The Ultimate Immunology Study Guide Part 1

1 Overview → Immunology = defense mechanisms developed against intra- threats A. Threats to Living Organisms 1. Inter - “outside” 2. Intra - “inside” B. Defense Mechanisms (RPR) 1. Recognition a) Negative - primary defense (1) Foreign substance that isn't not self cell => kill b) Positive - immunological memory (1) Familiar encounter a/ foreign substance => knows how to react *Only self is your “friend”; recognition of self & non-self 2. Processing information 3. Response C. Traits of Immune System 1. Adaptivity 2. Specificity 3. Memory *Organs/immune systems memories so they can adapt/prevent from occuring again D. Mammalian Immune System (dual character - two connected parts) ● Neither B/T cells capable of recognizing forgen antigens & accessory cells ● B/T/Accessory cells have cell-cell contact & release cytokines needed to initiate immune response 1. Humoral immunity ○ Carried out by antibodies secreted by bone marrow from white blood cells (B lymphocytes cells) ○ Present in all bodily fluids ○ Antibodies recognize antigens & bind them for destruction *Effective against extracellular bacteria & viruses in extracellular phase 2. Cellular immunity *More effective against intraceullar bacteria & viruses in intercelluarphase ○ Carried out by thymus-derived lymphocytes (T cells) circulating in blood & lymph & recognizing self cells that are infected/transformed … then destroyed II. Foreign Antigens A. Molecular Level Antigens ● Recognition of foregin entities involve recognition of some molecules on their surfaces 1. Good Antigens a) Large size b) Complexity c) Some structural stability d) Degradability e) Foreignness (tested before) B. Proteins ● Present on surfaces of invading microorganisms & important targets for immune systems ● Few parts of large antigen can be recognized by immune system independently ● Smallest part of antigen cable of inducing immune response (antigenic determinant - epitope ) III. Basic Antibody Structure A. Searching for antibody in Serum 1. Albumin ( simple soluble protein in water & coagulable by heat ) & globulin (insoluble proteins: 50% (NH 4 ) 2 SO 4 --retains antibody activity) → Immunoglobulins = antibodies; glycoprotein produced by plasma cells (white blood cells) 2. Electrophoresis (separates protein by charges) - antibody activity is retained in γ-globulin fraction *γ used in antigens 3. Molecular weight of antibodies a) IgG (γ) MW: 150k (7S) b) IgM (macro) MW: 900k (19S) I. 2 Papain digestion of IgG = fragment antigen binding (Fab) & fragment crystalline (Fc); IgG = (Fab)2Fc Determination of polypeptide chain composition of IgG = breaking disulfide bridges by 2-ME +gel filtration a) Heavy (H) & Light (L) chains ; IgG = H2L2 6. Prep. of anti-Fab & anti-Fc antibodies cross react w/ antibodies w/ separated H & L chains B. Myeloma proteins = abnormal immunoglobulin fragment that is produced in excess by an abnormal monoclonal proliferation of plasma cells ● Secreted in large amounts by patients w/ multiple ● Homogeneous immunoglobulins C. Bence-Jones Proteins (B-J) = monoclonal globulin protein /immunoglobulin light chain found in the urine ● Light chain dimeric proteins (MW: 25k) -- peptide mapping ● Secreted by multiple myeloma patients D. Two Classes of Light chains [Korngold (κ) & Lipari (λ)] 1. Taking particular B-J 2. Injecting into animal => anti-B-J 3. React w/ other B-J E. Constant & Variable Domains ● Discovered by Putnam ● Homologous segments of cain ● X-ray crystallography shows domains have common characteristic structure - immunoglobulin fold *Fab fragments of antibodies have to be able to bind many different antigens - and for this they need variability of their Ag binding sites. You see it CDR. They also have to trigger relatively few biological functions, and have similar conformations - achieved by less variable regions F. Basic Structure of Immunoglobulin 4. 