/HLA. Obviously, this helps the immune system identify cells that are infected. Similarly, cancerous cells can be recognized in the same manner. Cells that become cancerous likely make abnormal proteins which can and will be processed, presented, and recognized as foreign by our immune system. Class II MHC/HLA proteins are found on antigen-presenting cells, and display proteins that originate from outside the cell. APCs endocytose foreign antigen and break it down into fragments to be combined with MHC/HLA Class II proteins. These complexes are then transported to the cell’s surface where they are displayed to other immune cells. Only monocytes/macrophages, dendritic cells and B cells can present foreign antigen in a Class II MHC/HLA and Figure 11. The capture and presentation of protein antigens by are hence referred to as “professional antigen-presenting cells.” antigen-presenting cells (APCs). Antigen-Presenting Cells In order for T and sometimes B cells to recognize an immunogen, it must first be processed and presented on an MHC/HLA molecule. Upon exposure to an antigen or infection, APCs capture the molecule, transport them to peripheral lymphoid tissues, and present them to naive lymphocytes (i.e., cells that have not yet been activated by an antigen). Figure 11 illustrates how dendritic cells in the skin (Langerhans cells) capture antigen and migrate via the lymphatic system to draining lymph nodes where they stimulate lymphocytes. Cells of the Adaptive Immune System There are two major populations of cells that constitute the adaptive immune system: lymphocytes (B and T cells) and antigen- presenting cells. As APCs were introduced above, the following sections will focus on B and T lymphocytes. Like all blood cells, lymphocytes are derived from hematopoietic stem cells resident in the red bone marrow. As newly formed lymphocytes mature, they MUST NOT recognize host molecules as antigens to ensure that the immune system does not attack the body’s own cells (i.e. must be self-tolerant). Furthermore, each lymphocyte must be able to recognize one specific antigen (i.e must be immunocompetent). The maturation process of lymphocytes ensures that mature cells released into circulation from either the bone marrow (i.e. B cells) or thymus (i.e. T cells) are both self-tolerant and immunocompetent. Integrated Pharmacotherapy 3 7 Introduction to Immunology Both T and B cells express a unique type of receptor on their cell membrane. The receptors are unique because they bind to one specific antigen. All of the receptors on that particular cell are exactly the same so they all recognize the same antigen. There are approximately 105 receptors per cell. The receptors on B cells are membrane-bound antibodies, and will have the same specificity when secreted as antibodies following B cell activation and differentiation into a plasma cell. T cell receptors are specific proteins designed to recognize antigens, but will not be secreted as antibodies. Remember that T cells combat intracellular pathogens, so when they become activated, they either kill the cell on target (CD8 T cells) or activate and recruit other immune cells (CD4 T cells). B Cell (Humoral) Immune Response B cell activation provokes the humoral immune response. It is important to point out that B cells can be activated by APCs OR through direct binding of an antigen to membrane-bound antibodies. However, B cell activation mostly occurs without any requirement for antigen processing and displaying (see Figure 12). The activation process begins when the antigen binds to and cross-links adjacent membrane-bound antibodies. The antigen-antibody complex is endocytosed and processed triggering the B cell to grow and multiply. (In cases where the antigen is a protein fragment, or peptide, B cell proliferation and differentiation requires a second stimulus from helper T cells that have been activated by the same antigen...see Figure 12. B cell activation and generation of plasma cells. Taken from Abbas & costimulation section). Lichtman: Basic Immunology, 3rd Edition. This expansion of a single B cell is called clonal selection. An army of B cells, or clones, exactly like the original cell is produced bearing the same antigen-specific receptors. Many of the clone cells become plasma cells which are elaborate and efficient manufacturers of antibodies-- producing nearly 3000 antibody molecules per second! Each plasma cell only exists for 4 to 5 days and then dies. The remaining clone cells do not differentiate into plasma cells and, instead, become memory cells. The memory cells are important in mounting a humoral response upon subsequent exposure to the same antigen. Antibodies As discussed above, antibodies are produced by activated B cells that differentiate into effector plasma cells. However, some B cells may begin to produce antibodies during their differentiation, prior