24: Vertebrate Immune System

1. 24: Vertebrate Immune System Dr. Jason R Mayberry Castle View High School

2. Some Introductory Terms • Disease: abnormal function of the body • Pathogen: Organisms that inhabit the bodies of other organisms causing a disease • Infection: invasion of an organisms body by a pathogen • Immune System: Mechanisms for preventing and ending an infection • Sickness/Illness: the abnormal, negative feelings often associated with disease

3. How do Pathogens cause Disease? • Living Pathogens • Bacteria, Fungi, Protozoans, Parasites. • Release toxins that harm surrounding cells and interfere with normal metabolism • Utilize resources intended for normal function. • Sometimes get inside of our cells preventing their normal function or killing them. • Viruses • Particle consisting of a Protein capsule surrounding genetic material (DNA or RNA) and a few enzyme • Inactive when not in a living cell • In a living cell, co-opt the enzymes for Gene Expression and Replication to make copies of itself • destroys the cells ability to function normally • Kills cells

4. Viruses • Made of a Protein shell protecting some type of genetic material (DNA or RNA) which codes for the virus’s proteins, and a few viral proteins • Infect cells by “injecting” their contents into the cell and “Commandeering” our cells protein-making machinery so that viral proteins are produced and viral genetic material is replicated • Retroviruses (such as HIV) house RNA, and contain an enzyme called “reverse transcriptase” which makes DNA from RNA, usually inserting the DNA into the host cells own DNA. • When infected, cells don’t perform their specified functions properly • As Viral materials are made new Viruses assemble inside the cell • Virus usually spreads by breaking open the infected cell and releasing all the new viruses. HIV Virus Binds to Cell surface and releases contents Reverse Transcriptase makes Viral DNA out of RNA Integrase inserts Viral DNA into the Genomic DNA Cell spends energy making Viral Proteins and DNA

5. Divisions of the Immune System INNATE Inherited ability to recognize and fight a broad range of pathogens ACQUIRED Ability to generate new receptors and select for those that fight a specific infection Barrier Defenses Physical features that deny pathogens entry into an organisms body Cellular Defenses Cytotoxic Mobile cells specialized for recognizing and killing Pathogens and Infected Cells T-Cells recognize cells infected with a specific pathogen and kills them Peptides Humoral • Secreted proteins that aid the function of the immune cells. • Cytokines: cell signaling molecules that mobilize other immune cells. • Interferon: secreted by infected cells • Complement: blood born proteins • Histamine: induce inflammation • Etc. B-Cells produce antibodies that recognize specific pathogens

6. Recognizing Pathogens A B B A A B + + + “Self” proteins Antigens: Proteins unique to pathogens; used to recognize non-self • The immune system works by recognizing foreign protein markers • During development immune cells that recognize “Self” proteins are eliminated Cellular Defenses Cytotoxic Antigen binds to Antigen Receptors on Immune Cells – initiates mechanisms to destroy the pathogen. • Free Immune Proteins bind to antigens • Inactivates pathogens • labels pathogens for destruction. Antigens Pathogen Pathogen Antigen Receptor Antigens Antigen Receptor Immune Cell Pathogen Killing Response Immune Cell

7. White Blood Cells and Other Immune Cells • White Blood Cells (Leukocytes): • Originate in the bone marrow • Different types have specific functions. • B and T Lymphocytes function in adaptive immunity • Others function in Innate immunity • Microglial Cells: specialized immune cells of the CNS • Dendritic Cells: specialized immune cells of epithelial tissue (e.g. skin) • Mast Cells: specialized immune cell of connective Tissue and mucous membranes Hematopoiesis B-Lymphocytes Stem Cell T-Lymphocytes Natural Killer Mast cell Eosinophils White Blood Cells (Leukocytes) Basophils Neutrophils Monocytes Platelets Red Blood Cells Dendritic Cells Microglial Cells

8. FYI: Summary of Immune Cells Type and Function

9. Blood and Lymph Vessels and Circulation CARDIOVASCULAR System with Arteries, Capillaries and Veins Lymph vessels pick up fluid leaked from capillary networks, and digested lipids from small intestines. Lymph vessels return Lymph fluid to the Cardiovascular system into veins just before they enter the heart. LYMPHATIC SYSTEM With Lymphatic Vessels and Lymph Nodes

