Copy of Immuno Review Questions Test 2.pdf

Lecture 11 Why do we need MHC? We need MHC so that the immune system can distinguish self from non-self and present antigens to T cells (Also called Human Leukocyte Antigen (HLA)) Major histocompatibility complex (MHC) pro...

Lecture 11 Why do we need MHC? We need MHC so that the immune system can distinguish self from non-self and present antigens to T cells (Also called Human Leukocyte Antigen (HLA)) Major histocompatibility complex (MHC) proteins: ○ Elicit strong immune responses ○ Major determinant of whether organ transplants are accepted or rejected Minor histocompatibility complex: ○ much less immunogenic, do not elicit antibodies ○ still involved in self/non-self recognition and important for graft vs host disease What do the 3 sub-loci of MHC genes encode? MHC genes located on chromosome 6 (more than 200 genes) Class I MHC genes ○ Present endogenous peptide to CD8 ○ Encode for classical: HLA-A, HLA-B, HLA-C ○ nonclassical : HLA-G in fetal cells Class II MHC genes ○ Present exogenous peptide to CD4 ○ Encode for classical: HLA-DP, HLA - DQ, HLA - DR ○ non-classical : HLA-DM and DO Class III MHC genes ○ Complement proteins and inflammation proteins (TNF-a) Define haplotype, codominance, polygenic, and polymorphism. Haplotype is a group of alleles that are inherited together from one parent ○ Allelic forms of MHC genes are inherited in liked groups called haplotypes MHC alleles are codominantly expressed, meaning both maternal and paternal proteins are expressed on each cell ○ Neither allele is recessive, and both phenotypes are expressed Polygenic means that the MHC proteins are the cumulative effect of many genes (ex. Height, weight, skin) ○ Not controlled by a single gene and does not follow mendelian inheritance patterns Polymorphic means that there is lots of diversity of the gene within the population ○ MHC is the most polymorphic region in the genome = HIGH AMOUNT OF DIVERSITY  ○ MHC polymorphisms provide a survival advantage to outbred species What are anchor residues? Anchor residues hold the peptide into the binding groove of the MHC pocket These are found on both MHC I and MHC II peptide interactions What is MHC restriction? MHC restriction occurs during T cell development in the thymus T cells only respond to antigen when presented by MHC Also called positive selection - allows for weeding out of T cells that are unable to recognize MHC What are the characteristics of MHC peptide binding? Only peptides have the structure to be presented by MHC proteins Broad specificity: Different peptides bind to same MHC molecule ○ Serial monogamy: Each MHC protein can only present one peptide at a time because of the binding groove Peptides added to MHC inside host cell (even though they may have come from somewhere else) Peptides and MHC have a stable interaction and very slow off-rate so T cells have time to see MHC complex Very small number of peptide-MHC complexes to activate specific T cell (few as hundred) On which cell types do you find MHC class I and MHC class II? Non professional APC can express MHC I ○ All nucleated cells Professional APC express MHC II all the time ○ DCs, macrophages, B cells How are the structures of MHC class I and II similar and different? Class I molecules ○ Has a single transmembrane domain ○ 45 kDa glycoprotein alpha chain ○ 12 kDa B2 microglobulin protein Class II molecules ○ Two transmembrane domains ○ Heterodimeric: 33kDa alpha chain and 30 kDa B chain Similarities: both member of Ig superfamily (Ig Fold), both have at least 1 transmembrane domain Which cytokine increases MHC expression? IFN-y and other inflammatory cytokines What is the rule of 8?  MHC I binds with CD8 T cells (1 x 8 = 8) MHC II binds with CD4 T cells (2 x 4 = 8) How do different types of immune cells and molecules recognize microbes in different locations? How do innate immune cells direct/respond to adaptive immune Describe the sequence of events (antigen processing pathway) by which class I and class II MHC molecules acquire antigens for display MHC Class I: Endogenous Pathway ○ Proteolysis of cytosolic proteins (digested by ubiquitin-proteosome pathway) ○ Peptides transported to ER by transporter associated with antigen processing (TAP), which is linked by Tapasin inside the ER ○ Transport of peptide-MHC complex to cell surface MHC Class II: Exogenous Pathway ○ Internalization and digestion of antigens in endosomes/lysosomes (digested by proteases that work best by acidic pH) can be inh by protease inh or basic stuff ○ Peptides bind to class II MHC molecules in endo/lyso vesicles Invariant chain (I) attached to new class II MHC Class II invariant chain peptide (CLIP) is what binds to MHC peptide binding groove HLA-DM (non-classical class II protein) exchanges clip for other proteins ○ Transport of peptide-MHC complex to cell surface List the functions of TAP, tapasin, CLIP, invariant chain, and HLA-DM, as well as which pathway they are associated with TAP- part of MHC Class I is what transports peptide to the ER Tapasin - Keeps Class I MHC and TAP linked Invariant chain- attaches to class II MHC CLIP- part of invariant chain that binds to MHC peptide binding groove HLA-DM- removes CLIP and and binds other peptides to MHC class II in endosome What is the role of MHC molecules in the recognition of infected host cells by NK cells? What is the physiologic significance of this recognition? Something to do with MHC Class I?? And self hypothesis?? Does this have to do with Bare Lymphocyte Syndrome Type I?? ○ Decreased/absent expression of MHC Class I on cell surface because of defects in TAP or tapasin which can cause low CD8+ T cells ○ This would cause decreased NK cell killing activity Define cross-presentation. Why is it significant? Cross presentation (or cross priming) is needed for cells that only express MHC I that need to elicit a cytotoxic response to an exogenous antigen ○ Exogenous antigen is usually presented on MHC II ○ Important for CD8 T cell responses without having to have an APC infected ○ Remember: only APC cells express MHC II, all nucleated cells express MHC I Which molecules present antigen to NKT cells? What type of antigen? NKT cells recognize lipids and glycolipids displayed by CD1 ○ Important for mycobacterial and fungal infections  CD1 is an MHC like molecule that loads the antigen via endosomal processing pathway but with different adaptor and chaperone proteins What is the role of MHC expression on B cells? B cells are APC so work best with effector T cells and must be activated to present Ag to T cell B cells also receive CD4 T cell help ○ Activated Helper T cells bind peptide-MHC complex on B cell to release cytokines (IL-4) to drive B cell functions ○ Can lead to clonal expansion and differentiation into plasma cell Therefore, B cells have MHC Class I and Class II How do superantigens bypass normal T cell activation? List examples of superantigens. Superantigans activate helper T cells by binding to the outside of TCR B chain and MHC Class II molecule ○ Instead of a small amt of T cells activated during a response, 20% of T cells can become activated ○ This induces a cytokine storm which can lead to Systemic Inflammatory Response Syndrome (SIRS). SIRS progresses to shock and multi-organ dysfunction Majority of superantigens are bacterial exotoxins, but there have been viral and fungal superantigens identified ○ Ex: Toxic shock syndrome, staphylococcal, clostridium enterotoxin Lecture 12 What are the functionally distinct domains (regions) of BCR and TCR molecules? ○ BCR molecules: membrane-bound antibodies associated with two signaling chains Antibodies are secreted forms of the BCR Ab molecules have two functional regions Two Fab regions - identical arms (specificity for aa) Two Stem Fc regions - mediates function ○ TCR: 2 chains (either ab or yd) Ab is more common Each chain has a variable and constant region Do NOT interact with free antigen (there is APC on MHC molecules) Compare and contrast BCR and TCR structure and function ○ BOTH are NOT germline encoded ○ BOTH are highly specific for individual regions ○ BOTH are assembled by rearrangement because of Variable regions during development in primary lymphoid organs VDJ recombination and changing order of genes to create diversity  Each gene segment is flanked by RSS (signal sequence, which direct RAG where to cut the DNA) and that is joined by RAG 1-2 Recombinase Each gene segment is flanked by RSS which direct RAG where to cut