5. - NH 2 & COOH Intra- & Inter- chain disulfide bridges Domains Chains Fragments Constant & variable domains Hinge Antigen-binding site Carbohydrates IgG - Basic 4-chain - Most common w/ few IgM - Second most common (in humans, IgA has IgA - Second most common in humans IgE - Serves as B-cell receptor IgD - Basic 4 chain structure w/ additional CH 4 3 subclasses - Secondary immune response - Crosses placenta to protect fetus additional CH 4 ) - Extra J chain (15 kDa); pentamer - Major in primary immune response - Large size (found in blood) - Mostly oligomers (dimers) -- 4 basic unit joined by J chain - Serum IgA & secretory IgA (sIgA) -- has extra secretory component (71k) that protects from digestion in intestine - Found In serum & body fluids - No hinge - Very low [serum] - Mediates hypersensitivity rxns (allergies) IV. Biological Functions of Different Immunoglobulins *cross-reactivity is the interaction of the antibody not only with the intended target antigen, but also with unrelated antigens. Cross-linkage is a different system altogether A. IgG 1. Protection by binding a) Neutralization of toxins (binds to cells in body & changes function) b) Covering attachment sites of microorganisms (1st penetrate & stay in host) 2. Opsonization = bind to specific epitopes on bacterial surface antigens through antigen-binding site => activates complement system -- utilizing low efficient Fc receptors of phagocytes a) Bacterial organism foraging around w/ protein surfaces b) Macrophages w/ receptors attracts phagocytes & eat microorganism that invades 3. Complement Binding a) Opsonization of activating surface by products of enzymatic cleavage of some complement proteins b) Perforation of cell surface of an invading cell by complement proteins 4. Homocytotropic Binding = any antibody that attaches to origin species cells, not others ( binding to the cell of the same organism (eg, IgE to mast cells)) a) Binding Ig to high affinity Fc receptor on surface of leukocyte of same organism in absence of antigen * high affinity = strong binding - to be ready when the antigen comes (or allergen) b) Cross-linking of Ig by antigen c) Degradation of cells & release factors => damaging &/ expulsion of parasite (chemical warfare) 5. Immunoglobulins as Receptors a) Secretory: soluble & membrane bound w/ extra hydrophobic segment used to anchor Ig in B cell membrane b) Activates antibody production by B cell after binging specific antigen * need the secretory form to go out there and catch antigens, and the membrane form to activate production of this particular immunoglobulin by B cells IgG - Most common Ig (80%) - Produced in secondary response - Small size allows cross blood to lymph to placenta - Particulates in protection by binding, opsonization, complement binding - 20 days in serum - 150 kDa IgM - 1st Ab produced by immunization - Multivalency makes effective for agglutination of microorganisms - most efficient complement binding - second most common only in blood - 900 kDa IgA - Present in serum, lymph, & other fluids - Protects epithelial surfaces by preventing attachment of microorganism - Secretory protects G.I. - Usually dimer, J-chain, 15% human serum - secretory component 71 kD IgE - E-antigen of ragweed - Homocytotropic binding - Allergies, parasites - Low in serum - 190 kD - Extra 3 domains IgD - B cell receptor *Each class can & does serve as B cell receptors B. Fine Structure of Immunoglobulins 1. Variability accomiate diff. antigens 2. Constants perform relatively few bio. functions *Plots of variability vs. position # reveal regions w/ higher variability, hypervariable regions (20%), framework regions (80%) *Framework regions responsible for maintaining stable, similar conformation of VH & VL V. Antigen-Antibody Interaction *Hypervariable regions of Ab reversibly interact w/ Ag by forming non-covalent bonds 4 A. Forces Contributing to Interactions 1. Electrostatic 2. Hydrogen bonding (b/w N, O, F) 3. Hydrophobic interactions 4. Van der Waals Forces (VDW) *Each for weak & act on short distances: closeness & close fit on Ab & Ag surfaces needed for strong binding → Affinity = association constant for Ag-Ab interaction → Avidity = overall strength of binding between an antibody and an antigen K = [AbAg]/[Ab][Ag] ΔG= - RTlnK Lowest specific: ~ 10 -4 M -1 Highest found: ~ 10 -9 M -1 *Multivalent binding can inc. association constant by 2-3 order of mag. B. Antibody Heterogeneity ● Same antigen can fit & bind to diff. Antibody combining sites albeit w/ diff. Affinities ● Each reasonable fit will induce production of corresponding antibody by B cell ● Normal immune response => diff. antibodies w/ same specificity are produced C. Production of Monoclonal Antibodies 1. Primed B cells a. Cannot grow indefinitely in tissue culture b. Secret Ab of interest + many other Ab c. Not defective in nucleotide syn. (can grow in HAT medium) D. Fusion Partner 1. Myeloma line which can grow almost indefinitely in culture 2. Doesn’t secret Ab 3. Has defective nucleotide syn. (cannot grow in HAT medium) - HAT = hypoxanthine, aminopterin, thymidine - HGPRT = hypoxanthine guanine phosphoribosyltransferase E. Hybridomas = hybrid cell used as the basis for antibody production in large amounts for diagnostic/therapeutic use ● Produced by injecting a specific antigen into a mouse ● Collecting an antibody-producing cell from the mouse's spleen ● Fusing it w/ a tumor cell called a myeloma cell a) Can grow almost indefinitely in culture b) Secrete Ab of interest + other Ab c) Are not defective in nucleotide syn. (can grow in HAT medium) *After fusion, hybridoma cells cloned & residue tested for required reactivity F. Detection of Ag-Ab Interaction = interaction b/w antibodies produced by B cells of the white blood cells & antigens during immune rxn → Precipitin rxn = antibody which can precipitate out of a solution upon antigen binding 5 Ouchterlony Double Diffusion = used in the detection, ID, & quantification of antibodies & antigens (immunoglobulins & extractable nuclear antigens) Immunoelectrophoresis = # of biochemical methods for separation & characterization of proteins based on electrophoresis & rxn w/ antibodies Immunofluorescence = assay used primarily on bio. samples; procedure to detect antigens in cellular contexts using antibodies Radioimmunoassay = technique for determining antibody levels by introducing an antigen labeled with a radioisotope and measuring the subsequent radioactivity of the antibody component G. Enzyme-Linked Immunosorbent Assay (ELISA) & glycoproteins in bio. samples = immunological assay used to measure antibodies, antigens, proteins ● ● ● ● ● VI. 6 Can detect antigen/antibodies present in nanogram & picogram quantities Requires specific antibody to pathogen ○ Primarily antibody is used to detect specific antigen Samples serially diluted & bound to plate Non-specific interactions are removed by washing the plate ELISA used to quantify levels of target based on strength of antibody binding Major Histocompatibility Complex A. Important Part of Immune System 1. Self from non-self 2. Alter infected/transformed self *Some molecules on cell surface recognizable by immune system of organism as self, others recognize by another organism as non-self → Histocompatibility Antigens = group of antigens in tissues that provoke rejection response if tissue contain antigens are genetically dissimilar to recipient → Major Histocompatibility Antigens = strongest indicator for inducing allograft rejection - Rapid/strong graft rejection → Major Histocompatibility Complex (MHC) = allows large proteins in immune system to ID compatible/forgein proteins => matching potential organ/bone marrow donor w/ recipient → Minor Histocompatibility Antigens = receptors on cell surface of donated organs that give immunological response in some organ transplants - Slow, weak, chronic graft rejection B. Concept of Inbred Strain of Mice: strains maintained by successive mating 20+ generations; repetitive inbreeding => removes heterogeneity = identical to each other 1. Outbred (wild) 2. Inbred (genetically identical) 3. Congenic (identical, expect MHC) = 2 