10. Lymphatic System and Other Immune Organs Interstitialfluid • Immune cells are not organized into discrete tissues and organs like other body systems, but are distributed throughout the tissues of other systems. • Most Immune Cells (e.g. WBC) originate in the bone marrow then circulate through the body tissues via the blood and lymph vessels • Lymph nodes are organizing and integration centers for the innate and adaptive immune systems Blood capillary Adenoid Tonsils Lymphatic vessels Thymus Lymphatic vessel Tissue cells Lymphatic vessel Spleen Peyer’s patches (in small intestine) Lymph nodes Appendix (cecum) Masses of defensive cells Lymph node

11. Innate Immune System

12. Divisions of the Immune System INNATE Inherited ability to recognize and fight a broad range of pathogens ACQUIRED Ability to generate new receptors and select for those that fight a specific infection Barrier Defenses Physical features that deny pathogens entry into an organisms body Cellular Defenses Cytotoxic Mobile cells specialized for recognizing and killing Pathogens and Infected Cells T-Cells recognize cells infected with a specific pathogen and kills them Peptides Humoral • Secreted proteins that aid the function of the immune cells. • Cytokines: cell signaling molecules that mobilize other immune cells. • Interferon: secreted by infected cells • Complement: blood born proteins • Histamine: induce inflammation • Etc. B-Cells produce antibodies that recognize specific pathogens

13. Barrier Defenses • Physical features that deny pathogens entry into an organisms body • Skin • Physical barrier • Acidity of sweat and oil glands (pH 3 to 5) • Enzymes in body secretions • saliva, gastric juices , saliva, tears • destroy bacteria and digest proteins. • Mucous and cilia in respiratory tract • Acidity of stomach, urine, and vagina

14. Divisions of the Immune System INNATE Inherited ability to recognize and fight a broad range of pathogens ACQUIRED Ability to generate new receptors and select for those that fight a specific infection Barrier Defenses Physical features that deny pathogens entry into an organisms body Cellular Defenses Cytotoxic Mobile cells specialized for recognizing and killing Pathogens and Infected Cells T-Cells recognize cells infected with a specific pathogen and kills them Peptides Humoral • Secreted proteins that aid the function of the immune cells. • Cytokines: cell signaling molecules that mobilize other immune cells. • Interferon: secreted by infected cells • Complement: blood born proteins • Histamine: induce inflammation • Etc. B-Cells produce antibodies that recognize specific pathogens

15. Innate Recognition of Pathogens EXTRACELLULAR FLUID • Toll-Like Receptors (TLR) • Similar to Toll proteins in invertebrates • Recognize structurally conserved molecules derived from pathogens • Found in macrophages, dendritic cells, and others. • Many different kinds • Cell surface TLR • Initiate phagocytosis • Initiate release of enzymes that lyse the pathogen • Intracellular TLRs • Secrete “warning” signals to neighboring cells • Recognize when cell is infected, initiate apopotosis Lipopolysaccharide Helper protein Flagellin Flagellin TLR4 PHAGOCYTIC CELL TLR5 CpG DNA TLR9 Innate immune responses VESICLE dsRNA TLR3

16. Innate Recognition of Infected Cells • Major Histocompatibility Complex Type 1 • The most significant human cell marker (but not the only one!) • Transmembrane protein with a receptor-like notch on the external face. • “Notch” is filled with a small Peptide resulting from the breakdown of proteins inside the cell before it is put on the cell’s surface • Millions of genetic combinations are possible • Found on all cells • If Immune Cells encounter Type 1 MHC with non-self protein, the cell is destroyed. “Non-Self” Antigen; Cell will be destroyed Antigens “Self” Peptide Cell’s Own Protein

17. Phagocytosis • White Blood Cell Phagocytes • Neutrophils become active quickly when exposed to pathogens • Monocytesdevelop into macrophages more slowly when exposed to antigens but become the dominant phagocyte • Eosinophilsare weakly phagocytic, mostly fighting parasitic worms. • Basophils are weakly phagocytic • Other Phagocytic Cells • Dendritic cells of the epidermis (mature forms of Langerhans cells) derived from mesoderm below the epidermis) • Mast Cells form in the bone marrow along side the white blood cells but are of different origin; have functional similarities to Neutrophils and Basophils. • Microgliacells of the brain • When an injury occurs and cells are destroyed, phagocytes also digest the cellular debris Pseudopodia surround pathogens. 1 Pathogen Pathogens engulfed by endocytosis. 2 Vacuole Vacuole forms. 3 Lysosome containing enzymes Vacuole and lysosome fuse. 4 Pathogens destroyed. 5 Debris from pathogens released. 6

