Exam 1 Summary Charts-compressed PDF

Overview Of Immune System Immunity definitions Immunity = protection against foreign pathogens (invaders/danger) or substances (antigens) ○ CRUCIAL FOR SURVIVAL: microbiome mostly commensal organisms in our normal flora, but changes in our environment can lead...

Overview Of Immune System Immunity definitions Immunity = protection against foreign pathogens (invaders/danger) or substances (antigens) ○ CRUCIAL FOR SURVIVAL: microbiome mostly commensal organisms in our normal flora, but changes in our environment can lead to dysbiosis = imbalance → immune overstimulation and inflammation ○ We are able to recognize self by tolerance – we do not react to ourselves (when mixing self “id” (MHC) then we react to the cells!) Father of immunology = Edward Jenner (used cows to stop smallpox → made vaccinations) Pathogens fall into four major categories ○ Viruses (e.g. Influenzas) ○ Bacteria (Salmonella) ○ Fungi (Ringworm) ○ Parasites (Malaria, Pinworms) Microbes are pathogenic when they manage to penetrate the immune defense → strategies of pathogens: ○ Disable host’s immune weapons (inactivate immune proteins) ○ Resist immunological weaponry (bullet proof membrane); immune system won’t work on them ○ Deception: camouflaged, invisible to host (sheepskin- covered with host’s molecules) ○ Hide identification invasive molecule in folds (stealth bomb) – hide in cells and go undetected ○ Intracellular parasitism ○ Distraction: Decoys (variable surface glycoprotein) – our normal systems will bind to them instead of our own stuff ○ Mimicry: invading molecule resembles hosts molecule (disguise, rhinovirus - adhesion molecule ICAM, SAR-COV-2 - angiotensin-converting enzyme (ACE)) INNATE IMMUNITY ADAPTIVE IMMUNITY FIRST LINE OF DEFENSE (relays info to adaptive ONLY on jawed vertebrates (from recombination pathway) – always present and ready activating gene (RAG) = genes for somatic recombination of gene segments that encode B/T cell Reacts on short notice and responds in minutes or receptors. hours with low/fixed specificity (usually responds this way to anything) → response to the repeat infection is We get this from experience = learned experience – the same each time (non-specific, generic strategy antigen specific that makes antibodies = memory for everything) Response takes days and the specificity is every diverse - Barriers: physical (skin, mucus, coughing), (adapts to improve during the time course of immune chemical (mucus, sweat, stomach acid, response) → more rapid and effected with subsequent lysozymes), or cellular barriers exposures - PAMPs - Components: - B lymphocytes (humoral) → make antibodies - Epithelial cells, Phagocytes (ingest (beta/immunoglobulins) from memory = long-lasting pathogens) - T lymphocytes (cell-mediated) = pathogen-specific T - Dendritic cells – most important for cells bridging between innate and adaptive - Cells isolated from blood for immunity immunity - Treg cells (guide other cells) → CTL cells, B - Natural killer cells lymphocytes/cells, Th cells, macrophages, - Complement (proteins) Innate and Adaptive Immunity INNATE IMMUNITY Innate molecules use two methods to recognize foreign material: ○ Microbial non-self – recognize things on all microorganisms or not made by host (activate pathogens) ○ Missing self – recognize missing MHC structures expressed on our cells (activate natural killer cells) These are done through cellular receptors (receptors bind molecules on pathogens for activation) Phagocytes use pattern recognition receptors (PRRs) to bind to pathogen-associated molecular patterns (PAMPs) ○ Microbes/pathogens have PAMPs = conserved molecular structures (invariant among pathogens of given pathways) → these are products of metabolic pathways and made to perform unique functions essential for the microbe’s survival (microbes make them NOT us/the host – host has MHC) Examples of PAMPs: Gram-negative bacteria have lipopolysaccharide on them Gram-positive bacteria have lipoteichoic acids, peptidoglycan, palmitoylated lipoprotein Lipoarabinomannan (much more lipids) on the mycobacteria Beta-glucans and mannans in fungal walls Double-stranded RNA produced by most viruses Unmethylated CpG DNA from bacteria ○ PPRs are on the host and recognize the PAMPS – there are three locations/forms: Intracellular (dsRNA) → almost every cell type On surface (TLR) → on the surface of macrophages, neutrophils, and dendritic cells Toll-like