Biology of Immunology

Biology 320 (Immunology) Gregory E. Steinkraus, Ph.D., ABMM Clinical Director and Chief Scientific Officer Department of Pathology and Laboratory Medicine New Hanover Health Network and Wilmington Pathology Associates and NextWave Diagnostic Laboratories Wilmington, North Carolina Clinical Associate Professor Coastal Area Health Education Center Department of Pathology and Laboratory Medicine University of North Carolina Hospitals and School of Medicine Chapel Hill, North Carolina Adjunct Faculty Department of Biology and Marine Biology University of North Carolina at Wilmington Wilmington, NC INTRODUCTION TO IMMUNOLOGY IMMUNOLOGY AND THE IMMUNE RESPONSE * Immunology * Study of the components and function of the immune system * Immune System * Molecules, cells, tissues and organs which provide non-specific and specific protection against * Microorganisms * Microbial toxins * Crucial to human survival * Immune Response * A non-specific and specific defensive response involving antibodies (other proteins) and specialized lymphocytes to * Microorganisms * Microbial toxins IMMUNOLOGY AND THE IMMUNE RESPONSE * Antigens * Molecules which provoke immune response * Name derived from ―antibody generators‖ * Immunity * State of specific protection against * Microorganisms * Microbial toxins * Acquisition of Immunity * Natural * Active or Passive * Artificial * Active or Passive NATURALLY ACQUIRED IMMUNITY * Active * Antigens enter body naturally and body produces * Antibodies (Immunoglobulins) * Specialized lymphocytes * Passive * Antibodies pass from mother to * Fetus across placenta * Infant in breast milk * Provides immediate protection ARTIFICIALLY ACQUIRED IMMUNITY * Active * Antigens enter body through vaccination and body produces * Antibodies (immunoglobulins) * Specialized lymphocytes * Passive * Antibodies from immune individuals injected into body * Referred to as * Immune Serum Globulins (ISG) * Immune globulins * Gamma globulins * Provides immediate protection PRINCIPAL FUNCTION OF THE IMMUNE SYSTEM * To protect and defend humans from pathogenic microorganisms * Pathogen * Microorganisms capable of causing infection and/or disease * Infection * Ability of pathogen to enter host, multiply and stimulate an immune response * Disease * Clinical manifestations associated with infection * Classification of pathogens * Bacteria * Viruses * Fungi * Parasites BACTERIA, VIRUSES, FUNGI, PARASITES—OH MY! * * * * * * * * * * * * Streptococcus pyogenes (Group A Streptococcus) Klebsiella pneumoniae Mycobacterium tuberculosis Ebola virus Human Immunodeficiency Virus (HIV) Aspergillus fumigatus Candida albicans Cryptococcus neoformans Cryptosporidium parvum Stronglyoides stercoralis Ascaris lumbricoides Plasmodium falciparum DEFENSE MECHANISMS OF THE HUMAN HOST * Innate Mechanisms (Innate immunity) * Non-specific * First line of defense * Fast to start but limited * Adaptive Mechanisms (Adaptive immunity) * Specific * Second line of defense * Slow to start, becomes powerful with fast recall * Cooperation between mechanisms ORIGIN OF CELLS OF THE IMMUNE SYSTEM * Derived from common progenitor cell in bone marrow * Pluripotent hematopoietic stem cell * Progenitor Stem Cells * Erythroid lineage * Erythrocytes and Megakaryocytes * Myeloid lineage * Monocyte/macrophage, dendritic cells, PMN’s, mast cells * Lymphoid lineage * Small and large lymphocytes CELLS OF INNATE AND ADAPTIVE IMMUNITY * Myeloid Lineage (Granulocytes) * Neutophil * Principal phagocytic cell of innate immunity * Eosinophil * Principal defender against parasites * Basophil * Initiator of IgE mediated hypersensitivity * Referred to as * Polymorphonuclear leukocytes (PMN’s) * Nuclei are multilobed (2 to 5) * Neutrophils primarily * Granulocytes * Cytoplasmic granules CELLS OF INNATE AND ADAPTIVE IMMUNITY * Myeloid lingeage (Monocytes /Macrophages) * Monocytes * Leukocytes with bean shaped or brain-like convoluted nuclei * Circulate in blood with half life of 8 hours * Precursors of tissue macrophages * Macrophages * Mononuclear phagocytic cells in tissue * Derive from blood monocytes * Participate in innate and adaptive immunity CELLS OF INNATE AND ADAPTIVE IMMUNITY * Myeloid lineage * Dendritic cells * Cells with dendriform (star shaped) morphology * Interdigitating