18. Divisions of the Immune System INNATE Inherited ability to recognize and fight a broad range of pathogens ACQUIRED Ability to generate new receptors and select for those that fight a specific infection Barrier Defenses Physical features that deny pathogens entry into an organisms body Cellular Defenses Cytotoxic Mobile cells specialized for recognizing and killing Pathogens and Infected Cells T-Cells recognize cells infected with a specific pathogen and kills them Peptides Humoral • Secreted proteins that aid the function of the immune cells. Example: • Cytokines: cell signaling molecules that mobilize other immune cells. • Perforins: break down plasma membrane of pathogens and infected cells. • Interferon: secreted by infected cells • Complement: blood born proteins • Histamine: induce inflammation B-Cells produce antibodies that recognize specific pathogens

19. Killing Infected and Cancerous Cells • Natural Killer Cells (large granular lymphocytes): A unique group of lymphocytes fight cancerous and virus infected cells • Participate in both innate and acquired immunity. • Innate: recognize general features associated with cancerous or virus infected cells • Acquired: develop antigen-specific response and memory • Recognition of a cancerous/infected cell causes them to release granules on the cell • Granules contain enzymes called Perforins which break open the plasma membrane and nucleus of effected cells. Natural Killer Cell Granules containing Perforins “Self” Peptide Cancer cell Antigen Cancerous Cell

20. Antimicrobial Proteins: Interferon PKR • Interferon is a family of proteins secreted by cells infected with a Virus • Stimulates nearby cells to produce PKR, a protein which interferes with viral replication by blocking protein synthesis • Mobilizes Natural Killer cells • Promotes the development of macrophages from monocytes Toll-Like Receptor stimulates Interferon Natural Killer Cells Monocyte → Macrophage

21. Antimicrobial Proteins: Complement Secretion Inactive Complement Proteins in Blood • Family of ~20+ proteins produced by the Liver and then released into the blood • Proteins are inactive until they encounter specific antigens (Alternative Pathway) • Can also be activated by antibody-antigen complexes as part of the adaptive immune system (Classical Pathway) • Opsonization: Compliment proteins coat pathogens assisting in Adherance of phagocytes • Membrane Attack Complex: a group of complement proteins which punch a hole in the plasma membrane of pathogens • Stimulates parts of the inflammatoryresponse. Classical Pathway Alternative Pathway Antibody-Antigen Complex Specific foreign polysacharides Active Complement Proteins Inflammation Opsonization MAC Inflammation

22. Fever • Caused by signaling molecules called Pyrogens(a cytokine) • Released by Leukocytes exposed to foreign substances in the body • Target the Hypothalamus to re-set the body’s thermostat to a higher level. • Benefits • Causes the liver to sequester iron and zinc, which bacteria need to multiply • Increases the metabolic rate of tissue cells, speeding up the repair process • Dangers • High fevers can denature the body’s own proteins, causing cells to cease their normal functions The key to faking out the parents is the clammy hands. It's a good non-specific symptom. A lot of people will tell you that a phony fever is a dead lock, but if you get a nervous mother, you could land in the doctor's office. That's worse than school. What you do is, you fake a stomach cramp, and when you're bent over, moaning and wailing, (confidentially) you lick your palms. It's a little childish and stupid, but then, so is high school.

