Inflammation and Immunity
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What is the core function of the immune system?
- Protect hsot against invading viruses, bacteria, fungi, and parasites
- Also protects against malignant cells it considers "nonself"
- Also responds to damaged tissues
Describe the 2 major arms of the immune system and outline key differences between them
- Adaptive immunty: develops in response to challenge; acquired; induced by antigen (pathogen); mediated by lymphocytes (not phagocytic cells); primitive and sophisticated effector mechanisms; highly diverse; immune memory cells; protection is delayed; antigen recognized by antibody, B-cell Ag receptor (BCR), T-cell Ag receptor (TCR)
- Innate Immunity: recognizes foreign patterns with pattern recognition receptors; basis for self v. nonself discrimination; immediate response to foreign invaders; present at birth; primitive effector mechanisms; can also target non-immunogenic epitopes
LPS on gram-negative bacteria is more closely correlated with innate or adaptive immunity?
Innate immunity; LPS is an endotoxin that activates pattern recognition mechanisms
What is the primary function of an antibody?
- To bind antigen
- If done ion the presence of serum proteins, complement will be activated resulting the formation of pores that initiate lysis of invader
What is the primary function of macrophages?
To phagocytose and kill invaders
What is the primary function of cytotoxic T cells?
To bind to and directly kill foreign cells; this includes "nonself" tumor cells
What are the primary cells involved in innate immunity and what are their principal responses?
- Macrophages, neutrophils, and natural killer cells
- Lysis, phagocytosis, and altering other cells to presence of pathogen (danger signaling)
What are the primary cells involved in adaptive immunity and what are their principal responses?
- B lymphocyte: produces anitbody
- T helper cells: promotes antibody production; antigens presented to Th cells are exogenous
- Cyt T lymphocyte: lyses tumor cells or foreign transplants; antigens presented to Tc cells are endogenous
What are the 6 major categories of immune-mediated human disease? (Give an example of each)
- Allergies and hypersensitivity conditions: bee sting or nut allergy; an inappropriate and exessive response to environmental antigen
- Autoimmune disease: Lupus and Type I diabetes; tissue mediated by immune attack on self
- Transplantation rejection: kidney and pancreas; has to do with types and amount of receptors on donor organ
- Cancer: lymphoma and myeloma
- Immunodeficiency: SCID and AIDS; HIV targets CD4 Th cells; patients present with recurrent infection
- Infection-related: sepsis and toxic shock; over-stimulation of immune system with microbial superantigen
What are the different categories of innate immunity?
- Skin and mucous membranes
- Mucociliary movement (lungs) and peristalsis (gut)
- Lung surfactants (low surface tensions promotes physical clearance; also has opsonins to promote opsonophagocytosis by macrophages)
- Low pH (sstomach)
- Fatty acid content (skin)
- Mucins (GI tract and lung)
- Competition by normal microbial flora
- Iron-binding proteins (compete for iron at sites of infection)
- Enzymes (pepsin, trypsin, and chymotrypsin in GI tract; lysozymes in slaiva, sweat and tears; degrades bacterial cell walls)
What are the 3 innate immune mechanisms of host defense against microbes that have penetrated the first lines of defense?
- Direct lysis of microbe
- Phagocytosis and intracellular killing of the microbe
- Danger signaling to other immune cells
- Pathogen associated molecular patterns
- Widely distributed structures found on many diverse organisms
- Essential to microbial survival (microbes really don't mutate)
- Host's ability to recognize PAMPs is highly conserved and encoded by germ line genes; primitive (i.e. cell wall damage)
How is LL37 related to innate immunity?
- Located in the vernix caseosa of newborns (sticky stuff on skin)
- Disinfectant function; non-specific microbicidal function
- Member of cathelicidin family; containes leukocyte granules
- Recognizes negative charge on the outside membrane of cells (in humans, negative charge is only on the inside); charge pattern recognition
- Intercalate in membrane and cause damage
Hos is LPS related to innate immunity?
- Lipopolysaccharide is an endotoxin located on the outer membrane of gram-negative bacterial cell walls
- Recognition of LPS leads to danger signaling; LPS is the leading cause of septic shock
- LPS bidns to soluble host LBP (LPS-binding protein); this complex binds to CD14 on host cell membrane; CD14 passes it off to TLR4; intracellular signaling results in danger signaling
What is the function of TLR4?
- Toll-like receptor 4 interacts with the LBP-LPS complex through the help of CD14
- It is linked to transcription factors via adaptor proteins (MyD88), signaling kinases (IRAK-interleukin-1 receptor associated kinase-phosphorylates molecules to activate transcription factors) and transcription factors (NFkappaB and IRF-3; initiate transcription of inflammatory genes)
- TLRs also trigger phagocytosis and intracellular killing of microbes
What is the function of TLR9?
- Located on the endosome/phagosome membranes within the cytosol
- It can distinguish bacterial DNA with hypomethylated CpG motifs
- Adaptor protein (similar to MyD88)-->IRAK-->NFkappaB pathway
Define NLR and function
- NOD-like receptors
- Cytosolic proteins that recognize intracellular pathogens that may have escaped the phagosomes
- Recognize conserved patterns (i.e. bacterial flagella)
- Trigger inflammatory gene expression and possible cell death of infected cells
- Cytosolic equivalents of membrane TLR
- Binding enhancer that promotes phagocytosis; binds to negative charge on invader's membrane and then binds to phagocytic cells
- Usually neccessary for phagocytosis to occur (opsonophagocytosis)
- CD14 receptor for MBP binds to properly spaced microbial mannose residues to promote phagocytosis
- Complement receptors bind complement peptides
- Fc receptors bind antibodies
- Process of engulfing the foreign particle
- Usually attracted to the site of infection by chemotaxis
- Steps: Diapedisis and chemoattraction brings the cell into contact with the foreign body; recognition and attachment; ingestion into the phagosome; fusion of pahgosome with lysosome (phagolysosome); killing and digestion; release of particles or display on MHC
- Receptors on the membrane surface of neutrophils and macrophages bidn to chemoattraction molecules from the site of infection or injury
- Movement is directed toward the area that has the highest gradient of chemoattractants
- Microbe-derived examples: lipotechoic acid (bacterial cell wall) and bacterial formyl-methionyl peptides
- Host-derived examples: antimicrobial peptides (defensins), fibrinogen-derived peptides, complement peptides (C5a), inflammatory mediators (LTB4), and chemokines (IL-8)
What are the 3 principal mechanisms of intracellular killing by professional phagocytic cells?