reticular cells (synonym) * Capture and present antigens to T lymphocytes * Mast cells * Located in mucous membrane and connective tissue throughout body * Display high affinity receptors for IgE * Effector cell of immediate hypersensitivity CELLS OF INNATE AND ADAPTIVE IMMUNITY * Lymphoid Lineage * Large lymphocytes (large granular lymphocytes) * Natural killer (NK) cells (CD16 + CD56) * Innate immunity to viruses and other intracellular pathogens * Participate in antibody-dependent cell-mediated cytotoxicity (ADCC) * Small lymphocytes * B cells (CD19) * T cells (CD3 + CD4 or CD8) * Adaptive immunity * Lymphocytes refers to small lymphocytes CLUSTER OF DIFFERENTIATION (CD) MOLECULES * Molecules on the surface of cells as recognized by specific sets of monoclonal antibodies * Employed to * Identify cell type * Stage of differentiation * Activity of cell * CD nomenclature established in 1982 * 1st International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA) * CD assigned on basis of 1 antigen by 2 monoclonal antibodies CASE STUDY - SEPTEMBER OF 2006 * 21 year old female college student presents to ER with 36 to 48 hour history * * * * * * * * * Fever (40 C) Headache Irritability Nausea Vomiting Nuchal rigidity Maculopapular rash on face and trunk Sore throat Photophobia CASE STUDY – CLINICAL DIAGNOSIS * Viral (aseptic) meningitis * Etiologic agents * Enteroviruses * Human immunodeficiency virus (HIV) CASE STUDY – LABORATORY DIAGNOSIS Cerebrospinal fluid (CSF) Patient Cell # 410 / uL Reference 0 - 8 / uL Cell type Protein Glucose 98% lymphs 50 mg/dL 50 mg/dL 15 – 45 mg/dL 40 – 70 mg/dL CASE STUDY – SEPTEMBER OF 2006 * 18 year old male college freshman presents to ER with 15 to18 hour history * * * * * * * * * Fever (40 C) Headache Irritability Nausea Vomiting Nuchal rigidity Petechial rash on trunk and limbs Sore throat Photophobia CASE STUDY – CLINICAL DIAGNOSIS * Bacterial meningitis * Etiologic agents * Streptococcus pneumoniae * Neisseria meningitidis CASE STUDY – LABORATORY DIAGNOSIS Cerebrospinal Fluid (CSF) Patient Cell # 2,000 / uL Reference 0 – 8 uL Cell type Protein Glucose PMN 125 mg/dL 20 mg/dL 15 – 45 mg/dL 40 – 70 mg/dL LYMPHOCYTES IN BLOOD, LYMPH AND LYMPHOID TISSUES * Lymphocytes originate in bone marrow * Circulate in blood / lymph and congregate in lymphoid tissues * Primary lymphoid tissues * Development and maturation of lymphocytes * Bone Marrow (B cells) and thymus gland (T cells) * Secondary lymphoid tissues * Mature lymphocytes meet pathogens * Spleen, adenoids, tonsils, appendix, lymph nodes, Peyer’s patches, mucosa-associated lymphoid tissue (MALT) THE LYMPHATIC SYSTEM * Lymphatic vessels * Originate in connective tissue * Collect plasma which leaks out of blood capillaries and returns it to blood * Lymph * Fluid and cells in lymphatic vessels * Lymph nodes * Kidney shaped organs of lymphoid tissue located at intervals along lymphatic vessels LYMPHOCYTES AND THE LYMPH NODES * Naïve lymphocytes circulate between blood, lymph and secondary lymph nodes * Pathogens from infected tissue sites are picked up by lymphatic vessels and arrive at closest lymph node * T and B cells congregate at specific regions of nodes * Architecture and size of nodes change in response to activation of lymphocytes LYMPHOCYTES AND THE SPLEEN * Spleen * Lymphoid organ in upper left abdomen * Functions * Remove damaged or old erythrocytes * Activation of lymphocytes from blood borne pathogens * Architecture of Spleen * Red pulp * Erythrocytes removed * White pulp * Lymphocytes stimulated SECONDARY LYMPHOID TISSUES ASSOCIATED WITH MUCOUS MEMBRANES * Primary portals of entry for pathogens * Respiratory tract * Gastrointestinal tract * Secondary lymphoid tissues * Bronchial-associated lymphoid tissue (BALT) * Gut-associated lymphoid tissues (GALT) * Tonsils, adenoids, appendix, Peyer’s patches * Pathogens are directly transferred across mucosa by “M” cells THE INNATE IMMUNE RESPONSE * Mediated (initiated) by phagocytes, NK cells and soluble proteins * Phagocytes * Cells specialized in the process of phagocytosis * Macrophages * Reside in tissues and recruit neutrophils * Neutrophils * Enter infected tissues in large numbers * Recognize common molecules of bacterial cell surface using a few surface receptors * Phagocytosis * Capture, engulfment