organisms that differ in only 1 locus & linked segment of c’somes 4. Recombinant congenic (identical, except part of MHC) = strains formed by crossing 2 inbred strains => backcrosses to 1 parental strain w/ inbreeding w/o selection for specific markers 7 *Loci in MHC are polymorphic & close together: crossover frequency ~.5% (inherit two sets, 1 from each parent) → Haplotype = each set of alleles → Codominantly Expressed = neither allele can mask expression; alleles expressed equally Class I Antigens MHC = pathways that alters immune system to virally infected cells FUNCTION: expresses on cell surface on nucleated cells & bind peptide frag. derived from proteolytically degraded proteins internal syn. by cell & protect cytotoxic tc lymphocytes => consent survey of cell surfaces & kill cells sheltering metabolically active microorganism Class II Antigens MHC = heterodimeric glycoprotein composed of 𝛼 & β chain restricted to antigen presenting cells (APCs) FUNCTION: healer th lymphocytes locally activated wherever cells encounter macrophages, dendritic cells, or B cells that have internalized & processed antigens produced by pathogenic organism - - Membrane-bound single peptide chain glycoprotein MW: 45k Noncovalently bound to 12k MW protein β2 microglobe not encoded by MHC (30% homology w/ human CH 3 - single Ig domain) Heavy chain has 5 domains (3 have ~90 AA residues/domain) Both heavy chain & β2 syn. by all cells Located on cells of immune system & serve for cell-cell communication 8 VII. Cells & Organs of Immune System → Cytotoxicity = quality of being toxic to cells A. Cells ● All blood cells (including immune system) derive from stem cells in bone marrow ● Stem cells are unspecialized & undifferentiated -- provide supplier for short-lived mature cells B. Stem Cells 1. Proliferation 2. Self-renewal 3. Differentiation a) Myeloid Lineage = during the process of myelopoiesis and include Granulocytes, Monocytes, Megakaryocytes, & Dendritic Cells ○ Erythrocytes ○ Platelets ○ Granulocytes - Majority of white blood cells - Classified by giemsa: basic dye (me blue) & acidic dye (eosin) b) Lymphoid Lineage = during the process of lymphopoiesis and include B Cells, T Cells, Natural Killer (NK) Cells, & Dendritic Cells ○ Natural Killer (NK) cells = = component of innate immune system; functions as host-rejection of both tumours and virally infected cells - Comprised of 5-10% lymphocytes in peripheral blood - Recognize altered surface antigen profile of some tumor & virus-infected cell - Participate in antibody-dependent cell-mediated cytotoxicity (ADCC) ○ T cells = component of adaptive immune system that directly kills infected host cells activating other cells producing cytokines & regulating response - 70-90% lymphocytes 9 Originate in bone marrow & mature in thymus THY-1 antigen on surface & TH, TC, Treg (subsets) Participate in cell-mediated cytotoxicity (TC cells) & regulation of immune response (TH- & Treg) B cells = WBC functioning in humoral immunity by secreting antibodies - Originate & mature in bone marrow & reside in secondary lymphoid organs - Immunoglobulin molecules on surface as antigen receptors - After stimulation (presence of specific antigen & help from TH cells) => transformation & start secreting large amounts of antibodies (plasma cells) Dendritic cells = antigen-presenting cells (accessory) of mammalian immune system that processes antigen material & present it on cell surface to T-cells - messengers b/w innate & adaptive immune system - Lymphoid origin; “tree” shaped - Express high levels of MHCII - Function: presenting Ag to lymphocytes - ○ ○ i. Lymphoid = found in secondary lymphoid organs; particulates in graft rejection ii. Follicular = located in lymph nodes; do not have MHCII, but high levels of FcR - stimialte B cells iii. Interdigitating = close interaction w/ T cells iv. Langerhans Cell = found in epidermis; interact w/ T cells C. Neutrophils (PMN) = WBC in innate immune system that circulate in bloodstream & senses infection - migrates