Immunology Exam 3 Learning Objectives.pdf

Lecture 20 – Antibodies 1. Humoral immunity a. Acellular response utilizing macromolecules in body’s fluids b. Primary target à extracellular pathogens c. Types à complement and Ab 2. Antigen a. Ag à substance that specifically binds Ab or TCR b. Immunogen à Ag that elicits a T or B cell response i....

Lecture 20 – Antibodies 1. Humoral immunity a. Acellular response utilizing macromolecules in body’s fluids b. Primary target à extracellular pathogens c. Types à complement and Ab 2. Antigen a. Ag à substance that specifically binds Ab or TCR b. Immunogen à Ag that elicits a T or B cell response i. Immunogen ALWAYS Ag, but Ag not always immunogen c. Epitope/determinant à smaller portion of Ag specifically bound by T cell or Ab i. Single Ag usually has multiple epitopes ii. Immunodominant epitopes à expressed at higher levels and elicit greater responses (greater magnitude) d. Ag may or may not elicit adaptive immune response e. Typically, protein or glycoprotein, but can be polysaccharide or glycolipid f. Order of immunogenicity i. Protein >> carbohydrate >> lipid, haptens, amino acids, DNA g. Good Ag à highly folded, unevenly charged, and greater than 1 kD (most over 100 kD) 3. Haptens a. Small part of Ag that is bound by Ab (< 1 kD in size) b. Unable to initiate immune response by itself i. Lack of sufficient avidity to trigger B cell activation c. MUST be attached (conjugated) to larger molecule (carrier – usually protein) d. Common haptens à penicillin, warfarin, dander, industrial chemicals, sodium benzoate, titanium oxide, polyethylene glycol e. Carrier proteins à albumin (BSA, ovalbumin), keyhole limpet hemocyanin f. Natural reaction à drug binding to host protein g. Designed reaction à vaccine Ag 4. Valency vs Affinity vs Avidity a. Valency à number of binding sites b. Affinity à binding strength between one Ab-epitope i. Not all binding sites have same affinity c. Avidity à total binding strength between all Abepitope pairs in multivalent Ag-Ab interactions i. More binding sites, potentially more avidity 5. Antibodies a. Structure i. Immunoglobin (Ig) domain à globular domain containing disulfide bond between cysteine residues ii. Fab region à variable region that binds Ag 1. Formed by both heavy and light chain 2. Ag-binding site à highly variable region that make direct contact à CDR/HVR a. Lock and key fit iii. Fc region à constant region 1. More conserved, mediates Ag-independent functions iv. Hinge region à structural flexibility that helps in binding v. Two heavy chains, two light chains 1. Valency = 2 b. Cross reactivity / Molecular mimicry i. Ab produced against one Ag can bind different but structurally similar Ag ii. Enough contact in binding to generate sufficient affinity to activate B cell iii. Can play role in autoimmunity c. Functions (Lecture 21) i. Indirect à activating complement which generates anaphylatoxins and opsonins that promote inflammation and phagocytosis (respectively) ii. Direct à neutralization d. Generation of diversity i. Combinatorial diversity à somatic recombination (exam 2) 1. Different combinations of V, D, and J genes 2. Allelic exclusion (exam 2) ii. Junctional diversity 1. Gaps left by RAG recombination filled by nucleotides 6. BCR complex a. Cytoplasmic tail too short to allow docking and assembly of signaling molecules b. Thus, BCR must be able to associate with accessory molecules and transmit Ag binding signal to them for B cell activation i. Accessory molecules à Ig alpha and Igß 1. ITAM domain allows for activation signal transmission 2. Interact with Fc portion of BCR 7. Recall from Exam 2 a. B cell development process b. Receptor editing checkpoint à make sure BCR doesn’t recognize self-Ag c. Phases of B cell response i. Impact on Ab à optimize Ab functionality à most effective/efficient clearance of pathogen 1. Clonal expansion (lecture 21) 2. Affinity maturation à somatic hypermutation a. Spontaneous mutations that increase affinity b. Doesn’t change antigenic specificity c. Mutations occur across time of response and with each exposure to Ag 3. Isotype switching à specialization of function a. Activated B cell secrete IgM à B cell encounter CD4 Th cells à receive costim and cytokine à AID (activationinduced cytidine deaminase) produced i. AID functions like RAG b. Isotype of BOTH BCR and Ab are permanently changed i. Due to loss of DNA c. 3 signal activation (lecture 21) 8. Ab Isotypes Monomers Dimers Multimers Ab isotype IgG, IgE, IgA, IgD IgA IgM pentamers à bound by J (joining) chain Location Blood Mucosa Circulation Valency 2 4 10 Avidity Low Medium High a. Affinity i. More mature Ab have higher affinity ii. IgG, IgE, IgA > IgM, IgD 9. T cell dependence in B cell response a. T-dependent Ag à protein or glycoprotein i. Class II MHC pathway (exogenous) à Ag-loaded B cell binds activated CD4 Th cell that provide costim and cytokines