Lecture 1 Flashcards

Question

Describe the TLR Signalling Pathway.

Answer 1

OVERVIEW: TLR binds PAMP > creation of TIR complex > Kinase cascade degrades IkB > NKfB binds to promoters > release of inflammatory cytokines 1. TIR domain of TLRs helps to transduce an intracellular signal by interacting with MyD88 (an essential signaling adaptor for many TLR signalling events; also has its own TIR domain) 2. homotypic interactions of the two TIR regions lead to creation of a complex Complex will cause a kinase cascade that results in degradation of IkB, which inhibits NFkB 3. Once no longer inhibited, NFkB will bind to promoters and turn on gene expression, leading to release of inflammatory cytokines

Answer 2

Both have innate immunity BUT flies also have embryonic development Mammals have TLR1-12 while Flies only have Tolls Mammals have adaptor MyD88 while Flies have dMyD88 Mammals have inhibitor IkB while Flies have cactus Mammals have transcription factor NFkB (p60/p65) while Flies have Dif/Dorsal The target genes of mammals are inflammatory cytokines, antimicrobial products, etc ;; while those of flies are antimicrobial products, etc

Answer 3

type of cytokine that is specialized to fight viruses; named for their ability to INTERFERE with viral replication in vitro Functions: - Makes the host an inhospitable place for the virus to replicate by blocking protein synthesis of the host cell → now virus can’t hijack machinery for replication bc machinery is no longer working - Increase MHC class 1 expression and antigen presentation in all cells - Activate dendritic cells and macrophages - Activate NK cells to kill virus-infected cells

Answer 4

Viral infections induce strong expression of Type 1 interferons aka IFN-alpha and IFN-beta, which promote potent antiviral effector mechs Vacuolar TLRs (involved in nucleic acid sensing) often respond to viral PAMPs and trigger production of type 1 interferons TLR3 can link recognition of viral PAMPs to activation of the IRF and induction of antiviral cytokines (Type 1 interferons, alpha and beta) via the transcription factor IRF3 - IRF transcription factors are inactive, cytosolic in resting cells - TLR3 signaling thru TRIF leads to phosphorylation of IRF3, leading to nuclear transport and activation of the target gene, IFN-beta (a type 1 interferon)

Answer 5

TLR helps to initiate inflammation in response to local infection Systemic TLR activation can have disastrous consequences (esp w gram negative bacteria, ie LPS), such as release of inflammatory cytokines into the entire body’s bloodstream → leads to vascular permeability throughout the body, leading to decreased blood volume overall and subsequent collapse of vessels, eventually leading to organ failure and death Mice lacking TLR4 are resistant to LPS-induced shock Mice lacking TLR4 are also more sensitive to localized infection with gram negative bacteria

Answer 6

large family of mammalian cytosolic innate immune sensors that contain nucleotide oligomerization domain (NOD) domain, aka nucleotide binding (NBD) domain NOD1 and NOD2 (aka NLR with card domain, NLRC) mediate cytosolic sensing of bacterial PAMPs Functions - LRR domains involved in sensing PAMPs / DAMPs - Some activate via NFkB transcription factors - Others activate a multi protein complex called inflammasome (contains enzyme caspase, which can be activated to process cytokine proproteins)

Answer 7

Defined as NLRs that activate inflammasomes have PYRIN domains, aka NLRP / NALP Senses eflux of potassium out of the cell (indicating infection or cellular damage) via cytosolic sensors Can be triggered by crystalized molecules - Uric acid crystals from Gout - Aluminum hydroxide crystals from Alum adjuvant - Cholesterol crystals from artherosclerosis NLRP3 activation is associated w inflammatory response via association with adaptor protein ASC (aka PYCARD) and cleaving of Caspase 1 (1) Potassium efflux induces dissociation of chaperones that keep NLRP3 in an inactive conformation (2) NLRP3 forms oligomers with ASC causing proteolytic cleavage of pro-caspase 1 (3) Caspase 1 releases mature inflammatory cytokines from their proproteins

Answer 8

When innate immune signaling is insufficient to clear a pathogen, the adaptive immune system kicks in → thus, innate immune signaling turns ON the adaptive immune response (innate imm response overlaps in time with the initial triggering of the adaptive immune response) Stimulation of dendritic cells by PAMPs promotes their ability to turn on immune cells (ie T cells) by: - Inducing DC migration from sites of infection to lymph nodes - Up-regulation of molecules on DC involved in APC function

Answer 9

Behring and Kitasato Experiment: immunize rabbits with tetanus bacteria (treated)→ isolate serum from immunized rabbits and inject into naive rabbit (create treated naives); have control group of untreated naives → challenge all three groups with lethal dose of live tetanus bacteria → found that only treated and treated naives survived Active immunity: animal that was directly injected with the killed pathogen; has immunity for life Passive immunity: short lived and taken from an actively immunized organism; very specific Active substance in the “immune serum” that provides protection is ANTIBODIES.

