Elizabeth's Immunology One Pagers PDF

Immune response for too long = tissue remodeling Leukocyte extravasation Too vigilant = hypersensitivity (allergic rxn) 1. Rolling (selectins) Too effective = graft rejection...

Immune response for too long = tissue remodeling Leukocyte extravasation Too vigilant = hypersensitivity (allergic rxn) 1. Rolling (selectins) Too effective = graft rejection 2. Firm adhesion, arrest (integrins) 3. Extravasation (unzips between endothelial cells) Path of pathogen (innate, adaptive) 4. Chemotaxis Epidermis/mucous membranes | Adaptive Immune System Hostile milieu (serum, lymph) Lymphocytes | o Compact nucleus expands when activated Dermal/submucosal tissues (cells for o Proliferate and differentiate into T and B cells engulfing, cells for alarm) Not limited to recognition of shared molecular patterns, can distinguish | facets of a unique single pathogen Peripheral tissues (reconnaissance, Antigen: target recognized by lymphocyte show invaders to central system) Each new lymphocyte has a unique antigen receptor | o Generated randomly by genomic DNA rearrangement Secondary lymphoid organs (LN, Naïve lymphocytes patrol secondary lymphoid organs spleen, MALT; brain of immune o Antigen encounter —> activation and proliferation —> clonal system, builds responders and expansion memory cells) B lymphocytes Develop in bone marrow with unique B-cell receptor (antigen receptor) Innate Immune System o BCR = surface immunoglobulin = antibody Soluble: complement cascade Antigen binding by BCR —> clonal expansion into two types Cellular: phagocytes, NK cells o Plasma cells: live in bone marrow, long lived, secrete Pathogen-associated molecular patterns (PAMPs): immunoglobulin o LPS o Memory cells: long-lived, capable of rapid response when o CpG DNA (prokaryotic) antigen is seen again o Mannose (lots in bacteria, covered in humans) Immunoglobulin structure o Flagellin Tetramer with 2 heavy and 2 light Rapid activation of the system, no prior contact needed chains with interchain disulfide bonds Complement System Ends are highly variable The pre-existing hostile milieu Constant end determines class Serum proteins (approx. 25) identity (IgM, IgD, IgG, IgA, IgE) Proteolytic cascade at the cell surface has two possible outcomes and function 1. Generation of cell-bound fragments —> lysis of pathogen OR tagging Hinge region made of random coil for phagocytosis (opsonization) peptides, highly flexible 2. Release of soluble fragments —> recruit phagocytes T Lymphocytes Cells of innate system = phagocytes and NK cells Origin in bone marrow but develop in the thymus Common lymphoid derived: B-Cell, T-Cell, NK Cell T Cell Career Paths Common myeloid derived: neutrophil, dendritic cell, macrophage o CD8+ T cells = cytotoxic T cells, recognize MHC Class I and Common erythroid derived: erythrocyte, platelets kill cells expressing abnormal cytoplasmic proteins o CD4+ T cells = helper T cells, recognize MHC Class II, signal Macrophages B cells, CD8+ T cells, phagocytes (soluble OR surface signals) Tissue-resident, collect debris PAMP, complement, antibody, chemotaxis receptors Both B Cells and T Cells… Reorganize cytoskeleton—seek and engulf Have a surface antigen receptor (BCR vs. TCR) Neutrophils (PMNs) Have extreme diversity of antigen binding Most abundant blood leukocytes, ↑ with infection Require antigen triggering to initiate clonal expansion Demarginization: entry of neutrophils into blood from reserve (in liver, Antigen activated cells produce effector T cells and memory cells spleen) Only T cells… Engulf + kill with myeloperoxidase (HCl producers) Never secrete their antigen receptors Dendritic Cells (DCs) Cannot bind free antigen molecule—only peptides of 8-25 AAs Tissue-resident sentinels Require antigens be presented to them via major histocompatibility Pathogen contact —> migration from tissue to LN molecules (MHCs) Inform the adaptive response (professional APC) Major Histocompatibility Molecules Definitions: Two classes I and II Cytokines: secretory proteins that mediate immune cell Highly polymorphic, varying greatly among individuals, basis for organ developments/career choice; direct lineage, localization, effector functions rejection between non-twins of recipient cell Also called human leukocyte antigens (HLAs) Interleukins: subset of cytokines that communicate between leukocytes o MHC Class I Chemokines: small cytokines used for leukocyte chemotaxis ▪ Expressed on all nucleated cells ▪ Alpha chain (alpha 1, 2, and 3 domains) + beta Innate Call for Help microglobulin (no polymorphism) PAMP recognition —> macrophages —> ALARM ▪ Antigenic peptides derived from cytoplasm o Release of IL-1 and TNF-a o MHC Class II ▪ Activate neutrophils and increase their phagocytosis ▪ Expressed on APCs (ex. DC, macrophage, B cell) and ROS ▪ Alpha and beta chains ▪ Alter vascular epithelium to recruit more PMNs ▪ Antigenic peptides derived from endocytic ▪ Signal DCs to mature, migrate compartment (from proteins external to the cell) ▪ Signal hypothalamus to increase temp Binding of BCR vs TCR ▪ Chemokines (ex.CXCL8 attracts PMNs) BCR: native, unprocessed, or free-floating antigen; monovalent or o Complement derived chemoattractants (C3a, C5a) polyvalent; protein, lipid, or carbohydrate o Pathogen derived chemoattractants (f-mlp) TCR: only processed antigens presented by another cel Complement System TERMINAL COMPONENTS APPLYING TO ALL PATHWAYS Three primary initiation routes: Classical, MB-Lectin, Alternative C5 convertase cleaves C5 into C5a and C5b Three results following activation: recruitment of inflammatory cells, o C5a floats away opsonization of pathogens, killing of pathogens o C5b non-covalently binds cell surface, associates with C6,7,8 Remember: first activate lots of C3, then activate lots of C5 C5678 catalyzes formation of C9 —> membrane attack complex (MAC) MBL (MB-lectin) likely gave rise to C1q (classical) MAC leads to formation of a pore in the cell membrane that allows free exchange of ions and water —> CELL LYSIS Classical pathway and MBL Collagen-like stalks serve as springs, bouquet appearance Small Complement Cleavage Products o Initiation molecule of Classical = C1q C3a, C4a, and C5a increase vascular permeability to serum in endothelial ▪ Lectin-like heads bind Fc of antibodies IgG/IgM cells o Initiation molecule of MB-Lectin Pathway = MBL o Increased flow of immunoglobulins and lymphatic drainage ▪ Lectin heads bind mannose on bacteria Chemoattractants—C5a most potent for neutrophils and monocytes Serine proteases activate when spring is deformed by lectin binding o C3a attracts eosinophils (allergies) o C1q serine proteases = 1 C1r and 1 C1s Complement system also removes immune complexes that can cause o MBL serine proteases = 2 MASP-2, 1 MASP-1 inflammation—protects the glomerulus SHARED FUNCTION OF C1q AND MBL o Remember: kept portion of cleaved molecule binds to previous Complement deficiencies bound molecule (i.e. C2a binds to previously kept C4b) C1q, C2, C4 —> autoimmune disease, increased bacterial infection o Serine proteases (C1r/s and MASP1/2) catalyze cleavage of C4 C3–> susceptibility to bacterial infection, glomerulonephritis into C4a and C4b—remember: C1q requires previous contact C5, C6, C7, C8–> susceptibility to Neisseria infection (antibodies!) ▪ C4a