4 Basic Principles of Immunology

1. 4 Basic Principles of Immunology I. Natural or Nonspecific Immunity A. Antimicrobial Agents B. Phagocytic Cells C. Nonphagocytic Cells D. Natural Killer Cells E. Inflammation and Fever II. Acquired or Adaptive Immunity A. Lymphocytes: 1. T cells 2. B cells B. Major Histocompatibility Complex C. Antibodies III. Overview of Cell- and Antibody-Mediated Immune Responses IV. Vaccines Cause for Concern? New Vaccines for the Developing World? V. Immune System Disorders A. Hypersensitivity 1. Allergies 2. Autoimmune Disorders 3. Immunodeficiencies - HIV and AIDS VI. Monoclonal Antibodies A. Biotech Revolution: Future of Monoclonal Antibody Production Tools of Immunology: A. Western Blotting B. Fluorescent Antibody Technique C. Enzyme-Linked Immunosorbent Assay

2. I. Natural or Nonspecific Immunity A. Rapid way for the body to fight off organisms before the specific mechanisms of acquired immunity are activated (Figure 4.1, Table 4.1). B. Physical barriers are present, such as: 1. Skin and mucous membranes, which block entry of pathogens. 2. Sweat gland and tear duct secretions that contain lysozyme. 3. Blinking bathes eyes in tears that wash away pathogens. 4. Perspiration is acidic and prevents growth of microorganisms. 5. Stomach acid and other digestive enzymes. 6. Cilia in the respiratory tract.

3. C. Antimicrobial Agents 1. Act to deter or destroy microorganisms. Examples include: 2. Interferon (Figure 4.2): a) Called “cytokines” and serve as signals during the immune response. b) Many different types generated, but mostly act against viruses. c) Activates a series of reactions that trigger the synthesis of antiviral proteins (AVPs) that affect the assembly of virus particles within the cell. 3. Interleukins: a) Produced by cells of the immune system and regulate interactions of immune cells with other body cells. 4. Lactoferrin and transferrin: a) Bind and sequester the element iron to reduce its cellular amounts. b) Inhibits bacterial growth because iron is needed for bacterial growth. c) Transferrin is in blood, and lactoferrin is in milk, saliva, mucus, and tears. 5. Complement (Figure 4.3): a) A family of more than twenty proteins in blood serum. b) Complement actions of other components of the immune system in the immune response. c) Activated by a complex series of reactions, working in four ways: (1) They can form a coating on the pathogen surface so that phagocytes (macrophages and neutrophils) can engulf easily. (2) Lyse the cell wall of microorganisms. (3) Release substances like histamine that increase inflammation, causing increased capillary permeability and dilated blood vessels. (4) They can attract lymphocytes (white blood cells) to an infection site.

4. Phagocytic Cells 1. These are cells that destroy pathogens if they cross the physical barriers. 2. Contain many enzymes for digestion such as lysozymes, proteases, nucleases, and lipases. They also produce toxic substances such as hydrogen peroxide. 3. Two types of phagocytes (Figure 4.4 & 4.5): a) Stationary phagocytes (macrophages): (1) Large cells made in the bone marrow that live in two phases: a mobile phase in the blood when they are called monocytes, and a stationary phase where they are called macrophages. (2) Engulf and consume bacteria, cancer cells, and virus-infected cells. b) Wandering phagocytes (leukocytes): (1) Circulate in the blood, and are two types: (a) Neutrophils (Fig 4.6)—best on bacteria, not used in sustained responses, increase in response to infection, involved in inflammation. (b) Monocytes—the mobile phase of the macrophage.

5. E. Nonphagocytic Cells 1. Three types: a) Basophils—involved in inflammation and allergies. b) Eosinophils—somewhat phagocytic and active against parasitic worms. c) Lymphocytes—large cells that respond to specific organisms and are involved in acquired immunity. Include B cells and T cells. F. Natural Killer (NK) Cells 1. Non-phagocytic cells that attach to cell surfaces and produce enzymes that destroy cells infected with viruses or microorganisms, even cancer cells. 2. They don’t attack the pathogen, but cells infected with the pathogen. 3. Cells are coated in antibodies and are then recognized and killed by NK cells. G. Inflammation and Fever (Figure 4.7) 1. Nonspecific responses to infection by microorganisms and tissue damage. 2. Inflammation: a) Chemicals such as prostaglandins (cause vasodilation) and histamine (causes increased capillary permeability) are produced. b) Characterized by swelling (by an influx of cells and plasma fluid), pain (caused by swelling), warmth, and redness (by increased blood flow). c) Blood cells, dead cells, and microorganisms are trapped, and phagocytes destroy and remove invading microorganisms. • 3. Fever: • a) Induced by substances produced by pathogens, and causes body temperature to rise. • b) Kills pathogens, increases inflammation, and stimulates phagocyte activity. • c) Also reduces amount of iron in blood to inhibit bacterial growth.