Answer 10

Made of non covalent bonds that are relatively long and low energy (compared to a covalent bond) → less energy needed to break non covalent bonds BUT because there’s a lot of interactions btwn non covalents, it can lead to a very stable structure overall Primary structure: amino acid sequence of polypeptide chain Secondary: alpha helix and beta pleated; more interactions Tertiary: more of a 3D structure that has coalesced due to the interactions btwn them - Domain: portion of a protein that can form a compact 3D structure - Can be covalently linked to other domains via flexible region of peptides; all part of the same peptide chain tho Quaternary: two (or more) folded polypeptides that come together to interact, leading to a dimeric protein molecule

Answer 11

(1) immunized rabbits with the purified protein chicken ovalbumin (2) serum taken from immunized rabbit contains a substance with the capacity to bind to ovalbumin, creating a precipitate (3) isolate serum into two parts for comparison, one via electrophoresis for identification of different protein fractions (aka starting serum; found albumin, alpha, beta, and gamma peaks) and the other for removal of ovalbumin-binding materials (aka depleted serum) (4) compared starting v depleted serum, found that gamma had most absorbance / binding activity, THUS must contain antibody (termed it “immunoglobulin”)

Answer 12

Comprised of two types of polypeptide chains: 2 Heavy Chains + 2 Light Chains = 4 chains total Chains are held together by interchain disulfide bonds and non-covalent interactions Ig Domains are structures made up of repeating structural units of ~110 amino acids. Each antibody block makes up one Ig domain, thus there are multiple Ig domains in a chain. Heavy has 4 domains, Light Chain has 8. C terminal Ig domains are constant and encoded by the germline, thus involved with effector functions. Variations here are limited BUT are responsible for different antibody isotypes and whether they are membrane bound or secreted. N terminal Ig domain is variable and generated by somatic DNA rearrangements, thus acts as the antigen binding domain bc have essentially unlimited diversity. Antibodies are BIVALENT, thus have two identical antigen binding sites. More binding sites induce cooperative binding, leading to stronger binding relationships and less dissociation (compared to monovalent) Hinge region allows flexible movement of antigen binding arms

Answer 13

Commonly used structural motif amongst cell surface protein; used throughout the body to maintain good protein-protein interactions All Ig domains have a similar 3D structure → 2 beta pleated sheets come together to form a sandwich, held together by disulfide bond and hydrophobic interactions, yielding three flexible loops at the end (aka hypervariable regions 1-3) HV1-3 (aka complementary determining regions, CDR 1-3) is located at each N terminal tip, thus there are 6 CDRs per chain → high variability means that each antibody molecule has a unique antigen binding site with its own dimensions and complementarity (aka specificity) Intervening framework regions (FR1-4), aka non hypervariable regions, will make up the rest of the structure (ie region of the C terminal)

Answer 14

Three dimensional face of an antigen which makes physical contact with the antibody molecule → epitopes and antigen binding sites form complementary surfaces (in terms of shape, charge, and hydrogen bond donor / acceptors) Most antibodies can recognize “conformational” or “discontinuous” epitopes → ie regions that are separated in primary but together in tertiary Some antibody can recognize “linear” or “sequential” epitopes → ie residues that are adjacent to each other in the primary Conformational epitopes are lost upon denaturation while linear epitopes are preserved.

Answer 15

Isotypes arise due to differences in HC or LC constant region sequences → Heavy comes in five major types that have different tissue distributions, effector functions, and characteristic carbohydrate attachment sites IgM (mu): first antibody secreted in an immune response, thus found in serum; can form higher order multimers via J chain, thus can have 5 units with total of 10 identical antigen binding sites (pentameric / decavalent structure); will class switch to produce IgA / G / E IgG (gamma): predominates in serum; found in tissues and can cross the placenta to provide protection to the fetus; production requires class switching IgA (alpha): can form higher order multimers via J chain, thus can have 2 units with total of 4 identical antigen binding sites (dimeric / tetravalent);; predominates in secretions; production requires class switching IgE (epsilon): binds to Fc receptors on mast cells during allergic reactions; production requires class switching IgD (delta): no known effector function; will class switch to produce IgA / G / E

Answer 16

Neutralization: binding itself prevents pathogenesis; prevents release of exotoxins and endotoxins that can induce tissue damage → bacterial toxin can be stopped from binding to host receptors if already bound by an antibody Opsonization: enhancing phagocytosis (usually spontaneous) by coating the pathogen entirely with antibodies → makes the pathogen more tasty Complement activation: antibodies will bind to bacteria and will attract complements that target the bacteria for destruction