receptors = type I transmembrane receptor, at least 10 types in mammals These recognize more than 1 ligand that are structurally unrelated They recognize more than one ligand that may be structurally unrelated with each other Most known: ○ TLR2 – binds parasite/yeast molecules ○ TLR4 – binds lipopolysaccharides in gram(-) and lipoteichoic acid on gram(+) Or secreted (mannan-binding lectin, collectins, acute phase reactants) → produced by macrophages and epithelial cells (complement system you will see proteins and know the receptors were secreted) To recognize damages cells, innate immunity uses Damage-associated molecular patterns (DAMPs) ○ These are released by stressed cells undergoing necrosis → these engage TLRs to promote and exacerbate the inflammatory response (initiate non-infection inflammatory response) ○ DMAPs are activated via NKG2D (natural killer cell receptor!) Other activation mechanism = death receptors that induce cell death: ○ Fas (CD95) ○ TNF-related apoptosis-inducing ligand receptor (TRAILR) INFLAMMATORY RESPONSE (always when immune response is activated this is upregulated!): ○ Increased phagocytosis ○ Increased Fc receptors for opsonization ○ Increased production of digestive enzymes (lysozymes, acid hydrolase), antimicrobial peptides, free radicals, vessel dilating substances (nitric oxide), and pro-inflammatory cytokines (M1) ○ Link innate to adaptive immunity  Increased expression of MHC molecules for presenting antigens to T cells Increased differentiate into dendritic cells → present antigens to T lymphocytes Production of MORE inflammatory cytokines and chemokines LINK between Innate and Adaptive Immunity Innate system using natural killer cells, which expression FcγRIII (Fc receptor for IgG) → bridges to the adaptive immunity because they do ADCC where IgG is the target for the NK cells to activate cytolysis ○ NK will lyse target cells = “antibody cell-mediated toxicity” (ADAPTIVE = HAS SPECIFICITY!) Dendritic cells present antigens to T cells and provide signals to stimulate lymphocyte proliferation/differentiation ○ Two types of dendritic cells from hematopoietic progenitor cells in the blood: Conventional (Classical) Dendritic Cells – recognize PAMPS, so activated by microbes → display the microbial antigens to T lymphocytes (first responders?) Most potent APC to activate naive T cells during the primary response Plasmacytoid Dendritic Cells – recognize nucleic acids of viruses, so respond to viral infections → make lots of type I interferons (prevent virus from infecting out cells) ○ Follicular dendritic cells – mesenchymal-derived to regulate structure of germinal centers (FOR ADAPTIVE IMMUNITY). Present to B cells = B cell development → debris removal and B cell memory ADAPTIVE IMMUNITY Uses antigen recognition = antigen specificity for acquired immunity (recognizes different pathogens!) ○ Specific B- and T-cells make receptors (BCR & TCR), which bind foreign molecules, if they come along → have this receptor before they see pathogens!! Antigens = foreign substances that activate the immune system ○ Antigens can be: Carbohydrates: polysaccharides of bacterial cell wall, glycoproteins Lipids: glycolipids (blood group antigens) Nucleic acids: autoimmune diseases (do want this bc cells degrade into this and if not properly removed we activate our own immune response = auto-immune disease) Proteins: high degree of complexity (many structures/dimensions that can trigger immune response and immunogenicity) – proteins are easiest to study… ○ Lymphocytes are activated via receptor-ligand (antigen) binding Binding → dimerization or multimerization = cluster together and make lipid rafts (these activate cells and induce cascades of intracellular events) AFFINITY = the strength of reactions between a single antigenic determinant and a single receptor combining site → better fit = more likely the cell will be activated ○ Requirements for Immunogenicity: 1. Foreign 2. Complexity (poly-glutamic acid of Bacillus anthracis = not immunogenic) 3. Time of exposure (quick elimination, degradation) – have to give the immune response time to react (right in/out = not enough time for the immune system to get it) 4. Size > M.W. of 6000 daltons a. Small molecules (drugs ~ 400 MW) attach onto larger molecules = makes a big carrier molecule; small molecules on it are antigen determinants (epitopes) Intracellular events: leads to tyrosine phosphorylation (early step in