ii. B cell fully activated à secretes IgM, isotype switching, high affinity Ab memory B cell, long-lived plasma cells b. T-independent Ag à polysaccharide, glycolipid, or rarely protein i. Repetitive structures ii. Mainly IgM, low affinity Ab, and short-lived plasma cells iii. Crosslinking of receptors activates B cell directly à secretes IgM iv. Doesn’t isotype switch or differentiate to memory cells Lecture 21 – Humoral Immunity 1. Recall – B cell development a. Starts in bone marrow à VDJ recombination à checkpoints à leaves bone marrow immature à completes maturation in 2º lymph tissues à optimization of effector function à plasma/memory cell b. Primary sign of maturity à expression of surface IgD 2. Direct Ag Acquisition and Activation a. Whole Ags bind directly to BCR WITHOUT processing b. Can be small Ags à bind single BCR à not a lot of signal generated à anergy c. Can be large, repetitive AG à crosslinks multiple BCR à B cell activated 3. Indirect Ag Acquisition and Activation a. Ag are captured by APCS à Ag NOT endocytosed or processed à stays on APC surface à transferred to BCR b. Two specialized APCs i. Subcapsular sinus macs à bind larger Ag and Ag-Ab complexes ii. Follicular DCs à bind smaller Ag that filter into follicles 1. DCs in medulla à bind Ag and can traffic to follicle 4. T-independent: B cells as APCs a. B cell binds native, conformational Ag à exogenous pathway b. Process doesn’t activate B cell 5. T-dependent: B cell as recipients of T cell help a. B cell binds unprocessed Ag à processed and displays MHC II peptide à B cell and CD4 T cell (activated elsewhere) bind à B cell activated b. CD4 T cell help critical for long-term production of Ab to polysaccharide Ags c. T-B cell interactions and follicles i. Activation à T cell activated in T cell zone of LN; B cell activated in B cell zone ii. Migration à T and B produce chemokines and move toward each other iii. Contract à meet in parafollicle; B cell gets Th help, Abs made iv. Outcomes à 1. B and T cells stay outside follicle à B cell becomes short-lived plasma cell à low level Ab production 2. B cells return to follicle à proliferate/differentiate to plasma cell à germinal center forms à high level Ab production d. Germinal center à highly organized area within lymphoid follicle formed by T-dependent activated B cells i. Naïve B cells à mantle zone ii. Activated by CD4 à parafollicle iii. Reenter follicle and proliferate à dark zone iv. Non-dividing B cells interact with follicular DC (FDC) and CD4 until selected à light zone v. Repeated trips to dark zone à become high affinity plasma/memory cell 1. Somatic mutation, affinity maturation, and isotype switching vi. B cell exits germinal center 6. 3-Signal Hypothesis a. Specific Ag à CD4 TCR must bind MHCII-peptide complex b. Costimulation à CD28 or CD40L (CD4) binds to CD80/86 or CD40 (B cell) c. Cytokine à for proliferation and differentiation (isotype switching) 7. Phases of B cell response a. Clonal Expansion i. Massive proliferation 1. Driven by Ag specific for BCR 2. Specificity for single Ag based on BCR ii. Immunodominant epitopes b. Immunoglobin secretion i. Activated B cell starts secreting IgM ii. Surface Ig (BCR) and secreted Ig translated from same mRNA 1. Variable regions are identical 2. Constant chain isotype also same c. Affinity maturation i. B cell transition from dark to light zone ii. Contact FDC presenting Ag and mutate Ab binding site iii. If affinity increases à B cell selected to survive 1. Can re-enter dark zone and proliferate à whole process repeats d. Isotype switching i. Occurs in light zone ii. Cytokine signal from CD4 Tfh (major source) and FDC (minor source) 1. IgG à induced by IFN-gamma 2. IgE à induced by IL-4 and IL-13 3. IgA à induced by TGF-ß iii. Recombination mediated by AID (Lecture 20) 8. Ab functions Type Degranulation Ab Isotype IgE Ag type Ag that are too large to be endocytosed à allergens and parasites Effector function - IgE binds Ag à eosinophil or mast cell binds IgE through FceR - IgE binds FceR on mast cell or eosinophil à binds allergen Result Crosslinking of FceR activates cell à degranulation and killing of Ag Complement IgM > IgG3 Mediated Lysis > IgG1 > IgG2 Opsonization IgG or C3b Ag on cell or pathogen surface Neutralization IgG Microbes and toxins Ab-dependent cellular cytotoxicity (ADCC) IgG Ag on surface of infected cell Microbe Classical C’ Pathway activated when C1q binds Ab Fc region Microbe opsonized by IgG à binds phagocyte Fc receptor (FcyRI) or complement receptor Binding of Ab prevents binding of pathogen IgG binds Ag à FcyRIII (CD16) on NK cells and CD8 binds IgG Fc region 9. Fc Receptors (FcR) a. Different classes à based on affinity for various Ig isotypes b. Different roles for each class c. ITAM or ITIM domain in cytoplasmic tail d. FcRN à neonatal FcR (Lecture 23) i. Neonates à expressed on placenta and gut ii. Adults à expressed on endothelial cell and macs iii. Function à transfers IgG across cells while protecting it from lysosomal degradation 1. Delivers maternal IgG to baby’s circulation 2. Prolongs half-life of IgG MAC complex formed and osmotic lysis kills cell Phagocytosis triggered à microbe degraded - blocks infection by pathogen - inhibits toxic effect Target cell killed by cytolytic processes (i.e., perforingranzyme) 10. Ab Feedback a. Production of Ab downregulates continued Ab production and secretion b. Binding process i. Ab binds polyvalent target Ag ii. Ag in Ag-Ab complex binds BCR iii. Simultaneously Fc portion of Ab binds CD32/FcyRIIB 11. 