and breakdown of bacterial pathogen THE INNATE IMMUNE RESPONSE * Phagocytosis triggers the inflammatory response through cytokines * Inflammatory response enhances phagocytosis through acute phase proteins * Mannose-binding lectin (MBL) * C-reactive protein (CRP) * Bind to bacterial surface acting as opsonins and activating complement * Complement * Set of proteins which enhance phagocytosis * Inflammatory response * Accumulation of fluid and cells at infection site (swelling, redness, heat and pain) THE ADAPTIVE IMMUNE RESPONSE * Creates millions of different B and T cells for specific antibody-mediated and cell-mediated immunity * Antibody-Mediated Immunity (AMI) * Involves B lymphocytes, plasma cells and antibodies * Humoral immunity * Name derives from antibodies found in body fluids (humors old medical term) * Cell-Mediated Immunity (CMI) * Involves T lymphocytes, antigen-presenting cells and MHC (major histocompatibility complex) molecules * Cellular immunity ANTIBODY-MEDIATED (HUMORAL) IMMUNITY * Directed against extracellular microorganisms and toxins * B-lymphocytes (B cells) * Differentiate into plasma cells which produce antibodies * Function as antigen-presenting cells (APC) * Classification of Antibodies (Immunoglobulins) * * * * * Immunoglobulin M (IgM) Immunoglobulin G (IgG) Immunoglobulin A (IgA) Immunoglobulin D (IgD) Immunoglobulin E (IgE) CELL-MEDIATED IMMUNITY (CMI) * Directed against intracellular microorganisms * Phagocytic cells and nonphagocytic cells * T-lymphocytes (T cells) * Differentiate into effector cells following antigen presentation by antigen presenting cells (APC’s) * Activate B lymphocytes * Functional types of T cells * Helper (CD4 T cells) * TH1 and TH2 cells * Cytotoxic (CD8 T cells) * Regulatory (Suppressor) * CD4 Tregs * CD8 Tregs THE NATURE OF ANTIGENS * Historically named as antibody generators * Molecule which stimulates production of and binds specifically to an antibody * Contemporary view distinguishes between * Antigen * Molecule which can bind to specific antibody and specific BCRs and TCRs * Immunogen * Molecule which can stimulate immune response * Best antigens / immunogens are proteins with MW > 10,000 THE NATURE OF ANTIGENS * Carbohydrates, nucleic acids and lipids are potential antigens / immunogens * Hapten * Small (low MW) molecule unable to elicit immune response * Combines with larger carrier molecule which together function as antigen/immunogen * Antibody may react independently with hapten following hapten/carrier antibody induction * Example * Penicillin G (MW of 372) * Albumin (MW of 66,000) THE NATURE OF ANTIBODIES * Also referred to as * Immunoglobulins (IG) * Immune serum globulins (ISG) * Gammaglobulins * Contemporary immunology * Antibody * Secreted form of IG made by plasma cells * Immunoglobulin * Antigen binding molecules of B cells * (B cell antigen receptors) CLASSIFICATION OF ANTIBODIES (IMMUNOGLOBULINS) * Five (5) classes (isotypes) * * * * * Immunoglobulin A (IgA) Immunoglobulin G (IgG) Immunoglobulin M (IgM) Immunoglobulin D (IgD) Immunoglobulin E (IgE) * Based on structural differences in constant regions of heavy chains * Classes have specialized effector functions B LYMPHOCYTES AND HUMORAL IMMUNITY * B lymphocytes originate from stem cells in bone marrow * Maturation takes place in bone marrow followed by migration to secondary lymphoid tissue * Antigen exposure in secondary lymphoid tissue * Following exposure to antigen, B lymphocytes differentiate into plasma cells and memory cells * Plasma cells produce antibodies of all IG classes ACTIVATION OF ANTIBODY PRODUCING CELLS BY CLONAL SELECTION * B lymphocytes recognize intact pathogenic microorganisms and toxins * B lymphocytes possess specific surface receptors for recognition of specific antigen * IgM and IgD * B lymphocyte population makes millions of different IG’s * Binding of specific antigen results in proliferation of a clonal population of cells * Antigen determines clonal proliferation ACTIVATION OF ANTIBODY PROCDUCING CELLS BY CLONAL SELECTION * Proliferation of activated cells is followed by differentiation into * Plasma cells * Life span of * 4 to 5 days * 1 to 2 months * Produce 2,000 antibody molecules / second * Memory cells * Life span of years to decades * Differentiate into plasma cells