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Paremlys Top 10 1. Isohemmaglutinins = ABO antibodies Natural antibodies against CHO antigens 2. IgG half life = 3 weeks 3. IgA deficiency is the most common congenital immune defect, usu compensated by IgM translocation by poly-Ig receptor 4. Antibodies are not normally found on RBCs. Coombs test detects antibodies on RBCs 5. Poison Ivy Rash is an example of cellular immunity (contact allergy) 6. IVIG is used to treat antibody deficiencies 7. IVIG is contraindicated for IgA deficiency 8. Rheumatoid factor for rheumatoid arthritis is tested by IgM anti-IgG antibody that recognizes Fc of IgG 9. Major Cross match is one of 4 tests for organ transplant Dectects preformed recipient anti-donor antibodies 10. Requirements for GVH disease: a) recipient must have HLA antigen b) donor must have CD4+ T cells c) recipient unable to reject donor T-cells Objectives 1-1: First line of defense The intact skin mucous membranes peristalsis anti-microbial proteins (LBP and receptor, CD14) (Toll receptors) (MBP) (transferin/lactoferrin, chelate Fe) (lysozyme) microbial gradient mucocilliary movement surfactants. 1-2: List props of WBC's that contribute to central role in host defense: 1. quickly mobilized from blood/tissues 2. ability to phagoctytozize 3. bear receptors that increase phago 4. generate high levels of intracellular oxidants and radicals to kill 1-3: How does inflammation contribute to host defense: Inflammation enhances phagocytic cell mobilization (leukocyte migration, adhesion, and transendotheliar emigration) provides chemotaxis for neutrophils and macrophages activates phagocytosis and intracellular killing Produces vascular changes to allow extravasation of plasma proteins into infection sites 1-4: How does complement system amplify innate immunity: -Produces chemotactic peptides that attract neutrophils -Chemotactic peptides attach covalently to microbe surfaces and act as opsonins which the phagocytic cells receptors for. - stimulate release of neutrophil from bone marrow

have

1-5: Innate vs. Acquired Immun--list 5 props of acquired immunity to distinguish from innate: Innate Immunity is the "natural" immunity which is inborn and pre-existant in that it doesn't require a previous exposure to a foreign invader. Functions to prevent microbial entry, limit microbe growth, kill and remove microbes, neutralize foreign toxins. 5 props of acquired immunity include: 1. Memory for prior antigen exposure 2. High degree of antigen specificity 3. Capacity to recognize many diverse antigens 4. Ability to recognize between self and foreign 5. Transferability

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1-6: Define the following terms: Active Immunization: resistance induced after contact w/ foreign antigens; long term, slow onset Passive Immunization: resistance based on antibodies preformed in another host; (given drugs) Primary immune response: first encounter in which IgM antibody is produced Secondary (anamnestic) immune response: IgG produced, longer duration, less "expensive", higher antibody titer/affinity, new functions 1-7: List various lymphocyte populations and functions 1. B-Lymphs--have a membrane bound Ab BCR, recognize native conformation of Antigen, no need for APC, signals through receptor complex, produce secreted form of Ab 2. Th lymphs--TCR is Ab like molecule, recognized peptide antigens and MHC, APC needed, signals through receptor complex, produces cytokins (Th) 3. Tc lymphs--produces cytotoxic products 1-8: How do lymphs recognize foreign antigens: B-lymphs have a membrane antigen binding receptor (antibody) that recognizes the native antigen conformation. T-lymphs have an antibody like T cell receptor in the membrane. The T cells only recognize antigen which are presented by APC. These cells have a MHC on their membrane which binds the antigen and then the T-cells bind to the MHC-antigen complex via their TCR ab like receptors and the co-receptors CD4 (Th cells) and CD8 (Tc cells) 1-9: Describe fx of following surface markers on immune system cells: BCR- B cell antigen receptor TCR- T cell antigen receptor FcR- Receptors on Phagocytic cells that binds antibodies (opsonic receptor) CR (1-4)- Complement recept. on phago. cells that bind complement peptides (opsonic receptors) CD3- Signalling component of TCR complex CD4- Marker for Th cells (co-receptor) CD8- Marker for Tc cells (co-receptor) CD28- Co-receptor on T cells which promotes activation MHC- Molecules on APC that present peptide antigens to T cells 2-1: 2 opsinins and their receptors: IgG antibodies and C3b peptides. The antibodies bind to the Fc receptors and the C3bs bind to CR1, CR3, and CR4 The oxygen dependent mechanism used by phagocytes involves a "respiratory burst" (the single electron reduction of molecular oxygen) accomplished via the activation of a membrane bound oxidase (NADPH oxidase). This superoxide anion then generates other agents such as hydroxyl radicals and hydrogen peroxide. When a lysosome fuses with the phagosome, myeloperoxidase produces hypoclorite( household bleach) from hydrogen peroxide and chloride ions. The nitrogen dependent mechanism utilizes nitric oxide synthetase (NOS) which yeilds L-citrulline and NO. Other reactive nitrogen intermediates include NO2 and HNO2 (nitrous acid). The oxygen independent mechanisms involves intracellular components such as lactoferrin and lysozyme. Defensins are also produced. These are cystein rich cationic peptides that d/n require oxygen. Tumor necrosis factor alpha is also active in this mechanism. The colonal selection theory states: 1. All antibody-forming cells are pre-committed in their specificity. 2. One cell is specific for 1 antigen 3. Cell activation begins with Ag binding to a surface receptor 4. This activation leads to the expansion of a clone of cells all with the same specificity 5. The antigen receptor on a cell is uniform and identical with the antibody the cell secretes This theory can be related to the 5 properties of innate immunity???????

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Acquired immune responses can amplify innate defense mechanisms in the following manner 1. Antibody recognition promotes phagocytosis 2. Compliment activation by Ab-Ag complexes recruits neutrophils 3. T-cell derived cytokines activate macrophages for intra cellular killing of microbes 4. Autoantibodies target host tissues for neutrophil-mediated damage The lymph node structure is as follows: Lymph from the surrounding tissue arrives via afferent lymph vessels. 1. The lymph/lymphocyte first encounters and must pass through the high endothelial venules. 2. Antibody production occurs in the medullary cords 3. Memory B cells are produced in the germinal centers 4. Lymph flow is from afferent lymph vessels, into the lymphoid follicle, to the medullary sinus, and out the efferent vessel. The spleen structure and fx is as follows: 1. The splenic artery enters at the hilum and divides into progressively smaller arterioles 2. The red pulp is filled with erythrocytes and this pulp surrounds the sinusoids 3. The white pulp forms the periarteriolar lymphoid sheath (PALS) around the arterioles 4. T cells are contained in the PALS 5. The marginal zone, closely associated with the PALS is rich in B cells. It also contains lymphoid follicles that develop into secondary follicles containing germinal centers. Antigens show antigenicityie the have the ability to react with antibodies or T-Cells. The antigenic determinant, or epitope, of the antigen is the region of the antigen that binds to the Ab or TCR. If the antigen is capable of inducing an immune response, it is termed immunogenic. A Hapten is a molecule which lacks immunogenicity (b/c too small) but shows antigenicity. The production of anti-hapten antibodies is accomplished by covalently coupling haptens to carrier molecules, eg. Hapten-protein conjugates. An example is a conjugate vaccine (Hib-TT) An adjuvant is a substance which enhances the immunogenicity of an antigen. This is accomplished by: 1. causing antigen aggregation 2. facilitating uptake of antigen by APCs 3. promoting the long-term survival of an antigen 4. co-activating immune cells examples of adjuvants are: aluminum salts, oil-water emulsions, and microbial glycolipids. The differences between T and B cell anigenic determinants are as follows: T-Cells: Ex. Peptides 1. The antigen must bind to the surface of the MHC molecules. 2. The binding is conformation-independent. 3. T cell binding is never nonsequential 4. Often involves internal domains of macromolecules B-Cells: Ex. Native proteins and carbohydrates 1. Hydrophillic or charged groups 2. Conformation-dependent 3. Sequential and non-sequential determinants Natural Antibodies have the following attributes: 1. Acquired early in life 2. Generally are specific for carbohydrate antigens 3. Possibly induced by normal microbial flora 4. Are of the IgM class Examples: antibodies to ABO antigens, isohemagglutinins (TOP 10 LIST)

