Lecture 1 Flashcards
Four Main Classes of Pathogens
- Extracellular bacteria / parasites / fungi
- Intracellular bacteria / parasites
- Viruses (intracellular)
- Parasitic worms (extracellular)
Most cells are better equipped to fight EXTRACELLULAR pathogens.
Define the following terms:
- Commensal bacteria
- Pathogen
- Antigen
- Leukocyte
- Lymphocyte
Commensal bacteria: complex community of microorganisms that live in the mucosal surface of our digestive tracts; 10x more numerous than our own cells; protects us by maintaining the barriers to outcompeting pathogenic bacteria
Pathogen: microbe that can cause disease (= symptoms)
Antigen: material (from pathogen) that induces an immune response
Leukocyte: white blood cells (incl lymphocytes and myeloid cells)
Lymphocyte: specialized white blood cells that mediate adaptive immunity
Compare Innate and Adaptive Immunity on the following:
- Response time
- Response to repeat infection
- Found in (what?)
- Ligands / inducers
- Receptors that mediate pathogen recognition
- Receptor diversity
Response time: (i) hours vs (a) days
Response to repeat infection: (i) identical to primary; no memory vs (a) stronger response upon second exposure; memory
Found in: (i) all multicellular orgs vs (a) vertebrates only; came later in evolution
Ligands / inducers: (i) pathogen associated molecular patterns vs (a) virtually any component of pathogen
Receptors that mediate pathogen recognition: (i) pattern recognition receptors, toll like receptors vs (a) antibodies and t cell antigen receptors
Receptor diversity: (i) limited bc fixed in germline vs (a) unlimited bc generated by V(D)J recombination
What are the 5 types of lymphocytes?
T helper cells: regulate other immune cells → THE GENERALS
T cytotoxic (killer) cells: kill infected cells → THE SOLDIERS
B cells: produce antibodies (immunoglobulin); can also produce cytokines → THE MEDICS
Dendritic cells and macrophage: can directly kill microbes by phagocytosis and other mechs BUT also help to activate T cells (connection btwn innate and adaptive imm)
NK cells: lymphocytes that have characteristics of innate and adaptive immunity; kills virus-infected cells by lysing them; involved with antibody-dependent cell-mediated cytotoxicity (ADCC)
Provide two theories of immune recognition.
Pattern Recognition theory: pathogens contain molecular patterns that differ from host cells “pathogen-associated molecular patterns) (aka PAMPs) ;; recognized by immune cells using specific receptors → FOREIGN
- Pathway: TLR4 → NFkB → inflammatory cytokines
- Eg. lipopolysaccharide LPS in gram negative bacteria
Danger theory: Pathogens cause damage to tissues which leaders to the release of alarm signals that trigger immune responses, sometimes called “damage associated molecular patterns” (aka DAMPs) → NATIVE; released by dying cells / tissues
- Pathway: NALP3 inflammation → pyroptosis
- Eg. uric acid release from dying cells
Define the following:
- Innate Immune System
- Adaptive Immune System
- Immunological Memory
Innate: rapid and non specific; initiated for every injury / infection that occurs to the body; use toll like receptors (TLRs) to mediate response → FIRST LINE OF DEFENSE
Adaptive: develops over time but very specific; initiated only for pathogens; retains memory for faster response in case of future infections; use antibodies and T cell receptors (TCRs) to mediate response → SUIT UP FOR WAR
Immunological memory: core part of adaptive immune system; develops over time but can be a lifelong response ;; usually in regards to the maintenance of memory B and T cells and high serum or mucosal antibody levels that are intended to protect against reinfection
What are the 2 fluid systems. Describe the connection.
Lymph and blood.
Lymphatic system collects lymph (extracellular fluid) that drains from tissues into LYMPH NODES, and eventually returns to blood via thoracic duct.
Naive lymphocytes enter lymph nodes from the blood. Pathogens and their antigens are transported from tissues via lymphatic vessels to the lymph nodes, where they encounter immune cells and activate them.
Contrast primary vs secondary lymphoid organs. Provide examples of each.