6. Acquired or Adaptive Immunity (Figure 4.8) A. Recognizes foreign invaders and responds to the invader, called an “antigen” (Figure 4.9). B. Can also recognize the body as self and the tissues of others as non-self. C. Antigens can be protein, glycoprotein, polysaccharides, and nucleic acids, and small parts of the antigen can trigger a response (called the “antigenic determinant”). D. Based on the complex interactions of different types of cells and other components (Figure 4.10). There are two types: 1. Cell-mediated—mediated by cells called “T lmphocytes” (T cells) (Figure 4.11) • Antibody-mediated—mediated by cells called “B lymphocytes” (B cells) (Figure 4.12). • E. Lymphocytes are circulated in the blood (by blood vessels) and lymphatic system (by lymphatic vessels) and in organs such as the spleen, tonsils, and thymus gland.

7. F. Lymphocytes 1. Have specific receptors on the surface to recognize antigenic determinants. 2. The primary cells are T cells, B cells, and NK cells. • 3. T cells (Figs 4.13, 4.14, 4.15): a) 60% of all lymphocytes; they develop in the bone marrow and mature in the thymus gland. b) Four major types: (1) CD8—cytotoxic T cells. Directly kills cancer, infected, or foreign cells. (2) CD4—helper T cells. Secretes growth factor that stimulates B cell reproduction, antibody production, and activity of CD 8 T cells. Two types—T helper 1 (TH1) is involved in cell-mediated immunity, while T helper 2 (TH2) is involved in antibody-mediated immunity. (3) Suppressor—inhibit immune reactions by decreasing the activity and division rates of B and T cells. (4) Memory—waits reintroduction of antigen, when they quickly divide and differentiate into CD8, CD4, and suppressor T cells. c) Contain the T cell receptor, a glycoprotein that recognizes and binds to antigens on infected cells or on cells that present the antigen, called “antigen-presenting cells” (APCs), of which macrophage is an example. 4. B cells (Fig 4.16): a) Develop and mature in the bone marrow. b) Three types of B cells, based on function: (1) Naïve B cells—not yet been exposed to an antigen, contain about 100,000 antibody molecules on the cell surface. They move into the lymph nodes. When exposed to an antigen, they become plasma cells, producing one specific antibody. (2) Plasma cells—makes antibodies specific for antigen it was exposed to. Also called effector B cells. (3) Memory B cells—do not die like plasma cells; produces small amounts of antibodies waiting for reintroduction of the antigen. They then divide quickly and differentiate into plasma cells.

8. Major Histocompatibility Complex (Figure 4.17) 1. Body cell antigens that identify the cells of the body as self. 2. APCs present antigens as complexes with MHC, activating T and B cells. 3. Three classes of proteins make up the MHC: a) MHC class I—on most cells, involved in self-recognition. Can bind to antigens. b) MHC class II—found on macrophages, B cells, dendritic cells, and some T cells. Regulate interactions among APCs, B cells, and T cells. Bind to antigens that APCs have engulfed and destroyed. c) MHC class III—make up some of the proteins in the complement system.

9. Antibodies (Figure 4.20) 1. Made of four polypeptide chains: a) Two heavy chains and two light chains. b) Each chain has a constant (C) region and a variable (V) region, which are also called “Fab fragments” and can change during the differentiation and activation of B cells. • Bind to antigens and trigger several responses: • a) Inactivation of a pathogen to prevent a cell from infection. b) Activation of phagocytes that ingest pathogens. c) Activation of complement proteins that destroy the pathogen. d) The constant region, called the “Fc region,” bind stoFc receptors on phagocytes. 3. Classified based on the amino acid sequences on the C region of the heavy chain: a) IgA—prevents pathogens from binding to epithelial surfaces. Found in mucus, tears, saliva, and breast milk. b) IgD—on the surface of B cells, activates B cells after they attach to antigens. c) IgE—binds to mast cells, causing them to release histamine, causing inflammatory responses. Also involved in parasitic worm infections. d) IgG—found in plasma and involved in long-term immunity. Helps provide immunity in newborns (called “passive immunity”). e) IgM—produced during the first encounter with an antigen and are found on the surface of B cells. 4. Have a large diversity, generated during the differentiation and maturation of B cells. DNA that codes for the constant and variable regions of the heavy and light chains of the antibody are randomly arranged to form a unique antibody. Can possibly create up to 100 million different antibody genes (Figure 4.22). 5. Overall, the specific and nonspecific immune responses work together to provide immunity, as seen in Table 4.9.