floats away Sensing danger ▪ C4b remains bound to proteins on bacterial surface Toll-Like Receptors (TLRs) o C4b cleaves C2 into C2b (floats away) and C2a (binds C4b) Located: cell surface (membrane), endosomes o New C4b2a is a C3 convertase, cleaves C3 into C3a and C3b o Transmembrane with two domains: ▪ C3a floats away ▪ Ectodomain: leucine-rich repeats ▪ C3b many bind to surface, one binds to C4b2a ▪ Cytoplasmic domain: Toll/IL-1R containing domain Many C3b = opsonization! (TIR) initiates intracellular signaling o New C4b2a3b = C5 convertase Signal danger from: bacteria, fungi, viruses, protozoa Signal via: cytokine transcription o NF-kB: promotes inflammation alarms o MAPK (AP-1): produce more pro-inflammatory cytokine o Interferon response factors (IRFs) Alarm: Pro-IL-1B, chemokines, interferons (type I) ALL TLRs act as dimers ALL TLRs detect microbial PAMPs Results of TLR alarms: recruitment of PMN, NK cells; activation of macrophages and DCs; increase PMN and NK production, increase body temp; production of serum proteins (acute phase response) NOD-Like Receptors (NLRs) Located: cytoplasm o Modular domain structure and sense with LRR Alternative Pathway o NOD domain allows oligamerization Not reliant on a surface trigger Signal danger from: bacteria, viruses, fungi, protozoa, cell distress Activation based on instability of C3 (conversion between hydrolysis and o Activating signals are PAMPs, Damage AMPs (DAMPs) intact state) Signal via: cytokine transcription and maturation o In hydrolyzed form, C3 binds factor B Alarm: Pro-IL-1B o Factor B is able to be cleaved by factor D into Ba and Bb Two main types of NLRs o Factor Bb binds C3(H2O)—> C3(H2O)Bb is a soluble C3 o NOD receptors (NOD-1 and NOD-2): activate NF-kB convertase ▪ CARD domains that mediate binding with other o This C3 convertase produces C3a (floats away) and C3b (bind molecules with homologous CARD to surface) ▪ Resting = monomeric, in presence of PAMP = o Factor B associates with surface-bound C3b and product is dimerize via CARD domains cleaved by Factor D to make C3bBb ▪ Ends in activation of NF-kB o C3bBb is a cell bound C3 convertase and more C3b o Inflammasome Assembly NLRs (IPAF and NLRP) accumulates ▪ NLR senses PAMP/DAMP —> assembly of protein o Cell-bound C3 convertase + C3b —> C5 convertase complex —> aggregation of pro-Caspace-1 —> autocleavage to Caspace-1 —> activates pro-IL-1B into IL-1B ▪ Assembly: NOD domain (oligamerization) + LRR domain (sensing) + dimerization domains (pyrin domain and CARD domain) o NLRP3 is a player in gout RIG-Like Receptors (RLRs) Located: cytoplasm o Two domains: ▪ Helicase-like domain binds viral RNA ▪ Two CARD domains interact with adapters Signal danger from: viruses Signals via: cytokine transcription Alarm: interferons Result of RLR alarms: activation of NF-kB and IRF-3 Two RLR family members o RIG-1: recognizes short blunt-ended dsRNAs o MDA-5: recognize long dsRNAs o Both detect COVID-19 Interferons Type I = IFN-a and IFN-B o Produced in response to viral infection o Produced by all nucleated cells in response to detection of their own infection by viruses ▪ Autocrine and paracrine signaling occurs o Induce resistance to viral replication ▪ Induction of RNAases ▪ Inhibition of ribosomal protein translation o Activate NK cells to kill virally infected cells o Increase expression of MHC I on cell o Recruit other leukocytes Phagocytosis Type of receptor-mediated endocytosis Cathrin-coated pits internalize the phagosome —> clathrin uncoats from the endosome —> endosome traffics into the cell and fuses with lysosomes Opsonized (coated) or non-opsonized (engulfed without coating) Scavenger receptors (SRs) mediate non-opsonic phagocytosis o Ligands are usually lipid-related o When healthy, help with recycling of apoptotic bodies (efferocytosis) and HDL o Bacteria respond to SRs with a capsule (dense polysaccharides) ▪ Overcome by opsonization Opsonins: o Collectins: C1q and MBL, surfactant proteins (detergent-like) ▪ Collagen stalks + lectin heads o Non-collectins: IgG and C3 ▪ Bind to determinants on capsule to give phagocytes a way to grip the capsule Opsonic phagocytosis o Bacterium coated with complement (CR1) and IgG antibody (FC receptor) Lysosomes o Extremely acidic o Lysozyme disrupts peptidoglycans o Human beta-defensins (HBDs) disrupt pathogen cell membrane Oxidative Killing Lysosome makes reactive oxygen species (O2-, HOCl, H2O2) and reactive nitrogen species (ONOO-) o Chronic granulomatous disease occurs due to NADPH oxidase ▪ Recurrent Staph infections (skin, dental, lungs) ▪ Mostly x-linked Leukocyte extravasation 1. Rolling (selectins) o Propelled by shear forces o Low affinity interactions 2. Firm adhesion, arrest (integrins) o Leukocyte detection of chemokine —> LFA-1 high affinity state o Tight adhesion to endothelial ICAM-1 o Inside-out signaling: internal cytoskeletal changes lead to external conformational changes of binding site 3. Extravasation (unzips between endothelial cells) o Leukocyte PECAM-1 binds endothelial PECAM-1 4. Chemotaxis (via chemotactic gradients) Receptor Features and Types Remember: B-cell surface immunoglobulins are identical to antibodies Organization of the Heavy and Light Chain Loci except that antibodies are soluble V region = variable gene segments BCR has 2 heavy and 2 light chains D region = diversity gene segments o To become soluble, transmembrane domain is removed J region = joining gene segments o Has a hinge to give flexibility o Fab and Fc domains Heavy chain locus: piece of chromosome 14 that encodes the heavy TCR have alpha and beta chains (same size) chain of the antibody o Always anchored to the surface of the cell One maternal and one paternal allele Both TCR and BCR have constant and variable regions Each allele has 40 V, 23 D and 6 J segments (from 5’ to 3’) Remember: goal is to pick one V, one D, and one J and join them Hypervariable Regions together Variability at a given position = This makes combinatorial diversity: diversity by choosing one # 𝑜𝑓 𝑑𝑖𝑠𝑡𝑖𝑛𝑐𝑡 𝑎𝑚𝑖𝑛𝑜 𝑎𝑐𝑖𝑑𝑠 from three different groups 𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑜𝑓 𝑚𝑜𝑠𝑡 𝑐𝑜𝑚𝑚𝑜𝑛 𝑎𝑚𝑖𝑛𝑜 𝑎𝑐𝑖𝑑𝑠 D joined to J first, DJ joined with V Hypervariable loops contribute to the specificity of the antigen Light chain locus binding site Joining one V and one J All heavy and light chains and TCR alpha and beta chains have distinct framework regions and hypervariable regions at In a linear stretch of DNA, the cell loops out part of the DNA to bring predictable locations together two segments; intervening DNA makes an excision circle and Interaction of AA side chains on the antigen and antibody via is cut out electrostatic forces, hydrogen bonds, Van der Waals forces, and Constant regions are not changed throughout this process hydrophobic forces holds the complex together Large number of variable V, D, and J regions likely due to ancient gene duplication events Recombination signal sequences (RSS): palindromic regions of 23 Human Immunoglobulin Classes (Isotypes) or 12 base pairs indicate where the RAG should cut the DNA to Isotype determined by heavy chain constant region (Fc region) recombine it Naïve B cells express IgM and IgD on cell surface A 23 will always be joined to a 12 (never 12-12 or 23-23) IgD disappears as cell becomes experienced—naïve B