the DNA ○ BOTH have clones that have unique specificity Antibody - 10^11 specificities; TCR has 10^16 specificity ○ Effector T cells directly interact with target cells ○ TCR do NOT interact with free antigens ○ Different chains What types of antigens are recognized by BCR and TCR? ○ B cells – extracellular pathogens ○ T cells – intracellular pathogens Explain positive and negative selection, anergy, and clonal deletion. Where do these events occur? ○ For TCR positive selection is when thymocyte receptors are capable of binding MHC, which results in death by neglect (if they fail) or MHC restriction (if they pass). This occurs in the cortex. Negative selection of T cells also occurs in the medulla ○ Negative selection is when self antigens are highly bound to surface receptors, resulting in cell death. This occurs in the periphery for B cells and medulla for T cells. ○ Anergy is functional unresponsiveness. The receptor just has too low of an affinity for self antigen. ○ Clonal deletion is death by apoptosis What mechanisms contribute to the diversity of Ig molecules, and how? ○ Combinatorial diversity (generation of diversity by random formation of different VDL combinations) is limited by number of V,D,J segments (millions) ○ Junctional diversity (imprecise joining of gene segments and addition of DNA nucleotides) - almost unlimited (10^11 in B and 10^16 in T) 3 mechanisms of action Exonucleases - remove nucleotides P nucleotides - before breaks happen, there is template of where to place it via overhanging breaks of DNA at site of recombination Terminal deoxyribonucleotidyl transferase (Tdt) adds random nucleotides untemplated What is V(D)J recombination? What is its function? Where in the body does it occur, and in what cell types? ○ VDJ recomb is when cells use groups of parts of genes to create possible receptors. These are pieces of DNA being arranged to generate a functional variable region gene ○ Requires different genes on different chromosomes (alot of work)  ○ This is just to increase the diversity of receptors because there are not enough genes in the genome for every antigen receptor to be encoded by a separate gene. This occurs during B and T cell development when they are pro-lymphocytes(primary lymphoid organs: B- bone marrow; T- thymus) List events that occur during V(D)J recombination and which enzymes are necessary ○ VDJ is directed by receptor signal sequences (RSS) that are at the end of each AB gene segment. They are joined together by RAG 1-2 Recombinase ○ Tdt (terminal deoxynucleotidyl transferase) is a template independent enzyme that adds on nucleotides to the 3’ end of a DNA molecule (helps with junctional diversity) What is receptor editing? ○ Receptor editing occurs when the self antigen has too high of an affinity to the surface receptor, so the RAG genes are reactivated. This helps produce a new light chain and they are retested. If that fails, they will be clonally deleted. What are the checkpoints in lymphocyte development? What is their functional significance? ○ Checkpoints are just to make sure things are working properly (need to ensure that there is expression of functional antigen receptor) ○ The first checkpoint: signaling through the pre-B/T cell. If it is successful, heavy chain on B cells get turned off and beta chains on T cells get turned off. This results in allelic exclusion. Now light chains and alpha chains can undergo rearrangement. ○ Second checkpoint: The other chains are successful and signaling through the BCR/TCR means that it is successful. List steps of T cell development and where they occur in the thymus ○ VERY early thymocyte (HSC precursor) occurs in bone marrow. Then migrate to thymus. ○ In the cortex, they undergo Double negative, which is no CD4 or CD8 and it is where rearrangement occurs. It is also where they are committed to T cell lineage by the Notch receptor. ○ Then Double positive happens when CD4 AND CD8. ○ Positive/negative selection stages to become Single Positive: CD4 OR CD8. ○ In the medulla, negative selection occurs to remove autoreactive cells Tissue-restricted self-antigens are expressed in the thymus due to the action of autoimmune regulator (AIRE) ○ Mature Cells are eventually released into peripheral circulation List differences in B and T cell maturation ○ HSC commit to B and T linease ○ Progenitors proliferate ○ Antigen receptor genes are rearranged and expressed ○ Both have two checkpoints ○ Both undergo negative selection  ○ T cell- thymus; B cell - bone marrow ○ T cell- CD3, CD25; B cell- CD19/20 Lecture 13: CMI I What is the function/purpose of cell-mediated immunity? ○ An immune response that does NOT involve antibodies ○ CMI kills infected cells, kills cancerous cells, and can also reject transplant organs Which cell types are involved in cell-mediated immunity? ○ T cells: CD4+ (secrete cytokines), CD8+ (directly kill infected cells), NKT cells (lyse target cells) ○ NK cells (Kill target cells) ○ Macrophages (phagocytose and kill ingested microbes) What are ITAMs and ITIMs? What do they do? ○ ITAMs/ITIMs are motifs on receptors that regulate immune cells ○ ITAMs- activate signal on NK to kill infected cell and produce IFN-y ○ ITIMs- inhibitor signal on NK to not kill cell (remove phosphate) if they recognize a normal cell How are macrophages activated? ○ NK recognizes infected, damaged, or tumor cells. The receptors bind to killer immunoglobulin-like family (KIRs) Humoral immunity and CMI work together: CD16 binds IgG, which leads to antibody dependent cell-mediated cytotoxicity ○ Macrophages are activated by IFN-y from T cells. Kill ingested microbes Enhance Ag presentation by increasing expression of MHC Class II (CD80/CD86) List the phases of T cell mediated immune responses. ○ Antigen Presentation - APC present Ag to naive T cell ○ Activation - Recognize Ag as foreign and receive APC signals ○ Proliferation - clonal expansion ○ Differentiation - Become right kind of effector cell ○ Migration - get to site of infection  ○ Effector function - Apoptosis ○ Contraction - Homeostasis, response to diminish ○ Memory- Surviving cells leave antigen specific T cell What is the functional outcome of the 3 signal hypothesis? Why does your immune system do this? ○ Must receive 3 signals during activation, which is called VERIFICATION. Immune system does this because want to make sure T cells are responding to the correct antigen. ○ If all 3 signals are not received then either anergy (functional unresponsive) or very weak response will result List the 3 signals of the 3 signal hypothesis, the proteins involved in each signal, and on which cell they are found. ○ TCR binds to MHC-peptide complex. Needs CD4 or CD8 as coreceptor. This is first signal and the first event for adaptive response. Activation is triggered by coreceptors AND proteins part of TCR complex TCR, CD3, and zeta chain TCR binding to MHC-Ag complex causes conformational change in coreceptor Lck kinase on coreceptor phosphorylases CD3 and zeta chain ITAM domains Integrins stabilize APC-T cell interaction and facilitate T cell trafficking ○ 2nd signal = Costimulation can be activating or inhibitory Activating: CD80/86 on APC bind CD28 on T cell; or CD40 on APC/B cell binds to CD40L on T cell Inhibitory: CD80/86 on APC bind CTLA-4 on T cell; PD-L1 on APC binds to PD-1 on T cell CTLA-4 has higher affinity for CD80/CD86 at resting states, but when a foreign body is ingested, the expression of CD80/CD86 is increased, allowing the odds of CD28 (low affinity) to increase for binding ○ 3rd signal = Cytokines Secreted by the APC or that present in the environment so the T-cell can respond to it Ex: IL-12, IL-6, TGF-B Why are adhesion molecules necessary for cell activation? List the proteins involved and their locations.  ○ Adhesion molecules stabilize the APC-T cell interaction and help facilitate T cell trafficking Basically keep cells close together to allow time for activation signals Cytokine secretions finds this helpful because of vectorial redistribution (aims secretions towards targets), cytotoxic cells use this too LFA-1 (CD11a/CD18) on T cell binds to ICAM-1 (CD54) on APC VLA-4 on T cells binds to VCAM-1 on endothelium What is the immune synapse? What is its function? ○ Immune synapse is the point of interaction between the T cell and APC or target cell ○ TCRs, Integrins, and other proteins move through plasma membrane and localize to lipid rafts on activation ○ Also called Supramolecular Activation Clusters (SMACs) which help strengthen signals What are NK-B, NFAT, AP-1, Ras/Rac, and PI3-Akt? What do they have in common? ○ NK-B, NFAT, AP-1 are transcription factors that are activated through the signaling cascade in T cells ○ NF-kB, NFAT, and AP-1 leads to gene transcription for cytokines, proliferation, and differentiation ○ Ras/Rac are GTPases ○ PI3-Akt is a signaling pathway that leads to transcription of anti-apoptotic factors Explain the mechanism of action of cyclosporine and rapamycin ○ Cyclosporine is a drug that is meant to decrease cytokine production (IL-2 specifically). It binds to Calcineurin, which prevents the binding of Calcineurin (a phosphatase) to NFAT. Calcineurin dephosphorylates cytoplasmic NFAT (which activates it) ○ Rapamycin is a drug that is meant to bind to mTOR kinase, which would prevent protein production. This would decrease cell size and growth. This is also useful in graft rejection/organ transplants as an immunosuppressant. Explain the components and structure of the IL-2 receptor and how it changes during an immune response ○ IL-2 receptor is composed of IL-2Ra, IL-2RB, and yC. IL-2Ra (CD25) has no signaling function IL-2RB and yC (common gamma chain) have all the signaling capability Naive T cells have a low affinity IL-2R complex (NO ALPHA) ○ Naive T-cells express low affinity IL-2 Receptors. Activation of T cells causes IL2 genes to be secreted and expression of IL-2Ra chains. Addition of CD25 creates a high affinity  IL-2 Receptor. Activated T cells have a better shot at competing for IL-2 than Naive T cells. They are more likely to proliferate because better to bind to IL-2 causing clonal expansion to be induced. T-reg expresses high affinity IL-2Ra at rest, which causes more Tregs to be proliferated at rest from IL-2. A good thing because we do not need other T cells to be activated at rest. What are the main types of conventional T cells and their functions? ○ Helper T cells (CD4): Produce cytokines that help other cells differentiate and function efficiently. Binds to MHC Class II ○ Cytotoxic T cells (CD8): Kills altered self cells and produce IFN-y to activate macrophages and NK cells (IL-2) Binds to MHC Class I ○ Regulatory T cells: Immunosuppressive, turn off CMI, and express high levels of CD25 so they can out compete other T cells for IL-2 ○ NKT cells: produce cytokines and direct cytotoxic activity and control pathogens expressing lipid antigens Lecture 14 ​What are the major cell types and effector functions of cell mediated immunity? Are these functions performed directly or indirectly by effector cells? Major cell type = T cells Effector function ○ Promote immune response (humoral and cellular) ○ Killing pathogens (CD8 T cells are main but not only effector for cytolysis) ○ Regulating immune response Functions can be performed by T cells contact directly or mediated indirectly by secretion like cytokines What are the 3 distinguishing properties of Th subsets? Distinct polarizing cytokine set Master gene regulator Signature set of effector cytokines produced by each subset What are the main functions of each Th subset? (what are their jobs?) CD4 Th1 cells: main function is to activate macrophages with cytokine IFN-gamma CD4 Th2 cells: main function is to promote destruction of helminths (activate eosinophils) and inhibit classical macrophage activation (alternatively activate macrophages), basically opposite of Th1 CD4 Th17 cells: main function is to destroy extracellular bacteria and fungi by recruiting neutrophils and monocytes to sites of infection/inflammation T regulatory cells: main function is to regulate immune response / prevent autoimmunity (inactivate activate or self-reactive T cells & cytokine or cell mediated cytolysis) CD4 Tfh cells: main function is to provide cytokine signals to B cells during antigen presentation by follicular DC What are the functions of the effector cytokines of Th subsets? CD4 Th1: effector cytokines are INF-gamma, TNF → cell mediated immunity, macrophage activation, inflammation CD4 Th2: effector cytokines are IL-4, IL-5, IL-13 → allergic and anti-helminth responses CD4 Th17: effector cytokines are IL-17A, IL-17F, IL-22 → inflammation T reg: effector cytokines are IL-10, TGF-B → regulation, suppression of immune and inflammatory responses Tfh: effector cytokines are IL-4 and IL-21 → B cell help in germinal centers What is the immunological synapse? Why is it important in cell mediated immunity? Immunological synapse is a stable cell junction between a CTL and APC target cell Formation of the synapse depends on interaction between accessory molecules ○ LFA-1 (CTL) and ICAM-1 ○ CD8 (CTL) and MHC I This is important for cell mediated immunity because without interaction between accessory molecules in the synapse, the T cell activation process cannot begin What are the effector functions of NK and NKT cells? How are these cell types similar and different? NK cells don't recognize specific antigens, they recognize death receptors upregulated on infected or damaged host cells NKT cells are specialized subset of T cells with BOTH T cell and NK cell markers ○ They respond to lipid antigen presented by CD1 (an MHC class I like molecule) ○ T cell maker: CD3 ○ NK cell markers: CD56, CD16, NKG2D Similar because they are both involved in killing of damage/infected cells What are the pathways used by CTLs for killing target cells? Explain how each works. Cytolysis: Perforin/Granzyme pathway ○ CTL releases granule contents into immune synapse ○ Perforin induces uptake of granzymes into target cell endosome and release into cytosol, activating caspases Perforin is a pore forming protein Granzymes are serine proteases ○ Leads to apoptosis of target cell Cytolysis: Fas/FasL pathway ○ Activated CTL upregulate FasL expression ○ Fas expression is induced by cellular stress ○ Fas binds FasL, initiating a death signal and leading to apoptosis of target cell Fas (CD95) is receptor found on surface of target cells FasL (CD95L) is on surface of CTL ○ Results in apoptosis by activating the caspase cascade and cytochrome C pathways Cytolysis: TNF receptor family pathway ○ TNFa is made by activated CTL and NK cells ○ Binding of TNFa to its receptor on target cells triggers signaling cascade that activates caspases  TNFR family contains death domains (DD) that allow assembly of activating factors in apoptotic cascade Leads to cytochrome C release from mitochondria and increases Ca2+ which activates caspase ○ Target cell dies by apoptosis How are host cells protected from CTL? In perforin/granzyme pathway, CTLs have serpins (serine protease inhibitors) that protect them from granzymes and cathepsin B degrades perforin What is the role of macrophages in CMI? Macrophages kill intracellular pathogens using ROS and NO after phagocytosis Macrophages also amplify CMI responses ○ IL-12 from macrophages activates naive T cells ○ IFN-gamma from activated T cells increases killing capacity of macrophage ○ Establishes a feedback loop List the humoral mediators that participate in CMI C’ opsonization ○ C3b stuck to target binds CR, triggers phagocytosis of target by CMI effector cell Complement dependent cytotoxicity ○ C1 binds Fc portion of Ab ○ C1 binds CR on CMI effector cell, triggers cytolysis Ab opsonization ○ Fab portion of Ab binds to target ○ FcR on CMI binds to Ab Fc, triggers phagocytosis Antibody dependent cellular cytotoxicity (ADCC) ○ Fab portion of Ab binds to target ○ FcR on CMI cell binds to Ab Fc, triggers cytolysis What is the contraction phase? What is its function? The contraction phase is the decrease in lymphocytes after an infection is cleared Results in resolution of inflammation Contraction returns host response to homeostasis How are memory cells different from effector cells? What is their function? Memory cells can rapidly make Ag-specific responses upon re-exposure Memory t cells can live for years, are found throughout circulation and in tissues, and have different phenotypic markers compared to effector cells Which steps in T cell activation and function are targets for drug therapy? Cyclosporine inhibits calcineurin, blocking TCR signaling IL-2 receptor antibodies or antagonists prevent IL-2 signaling, no T cell growth JAK inhibitors block cytokine signaling TNFa inhibitors (Humira, adalimumab, etanercept) CTLA-4-Ig blocks CD80/CD86, no costimulation during Ag presentation Anti-CD3, anti-CD52 blocks MHC-TCR signaling or deplete T cells Azathioprine inhibits cell cycle progression, no T cell