10. III. Overview of Cell and Antibody-Mediated Immune Responses A. During the cell-mediated immune response (Figure 4.23): 1. Virus invades cells of the body. 2. Foreign antigens combine with MHC and form foreign antigen-MHC (class I) complex that is displayed on surfaces of antigen-presenting cells. 3. Specific T cells are activated by foreign antigen-MHC complex. 4. T-cell clone produced by cell division—some become cytotoxic T cells. 5. Cytotoxic T cells migrate to infection site. 6. Cytotoxic cells secrete proteins that destroy target cells—those infected by virus. 7. Helper T cells secrete substances that attract macrophages and other cells to help fight infection.

11. During the antibody-mediated immune response (Figure 4.18): 1. A pathogen with a foreign antigen penetrates body. 2. Antigen-presenting cells (APCs) (for example, macrophages) phagocytize (ingest) the pathogen and display the antigen on their surface so they are recognized by the immune system. 3. The foreign antigen along with the MHC (antigen-MHC complex) is displayed on the surface of the phagocytes. 4. Helper T cells bind with foreign antigen and become activated. 5. B cells display the antigen of the infecting microorganism to activated helper T cells. 6. New B cells are produced through cell division. 7. B cells differentiate and become plasma cells. 8. Plasma cells secrete antibodies. 9. Antibodies attach to the pathogen. 10. Antibody-pathogen complexes trigger the destruction of the pathogen by other cells and chemicals.

12. Vaccines A. Provides active immunity (the full immune response) to a pathogen, which is usually provided through exposure to a pathogen (naturallyor artificially by vaccination). Table 4.10 lists some current vaccines. 1. Protection by a vaccine occurs in several ways: a) Weakened virus (live, attenuated vaccine) that do not cause disease. b) Killed pathogens (inactivated vaccines). They cause an immune response because the antigens are still present to cause an immune response. Examples are the typhoid, whooping cough, and rabies vaccines. c) The toxin isolated from a pathogen can be modified to eliminate the toxic properties of the substance; however, it still remains antigenic (toxoid vaccine). The tetanus vaccine is an example. d) Subunit vaccines are used to provide extra shield against side effects. Specific antigens from the pathogen are used in the vaccine. For example, one coat protein from a virus will produce an immune response. Do not stimulate the immune system as strongly as other vaccines, but recombinant DNA technology is increasing their ability to provide an immune response (mutations to increase antigenecity and the response of the immune system). e) Different antigens can be mixed to create a stronger immune response and, thus, better protect the individual. These are called “multivalent vaccines.”

13. B. Potential other developments in vaccine technology include: 1. Small synthetic peptides that elicit an immune response but lack parts that cause side effects. 2. Nucleic acid (DNA or RNA) vaccines made from the pathogens genome. DNA is injected in the patient, gets incorporated in their genome, then expressed to produce a polypeptide that can elicit an immune response. DNA vaccines for HIV are in clinical trials). 3. Therapeutic vaccines for cancer cells which have altered anitgensonn their surface that can hide from the immune system. Engineered antigens specific for binding to tumor cells can elicit a strong immune. 4. Production of edible vaccines in plants such as potato, tomato, and banana. Such vaccines don’t require refrigeration and are intended for the developing world. C. Cause for Concern? New Vaccines for the Developing World? 1. Vaccines that are currently developed will be of great benefit in the United States, but what about the developing world? 2. Will vaccines for diseases in the developing world be developed? Will those vaccines be tailored to places where refrigeration is not adequate? Will those vaccines be distributed to those who need it quickly?

14. Immune System Disorders • There are occasions where the immune system is overactive and attacks moleculesand cells produced by the body. A. Hypersensitivity. 1. Allergies. a) Occur in response to food, insect stings, antibiotics, and plant oils (e.g. poison ivy). b) Antigens that cause the allergic response are called “allergens.” c) Two types of immune responses to allergens: (1) Cell-mediated allergy—mediated by T cells and macrophages in response to contact with plant oils, chemicals, and latex, which bind to cell membrane proteins in cells that make up the skin. Delayed response. (2) Antibody-mediated allergy—induces hay fever, asthma, and reactions to insect stings. IgE is made and binds to basophils and mast cells, causing the release of histamine. Treated with antihistamine drugs, steroid hormones, or desensitization treatments.