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3-6 cont. The IgG molecule is a 4 chained molecule. It demonstrates bilateral symmetry. Consists of H and L chains which are bonded by disulfide interchain bonds. V and C regions are also present. Intra chain disulfide bonds within the H and L chains definie 110 aa domains. A hinge region is also present. Fab fragments consist of Vh, Ch1 + Vl, Cl which = antigen binding activity Fc fragments consist of the Ch2-3 domains and represent biological activity Function: The antigen binding portions of the IgG molecule are the Vh + Vl the Ch domains are determined by biological activities. V-regions: 1. Are more variable in aa sequence than C regions 2. The variability is clustered in Hypervariable Regions HV1, HV2, HV3 3. These HV regions comprise the "walls" of the biding region that react with the antigen. Referred to as the CDR or complementary determining region. 4. Interspersing regions are known as framework The L chains of immunoglobulins are heterogenous and exist as either Kappa or Lambda. The H chains are also heterogenous and exist in several forms including gamma 1-4, alpha 1and 2, delta, epsilon, and miu. IgG, IgD, and IgE are all monomeric immunoglobulins. IgM and IgA can exist as poymeric. 3-7: The polymeric immunoglublins (IgA and IgM) have the following structures: The IgA can exist as a dimer in which a J chain connects the two immunoglobulins and contains a hinge region. The IgM exists as a pentamer connected by disulfide bonds with a J chain between 2 of the immunoglobuins in the pentamer and contains NO hinge region. The J chain (Fc linked polypeptide) appears to be required for polymerization of the monomers to form pentameric IgM and is added just before secretion. In IgA, the J chain serves the function of facilitating the dimerization of both serum IgA and secretory IgA. The secretory component is a 70K MW polypeptide produced by the epitelial cells of mucous membranes. Consisting of 5 immunoglobulin like domains that bind the Fc region domains to the IgA dimer, this component is responsible for transporting polymeric IgA across cell membranes. Isotype refers to difference in C region sequences Allotype refers to inherited single amino acid substitutions

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Idiotype refers to differences in the V region of a single Ig molecule or monoclonal Ig; characteristic of a single B cell clone. 3-9: The principal functions of the 5 major classes of immunoglobulins are: 1. IgG--The IgG1, 3, & 4 molecules readily cross the placenta and play an important role in protecting the developing fetus. IgG3 is the most effective complement activator while IgG4 can not activate complement at all. IgG1 and 3 are the primary binders to Fc receptors on phagocytic cells and mediate opsonization. 2. IgM--The first immunoglobulin produced in a primary response to an antigen and the first to be synthesized in the neonate. 3. IgA-- The predominant immunoglobulin class in external secretions..ie breast milk, saliva, tears, and mucus of the bronchial, genitourinary, and digestive tracts. 4. IgE-- Mediate the immediate hypersensitivity response which are responsible for the symptoms of hay fever, asthma, hives, and anaphylactic shock. 5. IgD--Thought to function in the activation of B cells by antigen however no biological effector function has been identified. Monoclonal antibodies are antibodies derived from a single clone and thus are specific for a single epitope. Monoclonal antibodies are produced via hybridoma techonology. 1. Immunize a mouse with a desired Ag 2. Recover immune splenic B cells 3. Fuse B cells with a myeloma cell line 4. Screen the hybrids for Ab production and specificity 5. Clone the hybrids and grow them in vitro 6. Return the hybridoma cells to the mouse for high level Ab production The monoclonal antibody is used clinically in many diagnostic tests such as pregnancy tests, diagnosing numberous pathogenic microorganisms, measuring blood levels of various drugs, matching histocompatibility antigens, and measuring/detecting anitgens shed by various tumors. The structural features of IgG polypeptides that suggest the unusual genetic basis for diversity: 1. The vast diversity of antibody specificities (109 Abs would require 10-15% of the germ line being devoted to encoding Ig polypeptide chains) 2. The presence of a variable region at the amino-terminal end and of a constant region at the carboxylterminal end of the Ig heavy and light chains. 3. The existence of isotypes with the same antigenic specificity, which result from the association of a given variable region with different heavy-chain constant regions (biclonal antibodies) 4. Class switching of Ig in normal B cells retains the same L chain and idiotype (Vh + Vl) The genetic organization of the 3 gene families that encode Ig polypeptides are: 1. The K and lambda families (light chains) consists of variable gene segments, joiner gene segments, and constant gene segments. The VJ segments encode the variable region. 2. The H (heavy chain) family contains V, D (diversity), J, and C regions. The rearrangement of VDJ encodes the variable region of the heavy chain. The C regions (in the order of miu, delta, gamma, epsilon, and alpha) encode the constant region of the heavy chains. The process of light chain gene rearrangement and recombination is as follows: 1. 5'-3' VJC regions exist on the germ line K-chain DNA 2. The K-chain DNA is rearranged via V-J joining 3. The rearranged DNA is transcribed to produce a primary RNA transcript 4. Polyadenylation and RNA splicing occurs to produce an mRNA 5. This mRNA is translated to form a nascent polypeptide 6. The L portion is cleaved off to form the finished K-light chain The process of heavy chain gene rearrangement and recombination is as follows: 1. 5'-3' VDJC regions exist on the germ line H-chain DNA 2. The DJ regions join first and cause DNA rearrangement

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The V region joins with the newly joined DJ region to produce a V-DJ rearranged H-chain DNA This rearranged DNA is transcribed to produce a primary RNA transcript RNA splicing occurs to cut out various C regionspolydenylation also occurs The newly formed mRNA is translated into a nascent polypeptide The L portion is cleaved to form the finished miu, delta, or etc heavy chain depending on step 5

The order of gene rearrangements is H chain followed by Kappa, followed by lambda Non-productive rearrangements are the result of rearrangements between the V and J regions that result in AA sequences which encode for stop codons. The result is a variable which is not translated into protein. Allelic exclusion: since chomosomes exist in diploid state, there are 2 opportunities per locus for rearrangement. If the kappa rearrangement is Unsuccessful, then the lambda locus is rearranged. However, if the kappa rearrangment IS successful, then the lambda lucus rearrangement is suppressed and this is known as allelic exclusion. It ensures that only 1 H chain and only 1 L chain are synthesized by one B cell. The processes/events that contribute to the immune diversity of antibody molecules are: 1. Multiple germ-liune gene segments: DNA contains 51Vh, 27D, 6Jh, 40Vk, 5Jk, 30Vlambda, 4Jlambda 2. Combinatorial V-(D)-J joining: 8262 possible combinations, 200 possible at K locus, 120 possible at lambda locus. 3. Junctional flexibility: can lead to unproductive rearrangements, however variable productive rearrangements increase the diversity. 4. Somatic Hypermutation: occurs in rearranged Ig V genes, occurs in germinal center B cells, it is T cell and antigen driven, caused by random substitutions. The above processes allow the human immune system to exceed 10 to the 10 distinct antibodies/sequences. Isotype switching is an antigen driven and Th cell dependent, irreversible switch in immunoglobulins (ex. IgM to IgG) which involves a change in the Ch region only. The recombination events controlling this process are: 1. Switch region sequences are located upstream of each Ch region (except delta) 2. Class specific recombinases bind to these switch regions and facilitate DNA recombination. 3. When the switch sites recombine and are excised from the DNA, the remaining DNA and the remaining constant regions determine the new isotype. 4. This occurs in a specific order as was discovered by looking at the concentration of the excised circular DNA products. Existence of both membrane bound and secreted forms of Ig heavy chains with the same specificity can be explained in the following manner: 1. A primary H-Chain transcript exists. 2. This transcript contains a C miu region which consists of 4 exons. Immediately following the 4th exon is a nucleotide sequence (S)[site 1] that codes for the hydrophilic sequence in the Ch4 domain of the secreted Ig 3. Two additional exons, M1 and M2 are located just downstream to the Cmiu4 exon. [site 2] The M1 encodes for the transmembrane segment and the M2 encodes for the cytoplasmic segment of the membrane bound Ig. 4. If cleavage/polyadenylation of the primary transcript occurs at site 1, the M1 and M2 regions are lost and after mRNA processing an excreted form of the heavy chain is produced. Vice versa for site 2 cleavage. co-expression of membrane forms of IgD and IgM can be explained in that the constant regions are the only regions that differ. The variable regions of the Heavy and Light chains are the same, thus the idiotypes are the same. One B cell can express multiple membrane Ig isotypes. Antibody affinity refers to the measurement of the behavior of ONE Ab combining site Antibody avidity reflects the cumulative binding activity of ALL combining sites on the molecule. IgM >>IgG