- Oxygen-dependent killing (respiratory burst): NADPH oxidase turns O2 into superoxide; superoxide dismutase turns this into hydrogen peroxide; myeloperoxidase turns this into hypochlorite (bleach); hydrogen peroxide can also undergo Fenton reaction to produce hydroxyl radicals; NEUTROPHILS use this more than macrophages
- Nitrogen dependent killing: NO synthase converts L-Arginine, NADPH and O2 to nitric oxide (NO) and L-citrulline; iNOS isoform is used; NO effective against certain intracellular bacteria and parasites
- Oxygen and nitrogen independent killing: Important for anaerobic condition (i.e. deep tissue abscesses) or when resp. burst fails (catalase wielding bacteria); includes defensins and cathelicidins (LL37), bacterial permeability-increasing protein (BPI), lysozyme, lactoferrin, lysosomal enzymes (cathepsin G, lipases, and nucleases)
When is oxygen-dependent killing ineffective?
In anaerobic conditions and with bacteria that have mechanisms to overcome it (i.e. catalase (anti-oxidant) that converts hydrogen peroxide to water and oxygen)
What is the function of epithelial cells in innate immunity?
- Barrier function
- Mucus secretion (goblet cells-apical side)
- Defensin production (paneth cells)
- Respond to microbial flora (produce chemotactic factors to attract lymphocytes and leukocytes-basal side)
What is the function of intra-epithelial lymphocytes in innate immunity?
- Present in intestinal and lung epithelium; primarily T cells
- Express Ag receptors with limited diversity
- Recognize conserved microbial structures
- Produce cytokines that activate inflammatory cells
What is the function of natural killer cells in innate immunity?
- Bind to conserved ligands on microbes and produce ctyokines
- Recognize microbe-infected host cells and lyse them
- Also recognize damaged or stressed host cells
What is the function of chemokines in innate immunity?
- Bind to surface receptors and direct migration of immune cells
- Operate in a gradient concentration fashion
What is the function of cytokines in innate immunity?
- They are the hormones of the immune system
- Bind to cell surface receptors and mediate cell signaling
- TNFalpha released from macrophages binds to neutrophils and induces upregulation
How does complement function in the innate immune system?
- Through 3 pathways: Classical (think of this more as adaptive), alternative, and lectin
- Activation induces lysis of microbes, neutrophil chemotaxis, opsonophagocytosis, and increased vascular permeability
How does coagulation function in innate immunity?
- It functions to limit the spread of infection
How does fibrinolysis function in innate immunity?
- Produces plasmin which can cleave and activate complement
- Fibrinolysis also works against coagulation; regulation
What do innate immunity and acute inflammation have in common?
- Pattern recognition
- Rapid response
- Danger signaling
- Leukocyte mobilization
- Membrane attack
What are the key differences between innate and adaptive immunity?
- Innate immune response is rapid (seconds to days) and the same each time the antigen is encountered; includes lysis and phagocytosis of microbes, complement activation, NK cell activation, danger signaling, and cytokine production
- Adaptive immunity has a small initial response that primes it for a much larger and quicker response upon a second encounter; can takes days to weeks to become establised; memory cells are primed for another encounter; includes antibody production, memory cell idnuction, T cell cytokine release, macrophage activation, cytotoxic T cell activation
Products released in the _______ tend to influence the _________
- Innate response and adaptive response
- Both systems utilize complement and phagocytosis
- Systemic inflammatory response; extreme response to PAMPs or DAMPs; leading cause of death in the ER
- Initiated by infection, acute trauma, or massive tissue damage
- Characterized by fever (pyrogens), leukocytosis, production of acute phase proteins and mediators of tachycardia, tachypnea, and hypotension, decreased sweating, chills, and loss of conciousness
- Watch the lungs, blood, and urniary tract
Define bacteremia and endotoxemia
- Bacteremia: bacteria in the blood
- Endotoxemia: endotoxins in the blood
- SIRS caused by an infection
- If the systemic response is due to hypersensitivity or allergen, it is not sepsis
What pathology makes SIRS and sepsis different from a normal immune response?
- Overproduction of cytokines (TNFalpha, IL-1beta, and IFNgamma)
- Thrombosis, disseminated intravascular coagulopathy (DIC)
- Hypotension-->hypoxia-->decreased tissue oxygenation-->ischemic injury
- Acute respiratory distress syndromes (ARDS), cardiac suppression, renal damage
- Organ damage and death
What are the 5 specific functions of the complement system?
- Lysis of cell membranes: MAC complex (C5b6789n is fast, C5b678 is slow, but still effective)
- Opsonization: C3b and C4b (iC3b and iC4b)
- Inflammation: Leukocyte chemotaxis, increased vascular permeability, vasodilation
- Solubilization and clearance of immune complexes: C3b binds to Ag-Ab complexes and solubilizes them; phagocyte and RBC-mediated clearance; deposition of these leads to disease (RA)
- B lymphocyte co-activation: C3d bound to Ag co-activates B cells through CR2
At what point do all complement pathways converge?
- C3 convertase cleaves C3 to form C3a and C3b
- C3 convertase + C3b-->C5 convertase followed by noncatalytic assembly of the MAC complex
Define classical complement pathway
- Activated by Ag-Ab complexes; adaptive immunity
- C1q binds to CH domains of mu and gamma heavy chains (cross-links 2 adjacent sites, meaning that IgM is more efficient than IgG because it's pentameric); C1q recruits 2 copies each of C1r and C1s to form active enzyme (C1qr2s2); active enzyme cleaves C4 and C2 to form classical C3 convertase C4b2a; goes on to cleave C3 to form C3a and C3b
Define lectin complement pathway
Initiated by MBL bidning to microbial carbohydrate (mannose); recruits a C1-like protease (MASP); MASP cleaves C4 and C2 to yield C4b2a (lectin C3 convertase); goes on to cleave C3 to form C3a and C3b
Define alternative complement pathway
- Constitutively active; can be further activated by production of C3b in classical and lectin pathway, spontaneous C3*H20 hydrolysis to form C3B, or surface binding (pattern recognition of microbes=innate) to form C3
- C3B is cleaved by Factor D (enzymatic activity) to form Bb and Ba
- C3(H2O)Bb is unstable and falls apart
- C3bBb is unstable and falls apart unless Factor P binds it (highly stable C3 convertase=C3bBb)
In general, what are the similar processes that all 3 complement pathways go through?