to site to kill invading microbes ● Stain w/ both acidic & basic dye ● ● ● Formed in bone marrow → blood circulation for ~ 10 hours → tissues where they function for a few days & die 50-70% of leukocytes in blood Function: phagocytosis = uses its plasma membrane to engulf a large particle ○ Usually die after 1 phagocytosis, but large # & fast speed movements makes them most important part of defense against infection ○ W/ death = release granules, chemoattractants, & inflammatory agents D. Eosinophil = WBC responsible for combating multicellular parasites & certain infection in vertebrates ● Stained w/ acidic dye eosin red ● Granules are basic E. Basophil = WBC appears in specific inflammatory rxn (allergies) - prevents blood clotting too quickly & promote blood flow to tissues ● Stained w/ basic dye MET blue ● Granules are acidic 10 ● Comprised of 1% leukocytes in normal blood ● Found in many tissues ● Partially responsible for allergic rxns & functions against parasites F. Mononuclear Cells (aka: macrophages & histiocytes) = blood cells that have a single, round nucleus, such as lymphocytes and monocytes ● Originate in bone marrow (promonocytes) → blood (monocytes) for ~ 8 hours → tissues G. Primary Functions of Macrophages → Mass Cell = a cell filled with basophil granules, found in numbers in connective tissue and releasing histamine and other substances during inflammatory and allergic reactions → Macrophages = large cells & have many lysosomes (RER & golgi apparatus) - Comprised of 5% leukocytes in normal blood - Highly mobile & long-lived (2-3 months) 1. Phagocytosis 2. Processing of Antigen & presentation to T cells 3. j Secretion of Monokies 11 H. Mast Cells = cell filled w/ basophil granules (found in #s in connective tissue & releasing histamine & others during inflammatory and allergic reactions) ● Originate in bone marrow & found in variety of tissue (including connective, skin, & mucosal epithelial of respiratory, genitourinary, & digestive tracts) ● During parasitic infections accumulates in lymph nodes & cut ● Degranulate upon exposure to allergies & release histamine & enzymes causing allergic rxn I. J. Lymphocytes = small leukocyte (WBC) w/ single round nucleus (occurs in lymphatic system) 1. Comprised of 20-40% blood leukocytes 2. Originate in bone marrow & found in blood, lymph & all lymphoid organs & tissues 3. Undergo activation & transformation into effector form in course of immune response 4. Carry immunological memory Lymphoid Organs 1. Primary: cells are produced/differentiate 2. Secondary: cells reside & participate in immune response a. Bone marrow (where B-cells come from) 12 - Majority hematopoietic & vascular, intersliced → Hematopoi etic = stem cells that give rise to other blood cells - 2-5% adult body weight - Large pop. of lymphocytes (10%) & major site of antibody production by B cells b. Thymus (where T-cells come from) - Site of maturation of thymocytes originating from bone marrow → mature T cells - Thymocytes attracted to thymus by hormones secreted by thymus epithelial cells - Thymus large in infants & shrivels in adults - Divided into cortex (outter) & medulla (inner) - Thymocytes enter cortex & rapidly divide 90-95% die - Rest migrate to medulla & secondary lymphoid or K. Lymph Nodes 1. Lymph nodes are bean shaped (~ 1 cm in health) located directly in path of lymphatic vessel throughout body 2. Leukocytes enter the lymphatic system from blood in lymph nodes: trap for antigens! 3. Germinal centers are locations of Ab production ● Have concentrate pop. of macrophages (medulla), T cells (paracortex), & B & dendritic cells (cortex) = efficient/strong immune response L. Spleen 1. Removal of old erythrocytes by macrophages in red pulp region of organ 2. Immune response site similar to lymph node carried by white pulp region M. Mucosa-Associated Lymphoid Tissue (MALT) respiratory, & urogenital tissues 1. Peyer’s Patches 2. Tonsils 3. Appendix = series of cells, follicles, & nodules of lymphoid tissue lining digestive,