to becoming a plasma cell. Antibodies bind specifically to an antigen and form an antigen-antibody complex. Antibodies may be membrane-bound antigen receptors (as found on B cells) or secreted proteins (by B cells or plasma cells). There are five classes of antibodies/immunoglobulins, each slightly different in structure and function. Figure 13. Antibody structure. Basic Antibody Structure Regardless of its class, each antibody has a basic structure consisting of four looping polypeptide chains linked together by disulfide bonds (see Figure 13). Two of the chains, the heavy chains, are identical to each other and contain about 400 amino acids each. The other two chains, the light chains, are also identical to each other but only contain half the number of amino acids as the heavy chains. Collectively, the four chains form an antibody monomer assembled to form a Y-shaped molecule. Each light chain is attached to a heavy chain, and the two heavy chains are attached to each other. Each antibody contains a variable domain at the amino (N)- terminal of each chain. The variable domain of an antibody is the antigen-binding site. This domain gives antibodies their specificity to a unique antigen. A much larger constant domain is located at the carboxyl (C)-terminal end of each chain. A heavy chain has one variable domain and three or four constant domains, and a light chain is made up of one Taken from Human Anatomy & Physiology, 7th Ed., Marieb. variable domain and one constant domain. There are two regions of an antibody named based on the functional properties of proteolytic fragments of immunoglobulins. The first region called Fab (fragment antigen binding) is the fragment that contains the portion of the antibody required for antigen recognition. Fab consists of a whole light chain attached to the variable and the first constant domain of a heavy chain. The second Integrated Pharmacotherapy 3 8 Introduction to Immunology region consists of the remaining heavy chain constant domains Figure 14. Binding of an antibody to antigen called the Fc (fragment crystalline), and is responsible for most of the biological activity and effector functions of the immunoglobulin. Each immunoglobulin molecule contains two identical Fab regions that bind antigen and one Fc (the base of the “Y”) region. Between the Fab and Fc region exists a flexible portion called the hinge region. The hinge region allows each Fab region to move independently allowing for simultaneous binding to the antigen that may be separated by varying distances. The C-terminal region of the heavy chain may either contain a membrane anchor and act as a B-cell receptor, or lack the membrane anchor so that the antibody may be secreted. Antibodies bind to antigens with noncovalent interactions (hydrogen and ionic bonds) in a reversible manner. Because the antigen-antibody binding site is very small, only a small portion of the antigen is recognized by the antibody. The antigenic determinant can be recognized based on a linear sequence or a conformational epitope (i.e. shape) which is complementary to the shape of the combining site on the antibody (see Figure 14). The strength to which an antibody binds with an antigen is called the affinity. This concept is exactly the same as a drug binding to a receptor and is expressed as the dissociation constant (Kd). That is, the lower the Kd, the higher the affinity. Antibodies typically have a Kd in the range of 10-6M to 10-9M and the affinity can increase to a Kd of 10-8M to 10-11M with repeated exposure to the antigen, a process referred to as affinity maturation. Antibody Classes There are two isotypes of light chains (k and l) and five isotypes of heavy chains (a, d, e, g and m). The five major immunoglobulin classes are classified on the basis of their heavy chain isotype. These five classes include IgA, IgD, IgE, IgG, and IgM (Figure 15). Each isotype has distinct physical and biological/effector functions. Typically B cells produce antibodies from one class, but under certain conditions, the Ig isotype (i.e. effector function of the Ig) can be switched. That is to say that the heavy chain C regions may switch during a humoral response, but the variable, and hence the antigen specificity, of the B cell clone does not change. IgD, IgE, and IgG have two antigen binding sites. IgA is a dimer containing four antigen binding sites and IgM is a pentamer with 10 antigen-binding sites. Antibody Functions Antibodies alone cannot destroy antigens, but they can bind to the antigen and inactivate it. Remember, the antigen-binding (Fab) regions recognize the antigen. The antigen-antibody complex is then designated for destruction through the following mechanisms (Figure 15): Figure 15. Classes of Antibodies and Their Functions (Taken from Seeley’s Anatomy and Physiology 9th edition) Integrated Pharmacotherapy 3 9 Introduction to