1º and 2º response a. Primary i. IgM produced 48-72 hours after Ag exposure ii. Relatively short-lived B cell 1. Undergoes affinity maturation à low average affinity 2. Long lived IgE/G/A made à 5-7 days later iii. Contraction and memory cells made b. Secondary i. Memory cell activated and proliferate à clonal expansion ii. Secrete IgG à more rapid and higher magnitude iii. Contraction à greater number of memory cells Lecture 22 – Epithelium and Cutaneous Immunity 1. Epithelium basics a. Three layers i. Outer epithelial layer à barrier 1. Skin, GI tract, respiratory tract, urogenital tract, ocular surfaces ii. Basal cell layer à regeneration iii. Underlying connective tissue layer à innate and memory tissue b. Function i. Barrier against infection and damage ii. Balance between local microflora and pathogenic organisms c. Epithelial surfaces i. Continual turnover of cells à new cells generated from stem cells at base of epithelial layer ii. Immuno-privileged cells à tissues not recognized as self and need protection 1. Anatomically isolated à eye, brain, testes, ovary, placenta d. Specialized structures i. Mix of cells and proteins 1. Several layers or single layer 2. Columnar or cuboidal epithelial cells 3. Ciliated or non-ciliated cells 4. Secretory cells ii. PRR expression à high levels and great diversity 2. Epithelial cells a. Epithelial APCS i. Must be able to differentiate harmless and harmful insults 1. Tolerant to flora and reactive to pathogens ii. Unique APCs in each epithelial barrier 1. Skin à Langerhans and dermal DC 2. Liver à Küpffer 3. Brain à microglia 4. Connective tissue à histiocytes b. Intraepithelial lymphoid cells (IELs) i. Includes: 1. Innate lymphoid cells (ILC) à Ag independent a. Look and act like T cell but don’t express TCR or CD3 b. Respond to PAMP/DAMP through TLR or lectin PRR c. Rapidly respond at site of infection d. Produce cytokines à IFN-gamma (intracellular pathogens) and IL-13 (parasites) 2. Conventional alpha-beta T cells à Ag dependent a. Most adaptive IELs are CD8 3. Gamma-delta T cells à Ag dependent a. Found in GI and respiratory tract and skin b. TCR-dependent i. MHC independent ii. Ags include proteins, lipids, and phoshomolecules c. TCR-independent i. Activated by TLR binding d. Effector function à cytokine production and direct cytotoxicity (Fas and ADCC) ii. Have activated or memory phenotype 3. Microbiome a. Homeostatic microbiome is commensal à as long as it stays on outer epithelial surface b. Advantages i. Outcompete pathogens à space and nutrients ii. Active defense against pathogens à produce products to kill invaders and stimulate immune response that protect against invaders c. Sources and adaption i. Initial inoculation à birth ii. Great diversity à changes with age, geography, exposures d. Specialized soluble mediators i. Defensins à help maintain balance between flora and pathogenic bacteria ii. Cytokines à originate from infected/damaged epithelium itself or resident APCs, ILCs, or T cells 1. IL-17 à produced by epithelium and T cells à PMN recruitment 2. Acute inflammation and chronic inflammation 4. Cutaneous Epithelium a. Epidermis à outermost layer i. Basal keratinocytes à most undifferentiated stem cells 1. Stratum corneum à keratinocytes that undergo apoptosis a. Form layer of keratin and lipid rich barrier cells ii. Keratinocytes secrete connexins/filaggrins for development and cytokines for protection à innate and adaptive immunity 1. Connexins a. Structure and communication between epithelial cells b. Form homo- and heteromeric aggregates à gap junctions c. Defined role in wound healing and diseases 2. Filaggrin a. Formed from profilaggrin b. Filaggrin monomer binds keratin and aggregate to form macrofibril around terminally differentiated keratinocytes c. Macrofibrils pack tightly à form stratum corneum b. Dermis à underlying layer of connective tissue c. Cells i. Specialized DCs Phenotype Location Function Langerhans Epidermis Development of CD4 Th1 and CD8 CTL Dermal DC Dermis IL-4 secreting CD4 Th2 and Ab-producing plasma cells ii. Innate lymphoid cells (ILCs) iii. Specialized T cells à gamma-delta 1. Can be activated by TLR or TCR generated signals 2. Direct à cytolytic activity (Fas pathway) 3. Indirect à cytokine production à IL-17 