- Initiation (assembly of a serine protease or upregulation of a constitutively active one)
- Sequential cleavage of inactive zymogens to form proteolytic enzymes (convertases)
- Formation of a C3 convertase is a key step; this is where the most control is
- Amplification at each step results from cleavage of multiple substrate molecules
What is the positive feedback associated with the alternative pathway of complement?
formation of C3b in the lectin and classical pathway feeds into the alternative pathway to further activate complement
What are the C3 and C5 convertases of the lectin and classical complement pathways?
C4b2a and C4b2a3b
What are the C3 and C5 convertases of the alternative pathway, respectively?
C3bBb (C3 convertase) +C3b + factor P=C5 convertase
What does C3 tickover refer to in the complement cascade?
The internal positive amplification loop built into the alternative pathway
What is the significance of the thioester bond in C3b and C4b in the complement cascade?
The exposed internal thioester bond of these two molecules is formed between the glutamine and the sulfide of serine; it is unstable and readily rearranges to form an amide and ester linkage site; the binding of an Ag site stabilizes C3b and C4b for continued complement activation
What is the structure and function of MAC?
- Membrane attack complex; relevant in complement
- C5b + C567, C8, and C9n forms pore in gram negative bacterial cells that is known as MAC
- Causes lysis of the cell
- Could attack gram positive bacteria if there was lysozyme around to degrade the peptidoglycans surrounding the membrane
- Can have lysis of a host cell membrane if DAF is deficient (autoimmune Ab-mediated hemolysis is an example)
In the complement cascade, which peptides are involved in opsonization?
- Complement Mediator: C3b and C4b (iC3b and iC4b)
- Complement receptor: CR3 and CR4>CR1 (phagocytes)
- Diseases Associated: LAD-1 and Factor I deficiency
In the complement cascade, which peptides are involved in lysis?
- Complement mediators: All
- Inhibitory factors: Protein S, HRF, and MIRL
- Disease Associated: any that result in complement peptide deficiencies
In the complement cascade, which peptides are involved in clearance?
- Complement Mediators: C3b
- Complement Receptors: CR1 (mostly erythrocytes, some neutrophils and macrophages)
- Inhibitory Factors: Factor I
- Diseases Associated: RA
In the complement cascade, which peptides are involved in inflammation (Chemotaxis)?
- Complement mediators: C5a
- Complement Receptors: C5aR (neutrophils)
- Inhibitory factors: AI
- Diseases Associated: Hereditary Angioedema
In the complement cascade, which peptides are involved in inflammation (anaphylaxis)?
- Complement Mediators: C5a>C3a and C4a
- Complement Receptors: C5aR> C3aR and C4aR (mast cells, basophils, smooth muscle cells)
- Inhibitory factors: AI
- Diseases Associated: Hereditary angioedema
In the complement cascade, which peptides are involved in B cell co-activation?
- Complement Mediators: C3d
- Complement Receptors: CR2 (B cells)
What are the regulators of complement, and how do they function?
- Factor P: stabilizes C3 convertase C3bBb
- C1 Inhibitor: Blocks the classical pathway by dissociating C1qrs; this is deficient in hereditary angioedema
- C4b Binding protein (BP): inhibits the formation of the classical and lectin C3 convertase through the separation of C4b and 2a
- CR1: blocks formation of C3 convertase
- S protein: blocks assembly of MAC (protects host cells)
- DAF: decay accelerating factor: membrane protein widely distributed on our cells but not on bacteria; induces decay of MAC on host membrane
- Factor I: constitutively produced factor; cleaves and inactivates C3b and C4b to iC3b and iC4b (still opsonins)
Which lab tests are diagnostic for complement deficiencies?
- Immunoassay (ELISA): checks C1, C2, C3, C4 and Factor B levels; can be used to find systemic lupus (SLE)
- Total Hemolytic complement: measuers the classical pathwya components and the activity of the complement system; can be used to detect hereditary angioedema (HAE) and paroxysmal nocturnal hemaglobinuria (PNH)
- Opsonophagnocytic activity: checks uptake of antibody coated bacteria by neutrophils in the presence of fresh nonimmune serum; can be used to detect lekuocyte adhesion defect (LAD)
LAD, PNH, HAE, and SLE are disease most closely associated with what?
Defects in the complement pathways
- leukocyte adhesion defect
- Recurrent bacterial infections with poor pus formation; neutrophils cannot get to infection site because of deficiency in opsonophagocytosis
- Deficiency in CD18 gene (member of beta2 integrin family); forms heterodimers with alpha-beta receptors
- CD11a/CD18=LFA-1; cell adhesion
- Paroxysmal nocturnal hemaglobinuria
- Absence of DAF, CD59, HRF, etc. (phosphotidal-inositase (PI) linked proteins; non-transmembrane, they don't signal
- Results in stable membrane bound C3 convertases and sporadic hemolysis due to spontaneous C3 activation on membranes and resulting host cell lysis; erythrocytes are highly susceptible
- Hereditary Angioedema
- Acute, nonpainful, nonpruitic and nonerythematous swelling of the skin and mucous membranes; edema of the bowel wall; laryngeal swelling is potentially life threatening due to asphyxiation
- Unregulated classical complement activation; C1 inhibitor deficiency (serine protease that regulates C1qr2s2, Hageman factor, and plasmin)
Patients with deficiencies early in the classical complement pathway have an increased risk for what type of disorder?