cell marker IgM: BEST at fixing complement and initiating classical cascade Recombination activating gene (RAG) enzyme is a heterodimer of o Can opsonize a little bit by clumping things together Rag1/Rag2 o Has low affinity but high avidity in its pentameric form DNA is open and RAG is expressed at specific times in B and T IgG: most abundant, main protector—does everything! Cell development Neutralizer, sensitizing for NK cells, activates complement, RAG binds a 23 and a 12 and brings them together (synapsis) and diffuses into all interstitial spaces cuts exactly between the exon and the palindromic region o Remember: IgG = I Go across the placenta o Prevents free base pairs on the ends of the DNA that IgA: secreted into epithelial lumen, protects GI tract and lungs may disrupt the reading frame o Good at neutralizing in the lumen Cut DNA ends are bound by DNA repair enzymes o Usually dimeric Junctional diversity: TdT adds random bases between cut ends IgE: allows mast cells to recognize allergen and degranulate to (approx 2-6 bases) and repair enzymes fill in the rest release histamine o Can cause frameshift mutations Affinity: strength of binding at a single site o Creates even more diversity Avidity: overall strength of binding at multiple sites Excision circles are also fused o Generated with every TCR and BCR Mechanisms by Which Antibodies Combat Infection o Remain in lymphocytes, can be assayed to test for SCID 1. Neutralization: Antibody binds very tightly to pathogen to o SCID = no B cells or T cells due to RAG deficiency prevent it from causing damage Mediated by an Fab arm Third hypervariable region (CDR3) is the joint between DJ or VDJ Prevents toxin from getting to the receptor The most diverse due to TdT 2. Opsonization: antibodies bind to surface of bacteria/virus and a phagocyte with Fc receptor Three processes establish diversity 3. Complement: fixed by Fc regions on IgG and IgM leads to 1. Combinatorial diversity (VDJ assortment) C3b deposition and subsequent lysis 2. Junctional diversity 4. Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) 3. Combinatorial diversity through heavy chain and light chain NK cells find pathogen and kill it by inducing apoptosis combinations NK cells recognize distress markers on cell surface Monoclonal antibodies: soluble protein in unlimited quantities, with Differences Between B and T Cell Receptors exquisite specificity and high affinity for any ligand you choose Epitope: antigen region physically interacting with the antigen receptor Neutralize toxin of choice or viral infection after exposure B cell recognizes surface region on the antigen Target pathologic cells (i.e. malignancy) T cell recognizes a peptide within the folds of the internal Identify cells with specific surface protein markers antigen molecule (i.e. antigen needs to be broken down to be recognized) B Cell development: in the bone marrow, antigen-independent T Cell development: in the thymus, self-antigen dependent Antigen-driven maturation of B and T cells occurs in the lymph nodes, spleen Features of Each Phase B Cell Tolerance B-Cell Development: in the bone marrow, antigen-independent Central tolerance: in the bone marrow (B cells) or thymus (T cells) Generates diversity Check against reactivity at the immature B cell stage (IgM+/IgD-) Enormous cell wastage Mechanisms: Selection against self-reactivity A. Strong self-affinity reactivates RAG —> attempt to edit B-Cell Maturation: in the secondary lymphoid tissues, antigen-driven L-chain, if unsuccessful apoptosis occurs (deletion) Somatic hypermutation (aka affinity maturation improved antigen B. Intermediate self-affinity —> state of relative BCR binding) unresponsiveness (anergy) Class switch recombination—vary antibody effector function Peripheral tolerance: maintenance of tolerance in peripheral tissues B cell dependence on T cell help, not available for anti-help response Challenges of B Cell Assembly VDJ Recombination Naïve B Cells Some V gene segments are incomplete Go to secondary lymphoid tissued to undergo antigen-dependent maturation in Rearranging can lead to frame shifts (1/3 are viable) germinal centers = germinal center reaction Autoreactive BCRs must be removed Germinal centers form within B cell follicles in LN, spleen, MALT Generation of B Cells in the bone marrow Lymph node structure Orderly recombination Lymphatics drain into subcapsular sinus Allelic exclusion ensures that each clone expresses one antibody (one Follicles in cortex of (B Cell zone) heavy chain and one light chain only) o Follicles always occupied by resting B cells performing o Prevents energy wasting antigen surveillance o Two or more distinct receptors on the same cell mean every Paracortex (T Cell zone) activation = production of TWO types of antibodies (anti-X + o T Cells looking for antigens on dendritic cells live here anti-Y) Germinal center arises in the follicles when naïve B cell recognizes Allelic exclusion properties: antigen and finds T cell help o Reduces expression of RAG-1 and RAG-2 o RAG-2 targeted for degradation Antigen-Dependent B Cell Maturation o HC locus no longer accessible for recombinase machinery Steps Deletion of self-reactive clones via apoptosis, establishes central tolerance 1. Antigen-Specific B cell maturation o Only prevents auto-immunity to cells found in bone marrow 2. T-Cell and B-Cell recognition and pairing a. Naïve B cells sample antigen flowing in lymph in follicle Key Steps of B Cell Development in the Bone Marrow b. B cell recognizes antigen via BCR and antigen receptors Pro B Cell —> Pre B Cell indicates production of a full heavy chain cluster on surface of cell—> activation signal Pre B Cell checkpoint occurs at this step (see steps below) c. Clustering leads to endocytosis of BCR and antigen o Prevents more heavy chain rearrangement when finished d. B Cell displays MHCII peptides at B-T border 1. DJ rearrangement occurs e. Effector CD4+ T cells patrol B-T border for MHCII, 2. Completed DJ rearrangement allows RAG to bring VDJ segment when recognized, T Cell receives activation signal 3. If complete a VDJ segment with an open reading frame is created, f. Partnership! translation occurs g. LFA-1 on T cell binds ICAM-1 on B cell 4. Translated polypeptide = mu chain because first heavy chain h. While paired, T cell expresses CD40-ligand and B cell constant region used by all B cells is for IgM CD40 binds it to complete B cell activation 5. Mu chain combines with surrogate light chain: VpreB and λ5 i. Activated B cell migrates to the follicle and proliferates (germline encoded, no rearrangement) 3. Germinal center formation 6. Entire mu chain with surrogate light chain is exported to B cell Somatic hypermutation (SHM)/affinity maturation: antigen binding surface as pre-B cell receptor surface (complementarity determining region) is switched 7. Pre-BCR associates with Igβ and Igα which initiate intracellular o Iterative process where highest affinity antigen binders signaling for mature and pre-BCR are kept, others are apoptosed Complete Pre-BCR = mu heavy chain + surrogate light chain (VpreB o GC dark zone (centroblasts): B cells proliferate while and λ5) + Igβ and Igα introducing mutations into variable regions Tonic signaling: no known ligand, cell has succeeded in creating a o GC light zone (centrocytes): B cells undergo affinity normal heavy chain, pre-BCR checkpoint should be passed checking, only the highest affinity kept X-linked agammaglobulinemia: no circulating B cells because tonic o Activation induced deaminase (AID) helps introduce mutations by converting cytosine to uracil signal never makes it into the nucleus (lack of BTK) ▪ Repair of excision sites is error-prone, therefore mutations occur Pre B Cell —> Immature B Cell indicates production of a full light chain Immunoglobulin class switch recombination (CSR): entire heavy Proper BCR checkpoint occurs here chain variable region switched onto a different heavy chain constant VJ joint creation for light chains allows multiple attempts for correct region (IgM—> IgG, IgA, or IgE) rearrangement o Happens once or twice, usually early in maturation o Two attempts at κ and λ locus (one per chromosome) o IgM to IgD via differential RNA splicing o κ success = IgMκ receptor, λ success = IgMλ receptor o IgM to IgG, IgA, or IgE o by DNA rearrangement BCR Signal Induction o Each alternative constant-region encoding gene is Igβ and Igα each have a conserved immunoreceptore tyrosine-based preceded by a switch region, which is a target for the activation motif (ITAM) sequence class switch recombination machinery o Tyrosine side chains stick out from receptor, hydroxyl groups ▪ Cytokines determine switch region targeting are phosphorylated by Src-family tyrosine kinases o Activation-induced deaminase (AID) is key enzyme Tyrosine side chains stick out, act as docking site for signaling molecules o Hyper IgM syndrome: due to failure of B cell to get SH2 domains attach to pair of phosphorylated tyrosines on ITAM contact-dependent help signal (usually deficiency of o Remember: SH2 (signaling molecule) + ITAM (receptor) is a CD40L (x-linked recessive), CD40 (autosomal recessive), very common motif for signaling! or AID (autosomal recessive); NO class switch occurs Syk: kinase family with SH2 domain that mediates BCR signaling 4. Differentiation of B cells into plasma cells and memory B cells Improving Affinity with Exposure Increase in antibody level and affinity for antigen over time With a booster, IgM levels rise a bit and IgG levels rise a lot—class switched antibodies are the highest affinity ones, so more GC time = more class switch How T Cells Recognize Antigen T Cell Surveillance TCRs recognize AA sequences of peptides 9-16 AAs long T cells must respond differently to viruses and bacteria T cells respond to TCR engagement by activation, clonal expansion, Virus: viral peptide on a cell’s MHC molecules indicates to a and differentiation T cell that it is infected and should be killed Remember: mechanisms are based on TCR recognition of pathogen o Peptide presented on MHC Class I molecules peptides bound to MHC (aka HLA) molecules o Response: KILL the APC P-MHC is recognized by T cells o CD8 = kill o Peptide is “embraced” by HLA molecule based on its Bacteria: bacterial peptide on phagocytic cell signals to a T chemical structure of alpha helices and beta pleated cell that the phagocyte has ingested a foreign substance and sheets must be helped to eliminate the pathogen o Side chains of peptide bind receptor site o Peptide presented on MHC Class II o Response: HELP the APC T Cells Must Recognize Many Pathogen Peptides ▪ Endocytic pathogen/ingested bacteria = Somatic recombination mechanism randomly generates the antigen activation of macrophages and dendritic recognition portion of TCR cells to enhance pathogen killing Genome is not large enough to encode this many genes ▪ Extracellular pathogen/toxin = provision Selection mechanism must identify T cell clones that can recognize of help for production of antibodies pathogenic peptides and eliminate clones that recognize self o CD4 = help Diversifying MHCs How Peptides Bind Microbial pathogens can mutate around a defense recognition system Peptides bind differently to Class I and Class II MHC molecules that is highly stereotyped (binds in only one way) Peptides binding to Class I: originate from protein making Therefore…MHCs themselves must be diversified and degradative machinery in cell’s cytoplasm Make everyone have different types of MHC o Clusters of tyrosine residues on MHC molecule Increase the number of MHC structures in individuals (multiple interact with N-terminal AA side chains loci) o Positive regions of MHC molecules attach to C- The peptide-presenting MHC molecules are changed from individual to terminal AA side chains individual, so each person binds different kinds of peptides o Binding must occur this way for specificity Allows species to survive because everyone has unique MHC o Usually peptides are 9 amino acids long ▪ Always oriented with NH2 to the left MHC DNA Structure ▪ Side chains 2 and 9 usually anchor Sits on short arm of chromosome 6 peptide to MHC pocket (determines Class I and Class II regions with different regions in them affinity) o Class I: HLA-A —> HLA-C —> HLA-B ▪ 2 and 9 are anchoring sites (similar every o Class II: HLA-DR —> HLA-DQ —> HLA-DP time) with different intervening regions o Thousands of alleles for each region = extreme diversity Peptides binding to Class II: synthesized by pathogen and Pathogen drive equips species with lots of polymorphic MHC degraded imprecisely by enzymes in endocytic vesicle during molecules phagocytosis o Length of peptide is unknown Selecting T Cells o Class II tolerant to peptides of any length T cells must recognize own MHC molecules and specifically bind and o Tightly regulated middles (1, 4, 6, 9) recognize pathogen peptides prior to encountering the pathogen Self-peptides are used as a surrogate for pathogen peptides to Structures of Class I and Class II Molecules select one’s T cell clones on self-MHC molecules Homologous domain organization with different chain structure Since the system is selected on self-peptides and self-MHC, MHC I it is inherently autoreactive o Antigen binding domain has an α1 and α2 domain Clonal selection must occur in the thymus to select TCRs non-reactive which are coupled to stalk-like α3 domain with a to self and reactive against non-self transmembrane piece Steps: o Extrinsic β2m domain locks the remaining structure 1. Positive Selection: selects T cell clones that recognize self- in place peptide in an individuals own MHC molecules MHCII 2. Negative Selection: deletes overtly self reactive clones with o Peptide binding region has an α1 and a β1 domain high affinity for self-peptide MHC (central tolerance) o α1 binds to α2 domain with transmembrane region 3. Negative selection leaves T cell clones that recognize self- o β1 binds to β2 domain with transmembrane region peptides with moderate affinity o Symmetry! Individuals of the same species have different MHC alleles and others’ cells and tissues are recognized as non-self and attacked by the person’s T cells as if they were pathogens Differences Between B and T Cell Development TCR Development BCR have no antigen binding requirements other than being non self-reactive Variable and constant regions TCR must surveil antigen presented in the context of MHC molecules TCR is never secreted One selection step for B cells, two selection steps for T cells TCR is analogous to one arm of the BCR o α and β chains (instead of heavy and