d. Compromised cutaneous barriers i. Physical (trauma), chemical (irritants, UV), biologic (microbial, allergens) ii. Result à altered integrity à increased permeability 5. Cytokine Sources a. Keratinocytes à activate resident DCs and macs b. Langerhans and dermal DCs à activate resident macs and memory T cells and traffick to regional LN to activate naïve T cells c. Effector CD4 T cells i. Th1 à for intracellular infections ii. Th17 à for extracellular infections; stimulate defensin and cathelicidin production in keratinocytes iii. Th2 à IL-4 and IL-13 suppress defensin and cathelicidin d. Key effect à trafficking of cells 6. Cutaneous Immunity a. Vitamin D à critical in generating homing signals UV B radiation triggers previtamin D synthesis pre-vitamin D taken up by dermal DC and processed to active form active form presented to T cell in draining LN induces expression of homing receptors (chemokines and adhesion molecules) direct activated T cell to traffic back to skin b. Microbiota and immunoprotection i. Commensal bacteria can induce 1. Innate immune responses à healthy wound repair 2. Produce anti-microbial substances (defensins) à kill pathogen ii. Commensal microbiota à induce cytokines à prevent and clear infection 1. Dysregulated cytokine production à immunopathogenic 2. Pathogenic microbiota à induce inflammatory mediators which exacerbate cutaneous diseases and promote infection Lecture 23 – Mechanisms of Mucosal Immunity 1. Mucosal Tissue a. Major sites of mucosal-associated lymphoid tissue (MALT) i. Gut associated (GALT), Bronchus associated (BALT), Nasal associated (NALT), Genitourinary associated, Mammary glands b. Specialized structures i. Epithelial structures 1. Villi (gut) and cilia (lung) à function specialized to surface ii. Lymphoid structures 1. In submucosal layer under epithelium 2. Organized but not encapsulated like LN 3. Contains T/B cells, ILCs, DCs and macs à efficiency of response 4. Functions a. Generate specialized adaptive immune responses b. Regulate local immune responses c. Homing of adaptive lymphocytes and DCs back to particular mucosal surface after entering circulation 2. GALT a. GALT development overview Role Location b. Challenges i. Large surface area to protect ii. Discrimination between microbial communities iii. Solution à highly specialized mucosal immune system c. GI epithelium i. Only one layer ii. Specialized epithelial cells 1. Arise from stem cells in crypt à at base of epithelial layer à High turnover 2. Absorptive epithelial cells à absorb nutrients and secrete cytokines Goblet Cells Microfold (M cells) Secrete mucin to form Transport of whole mucus layer microbes and microbial fragments (Ag transport) (raspberry color stain) Mucin forms two layers à more and less viscous Located in small intestine close to Peyer’s patch Paneth Cells Secrete antimicrobial peptides Defensin action à direct cytotoxic and activate local innate immune cells (PMN, NK, etc) Base of mucosal epithelium Components Membrane bound mucin à associate with glycolipids à form glycocalyx Mucin à glycoprotein with O-linked oligosaccharides Mucus à buffer zone à helps prevent contact of microbes with epithelium Different mucins à different types à different levels à different glycosylation patterns Blunted microvilli (microfolds) à help capture Ag for internalization à Endocytosed material transported through M cell cytoplasm without processing à transcytosis à Material exocytosed at basolateral surface and delivered to DCs in Peyer’s patch or lymphoid follicle Alpha-defensin in small intestine and ß-defensin in colon Alpha-defensins à contained in PMN, NK, CD8 granules ß-defensins à no Paneth cell à want bacteria removed by the time it reaches colon iii. Specialized molecules 1. Secretory IgA à predominant Ig produced at mucosal surface a. Neutralizes organisms and toxins à prevents attachment b. Dimerizes and binds J chain c. IgA-producing B cells generated in Peyer's patch and mesenteric LNs Ag taken up by M cells --> presented to naive CD4 T cell and B cells Ag-specific B cells activated DCs secrete RA (homing) and factors that trigger IgA isotype switching 2. Poly-Ig receptor a. Transcytosis of immunoglobin across epithelial layers Poly-Ig receptor binds sIgA dimer and IgM pentamer through J chain domain receptor and Ig endocytosed and transcytosed at apical surface, polyIg receptor proteolytically cleaved portion of poly-Ig reeceptor remains bound to Ig = secretory component b. secretory component thought to protect sIgA and IgM from degradation in intestinal lumen 3. Mucins à carbs that make mucus (above table) 4. Defensins and other antimicrobial peptides (above table) d. TLRs i. Make sure they aren’t constantly activated and secreting cytokines ii. Location of PRR 1. Limited number on apical surface 2. Higher conc in cytoplasm and on basolateral surface à detect pathogens that have invaded iii. Threshold of