proliferation  Lecture 15 Define epitope, affinity, avidity, antigen, immunogen, hapten, T-dependent antigen, and T-independent antigen ○ Epitope- also known as antigenic determinant. It is a small piece of the ANTIGEN and where receptor/antibody binds. Can be conformational or linear ○ Affinity- Binding strength between antibody and antigen (epitope). Represented by Kd IgG has higher affinity ○ Avidity- Overall strength of antibody attachment, including number of binding sites. Antibodies with more binding sites (IgM has higher avidity because it has more binding sites) ○ Hapten- Small chemical or epitope (such as several drugs like Penicillin) that binds to antibody but antibody production is not elicited (NOT IMMUNOGEN) ○ Immunogen- antigens that generate an immune response. In order to become immunogenic, haptens must bind to a carrier proteins ○ T-dependent antigen - Require T cells to help B cells. Elicited by protein antigens and involve follicular B cells that reside in follicles of secondary lymphoid organs ○ T-independent antigen - do NOT require T cells to help B cells. Elicited by polysaccharide, lipid, and nucleic acid antigens. They involve B-1 B cells in mucosal tissues and peritoneum and marginal zone B cells in spleen Explain the structure of antibodies; include heavy chain, light chain, constant region, variable region, hinge region, Fab, Fc, hypervariable region, complementarity determining region ○ Antibodies have two functional regions (Fab and Fc) and shaped like a Y Fab regions bind to specific antigens on the tip of the fork of the Y. It contains both heavy and light chains, which have both a variable and constant region. Within the variable region, there is a hypervariable region, also known as complementarity determining regions, that form the antigen binding site. The Fc region interacts with complement components and Fc receptors on cells. It determines the antibody’s effector functions (what will happen after ab binds antigen) What’s the difference between linear and conformational epitopes? Which types of antigen receptors recognize which types of antigens? ○ Linear epitopes consist of continuous residues on a protein sequence ○ Conformational epitopes consist of residues that are discontinuous in protein sequence but come within close proximity to form an antigenic surface ○ B cells recognize native conformation and T cells recognize linear  What is a hapten? Give some examples ○ Hapten- Small molecules that only illicit an immune response when attached to a carrier protein Urushiol is a well known example that is found in poison ivy Commonly used in molecular biology applications: fluorescein, biotin, digoxigenin, and dinitrophenol. What defines a mature B cell? ○ Express IgM and IgD on cell surface What types of B cells respond to T-dependent antigens? Which types respond to T-independent antigens? Where can you find each type of B cell? ○ T cell independent regions B-1 B cells found in peritoneum and mucosa that make mostly IgM Marginal zone B cells found in white pulp of the spleen that is specialized for blood-borne Ag recognition ○ T cell dependent regions Follicular B cells of secondary lymphoid organs What signals are necessary for B cell activation? How are these signals different in T-dependent and T-independent antibody responses? ○ Signal 1: BCR signaling with BCR complex (BCR+Iga and IgB) Can be enhanced by CD2/CD21 coreceptor that recognize C3d bound to antigen or TLR5,7,9 that expressed by B cells that PAMPs recognize Induces some B an and secrete IgM ○ Signal 2: provided by helper T cells (CD40L on Th cell interacts with CD40 on B cell) X-linked Hyper IgM syndrome = mutations in CD40L Cause clonal expansion, differentiation, and antibody response to protein Ag B cells responses depend on signals in location (HUGE TABLE ON SLIDE 28) o T cell dependent antibody response: requires T cells to help B cells Elicited by protein antigen Involve follicular B cells Signal is provided by helper T cell o T cell independent antibody responses: do NOT require T cells to help B cells Elicited by polysaccharide, lipid, and nucleic acid antigens Signal 2 is provided by antigen itself ○ List the components of the BCR complex. Which proteins enhance BCR signaling without being part of the BCR complex?  ○ BCR signaling with BCR complex (BCR+Iga and IgB) Can be enhanced by CD2/CD21 coreceptor that recognize C3d bound to antigen TLR5,7,9 that expressed by B cells that PAMPs recognize What are the phenotypic changes and functional consequences of those changes that result from BCR signaling? ○ These effects are the same for both T cell independent and T cell dependent ○ Inc exp of CCR7 → Migration toward T cell zone ○ Inc exp of B7 costimulators → Enhanced ability to activate Th cells ○ Inc exp of T-cell cytokine receptor → Inc responsiveness to signals from Th cells ○ Inc exp of anti-apoptotic proteins → increased survival of B cells How do B cells present antigen to helper T cells? ○ Antigens bind to BCR and that is endocytosed. Protein Ag are processed and loaded onto class II MHC molecules and presented to helper T cells ○ They do not have be specific for the same epitope on the antigen, but just bind to same antigen Define somatic hypermutation, clonal selection, and affinity maturation. Explain how these processes contribute to the differences between primary and secondary immune responses. ○ This occurs in the germinal center ○ Somatic hypermutation is numerous random mutations (at V regions of heavy and light chains) that do not guarantee a higher affinity. ○ Clonal selection is when the cells expressing mutations with the higher affinity are selected and proliferated, and the cells with lower affinity mutations are eliminated ○ Somatic hypermutation and clonal selection produce affinity maturation. The more mutations that occur, the higher the affinity. This is why primary immune response takes longer and less antibodies are secreted because B cells are still determining the best mutations to clone. During the secondary response, the affinity is higher, and there are more memory cells that can be reactivated to produce antibodies, so the response is faster and stronger Explain somatic hypermutation. What is the key enzyme? ○ Somatic hypermutation is when the BCR locus undergoes extreme mutations in the CDRs ○ Activation induced deaminase (AID) is the key enzyme that turns cytosine to uracil, mutation is removed by BER enzymes, then DNA pol fills in gaps and creates point mutations What is class or isotype switching? How does it happen?  ○ Isotype determined by heavy chain constant regions ○ Switching is regulated by Th signals. CD40 signals lead to expression of AID ○ DNA recombination at Ig heavy chain gene locus AID generates dsDNA breaks at switch regions upstream Break is joined to another break (looks kinda like a hairpin loop) Cytokines determine which region undergo transcription ○ END RESULT: different Ab isotype that recognizes same Ag as original IgM What are the functions of plasma and memory B cells? Where can you find them? ○ Plasma cells: terminally differentiated B cells that are commuted to abundant antibody production. Large cells with highly developed ER and Golgi with no surface Ig Short lived: found in periphery during T-independent Long lived: found in bone marrow in T-dependent germinal center and maintained by BAFF cytokines that bind to BCMA ○ Memory B cells: produced during germinal center reaction and inherit all those changes Express surface Ig and don't secrete antibody Long lived and express high levels of Bcl-2 RAPID, HIGH AFFINITY, HIGH TITER response during secondary exposure What is antibody feedback? Which receptors are involved? ○ Feedback loop through Fc receptors. If there is an increase IgG secretion, antibody response will decrease ○ Ab-Ag complexes bind to BCR and FcyRIIB. It is the only inhibitory receptor. The ITIM domain is phosphorylated. This causes inhibition of BCR signaling and B cell activation How are T cell-dependent antibody responses different from T cell-independent antibody responses? ○ T dependent: Antigen is a protein Antibody has high level of isotype switching and affinity maturation Long lived plasma cells Secondary response is there due to memory B cells ○ T independent: Antigen is polymeric (polysaccharides, glycolipids and nucleic acids) Antibody has low level of isotype switching (mainly IgM) and barely any affinity maturation  Short lived plasma cells Secondary response