light) because they are the Environment for T Cell Development same size T cells develop in the thymus from progenitors that are derived from o Recombination starts with β chain pluripotent stem cells located in the bone marrow ▪ Successful recombination —> allelic exclusion and Development takes three weeks and generates mature T cells which recombination of the α chain populate peripheral lymphoid organs Double Negative Stages o Lots of cell wastage Distinguished with cell surface molecules Thymus has lobules 1. DN1 = proliferative stage, no DNA recombination, T cell is not committed o Peripheral cortex: epithelial stroma, densely populated with 2. DN2 = recombination of D and J segments at β locus, commitment to lymphoid cells lineage o Central medulla: less lymphocyte rich, contains dendritic cells o Recombination simultaneously initiated at gamma, delta, and and macrophages (lighter staining) beta TCR loci No thymus = no T lymphocytes o If gamma and delta loci recombine before beta, they pair at cell o Lymphoid progenitors enter in cortico-medullary region surface and drive cell to gamma-delta lineage o Signals from stroma promote differentiation and proliferation ▪ Not favored because have to have success at two o Activation of NOTCH1 receptor drives T cell fate loci simultaneously ▪ Mediates cell contact dependent lineage o If beta combines first, gamma and delta loci can compete with determination alpha chain production o Ex. SCID mouse with no lymphoid progenitors can be rescued o Two D-J clusters = two full rounds of recombination by bone marrow from nude mouse; Nude mouse with no 3. DN3 = β chain V to DJ joining, chain incorporated into a pre-TCR thymic epithelial cells can be rescued by SCID thymus cells o β chain paired with surrogate α chain called pTα = pre-TCR o Pre-TCR associated with CD3 signaling complex to give off Broad Summary of T Cell Development signal that… Remember: thymocytes migrate as they mature ▪ Ends further beta, gamma, and delta rearrangement o Enter through venules at cortico-medullary junction ▪ Induces DN4 (proliferation of single beta chain o Migrate to peripheral cortex and subcapsular region for double cells) negative stage ▪ Induces transition to double positive stage o Migrate back toward medulla during double positive stage o Transition into DN4 shuts down TCR-β rearrangement via o Complete negative selection in the medulla and cortico- degradation of RAG proteins and closing of chromatin of β medullary junction chain loci on both alleles o Graduation from thymus happens at the cortico-medullary 4. DN4 = proliferation rebuilds cell numbers because so many are apoptosed junction 5. Successful β chain production = movement out of DN stage into DP stage 1. Pluripotent stem cell —> lymphoid progenitor (in bone marrow) 2. Lymphoid progenitor cells move to thymus via blood, become Double Positive Stages thymocytes TCR-α recombination occurs 3. Double negative stages 1-4 (T cells are CD4-/CD8-) Only one V-J joint 4. Double positive stages (CD4+/CD8+) Multiple rounds of recombination 5. Stringent selection process Successful TCR-α rearrangement does NOT block recombination until the a. Positive selection: TCR and MCH-p must interact at least thymocyte receives signal via CD3 that TCR has affinity for MHC peptide very minimally Gamma-delta recombination is also ongoing at this step b. Negative selection: TCR and MCH-p cannot bind too o Delta locus is located between Vα and Jα segments tightly o First VJ recombination event deletes the entire α locus 6. Single positive stage (either CD4+ OR CD8+) Two types of T Cells develop in competition Positive selection o Alpha-beta T cells (alpha-beta TCR on surface) Double positive T cells test interaction with MHC-p in the thymic cortex ▪ Most abundant, either CD4+ or CD8+ Coupled with CD4 and CD8 cell specification (see below) ▪ Responsible for adaptive immune response Unsuccessful interaction (ignoring MHC) allows continues rearrangement o Gamma-delta T cells (gamma-delta TCR on surface) of the alpha chain to make a new TCR and try again ▪ Minor population, most are CD4-/CD8- ▪ Recognize lipids in the gut, function without MHC CD4 or CD8 Cell Fate Specification presentation (innate-like) If TCR positive selection go ahead signal is a result of interaction with an epithelial cell expressing MHC I, double positive cell must maintain CD8 co-receptor expression o Rescue from apoptosis occurs, generates CD8 single positive (cytotoxic) T cell If TCR positive selection go ahead signal is a result of interaction with an epithelial cell expressing MHC II, double positive cell must maintain CD4 co-receptor expression o CD4 single positive (helper) T cell is generated Negative selection: double positive CD3+ thymocytes throughout cortex and especially at the cortico-medullary junction Thymic medullary epithelial cells and dendritic cells in cortico- medullary region express AutoImmune REgulator (AIRE) o Encodes transcriptional activator to induce expression of tissue-restricted proteins (ex. Retina- and ovary-specific), so T cells that react to them can be screened out— prevents autoimmunity to peptides not seen in CM region o AIRE dysfunction = inflammatory glandular destruction ovarian/testicular failure, vitiligo (melanin loss), alopecia Eliminates 60% of cells that pass positive selection Cytokines Systemic Infections Small, usually secreted proteins Infection of an organ or system Bind specific receptors with high affinity WBC elevates, fever occurs Expression is usually very tightly regulated If enough tissue is infected, enough macrophages begin secreting TNF, IL- Two families 1β, and IL-6 o Four-helix bundle family: interleukins o Blood vessels dilate, blood pressure is reduced ▪ Homo or hetero dimers that bring together two o Endothelial cells promote thrombosis, can get disseminated receptor chains intervascular coagulation, anoxia in tissues o TNF Family: TNF-α, CD40L, BAFF, FAsL o Preventing this is why preventing sepsis, etc. is important ▪ Trimer that brings together three receptor chains Cytokines driving systemic infections = TNF, IL-1β, and IL-6 Function at three ranges: o Promote mobilization of neutrophils o Autocrine (self): IFN-β and IL-2 o Stimulate hypothalamus to elevate body temperature o Paracrine (adjacent cells): IFN-α, most T-cell effectors ▪ NSAIDs block generation of prostaglandins o Endocrine (circulatory system): IL-1, IL-6, TNF-α stimulated by TNF, IL-1β, and IL-6 o Promote mobilization of energy stores from fat and muscle Chemokines (primarily IL-6) Functionally distinct, work with cytokines ▪ This also elevates body temperature Signal through GPCRs o Stimulate liver to up-regulate inflammatory mediators (acute Generate chemo-attractive gradients for cells with cognate receptor phase response-APR) ▪ IL-6 is the most potent at stimulating CRP, MBL, IL-1 complement components, etc. Product of the inflammasome Dendritic Cell Activation Member of the TLR family Has pattern recognition receptors, some cytokines and chemokines Potent inflammatory cytokine Best at taking up the antigen that it sees and delivering it to LN through lymph Directed by CCR7 chemokine receptor (recognizes two chemokines in T Signal through NF-kB and MAP kinase (AP-1) pathways cell zones of LN) TNF Family Requires a threshold of antigen