Answer 17

Fc receptors: found in HC ;; contrast to Fab found in LC - Mediates many antibody effector functions by binding to Fc receptors on effector cells - ie Antigen binding regions will attach to pathogen VERSUS Fc region will attach to an effector cell via Fc receptor Opsonization: aggregation of Ig on bacterial surface promotes aggregation of Fc receptors → activation of macrophage, thus destruction of bacterium Another end to opsonization is that antibody coating enhances killing of target cells via Antibody Dependent Cellular Toxicity (aka ADCC) (1) Antibody binds antigen on the surface of target cells (2) Fc receptors on NK (natural killer) cells recognize and bind to antibody (3) Cross linking of Fc receptors signal NK cell to kill target cell (4) Target cell dies by apoptosis and membrane damage, caused by the lysing action of the NK cell

Answer 18

IgM: good at complement activation IgG: important for opsonization, complement activation, and ADCC IgA: transported across epithelial cells via poly-Ig receptor; found in breast milk to supply passive immunity to baby IgE: important for binding to Fc receptors present on mast cells and basophils; levels increase in setting of parasitic infection (specifically, multicellular parasites, eg worms); can transfer allergy btwn individuals by injecting (ie pass a peanut allergy) IgD assumed to have no effector function, thus will have no relationship to Fc receptors.

Answer 19

First antibody found in fetus BUT is not made by fetus → IgG passively transferred from mother via placenta After birth, maternal IgG levels gradually decline bc no longer passively transferring IgG antibodies by placenta ;; instead, transfer IgA is passively transferred to fetus via breastmilk ``` Newborn will begin production of IgM after birth → IgM can class switch to the other antibody classes Transient period: where the both the maternal IgG levels are low and the newborn ``` IgM levels are not high enough, which is an extremely fragile / dangerous time for the infant as it as virtually no immune system

Answer 20

Antibody: protein / immunoglobulin that binds an antigen; produced by B cells Antigenicity: ability of a particular substance to be recognized by both B and T cells Immunogen: any molecule that can elicit an adaptive immune response upon injection into a person or animal ;; similar to antigen → high antigenicity Hapten: small inorganic molecule that can be recognized by a specific antibody but cannot by itself elicit an immune response; recognized only when chemically linked to a protein molecule → low antigenicity

Answer 21

Both encode genes that undergo somatic DNA rearrangement (aka VDJ recombination) during development Both are composed of repeated Ig domains and have N terminal variable domains that contribute to the specificity of the antigen binding site. Both are disulphide linked heterodimers → HC/LC vs alpha/beta Antigen receptors of B cells can exist in both a transmembrane receptor and secreted form (thus can bind to soluble antigens) while that of T cells exists only as a transmembrane form (thus cannot bind to soluble antigens) AR of B cells will bind DIRECTLY to the antigen while that of T cells will bind to a processed antigen on the surface of an Antigen Presenting Cell (APC) Most antibody responses require that the antigen is recognized by BOTH B and T cells

Answer 22

Ability of a particular substance (ie protein) to provoke an immune response in a person or animal Factors: - Foreignness: how similar is it to the self protein? greater difference from the host yields an increased immunogenicity - Size: how big it is? Larger proteins yield an increased immunogenicity - Complexity: more complex molecules yield an increased immunogenicity bc they need more time / cells to kill it - Susceptibility to phagocytosis: proteins that are particulates or denatured will yield an increased immunogenicity versus proteins that are soluble or native - Genotype of the host (ie interactions with host MHC): refers to whether we have the correct receptors to bind to the antigen, or else we can’t even trigger an immune response - Route of administration: subcutaneous > intraperitoneal > intravenous or intragastric - Dose: intermediate dosage will yield an increased immunogenicity → too high of a dosage means that the immune system cannot keep up ;; too low may not even trigger the immune system at all

Answer 23

- Trigger innate immune system as many are TLR agonists - Slow release of antigen - Promote phagocytosis The most effective adjuvants cannot be used in humans due to toxicity → the more effective an adjuvant is in triggering the immune system, the more toxic it is in the body Eg. Fruend’s complete adjuvant consisted of emulsified mineral oil and mycobacterial extract → the oil delayed uptake of antigen by macrophages due to emulsification, led to induction of co-stimulation in macrophages Eg. Alum (aluminum hydroxide) crystals activate NLRP3 inflammasome (important DAMP for triggering immune responses) by same mechanism

Answer 24

Strength is determined by the sum of multiple non covalent bonds; strength of interaction btwn a single epitope and antigen binding site is called its AFFINITY Measure affinity by equilibrium dialysis: once you know the concentration of free and bound ligand at equilibrium for diff ligand concentrations, you can calculate the equilibrium binding constant (K), which provides a quantitative measure of the affinity of the interaction - Ka = [concentrations of product] / [concentration of reactants; if multiple, should multiple concentrations] - If binding is weak, K2 (off rate) is high, and Ka (association binding constant) will be low → equilibrium shifted to left - If binding is strong, k2 is low, and Ka is high → equilibrium shifted to the right Measure binding strength by Kd (dissociation equilibrium constant) = basically 1 / Ka - The ligand concentration at which HALF the antibody is bound, is close to the Kd - Stronger binding corresponds to lower Kd