- Autoimmune; especially lupus-like nephritis; inability to clear immune complexes that contain C3b generated by the classical pathway
- Nephritis from complexes blocks up the small glomeruli
- Systemic Lupus Erythematosus
- Depressed levels of C1, C2, C3, and C4 during acute exacerbations
- Normally functioning complement system deposits intermediates on immune complexes; these complexes are cleared by CR1-expressing RBCs; results in depletion of classical pathway components from circulation
What properties of adaptive immunity distinguish it from innate immunity?
- Specificity: B cell expresses one Ag receptor
- Diversity: 4 billion varieties
- Self-nonself discrimination
- Memory: expansion involves creating memory B cells specific to single antigen
- Transferable: passive immunization; transfer serum
What is the difference between the primary and secondary responses in adaptive immunity?
- Primary response: predominated by IgM; macromolecular response
- Secondary response: predominated by IgG; smaller reaction of IgM
- IgG is much less expensive (biologically speaking) for the cells to make; smaller in size; half life of IgG is much longer in serum; affinity is higher than IgM
- IgG can serve as opsonin (has receptors on macrophages) and can cross placenta to the fetus while IgM cannot
What is the difference between active immunization and passive immunization?
- Active: like a vaccination, infection, transfusion, transplantation, or pregnancy
- Passive: requires transfer of Ab; gammaglobulin therapy, transplacental or translacteal passage of Ab
What is the clonal selection theory of adaptive immunity?
- all Ab forming cells are pre-committed in their specificity; one cell is specific for only one Ag; cell activation begins with Ag binding to a surface receptor and leads to the expansion of a clone of cells (all have the identical specificity of progenitor cell)
- Ag receptor on a given cell is uniform and identical witht he Ab the cell secretes; only B cell that would be activated was the one that had Ag bound to it
Which cells are most commonly associated with the humoral immunity pathway?
B cell lymphocytes; most are terminally differentiated plasma cells; produce antibodies; have a BCR (membrane form of immunoglobulin) and several co-receptors (including CR2)
Which cells are most commonly associated with cell-mediated immunity?
T cell lymphoctyes; thymus derived T cells; 2 major subsets include T helper (TCR and co-receptor CD4 which operates in the activation of the cell) and Cytotoxic T cells (TCR and co-receptor CD8 which operates in the activation of the cell as well)
Compare and contrast B and T lymphocytes
- Similarities: diverse Ag-binding regions; expressed in clonal fashion (ever BCR/TCR ona single cell is identical); expressed in a complex of other proteins that transmit signals
- Differences: BCR recognizes determinants on native antigens (hydrophillic, highly charged peptides) while TCR recognizes antigenic peptides presented by APC (requires cellular uptake and processing of protein Ags via proteolysis, subsequently presented on MHC molecules)
Which cells are APCs?
- All cells
- Professional APCs include dendritic cells (not neuronal), macrophages, and B cells
Which type of T cell recognizes endogenously and exogenously derived pathogens, respectively?
- Endogenously derived (i.e. viruses) are recognized by Cytotoxic T cells (CD8 co-receptors); involves the MHC 1 receptors on APCs
- Exogenously derived (taken from outside cell and processed within APC) are recognized by T helper cells (CD4 co-receptors); involves the MHC II complex
Cells that respond to antigenic peptides (proteins) presented on MHC molecules of APCs are
- T cells; they do not recognize carbohydrates and lipids because these do not stick well to MHCs
- 2 classes of MHC exist: MCH I recognized by CD8 co-receptor of Cyt T cells (endogenous) and MHC II recognized by CD4 co-receptor on Th cells (exogenous)
Co-receptors, cytokine receptors,c ytokines, and adhesion molecules are key for waht process in immune response?
CD4, TCR, CD3, CD28, CD40L/CD154, and CD45 are proteins on the surface of what type of cell?
Helper T cells
CD8, TCR, CD3, and CD45 are proteins on the surface of what type of cell?
Cytotoxic T cells
IgM, IgD, B7, CD19, CD20, CD21, CD40, MHC II, and CD45 are proteins on the surface of what type of cell?
MHC II, B7, CD40, CD14, and receptor for Fc and C3b are proteins on the surface of what type of cell?
CD1, Receptors for MHC I, CD16, and CD56 are proteins on the surface of what type of cell?
CD18 combined with CD11a, b, and c are proteins on the surface of what type of cell?
MHC I surface proteins can be found on what type of cell?
All cells except mature red blood cels (anucleated)
CD34 is a surface protein found on what type of cells?
Hematopoietic stem cells (and all cells derived from this population)
What is Burnett's Rule?
- A single antibody only has one specificity; one B cell will not be able to make different variable Ag binding sites\
- 10^8-10^9 antibodies=diverse repertoire
Describe the germling genetic organization of the 3 gene loci that encode Ig polypeptides
- Found in all cells of the body; however, only B cells are capable of rearranging these genes (pre-B cells or immature ones)
- 3 Ig gene families at separate loci
- Kappa locus: 1 constant gene segment; over 40 variable gene segments; handful of joiner (J regions) gene segments; L chain
- Lambda locus: L chain; similar to kappa
- Heavy chain locus: Many variable gene segments (>40); handful of joiner gene segments; ~25 diversity gene segments; 8-9 C gene segments (code for the variety of isotypes)
Define V, D, J, and C gene segments
- At the various Ig gene loci
- C gene segments: constant regions; only one in light chain segments (either kappa or lambda version); 8-9 in heavy chain segments; determines the isotype
- D gene segments: diversity segments
- J gene segments: joiner region segments
- V gene segments: variable region genes
What is the order of alleles in the Ig gene loci?
- mu, delta, gamma3, gamma1, gamma2b, gamma2a, epsilon, alpha
- Mu does not have a transcriptional stop site so whenever mu is transcribed, so is delta; making either mu or delta heavy chain depends on polyadenylation RNA splicing
How are heavy and light chains of the Ig molecule joined? (What is the order?)
- Heavy chain: D-J joining, V-DJ joining, VDJ-C joining
- Light chain: V-J joining, VJ-C joining
- Activation of transcription by rearranged promoters and enhancers, synthesis of a primary RNA transcript, RNA processing and polyadenylation, protein translation on polyribosomes, assembly of intact Ig (2H and 2L chains)
What is the function of RSS in Ig formation?