PRR activation 1. Higher level of stimulation à proinflammatory response 2. Low to moderate stimulation à triggers IL-10 and TGF-ß proliferation à Treg differentiation e. APCS i. DCs continuously sampling the environment ii. Differentiate harmless and harmful insults iii. Küpffer cells in liver iv. Key interaction à activation of DCs and gut epithelium = TLR binding PAMP/DAMP f. T cell homing i. Process regulated by retinoic acid (RA) dietary vitamin A absorbed by DCs and converted to RA APCs traffic to regional mesenteric LN to activate T and B cells during activation, DCs secrete RA RA induces alpha4ß7 (integrin) and CCR9 g. T cell i. In epithelial barrier à CD8+ gamma-delta T cells ii. In lamina propria à most are CD4 memory T cells 1. Elevated CD4 Th17 à protection against bacteria 2. 2x number of Treg a. Suppress at low levels to protect commensal bacteria b. Both induced and natural i. Differentiation induced by TGF-ß and RA from resident DCs ii. Proliferation triggered by fermentation metabolites c. Major protective mechanism à IL-10 production h. Gut microbiome regulation i. Normal flora à induce mucin, antimicrobial peptides, and cytokine production à differentiates Tregs and IgA-producing B cells ii. Changes in microbiome à activation of more DCs and macs to produce more Th17 and Th1 cells à excessive inflammation and disease 1. More Th17 à more IL-17 production à more IL-8 2. Decreased number of Treg à Th17 counterbalance i. Th2 Responses i. Intestinal parasites 1. Secretion on IL-4 and IL-13 integrin allows T cell to home back to intestinal mucosa a. Enhance fluid and mucous secretion b. Increase smooth muscle contraction and bowel motility c. Induce alternatively activated macs and fibroblasts d. Recruits and activates mast cells and eosinophils 2. Isotype switching to IgE a. Binding to FceR on mast cells and eosinophils à crosslinking leads to degranulation ii. Food allergies 1. Allergen leads to IgE responses (induced by IL-4) à IgE binds FceR on mast cells 2. On re-exposure à mast cells degranulate to cause local symptoms 3. BALT a. Subject to continuous exposure of environmental and pathogenic substances i. Microbes, pollen, dust particles, pollutants b. Shared traits with MALT i. Specialized epithelium 1. Goblet cell and M cells 2. Mucin and defensin production ii. Unique DCs and macs iii. Resident innate and adaptive immune cells à CD4, CD8, gamma-delta T cells, ILCs iv. Lymphoid structures à follicles v. Dedicated regional LN vi. Unique microbiome à less diverse than GI vii. Production of IgA, FcRN, and poly-Ig receptor c. Goblet cells, mucus, cilia i. Mucus (produced by goblet cells) traps foreign material before it can contact epithelium ii. Cilia beat then sweeps contaminated mucus up and out of respiratory tract iii. Mucus plug à mucus and dead epithelium that is shed into airway from infection or chronic inflammation iv. Hypoxia à overproduction of mucus à interfere with gas exchange d. Pulmonary surfactants i. Phospholipid-protein complex produce by alveolar epithelial cells ii. Lung physiology function à reduce surface tension in alveoli to maintain lung compliance and fluid balance iii. Lung immunology function à SP-A and SP-D 1. PRRs à collectins that bind carb PAMPs 2. Increases phagocytic activity of alveolar macs 3. Sometimes enhances or sometimes suppresses inflammatory signal e. Alveolar macs i. Majority of free cells in alveolar space ii. Can divide and repopulate themselves iii. Express large numbers of inhibitory receptors (and stimulatory receptors) iv. Normal phenotype if anti-inflammatory à secrete IL-10, TGF-ß, and NO v. Inhibit activation of pulmonary DCs and T cells vi. Poorly phagocytic f. Dysregulated immunity i. Occurs with chronic antigenic stimulation, chronic inflammation, or viral infection ii. Lead to à expansion of BALT and lymphoid follicles, excessive mucus production, and airway and alveolar fibrosis 1. Difficulty/restricted breathing g. Microbiome i. More limited than GALT ii. Gut-Lung axis à Responses triggered by gut microbiome affects immune function in respiratory tract 1. Proposed mechanism à by products of microbiome metabolism, GALT cytokines, GALT-induced CD4 Th cells 4. Genitourinary Mucosa a. Vagina, endocervix, urethra, penis, and anorectal canal b. Difference from other MALT i. Multi-layered with stratified squamous cells ii. No lymphoid follicles iii. Little IgA à predominantly IgG c. Similarities to other MALT i. Goblet cells and production of antimicrobial peptides ii. Normal flora iii. Regional DCs (Langerhans) and T and B cells 1. DCs phenotypically different from other MALT iv. IELs (gamma-delta CD8 T cells) 5. Mucosal immunization and mucosal tolerance a. Mucosal tolerance à turn down immune response so unresponsive i. Balance of stimuli and cytokines, PR induce Treg, tolerogenic DCs, and sIgA in response to normal flora ii. Generate tolerance à high doses of