to initiate this response In T Cell zones, present antigen on MHCs on cell surface Activation entails trimerization If T cells recognize an antigen, they begin to proliferate Activate either NF-kB or caspase cascade (apoptosis) Important members of the family When a CD4 T Cell Recognizes an Antigen… o TNF-α TCR receptor recognizes the peptide and the MHC, signaling response initiated ▪ Potent inflammatory cytokine LFA-1 is activated and tightly binds ICAM-1 to stabilize interaction ▪ Activates NF-kB o CD40L TCR-MHCII complexes cluster in the middle of the immune synapse and ▪ Found on APCs, including B cells adhesion molecules cluster on the outside ▪ Activated NF-kB T cell α and β chains cannot signal, must associate with CD3 ITAM motifs o FasL Lck activates ITAMs which are only recognized by ZAP-70 ▪ Promotes apoptosis ZAP-70 has SH2 domains which associate with ITAMs, and it ▪ Signals through caspases becomes capable of signaling TCR activates four signaling molecules Four-Helix Bundle Cytokines Module 1: recruitment and activation of Akt (serine kinase) Signal through JAK/STAT pathway o Activates mTOR and blocks apoptosis o JAK3 is only found in leukocytes ▪ Rapamycin blocks mTOR—>immunosuppression Most interleukins, IFNs, some growth factors Module 2: IL-2 is secreted and promotes rapid proliferative expansion o Pathways which produce: NFAT, NF-kB, and AP-1 Cytokines in Localized Infections For full activation of naïve T cells, two signals are required When the epithelial barrier is breached, immune effectors and immune sentinels Signal #1: TCR/CD4 engage MHC-II peptide (macrophages and dendritic cells) which detect and respond Signal #2: CD28 engages B7 on APC Receptors are activated when they detect molecular patterns associated o Restricts activation of naïve cells to activated professional with pathogens antigen presenting cells (DCs >> macrophages) o Macrophages become phagocytic —> release inflammatory o Without Signal #2, T cell becomes anergic cytokines Signal #3: defines the CD4 T cell’s effector fate o Dendritic cells —> leave local site and head to lymph node to o TH1 Cells: help macrophages by making them more signal adaptive immune response inflammatory, secrete cytokines for infections (TYPE 1) Cytokines (like IL-1 and TNF) activate endothelial cells ▪ Secrete IFN-gamma o Promote vascular permeability to allow in things that are o TH2 Cells: important in response to helminths, recruitment of attracted by chemokines eosinophils, basophils, mast cells (TYPE II) o Promotes thrombosis to clot local vessels and prevent ▪ Secrete IL-4 spreading of the infection o TH17 Cells: important for recruiting neutrophils, important for Chemokines establish chemoattractive gradient to recruit neutrophils then extracellular bacteria and fungi (TYPE III) monocytes ▪ Secrete IL-17–> drives local cells to make signals that recruit neutrophils and activate them at Macrophage Activation infection site Activation through pattern recognition receptor, release inflammatory mediators o TFH Cells: never leave LN, move from T cell zone to interface Activation of one macrophage encourages others to release cytokines with B cell zone to provide help to B cells for somatic Types of cytokines include… hypermutation and class switch recombination IL-1β o Treg Cells: peripherally generated, largely found in GI tract; TNF-α suppress immune response to peripheral antigens Summary of Types of Immune Response IL-6: Role in systemic infections, activating lymphocytes Type I immunity: targets intracellular microbes, generate CXCL8 (chemokine): Recruits neutrophils inflammatory macrophages which secrete inflammatory cytokines CCL2: Recruits monocytes Types II immunity: targets helminths/tissue repair; signaled by IL-4; IL-12: Important for activating NK cells oxidative metabolism; eosinophils, basophils, mast cells IL-10/IL-1R: Dampen the inflammatory response Type III immunity: targets extracellular bacteria and fungi; response to mucosal barrier breach; glycolytic metabolism Cell Surface Signals of Trouble in the Cell o Cross-presentation must occur for the DC to bind both MHC I presentation of foreign peptides (CD8 CTL) CD4 and CD8 at the same time Foreign proteins in cell membrane (NK) ▪ Endocytosed protein is allowed to leak out of o Viral envelope, mutated self (cancer) the endosomal system to allow be processed Cell surface distress proteins: MICA, MICB (NK) by the proteasome and loaded onto MHC I for o MHC I-homologs which don’t present antigen, no β2M presentation to the CD8 cell o Cell stress, malignant transformation Loss of MHC I expression (NK) Virus Evasion of CD8 CTLs o Pathogens have developed ability to interfere with MHC- Latency: minimizing viral gene activity, minimize protein production to I expression, so this prevents this loophole hide with minimal peptide presentation to cell surface Modulation of molecules involved in antigen presenting CD8 Cytotoxic T Cells o LowerMHC Class I expression, interfere with antigen Induce apoptosis in target cells processing in ER, interfere with loading Destroy cellular and viral genetic material Antigen variation: rapid mutation of viral genome (ex. HIV) Blebbing: fragmentation into apoptotic bodies that can be easily o “CD8 T cells developed last week are useless this week” phagocytosed Infection of immune privileged sites, such as CNS (ex. Herpes) Source of inflammatory cytokines o Protects terminally differentiated tissues but also allows safe IFN-gamma (activates macrophages) haven for some types of viruses TNF- α Resistance to apoptosis Antigen-specific recognition by the TCR is the activating trigger o Ex. Mimicking the cell’s own anti-apoptotic factors Antigen is presented by the target cell MHC I Can take on more than one target simultaneously Natural Killer Cells Relatively small cells First responders for infection Not antigen specific, so does not require clonal expansion Method of CD8 T Cell Killing Once activation threshold is released, acts just like CD8 CTLs Exocytosis of cytotoxic granules (preformed and mobilized at the surface of the o Adhesive immune synapse between LFA-1 and ICAM-1 cell when activated) contain: o Perforin/granzyme and FasL secretion Perforin: forms pores in target cell membrane and endocytic vesicles Germline encoded receptors recognize certain targets to help “accounting” o Homology with C9 (complement-mediated membrane 1. Inhibitory receptors: stand down attack complex) o Provide target cell with a “credit” Granzyme B: serine protease that enters through perforin pores and o Killer Ig-Like Receptor (KIR) scans target for presence of cleaves caspases to induce apoptosis MHC I, which is a major credit Surface expression of FasL ▪ If found, KIR binds MHC I Engages target cell fas (on most nucleated cells) —> apoptosis ▪ KIR then signals through ITIM, which recruits May be cleaved for secretion phosphatases to the signaling complex and Cytokine production of IFN-gamma and TNF-α dampens kinase signals from excitatory receptors Friendly fire is prevented by requiring intimate contact between CD8 T Cells 2. Excitatory receptors: attack and the target o Target gets a “debit” and is one step closer to apoptosis Synapse forms between LFA-1 on T Cell and ICAM-1 on target o NKG2D (Natural Killer, Going 2 Die) scans the cell for Signaling