Answer 25

Can be generated by repeated immunization of animal (rabbit) with antigen (w adjuvant) → thus leads to many instances of exposure, allowing for memory to be built Polyclonal antibodies: antibody preparations from immunized animals; consist of complex mixtures of different antibodies produced by many different B cell clones → many varieties of fine specificity can respond to the target antigen Monoclonal antibody: homogeneous antibody preparations produced in the laboratory; consist of a single type of antigen binding site, produced by a single B cell clone

Answer 26

Avidity (strength of binding) is influenced by both affinity (Ka of a single binding site) and the valence of the interaction (number of interacting binding sites) Each antibody has at least 2 antigen binding sites, therefore antibodies are bivalent to multivalent → avidity (functional affinity) is the accumulated strength of multiple affinities Compare IgM and IgG → similar avidity but IgG is a more effective antibody - IgM has 10 antigen binding sites per molecule VS IgG has 2 - IgM: low affinity interactions BUT can have high avidity → tends to bind tightly but has less specificity - IgG: high affinity, thus binding tends to be more specific (aka more of a “perfect fit” btwn antigen binding site / antigen)

Answer 27

Defined as, Binding of antibody or T cell to an antigen NOT used to elicit that specific antibody Used in vaccines: vaccination to one type of influenza virus provides resistance to other forms of influenza → banking on cross reactivity to “infect” you with a virus similar enough for you to create immunity to the actual virus that the vaccine is aiming to protect you from Used in self tolerance: bodies contain many epitopes that resemble those found on pathogens, thus must restrict ability of our immune system to recognize certain self-antigens

Answer 28

Presence of antibodies cross react w polysaccharide antigens on red blood cells Individual will not produce antibodies that react w own RBC (Self tolerance) Blood type of individual can be determined by an agglutination assay: look for ability of serum to bind to, and agglutinate RBC from another individual Eg. Blood Type A will have A antigens on RBCs and Anti-B serum antibodies O is Universal donor bc RBC doesn’t interact w any other blood types AB is Universal recipient bc doesn’t have any antibodies to reject any blood donations

Answer 29

Clone: population of cells derived from a single progenitor Hybridomas: hybrids generated by fusion btwn a non transformed antibody producing B cell and a transformed cell (myeloma) that can grow continuously in culture → takes advantage of the properties of myeloma cell (unlimited growth capacity and cellular machinery to produce antibodies) and the antigen specificity of primary B cells

Answer 30

(1) Inject rate with antigen (2) isolate serum to yield polyclonal antiserum ; also isolate spleen cells to yield plasma cells ( B cells) and myeloma cells (3) Hybridize the components from the later isolation via polyethylene glycol (PEG; works as a fusing agent for the plasma membranes of adjacent cells) (4) Transfer to medium for HAT selection (used to select for growth of hybrids and against growth of parental myeloma) → allows for selection and isolation of specific hybridomas while letting unfused myeloma cells die (5) After matching antibody that is specific to antigen, create clones to yield monoclonal antibodies Advantages: consistent, limitless supply of specific reagent ;; can be more easily tested for cross reactivity

Answer 31

Early quantifications of these interactions relied on the the tendency of antibody - antigen complexes to come out of solution (precipitation of immune complexes) or the ability of antibodies to stick to antigens (agglutination) Precipitation refers to linking interactions w protein antigens ;; agglutination refers to linking interactions w cells. However, both have relatively low sensitivity and thus work well when the antigen is relatively abundant; precipitation in particular works best at a ratio of 1:1 antibody:antigen

Answer 32

Basically: Polyclonal antibody and antigen w multiple distinct epitopes → FORMATION OF A LATTICE DUE TO THE MIX OF INTERACTIONS Monoclonal antibody and antigen w repeating pattern of identical epitopes → LATTICES DO NOT FORM W MONOCLONAL ANTIBODIES AND ANTIGENS W MULTIPLE DISTINCT EPITOPES Polyclonal antibodies can form lattices w homogenous, monomeric protein antigens bc each antibody can interact with a diff epitope on the antigen - Formation of lattice bc have a mix of binding to the whale myoglobin’s epitopes that antibodies can bind to, leading to a lot of binding to the myoglobin that will eventually precipitate out due to the lattice structure Monoclonal antibodies do not form lattices w homogenous, monomeric proteins bc only they can bind to only one epitope on the antigen - Can cross link but no lattice will form / structures will remain soluble ;; monomeric protein doesn’t allow cross linking past the first binding