- Recombination signal sequences
- Directs the cleavage of DNA
- Imprecise re-joining adds diversity: for examples, D-J joining (same D and J could create different Abs by joining flexibility, just one nucleotide deletion can alter whole structure) and V-DJ joining (additional nucleotides can be added at joining sites)
What is the function of RAG in Ig formation?
- Plays an important role in gene recombination during VDJ recombination (H and L chains)
- Deficiency in this leads to severe combined immune deficiency (SCID); dramatically alters lymphocyte differentiation; severe lymphopenia; no B or T cells
In reference to Ig formation, what is meant by allelic exclusion?
- If a locus rearranges productively, rearrangement on the other chromosome is suppressed; this ensure that only one H and one L chain are synthesized by one B cell
- A diploid chromosome means that 2 opportunities are offered at each loci to create a successful rearrangement (if kappa rearrangement doesn't work, try other chromosome; if both kappas are unsuccessful, try the lambda locus)
How does somatic hypermutation contribute to diversity of the V regions of Ab molecules?
- Really only contributes to the diversity of memory B cells; only occurs in previously rearranged IgV genes; Ag-driven; T-cell dependent; occurs in germinal center B cells
- Random mutations to increase affinity; must occur after class switching from mu (pentameric) to G
How does BCR editing contribute to diversity of the V regions of Ab molecules?
Give autoreactive immature B cells another chance to rearrange their Ig chains (L chain only); if unsuccessful (95-98% of the time), clonal deletion
How does affinity maturation contribute to diversity of the V region in Ab molecules?
Small point mutations (somatic hypermutation) can be either tighter or looser binding to Ag; tighter binding Abs will proliferate and take over the attack
What is the difference between the potential repertoire and the utilized repertoire in Ig formation?
- The utilized repertoire excludes nonproductive rearrangements, deleted Igs that show self-reactivity, nonfunctional pairings of H and L chains, and deleted Igs that bind to tightly to Ags
- Thus, utilized repertoire=potential minus those that are actually deleted
Evidence shows that a single B cell can express multiple Ig molecules. Explain this
- Although a single B cell may experss mutliple class of Ig molecules (i.e. mu and delta transcribed together, and class switching), it will express molecules that are all specific for only one type of Ag
- Only the heavy chain constant domains differ: (variable regions express the same antigen specificity)
Define Ig Class Switching
- Ag-drive and Th cell-dependent
- Switch is irreversible (if you have an IgG cell, means that IgM has had its DNA looped out and cannot reform IgM); can have IgM-->IgG1 switching (excision of Cm, Cdelta, Cgamma3 all in one fell swoop)
What is the molecular basis for biclonal and monoclonal myeloma?
- In multiple myeloma, plasma cell tumors crowd out normal components of the marrow resulting in decreased RBCs, platelets, and WBCs
- Clinically, shortness of breath, fatigue, excessive bleeding, and increased susceptibility to infection
- Production of M protein (monoclonal, abnormal immunoglobulin) by these plasma cells causes high protein levels in the blood; these can lodge in the kidneys and mess with blood flow
- Very rarely, the patient may produce two different M components in their plasma (biclonal)
What is the differnce between membrane and secreted Igs in B cells?
- Both are produced by a single B cell
- The 2 differ only at their C-terminal ends where the membrane Ig has a membrane spanning region; difference is determined at teh RNA level
- One RNA can encode btoh membrane and secreted forms of mu heavy chain; there are 2 poly-A sites; if cut at the first adenylations ite, secreted; if cut at the second adenylation site, transmembrane domain is added and membrane receptor is formed
What are the key steps in the differentiation of B cell?
- Start with hematopoietic stem cell
- Stimulation makes it into lymphoid stem cell (pro-B cell; no Ab receptor)
- Ig Heavy chain rearrangment (pre-B cell; VDJ chain arrangement; RAG 1/2 expression and Tdt expression)
- Expression of the Pre-BCR; contains surrogate light chain (helps it get to the surface), heavy chain, and Ig alpha/beta which is involved in intracellular signaling to begin L chain rearrangement
- Positive selection
- Ig L chain rearrangment and expression of the BCR (IgM); RAG 1/2 expression, BCR editing, clonal deletion
- Imamture B cells exit from bone marrow
- Additonal maturation in the periphery (somatic hypermutation, affinity maturation)
- mIgM and mIgD co-expression in mature B cells
- Positve and negative selection (recognition of self-Ags) throughout last couple steps
Positive and negatiev selection in B cell maturation involve signaling through which receptors, respectively?
- Positve: Pre-BCR and BCR
- Negative: BCR only (mIgM)
What occurs when a B cell confronts a T cell-dependent antigen?
- Binding native antigen to BCR
- Activated Th cell contact with B cell (MHC II-TCR; must involve contact with co-receptors on Th cells)
- Th cell help via Th released cytokines (IL-2, 4, 5, 6); cytokine receptor stimulation and activation on B cells
Define memory B cells
- High affinity, long-lived B cells
- Have undergone class switching, somatic hypermutation, and affinity maturation
- Develop in the lymph node germinal centers
Describe intracellular signaling that accompanies B cell activation through BCR
- BCR binds Ag; Src kinase phosphorylates ITAM motif which recruits and binds to Syk (similar to Zap-70 in T cells); phosphorylates and activates Btk; activates PLC gamma; PLC cleaves PIP2 to make DAG (NkappaB activation) and IP3 (NF-AT activation)
- BCR signaling also involves GEF activation and small G protein pathways involving Rho, Rac, and Ras
How does CR2 on the surface of B cells affect intracellular signaling?
Recognizes C3d antigens and activates the Src kinase pathway
Which cytokines are important for B cell maturation and activation?
- IL-2: Growth (cell cycling) and increased IL-4 receptor
- IL-4: Switching from IgM to IgG and IgE and increased MHC class II expression
- IL-5: Switching to IgA and increased Ig synthesis
- TGFbeta1: switching to IgA
- IL-6: differentiation into plasma cells
- IL-13: switching to IgE
What is the function of the various receptors on the surface of B cells?
- Fcgamma: receptor for Ab-Ag complexes
- MHC II: presentation of Ag to CD4 T cells (Th)
- CD40: co-receptor that binds CD154 on T cells
- B7: co-receptor ligand that binds CD28 on T cells
- CR2: co-receptor that binds C3d on Ags
- Cytokine receptors: IL-2, 4, 5, 6 etc.