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Cross reactivity is the reactivity of an antibody with more than one antigen this can be explained if the antigens have chemically similar epitopes or if sharing of the same epitope is present. Both B and T cell antigen receptors can show cross reactivity. Ex. Antibodies to ABO antigens. 5-2: Precipitation reactions in Agar gels can be used to define the following: 1. Identity--this occurs when two antigens are placed in wells above/below one antibody. In a pattern of identity, the diffusing antigens and the diffusing antibody produce a fused line of precipitation. In other words the two antigens cross-react. 2. Non-identity--occurs when two non-crossreacting antigens are present on an agar plate. In this case when the precipitation lines from the diffusing antigen and antibody form, precipitation spurs are visible. 3. Partial-identity--occurs when both a fused line AND a spur are visible. This indicates that crossreactivity was present, however, there was also unique determinants of each antigen. Agglutination occurs when antibodies that recognize cell surface antigens cross-link cells. This can be used to "type" the antigens on erythrocytes OR measure the titre of an antibody to a known erythrocyte antigen. The typing is done by mixing RBC's on a slide with antisera to the A or B blood-group antigens. If the antigen is present on the cell, they agglutinate. 1. RIA (Radioimmunoassay)- Used to measure IgE antibodies to allergens a. a microtiter plate well is coated with antibody b. sample is added and the antigen binds. c. A radioactive antigen is then added d. The radioactivity is then measured. e. If serum is infected, there is decreased radioactivity, due to competition b/w antigen and radioactive antigen f. If serum not infected all bound antigen is radioactive and the radioactivity is high 2. RAST (radioallergosorbant assay)-a. detects the serum level of IgE specific for a given allergen. b. The allergen is coupled with beads or disks c. Patients serum added d. Unbound antibody washed away e. Amount of specific IgE bound to solid-phase allergen is measured with radioactive anti-IgE f. Beads washed g. Radioactivity counted h. In this assay the radioactive anti-IgE binds to the IgE which bound to the allergen. So if the patient has an IgE for a specific allergen it will become radioactive when the anti-IgE is bound to it 3. ELISA (enzyme linked immunosorbant assay) used in home preg. Tests and measurement of serum complement and antibodies to specific infections a. A well is coated with antibody and washed b. Pts serum/antigen is added and binds to antibodies in well if present c. And an enzyme-conjugated secondary antibody to the antigen of interest d. Add the substrate for the enzyme and measure the color change in the solution Western blotting aka immunofixation used in characterizing myeloma Igs, and confirming the specificity of anti-HIV antibodies a. SDS-PAGE of protein mixture b. Protein bands transferred to nitrocellulose by electrophoresis c. Bands identified by flooding membrane with radiolabeled or enzyme-linked polyclonal or monoclonal antibody specific for the protein of interest. d. Expose membrane and bands of interest appear Immunofluoresence 3 types used to detect deposition of IgG or complement proteins in the kidney and the binding of auto-antibodies to tissues (ex. Anti-dna antibodies in lupus)

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a. b. c. 6.

Direct--membrane antigens on cells are exposed to antibody tagged with fluorochrome. Indirect 1-a labled anti-isotype antibody is made that binds with the antigen/antibody complex on the cell. Indirect 2 - a labled protein is made which binds with the primary antibody/antigen complex on the cells of interest.

Flow Cytometry--Used to measure total number of circulating T and B cells (CD3, CD4, CD8, CD19) and can be modified to purify cell subsets a. Cells are stained with antibodies (results ex anti a, anti b, mixed, and unstained) b. One drop at a time is passed through a laser c. The computer measures the fluorescense caused by the activation by the laser d. The cells are charged and can be separated based on this charge into separate solutions e. Results are printed on the computer screen see KUBY FIG. 6-16

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Which test should I use for: 1. Anti-rbc antibodies in autoimmune hemolytic anemia =Hemagluttination possibly RIA 2. Immune complex deposition in the kidney in SLE= Immunofluorescence 3. Serum anti-HIV antibodies= Western Blotting (immunofixation) 4. A monoclonal serum Ig (M component) in multiple myeloma= Western Blotting 5. Serum IgE antibodies to ragweed pollen= RAST 6. HCG in the urine of a pregnant woman=ELISA

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The structure of the B-cell antigen receptor complex (BCR) is as follows: 1. A membrane bound immunoglobulin (mIg) 2. Disulfide linked heterodimers called Ig-alpha/Ig-beta. 3. Each heterodimer contains the immunoglobulin-fold structure and cytoplasmic tails (longer than the tails of the mIg. 4. Each mIg is thought to be associated with two heterodimes 6-1: The T-cell antigen receptor (TCR): 1. Has transmembrane heterodimers with V and C regions 2. Has two types of receptors alpha-beta(95%) and gamma-delta (5%) 3. Has a single (fab like) ligand-binding site 4. Has specificity for self MHC + Peptide 5. Is expressed as a complex with CD3 peptides which transmit signaling

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Genetic mechanisms for generating diversity in the TCR include: 1. The expression requires gene rearrangements similar to antibodies except NO somatic hypermutation 2. V, D, J, C gene segments present 3. Both germ-line alpha and beta chain DNA exists 4. Gene rearrangements occur in the DNA

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The DNA is transcribed and translated to produce the alpha and beta chains There is combinatorial joining Imprecise joining possible Nucleotide addition possible Combinatorial association between alpha and beta chains

The TCR ligand properties are as follows: 1. The TCR does not recognize native antigens 2. The TCR binds to peptide and MHC complex in a trimolecular complex 3. CD3 always present 4. MHC class I on the Tc cell 5. MHC class II on the Th cell The ligands for the following are: 1. CD4=class II MHC [presented by b cells, macrophages and dendritic cells] 2. CD8=class I MHC [presented by nearly all nucleated cells] 3. CD28=B7 [presented by macrophages, dendritic cells, subsets of T/B cells] 4. CD154=CD40 [presented by same cells as CD4] 5. LFA-1=ICAM-1 (CD54) [presented by endothelial cells, macrophages, others] Superantigens bind simultaneously to a class II MHC and to the Vbeta domain of the TCR activating all T-cells bearing a particular V beta domain. This binding occurs outside of the peptide binding groove. The binding produces polyclonal T cell activation and extensive cytokine production. This can cause shock in infected patients. Examples include TSST1 (from staph aureus), pyrogenic exotoxins from strep pyrogens, and MAS (mycoplama arthritidis supernatant) T helper cells can amplify innate immune mechanisms via the soluable mediators activated Th cells produce. These can be found on figure 12-5 of Kuby. Activated Th cells produce: (see kuby p. 311) 1. IL-3 (stimulates histamine secretion in mast cells) 2. IL-4 (upreg. Class II mhc expression in macrophages, inc. phago activity, stimulates mast cell growth) 3. IL-10 (opposes effects of IL-4) 4. IL-13 (inhibitory to macrophages, elicits control) 5. IFN-alpha (inhibits viral replication) 6. IFN-gamma (same as above) 7. TNF-beta (enhances phagocytic activity in macrophages) For cytokines produced by Th1 and Th2 cells see Kuby table 12-4 pg. 319

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IL2: leads to positive effects on B cells (IgG2a) and Tc activation IFN-gamma: same as above, plus activates macrophages TNF-beta: activates macrophages IL3: leads to mast cell growth IL4: Bcell activator to produce IgM, IgG1, IgA, IgE, and mast cell growth factor, activates macrophages IL5: Eosinophil growth and differentiation and bcell activator as above IL10: mast cell growth factor, macrophage activation IL13: macrophage activation and bcell differentiation 6-7: T cell cytotoxicity is mediated by CTL cells in the following manner: 1. Initiated by recognition of peptide + MHC class I 2. Involves CTL-target conjugation formation 3. Granules containing perforin (pore formation) and granzymes (caspase activation) are released 4. Fas-Fas ligand interactions lead to apoptosis via the caspase activation pathway TSS is a systemic condition characterized by fever, rash, multi-organ failure, and hemodynamic shock. This is mediated by superantigens (TSST-1) and leads to polyclonal T cell and macrophage activation SCID (severe combined immunodificiency disorder) is a lymphopenia associated with the failure to differentiate lymphoid progenitor cells in the bone marrow. Leads to death by opportunistic infections early in life (ie. Viral and fungal). Multiple etiologies including failure to rearrange TCR genes (Rag 1,2 deficiency)

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The functions of the MHC are as follows: 1. Shapes the T cell repertoire during T cell differentiation in the thymusdeletes self-reactive T cells 2. Mediates antigen presentation to T-cells in peripheral lymphoid tissues 3. Serves as principal barrier to tissue tansplantation b/w different members of a species 4. Structurally, there are two classes of genes and gene products based on structure and function 5. Class I and II are cell surface heterodimeric glycoproteins 6. Each polypeptide is comprised of Ig-like globular domains CLASS1: 1. Regulate activation of CD8+ Tcells. 2. heterodimers; alpha chain (transmembrane with 3 globular domains) plus beta2mi croglobulin chain (invariant polypeptide encoded by gene outside the MHC) CLASSII: 1. Restrict antigen recognition by CD4+ T cells 2. Have a limited tissue distribution (macroph, monocytes, b cells, dendritic cells) 3. consist of alpha and beta chains 4. both chains span membrane 5. Ig-like globular domains 6. Greatest sequence variability exists in N-terminus 7-2: MHC Class I genes: 1. Have NO distinct C and V regions 2. NO gene rearrangements occur 3. Exons of MHC genes correspond to domains of MHC proteins 4. Nucleotide differences in the genes cluster at the amino terminus 5. Products of HLA class I loci are HLA-A, HLA-B, HLA-C 6. Kuby p. 174 "class I MHC genes encode glycoproteins expressed on the surface of nearly all nucleated cells; the major function of the class I gene products is presentation of peptide antigens to Tc cells." MHC Class II genes: 1. There are three major regions; HLA-DP, HLA-DQ, HLA-DR 2. Each region contains one or more alpha and beta chain-encoding genes 3. Multiple alleles exist at each locusex 214 alleles at beta chain locus of HLA-DR 4. Polygenic and highly polymorphic 5. Kuby p. 174 "class II MHC genes encode glycoproteins expressed primarily on APCs where they present processed antigenic peptides to Th cells"