Primary: where immune cells develop → creation of naive cells
- Thymus: primary lymphoid organ for T Cell Development
- Bone Marrow: … B cell development
Secondary: where immune cells encounter antigens → activation of naive cells
- Lymph nodes: collect antigens from tissues
- Spleen: … blood stream
Define leukocytes. Which of them belong to the innate v adaptive immune system? What’s the special case?
Leukocytes: white blood cells that derive from hematopoietic stem cells
MYELOID LINEAGE = INNATE → white blood cells other than lymphocytes BUT incl phagocytes
Ie. Monocyte / macrophage, neutrophil, eosinophil, basophil
LYMPHOID LINEAGE = ADAPTIVE → aka lymphocytes; mostly small, relatively inactive cells BUT can be activated / proliferate when triggered by an immune response
Ie. Natural Killer cell, T cells (helper and cytotoxic), B cells
SPECIAL CASE = DENDRITIC CELLS → can arise from either myeloid or lymphoid progenitors
Define the following:
- Bactericidal Mechanisms
- Effector Cells
Bactericidal mechanisms: any mechanisms which eradicates or kills bacteria
Effector cells: perform functions of an immune response, such as cell killing / activation, that can clear the infectious agent from the body
What are the functions of the five myeloid cells?
Macrophage: phagocytosis and activation of bactericidal mechanisms; can present antigens to naive cells to trigger an immune response
Neutrophil: most common white blood cell; phagocytosis of extracellular pathogens; activation of bactericidal mechanisms
Eosinophil: combat parasitic infections via opsonization; effector cell in allergic reactions
Mast cell: releases of granules containing histamine and active agents; has Fc Receptors
Basophil: has Fc receptors → textbook says similar function as mast cell but slide says “unknown activated function”
What are the functions of dendritic cells?
Important bridge btwn innate and adaptive immunity
Myeloid function: antigen uptake in peripheral sites for presentation → early responders to infection
Lymphoid function: antigen presentation to naive lymphocytes for activation → turns on adaptive immunity
Explain how adaptive immunity works in terms of “clonal selection”.
Each lymphocyte bears a single type of receptor w a unique specificity → over time, diversity of these receptors ensures that the immune system can respond to any pathogen
(1) High affinity binding btwn a foreign molecule and lymphocyte receptor leads to lymphocyte activation
(2) This specific lymphocyte with its receptor is now “selected” for “clonal expansion”, referring to the antigen-driven proliferation of mature naive clone of the chosen lymphocyte that can target the foreign antigen
(3) Activation of these clonal cells can lead to eradication of the antigen.
Define the following:
- PAMPs
- DAMPs
- PRRs
PAMPs: aka pathogen associated molecular pathways; molecules specifically associated with groups of pathogens that are recognized by cells of the innate system
DAMPs: aka damage associated molecular pathways → ie efflux of potassium detected in the cytosol indicates cell damage
PRRs: aka pattern recognition receptors; receptors of the innate immune system that recognize PAMPs
Define the following:
- Complements
- Cytokines
Complement: set of plasma / serum proteins that act together against pathogens in extracellular settings; generated in the liver but circulate in the blood as inactive forms; can coat pathogen to facilitate removal or directly kill certain pathogens
Cytokines: aka interleukins; small secreted peptides used for intracellular communication btwn cells in order to influence cell behavior (ie turn on / off immune responses); requires a receptor in order to induce new activity in a cell
What events can arise once a PRR has bound a PAMP?
Increased secretion of antimicrobial products
Increased microbial killing in phagosomes
Production of inflammatory mediators
Increased ability to turn-on T cells
What is inflammation and how does it happen?
Complex series of events induced by infection or tissue damage; one possible end of the complement cascade
Symptoms: redness, pain, swelling, heat
Steps
- Bacteria triggers macrophages and mast cells to release cytokines and chemokines (ie histamine)
- Vasodilation occurs, leading to increased vascular permeability and blood flow to affected area → causes redness, heat, and swelling
- Inflammatory cells migrate into tissue via chemotaxis, releasing inflammatory mediators that cause pain
Chemotaxis: directed migration of cells from blood stream to site of injury via concentration gradient of extracellular signals (in this case, signals are chemokines OR the structurally similar C3A and C5A anaphylatoxins)
What mechanisms make up cell-mediated immunity vs humoral immunity?