- BCR: membrane IgM and IgD in complex; binds Ags to activate cell
What is a T independent Ag and how do B cells respond to such crap?
- TI Ag: repeating identical determinants (i.e. polysaccharides)
- Pattern recognition receptors detect these
- B cells respond to these with less Ig class switching (less cytokines present) and mostly IgM response (some IgG2); little somatic hypermutation and no memory cells formed
What are asplenic patients at risk for?
- T independent pathogens; esp. polysaccharide encapsulated bacteria
- The spleen cotnains a unique population of B cells that recognize TI Ags
Which type of vaccine is better, TI or TD?
- A T independent vaccine induces IgM (low affinity for Ab); no memory is developed, rendering the vaccine virtually useless
- A T dependent vaccine induces IgG1-4 response with a much higher affinity for the Ag; memory B cells are produced
- To turn a TI pathogen into a TD pathogen for vaccines, covalently link the polysaccharide to a protein (with flu example, we talked about linking it to diptheria toxin which is internalized by ACP, degraded and presented on MHC)
Describe the process of differentiation and maturation of T cells
- Pluripotent hematopoietic stem cell in bone marrow recieves stimulation to becomes a lymphoid progenitor cell
- T cell precursor migrates to the thymus
- Decides to be alpha-beta or gamma-delta T cell (95% are alpha-beta)
- TCR beta gene rearrangement (Tdt expressed; RAG1/2)
- Expression of pre-TCR
- Positive selection (cells become permissive for alpha-chain locus arrangement and stops additional beta chain rearrangement)
- TCR alpha gene rearrangement and expression of TCR
- Positive and negative selection (screen for functionality and autoreactivity; if a TCR can not bind its own MHC it is thrown away)
- CD4/CD8 lineage committement (RAG 1/2 expressed)
- Exit from the thymus into the periphery
In reference to thymocyte (T cell) development, what are the DP, DN, and SP stages?
- Double Negative: Pre-TCR; positive selection; RAG 1/2 turned on for rearrangements
- Double positive: TCR alpha/beta; CD4 and CD8 expressed; undergoes positive and negative selection, RAG 1/2 not expressed; part of allelic exclusion
- Single Positive: TCR alpha/beta; either CD4 or CD8 expressed; RAG turned back on to select for CD4 or CD8
What is the role of MHC in defining the repertoire of T cells?
- Happens in the thymus
- Has a role in positive selection; only cells expressing TCR that can bind MHC peptide complexes will proliferate; cells that fail to bind undergo apoptosis
- Different individuals express different MHC genes; mature T cells bear receptors restricted to self MHC of individual person; cause of transplant rejection (i.e. someone may express HLA-DR2 receptor but not HLA-DR4)
What are the 3 possible outcomes of the DP stage of T cell development?
- TCR binds MHC class I; differentiates into SP CD8 T cell
- TCR binds MHC class II; differentiaties into SP CD4 T cell
- TCR binds MHC peptide complexes in the thymus; results in elimination of autoreactive T cells
What are the 3 possible outcomes of positive and negative selection in T cell development?
- Weak MHC binding: positive selection
- No MHC binding: death by neglect
- Strong MHC binding: negative selection; leads to autoimmune disease if not eliminated
What must happen for optimal T cell activation by an antigen?
- TCR activation
- Co-receptor activation (CD4 binding MHC II, CD8 binding MHC I adn CD28 binding B7)
- Cytokine-mediated activation (makes IL-2 and IL-2R; autocrine signaling and propogation of memory/effector cells; IL-4, IFNgamma, IL-10)
What kind of T cells do professional APCs activate?
- Macrophages: effector and memory
- B cells: naive, effector, and memory
- Dendritic: naive, effector, and memory
CD4 T cells can differentiate into Th1, Th2, Treg, and T17 cells. What are the differences between these?
- Th1: promotes lymphocyte growth; affects macrophages and Tc cells; functions in cell-mediated response; utilizes IFNgamma and TNFalpha (when an APC presents to a Th cell, it's not a Th1 or Th2 yet; becomes one of the other based on what the cell needs)
- Th2: promotes lymphocyte growth; affects B cells; functions in humoral immunity; utilizes IL-4, IL-5, IL-13
- Treg: secretes IL-10 and TGFbeta to inhibit lymphocyte and macrophage activation; expresses CD25 and FoxP3; defects in this may lead to autoimmune disease (IPEX and APEDCED)
- Th17: found in intestines; induces inflammation (implicated in Crohn's disease); produces IL-17; differentiation is dependent upon TGFbeta and IL-6 (balance between these and Tregs)
How do CD8 T cells function?
- Activated by endogenous pathogens (viruses) and Th1 subset
- Kill by perforin/granzyme, Fas/Fas-ligand, TNF mediated mechanisms
How do NKT cells function?
Specfic for glycolipids and CD1; express invariant TCR alpha chain (important marker) and utilize the same killing mechanisms as cytotoxic T cells
How do NK cells function?
- Cytotoxic lymphocyte (not T cells) that kill rapidly and without proliferation; kill by perforin/granzyme, Fas/FasL, or TNF mechanisms; recognize targets that lack MHC class I (missing self; many tumor cells and some viruses will downgrade their MHC receptors and NK cells recognize this and kill them)
- Produce IFNgamma cytokines
- 2 types of receptors: activating receptors (recognize a variety of ligands on target cells) and inhibitory receptors (recognize non-polymorphic residues on MHC class I); the inhibitory signal is dominant over the activating signal
What is the molecular basis of SCID?
- Severe combined immunodeficiency
- Mutations in the genes for RAG recombinases, CD3, ZAP-70, TAP proteins, and IL-2 receptor
- Maternal IgG provides immune protection for the first few months of life
- Hematopoietic stem cell transplantation is the only practical cure
What is the molecular basis of oral thrush?
caused by infection by opportunistic yeast Candida albicans; most frequent immune deficiencies that can lead to oral thrush include SCID, seletive T cell deficiencies (DiGeorge syndrome, CD3 mutation) and MHC deficiencies affecting T cell differentiation
What is the molecular basis of MHC deficiency?