23. Inflamation Systematic Response of the Innate Immune Response to Infection (real or potential)

24. Inflammation: innate local response to infection or injury Bacteria Splinter Bacteria Mast cell Histamine Neutrophils Vascular Changes Phagocyte Mobilization Repair Neutrophil • Increased blood flow favors arrival of immune cells and complement • Also causes swelling and increased temperature • Nitric Oxide from Endothelial cells causes vasodilation • Histamine from Mast Cells increases capillary permeability. • Neutrophils • Usually First immune cells to encounter pathogens and release Cytokines • Begin phagocytosing pathogens and debree • Cytokines • act as chemoattractant for other immune cells (monocytes) • Stimulate maturation of macrophages • Complement • aid in pathogen destruction • promotes histamine release • Fibroblasts (connective tissue cell) secrete large amounts of collagen • Endothelial cells proliferate to restore blood vessels and thus blood supply

25. Adaptive Immune System Canisius College Bio 112 Jason Mayberry

26. Divisions of the Immune System INNATE Inherited ability to recognize and fight a broad range of pathogens ACQUIRED Ability to generate new receptors and select for those that fight a specific infection Barrier Defenses Physical features that deny pathogens entry into an organisms body Cellular Defenses Cytotoxic Mobile cells specialized for recognizing and killing Pathogens and Infected Cells T-Cells recognize cells infected with a specific pathogen and kills them Peptides Humoral • Secreted proteins that aid the function of the immune cells. • Cytokines: cell signaling molecules that mobilize other immune cells. • Interferon: secreted by infected cells • Complement: blood born proteins • Histamine: induce inflammation • Etc. B-Cells produce antibodies that recognize specific pathogens

27. White Blood Cells and Other Immune Cells • White Blood Cells (Leukocytes): • Originate in the bone marrow • Different types have specific functions. • B and T Lymphocytes function in adaptive immunity • Others function in Innate immunity • Microglial Cells: specialized immune cells of the CNS • Dendritic Cells: specialized immune cells of epithelial tissue (e.g. skin) • Mast Cells: specialized immune cell of connective Tissue and mucous membranes B-Lymphocytes Stem Cell T-Lymphocytes Natural Killer Mast cell Eosinophils White Blood Cells (Leukocytes) Basophils Neutrophils Monocytes Platelets Red Blood Cells Dendritic Cells Microglial Cells

28. B-cells and T-cells Maturation and Generally How they fight infection Blood Stem Cells are found in Red Bone Marrow • Immature(incapable of binding antigens) Lymphocytes form in bone marrow from hemocytoblasts • Lymphocytes become Immunocompetent, (capable of binding to antigens) by forming unique cell surface receptors capable of binding antigens • B-cells in the Bone Marrow • T-Cells in the Thymus • Lymphocytes are Naive until they encounter an antigen and are subsequently Activated. Maturation and Selection of B-Cells and “non-specific” white blood cells in bone marrow Maturation and Selection of T-Cells in Thymus • Humoral Immunity: B-Cells (B Lymphocytes) • Fight infections by releasing antibodies • Antibodies are like antigen receptors which are secreted out of the cells. • Cell-Mediated: T-Cells (T Lymphocytes) • Fight infection by binding to infected cells and releasing chemicals which destroy the cell. • Do not produce antibodies, but have antibody-like cell surface receptors

29. How T and B Cells “Learn” to Recognize an Antigen The development and activation of T and B cells is essentially an evolutionary Process: Immature Lymphocytes 1) Variation: Random Genetic Recombination and Mutation in each cell produces a unique antigen receptor 2) Differential Reproductive Success: If and Only If one of the randomly generated receptors matches an antigen it is activated: proliferates by Mitosis and starts fighting pathogens. Immunocompetent Lymphocytes Active Lymphocytes 3) Heredity: all progeny of activate lymphocytes express the same receptor and target the same antigen/pathogen.

30. B-Cells and Humoral immunity Antibodies

31. Antibodies Also forms monomers • Made of four protein chains bound together, two identical pairs. • Heavy chains are Longer • Light chains are Shorter • Constant regions (CH and CL) determines the class of the antibody (M, A, D, G, or E), each having unique properties. • Variable regions (VH and VL) • Come together to form 2 identical receptors which bind to antigens • Unique in each B-cell so that many different antibodies can be produced to fight different antigens. • Because pathogens may have many antigens, many different antibodies can combine in their fight against a single pathogen. • Note: Antibodies are also called Immunoglobulins or gamma-globulins