Immunology  neutralization--an antibody binds to and inactivates an antigen, opsonization and phagocytosis--an antibody crosslinks an antigen and a macrophage, which stimulates the macrophage to endocytose the antibody-antigen complex, antibody-dependent cellular cytotoxicity--some innate immune cells are activated by pathogens opsonized by IgE (mast and eosinophils) or IgG (NK cells), and release the contents of their granules that kill the opsonized target, or complement activation--binding of antibody to antigen can activate the complement cascade which can result in lysis, phagocytosis via complement proteins, and inflammation. B Cell-Mediated Immunological Memory The primary immune response involves an initial cellular differentiation and proliferation upon exposure to a particular antigen. During this period, antibody production by plasma cells typically takes about 3 to 6 days following exposure to the antigen. Antibody levels then rise and peak at about 10 days. When a person is exposed to the same antigen again, a secondary immune Figure 17. Primary and secondary humoral responses. (Taken from Human response occurs. This response is faster, more effective, and more prolonged because the immune system has already been primed by the initial exposure to the antigen. Within just a few hours of reexposure to the original antigen, a new set of plasma cells are produced, and within 2 to 3 days antibody production is maximal, with blood levels remaining high for weeks to months. Furthermore, antibody production following this subsequent exposure often involves more heavy chain class switching as well as affinity maturation. In other words, memory cells are more efficient at tailoring the response to antigen in addition to having a stronger affinity for the antigen than the original naive B cells. Memory cells persist for long periods of time and often for life (Figure 16,17). Please review this process to understand how memory cells are the basis for adaptive immunity. Figure 16. Clonal selection of a B cell. Taken from Human Anatomy & Physiology, 7th Ed., Marieb. The primary response includes a series of cell divisions, cell differentiation, and antibody production. During differentiation, B cells grow in size and become plasma cells, which produce antibodies. Others remain small lymphocytes and become memory B cells. In the secondary/memory response, memory B cells divide more rapidly to produce plasma cells. This rapid cell division produces many more plasma cells in the same amount of time, and therefore much more antibody than the primary response. As a result, the antigen is specifically and rapidly cleared, often before disease symptoms develop. Lastly, plasma cells die and antibodies are degraded following destruction of antigen, but the newly-formed memory B cells remain. The longevity/health of these memory cells determines whether repeated infections by the same antigen are possible. Integrated Pharmacotherapy 3 10 Introduction to Immunology T Cell (Cell-Mediated) Immune Response As previously mentioned, humoral immunity only protects the body against extracellular pathogens since antibodies can not enter cells. In these cases, cell-mediated immunity is required. T cells play a major role in cell-mediated immunity. There are two major types of T cells, CD4 and CD8. The letters “CD” stand for cell differentiation glycoprotein, a family of glycoprotein surface receptors residing on mature T cells. These receptors help the T cell to interact with other cells or foreign antigens. CD4 cells are primarily helper T cells and have roles in helping to generate some B cell responses and coordinate other immune responses. CD8 cells are cytotoxic T cells and destroy cells in the body that are infected with anything foreign. T Cell Antigen Recognition and MHC Restriction Figure 19. T cell maturation, activation, and clonal selection. T cells are derived from red bone marrow and migrate to the thymus where they mature into CD4 and CD8 cells. Immunocompetent and self tolerant T cells constantly circulate through peripheral lymphoid organs in their search for foreign antigens. These cells however are not able to perform the effector functions to eliminate pathogens. They must first be presented an antigen by an APC and differentiate into effector cells. This process occurs in the lymphoid tissues and organs. T cell maturation, activation, and clonal expansion is depicted in Figure 19. CD8 T cells specifically recognize antigens of intracellular pathogens presented on Class I MHC/HLA molecules. As described earlier, small parts of an antigen are displayed on MHC/HLA proteins. Class I MHC/HLA proteins, which are present on all nucleated cells, display small protein fragments from proteins that originated within the cell. When class I MHC/HLA proteins display fragments of its own (self) proteins, cytotoxic T (CD8) cells