zone at the center, adhesion zone formed by LFA-1 on the distress markers (MICA and MICB) periphery, and secretory zone in the middle ▪ If found, activating signal via kinase recruitment (like CD28 on a T cell) promotes Secretory granules and Fas-ligand secreted in secretory zone and killing the target pool there as they wait to enter the target cell via the synapse o Fc(gamma) Receptor III (FcyRIII) ▪ Binds IgG coating on the target cell which is not usually present in healthy cells ▪ ITAMs recruit kinases and favor NK activation o TLR-3, TLR-7 ▪ Detect viral RNA ▪ Contribute NK activating signals If activating signals outweigh inhibitory signals…RESPONSE—> immune synapse formation, expression of FasL, perforin/granzyme B Subset of NK cells don’t kill and don’t have perforin or granzyme o Instead, they induce rapid and copious cytokine production ▪ IFN-gamma How CD8 T Cells Are Made ▪ TNF-α Naïve CD8 T cells are CTL precursors o Inhabit the secondary lymphoid tissue (instead of peripheral tissues) No perforin, granzyme, or FasL expression to survey lymph draining from periphery Very high threshold for optimal activation o Dense antigen presentation, abundant TCR cross-linking Time Course of Viral Response o Strong co-stimulation with licensed dendritic cell 1. Earliest response is burst of cytokines Licensing: dendritic cells are approved to be allowed to activate a CD8 T cell from alarm NK cells DC licensed after presenting an antigen to a CD4 T cell and getting a IFN-gamma lowers threshold for killer response in return NK cells (on alert) o Ex. DC CD40 ligation by TH cell CD40L TNF-α activates the endothelium, o Ex. TH cell cytokine production facilitates lymphocyte trafficking, Dendritic cell interacts with CD4 and CD8 cell simultaneously causes feeling of malaise during licensing and activation 2. Rapid rise in NK Cell killing of virally o Because the dendritic cell loads viral peptides from the infection cells dead cell onto MHC II (following endocytosis and As this peaks, the trend changes degradation), the dendritic cell can obviously present to and virus activity decreases CD4 3. Naïve host, so several days until CD8 ▪ Remember: T Cell co-receptor for MHC I is CTLs are detectable, 7-9 days to peak CD8, T Cell co-receptor for MHC II is CD4 Nomenclature Peptide Loading onto MHC Molecules Alleles: different forms of a gene Synthesis of Class I MHC Molecules Allotypes or allomorphs: different or alternative protein forms 1. MHC Class I formed with the aid of chaperone molecules encoded by alleles 2. Proteasome digests peptide fragments Genotype: collection of genes in an individual, referring to the two 3. MHC Class I molecules bind peptides derived from cytosolic alleles of a locus molecules during assembly within the ER Haplotype: the genes (alleles) on one chromosome, contributed by 4. Peptide fragment comes into the outer domain and the one parent, usually referring to alleles of both class I and class II extrinsic β2m domain locks the entire structure in place loci a. Stabilizing by peptide and β2m is required to prevent Linkage: measure of the genetic distance between gene loci killing of bystander cells by random peptides being Linkage disequilibrium: alleles making up a haplotype are found taken up by empty MHC molecules together significantly more or less frequently than expected by chance Synthesis of Class II MHC Molecules Synthesized in the ER and need to be directed to the endosome MHC Alleles and Their Diversity without binding any cytosolic-derived peptide Different MHC alleles confer different functional properties on the 1. Invariant chain (Ii): chaperone that complexes with MHC adaptive immune system Class II molecules during their synthesis in the ER o Blocks binding groove of MHC Class II Naming: molecule to prevent loading of wrong peptides o Recognition sequence on Ii redirects new MHC Class II molecule to the endosomal compartment for loading 2. Within acidic endosome, Ii is degraded to Class II associated invariant chain peptide (CLIP), which associates with the pocket (does not bind) 3. HLA-DM relaxes the structure of the Class II MHC to allow it to bind the highest affinity peptide The genetic unit is the allele, but because of their variety, they 4. Without Ii, MHC Class II can now traffic to the cell are grouped into families (ex. HLA-B*27) membrane Wide variety of peptides are found on MHC II Remember: MHC Class I has loci HLA-A, HLA-C, and HLA-B MHC Class II has loci HLA-DR, HLA-DQ, and HLA-DP Codominant Inheritance of MHC Alleles Four combinations of parental haplotypes MHC polymorphism is due to survival—evolutionary response o Ex. paternal (a/b) and maternal (c/d) can to structural diversity and mutation potential of combine into ac, ad, bc, or bd microorganisms o Parents are always a one haplotype mismatch for o Frequency-dependent selection: individual with the donation rarest allele has best chance of survival o Heterozygous advantage: the individual with more Expression of MHC on the Cell Surface MHC structures can present more, different pathogen Six Class I molecules are on the surface of each cell peptides Maternal and paternal A, B, and C o No limit on number of alleles that a species can have Each allotypic molecule can be separately loaded with Variety is advantageous, specific alleles are not better or worse different pathogen peptides MHC Class II molecules have highly variable HLA-DRβ, HLA- Origins of MHC Alleles DQα HLA-DQβ, HLA-DPα, and HLA-DPβ (NOTE: only β chain Many of the modern immune system molecules descend from of HLA-DR is highly variable neanderthals Codominant expression of MHC II genes gives two o Different populations have differing amounts of different HLA-DR when alpha chains are the same in neanderthal alleles maternal and paternal HLA alleles have very distinctive geographical distributions Presence of different alpha and beta chains for maternal due to… and paternal gives four different HLA-DP and HLA-DQ o Diversifying selection related to their immune Each MHC molecule selects its own T cell repertoire that only function in response to local pathogens recognizes peptides presented by that particular type of MHC o Demographic factors (trade, migration, etc.) molecule Features of Different HLA Alleles Remember: Different HLA types bind different peptides from the Molecules encoded by each MHC allele differ in the AA sequence same viral protein around the anchoring peptide binding pockets Each allotypic MHC binds a different set of peptides Ex. AA composition on HLA-B27 prefers positively charged, bulky amino acid like arginine or lysine MHC Class I pocket shapes confer specificity for peptides B pocket binds P2 AA side chain of the peptide F pocket binds P9 AA side chain of the peptide Tolerance Basics Clonal deletion: occurs after successful T cell activation; deletion via Discrimination between… activation induced cell death (AICD)—mechanism below Self and non-self (virally infected vs. healthy) o Eliminates auto-reactive cells that are too self-reactive Harmless and dangerous ▪ If antigen is a virus that can’t be eliminated Adaptive immune system primarily responds to/enforces tolerance (HIV, EBV, etc.) —> pathologic T cell loss Remember: innate cells naturally focus on pathogen-associated o Eliminates clonally expanded