32. Antibody Diversity J3 J3 J4 J4 J1 J1 J2 J3 J4 • Antibody Genes • Heavy Chain • ~300 Variable Regions • 4 Joining regions • 1 Constant regions for each class • transmembrane region • Short chain: fewer Variable, one constant • Generating Antibody Diversity • Somatic Recombination: Enzymes (RAG-1 & RAG-2) randomly and sloppily remove a segment of DNA placing one of the V regions next to one of the J regions • Somatic Mutation (hypermutation): Enzymes randomly convert C  to U • Heavy + Light Combination: H. and L. independently affected by 1&2, leading to greater diversity when they come together • Antibody mRNA • V-Jsplice • One of the C regions • Transmembrane segment in naive cells • *Other regions are spliced out after transcription. • The genetic changes resulting from Recombination and Hypermutation are inherited by daughter cells • *T-cell diversity is similarly generated Heavy Chain Gene ... V1 V2 V3 V4 CM CM CM CA CA CA CD CD CD CD CG CG CG CE CE CE TM TM TM TM Somatic Recombination V3J2 V300 V300 V1 V2 V4 ... Somatic Mutation Translation and Processing Transcription and mRNA Processing V3J2 V1 V2 Identical Antigen Binding Sites Light Chain Heavy Chain V3J2 Variable Region Variable Region Joining Region Joining Region Constant Region Constant Region Transmembrane domain

33. B-Cell Receptors Antigen- binding site Antigen- binding site Disulfide bridge V V V V Variable regions C C B cell antigen receptor Constant regions C C Light chain Transmembrane region Heavy chain Plasma membrane B cell Cytoplasm of B cell

34. Overview of B-Cell Activation Mutations produce B-cells with random receptors If receptors recognize Antigen it is Endocytosed Antigen displayed on MHCII B-Cell awaits confirmation until becoming active C1) Pathogen Phagocytosed and antigens displayed on MHC II C2) Phagocyte travels to Lymph Node as professional Antigen Presenting Cell C3) Mutations previously produced Helper T-cells with random receptors C4) APC Activates Helper T-Cells whose receptors recognize antigen Activated Helper T cell binds to MHCII+antigen on B-cell Activates B-Cell with Cytokines B-Cell proliferates into antibody producing Plasma cells and Memory Cells Macrophage or Dendritic Cell Travels to Lymph Node Cytokines Class II MHC Helper T-Cells Plasma Cells Memory Cells

35. MHC: Class I vs. Class II • All cells display Type 1 MHC • Macrophages, Dendrocytes, B-Cells, and a few other cells also display Type 2 MHC • When pathogens/antigens are phagocytosed, fragments (epitopes) of the antigens are displayed on Type 2 MHC • Antigen’s presented with Type 2 MHC are not attacked by other immune cells • These cells then become professional Antigen Presenting Cells (APCs): • migrate to lymph nodes (if not already there) • present the antigen to cells of adaptive immune system “Non-Self” Antigen; Cell will be destroyed “Non-Self” Antigen on MHC II; not destroyed; stimulate T cells Antigens Antigens Non-Immune Cell Macrophage, Dendrocyte, and B-Cells “Self” Peptide “Self” Peptide Class II MHC Cell’s Own Protein Cell’s Own Protein

36. Antigen Presentation B Cell acting as an APC TC Cell TH bound to APC TH cell activating the B-Cell B Cell • Macrophages phagocytose pathogens and degrade them in a phagolysosome • Antigen fragments from the pathogen are diplayed on Class II MHC receptors • T-Cells bind to the APCs via • T-cell antigen Receptor • Non-antigenic Receptor (Co-stimulus) • CD4 Protein (CD8 for cytotoxic-T cells) • APC stimulates T-cell via secretion of cytokines: Interleukin 1 and Tumor Necrosis Factor • Ativated Helper T-Cell • Release cytokines that stimulate Cytotoxic-T cells and B-Cells • Bind to B-Cells bound to antigen via Type II MHC and stimulate with cytokines inducing mitosis. Macrophage Class II MHC protein within endosome Pathogen Antigen fragment (epitope) Class II MHC protein Antigen fragment (epitope) Co-stimulus CD4 Secretion of IL-1 and TNF Helper T-cell receptor Helper T cell • Antigen Presenting Cells: • Dendritic Cells • Macrophages • B-Cells • A handful of other cells only after cytokine stimulation Cytokines

37. Means of Antibody Function • Neutralization: antibodies block sites on viruses or bacterial toxins preventing them from being active • Agglutination: Antibodies form cross links with many pathogens and antibodies • Precipitation: Antibodies bind to water soluble molecules and cause them to come out of solution • Complement Fixation: Antibodies bound to the cell surface of pathogens act as a signal triggering complement fixation. • Antibodies can disable pathogens/antigens their major function is to enhance the non-specific defenses.