- Bare lymphocyte syndrome 1 and 2
- Inefficient positive selection onf thymocytes in the thymus; congenital; MHC classes I and II are not expressed
- BLS2 patients are lymphopenic because CD4 T cells would normally constitue a majority of T cells in the blood; before diagnosing this, must eliminate HIV-1 infection (much more common cause of decreased CD4:CD8 ratios)
What is the molecular basis of ALPS?
- Autoimmune lymphoproliferative syndrome
- Defect in apoptosis signaling in activated T cells because of mutation in Fas; apoptosis is central to thymocyte selection
- Patients present with enlarged lymph nodes, splenamegaly, and autoimmunity (i.e. Coombs positive hemolytic anemias); show lymphcytosis and elevated numbers of unusual double negative T cells
How are TCRs and BCRs similar?
- Members of the immunoglobulin superfamily
- Variable and constant regions present
- Transmembrane spanning regions with short intracellular tails that don't do anything on their own (associated with accessory proteins; charges between receptors and accessory proteins keep them next to each other)
- Alpha and beta chains of T cells are similar to heavy and light chains of B cells
A patient missing CD3 would have what problem?
- The TCR complex would fall apart
- Lymphopenic (with B cell abnormalities as well; Th2 cells activate B cells)
What is the difference between alpha/beta and gamma/delta in teh TCR?
- alpha/beta are primarily in the lymphoid tissue and comprise 95% of T cells
- gamma/delta T cells primarily in the skin/gut; little variability in comparison to the alpha/beta pool
What are the genetic mechanisms for generating diversity in the TCR?
- Same VDJ recombination that generated Ab diversity is present in TCR diversity
- D-J joins; V-DJ joining; VDJ-constant joining
- Junctional diversity (additon and deletion of nucleotides at the junction regions)
- Alpha of TCR is similar to light chain of BCR; however, there are no D segments in the alpha locus region; BUT, there is a delta locus interlaced in the alpha locus that does contain D segments; if you're making an alpha/beta TCR, you loop this delta region out
- No somatic hypermutation
- TCR editing is almost non-existent
- No isotype switching because there are no isotypes
What are the natural ligands fo TCR?
T cells recognize and bind short peptides and they only recognize these when they are processed and presented on the MHC (peptide/MHC complex)
Define superantigen and relate it to toxic shock syndrome
- A superantigen binds the constant regions of the TCR and MHC; thus, TCR/MHC binding independent of antigen; microbial antigens can cross-link TCR and MHC in this way; leads to polyclonal T cell responses (nonspecific) and cytokine storm
- TSS is caused by exotoxin TSST-1 (Staph) which is a superantigen; leads to polyclonal activation of T cells, massive cytokine production, activated macarophages and endothelial cells, production of inflammatory mediators, hemodynamic shock, organ failure, and eventual death
What is unique about the TCRs located on NKT cells?
recognize glycolipids instead of peptides
Why are co-receptors important in MHC-TCR interaction?
necessary to overcome the low affinity binding that occurs; sticks cells together long enough for MHC and TCR interactions to result in intracellular signaling; provide additional signaling
Where would you find MHC I that you wouldn't find MHC II?
- MHC I expressed ubiquitously on all nucleated cells
- MHC II only on B cells, dendritic cells, and macrophages
- B7 co-receptors are expressed on B cells, dendritic cells, macrophages, and some T cells as is CD40
LFA-1 is a co-receptor on T cells that binds to what?
ICAM-1 on APC (present on almost all cells)
When a TCR is bound by APC, what happens?
- Must first cluster receptors (brings the kinases into same place); phosphorylation of CD3 domains; recruitment and activation of ZAP-70 (tyrosine kinase) which leads to the following intracellular responses
- PLCgamma (phospholipase C)-->DAG-->PKC-->IkK which phosphorylates inhibitor of kappa B (IKB) which activates NFkappaB (transcription factor)-->production of IL-2, IFNgamma, TNFalpha
- PLCgamma-->IP3-->binds to IP3 receptor on ER-->increased calcium in cytosol-->activation of calmodulin/calcineurin-->dephosphorylates NF-AT which frees it to enter the nucleus and transcribe IL-2, IL-4, IFNgama, and TNFalpha
- Mediated by preformed transcription factors
What does the drug cyclosporing target?
- The activation of NF-AT in T cells that interact with APCs (inhibits de-phosphorylating of NF-AT by calcineurin)
- Very important for transplant surgery
When does anergy occur?
- Stimulation of TCR by APC alone
- State of unresponsiveness
- TCR + co-receptor (i.e. CD28) results in activation and differentiation
What is the mechanism of killing for cytotxic T cells?
- Once bound to a receptor on an infected cell, it becomes activated; granules line up along the membrane (cytoplasmic rearrangement); CTL then exocytoses these granules (granymes and perforins); perforins make pores in membrane and granzymes go into cell and trigger the caspase cascade; results in apoptosis
- Activation also causes Fas ligand to be expressed on T cell; Fas is a death receptor present on the target cell that bidns to fas ligand and results in the caspase apoptosis pathway
Which type of cell recognizes peptides/MHC complexes, has enhanced activation by Th1 cytokines, and requires direct contact and conjugate formation for killing?
Cytotoxic T cells (CTLs)
What is the difference between antigenicity and immunogenicity?
- Antigenicity: reactive with Ab (BCR) or T cells (TCR); antigenic determinant or epitope
- Immunogenicity: ability to induce an immune response (immunogen)
- Molecular mass (>10kDa), molecular complexity, conformation, and phylogenetic disparity from the host (the more similar it is) are the key factors that determine which it will be
- Most natural (medically important) antigens are macromolecules; capable of covalent bonding; include proteins, carbohydrates, lipids, and nucleic acids
- Non-immunogenic antigens; react with Abs, but do not induce their production; lack immunogenicity, but display antigenicity
- Very small
- Anti-hapten Abs are produced via hapten-carrier complexes or conjugates (covalently attached to larger macromolecules); conjugate vaccines are an examples
- any substance that nonspecifically increases immunogenicity of an antigen
- includes substances that: aggregate antigens, facilitate uptake of antigen by APCs, promote long-term survival of Ags, induce co-receptors on immune cells (i.e. CD28), serve as ligands for co-receptors (i.e.C3d), or induce cytokines; adjuvants allow us to make certain recombinant vaccines
What is the difference between a sequential determinant and a continuous conformational determinant?