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The genetic basis for HLA diversity can be attributed to the number of alleles found at each locus encoding for the structure of the HLA. Each individual has a maternally and paternally derived allele at each locus. Each diploid individual therefore has 1 (homozygous) or 2 (heterozygous) alleles at each locus. Maximum HLA class 1 variants for individual is 3 X 2=6. For species, 250 +70 + 120= 440 For class II, there are three gene regions each with multiple alleles. For example, there are 214 alleles at the beta chain locus of HLA-DR Polymorphism is when one finds many alternative forms of the gene, or alleles, at each locus. Secondary and crystallographic structures of HLA class I and II

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HLA molecules contain transmembrane and cytoplasmic domains. Two membrane proximal domains stabilize the molecule. A peptide binding groove is formed by two membrane-distal domains, The binding groove is composed of a beta pleated sheet platform and 2 anti-parallel alpha helices. The variability in the amino acid sequences of the HLAs is found at the residues of the platform and the inward or outward pointing residues of the alpha helices. These variable residues determine the affinity of the MHC for peptide and TCR. Polymorphic residues determine fx. 7-5: Draw a map of the HLA gene complex that shows the positions of at least 12 different loci:

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HLA shows a mendelian inheritance pattern with autosomal, co-dominance expression. In other words both alleles are expressed in heterozygotes. Class I is expressed on nearly all nucleated cells. Class II is expressed on monocytes, macrophages, B cells, and dendritic cells. Specific mediating cytokines include: 1. IL-4; Up-regulates class II MHC expression on resting B cells/macrophages

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IL-10; Down-regulates MHC class II on APCs IL-16; induces expression of MHC class II on CD4+ T cells, and monocytes IFN-gamma; increases expression of class I and II MHC on many cell types

The cytosolic pathway (class I) of antigen processing presents endogenous antigens present inside the APC Cytosolic protein antigens are ubiquinated (marked for degredation by small protein called ubiquitin and digested by multicatalytic proteasome (large 26s cylindrical particle consisting of 4 rings of protein subunits with a central channel of 10-50 Angstroms which cleaves peptide bonds between 2 or 3 different amino acid combinations in an ATP dependent process) -Processed peptides are transported into cisternae of ER by "transporter of antigenic peptides" (TAP). -Peptide binds to groove of MHC class I -Transport of cytosolic peptides to ER lumenby TAP -Synthesis of class I polypeptides is occuring on polyribosomes -Folding and transport of class I to TAP occurs via chaperones (calnexin, calreticulin, tapasin) -Class I binds to TAP and capture of peptide occurs -Golgi moves to cell surface -The antigen is presented to the CD8+ T cell -Step 9 occurs by vitually all cells capable of synthesizing class I -Presentation is primarily of protein antigens, but some glycolipid presentation occurs -Examples are viral glycoproteins and nucleoproteins syntesized in virus-infected cells The class II pathway, the endocytic pathway, involves the following steps and components: 1. Antigen is taken up by pinocytosis,phagocytosis, or receptor-mediated endocytosis 2. Acidic endosomal proteases degrade protein antigens 3. Endosomes fuse with vesicles containing newly synthesized MHC class II 4. Fused vesicles move to cell membrane 5. The class II molecules are synthesized on polyribosomes 6. The antigen-binding groove is protected by an invariant chain 7. The invariant chain is cleaved CLIP (cleavage of invariant chain) 8. Step 3 above occurs simultaneously with step 7 above. 9. The processed peptides bind to the MHC 10. The entire complex moves to the cell membrane for presentation 11. Complex presented to CD4 + T cells 12. Presented by macrophages, b cells, and dendrocytes 13. Polysaccharide and lipid antigens are NOT presented in this pathway 14. This explains why some antigens do not stimulate helper T cells Summary: The unique steps in each are: Cytosolic (class I) Pathway: Ubiquitin, Proteosome, TAP, Chaperones, CD 8+ Endocytic (class II) Pathway: Invariant chain, CLIP, CD 4 +

8-1: B-Cell development: 1. The lymphoid cell becomes the Progenitor B-Cell (pro-B cell) when the expression of CD45R is present. [At this step, surrogate light chain present and no heavy chain] RAG-1/RAG-2 recombinase enzymes expressed during this stage.

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2.

3. 4. 5. 6. 7.

In the bone marrow still pro-B cells become precursor B-cells (pre-B cells). This only occurs in the presence of stromal cells. Stromal cells secrete IL-7 which is imp. for B-Cell development. [At this step mu heavy chain membrane Ig exists and surrogate light chain] ] RAG-1/RAG-2 recombinase enzymes expressed during this stage. Light chain gene rearrangement occurs leading to the development of an immature B-cell. [In this stage, mu heavy chain exists as well as either kappa or lambda light chains] These cells express membrane IgM (mIgM) on the cell surface. Change in RNA processing allows for distinction between immature and mature B-cell. The mature B-cell expresses both mu and delta mIg Apon antigen stimulation the mature B-cell becomes known as an activated B-cell. This process occurs in the peripheral lymphoid organs. This leads to class switching and further differentiation and the formation of.. Plasma Cells. These cells secrete Ig of various isotypes Plasma cells then lead to the development of Memory B-cells. These cells express mIG of various isotypes on their surface.

8-2:

Blocks in B-Cell differentiation can be caused by at least two immune abnormalities. 1. X-Linked (Bruton's) agammaglobulinemia--This disorder causes the failure of pre-b cell differentiation (Btk tyrosine kinase) Kuby 280-281 "clinical focus". Btk or Bruton's tyrosine kinase is

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2. 3. 8-3:

the gene product of the X-LA gene. This kinase is responsible for important roles in B-cell signalling and subsequent differentiation. Hyper IgM syndrome--This disorder is due to defective CD154 (CD40 ligand) expression. CD154 is an inducer of B-cell proliferation and activation Selective IgA deficiency--This is the failure to develop IgA plasma cells

Negative Selection refers to the following: 1. Occurs in the bone marrow 2. Large numbers of immature B cells are deleted 3. Recognition of self antigens occurs 4. Autoreactive cells are deleted 5. Signaling occurs through BCR (mIgM) Positive Selection refers to: 1. Selection occurring within the bone marrow at the pre-B cell stage 2. The promotion of differentiation into immature B-cells 3. Signaling accomplished through the preBCR The following surface molecules and co-receptors are expressed by a mature nave B cell: 1. BCR--mIgM, mIgD; complex with Igalpha and Igbeta 2. FcR--receptor for antibody-antigen complexes 3. MHC class II--presentation of antigen to CD4+ cells 4. CD40--coreceptor that binds CD154 (CD40L) on Th cells [CD154 defective in hyper IgM syndrome] 5. B7--co receptor that binds CD28 on T cells 6. Cytokine receptors; IL-2, IL-4, IL-5, IL-6, etc The process of B-cell activation by a T-dependent antigen is: 1. A native antigen binds to the BCR 2. Antigen is taken up as a receptor mediated process, processed endosomally, and presented on MHC class II to CD4+ T cells 3. CD40-CD154 co-stimulation occurs 4. B7-CD28 co-stimulation occurs 5. There is a conjugate formation with the T cell which leads to contact-dependent signaling through co-receptors 6. Signaling occurs via Th cell derived cytokines 7. Enhanced IL receptor expression 8. IL-2,4,5,6 receptor activation a. IL-2: Growth (cell cycle progression) b. IL-4: IgG-IgE switching; increased MHC class II expression c. IL-5: IgA switching d. IL-6: Differentiation into plasma cells e. TGF-beta1:IgA switching f. IFN-gamma: Inhibition of IL-4 effects; terminal plasma cell differentiation Define the following terms: 1. Memory--a property of the immune system in which a second encounter with the same antigen induces a heightened state of immune reactivity. Kuby, 10. 2. Ig Class Switching--Allows any given Vh region to associate with the constant region of any isotype. This enables antibody specificity to remain constant while the biological effector activities of the molecule vary. Kuby p. 294 3. Somatic hypermutation--excessive mutation in the heavy and light chain variable regions that has been shown to be the major factor in affinity maturation. 4. Affinity maturation--this is the increase in the affinity of the antibodies produced during the course of the humoral response (an increase as much as 100-10k fold) The properties of TI (T-independent) and TD (T-dependent) antibody responses are:

8-4:

8-5:

8-6:

8-7:

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TI 1. 2. 3. 4. 5. 6. 7. TD 1. 2. 3. 4. 8-8: Consists of a polysaccharide covalently attached to a TT Induces IgG more than IgM Is a high affinity antibody Memory B cells Consist of polysaccharides Repeating identical determinants leads to BCR cross-linking There is limited Ig class switching Thereis limited affinity maturation or somatic hypermutation There is no memory cell induction Repeated IgM antibody responses An example of a TI vaccine is pneumovax

Mucosal-associated lymphoid tissues are those in which the antibody response involves secretions. These tissues include GI, pulmonary, GU, salivary, and mammary. Secretory IgA (dimeric IgA + J chain + Secretory component) is formed in the plasma cell. The poly Ig receptor IgR is formed by the epithelial cell. IgA is then transported transepithelialy by vesicles from the submucosa to the lumen. Parenteral versus Mucosal immunization Parenteral: 1. Involves intramuscular challenge 2. Induces serum IgG antibodies 3. Neutralizes viruses in the blood 4. Protective; no CNS infection 5. Does not block viral entry 6. Permits carrier state Mucosal (Oral): 1. Oral challenge 2. Induces secretory IgA in gut 3. Neutralizes viruses within GI secretions 4. Protective; no CNS infection 5. Blocks viral entry and replication in gut 6. No carrier state