Answer 33

bind to the primary antibody to assist in detection, sorting and purification of target antigens Commercially available and thus used in many immunoassay techniques Eg. Use of anti-IG antibodies increase degree of crosslinking and can increase lattice formation

Answer 34

Very sensitive and can detect minute concentrations of material; utilizes standard curve with known amounts of unlabeled antigen to find unknown Can be used to measure the amount of AntigenA in solution Can also be used to measure ability of test sample to complete for binding with labeled antigen to antibody on dish → takes advantage of protein binding to plastic of tissue culture dish (1) AntiA antibody bound directly to plastic tissue culture dish (2) Followed by incubation w irrelevant protein (usually powdered dry milk) to block further non specific binding to plastic (3) Add labeled AntigenA (to be specifically bound by Anti A antibody) and unlabeled competitor antigen (4) Wash away any unbound antigens (5) Determine concentration of unknown samples determined by comparison to standard curve w known sample of AntigenA

Answer 35

Goal: determine quantity / presence of an antibody or antigen of interest in a serum sample Takes advantage of: - High degree of specificity of antibody binding → antigen will bind to plastic well instead of proteins - Non specific binding of protein to plastic - Colorimetric detection of antibody-antigen binding using covalently linked enzymes DIRECT: specific antibody is directly conjugated w an enzyme to facilitate detection and determination of how much you have → results in a more rapid assay bc primary antibody is conjugated; less background INDIRECT: specific antibody is unconjugated and is detected using an enzyme conjugated secondary antibody (antibody that binds to another antibody) → better amplification thus better response generated SANDWICH: well-bounded capture antibody binds antigen that can then be detected in a direct / indirect ELISA, leading to “sandwich” of antibodies → more sensitive and flexible than in/direct ELISA; can determine if cells in a blood sample express antigen on the cell surface also express a second antigen bc utilizes two antibodies and thus can have two different epitope recognitions

Answer 36

Direct and Indirect ELISA differ from SANDWICH bc the antigen of interest is bound directly to the plate rather than a capture antibody. DIRECT: Start with antigen of interest bound to well > add anti-A antibody that is covalently linked (therefore, directly conjugated) w enzyme > wash away any unbound antibodies > add colorless substrate and allow enzyme to make colored product > measure absorbance of light by colored product to determine presence / quantity of production INDIRECT: Start with antigen of interest bound to well > wash, then add specific antibody (unconjugated) to be measured > wash, then add enzyme-conjugated secondary antibody that will bind to primary antibody > wash, then add substrate and measure color - Wash out anything that did not bind at every step SANDWICH: Start with unconjugated antibody bound to well > add antigen to be bound > add antibody to be used in Direct / Indirect > follow respective steps above to finish

Answer 37

Goal: obtain quantification and molecular weight of the antigen; can also determine if antigen is nuclear or cytoplasmic Denature protein antigens in SDS → run mix thru SDS PAGE (SDS polyacrylamide gel electrophoresis) to separate based on their molecular weight → then transfer proteins from gel to a membrane sheet via electric current→ after transferring proteins from gel to membrane sheet, use labelled antibodies to your protein of interest to detect the relevant band → labelled antibodies bind to band containing your protein of interest → use colorimetric assay to detect this labelled antibody SOUTHERN BLOT: restriction endonucleases cleave at sequences in DNA and can be used to generate a physical map of DNA → basically compares different structures of antibody genes of the germline to other somatic cells

Answer 38

Goal: detect and physically isolate / purify a specific protein from a complex mixture based on specific antibody binding Start with mixture of proteins → add in anti-X antibody that will bind to AntigenX→ insert Protein A / G to extract Anti-X antibody and AntigenX complex - Protein A / G: bacterial cell wall proteins that bind to Ig / Antibody Variation: MAGNETIC PROTEIN BEADS coupled to secondary antibodies can be used to isolate the antibody-antigen complex from solution, or cells from a suspension

Answer 39

Goal: detect and physically isolate / purify a specific protein from a complex mixture based on specific antibody binding Magnetic bead approach: Start with a column of antibodies that have been bound to beads → add in the mixture of molecules → wash away any unbound molecules, yielding mixture depleted of antigenA → Elute specifically bound molecules, yielding purified antigenA

Answer 40

Goal: Determine if the antigen is nuclear or cytoplasmic; also determines location of antigen within the spleen by using a fluorescently labeled antibody Direct: labeled primary antibody that will bind to cell Indirect: labeled secondary antibody that will bind to the primary antibody

Answer 41

Goal: used to determine the NUMBER of cells and the level of their fluorescence within a sample that react w a particular fluorescently labeled antibody; determine if cells in a blood sample that express antigen on the cell surface also express a second antigen; determine if surface levels of antigen increase after treatment with TLR ligand Start off with mixture of molecules → add in fluorescently labeled antibodies so that each molecule is now an antibody-antigen complex → wash any unbound antibodies → run through laser, which scatters based on size and irregularity → use computer to plot the scatters for interpretation