protein Ags iii. Generate responsiveness à Ag delivery in context of innate immunity iv. No tolerance = disease b. Mucosal immunization i. Want no pro-inflammation à make sure disease doesn’t occur Lecture 24 – Tolerance 1. Potential results of lymphocyte activation a. Foreign vs Self Ag and Activation vs Tolerance i. Foreign and activation à control/clearance of pathogen ii. Foreign and tolerance à infection iii. Self and tolerance à no lymphocyte response iv. Self and activation à autoimmune disease 2. Tolerance a. Tolerance à immunologic unresponsiveness à lack adaptive immune response b. Self-tolerance à immunologic unresponsiveness to self Ag i. Prevents autoimmune disease c. Purpose à inactivation or elimination of lymphocytes which react to specific Ag d. Forms i. Central à occurs with immature lymphocytes in 1º lymphoid tissue ii. Peripheral à occurs when mature lymphocytes are made tolerant in circulation and 2º lymphoid tissue e. Pathways i. T cell tolerance à elimination or inactivation of selfreactive T cells ii. B cell tolerance à elimination or inactivation of self-reactive B cells iii. Leads to homeostasis à lymphocyte anergy and apoptosis iv. Tolerance induced by foreign Ag 1. High numbers or activation of APC à stimulation of immunity 2. Low numbers or little activation of APC à tolerance 3. T cell central tolerance a. Occurs in thymus b. Mechanisms i. Negative selection checkpoint à deletion of cells with TCR that strongly recognize self-Ag ii. Generation of nTreg c. Central selection factor à avidity of TCR binding to MHC-self peptide complex d. AIRE-ing out T cell central tolerance i. AIRE = autoimmune regulator protein ii. Transcription factor in mTREC that activates expression of peptide Ag for tissues outside thymus iii. Must induce tolerance to all self-tissues (inside and outside thymus) 4. T cell peripheral tolerance a. Determinants i. Three signal hypothesis à first chance to tolerize is determined by second signal binding event à B7 binds CTLA4 instead of CD28 ii. Immunologic tolerance is Ag-specific b. Anergy i. Cell rendered permanently unresponsive, but doesn’t die ii. Mechanisms 1. Lack of Costimulation à no B7 2. Binding of inhibitory receptors à CTLA4 and PD1 a. Competitive inhibition à B7 bound by another cell b. Direct inhibition à two mechanisms i. ITIM-like domain on CTLA4 overrides ITAM phosphorylation of tyrosine residues in ITIM recruits and activates SHP phosphatase activated SHP dephosphorylates proteins phosphorylated by ITAM ii. CTLA4-B7 binding induces endocytosis of B7 off APC into T cell iii. T cell exhaustion 1. Long-term Ag exposure induces anergy Long-term Ag exposure leads to induces anergy c. Suppression i. nTreg and iTreg suppress lymphocyte responses ii. Recall: FoxP3, high levels CD25, induced by TGF-ß, MHC-Ag dependent iii. Contact dependent mechanism 1. Treg expression of CTLA4 and PD1 à suppresses T cell and DC 2. Releaser perforin and granzyme granules iv. Contact independent mechanism 1. Secrete cytokines à IL-10 and TGF-ß à inactivate T/B cells, DC 2. Secretion of IDO à reduces ability of DC to function as APC v. Competition for resources 1. High levels of CD25 à soak up IL-2 before it binds CD4 or CD8 2. Bind MHC-peptide and B7 à crowds T cell so it can’t bind vi. Tolerize the APC 1. Binding of specific receptors (C-type lectin) on APC 2. Treg suppression à IL-10 and IDO production of inhibitory receptor binding vii. Tolerized DC à doesn’t respond to activation signal à anergy or die 1. Costim, MHC molecules, and cytokines not upregulated 2. Tolerogenic and killing proteins upregulated d. Deletion/Apoptosis i. Intrinsic pathway à mitochondria Triggered by strong binding of self ag or by growth factor deprivation (IL-12) Involves activation of pro-apoptotic proteins Bax and Bak which oligomerize and bind to mitochondria Increases mitochondrial permeability à activates caspase 9 and 3 ii. Extrinsic pathway à death receptors cell surface receptors bound by ligands (TNFR, Fas) activate death domains --> activated caspase 8 caspase 8 activates caspase 3 (or Bax, Bak and activate intrinsic) iii. Phagocytes remove cellular debris 5. B cell central tolerance a. Occurs in bone marrow b. If BCR recognizes self Ag with high affinity i. Receptor editing ii. Deletion c. If BCR recognizes self Ag weakly à B cell anergy induced i. Binds soluble Ag à insufficient BCR crosslinking ii. Binds with weak affinity à insufficient signal iii. B cell leaves bone marrow in unresponsive state 6. B cell peripheral tolerance a. Self-reactive B cells slips checkpoint and leave bone marrow not tolerized b. Inactive the cell i. Anergy 1. Weak binding of foreign Ag 2. B cell exhaustion ii. Inhibitory receptors 1. If BCR binds Ag with low avidity à inhibitory receptors triggered 2. CD32 – FcyRIIB à Ab-Ag binds BCR and CD32 simultaneously iii. Lack of T cell help and costimulation c. Delete the cell à apoptosis