8-9:

Hyper IgM syndrome occurs when there is a deficiency in the CD154 co-receptor. Allergic Rhinitis inolves the Th2 cytokine IL-4. IL-4 is an IgE switch cytokine. In other words, IL-4 promotes the switch to IgE which is used in mast cell response to trigger mast cell release of histamine in allergic responses. Thymocyte maturation pathways: 1. In the bone marrow, stem cells become lymphoid progenitor cells, which in turn become Progenitor Tcells. These Pro-T cells migrate to the thymus. 2. In the thymus the pro-T cell becomes a pre-T cell 3. The pre-TCR receptor is expressed which consists of a pre-Talpha + TCR beta (no CD4 or CD8this is known as the double negative stage) 4. CD4 and CD8 are expressed (this is the double positive stage) 5. Positive selection occurs as well as TCR alpha/beta expression 6. Positive and negative selection TCR alpha/beta-dep 7. Two subsets emerge CD4+ and CD8+ (either one or other, this is the single positive stage) 8. These exit to the periphery Correlations: Pre-TCR expression = double negative = early thymocyte = positive selection TCRalphabeta expression = double positive = cortical thymocyte = + and - selection Functional TCR alpha/beta expression = single positive = medullary thymocyte

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9-2:

Positive and Negative selection in the Thymus occurs as follows: Positive Selection; 1. Mediated by both pre-TCR and TCR-alphabeta 2. Pre-TCR induces TCRalpha 3. TCR recognizes self MHC; CD8 and class I, CD4 and class II 4. This functionally establishes MHC restriction specificity Negative Selection: 1. Mediated by TCRalphabeta 2. Recognition of self MHC 3. Elimination of autoreactive clones Since different individuals express different MHCs, the T cell repertoires of different individuals must be distinct due to the above selection processes. Major T cell subsets: CD4 subset: 1. TCRalphabeta/CD3 2. Primarily Th cells Th1 and Th2 subsets CD8 subset: 1. TCRalphabeta/CD3 2. Some TCR gammadelta/CD3 3. Primarily cytotoxic 4. Some cytokine (IFN-gamma) production NK subset: 1. Novel receptors 2. Cytotoxic and cytokine secreting (IFN-gamma) NK-T subset: 1. TCRalphabetalo/CD3 and NK markers 2. Cytotoxic 3. IFN-gamma production Gamma/delta subset: 1. TCR gammadelta/CD3 2. CD4-8- is greater than CD8+ 3. Cytotoxic and cytokine producing

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9-4: How do APCs differ: Mechanisms of Ag uptake: 1. Macrophages employ phagocytosis 2. Receptor mediated endocytosis (b cells) most efficient 3. Endocytosis MHC expression 1. can be constitutive as in b cells and dendritic cells 2. or inducible as in the macrophages B7 and other Co-receptor expression 1. Can be constitutive as in dendritic cell 2. Or inducible as in macrophages and B-cell Varying cytokine production 1. IL-1 in macrophage 9-5: IL-2 is a T cell growth factor. It functions in the following manner: 1. TCR and co-receptor activation leads to both autocrine and paracrine cytokine mediated activation 2. CTL precursor cell recognizes viral peptides + MHC class I 3. CD28-B7 co-receptors provide secondary signals 4. IL-2 from Th1 signals full activation for cytotoxicity Defective IL-2 is caused by defects in the IL-2Rgamma gene. This leads to defective T and B cell development. This condition is termed SCID severe combined immune deficiency.

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9-6:

Cytotoxic T lymphocytes recognize targets (endogenous foreign antigen) by binding the target + self MHC class I to the TCRalphabeta/CD3 receptor-co-receptor complex. They then kill by membranolytic granule constituents and receptor mediated apoptosis (Fas pathway) Natural Killer Cells are activated by the absence of self MHC class I in the NKIR (inhibitory receptor) These cells kill by the same mechanisms listed above.

9-7:

gamma-delta T cells have the following distinguishing properties: 1. They consist of less than 5% of the total peripheral T cells 2. The utilize CD3+ 3. CD4-8- is much more common than CD8+ 4. They are concentrated in the skin and mucosal epithelium of the lung and intestine 5. They have a limited TCR repertoire 6. Not limited to recognizing protein antigens; glycolipids 7. They are not always MHC-restricted 8. They respond to mycobacterium and heat shock protein antigens A Cytokine is a polypeptide-like signaling molecule which acts through high affinity cell surface receptors. They are important immune and inflammatory mediators. Cytokines show Pleiotropic, redundant, synergistic, and antagonistic actions. Examples are interleukins (Ils), interferons (IFN), colony stimulating factors (CSF), growth factors and chemokines. 1. Pleiotropic-refers to a "cytokine that has different biologica effects on different target cells" Kuby p. 304 2. Redundancy-refers to "two or more cytokines that mediate the same function" p. 304 3. Synergism--"occurs when the combined effect of two cytokines on cellular activity is greater than the additive effects of the individual cytokines" p. 304 4. Antagonism--refers to the action of "one cytokine inhibiting or offsetting the effects of another cytokine" p. 304

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10-2:

T-cells can regulate antibody production in response to "T-dependent" antigens in the following manner: 1. When T cells recognize a T-dependent antigen 2. Cytokines are produced that regulate B cell activity 3. IL-2 and IL-4 specifically activate latent B cell transcription factors 4. Many of the cytokine receptors found on the B cells belong to the class I cytokine receptor family (multimeric; shared gamma chain) 5. This signal transduction from the binding of the cytokine to the cytokine receptor on the B cell activates receptor associated kinases 6. The JAK/STAT pathway occurs; phosphorylation and nuclear localization of latent cytoplasmic transcription factors. Details: Cytokines involved: 1. IL-2: Mediates B cell growth 2. IL-4: Mediates B cell proliferation, class switching to IgG and E, and increased MHC class II expression 3. IL-5: Switching to IgA expression 4. IL-6: Differentiation into plasma cells 5. TGF-beta1: Switching to IgA expression 6. IFN-gamma: Inhibits IL-4 effects, plasma cell differentiation JAK/STAT Pathway details: 1. Dimerization of the IL-4 receptor on the B cell membrane leads to activation of the JAK (Janus Kinase) family tyrosine kinases 2. The JAK kinases phosphorylate the receptor 3. JAK also phosphorylates STAT (signal transducers and activators of transcription) 4. Once phosphorylated, STAT dimerizes 5. Specific gene transcription is activated

10-3:

Patterns of cytokines: Th1 cell cytokines: 1. Produce IL-2, IFN-gamma, GM-CSF 2. These activate macrophages, T cells, and NK cells 3. Induction by IL-12 and suppression by IL-10 4. Responsible for Cellular immunity Th2 cell cytokines: 1. Produce IL-4, IL-5, IL-10

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2. 3. 4. 10-4:

Responsible for B cell activation and class switching Induction by IL-4 and suppression by IFN-gamma Humoral immunity

10-5:

Chemokines are small, chemotactic peptides which also regulate leukocyte adhesion. 1. Two subfamilies exist: alpha (CXC --where C is cysteine residue, X is any amino acid, and C is cysteine residue found in that order first along the AA sequence) 2. Beta (CC -- classified as beta if two cysteine residues are found together first in the AA sequence) 3. Examples include: a. IL-8 (CXC): b. MIP-1 (macrophage inflammatory protein-1)[CC]: c. MCP-1 (monocyte chemotactic protein-1)[CC]: 4. Act through high affinity serpentine, transmembrane, G-protein coupled receptors 5. In COPD, IL-8 is a major neutrophil recruitment and activation chemokine Cell Mediated Immunity: This is immunity that is not dependent on Ab. It is mediated by T cells, NK cells, and macrophages. 1. Th cells or macrophages initiate the immunity. 2. Production of cytokines: IL-12, IL-1,IFN-gamma, and CC chemokines 3. These induce the Tc (CTL), NK cells, and macrophages Examples of cell mediated immunity providing protection against microbial pathogens is seen in 1. Tc cell lysis of virus-infected cells 2. Macrophage uptake and intracellular killing of bacterial and fungal pathogens. 3. NK killing of tumor cells 4. Tc and NK cell-mediated lysis of foreign tissues (acute allograft rejection) 5. Contact allergies to metals and chemicalspoison ivy rash (TOP 10 LIST!!!!!) Antibody production can be inhibited by products of immune responses. This includes: 1. Inhibitory Cytokine production: a. IFN-gamma:

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2. 3. 10-8:

b. IL-10: c. TGF-beta1: Antibody feedback by increased Ig catabolism Immune complexes are immunosuppressive (when the BCR and FcR are crosslinked, this tells the b cell to turn off)