Answer 42

Examine expression of T Cell activation markers by dendritic cells (Hemmi 2000) Identify different lymphocyte populations and also diff variations of diseases (ie leukemia) Can be used to sort different antibodies based off their staining, using deflection plates → fluorescence activated cell sorting, aka FACS (1) Start w suspension of cells labelled w antibodies (2) Go thru machine and break into droplets (3) Run through laser and collect information on computer screen (4) Deflect droplets of diff antibodies into diff wells based on expression as observed on the computer by the markers expressed Can also be used to purify cells by using magnetic beads → FACS alternative that can process larger number of cells BUT can be combined with FACs to improve sorting of cells by increasing the number of parameters

Answer 43

Antigen dependent vs independent phase Molecular (regarding DNA that encodes antibodies) vs cellular (regarding development of B cells) events ``` Dependent x Molecular = class switch, somatic hypermutation Dependent x Cellular = B cell activation, memory and plasma B cell differentiation ``` Independent x Molecular = VDJ rearrangement Independent x Cellular = proB > preB > mature B cell development

Answer 44

Heavy chains are encoded by a single gene locus with four kinds of gene segments (Variable, Diversity, Joining, Constant) Light chains are encoded by two gene loci (kappa and lambda) with three kinds of gene segments (V, J, C) Constant region encodes the antibody isotypes → mu, delta, gamma, alpha, epsilon Somatic DNA rearrangement occurs in both chains via “V(D)J Recombination”, which is done before transcription bc RNA splicing removes introns - Heavy chain: D and J brought together first, then V brought to the DJ complex - Light chain: V and J brought together Leader exon (L) will be removed / spliced out during transcription.

Answer 45

Combinatorial diversity: in humans, arise out of VDJ Recombination ;; number of different combinations can be calculated by multiplying the respective possibilities together → Eg. VkJk combinations = 5 * 35 = 175 different combinations Junctional diversity: uses enzyme terminal deoxynucleotidyl transferase (TdT); refers to variability / flexibility within the joining regions of the segments also contributes substantially to the total diversity of antibodies → addition of P (palindromic) and N (random) nucleotides vs removal / trimming away of nucleotides

Answer 46

Ig promoters are actively transcribed when they are brought close to enhancers due to gene rearrangement. Promoters: nucleotide sequences within ~200 bp of transcriptional start site that initiate transcription in a certain direction Enhancers: nucleotide sequences that activate the promoter in an orientation (thus position independent) manner

Answer 47

Rearranging gene segments are flanked by a conserved “REARRANGEMENT SIGNAL SEQUENCE” (RSS) Spacer = 12 or 23 nt → 12 for one turn, 23 for two turn 12/23 Rule: need a 12 in line w a 23 ; only gene segments flanked by RSSs w dissimilar spaces can undergo V(D)J recombination w one another → ensures that V segments don’t join w other Vs that VH don’t join w JH etc

Answer 48

Coding joints: contain imprecise fusion of V/D/J segments bc will add or subtract nucleotides; will encode CDR3 of antibody genes → leads to junctional diversity BUT only one-third of rearrangements preserve the correct reading frame of the J segment Signal joints: contain precise fusion of RSS ; non functional by-product of recombination - Precise: Always have RSSs joined to each other exactly CDR3 = most diverse / variabel of all the loops due to it being encoded btwn the junction of V/J CDR1/2 are diverse only bc there’s a lot of V segments

Answer 49

Recombination activating genes (RAG) are expressed only in developing lymphocytes (T and B cells) and target Ig and TCR gene segments for rearrangement DNA repair machinery is used by all cells to repair DNA damage; has been co-opted by VDJ Recombination to participate in BCR and TCR gene rearrangement RAG1 and 2 form a multimeric complex that binds to 2 compatible RSS sequences and brings them together via a process called synapsis → after synapsis, DNA is cleaved (also done by RAG1/2 complex) btwn the RSS and coding sequences to generate covalently closed DNA hairpin structures on coding joints, leaving a dsDNA break on the non coding (aka signal) joint → Components of double stranded break repair machinery assemble at the broken DNA ends and ligate them Mutations in either RAG or DNA repair machinery can lead to defective VDJ recombination and blocks in B and T cell development

Answer 50

Antigen Independent: development (1) Early differentiation in primary lymphoid organs (bone marrow for B cells, thymus for T cells) (2) Naive cell: aka resting or quiescent B or T cells; will express IgM and IgD on membrane Antigen dependent: class switching and somatic hypermutation (3) Effector cell: rapidly dividing, fully function (4) Cell fate: cell death or memory cell