Cell-mediated:
- Phagocytosis (cellular eating)
- Cytotoxicity (cellular killing)
Humoral:
- Complements: circulating proteins that can kill pathogens in various ways
- Antibodies: proteins secreted by B cells that assist in more efficient destruction by indirect means
What are the steps of phagocytosis and which cells perform it? What is the function of phagocytosis? What is the relationship between PRRs and phagocytosis?
Macrophages, neutrophils, dendritic cells (also eosinophils) – Provides protection against extracellular pathogens
- Bacterium attaches to membrane
- Bacterium is ingested, forming phagosome
- Phagosome fuses with lysosome
- Lysosomal enzymes digest the bacteria
- Digested material is released from the cell
Many phagocytic receptors are also PRRs involved in innate immune destruction
Encounters with pathogens via PRRs activate the phagocyte by triggering release of inflammatory mediators and increasing phagocytic activity (ie the ability to kill pathogens)
Define these terms. What do they have in common? What are their differences?
- Necrosis
- Apoptosis
- Pyroptosis
All forms of cell suicide, which is used to disrupt the spread of intracellular pathogens and help protect the host by causing an “early death” of the hijacked cell
Apoptosis considered “tidy” while Necrosis and Pyroptosis are “messy” as they rupture the cell membrane and release the foreign bodies
Necrosis: cell organelles and DNA swell and clump until the cell eventually bursts, releasing the foreign bodies → triggers inflammation
Apoptosis: aka “programmed” or “immunologically silent” cell death; broken down from the inside by nucleases and proteases, leading to nuclear fragmentation, proteolysis, blebbing, then death by phagocytosis
Pyroptosis: aka “fiery” death; mediated by catalytic activity of caspase-1 within inflammasome (which can be activated in response to PAMPs or DAMPs) → triggers inflammation and involved with microbial response
Define the following:
- Antigen
- Adjuvant
Antigen: something that generates an immune response by binding specifically to an antibody or by generating peptide fragments that are recognized by a T cell receptor (ie antigen presenting cell)
Adjuvant: any substance that enhances the immune response to an antigen with which it is mixed
What are three components of bacterial cell walls and why are they important to how our immune system functions?
Lipopolysaccharide, aka LPS
Peptidoglycan
Lipoprotein, made of lipopeptides
Are not found in mammalian cells → thus they are PAMPs that can trigger the immune system and can act as good adjuvants
What are TLRs and their functions?
Toll Like Receptors (TLRs) are subsets of PRRs; some can reside on the cell surface while others reside in intracellular spaces
TLRs on plasma membrane recognize bacterial components ;; TLRs on intracellular vacuoles respond to nucleic acid sensing from viruses or bacteria, and are found in endosomes
Ligand induced dimerization of Leucine Rich Repeat (LRR) domains leads to dimerization of the intracellular signaling Toll Interleukin 1 Receptor (TIR) domain
- LRR domains in extracellular domain form a horse-shoe like structure that can bind ligand to the inner or outer groove
- Genes containing LLRs and TIR domains separated by a transmembrane segment are VERY LIKELY to be TLRs
Provide the location, name, and specificity for 9 TLRs given in class. (Some of these TLRs may join together to form complexes.)
Found on PLASMA MEMBRANE:
- TLR-2 / 6 complex recognizes diacyl lipopeptides
- TLR-2 / 1 complex triacyl lipopeptides)
- TLR 4 / MD-2 complex recognizes LPS
- TLR 5 recognizes flagella, which is a bacterial component for movement (not part of cell wall)
Found on INTRACELLULAR VACUOLES:
- TLR 3 responds to dsRNA (produced during viral replication)
- TLR 7 responds to ssRNA (found within viral genomes)
- TLR 9 responds to CpG DNA (unmethylated bacterial DNA)
Describe the TLR Signalling Pathway.
OVERVIEW: TLR binds PAMP > creation of TIR complex > Kinase cascade degrades IkB > NKfB binds to promoters > release of inflammatory cytokines
- 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)
- 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 - Once no longer inhibited, NFkB will bind to promoters and turn on gene expression, leading to release of inflammatory cytokines
Compare the TLR Signalling Pathway of Mammals against that of Flies.