Immune complexes are immunosuppressivesee 3 above. Rhogam therapy works in the following manner: 1. Rhogam is an immune gammaglobulin (IgG Ab) to Rh antigens 2. If given to Rh- mothers during pregnancy and at parturition it works in the following way. a. Fetal Rh+ rbcs are eliminated from the maternal circulation b. Maternal B cells are immunosuppressed via BCR/FcR crosslinking which occurrs. Anti-idiotype antibodies are antibodies against idiotypic determinants that can specifically suppress immune responses. The idiotype network theory can be explained with an example. First an antigen induces the production of antibody 1. Idiotype 1 of antibody 1 is an autoantigen and induces Ab2 production. Idiotype 2 of antibody 2 is an autoantigen as well and induces production of Ab3. Idiotype 3 of Antibody 3 is also an autoantigen and induces Ab4 and so on. Since Ab1 and Ab2 are the result of autoantigens, they are capable of forming Ab1-Ab2 immune complexes. This Ab1-Ab2 complex crosslinks BCR and FcR and turns off Ab1 production by the B cell The overall biological outcomes of complement activation are as follows: 1. The lysis of cell membranes 2. Opsonization 3. Inflammation; leukocyte chemotaxis and increased vascular permeability 4. solubilization and clearance of immune complexes from circulation

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11-1:

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11-2:

Classical Pathway: 1. Utilized in acquired immunity 2. Immune complexes IgG and IgM involved 3. Complement involved: C1,C2,C4 4. C3 convertase (C4b2a) 5. Regulator (C1 inhibitor) Alternate Pathway: 1. Utilized as part of innate immunity 2. Activated by polysaccharides, LPS, 3. Convertase (C3bBb) 4. Regulator (Factor H) 5. Feedback amplification 6. C3 tickover Lectin Pathway: 1. Initiation via MBP (mannose binding protein) a. C1q-like b. Binds to mannose residues on microbial surfaces c. + MBL-associated serine proteases (MASP): Clr S-like Other acute phase plasma proteins Surfactants A & C 2. Cleavage of early components of the classical pathway occurs; ie C4 and C2 3. Often this pathway leads to lysis of microbes 11-3: Classical Pathway in Detail: 1. The classical pathway is initiated via antibody-antigen binding 2. Clq binds to 2 Fc to form and activate Clqr2s2

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3. 4. 5. 6. 7.

Clqr2s2 then activates C2 and C4 C4 activation leads to the formation of C3 convertase which is C4b2a in the classical pathway C3 convertase activates C3 and after formation of C3b.. C3 convertase combines with C3b to form the C5 convertase (C4b2a3b) The C5 convertase activates C5 and leads to the MAC

11-4: Alternative Pathway in Detail: 1. A surface bound C3, C3b, or C3(H20) initiates the pathway 2. B is bound and activated by Factor D 3. This leads to an unstable convertase C3Bb which combines with active B (Ba) [C3 tickover] 4. C3 cleavage leads to the formation of a stable convertase C3bBb which combines with C3a 5. C3bBb plus P leads to C3bBbP which is a very stable convertase An amplification loop exists in which C3b contributes to the formation of more C3 convertase (C3bBb3b) 1. C3b (any source) plus B (D) leads to C3bBb 2. C3bBb cleaves C3 to form more C3b 3. step 1 repeats 11-5: The membrane attack complex has the following structure: 1. It is an intramembrane pore formed via the complement pathways (C5b6789(10-12)) 2. It is similar to pores formed by perforin from cytotoxic T cells 3. Causes osmotic lysis 4. C5b678 causes slow lysis 5. C5b6789n causes fast lysis Had to consult Dr. Parmely on this one: Basically he told me the enzymes he was looking for are those found in the alternative and classical pathways of complement activation. The enzymes are: 1. Clqr2s2- This enzyme cleaves C4b to C4a as well as C2b to C2a 2. Factor IThis enzyme inactivates C3b and C4b conversion by cleaving them into inactive convertase components 3. AI (anaphylatoxin inhibitor)Cleaves C3a, C4a, and C5a at an Arg residue and leaves them inactive. 4. Factor DForms C3bBb convertase from C3b and B State the mechanisms by which the following regulate complement activation: 1. C1 Inh: This is a serine protease inhibitor that dissociates C1qrs (classical pathway) 2. CR1: This is a complement receptor that dissociates C3 convertases 3. AI: Anaphylatoxin inhibitor inactivates C3a, C4a, C5a by cleavage at the Arg residue 4. Factor H: induces the decay of C3bBb in solution (alt. Pathway) 5. Factor P: stabilizes the C3 convertase of the alt. Pathway 6. DAF: decay acceleration factor; induces the decay of C3 convertases on membranes 7. C4bp: C4 binding protein; induces decay of C3 convertase in classical pathway 8. Factor I: cleaves C3b and C4b and leaves them Inactive as convertase components Dissociators: C1 Inh, CR1 Decayers: Factor H, DAF, C4bp Stabilizers: Factor P Inactivators: AI, Factor I

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11-7:

11-8:

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11-9:

Cell surface receptors capable of binding complement peptides 1. CR1: Receptor for opsonization in phagocytes & clearance in erythrocytes 2. CR3: opsonization receptor in phagocytes, 3. CR4: same as above 4. C5aR: Anaphylaxis receptor in mast cells, basophils, and smooth muscle cells also a chemotactic receptor in neutrophils 5. C3aR/C4aR: anaphylaxis receptors

11-10: Lab tests: 1. ELISA: detects C1, C2, C4, C3, and B 2. Complement fixation 3. Activity Assay (not commonly used); total hemolytic complement (CH50) 4. Opsonophagocytic activity Human disease: SLE: Depressed C1, C2, C4, and CH50 Hereditary Angioedema: Absence of C1 inhibitor, depressed C2, C4, and CH50 Leukocyte adhesion defect: Absence of C18 on neutrophils, absent CR3 and CR4 Paroxysmal nocturnal hemaglobinuria: absence of DAF, CD59 Stable membrane bound C3 convertases cause sporatic hemolysis due to spontaneous C3 activation on membranes 12-1: Patients with immune deficiencies often present or have a history of frequent, severe, and complicated infections which are poorly resolved with antibiotic therapy. Age of onset and family history are two very important items to know. Pts may indicate they engage in high risk activities such as IV drug use and should be questioned about such activities. Occupational exposure is a possible risk factor. The patient should be questioned on the course of prior infections as well as their response to vaccines. They should also be questioned as to whether or not they are receiving immunosuppressive therapy, for a transplant as an example. In order to arrive at a differential diagnosis, several lab tests would be necessary. These include a CBC with diff, serum protein electrophoresis, quantitative Igs, and a T/B cell panel done by flow cytometry. With these lab tests as standard, a more complete work up could be done by ordering the following tests. 1. Isohemaglutinin titres (TOP 10 LIST!!!) 2. Look at antibody responses to immunization (DPT, Hib) 3. Opsonophagocytosis studies 4. NBT dye reduction (measure resp. burst) 5. Complement levels (individual comp. And CDH50) 6. Anti-HIV titres 7. Intradermal skin testing for type IV hypersen. 8. lymphoproliferation in vitro (Con A, PHA) 12-2----12-9: Disease specific findings: X-linked agammaglobulinemia:

Male (congenital disease) patients generally present with otitis media, diarrhea, pneumonia, and/or sinusitis at or around 6 months of age (when IgG from mother becomes low ie. Surpasses life in serum). Laboratory findings will show no IgM, IgA, and very low IgG (what remains from mother)
Cause/Pathogenesis: A defect in Brutons tyrosine kinase (Btk) expression in B-cells. This leads to reduced B-cells in peripheral lympoid tissue. Treatment: Selective IgA deficiency: (TOP 10 LIST!!!) Hx/presentation: Half of the patients asymptomatic most common immune deficiency 1/330

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Cause/Pathogenesis: There is membrane expression of IgA on B cells, but there is a block in secretion. Most of these individuals make compensatory secretory IgM. Treatment: Transient Hypogammaglobulinemia of Infancy: Hx/Presentation: Pts present with recurrent otitis media, pneumonias, and frequent diarrhea. These patients present late in infancy due to mother IgG half-life. Cause/Pathogenesis: There are normal numbers of T and B cells, however, the infants IgG synthesis does not compensate for the gradual decline in maternal IgG in the neonatal period. Treatment: Common Variable Hypogammaglobulinemia: (common variable imm.def) Hx/Presentation: Adult onset, presents secondary to viruses, splenectomy, lymphoma, certain drugs, protein loss (enteropathies, severe burns) Impaired CMI Cause/Pathogenesis: Abnormal plasma cell differentiation, antibody secretion, or Ig metabolism. Treatment: Treatment of Ab deficiencies: 1. Antibiotics prn 2. NEVER use stem cell reconstitution (TOP 10 LIST!!!!!!!) 3. IVIGuse sufficient amounts to achieve low normal levels, the amount is based on the serum life of IgG (TOP 10 LIST!!!) 4. Intravenous immunoglobulin contraindicated in selective IgA a. Can induce anaphylactoid reactions with repeated use b. Anti-IgA antibodies can be formed c. Fetal autotolerance to maternal IgG exists, but not IgA d. Fetal autotolerance to membrane IgM e. . (TOP 10 LIST!!!!!!!!!) DiGeorge Syndrome: Hx/Presentation: Patients will have early onset (first few weeks of life) of viral and fungal infections. Pts will exhibit lymphopenia and moderate hypogammaglobulinemia. Cause/Pathogenesis: Malformation of the 3rd/4th pharyngeal pouches leads to malfunctioning or non-existant thymus, parathyroids, heart problems, and large vessel disorders. Treatment: Thymus transplantation Severe Combined Immune Deficiency (SCID): HX/Presentation: Cause/Pathogenesis: IL-2Rgamma chain deficiency, Adenosine deaminase deficiency, RAG 1,2 deficiencies, or deficiencies in tyrosine kinases that mediate intracellular signaling in lymphoid progenitor or precursor cells. Treatment: Acquired Immune Deficiency Syndrome (AIDS): Hx/Presentation: Pts present with varying opportunistic infections:

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Cause/Pathogenesis: HIV-1 infection of the CD4+T cells, dendritic cells, and macrophages which leads to a severe depletion of CD4+T. There is a late shift from Th1 to Th2 cytokine response, a loss of cellular immunity and DTH responses. There is reduced cytotoxic T cell activity which leads to the presenting opportunistic infections. Treatment: Adenosine Deaminase Deficiency (ADA): Hx/Presentation: Same as SCID Cause/Pathogenesis: There is a deficiency in ADA (the enzyme which catalyzes the breakdown of adenosine to inosine) This leads to an increase in intracellular adenosine. This in turn leads to the inhibition of methyl transferases as well as T and B cell toxicity. ADA deficiency represents @ 50% of SCID cases. Treatment: Gene Therapy Bare Lymphocyte Syndrome (MHC Class II deficiency): Hx/Presentation: Lymphopenia Cause/Pathogenesis: There are blocks in the transcription of MHC Class II genes. There is subsequent poor development of CD4+T cells and absence of positive selection in the thymus. This leads to the lymphopenia. Poor activation of CD4+ cells in the periphery is also noted. Treatment: Wiscott-Aldrich Syndrome: Hx/Presentation: Often have infections with encapsulated extracellular bacteria. Cause/Pathogenesis: Defective Wiscott Alldrich Syndrome protein (WASP) which is a proline rich transcription factor. Initially the patients have poor antibody responses to T-independent polysaccharide antigens. The disease leads to progressive lymphopenia and Ab and CMI defect. Treatment: Leukocyte Adhesion Defect: Hx/Presentation: Cause/Pathogenesis: In LAD-1 there is defective CD18 expression which leads to the absence of LFA-1, CR3 and CR4. In LAD-2, there is a glycosylation defect affecting cell adhesion. These defects result in impaired host defense and pus formation. There is altered neutrophil extravasation and opsonization. Treatment: Chronic Granulomatous Disease (CGD): Hx/Presentation: Infections with catalase-positive bacteria common Cause/Pathogenesis: Genetic defects affecting NADPH Oxidase. Therefore, there is no superoxide formation. Intracellular microbes in the lungs, spleen, and liver are walled off in granulomas. Treatment: Myeloperoxidase (MPO) deficiency: Hx/Presentation: Similar to CGD Cause/Pathogenesis: Neutrophil specific absence of MPO, which is used to convert hydrogen peroxide to hypohalous acid. Treatment: C1q Deficiency: Hx/Presentation: Pyogenic infections common.

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Cause/Pathogenesis: Deficiency in complement leads to poor C3b-dependent opsonophagocytosis. This leads to pyogenic infections. These patients will often develop immune complex disease (50% will have lupus) They have poor clearance of immune complexes. Treatment: Fresh human plasma, recombinant complement components C5-8 Deficiencies: Hx/Presentation: Meningitis, disseminated Neisseria infections Cause/pathogenesis: Treatment: Hereditary Angioedema (HAE): Hx/Presentation: Cause/pathogenesis: Treatment: Paroxysmal Nocturnal Hemaglobulinuria: Hx/Presentation Cause/pathogenesis: Treatment: 12-10: The common therapies used in the treatement of immune deficiencies, the rationale for their use, and the contra-indications are as follows: 1. IVIG a. rationale: given to treat Ab deficiency b. CI: In selective IgAsee above 2. Antibiotics a. Rationale: treat opportunistic infections b. CI: 3. Stem Cell Transplant: a. Rationale: b. CI: 4. Thymus transplantation: a. Rationale: Thymus missing or non fx due to pouch defects in development b. CI: Rejection of transplant organ 5. Gene Therapy: a. Rationale: ADA deficiency b. CI: 6. Irradiation of all blood products a. Rationale: Graft vs. host disease b. CI: 7. Fresh human plasma a. Rationale: b. CI: 8. Recombinant complement components a. Rationale: b. CI:

13-1 & 13-2: Gell and Coombs Classification: Type I: 1. Immediate, occurs within minutes 2. initiated by soluble antigens/haptens 3. IgE antibody involvement 4. rapid activation of tissue mast cells with their release of a number of potent inflammatory mediators. 5. ex. Atopic Allergies seasonal pollenosis Type II (Cytotoxic):

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1. takes hours/days to occur 2. caused by cell/tissue bound antigens 3. response of IgG, IgM antibodies 4. Activation of the complement system 5. Neutrophil and macrophage involvement 6. Ex. Transfusion reactionsRBC lysis Type III (Immune complex): 1. Soluble antigens responsible 2. IgG, IgM antibodies major players 3. complement activation 4. neutrophil/macrophage recruitment 5. ex. Serum sickness, lupus (nephritis) 6. circulating immune complexes deposit in defined locations, activate complement and recruit neurtophils. Type IV (delayed type): 1. involves MHC bound antigenic peptides 2. No antibody is involved 3. CD4+T cells, APCs, macrophages, NK cells, and Tc cells 4. ex. Contact dermatitis 5. Activated Th1 cells release cytokines which recruit and activate macrophages and NK cells. These effector cells then cause tissue damage via inflamm. Mediators. 13-3: Type I hypersensitivity in detail using Atopic ragweed pollenosis as ex: Overview: A prior sensitization to ragweed caused the antigen to be taken up by APCs and presented to Th2 cells (MHC class II). IL-4 then induces IgE Ab production as well as memory T and B cells. The IgE binds to tissue mast cells. On second exposure, elicitation, the antigen binds to the IgE on the mast cells. This causes activation of the mast cells via the Fce receptor. Degranulation, phospholipid metabolism, and gene expression then occurs leading to inflammatory mediator release.

Mast Cell Activation in Detail:

Signaling in mast cells requires cross-linking of receptors. When the receptors are cross-linked, there is an increase in intracellular camp. This leads to an influx and mobilization of intracellular Ca++. Phospholipases are activated subsequently leading to fusion of granules with the cell membrane. Transcriptional activation of inflammatory genes also occurs. (Kd of FceRI = 10pM)

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See below picture for details of the Fc receptor cascade:

13-4:

Pre-formed (primary) mast cell mediators have the following characteristics: 1. They are stored in granules 2. Include a. Histaminecauses itching sensation, increased vascular permeability, vasodilation, & smooth muscle contraction b. Heparinanti-coagulation c. chemotactic peptides (ECF-A, NCF-A) d. proteases such as i. chymase and ii. tryptasemucus secretion Newly-synthesized (secondary) 1. Formed from membrane phospholipids 2. Examples are

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a. PAFincrease vascular permeability, smooth musc. cont., vasodilation b. PGsincrease vas. perm, vasodilation c. Leukotrienes--increase vasc.perm., sm. musc. cont., mucus sec 3. Also cytokines a. TNF b. IL-3 c. IL-4 Mucosal Mast cells: 1. Are IL-4 dependent 2. Have a low histamine content 3. Leukotrienes greater than prostaglandins Connective Mast Cells: 1. IL-4 independent 2. High hist. Content 3. PG>LT 13-5: Intradermal (ID) skin test: 1. Semi-quantitative ID test a. Injection of antigen of interest, saline, and histamine in same arm b. Measure wheel and flare result of each (edema and erythema) 2. Skin Prick Test Food Challenge: 1. To test for food allergies Radioallergosorbent assay (RAST): use of radioactive IgE for specific antigens to see if pts have IgE to specific antigens.

13-6: Therapies for IgE-mediated allergic conditions/rational for therapy: 1. 2. AVOID ALLERGEN Immunotherapy: a. Give the patient repeated intradermal injections of antigens b. Induces blocking of IgG and IgA antibodies and leads to diminished IgE synthesis. c. There is a Th2 to Th1 shift Desensitization a. This is a short term block of mast cell degranulation Block FcR signaling: a. Cromolyn sodium inhibits Ca influx b. Theophylline prevents drop in the intracellular camp

3. 4.

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5.

6.