is rare and only seem in some polysaccharide antigens ○ T dependent is better because independent does not get affinity maturation, somatic hypermutation, or long lived plasma cells Explain the structures of IgM and IgA; know what the J chain and secretory component do ○ IgM and IgA are both multimers (IgA is a dimer and IgM is a pentamer) ○ J chains holds the monomers together and is needed for mucosal transport ○ Cysteines in the Fc regions allow disulfide linkages to occur between monomers Understand the functions of specific antibody isotypes ○ IgG: most abundant serum Ig; monomer with 4 subclasses (Most versatile) Numerous effector functions and can exit vessels to enter tissues Opsonization of antigens for phagocytosis Activation of classical pathway ADCC Neonatal immunity Feedback inhibition of B cell activation Neutralization of microbes and toxins Longest lived because of FcRn (found in utero) ○ IgM: pentamer; ALWAYS first Ig produced during primary response; production begins at birth unless fetus has infection Found on surface of mature B cells Bloodstream infections Most efficient in triggering classical pathway ○ IgA: monomer in serum and dimer in secretions; Most abundant Ig produced (alot of mucosal tissue) Majority is dimer and produced by plasma cells in MALT Neutralizes toxins/viruses and interferes with attachment of microbes to host cells ○ IgE: Most stuck to Fc receptors on mast cells and basophils Acts as receptor and Ag binding triggers degranulation Fighting parasitic infections and allergic reactions  ○ IgD: barely know anything about this Found on surface of mature B cells Alternative mRNA splicing allows IgM and IgD to have same antigen specificity Development and maturation of antibody response Lecture 16 What are regional immune systems? List examples Regional immune systems serve specialized functions at particular anatomic locations, may have specialized cell types not present in other locations (e.g., Langerhans cells, M cells, Kupffer cells) They protect the body against pathogens and prevent unwanted immune responses Major regional immune systems ○ Mucosal ○ Cutaneous What are the functions of cytokine secreting epithelial cells, goblet cells, M cells, and Paneth cells? Where can you find each of these cells? Cytokine secreting epithelial cells ○ Cytokines recruit the necessary innate or adaptive cells to fight off pathogen??? ^not sure about this one Goblet cells ○ Make mucins (highly glycosylated proteins) which make up mucus forms a viscous barrier Paneth cells ○ Secrete defensins which are antimicrobial peptides and REGIII (c-type lectin) M cells ○ Microfold cells are specialized epithelium cells that are a major pathway for antigen delivery from gut lumen to GALT (without going through lymphatics) What are mucins and defensins? Mucins make up mucous and keep microbes away from epithelium and provide matrix for displaying antimicrobial substances ○ Most of layer is made of MUC2 ○ Membrane bound mucins + glycolipids from glycocalyx ○ Replaced every 6-12 hours  Defensins are small antimicrobial peptides that inset and disrupt outer membranes of pathogens to kill them (innate immune protection) What are the functions of innate lymphoid cells in the lamina propria and intestinal dendritic cells? ILCs contribute to defense against bacteria and parasites (mostly found in mucosal tissue); activated by alarmins which are released by epithelial cells in response to injury or infection ○ IL-17 promotes inflammation, IL-5 activates eosinophils to fight helminths, IL-13 increases mucus production ○ Promote epithelial barrier function (IL-17 and IL-22 stimulate defensin production and function of tight junction) Intestinal dendritic cells ○ Can extend dendrites between epithelial cells into gut lumen to capture antigen ○ Can also capture antigen that has crossed epithelial barrier into lamina propria ○ Processes and transport antigens via lymphatics to mesenteric lymph nodes for presentation to naive T cells Where are adaptive immune responses initiated in the GI tract? GI sites where adaptive immune responses are initiated ○ GALT (most important is peyer's patches in ileum) ○ Mesenteric lymph nodes What is the importance of IgA in mucosal immunity? IgA is the most abundant antibody isotype in body (not blood) Large number of IgA producing plasma cells in GALT IgA neutralizes microbes and toxins Explain how IgA is transported through the epithelium. IgA is made by plasma cells in lamina propria J chain of IgA binds to poly-IgR on basal side of epithelium Transcytosis: IgA + receptor complex is transported across epithelium Bound IgA is released into gut lumen by proteolytic cleavage Explain how maternal antibodies are transferred to the fetus and neonate. Before birth ○ Maternal IgG transported across placenta via FcRn into fetal blood After birth ○ Secretory IgA produced in lymphoid tissue in mammary glands  ○ Transcytosed into breast milk, ingested by breastfeeding infant ○ Smaller amount of IgG and IgM also secreted in breast milk What are the functions of intraepithelial lymphocytes and lamina propria T cells? Intraepithelial lymphocytes (IELs) ○ Mostly CD8 T cells, limited diversity of receptors ○ Can recognize and kill virus infected, damaged, or stressed mucosal cells Lamina propria T cells ○ Mostly CD4 T cells ○ Naive, activated effector, and memory T cells ○ Promote inflammation, epithelial barrier function, IgA responses What are the functions of Th17, Th2, and Tregs in the GI tract? Which cytokines are vital for these functions? Th17 cells ○ Abundant in intestinal mucosa ○ IL-17 and IL-22 induce expression of mucins and B-defensins ○ IL-17 induces cells to make chemokines and cytokines that recruit neutrophils and monocytes, leading to inflammation ○ Associated with some inflammatory diseases Tregs ○ Regulatory T cells (CD4+FoxP3+) ○ Abundant in intestinal mucosa ○ Suppress immune responses by producing IL-10 and TGF-B ○ Protect from inappropriate responses to commensal microbes Th2 cells ○ Key defense against intestinal helminths ○ Main cytokine produced are IL-4, IL-5, IL-13 IL4 simulates class switching to IgE, also growth factor for Th2 IL-4 and IL-13 increase peristalsis and eosinophil recruitment IL-13 increases mucus secretion IL-5 activates eosinophils How do mucosal pathogens exploit mucosal immunity to cause infection?  Some pathogens use M cells to cross epithelial barrier (poliovirus, some retrovirus, salmonella, shigella) Some pathogens directly infect epithelial cells, some use DCs to cross epithelium Some pathogens evade humoral immunity ○ Antigenic variation to evade Ab binding ○ IgA proteases ○ Mechanisms to hinder phagocytosis and complement activation What is tolerance? Systemic unresponsiveness to ingested/inhaled antigens Prevents inappropriate immune response to food antigens and commensal microbes Likely involves ○ Mechanisms of peripheral tolerance (deletion, anergy, Tregs) Can be induced with desensitization What are allergies? How do they relate to tolerance? Initial exposure to potential allergens occur during fetal development Environmental factors may hinder development of tolerance ○ hygiene hypothesis - the problem with extremely clean environments is that they fail to provide the necessary exposure to germs required to “educate” the immune system so it can learn to launch its defense responses to infectious organisms. Instead, its defense responses end up being misdirected that they actually contribute to the development of asthma or allergies Food allergies caused by Th2 dependent IgE mediated response to allergens ○ Activation of mast cells in intestine What are the functions of surfactant? Which cell types make it? Surfactant proteins (SP-A, SP-D) bind microbes and suppress inflammatory responses Surfactant helps with gas exchange by decreasing the surface tension of water and keeping alveoli open Produced by type II pneumocytes Explain the immune dysfunction in HIV/AIDS, allergies, IBD, celiac disease, and MALT lymphoma. HIV/AIDS ○ Cause by HIV virus, which infects and kills CD4+ T cells Loss of CD4 T cells causes immunodeficiency ○ Early after infection, there is loss of GI mucosal integrity (leaky gut syndrome)  ○ Sets up an immune response that allows more T cells to come so that it can infect more uninfected T cells ○ At sites of entry through mucosal epithelium HIV envelope protein gp120 disrupts epithelium barrier HIV infects and kills CD4 T cells Allergies ○ Food allergies caused by Th2 