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
What are interferons and their functions?
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
What kinds of PAMPs trigger Type 1 Interferons?
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)
What are the benefits and hazards of TLR signaling / activation in regards to shock? (Experiment with mice.)
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
Describe NOD-like receptors.
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)
Discuss NLRPs or NALPs.
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
Describe the connections between adaptive and innate immunity.
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
Discuss active versus passive immunity.
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.
Describe the various protein structures and the interactions within them.
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
What is the Tiselius and Kabat Experiment? Be able to draw the graph.
(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”)
Describe the structure of antibodies. Be able to draw and identify certain areas.
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
Define immunoglobulin fold. What is it associated with?
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)
What are epitopes?
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.
What are the different antibody isotypes and their functions?
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
List the three antibody effector mechanisms mentioned in class.
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
What are Fc receptors? What is its role in opsonization?
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
What is the relationship btwn effector functions of specific antibodies to Fc Receptors?
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.
What stages in our life do antibodies come about?
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
Define the following:
- Antibody
- Antigenicity
- Immunogen
- Hapten
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
Compare and contrast the antigen receptors of B cells and T cells.
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
Define immunogenicity. What factors influence it?
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
What are the functions of adjuvants? Provide examples.
- 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
How do you quantitate the strength of antibody-antigen interactions?
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
Compare and contrast polyclonal antisera vs monoclocal antisera.
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
What is the impact of valence on strength of binding? Provide binding examples using what you know about certain antibody structures
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)
What is cross reactivity and its importance?
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
How are blood types determined by antibodies?
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
Define the following:
- Clone
- Hybridomas
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
How do you generate monoclonal antibodies? What are the advantages of having monoclonal antibodies?
(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
Discuss the precipitation / agglutination reactions that arise out of antibody - antigen interactions.
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
What are the implications of lattice formation btwn polyclonal vs monoclonal antibodies?
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
Define secondary antibodies. Why are they important?
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
Describe Radioimmunoassay (RIA).
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
Describe Enzyme-Linked Immune Sorbant Assay (ELISA).
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
What are the steps of all three ELISAs?
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
Describe Western Blotting. Briefly describe the Southern Blot method.
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
Describe Immunoprecipitation.
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
Describe Immunoaffinity Chromatography.
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
Describe Immunofluorescence Microscopy.
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
Describe Flow Cytometry.
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
Why is Flow Cytometry important?
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
How can antibody responses be divided?
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
Describe the multigene organization of antibodies (Ig).
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.
Define the following:
- Combinatorial Diversity
- Junctional Diversity
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
What is the relationship btwn promoters and enhancers regarding gene rearrangement?
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
Why are certain DNA sequences within antibody gene loci targeted for rearrangement?
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
What are the reactants and products of V(D)J Recombination? What is the relationship btwn the CDRs?
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
What factors participate in VDJ Recombination?
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
How do lymphocytes develop? Describe the general path. Remember the locations of development are in primary lymphoid organs.
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
What are the stages of B cell development?
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)
What is allelic exclusion?
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
Define the following:
- Stromal Cells
- Knock Out mice
- Transgenic mice
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
What do preBCR signals lead to?
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
Where does the cell check for self-reactivity?
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
What are the T independent and dependent B cell responses?
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
How can a B cell clone produce antibodies w identical antigen binding sites but different constant regions?
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
How does Class Switch Recombination occur?
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
What are the activities involved in Class Switch Recombination?
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
Compare and contrast the two somatic DNA recombinations:
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)
Define the following:
- Germinal Centers
- Follicular Dendritic Cells
- Lectin
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
What are the cellular events that occur in germinal centers?
(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
Describe Affinity Maturation.
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
Describe Somatic Hypermutation.
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
Describe Error-Prone Repair.
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
What are the various roles and functions of complements? Provide examples.
- 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
Describe the different pathways of activating complements.
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)
What are the downstream events that can occur?
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
Why is IgM more effective at activating complements than IgG?
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.
What is the relationship between antigen presenting cells (APCs) and major histocompatibility complex (MHC)?
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