i. Fas on B cell binds FasL on CD4 à activate T cell extrinsic pathway 7. Immunoprivileged tissues (also lecture 22, part 1-c) a. Tissues that have developed mechanisms to prevent immune responses b. Mechanisms i. Physical barrier ii. Immune evasion à killing iii. Apoptotic receptors à PD1 iv. Inhibitory receptors à IDO, IL-10 v. Soluble immunosuppressive factor à HLA-G 8. Tolerance failure à autoimmunity a. Genetic polymorphisms and defects à prevent induction of tolerance b. Infections or environmental exposures à break tolerance i. Pathogen activated APC displaying self-Ag à self-reactive T cells are activated by APC à target self-tissue ii. Molecular mimicry: Pathogen protein epitope closely resembles selfpeptide à APC presents pathogenic peptide à self-reactive T cell activated à target self-tissue (following examples Lecture 25) 1. Guillain-Barré Syndrome à progressive weakness due to destruction of myelin sheath 2. Celiac disease à T cells that react to gliaden (peptide in gluten) c. Damage (physical or inflammatory) (Lecture 25) i. Sequestered Ag à exposure of Ags to which host hasn’t been tolerized ii. Neo-ags à sequestering leads to production modification of cellular proteins that are antigenically different iii. Immune response sees these Ags as foreign 9. Clinical application à immunotherapies a. Allergy shots induce tolerance b. Alloantigen (transplant) à Ab blockade of costim molecule CD40L on T cell 10. Tolerance vs immunosuppression a. Tolerance à Ag-dependent inhibition of Ag-specific lymphocytes only b. Immunosuppression à Ag-independent inhibition of broad set of cells Lecture 25 – Autoimmune Disease 1. Autoimmune Disease a. Diseases caused by auto-Abs or T cells that attack healthy molecules, cells, or tissues of the organism producing them b. Organ-specific or systemic c. Multi-factorial pathogenesis à breaking of tolerance d. Chronic, progressive, and self-sustaining e. Risk factors i. Family and medical history ii. Genetics iii. Environmental exposures à UV, toxins, silica, drugs iv. Gender à higher incidence in women v. Age à higher incidence the older one gets vi. Dysregulated immune reaction vii. Pre-existing defects in target organ 2. Molecular Immunology of Autoimmunity a. Failure of self-tolerance à lecture 24 part 8 b. T cells and autoimmunity i. Most have some component of T-cell mediated damage ii. Indirect damage à CD4 produced cytokines iii. Direct damage à CD8 CTL iv. Typically, proinflammatory response à CD4 Th1 and Th17, and CD8 c. Ab-Mediated Autoimmunity i. Abs are major effector molecule ii. Immune complex deposition à in joints/tissues leading to C’ activation iii. Ab binding to tissue à tissue damage 1. Opsonization à phagocytosis and contents of phagosome leak out 2. ADCC 3. C’ activation à generates anaphylatoxins that increase inflammation 4. If auto-Ag is receptor à Ab-Ag binding can either activate receptor or interfere with normal function/physiology 5. Any tissue damage à can expose neo-Ag a. More neo-Ag exposed à greater chance of breaking tolerance and self-reactive lymphs activated d. Anti-Nuclear Abs (ANA) i. Group of Abs specific for various Ags found inside nucleus of cell ii. Stain pattern in cells can sometimes help with disease identification e. HLA Genetic Polymorphisms i. Some diseases strongly associated with very specific HLA polymorphic alleles à expressing allele increases odds of developing disease ii. Most are MHCII molecules à activate CD4 f. Non-MHC Genetic Polymorphisms i. Not encoded in MHC locus ii. Many are involved in response in some way à cytokines, receptors, transcription and signaling factors, etc. iii. Have polymorphisms at point of induction (HLA protein) and also at effector phase (cytokines, C’ proteins, FcR) g. Single-Point Mutations i. Alter protein or cellular function ii. AIRE à 2nd checkpoint altered à self-reactive T cells not deleted iii. FoxP3 à transcription factor activity compromised à Treg not induced h. Cytokine dysregulation i. Proinflammatory innate response activated 1. Cytokines produced à IL-1, IL-6, TNF à Tolerance broken à Tissue destruction à amplifies innate and adaptive à more cytokines produced à leads to cycle of damage 3. Autoimmune disorders Disease Mediators Auto-Ag Triggers and Presentation Systemic Lupus Erythematosus (SLE) ANA and antidsDNA Ab Nuclear proteins (histones) and DNA Triggers - UV exposure and drugs - more common in women Presentation - malar or buttery rash - fatigue, fever, headache - Cutaneous rash and MS Myasthenia Gravis T cell-dependent Bcell mediated Acetylcholine receptor (AchR) à most common Muscle-specific kinase (MuSK) Rheumatoid Arthritis (RA) CD4 and CD8 T cells Lipoprotein receptor-related peptide 4 (LRP4) Rheumatoid factor à IgM Ab to Fc portion of IgG B cells and Ab Osteoclasts Macs Anti-CCP Ab (anticyclic citrullinated peptide) Multiple organs Risk factors - using penicillamine - family history