pass by and ignore the complex. However, when class I MHC/HLA display foreign antigen, CD8 cells recognize this and become Taken from Human Anatomy & Physiology, 7th Ed., Marieb. activated. Remember, CD8 cells destroy cells displaying anything foreign in their MHC. CD4 T cells specifically recognize antigens of extracellular pathogens presented on Class II MHC/HLA molecules. Class II MHC/HLA proteins are only expressed on the surface of “professional” antigen presenting cells (APCs), including dendritic cells, macrophages, and B cells, all of which present antigens to CD4 T cells. Class II MHC/HLA molecules work in a similar fashion as class I MHC/HLA molecules in that they display a protein fragment associated with the MHC II/HLA protein. Class II MHC/HLA differs as they display peptides from extracellular antigens that have been engulfed, broken down, and processed by the APC (Figure 10). In such, these APCs present extracellular pathogens to helper CD4 T lymphocytes, which in turn help B lymphocytes to produce antibodies and phagocytes to destroy ingested pathogens. Typically, proliferation and activation of B cells involve helper CD4 T cells. This “help” augments the humoral response to combat extracellular pathogens. Integrated Pharmacotherapy 3 11 Introduction to Immunology T Cell Activation T cell activation is a two-step process. Remember, T cells only recognize antigens Figure 20. T cell receptor associated with either class of MHC/HLA proteins on the surface of cells. First Step of T Cell Activation The first step to T cell activation is recognition of the antigen-MHC/HLA complex by the T cell antigen receptor (TCR) (Figure 20). Additionally, CD4 on helper T cells recognizes antigens associated with class II MHC/HLA proteins, and CD8 on cytotoxic cells recognizes antigen fragments associated with class I MHC/HlA proteins. Interestingly, this first step of T cell activation in itself induces the production of proapoptotic proteins within T cells. That is, if this is the only signal the T cell receives, it will die. Why? Because T cell development is not perfect, and some T cells that are released from the thymus to circulate may be autoreactive--can initiate an autoimmune reaction. If these T cells are stimulated by self-antigens from normal processing, they will die in the absence of a second costimulus (described next) which acts to verify the presence of foreign invaders. In such, this second signal induces anti-apoptotic protein expression, and enables the T cell to become fully activated. Figure 21. Costimulation Second Step of T Cell Activation--Costimulation Before a T cell is fully activated, it needs to receive the second signal called a costimulator (see Figure 21), or it will die (see first signal above). This often involves the T cell binding to a separate and different surface receptor on the APC. For example, APCs express B7 (CD86) proteins which bind to the CD28 receptor on a T cell. This second stimulus can also involve the release of chemicals or cytokines such as interleukin 1 and 2 by APCs and T cells. Cytokines are basically immune system-specific hormones. They mediate cell development and differentiation by inducing growth factor and anti-apoptotic protein expression, and elicit certain responses in the immune system. Once a T cell is activated, it will secrete one or more cytokines in order to orchestrate the response by the innate and adaptive immune system. As you can see, several stimuli are needed to activate T cells in order to prevent inappropriate activation of an immune response. You Figure 22. Clonal selection of T cells. can think of this as the APC having to present two forms of identification (MHC/antigen complex and another identifier--either cytokines secreted from cells or molecules attached to the surface of cells) to prevent inappropriate and unnecessary immune responses. Once activated by MHC/antigen recognition and costimulated, a T cell increases in size and then undergoes cell division forming multiple clones of itself (Figure 22). T cells continue this for approximately a week following exposure to antigen. This is called the primary response. Activated T cells then undergo apoptosis (programmed cell death or cellular suicide) once the antigen has been removed in order to limit the T cell response. Similar to B cell clones, a small population of helper and cytotoxic T cells remain memory cells. Of course, this enables the T cells to mount a more responsive and more prolific immune response upon subsequent exposures to an antigen. Taken from Human Anatomy & Physiology, 7th Ed., Marieb. Integrated Pharmacotherapy 3 12 Introduction to Immunology B cells also require costimulation to mount an antibody response to protein antigens, which if you recall are the most common type of antigen. They can initiate a humoral response prior to receiving costimulation (i.e. prior