Block mediator synthesis: a. NSAIDs such as aspirin and indomethacin block AA metabolism b. Corticosteroids such as hydrocortisone i. Inhibit histamine synthesis from histadine ii. Inhibit PLA2 iii. Inhibit cytokine gene expression: the decrease NF-kappabeta Target tissue intervention: a. anti histamines such as diphenhydramine are H receptor antagonists b. epinephrine is used to stimulate beta adrenergic receptors and increase cAMP levels c. corticosteroids block gene expression

13-7:

Pathogenesis of a transfusion reaction:

This reaction occurs when there is a mismatched ABO, Rh, or other erythrocyte antigen on blood infused into a recipient. Complement activation is initiated in response to pre-formed antibodies (isohemagglutinins TOP 10 List!!!) Rbc antigen + the antibody + C1-9 leads to MAC activation and hemolysis. Rbc antigen + antibody + C1-3b leads to phagocytic clearance 13-8: Pathogenesis of Serum Sickness: There are several causes of serum sickness; anti-toxin therapy with heterologous Igs, some infectious diseases such as hepatitis, and certain drugs such as penicillin. Ag + Ab + C leads to release of anaphylatoxins which lead to mast cell activation; possible systemic anaphylactoid reaction can occur producing fever, vasculitis and hemorrhage, lymphadenopathy, arthritis, glomerulonephritis, and hypotension. 13-9: Pathogenesis of Poison Ivy Rash:

13-10: DTH (delayed type hypersens) skin tests are used as a diagnostic tool. Microbial and environmental antigens such as plant extracts and tuberculin are used for the test. In those used in diagnosis of allergic conditions you look for the wheel and flare response which maximizes in about 20 mins. In DTH skin tests you will see indurated skin lesions due to infiltrating mononuclear cells which maximize at 48-72 hours.

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13-11: How are hypersens. Rxtns beneficial: 1. Type I; eosinophils and IgE Ab defend us against intestinal parasites (schistosomes). 2. Type II; mechanism of attack against numerous extracellular bacteria and fungi Type IV; intracellular killing of phagocytized bacteria and CTL killing of virus-infected cells 14-1: Self-tolerance is simply, according to Kuby p. 242-248,255,273, unresponsiveness to self antigens. In the thymus for example, during clonal deletion, two factors are at work. Positive selection is occurring in which the body is ensuring at all alpha-beta TCRs on the mature T cells will bind to self-MHC. If they can not, they are eliminated by apoptosis. Negative selection eliminates thymocytes bearing a high-affinity receptor for self-MHC molecules alone or self-antigen plus self-MHC. These thymocytes, if matured, would pose a threat of an autoimmune response. (Kuby p. 242) Clonal anergy is a state of inactivation marked by the inability of the cells to proliferate in response to a peptide-MHC complex (kuby 255). The typical features of auto-antigens that are involved in auto-immune diseases are: 1. Usually involve altered forms of self proteins such as (pen-rbc) 2. They are typically autoantigens that cross-react with microbial antigens (strep M protein, myocardium, and rheumatic heart disease) 3. they can be sequestered tissue antigens such as lens antigens in the eye

14-2:

14-3: Theories of Autoimmunity: 1. Molecular Mimicry Theory: a. Immunity to an infectious agent cross-reacts with self-antigens i. Ex. Syphilis T. pallidum antigens and fibronectin, collagen ii. Ex. Measles virus and myelin basic protein (MBP) iii. Papilloma virus and insulin receptor 2. Epitope Spreading Theory:

a. B cells which are immune to the antigen take up the antigen and present self proteins, which contain autoantigenic epitopes. b. Ex. Intra-molecular spreading of immunity c. Inter-molecular spreading 3. Polyclonal Lymphocyte Activation Theory: a. an autoantibody is induced by polyclonal selection of lymphocytes b. LPS, EBV, CMV, and B cells c. Staph superantigens and T cells d. HIV infections e. Autoimmune hemolytic anemia in GVHD 4. Sequestered Antigen Theory: a. Trauma to an organ releases a cryptic autoantigen b. Ex autoimmune uveitis and orchitis 5.Co-stimulator Theory: a. inappropriate or over-expression of co-receptors on target cells b. MHC class II on beta cells in IDDM c. Or clonal anergy on T cells in vitro induced by Ag-APC lacking co-receptors 14-4: Witebskys Postulates to determine if a disease has an autoimmune etiology: 1. Demonstrate the presence of autoantibody or autoreactive T cells in the disease 2. Identify the autoantigen 3. immunize a nave individual and produce both the autoimmune response and the disease 4. transfer the immunopathology from a diseased individual to a normal with autoantibody or T cells.

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14-5:

Diagnostic tests: Immunofluorescence: used to detect tissue bound autoantibodies ex. Goodpastueurs and lupus nephritis. Anti-nuclear antibody: used to detect circulating antibodies such as in lupus. A form of immunofluorescence includes antibodies to DNA, histones, and nucleolus ANCAanti-neutrophil cytoplasmic antigen: uses imm.fluoresc. found in certain forms of nephritis and vasculitis (Wegener Granulomatosis) Anti-double stranded DNA: lupus Rheumatoid factor (RF): the patients serum contains an IgM (G, A) anti-IgG Fc. Immune precipitation test is performed and RF is positive in a number of mixed connective tissue diseases not just RA. Coombs Test: Antibody to human IgG, agglutinates human IgG coated rbc. There is a direct and indirect test. Positive in drug induced hemolytic anemias and thrombocytopenias Direct: detects antibody present on patients rbc 1. Implies reactivity with endogenous or exogenous surface Ag 2. pt rbc + coombs rgt Indirect: detects antibody present in pts. Serum 1. antibody reacts with normal rbc antigens 2. pt serum + normal rbc +coombs rgt Serum CH50:

15-1 For each of the following mechanisms of immune tissue injury, list 2-3 human autoimmune diseases for which these provide the pathogenic basis for disease. Where possible, identify the auto-antigen and suggest a relevant therapy for each disease?

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a.

Antibody +/- complementmediated damage 1. autoimmune hemolytic anemia: antibody coats the RBCs and tehn Fc fragment of AB binds to the FcR on phagocytes. Phagocytosis can/cannot be complement mediated. Detected by Coombs test 2. autoimmune thrombocytopenia: The Ab is directed against the platelets. Same mechanism as 1. Marked drop in platelet count. 1. and 2. can be treated by blocking the Fc receptor using monomeric IgG. Give IVIG. 3. goodpasture syndrome: Auto Ab against the glomerular BM. Linear deposit pattern. Usually die of kidney failure because of rapid onset of anti-basement membrane AB. 4. pemphigus vulgaris: Skin disease from auto AB against epidermal cadherin (adhesion molecule). 5. neonatal lupus: Pregnant mother can pass on maternal autoantibodies (IgG) to fetus. Some develop heart block caused by SSA antibody.

b. Immune complex-induced injury 1. Systemic lupus: Anti-nuclear Ab (ANA). Phagocytosis of cells with damaged DNA. Deposits in kidney, joints, and skin. Ag-Ab complexes deposit at the junction of dermis and epidermis and glomerular basement membrane. 2. Mixed cryoglobulinemea: IgM-IgG (+/- hepatitis C) These complexes deposit in skin and cause vasculitis. They can then become necrotic. 3. Subacute bacterial endocarditis: bacterial Ag-Ab. Bacterial Ag-Ab deposits in heart valves and cause things like stenosis or regurgitation. c. Anti-receptor-mediated injury 1. Myasthenia gravis: Auto Ab to the Ach receptor in neuromuscular junction. Progressive muscle weakness due to lack of Ach reaching the receptors although enough is produced. 2. Graves disease: auto Ab stimulates TSH receptor by acting like TSH and serves as an agonist. Hyperthyroidism results. If pregnant during the disease, it will be passed on the fetus because IgG crosses placenta. 3. Insulin resistant diabetes: blocking AB against insulin receptor. Hyperglycemia results. Cant treat with coritcosteroids because it will make hyperglycemia worse.

d. Cell-mediated immune injury 1. Rheumatoid arthritis: (CD4, TNF, PG) IgG and rheumatoid factor (RF). Complement, cytokines, and inflammatory cells recruited. Destruction to synovial membrane, hypertrophy, and bone/cartilage damage. Formation of phagolysosomes which results in lysosomal enzymes being released injuring tissues. RF is Ab against Fc portion of IgG. So RF = anti IgM anti-Fc IgG. 2. Inuslin dependent DM: Pancreatic islet cell Ag. Islet cells destroyed and patient cannont make insulin. Bad. 3. Multiple sclerosis: (TH1, CD4, INF); myelin basic protein. Thats about all he said about this one. Sorry eh. 15-2 Describe the pathogenesis of the following specific autoimmune conditions: Coombs positive autoimmune hemolytic anemia: this one is covered in an earlier chapter objective, so why be redundant. Pernicious Anemia: In normal states, B-12 in diet binds to intrinsic factor (IF) made by parietal cells in stomach. This is necessary for absorption of B-12 in the ileum. In PA, patients develop auto Abs against own IF. They therefore cannot absorb B-12, and get anemic. Hashimotos thyroiditis: Patients are hypothyroidic because their tissue is being destroyed. Ab coat the thyroid cells recruiting cytotoxic T-cells which cause massive destruction. IDDM, Goodpasture disease, Rheumatoid arthiritis, Lupus, Myasthenia Gravis, Graves Disease are all covered above.