Answer 51

Asking about the antigen-independent stage and differentiation Normally in bone marrow BUT can be done in vitro by putting B cells and stromal cells in the appropriate environment (1) ProB cells: no HC or LC expression (2) PreB cell: HC (cytoplasmic) but no LC expression → heavy chain rearranged before LC; HC will be a part of the preBCR complex (HC + surrogate light chain) (3) (Immature) B cell: surface HC and LC expression but not yet activated by antigen / other signals → LC will replace surrogate LC of preBCR and complex will rise to the surface to become membrane bound ;; expression of IgM only (IgD expressed once it becomes mature / naive, ie after testing for self reactivity)

Answer 52

ensures that most B cell will express a single antibody specificity → only one is expressed within the heavy chains and light chains Related to junctional diversity because will exclude non productive rearrangements that do not fit into the reading frame Heavy chain has two opportunities for rearrangement (one for each chromosome) whereas the light chain has four (one for the respective kappa and gamma gene on each chromosome) (1) proB: D-J rearrangement occurs on both chromosomes → V-DJ rearranges on first chromosome, if fails then reattempt on second chromosome, if fails then cell is trashed (2) preB: V-J rearrangement on kappa gene of first chromosome, if fails then reattempt on kappa gene of second, if fails then reattempt on gamma gene of first, if fails then gamma gene on second, if fails then cell is trashed (3) light chain rearrangements determine what it expresses → ie if either kappa arrangements work, then B cell expresses kappa light chains

Answer 53

Stromal cells: provide secreted and cell surface factors that promote B cell maturation Knock out mice: have ENGINEERED MUTATIONS in endogenous genes ;; technique usually produces recessive mutations Transgenic mice: engineered to EXPRESS FOREIGN DNA inserted randomly into the genome ;; technique usually produces dominant mutations

Answer 54

Shut off heavy chain V to DJ rearrangement (allelic exclusion) Pro B cell > pre B cell transition - Start V to J arrangement in light chains (four attempts) - Alterations in gene expression (eg shut off TdT) - Don’t die - Proliferation

Answer 55

Twice (1) During Antigen Independent stage: btwn the immature to naive cell stage (2) During Antigen Dependent stage: post activation OR post somatic hypermutation checks for self reactivity after activation (bc might react to self over the antigen) → four outcomes: no self reaction yields mature B cell > multivalent self molecule undergoes receptor editing > soluble self molecule becomes anergic > low-affinity non-cross-linking self molecule becomes ignored

Answer 56

Two Signal Model: first signal is engagement of antigen receptor BUT that’s not enough! - For T dependent: signal 2 provided by helper T cells via CD40/L junction - For T independent: multiple pathways; can be activated by direct BCR crosslinking or by other receptors (ie TLR) CD40/L junction: activated Helper T cells will bind their CD40L receptor to the CD40 costimulatory protein of a B cell to activate it. BCR: antigen induced cross linking of BCR leads to phosphorylation of tyrosines in ITAMs

Answer 57

Alternative mRNA processing -- for secreted vs membrane Ig and for IgM vs IgD - mRNA processing, not DNA recombination, is occurring → both secreted and membrane forms of Ig HC can be made by the same B cell Class switch recombination: for IgG / A / E

Answer 58

Used in generation of other Ig isotypes (A / G / E) Utilizes “switch site” located 5’ to each CH segment to target recombination machinery → recombination occurs at these switch sites (ie breaking, rejoining) Once class switch has occurred, the B cell can no longer make IgM (bc the coding sequences have been deleted / broken off) → HOWEVER, progeny of single B cell can produce different isotypes ``` NOTE: there is no switch sequence in front of the delta units bc IgM and IgD are utilized together (therefore, expressed together) via alternative splicing When class switching occurs, can the B cell still express IgD?? → No, bc will also be spliced out ```

Answer 59

Require components of the dsDNA break repair (DSBR) machinery :: Ku70, Ku80 and DNA Requires at least part of each “Switch sequence” Requires the activity of Activation induced cytidine deaminase (AID) which initiates DNA damage and DNA repair that lead to double stranded breaks Cytokines produced by helper T cells regulate antibody class switching

Answer 60

VDJ Recombination vs Class Switch Recombination VDJ occurs as part of antigen-independent dev in primary lymphoid organs (bone marrow ) ;; CSR occurs as part of antigen-dependent dev in secondary lymphoid organs (lymph node, spleen) VDJ requires RAG 1/2 ;; CSR does not VDJ recom is targeted precisely via RSS ;; CSR occurs within simple repetitive DNA sequence via switch sequences Both require Ku70, Ku 80, and DNA-PK (dsDNA repair pathway)