dependent IgE mediated response to allergens Activation of mast cells in intestine – nausea, vomiting, diarrhea, abdominal pain ○ Can also see swelling of throat and difficulty breathing ○ Environmental factors may hinder development of tolerance ○ Without tolerance, there is “inappropriate” immune responses to food antigens or other antigens the body should not be reacting to IBD ○ Dysregulated response to commensal bacteria – abdominal pain, diarrhea, weight loss ○ Defects in innate immunity to gut commensals ○ Abnormal Th1 and Th17 responses ○ Defective Treg function ○ Ulcerative colitis vs Chrons Disease Ulcerative colitis restricted to colonic mucosa, more likely to have bloody stool Chrohn’s disease affects entire thickness of wall along entire GI tract Celiac disease ○ Chronic inflammation in small intestinal mucosa villus atrophy, malabsorption, nutritional deficiencies ○ Patients present with diarrhea or signs and sxs of malabsorption ○ Immune responses against gluten ○ Autoantibodies against gluten and transglutaminase 2A MALT lymphomas ○ Prolonged immune responses to GI microbes can lead to GI tumors ○ Gastric MALT lymphoma caused by H.pylori infection → leads to lymphoproliferation without Ag stimulus How are the respiratory and genitourinary regional immune systems similar to and different from the GI tract? Innate respiratory mucosal immunity ○ Commensal organisms more limited than in GI tract ○ Ciliated columnar epithelium has tight junctions and secretes mucus (similar)  ○ Immune responses in alveoli are controlled to preserve gas exchange (surfactant proteins) ○ Alveolar macrophages Anti-inflammatory, poorly phagocytic Inhibit T cell responses and DC Ag presentation Genitourinary mucosal immunity ○ Similar to other mucosal surfaces Mucus producing cells, normal flora, production of antimicrobial peptides, IELs, regional DCs, macrophages, T and B cells ○ Different Multi-layered epithelium (stratified squamous) Lacks MALT Little IgA, mostly IgG Lecture 17 What are the general roles of innate and adaptive immunity in immune responses? Think about what they do, types of pathogens, principal cell types and functions. ○ Innate immunity - quicker response, non-specific, no memory, always on and constant response, preformed components, germline receptors Phagocytes, granulocytes, etc. ○ Adaptive immunity - longer response, Diverse and specific, memory, faster and stronger every exposure, develops throughout life, antigen specific receptors; antibodies T and B lymphocytes Which innate and adaptive cell types are involved in immune responses to pathogens? Which cell types are most important for each type of pathogen? Bacteria Innate immunity to extracellular bacteria ○ Complement activation ○ Phagocytosis (neutrophils and macrophages) PRR recognize PAMPs ○ Inflammatory response DC and phagocytic cells secrete proinflammatory cytokines ILCs secrete IL-17, IL-22, and GM-CSF Adaptive immunity to extracellular bacteria ○ Humoral immunity is major protective immune response ○ Ab responses directed against cell wall antigens and toxins  ○ Spleen is essential to Ab response to polysaccharide Ag (encapsulated bacteria) ○ Ab effector mechanisms Neutralization (IgG, IgM, IgA) Opsonization and phagocytosis Complement activation (IgG, IgM) ○ CD4+ T cells contribute to immune responses ○ Cytokine secretion Plasma cell differentiation: IL-4 Isotype switching: IFN-y, IL-4 ○ Th17 recruits neutrophils to help promote inflammation Innate immunity to intracellular bacteria ○ Mediated mostly by phagocytes and NK cells First neutrophils, then macs and DCs NK cells recognize and kill infected cells ○ Amplification loop for activation Macs and DCs secrete IL-12 and IL-15 which activate NK cells NK cells secrete IFN-y which activates macs Adaptive immunity to intracellular bacteria ○ T cell mediated recruitment and activation of phagocytes ○ Th1 cells ○ CD8+ T cells ○ Amplification loop for activation Fungi Innate immunity ○ Phagocytes bind PAMPs using TLR and dectin PRR (lectin family) Binding leads to production of Th17 inducing cytokines ○ ILC makes IL-17 triggers IL-8 recruits neutrophils ○ Complement mediated opsonization Adaptive immunity ○ CD4 cells make cytokines for CD8 and B cell functions ○ Th17 cells make IL-17 ○ Ab neutralize, activate complement, act as opsonin ○ CD8 make IFNy and IL-17 Viruses Innate immunity ○ Responses aimed to block infection and eliminate infected cells ○ Type I IFN made by DC and infected cells: inhibit viral replication and spread ○ NK cells kill infected cells and secrete IFN-y Adaptive immunity ○ Antibodies: block virus binding and entry into host, Ab dependent cellular cytotoxicity ○ Th1 cytokines enhance CD8 killing activity ○ CTLs kill infected cells ○ Amplification of NK cell and Mac response through IFN-y Parasites Innate immunity to parasites  ○ Macs and PMNs activated by PRR binding ○ Direct phagocytosis for smaller parasites ○ Degranulation for larger parasites eosinophils important ○ Cytokines (proinflammatory like IFN-y) ○ Complement (all 3 pathways can be activated) ○ Innate immunity alone has little impact on clearance Adaptive depends on whether its extracellular or intracellular Intracellular parasites ○ Cell mediated immunity (mac activation by Th1) ○ CTL and Ab response similar to cytopathic virus ○ NK cells and gamma delta T cells kill infected cells Extracellular pathogen ○ Th2 activation production of IgE and eosinophils ○ Clearance requires cell mediated response (eosinophils are hallmark) ○ Ab can contribute to pathogen killing OR spread of infection How do encapsulated bacteria evade host immune responses? What is the most effective way for host immunity to respond to these pathogens? List pathogens. ○ Ab against polysaccharide Ag (encapsulated bacteria) are T- independent antigens that elicit antibody response but do not activate T cells. Humoral immunity is important, especially in the spleen. ○ Antiphagocytic and resistant to complement ○ Immune system kills with opsonins and phagocytosis ○ Examples: S. pneumonia, Neisseria spp., H influenzae, N. meningitidis, and C. neoformans How do toxins cause disease? What is the most effective way for host immunity to respond to toxins? List pathogens that produce toxins. ○ Toxins damage mucosa, are toxic to neutrophils or can act as a superantigen Ex: C. diptheriae, C. tetani, S. aureus, Streptococcus spp. ○ Ab responses and humoral immunity is the most effective way Because you’re damaging your innate immune system? List the injurious effects of immune responses to pathogens. ○ Lymphocytic choriomeningitis virus (LCMV) Virus infects meningeal cells but is not cytopathic CTL kill infected meningeal cells Immune response leads to meningitis ○ Hepatitis B virus (HBV) Circulating immune complexes lead to systemic vasculitis  Molecular mimicry leads to autoimmunity Hepatitis cause by CTL killing hepatocytes ○ Other examples of immunopathology Viral prodrome (headache, fever, malaise) Rash and arthralgias of parvovirus B19 Cytokine storm of pandemic influenza Explain antigenic variation and how it helps pathogens evade host immune responses. ○ Alterations to a protein or carbohydrate on the surface of the pathogen to help evade the host immune response ○ Ex: changing genes for pili helps bacteria escape antibodies What are antigenic drift and antigenic shift? ○ Antigenic drift and shift are when the antigens change and antibodies are no longer protective Antigenic drift is smaller changes or mutations in genes of the virus that accumulate over time Antigenic shift happens when there is swapping of genomic segments or reassortment that causes a new virus Explain the host immune response to Mycobacterium tuberculosis infection ○ Mycobacteria express glycolipids that facilitate binding and endocytosis by macrophages NKT cells recognize glycolipid Ag presented on CD1 TLR2 binds cell wall components ○ Mycobacteria prevent fusion of phagosome with lysosome to set up active infection ○ Immune response works to clear active infection or control latent infection through granuloma Active infection: CD8, NK, and NKT cells kill infected macs Latent infection: granuloma formed by infected, uninfected, and foamy macs, surrounded by Th1 and CD8 T cells IFN-y, CD4, CD8, TNF, and RNS required to maintain latency Describe the patient presentation for thrush. ○ Thrush can present on tongue (oral thrush) from the result of a fungal infection like candida albicans ○ Usually seen in immunocompromised