15. Autoimmune Disorders a) When the immune system malfunctions and attacks the body’s own cells. b) Two types of autoimmune disorders: (1) Cytotoxic hypersensitivity—the immune system attacks itself. IgM or IgG antibodies mistake the body’s own cells for invaders, bind to the cells, and the cells are destroyed by phagocytes or killer cells. Grave’s disease and Hashimoto’s thyroiditis are examples. (2) Immune-complex hypersensitivity—circulating molecules are attacked. Antibodies attach to antigens in the blood, and the antigens are then either removed from the blood or lodge in tissue, causing an inflammatory response. Lupus is an example.

16. 3. Immunodeficiencies a) Occurs when the immune system is unable to function at a proper level, and is defined by what is causing the deficiency (Table 4.12). b) HIV and AIDS. (1) Caused by a retrovirus (Figure 4.24) that binds to and attacks CD4 T cells. (2) The virus enters the cell and releases its RNA genome and the enzyme reverse transcriptase, which makes DNA from the RNA (Figure 4.25). (3) The DNA integrates into the host genome and can lie dormant for years. (4) The virus becomes active and reproduces, eventually killing CD4 T cells and weakening the immune response. (5) The immune system cannot attack HIV because HIV cannot be recognized inside of cells, the coat proteins of the virus mutate rapidly to allow HIV to evade the immune system, and other properties described in Table 4.13.

17. Monoclonal Antibodies A. Antibodies are used in biotechnology to detect diseases, as therapeutic agents, and tools in research. They can be produced in the laboratory against specific antigens, usually by a laboratory animal like a mouse. B. Antibodies can be: 1. Polyclonal—a mixture of different antibodies having different specificities to the molecule of interest. 2. Monoclonal—a mixture of one antibody that is specific for only one antigen. C. Monoclonal antibodies (MAbs)are produced in this fashion: 1. Mice are injected with the antigen, whch elicits an immune response in the mice. 2. The mouse’s spleen is removed, and B cells harvested. 3. The B cells are fused with cancer cells (usually a myeloma cell that would become a B cell) that will divide indefinitely and also mass-produce antibodies. The fused cell is now called a hybridoma. 4. The cells are separated and screened for production of the proper antibody. 5. The correct cell is obtained and its antibodies used.

18. D. Monoclonal antibodies have many uses in biotechnology, such as in immunoassays that detect molecules; therapeutic treatments such as possibly aiding in cancer treatments; and in protein purification (called “affinity purification”) that can remove one protein from a mixture. E. Biotech Revolution: Future of Monoclonal Antibody Production

19. VII. Tools of Immunology A. Western Blotting (Figure 4.26). 1. A technique used to detect a specific protein that is mixed with other proteins. 2. This is performed in a manner similar to Southern blotting. 3. The Western blot is performed in the following steps: a) Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is conducted to separate proteins by size. b) The proteins migrate into the gel, and the distance they move is inversely proportional to their size. c) The gel is blotted in a manner similar to Southern blotting so that the protein bands are transferred to a membrane. d) The membrane is soaked in monoclonal antibodies that are detected with a labeled secondary antibody that binds to the primary monoclonal antibody. e) The membrane is washed, and the places where proteins have bound with antibody are visible and show up as bands on the membrane.

20. Fluorescent Antibody Technique 1. Also called immunofluorescence microscopy, detects antigens in cells or tissues that are bound to a microscope slide. 2. Uses a fluorescent tag that is bound to an antibody that is detected with a fluorescent microscope. 3. The most common tag is fluoresceinisothiocyanate (FITC). 4. Two major methods (Figure 4.27): a) Direct assay—fluorescent antibodies are attached directly to the cell surface. This way examines tissues for infection. b) Indirect assay—unlabeled antibodies are first applied, followed by a secondary fluorescent antibody specific to the unlabeled antibody. Can detect whether antibodies are produced in response to an infection.

21. C. Enzyme-Linked Immunosorbent Assay (Figure 4.28) 1. Detects specific proteins, usually antibodies, in serum, and is extremely specific. 2. The label for the antibody is an enzyme that can catalyze a chemical reaction. 3. The steps of an ELISA are: a) Bind the antigen to a plastic microplate. b) Wash the wells of the microplate and add serum sample, so antibodies can react to the antigen if they are made. c) Wash wells and add a secondary antibody specific for the antibody in the serum to the plate. This antibody is labeled with the enzyme. d) Wash the wells and treat the plate with the enzyme’s substrate. e) A color change is a positive result, which can be quantified based on the intensity of color developed.