Answer 61

Germinal Centers: sites of intense B cell proliferation and differentiation Follicular Dendritic Cells: stromal cells (not blood cells) that reside in germinal centers and trap and train antigen containing immune complexes on their cell surface → binding of immune complexes via Fc receptors (FcR) and complement receptors (CR1) Lectin: carbohydrate-binding protein

Answer 62

(1) FDC present antigen to germinal center B cells in form of antibody-antigen complexes (2) B cells w highest affinity antibodies compete more effectively for supplies of antigen from FDC → competition ! (3) Successful B cells that pick up antigen from FDCs can interact w helper T cells and receive survival / differentiation signals (therefore, avoid apoptosis) Selected B cells give rise to high affinity plasma cells and memory B cells

Answer 63

Occurs due to Somatic HyperMutation (SHM) and B cell selection in the germinal center Defined as a “Mechanism that generates mutations does not discriminate mutations that increase / decrease affinity” Throughout the course of an immune response (or w successive immunizations), there will be an increase in the average affinity of an antisera (as a result of SHM influences) Sequence alignments of IgG isolated from B cells late in an immune response

Answer 64

Basically evolution in “real time” for the immune system → concept of CELLULAR SELECTION Antibodies / B cells want to survive and the only way they can do so is to receive help from the T cells (regarding differentiation signals) THUS must become better competitors and bind more effectively to antigens that are presented by follicular dendritic cells Hypermutation mechanisms generates point mutants in variable domains that generally increase affinity in B Cells, thus increasing their potential to bind and survive bc lesser affinity B cells will die by apoptosis Selected B cells will give rise to high affinity plasma cells and memory B cells that then undergo the same cellular selection → allows better response to repeat infections

Answer 65

Given dsDNA break, mutation introduced during DNA repair at a rate of 1 mutation per 1000 nucleotides per cell division - Normal mutation rate is 1 per 100,000,000 Requires the enzyme: AID, activation-induced cytidine deaminase → initiates DNA damage and DNA repair that lead to double stranded breaks - If break is in coding regions, leads to somatic hypermutation - If in switch sequences, leads to Ig class switching

Answer 66

- Opsonization of cellular (bacterial) antigens - Provoke inflammation - Poke holes in membranes leading to lysis of bacteria - Clear immune complexes - Activate antigen specific b cells Mannose-Binding Lectin (MBL) or ficolin binding to a pathogen surface triggers a complement cascade, ultimately resulting in the the creation of a pore in the bacterial membrane to cause the cell to lyse (one of three ends of the complement cascade) → thus, MBL and ficolin are examples of PRRs

Answer 67

Note: Each step of the complement cascade is regulated to inhibit activation on the host cell surface while allowing activation on pathogens LECTIN pathway: MBL and ficolins recognize and bind carbohydrates on pathogen surface → PRR example bc will recognize components of pathogenic origin (ie yeast) that differ from self surface components CLASSICAL pathway: utilizes antibodies that have bound to the surface of the pathogen ;; REMEMBER C1qrs + C3ab timeline → Binding of C1q to Ig activates C1r, which cleaves and activates the serine protease C1s → triggers cleavage of C3 molecule, C3b remains on surface of pathogen and the soluble C3a is released ALTERNATIVE pathway: remember C3BD-C3bBb → activated by the spontaneous hydrolysis of C3 to form C3(H2O)

Answer 68

All pathways generate a C3 convertase, which cleaves C3, leaving C3b bound to the microbial surface and releasing C3a → C3a and C3b lead to regulation of immunity and destruction of the pathogen through a variety of different mechanisms LECTIN: C3a recruit phagocytic cells to the site of infection and promote inflammation CLASSICAL: Phagocytes with receptors for C3b engulf and destroy the pathogen ALTERNATIVE: Completion of the complement cascade leads to formation of a membrane-attack complex (MAC), which disrupts cell membrane and causes cell lysis

Answer 69

IgM is soluble but can bind to antigens on bacterial surface and adopt “staple” form → thanks to its large pentameric structure, C1q binds to this “staple” configuration of the bound IgM molecule, triggering a strong signal for the cascade vs IgG is also soluble and will bind to antigens on bacterial surface BUT does not adopt any new formation → IgG is smaller than IgM and, thus, C1q needs at least two adjacent IgG molecules to bind (thus, likelihood of this occurring drops) THUS, IgM is more effective because C1q can bind more strongly and reliably than IgG.

Answer 70

Class 1 MHC: found on every nucleated cell; intracellular → hijacked machinery will presents peptides to cytotoxic T cells, which will lyse that cell and go hunting for other cells that present it Class 2 MHC: found on professional APCs; extracellular → present peptides from lysed cells to helper T cells THEN -- APCs will present antigens to T cells, activating them - Cytotoxic T cells hunt infected cells and lyse them → pathogen peptides are released and can be picked up by MHC Class 2 - Helper T cells will activate B cells via CD40L-CD40 junction

Lecture 1 Flashcards

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