- Sequential determinant: not dependent upon conformation, defined by sequence of AAs, typically recognized by T cells
- Continuous conformational determinant: AAs are continuous in a sequence, typically recognized by Ab
If a pathogen breaks past the body's first line of defense, will it be intercepted by B cells or T cells?
- Antigen will react with Ab or BCR if: has native conformation or the epitope is surface exposed and charged
- Antigen will react with T cells if: susceptible to internal antigen processing and presentation or it's a protein (less emphasis on the conformation and doesn't need exposed surface epitopes)
Define natural antibody
- First Abs produced early in life; induced by carbohydrate antigens of microbial flora ini GI tract (TI); induce IgM response (low titer and low affinity)
- Some of these Abs may cross-react with mammalian carbohydrates (i.e. ABO erythrocyte antigens; if you're type A, you have Abs against type B because of exposure from microflora)
agents that cause agglutination of RBCs; you develop antibodies against other human blood types because they are isoforms of your own blood type
What is the relationship between immunoglobulins and antibodies?
- Immunoglobulins are plasma proteins and antibodies are a group of plasma proteins; therefore, all antibodies are immunoglobulins
- Immunoglobulins are normally heterogenous in structure (polyclonal), but some are homogenous and monoclonal (i.e. multiple myeloma)
- A heterogenous increase in antibodies indicates infection
- A homogenous increase in antibodies indicates myeloma or a problem (can be seen on serum electrophoresis
What is the function of the disulfide bonds of antibodies?
- Hold heavy chains together
- Hold light chains together
- Intr chain disulfide bonds define domains
Which portion of the antibody is more responsible for the specificity seen in Abs, the heavy chain or the light chain?
Both contribute to the specificity
The binding pockets of antibodies are formed by what?
- Complementarity determining regions (hypervariable regions); CDR1, CDR2, CDR3; these are the 3 AA sequences in the L and H chain variable regions
- The rest of the variable region is only semi-variable and represents the framework
What is the function of the C-region, the hinge region, the Fab fragmets, the Fc fragments, and the oligosaccharide moieties of every antibody?
- C-region: heavy chain constant region is responsible for the biological activity of the antibody and the structural integrity
- Hinge region: allows for flexibility of molecule
- Fab: fragments of the antigen binding end of the arms
- Fc: fragements at the base of the heavy chain that have biological activity; these crystallize easily and are constant
- Oligosaccharide moieties: CHO regions; on the constant portion of the Ab heavy chain; make it a glycoprotein
Which terminal end of the Ab is variable?
The amino terminal halves of the kappa and lambda L chains are the variable regions while the carboxy ends are constant
Define polymeric immunoglobulins
- not monomers or simple 4 chains immunoglobulins (IgG, IgE, IgD, membrane form of IgM, and some IgA)
- IgA: dimer held together by J chain (polypeptide chain); can even form tetramers
- Igm: pentamer; results in 10 combining sites and very high avidity; also uses J chain to bind
What are the 5 different Igs?
- IgG: gamma heavy chain; 4 subtypes; half life is ~3 weeks; tends to activate classical complement pathway (not IgG4); and can cross the placenta; binds Fc receptors on phagocytes
- IgA: alpha heavy chains: 2 subtypes; half life is less than a week; does not activate classical complement; does not cross placenta; found in mucousal surfaces; can be monomer (mostly) or dimer depending on amount of J-chain synthesis; mucosal transport
- IgM: mu heavy chain; half life is 5 days; activates classical complement pathway; does not cross placenta; pentamer usually; present on membrane of naive and mature B cells; mucosal transport
- IgE: epsilon heavy chain; half life is 2.5 days; does not activate classical complement pathway cross placenta; parasites and allergic reaction; mediates type I hypersensitivity; induces mast cell degranulation
- IgD: delta heavy chain; half life is 3 days; does not activate classical complement or cross the placenta; not a major secreted immunoglobulin; major role in B cell Ag receptor; present on membrane of naive B cells
What is the difference between idiotype, allotype, and isotype?
- Isotypes: different classes of immunoglobulins
- Allotypes: inherited markers; single AA substitutions; distinguish individuals; why my IgG is different than your IgG
- Idiotypes: markers associated with the V regions; distinguihs the combining sites of different antibodies
How do Ag-Ab complexes activate complement?
- Ab undergoes steric change when it binds Ag; this reveals cryptic sites that can interact with Fc receptors or complement proteins
- IgM/G can activate classical complement pathway; C1q binding to 2 adjacent regions (CH) of Ag-Ab complexes; easier for IgM since in pentameric form; need higher concentration of IgG to make this happen
How do you clear immune complexes in the complement pathway?
C3b binds to CR1 on erythrocytes and gets sent to phagocyte in liver or spleen
Define monoclonal gammopathy
- Serum protein electrophoresis shows spike in gamma region=monoclonal gammopathy
- MGs are characterized by urinary Bence-Jones proteins; indicate excessive light chain synthesis (because they are just small enough to pass through renal filtration system); dimeric and precipitate upon heating
- If you find light chains in urine, you can tell they're monoclonal if a single L chain type is represented (kappa or lambda); normal ratio is 60kappa:40lambda; if polyclonal, you will see both in the urine
Multiple myeloma (plasma cancer cells), Waldenstrom's macroglobulinemia (IgM), and certain B cell lymphomas (non-hodgkins) are examples of what?
B cell hyperplasias and malignancies
MGUS and L-chain disease (production of excessive light chains) are examples of what?
- Nonmalignant B cell conditions
- MGUS=monoclonal gammopathy of undetermined significance
What is the molecular basis of myeloma kidney?
Overloaded with proteins; this leads to denatured cells, damaged renal tubule, and glomerular damage
Define hybridoma technology
- formation of hybrid cell lines through the fusion of a specific Ab producing B cell with a myeloma (B cell cancer); results in monoclonal antibodies (monospecificty against an antigen of interest)
- Used for research
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