and genetics Presentation - painless - weakness/fatigue of muscles, esp eyes - worsens through the day Organ specific à MS Risk factors - more common in women - genetic - environmental exposure Presentation - joint pain à starts monoarthritis - most often hands, wrists, and feet first - worse in morning - Ulnar deviation, Boutonniere deformity, Swan Neck deformity, Rheumatoid nodule Multiple organs (starts MS) Autoimmune Diabetes à type 1.5 à latent autoimmune disease of the adult (LADA) CD8 T cell specific ß-pancreas cell à insulin not made Risk factors - genetic and nongenetic - slowly progressive subtype of DM type 1 Presentation - excessive thirst and frequent urination - blurry vision - weight loss Lecture 26 – Immune Assays 1. Common Immunodiagnostic Tests a. Complete blood count with different à WBC count b. Biochemical profile à for total protein c. Serum protein electrophoresis à serum Igs d. Agglutination à particulate Ags e. Precipitation à soluble Ags, cells f. Immunoassays à quantify Ags and Abs g. Immunochemistry à find Ags in tissue sections 2. Blood Cell Analysis a. CBC à counts number of RBCs and WBCs b. CBC with differential à determines percentages of WBCs i. Neutrophilia vs neutropenia à high or low PMNs à bacterial infection ii. Lymphocytosis vs lymphopenia à high or low lymphs à viral infection, lymphoma, leukemia 3. Serum or Protein Analysis a. If total protein high à see what protein high b. General non-specific markers of inflammation à CRP and ESR c. Serum Immunoglobins i. First peak à albumin ii. Second peak à acute phase proteins 1. Positive acute phase proteins à CRP, SAA, fibrinogen, ferritin 2. Negative acute phase proteins à albumin and transferrin a. Downregulated to keep total protein concentration and blood volume at same level iii. Third peak à IgM iv. Fourth peak à IgG d. Serology i. Measure levels of Ag-specific Ab rather than measuring presence of Ag ii. Infectious disease à IgM (early response) vs IgG (late response) iii. Autoimmune disease à Ab mediated ones 4. Measuring Ab functionality a. Neutralization activity à how much Ag-specific Ab can neutralize target b. Complement fixing à Complement-Ab binding à caspase cascade activated c. Ab isotype à EIA/IFA à identify IgG, IgM, IgE 5. Production of Polyclonal and Monoclonal Abs a. Polyclonal Ab i. Host animal immunized à immune serum removed when Ab titers high ii. Many clones, so antiserum made with Ab representing many different B cells and clones b. Monoclonal Ab i. Host mouse immunized à make polyclonal Ab but want Ab specific for one epitope ii. Spleen removed à fused with myeloma (long-life) iii. Individual Ab-secreting cells isolated by culture à hybridoma cells iv. Humanizing MAbs à replace all sequences except most terminal regions of Fab 6. Purification Procedures with Ab a. Affinity Chromatography à bind Ab to column matrix, then purify Ag b. Immunoprecipitation à Ab mixed with Ag-containing solution or cell expressing Ag à centrifuge beads and elute bound material 7. Agglutination Assays a. Ab is used to crosslink Ag present on particle surface à form Ag-Ab complexes à produce visible aggregates b. Example à blood typing 8. Enzyme Immunoassay (EIA) or Immunofluorescent Assay (IFA) a. Used to detect either Ag or Ag-specific Ab b. Same process except last step à final detector Ab linked to enzyme or fluorescent c. Higher the signal à more Ag or Ab present 9. Lateral Flow Assays a. Rapid Ag test b. Sample added and pulled through test cartridge via lateral flow 10. Isolation Leukocyte Subsets a. By size/density à sample added to solution à centrifuged à separate based on density b. By Ag-ic specificity à FACs and MACs 11. Evaluate for Functionality a. Measuring Phagocytic Activity i. Label phagocytic cells with fluorescent molecule à measure level of fluorescence to see phagocytosis ability b. Measuring Oxidative Burst i. Labels ROS inside neutrophil with fluorescent marker à measure ability of PMN to kill foreign invader via oxidative burst c. Lymphocyte Proliferation i. Measure ability to proliferate when stimulated in cell culture ii. Measure how active cells are over time frame 12. Flow Cytometry a. Measuring Ag-specific cytokine production b. Immunotyping à determine what different cell populations are present c. FACS à isolating cell populations of interest for further study d. Epitope mapping of T or B cell responses i. Determine immune-dominant epitopes ii. Development of vaccines or diagnostic kits 13. Immunohistochemistry vs Immunocytochemistry a. Immunohistochemistry à looking at tissue section b. Immunocytochemistry à looking at single cells c. Use colorimetric or fluorescent labels on Ab 14. I want that cell a. Identification of cell by immunostaining à individually or from tissue section b. Remove cell à magnetic or flow sorting, affinity purification, laser capture microdissection c. Analyze à genomics, proteomics, etc.