to differentiation into a plasma cell), but it is not robust and is rather inefficient. They rely on help from CD4 T cells that have been activated by the same antigen. Thus, protein antigens constituteT-dependent antigens. T-independent antigens can elicit antibody responses without costimulation by CD4T cells, and include polysaccharides, lipids, and other nonprotein antigens. It is thought that the degree to whichT-independent antigens can cross-link B cell receptors is extensive enough to to stimulate B cell proliferation and differentiation in the absence of costimulation. Figure 23. Function of helper T cells in cell-mediated Specific T cell Roles and humoral immunity. Helper T Cells CD4 helper T cells become activated by professional APCs that present an antigen associated with the class II MHC protein. Once activated, helper T cells activate macrophages, facilitate T and B cell activation as well as induce T and B cell proliferation. Thus, CD4+ helper cells basically directs and facilitates the adaptive immune response (Figure 23). Figure 23 depicts the interactions between a helper T cell and an APC (23a) and an interaction between a T cell and a B cell (23b). CD4 helper T cells may differentiate into subsets of effector cells that produce specific cytokines and perform specific functions. There are at least four subsets of helper T cells, and their development is regulated by cytokines secreted by cells of innate immunity. Helper T cells 1 (TH1) stimulate phagocyte-mediated ingestion and killing of microbes, and play a major role in cell-mediated immunity. Helper T cells 2 (TH2) stimulate phagocyte-independent, eosinophil- mediated imunity. Recall that eosinophils combat helminthic parasites. TH2 cell secretions stimulate mast cells and eosinophils, as well as stimulate mucus secretion. Helper T cells 17 (TH17) mediate inflammation. This subset has been implicated in diseases such as multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. Regulatory Helper T cells (Tregs) develop in the thymus or peripheral lymphoid organs and recognize self antigens. They block activation of harmful lymphocytes that are specific for the same self antigens, and thereby maintain peripheral tolerance in an effort to prevent autoimmunity. Taken from Human Anatomy & Physiology, 7th Ed., Marieb Integrated Pharmacotherapy 3 13 Introduction to Immunology Cytotoxic T Cells CD8 cytotoxic T cells directly attack and destroy cells (virally-infected, tumor, or foreign tissues) that display foreign antigens associated with class I MHC/HLA proteins (Figure 25). Naive CD8 T lymphocytes that become activated by antigen/MHC(HLA) class 1 complexes and costimulated, differentiate into what are known as cytotoxic T lymphocytes (CTLs)--the effector cells. CTLs express molecules that can kill infected cells, and no longer require costimulation or T cell help for activation. Once a CTL recognizes a foreign antigen associated with a class I MHC protein, it can destroy the infected cell through two mechanisms: 1. Release of a molecule, perforin, that creates a transmembrane channel in the infected cell; the cytotoxic T cell then releases granzymes which are proteases that degrade cellular proteins, stimulating the cell to undergo apoptosis, or 2. Release of a molecule, Fas, which binds to a specific membrane receptor on the target cell, triggering the cell to undergo apoptosis. CTLs are able to kill a cell on target, detach, and go on to kill additional target cells. Furthermore, CTLs also secrete cytokines that activate macrophages to clean up the path of destruction by phagocytosing and destroying microbes, as well as enhancing the recruitment of additional immune cells. Figure 25. Effector Mechanisms of Cytotoxic T Cells (CTLs) T Cell-Mediated Immunological Memory Similar to the immunological memory imparted by memory B cells following the first exposure to antigen, the T cell response also remembers specific encounters with foreign pathogens by enabling survival of a fraction of antigen-activated T lymphocytes. These cells survive after an infection has been cleared--following antigen removal and decline of the innate immune response. Memory T cells circulate through lymphoid organs and mucosal tissues. It is unknown what factors determine whether a T cell will become a memory versus an effector cell, but it is known that the cytokine IL-7 is required to keep memory T cells alive. Following clearance of the initial infection, memory T cells go into a resting state in which they do not secrete cytokines or kill infected cells (there are none to kill at this point). A subsequent encounter with the same pathogen, however, initiates a rapid effector response from the memory cell(s). Integrated Pharmacotherapy 3 14 Introduction to Immunology  Phases of an Immune Response to Infection Exposure to Pathogen and Role of Innate Immunity An immune