effector T cells in the molecular patterns (PAMPs) “terminal phase” of response (after antigen is cleared) Components of tolerance happen in both innate and adaptive Mechanism of activation-induced cell death T cell tolerance is the most important because they activate B cells Cell death is apoptotic and mediated by Fas and FasL If T cells are self-tolerant, B cells will be too o Fas cytoplasmic tail has a death domain that mediates association with death domains on other intermediates Mechanisms Regulating Adaptive Response o Caspase 8 activated and activates caspase 3 —> apoptosis Central: Selection during development before they are mature lymphocytes Fas levels are low on resting T cells, induced upon T cell activation B cells (IgM+/IgD- immature B cell stage in bone marrow) FasL expressed later after T cell activation o Strong reactivity against self —> apoptosis o T cell’s FasL engages another T cell’s Fas and kills it o Weak reactivity against self —> anergy Anti-FAS or anti-FasL antibodies inhibit AICD T cells (thymic selection) Fas or Fas-L deficiency —> autoimmunity/lymphoproliferation o Negative selection —> too self-reactive —> apoptosis Peripheral: Other Dampening Molecules: CD28 Homologs Responder cell intrinsic (recessive): imposed by the cell itself CTLA-4: natural break on T cell response, negative regulator o Anergy, Ignorance, or Activation induced cell death Expression increases gradually (4-7 days) following T cell activation Responder cell extrinsic (dominant): regulatory (suppressor) T cells Outcompetes CD28 for B7, which inhibits costimulatory signal 2 impose regulation on the responder Recruits phosphatases to dephosphorylate ITAMs and ZAP70, reducing signal 1 Central Tolerance—Thymic Selection Does not directly promote apoptosis, but it removes anti-apoptotic Following successful rearrangement of TCRβ chain and passed pre-TCR signals (signal 1/signal 2), tipping balance in favor of apoptosis checkpoint, T cells are in the double positive stage (CD4+/CD8+) o No effect on anti-apoptotic factors themselves They are looking for self-peptide-bearing MHC complexes in the PD-1: expression promotes standing down immune response thymic cortex (positive selection) PD-L1 and PD-L2 are constitutively expressed on most tissues o Positive selection ensures that the new T cells have at o Guard against autoimmunity least mild affinity for self-peptide MHC Recruits phosphatases to the TCR signaling complex o Without positive selection, most T cells would have no Results in: inhibition of cell cycle, direct reduction in anti-apoptotic self-recognition factors, reduction in cell metabolism —> decreased survival o Positive selection opens the possibility of auto-immunity, Expressed rapidly after T cell activation (within 2 hours) but it also allows for the widest possible variety of TCRs o ↓ Calcium influx, ↓ NF-AT activation—> ↓ IL secretion Single positive cells must then pass negative selection —> ↓proliferation Characteristics of TCR thymic graduates: Defective PD-1 causes lupus, MS, rheumatoid arthritis, T1DM Anti-self affinity ranges from low to intermediate o Compounds pathologic loss of immune effector cells Anti-foreign affinity is entirely unfiltered, so affinity for any given (HIV) by being constitutively expressed on surviving peptide ranges from very low to very high anti-HIV; causes T-cell exhaustion, antigen o This is the importance of clonal expansion unresponsiveness o Monoclonal Ab block PD-1 inhibition of T cell response TCR Affinity vs. Antigen Abundance Affinity: length of time receptor and ligand are associated, relative to the Peripheral Tolerance—Extrinsic Mechanisms amount of time they are unbound Regulatory T Cells: when mixed with effector CD4 T cells, all types inhibit Higher affinity allows more time for the TCRs to cluster and activate response to antigen Lots of peptide/MHC complexes on APC = less time needed for clustering CD4+/CD25+ Treg: most prominent extrinsic regulatory cell, two types Low abundance = more time needed to achieve TCR clustering o “Natural” or “thymic”: develop in thymus during CD4 single- (therefore, higher affinity is important) positive stage, alternative to apoptosis for hyper-reactive cells Review: AIRE/APECED (Lecture 06, bottom of column 2) ▪ Decisive event: FoxP3 activation ▪ Lack of FoxP3 = autoimmunity/lymphoproliferation Peripheral Tolerance—Intrinsic Mechanisms ▪ Over 90% of FoxP3+ cells are CD4+/CD25+ Tregs Successful activation of a naïve T cell requires two signals: o “Induced”: start out as naïve CD4 T cells but are activated into 1. Signal 1: TCR cross-linking (antigen-specific) Treg in the periphery 2. Signal 2: Provided by APC, induced to mature by a danger signal ▪ Naïve CD4+ T cells —> CD4+CD25+FoxP3+ (TLR, NLR, RLR, etc.) when there is suboptimal antigen presentation OR Types of intrinsic responses TCR crosslinking in the presence of TGF-β Anergy: even if the presence of high concentration of antigen, the ▪ PD-1 ligation increases iTreg conversion by TGF-β cell remains a state of hypo-responsiveness o Constitutively express CTLA-4 and PD-1 o Caused by signal 1 without signal 2 o Dependent on IL-2 for survival o Cell resists activation throughout its lifetime o Require activation via TCR to implement suppression, then o Notable differences: less phosphorylation of CD3 suppress everything in the area (bystander suppression) ITAMs, less phosphorylation of ZAP70, less calcium ▪ Optimal suppression requires cell-cell contact flux, less NF-kB and NF-AT o Targets: CD4+ and CD8+ T cells, B cells, NK cell, mast cells, ▪ Overall: far less gene activation, including macrophages absence of IL-2 Treg direct suppressive mechanisms include: Ignorance: Low TCR-antigen/MHC affinity and/or low antigen o Secreting suppressor cytokines like IL-10 and TGF-β abundance result in failure to cluster TCR (signal 1) o Promote T-cell apoptosis by acting as IL-2 sponge o Important in immune-privileged sites (eye, CNS) which o Granzyme-mediated apoptosis have relatively low surveillance by immune cells, so little o Express on the surface and secrete Galectin which suppresses self-antigen from there is seen by T cells effector T cell proliferation o Ex. Sympathetic ophthalmia: drainage of lots of ocular Treg indirect suppressive mechanisms (via dendritic cell interaction): antigens into LNs; naïve anti-retinal T cells can achieve o Treg expresses LAG-3 which inhibits DC maturation clustering and signal 2 due to damage —> reject o Treg cells express CTLA-4 to engage DC B7, leading DC to undamaged eye downgrade surface B7 expression (inhibits costimulation) Hypersensitivity Overview Type II—Cytotoxic Type Harmful or non-harmful? Response to hapten-bound tissues (usually a drug) Originates in the adaptive immune system with T and B lymphocytes Damage mediated by tissue-specific IgG or IgM Hypersensitivity: aberrant or excessive response to foreign antigens Mechanism of sensitization o Damage mediated by the same mechanisms used for o Foreign agent acts as a hapten appropriate responses to pathogens o Conjugates to self protein —> modified self —> T cell/B Four main classes of hypersensitivity cell response —> high-affinity IgG or IgM against Type I—Immediate Type: mediated by IgE, histamine production drug/self protein combo (ex. Kid with a peanut allergy)