38. Antibody Mediated (Humoral) Immunity Secondary Immune Response (the person is “immune”) Primary Immune Response Antibody blood concentration 7 14 7 14 First Exposure Subsequent Exposure (perhaps years later) • When B-cells first become Immunocompetent but are still naive, • Very small amount of antibody secreted • Same genes from making the antibodies are used to make a cell surface receptor • B-cells only become active when they bind to an antigen (usually via a T-helper cell, but can be independent with some antigens) which just happens to complement their antibody receptors (notice that the antibody-antigen match is random!) • Once activated, the B-cells proliferate then differentiate into • Plasma Cells: secrete 2000 antibodies per second for 4-5 days before dying • Memory Cells: remain inactive but stand ready to respond to the same antigen at a future time during a Secondary Immune Response • The Primary Immune Response takes more time and is weaker than the Secondary Immune Response. • Because subsequent responses are so quick and overwhelming, the individual is said to be immune. Random Variety of B-Cells Clones of Activated B-Cell PrimaryResponse Memory Cells Plasma Cells SecondaryResponse

39. Classes of Antibodies - FYI • Almost always binds with the B-cell surface and is the most important antigen receptor involved in B-cell activation IgD • Monomer form binds to B-cell surface and act as an antigen receptor • Pentamer form is the first antibody released into the blood and is a powerful agglutinogen IgM • Dimer form is found in body secretions (saliva, sweat, intestinal juice, breast milk) • Monomer form is secreted in limited amounts IgA • Most abundant and diverse antibody secreted into the plasma • Crosses the placenta to give the fetus passive immunity which lasts for the first few months of life. IgG • Stem binds to Mast Cells and Basophils, recruiting them from the adaptive immune system • Responsible for most allergic reactions where the body mounts an immune response to a perceived threat which is really harmless (pet dander, pollen, etc.) IgE

40. T-Cells and Cell-Mediated immunity

41. T-Cell Receptors • T-Cell Receptor only has 1 antigen binding site per receptor • T-Cell Receptor diversity is generated very similarly to B-Cell receptor diversity • Many different types of T-Cells: Helper, Cytotoxic, Suppressor T-Cells, others Antigen- binding site Variable regions V V T cell antigen receptor Constant regions C C Transmembrane region Disulfide bridge β chain α chain T cell

42. Cytotoxic T Cell Stimulation and Action Macrophage Virus-infected cell Viral antigen Class II MHC protein Virus Virus Class I MHC protein IL-1 TNF Viral antigen CD8 T-cell receptor Helper T cell CD4 Cytotoxic T cell cytokines IL-2 and other cytokines Activation and proliferation • Activation of Cytotoxic T-Cell requires both stimulation by a Type 1 MHC bound to a foreign antigen and cytokine signals from Helper T-Cells Perforin Perforin Perforin Channels Water Infected cells

43. Cytotoxic T-Cells 3 2 1 Antigen • Cell Mediated Immunity: Cytotoxic T-Cells target and destroy infected cells with antigens on MHC Type I receptors displaying the foreign antigen that matches their antigen receptor. Variable region Constant region Transmembrane domain Granzymes Cytotoxic T cell Released cytotoxic T cell Accessory protein Dying infected cell Antigen receptor Class I MHC molecule Perforin Pore Infected cell Antigen fragment

44. Immune System Overview Immune System Non-Specific(Innate) Specific(Learned) Helper T-Cells Barriers Molecules Cells (WBC + Others) Humoral Cell-Mediated APC • Skin • Excretion (Enzymes, Acid, Mucous • Complement • Interferon • Pyrogens • Histamine • Etc. B-Cells produce Antibodies that disable or mark pathogens/ antigens for destruction by innate defenses Cytotoxic T-Cells destroy cells infected with a specific pathogen • Phagocytes(e.g. macrophages and dendrocytes) • Natural Killer Cells

45. Types of Acquired Immunity