response involves integration of events that occur both locally, at the site of an infection, as well as systemically. After entering tissues, many pathogens are recognized, ingested, and killed by macrophages. At this point, macrophages initiate the process of inflammation by releasing cytokines and small lipid mediators. Inflammation involves the accumulation of leukocytes at sites of infection, with simultaneous vascular dilation and increased leakage of fluid and proteins into the affected tissue. Manifestation of this process includes the following signs: rubor (redness) calor (heat) tumor (swelling) dolor (pain) functio laesa (inhibited or lost function) Each or all of these signs may be observed in certain cases, but none of them are always present in every case. Inflammation delivers additional effector molecules and cells (e.g. neutrophils, monocytes, eosinophils, natural killer cells) to the site of infection to aid macrophages in clearing the pathogen. Additionally, inflammation induces local blood clotting to prevent the spread of infection and repair of the tissue. Many infections are prevented from being established at this stage. However, if the innate immune system becomes overwhelmed and can not prevent a pathogen from establishing a focus of infection, innate immunity can still prevent the spread of the pathogen into the bloodstream while the adaptive immune response develops. Activation of Adaptive Immunity When innate immunity is insufficient to clear a pathogen, adaptive immunity is recruited. Antigen presenting cells, typically dendritic cells which reside in most tissues, take up antigen and present it on MHC/HLA II molecules. They migrate away from infected tissue via the lymphatic system to local lymphoid tissues where they activate naive T or B cells. Antigen-specific T cells and/ or antibody-secreting B cells clonally expand and differentiate over several days. Following differentiation, T cells and/or antibodies are released into the blood and are recruited to the site of infection by chemotaxis. At sites of infection, CD4 T cells activate macrophages to become more phagocytic; CD8 T cells, or cytotoxic T cells, kill infected cells on target, and antibodies activate complement as well as opsonize pathogens, enhancing their uptake by phagocytes. Integrated Pharmacotherapy 3 15 Introduction to Immunology  Figure 26. Immune System Interactions Integrated Pharmacotherapy 3 16 Introduction to Immunology  Decline of the Adaptive Immune Response As with any physiological system, the immune system must return to a steady state following an immune response. This enables immune readiness for encounters with future pathogens. During an immune response to invading pathogens, the survival and proliferation of both B and T cells is maintained by antigen, costimulation, and cytokines. When the infection is cleared, these stimuli disappear, and the immune cells die by apoptosis. This process takes 1-2 weeks following eradication of an infection, and only memory cells remain. B cells also employ a second mechanism of regulating the humoral response to antigen which involves feedback signaling from antibodies. Once antibody titers reach sufficient levels to combat antigen, the likelihood of the antibody-antigen complex encountering the B cell increases. B cell recognition of its own antibody bound to antigen results in inhibition of further antibody production. This serves to terminate the humoral response following production of sufficient quantities of antibody. IMMUNE SYSTEM SUMMARY Even though these course notes have divided the immune system into innate, antibody-mediated, and cell-mediated immunity, it should be apparent that an immune response often involves components of more than one of these divisions. Even with the great specificity and power of the adaptive immune system (B and T cell responses), innate immunity still plays a very important and rather large role in actually destroying the antigen. These efforts include inflammation and phagocytosis, among various others. Please review the interactions among the divisions of the immune system in Figure 26 below. This outlines the major interactions of key players in the immune system and their responses during the course of an infection. REFERENCES 1. Abbas AK, Lichtman AH. Basic Immunology: Functions and Disorders of the Immune System. 3rd ed. Updated Edition. Philadelphia: Saunders; 2011. 2. Actor JK. Elsevier’s Integrated Immunology and Microbiology. Philadelphia: Saunders; 2007. 3. Kuby J. Immunology. 3rd ed. New York: WH Freeman and Company; 1998. 4. Seeley R, VanPutte C, Regan J, Russo A. Chapter 22. Lymphatic System and Immunity. In: Seeley R, VanPutte C, Regan J, Russo A, eds. Seeley’s Anatomy and Physiology. 9th ed. New York: McGraw-Hill; 2011. Last Modified: January 6, 2023 2:46 PM Integrated Pharmacotherapy 3 17 Introduction to Immunology