following stimulation by same antigen PRIMARY AND SECONDARY ANTIBODY RESPONSE * Primary Response * Following exposure to an antigen, there is a slow rise in IgM followed by a slow rise in IgG * Secondary Response * Following exposure to previously encountered antigen, there is a rapid rise in IgG and slow or no rise in IgM * Memory or anamnestic response T LYMPHOCYTES AND CELLMEDIATED IMMUNITY * T lymphocytes originate from stem cells in bone marrow followed by migration to thymus gland * Maturation takes place in thymus gland followed by migration to secondary lymphoid tissue * T lymphocytes respond to antigens on the surface of antigen presenting cells (APC’s) * Antigen presenting cells (APC’s) * Macrophages * Dendritic cells * B lymphocytes T LYMPHOCYTES AND CELLMEDIATED IMMUNITY * Antigen presenting cells (APC’s) * Ingest and process antigens then display fragments (short peptides) on their surface in association with molecules of major histocompatibility complex (MHC) * Major histocompatibility (MHC) molecules * MHC class I molecules * Present antigens to CD8 T cells * MHC class II molecules * Present antigens to CD4 T cells * T cells which encounter antigen differentiate into effector T cells ROLES OF EFFECTOR T CELLS IN IMMUNE RESPONSE * CD8 cytotoxic T cells * Enter bloodstream and travel to infection site * Kill cells infected with viruses and other intracellular microorganisms * CD4 TH1 helper T cells * Enter blood stream and travel to infection site * Help activate macrophages * CD4 TH2 helper T cells * Work within secondary lymphoid tissues * Help activate B cells THE NATURE OF ANTIBODIES * Antibodies are glycoproteins * Proteins are polymers of amino acids joined together by peptide bonds * Basic antibody structure has 4 polypeptide chains * 2 identical light chains * 2 identical heavy chains * Regions of heavy and light chains * Variable * Constant DISORDERS OF THE IMMUNE SYSTEM * Hypersensitivity Reactions * Over-reaction of adaptive immune response to harmless antigens * Four Types of reactions (I- IV) * Autoimmunity * Misdirected adaptive immune response * Results from a loss of self-tolerance * Three Types (II, III, IV) of reactions * Immunodeficiencies * Components of immune system either absent or defective * Genetic or acquired etiology IMMUNOLOGY FOR DIAGNOSIS OF DISEASES * Analytical methods using either antibody or antigen with an indicator system for detecting specific * Antibodies * Detected using antigens or antibody * Antigens * Detected using antibody * Indicator systems * Latex particles (colored) * Antibody conjugated to dyes * Antibody conjugated to fluorochromes METHODS IN DIAGNOSTIC IMMUNOLOGY * Latex agglutination * Latex particles (dyed) coated with antigen or antibody * Read visually for clumping of latex particles * Immunochromatographic assays (Strip assays) * Antibodies * Stationary (Capture line and test line) * Mobile (Conjugated on dyed microspheres) * Membranes * Nitrocellulose, cellulose acetate * Read visually for colored test and control lines CASE STUDY – JANUARY OF 2006 * 17 year old female presented to family physician with * * * * * Very sore throat (pharyngitis) Fever of 102 F Malaise Fatigue Cervical lymphadenopathy CASE STUDY – CLINICAL DIAGNOSIS * Differential * Infectious mononucleosis (IM) * Group A streptococcus (GAS) pharyngitis * Etiologic agents * Epstein-Barr Virus (EBV) for IM * Group A streptococcus (Streptococcus pyogenes) CASE STUDY – LABORATORY DIAGNOSIS * Leukocyte count was 15,000/uL with * 62% lymphocytes * 15% atypical lymphocytes * Throat swab for GAS antigen and culture was negative * Monospot Test was positive * Test for heterophile IgM antibody IMMUNOLOGY FOR PREVENTION OF DISEASE * Hepatitis B * Pre-exposure prophylaxis * Vaccination with hepatitis B surface antigen (HBsAg) * Post-exposure prophylaxis * Administration of * Hepatitis B Immune Globulin (HBIG) Human * Purified IgG antibody from plasma of donors with high titer of antibody to the hepatitis B surface antigen (anti-HBs) IMMUNOLOGY FOR TREATMENT OF DISEASE * Rheumatoid Arthritis * Remicade (Infliximab) * IgG kappa monoclonal antibody against tumor necrosis factor – alpha (TNF-alpha) * Breast Cancer * Herceptin (Trastuzumab) * IgG kappa monoclonal antibody against human epidermal growth factor receptor 2 (HER2)