patients  Lecture 18 Define active, passive, natural and artificial immunity. Give examples of each ○ Active- host is making the immune response (antibodies); more reliable Getting COVID-19 from your friend ○ Passive- host is receiving the antibodies; only last until they are physically lost; can receive treatment before or after exposure No memory in passive immunity Baby getting antibodies from mom in utero ○ Natural- host is exposed to the antigen and starts making antibodies Getting an infection (active) or getting antibodies across placenta or breast milk (passive) ○ Artificial- Injection or external source Vaccination (active) or rabies, anti-venom/toxic (passive) Explain how maternal IgG is transmitted to a fetus and maternal Ig is transmitted to a neonate. You should understand the receptors involved and how they work. ○ Transplacental - Mom to fetus; FcRn is located on syncytiotrophoblast and transports maternal IgG (longest half life) and is endocytosed across into fetal endothelium ○ Colostrum- Mom to infant; IgA (J chain) binds to Poly-Ig receptor and that complex is transported across the epithelial cell. The bound IgA is still attached the secretory complex in order to protect against pathogens Smaller amounts of IgG and IgM can also be secreted into breast milk ○ FcRn- neonatal Fc receptor which is located in the placenta and the intestine What can artificial passive immunity be used to treat? How does it protect patients in these situations? ○ When there is no previous exposure to antigens or after exposure ○ Before exposure: vaccines to prevent disease to at-risk individuals or just anybody ○ After exposure: Not enough time to make antibodies so you get premade antibodies, such as anti-venom/rabies vaccine. IVIG is a pool of antibodies from numerous donors List advantages and disadvantages of passive and active immunity. Know when you would use each.  ○ Passive- No memory, preformed response; short lived; Immediate protection; Less immunity with more doses; protect immunodeficient people NO B OR T CELL ACTIVATION ○ Active- Memory; developed by exposure or immunization; make own antibodies; long lived; better defense with more exposure; not as helpful in protecting immunodeficient people B AND T CELL RESPONSE How does vaccination confer protection? (What does it do to your immune system?) ○ Prepare pathogens to induce active immunity. It can directly protect the individual or prevent spread in the population indirectly ○ Measure response by level of antibodies in blood What does prime-boost mean? What is the goal of this vaccination strategy? ○ Repeat the exposure (by infection or immunization) to increase the response time and magnitude of protective immunity Kind of similar to natural selection and you select the most efficient memory cells in order to boost your antibodies (affinity maturation) What are the advantages to the Salk and Sabin polio vaccines? When is each used? ○ Salk - inactivated; injection; good serum IgG, IgM, and IgA NO MUCOSAL IgA Given to people who do NOT live in endemic regions ○ Sabin- live-attenuated; oral; good serum IgG, IgM, and IgA AND GOOD MUCOSAL IgA in nasal and duodenal Hence oral = better mucosal IgA Pathogen can replicate and better for GI mucosa. Given to people Polio ENDEMIC regions to prevent fecal-oral transmission List factors to consider when deciding if a vaccine is appropriate for a patient, and understand how each factor can affect the immune response. ○ Must be sufficiently immunocompetent to generate the immune response and want to limit the replication and spread for the live-attenuated vaccines ○ Age: infants (immature immune) and Seniors (immunosenescence) ○ Pregnancy: Some can boost maternal immunity and protect infant (tetanus, pertussis, RSV) and some can be harmful (live-attenuated vaccines) ○ Immunosuppression (stress, malnutrition, chemotherapy, infection, etc.)  Is their immune response strong enough to spread attenuated pathogen ○ Allergies (eggs, antibiotics, yeast, preservatives (thimerosal)) Some vaccine strands are raised in eggs, if you have egg allergy you have to be careful ○ If travelling: Where and when is important Is the patient traveling to where something is endemic and needs protection? When are they going? Have to give enough time to mount immune response (usually at least 2 weeks before you leave) Define herd immunity and understand how vaccines relate to it ○ INDIRECT protection of unimmunized people ○ You try and stop the chain of infection to prevent spread and occurs when a large enough % of population become immune ○ Goal of mass immunizations (smallpox is only disease successfully eradicated) How do we know that vaccines are effective? ○ Efficacy - direct protection to vaccinated people measured by CLINICAL TRIAL (controlled environment) % reduction in spread comparing vaccinated and unvaccinated ○ Effectiveness - direct protection to vaccinated people measured in a FIELD STUDY (population) ○ From Dr. Morrow: “Math. If vaccines were not effective, the disease rates should show no differences between vaccinated and unvaccinated populations” What happens when vaccination rates decline? ○ Vaccine rates go down, you get outbreaks (measles recently in NYC) List some of the things that can cause vaccines to fail ○ Give to right group ○ Age, immune status Immunocompromised (pregnant, elderly, HIV, etc.) prefer passive immunization ○ Improper storage, timing, administration, antigen mismatch, extrinsic factors, toxicity Define antigen, stabilizers, adjuvants and explain their functions in vaccines ○ Antigen - comes from pathogen that is recognized by immune system (dead, alive, part, whole, etc.) ○ Stabilizers - Keep the vaccine effective during storage by stabilizing factors (pH, temp, etc.) ○ Adjuvants- enhance the immune response to an antigen but is not immunogenic  Enhance delivery Slow down absorption and make it last longer Increase antigen content and chaperone to prevent degradation Potentiation of immunity Set off danger signals (DAMPs/PAMPs) and set up innate response Types of adjuvants Inorganic- no carbon involved (aluminum salts, calcium phosphate OH) Organic- have carbon (squalene, saponin, Quil A) Immunologic/Biologic - things you find in immune response (TLR agonists, cytokines) TWO APPROVED: aluminum phosphate and squalene (cholesterol synthesis) ○ Antibiotics - prevent bacterial contamination during production ○ Preservatives- prevent bacterial/fungal growth in multi dose vaccines List advantages and disadvantages of attenuated and inactivated vaccines ○ Live-attenuated: weakened form of pathogen, CAN REPLICATE, single dose, immunize others, CMI response; require refrigeration, can cause disease in immunosuppressed, pathogenic reversion; IgG and MUCOSAL IgA (if oral/nasal) MMR, Sabin, varicella, yellow fever, smallpox, rotavirus, flu-mist ○ Inactivated: CANNOT REPLICATE, no infections or reversions; no amplification; booster is required; require adjuvant; no at-risk populations IgG A LOT of inactivated (injected influenza, Salk, typhoid, cholera, pertussis) Define and give examples of types of inactivated vaccines ○ Inactivated whole agent vaccines Influenza, rabies, Salk ○ Toxoids: inactivated toxin (make Abs) Diphtheria, tetanus ○ Subunit vaccines: Part of agent to make Abs Acellular (less side effects) pertussis vaccine ○ Recombinant vaccines: genetically engineered organism to make antigen  HepB vaccine ○ Virus-like particle (VLP) vaccine: empty capsids produced by genetically modified organisms HPV vaccine ○ Polysaccharide vaccines made from capsules Adult pneumococcal vaccine ○ Conjugate vaccine: Make T-dependent antibody response to T-independent antigens Polysaccharides (encapsulated bacteria) and attach to protein so T cells recognize proteins and B cell/Ab recognized capsule Hib vaccine (Haemophilus influenza) ← ear infection, has helped decrease cases of Hib meningitis in children S. pneumonia Understand the targets (antigens) of some common bacterial vaccines ○ Toxins and attachments factors such as flagella, pilli, fimbriae) and T-dependent antigens ○ Polysaccharides capsules are T-independent (IgM) and not strong responses ○ SO Combine Polysaccharide with the protein to elicit a strong response with the use of affinity maturation (somatic hypermutation + clonal selection) Why don’t we have vaccines for parasites? ○ Most parasitic infections happen in poorer countries (NO FUNDING for vaccine) ○ Parasites are super complex (like human cells); switch hotels, larger, different life cycles ○ Mass drug administration is easier (give them drugs) What are combination vaccines? Give an example. ○ Multiple antigens are combined (nice because one shot for multiple vaccines) ○ MMR is a good example (Pro-quad) ○ Influenza B and HepB (Comvax) List and be able to explain some of the controversies/objections about vaccines ○ Thimerosal - preservative in influenza and DTP vaccines; more thimerosal in a can of tuna Metabolizes to ethylmercury which clears body quickly ○ Vaccine failures, cautions, and recalls Rotashield in 1999 due to 99 children getting intussusception Smallpox in early 2000s with cardiac complications  SKB pulls LYMERix in 2002 because adverse reaction causing arthralgia? ○ Ethics- allocating influenza vaccine when short supply ○ Vaccine overload- too many vaccines Causes excess antigens and weakens immune system or misdirected immune response ○ Hygiene hypothesis Increased rates of asthma/allergies because immune systems are too good so they overreact to substances (in developed countries) ○ Fraudulent, evil pharma companies push unnecessary vaccines Lecture 19 What are the 3 main cell types in a CBC? What indicates anemia, infection, or clotting problems? ○ RBC (erythroctes): Anemia (less hematocrit, Hct) ○ WBC (leukocytes): infection (viral- lymphocytes; bacterial- neutrophils; asthma/allergies/parastitic - eosinophils) ○ Platelets: Coagulation (Thrombocytopenia (low plt) → BM problem or peripheral consumption → decreased production or increased breakdown) What is the difference between positive APP and negative APP? Give examples of each ○ Acute phase proteins - APP ○ Positive means acute inflammation (increased synthesis) CRP, serum amyloid A protein, Haptoglobin, fibrinogin ○ Negative means chronic inflammation (decreased synthesis) Albumin and transferrin What is the clinical importance of CRP? ○ C reactive protein is the most popular acute phase protein It increases at the fastest rate when inflammation occurs and decreases quickly after resolution  Detect occult (HIDDEN) infections (bacterial typically) and assess autoimmune disease activity (rarely elevated over longs period of times) without continued inflammation List the 5 major fractions of serum electrophoresis, and explain which proteins are found in each fraction. How do the typical shapes of the peaks change in acute inflammation, polyclonal gammopathy, and monoclonal gammopathy? What are the causes of these changes? ○ Larger band = more protein; taller size; ○ First region (one peak): Albumin Hyperalbuminemia- dehydration Hypoalbuminemia - increase in other proteins because of decrease liver production (antibodies), protein loss (Ascites), or inflammation ○ Second region (2 peaks): alpha globulins APP typical of acute inflammation A1: thyroid A2: haptoglobin (increased in acute phase) ○ Third region (2 peaks): beta globulins Transferrin, IgA, IgM Beta 1: transferrin Beta 2: beta like proteins ○ Fourth region (1 peak): gamma globulins Mainly IgG Large wide peak: Polyclonal gammopathy; infections M-SPIKE: Narrow peak: monoclonal gammopathy; multiple myeloma (cannot diagnose) ○ CRP located between beta and gamma globulins Define direct and indirect tests. What are each of them trying to detect? ○ Direct- looks for ANTIGEN ○ Indirect- looks for ANTIBODIES (serology); pathogen may not be there but antibodies indicate that it has been in the past What are the differences between polyclonal and monoclonal antibodies? ○ Monoclonal antibodies- Constant, renewable source; less background; homogeneity (same results multiple times); Specificity = extremely efficient; too specific = one epitope, Come from 1 clone; made in a lab  ○ Polyclonal antibodies- Inexpensive; High affinity; Multiple epitopes; denatured protein detection; higher tolerance; too much variability per batch; high background because non-specific; check for cross reactivity (multiple epitopes), Come from multiple B cells; normal response to infection Explain how monoclonal antibodies are produced. ○ Give animal antigen to produce antibodies ○ Collect B cells from spleen of immunized animal ○ Prepare myeloma cells for fused together with spleen cells to produce hybridomas with polyethylene glycol (PEG); immortal cell that keep dividing (cancer characteristic) ○ Screen clones and pick the specific one ○ Confirm and characterize through ELISA ○ Clonal expansion How does agglutination work? How can this be used to determine blood type? ○ Agglutination - detect Ab in patient’s serum or detect Ag ○ Indirect: Ag coated to latex bead and search for ANTIBODY ○ Direct: Ab is coated to latex bead and search for ANTIGEN ○ Hemagglutination (HA) used as DIRECT test for antigen (blood typing) or INDIRECT test for antibodies (pathogens) Blood type: clumps form when coated antibodies bind to surface antigens Anti-A sticks to antigen A = blood type A; Explain how ELISAs work. What can these assays detect? ○ ELISAs look for antibodies and antigen; Antibody detection: antigen on plate and then sample is added Antigen detection: antibody on plate and then sample is added Color change/fluorescence will be measured because an enzyme will act on an indicator to produce the signal/fluorescence You should be able to explain how a pregnancy test works. ○ Also called lateral flow (Snap) immunoassays ○ Antibody (anti-hCG with a dye) is on the plate and detecting the ANTIGEN. ○ Urine contains hCG if pregnant. And moves along plate through capillary action ○ First passes through mobile antibodies (thin strip with dye) and hCG will bind and move along ○ There is a second area containing anti-hCG antibodies that are immobile.  ○ Antibodies are stuck and hCG specific and they bind to antibodies and dye accumulates and becomes visible IF PREGNANT ○ Excess antibodies are on control zone and they just see if test worked properly How does Western blotting work? What is this assay used for? ○ Detect specific proteins and the higher the signal, the more proteins ○ Proteins separated through gel electrophoresis then put on a membrane for antibody specific detection of target protein Explain how flow cytometry/FACS works. What is special about this technique? ○ Used for immunophenotyping of cell suspensions Tells you what cells are present and what type Count and sort cell into different populations Antibodies have tags so each cell population you’re looking for has unique tag and cells go one a time through a laser to detect fluorescence FACS is the sorting process ○ It can show thymocyte progression by telling you what stage the T cell is in. Double negative to Double positive to single positive How does PCR work? What do the 2 different kinds of PCR detect? ○ PCR detects DNA ○ RT-PCR detects RNA (converts it to DNA) ○ Works by amplifying DNA ○ Heat the sample to denature, then Taq polymerase builds two new strands; Denature → Annealing → Elongation ○ Detect genetic material from pathogens before patients get sick It is sensitive and relies on primers. Can only find what you are looking for. List the methods used to isolate leukocyte subpopulations and the tests for leukocyte function. Understand how each of them work (which cell types they’re used to test and what results mean) ○ ISOLATE SUBPOPULATIONS ○ Density centrifugation (Ficoll/Percoll tubes) Separate cells based on density and take the portion you are interested with a pipette ○ FACS ○ Magnetic- activated cell sorted (MACS) Ab attaches to magnetic beads (similar to FACS except magnets not fluorescence) ○ Buoyancy-activated cell sorting (BACS) Ab attaches to glass microbubbles (bubbles float to cell surface) Leukocyte function TEST ○ DCF-DA assay: tests for neutrophils Measure oxidative burst of neutrophils (similar to NBT test) Oxidize DCF to fluorescent ○ Phagocytosis assay: neutrophils, monocytes, macrophages Phagocytic cells are incubated with fluorescent beads to see how many beads are ingested ○ Cytokine profiles: monocytes, macrophages, lymphocytes Cytokines can be trapped in cell and run through flow cytometer Stimulate lymphocytes to see what cytokines are produced Secreted cytokines can be measured with ELISA ○ Mitogen (cell proliferation) asay: lymphocytes Measures lymphocytes ability to proliferate by stimulate with mitogen to undergo MAPK pathway Checks clonal expansion during immune response ○ Cytotoxic assay (caspase activation): lymphocytes Checks the amount of apoptotic cells in sample by amount of AMC produced (increase AMC = increase apoptosis)