many signaling pathways) ○ ITAMs (“immunoreceptor tyrosine activation motifs”) phosphorylate CD3 (T cells) and Igα/β (B cells) ○ Phosphorylated tyrosines are docking points for adapter molecules and turn on gene expression CLONAL SELECTION → when the receptor is activated and cells expand ○ B and T cells have specific receptors that bind to antigens (if the cell has good affinity, it expands and makes many clones to “joint the fight”  When B/T cell receptors engage with that antigen the cell becomes activated = “selected” ○ Displace many copies of the unique receptors (that match antigens) → surface ○ Activation results in a proliferation, producing a large number of clones B cells recognize forgein 3D structures (several amino acids by polypeptides folding) and the 3D parts protrude for a B cell receptor/antibody to bond → activates antibody production ○ Ex. Sperm whale protein myoglobin (MW =17K, 156 aa) contains five linear B-cell epitopes (red), one of which is bound to the antibody-binding site of an antibody (purple) → the ab is specific for the amino acid residues 56–62 B-cell Receptor (BCR) T-cell Receptor (TCR) **has an antibody for a specific antigen **recognize peptides from antigens on MHC molecules (MHC has a groove for carrying the antigen BCR structure: quaternary protein with two identical heavy fragment and passing it to T cells - if right TCR then T cell is activated) chains and 2 identical light chains → chains make a 3D → bind processed antigens on MHC binding site for the antigen. Has antigen specificity due to the light/heavy chain interaction BCR has 2 forms: 1. Membrane Bound form – signal receptor on B lymphocytes (hydrophobic) 2. Secreted form – in circulation (hydrophilic segments so TCR structure: like N-terminal domains of antibodies – no cytosolic segments) resembles an Fab fragment with variable and constant regions (constant parts have transmembrane regions). Variable regions have 3 CDRs making a binding site: 1. Two TCR types: αβ (peptide) and γδ 2. Like the antibody “Y” but just one part of the small/heavy chains Co-receptors help with antigen recognition - CD3 has ITAMs that transmit signals to inside cell Co-receptors on antibodies for signal transduction (work - CD4, CD8 = increases avidity of peptide binding with B cells to be activated and do their function) by TCR to allow them to bind to the MHC - Igα, Igβ: transduce signals via ITAMs - CD28 = engages CD80/CD86 on APC to fully - CD19, CD81, CD21 = transmits signals to cell interior activate a naive T cell Lymphoid and Myeloid Origin Lymphoid Organs Primary organs = where immune cells develop → thymus and bone marrow ○ Hematopoiesis process starts in the yolk sac, then occurs in bone marrow ○ Hematopoietic stem cells (HSCs) seed the bone marrow late in fetal development; they expand and populate the bone marrow postnatally and interact with stromal cells in the basement membrane. These HSCs differentiate into many blood cells via hematopoiesis Pluripotent HCS → all white and red blood cells  ○ Stromal cells facilitate HSC proliferation, direct migration, and stimulate differentiation HSC’s constant reviews and directed into two major progenitor cells: Common myeloid progenitor cells Common lymphoid progenitor cells ○ Will only contain B or T cells Secondary organs = where lymphocytes encounter antigens and become activate (undergo clonal expansion = increase in number and differentiate into effector cells ○ These = lymph nodes, spleen, mucosa-associated lymphoid tissues (MALT), and other diffuse/ loosely organized areas (all connected to one another via blood and lymphatic circulatory systems) ○ Will contain both B and T cells and contant antigens Lymphoid Lineage Lymphocytes develop when they come into contact with stromal cells in the bone marrow B and T cells develop separately from the lymphoid progenitor cell (CLP): B cells T cells Mature and develop in bone T lymphocytes go from bone marrow → thymus (contact stromal cells marrow here to fully mature!) – for cell-mediated immunity (thymic cortex and medulla microenvironment direct stepwise changes) Cytokines and chemokines from TCR affinity of binding with MHC-peptides drives positive and negative stromal cells help selection (in thymus learn to recognize their ID / MHC) Grow and differentiate into plasma cells (these come out of antibodies - Cytotoxic T lymphocytes (CTL, Tc) = extracellular killing for humoral immunity - T helper cells = up-regulation of immune response Active during a secondary response of - T regulatory cells (Treg) = down-regulate (before were T suppressor an antigen → have B cell receptors cells) (BCR) that bind antigen and make - NKT cells = binds foreign and self lipids/glycolipids (mycobacterium → antibodies = develop memory tuberculosis) → properties of T cells and NK cells (NKC surface molecules) - Memory T cells Also make innate lymphoid cells (ILC) = detect changes in the environment through cytokine receptors and then secrete cytokines to quickly respond to pathogenic tissue damage = to shape adaptive immunity but these work with the INNATE immune system! ○ Abundant in mucosal surfaces of the intestine, lung, oral cavity and gingiva. ○ Three categories (ILC1, ILC2, ILC3) and Natural Killer Cells! ILC1 ILC2 ILC3 Type 1 response Type 2 Response Support barrier integrity (intestine Similar to NK cells: Repair tissue damage mucosal surface) → so pathogens - secrete IFN-γ (like NKs) to promote from helminth and viral cannot invade it (makes sure macrophages and dendritic cells to: infections type 2 response dysbiosis doesn’t happen!) - eliminate intracellular bacteria = repair / fighting off - present antigens by ↑ MHC and worms) Make cytokines (IL-22.IL-17) to: adhesion molecule expression Work with Th2 cells → - ↑ secretion of antimicrobial peptides - Combat intracellular pathogens; T. make cytokines and some from endothelial cells gondii, L. monocytogenes, Salmonella T cells - ↑ mucus production from goblet cells ○ Natural Killer Cells = tumor surveillance (cytotoxic killing of infected/damaged cells) → first ones that come in and do this! Cytotoxic T cells come in after training (they are adaptive), NK proliferate earlier  5-10% circulating lymphocytes in the bloodstream (NOT under mucosa really!) Do NOT have antigen-specific receptors like B/T cells (but HAS Fc receptor for IgG) DCC antibody-dependent cell cytotoxicity = Ab brings NK cells in contact with the target cells Make IFN-γ = connection to adaptive immunity With INNATE IMMUNITY – reacts to cells without an “ID” (no MHC I = find and lyse the cell) NK cells have two receptors that both bind ligands! Balance between inhibition/activation determines if an NK cells is activated (constantly working together to remove/put on a phosphate) → once activating signal molecules are engaged, NK cells use mechanisms very similar to CTLs to induce target cell death with release of perforins/granzymes at junction of two cells Our cells = NO Activation because Microbe = missing MHC (tumor removes it or they have MHC I tags that bind to invader without it) = inhibition turned off = inhibitory receptors activating signal stays on Inhibitory NK receptors (KIR3DL1) that bind Activating NK receptors (NKG2D) that bind to ligands to ligands MHC class 1 molecule NKG2DL (= MICA) **MICA = MHC class I-related chain in tumors (melanoma = NKG2D ligand) → looks like MHC but is not2 Immunoreceptor tyrosine-based inhibitory motifs (ITIMS) signaling – these recruit Turns on cells through immunoreceptor tyrosine based phosphatase that removes a phosphate from activation motifs (ITAMS) – tyrosine kinase is the tyrosine kinase enzyme = turns it off (no phosphorylated = makes IFN-γ = increase in IL-2 receptors → activation) activates the lytic machinery B cell Development B cell development starts in the bone marrow from Hematopoietic Stem Cells (HSC, the common ;lymphoid progenitor) → cytokines (IL-4) and chemokines form stromal cells in bone marrow drive development ○ Several stages before completely mature: (1) pre-pro-, (2) Pro-B, (3) Pre-B cells. (4) immature (mature once they finally get their BCR!) = antigen-independent phase ○ During these, spontaneous recombination occurs to make IgM BCR (diverse Ag combining sites!) For the immune repertoire, want to respond to any antigen = 1015-18 different antibodies with different specificities in 1 person (100 million billion!!). Each Eb has a different binding site ○ Once B cells express IgM receptors, they leave the bone marrow → spleen (still immature, finish developing in the spleen – before this has the right antibody though?? ontogeny?) Originally two mRNA transcripts: transmembrane and secretory domains (mu and delta domains) → H and L chains are on the protein level!! Heavy and light antibody peptides are assembled with different parts ○ Variable region = (aa 1-108) = made by DIFFERENT CLONES of B lymphocytes ○ Constant region = carboxyl-terminal = identical aa sequence made by the SAME B lymphocyte clones Clonal expansion = Ex. antigen with 3 epitopes (3, 4, 7) → activate clones to proliferate making anti-3, anti-4 and anti-7 Ig. Different locations for this to occur: ○ Bone marrow = antigen-independent ○ Peripheral system = antigen-dependent (clones that bind with higher affinity expand more) Isotype switching = making secreted Ab with different functions via CH (H chain constant region)  ○ These Ab have the same antigen-binding specificity (V regions) but different C region so they have different biological actions → via VDJ-C ○ Early in entogen (before encountering an antigen) B cells express both IgM and IgD on their surface, but C-region changes as it matures and proliferations → stimulation from an antigen with T helper cells allow B cells to switch isotypes (generate progenies of IgG, IgA, or IgE) C-regino gene ( Cμ, Cδ, Cɣ, Cα, Cε ) lies on 3’ JH gene (Cμ closest) T helper cells guide the switch of “C” genes via repetitive DNA switch regions (S) upstream of each C gene → after switching, coding region between JH/C is deleted Other B cells: ○ Memory B cells made in germinal centers: must bind to the antigen on follicular dendritic cells ( for selective debris removal from GC to protect autoimmune disease) or else does apoptosis ○ B-1 cells are a minor subtype from EMBRYONIC PRECURSORS in the peritoneal cavity (does self-renewal and does NOT have IgD). Make IgM from carbohydrates WITHOUT T cell help!! B-1 = T-indepepents, while B-2 = T-dependent! Steps for B cell Diversity (1) Selection of many Mini-genes encode variable regions (5 options = 5 x 41) – recombination of genes mini-gene segments to make different Ag binding sites (variable (V), diversity (D), joining (J), constant (C) (small parts) V, J, D mini-genes for diff peptides on chromosomes and recombined together: VDJ (H) and VJ (L) - L chains: V (aa 1-95) + J (aa 96-108) segments - H chains: V (aa 1-101) + D (102-106) + J (107-123) T cell receptors also use mini-genes!! (2) Recombination to V(D)J Recombination in variable regions = many antibody combining sites directed joint randomly selected by recombination signal sequences (RSSs) = conserved nonamer/ heptamer mini-gene segments sequence, between which is 12 or 23 bp spacer sequence that dictates a 12 bp RSS pairs with a 23 bp RSS for recombination to occur (“12/23 Rule”) RAG1/2 recombinase joins genes by cutting DNA at Ag-encoding region and the RSSS (3) Junctional Diversity During mini-gene joining, deoxynucleotide transferase (TdT) adds nucleotides (addition/deletion of while exonucleases remove nucleotides → frameshifts can happen (alter reading nucleotides during frame or disrupt coding sequences beyond joining site) mini-gene joining) 3rd hypervariable region (CDR3) in junction of VJ and DJ 1. VDJ heavy chain rearrangement 2. VJ alternation in light chain (κ or λ) chain → VDJ-Cμ, VDJ-Cδ 3. Allelic exclusion (1 per chromosome – paternal or maternal) = irreversibly inhibit Ig heavy chain locus arrangement on other chromosome = Pre-BCR (4) Association of Different H chains pair with different κ (200 x 6000 H = 1,200,00) or λ (160 x 6000 H = different L/H chains to 960,000) chains (same CH or CL can be connected to millions of VH and HL) make binding sites - Different β chains pair with different α chains → TCR αβreceptors - Different δ chains pair with different γ chains → TCR ϒδ receptor Elimination of self-reactive B cells = central tolerance – receptor editing to initiate a new round of VJ recombination (deletion = kill cells or anergy = shutdown function) In spleen: B cells undergo two transitional stages: 1. T1 (negative selection) – B cells with receptors that react to self (antigen / molecules) are deleted, the rest transit to T2  2. T2 (positive selection) – B2 cells fully mature with high IgD and IgM levels. BAFF (B cells activating factor) provides a signal, some B cells stay in spleen follicles and others migrate to lymphoid organs (nodes) and will be called follicular B cells RNA splicing for IgM/IgD → 2 mRNA transcripts made, split via polyadenylation – poly-A tail added to Cδ = will be on IgD In lymph nodes: mature B cells reside in LN follicle… contact an antigen here its recognizes and gets help from follicular T helper cells (TFH = CD4+ T cells in LN) - TFH engage B cells via CD40(L) co-receptor - Clonally expand and do isotype switching in germinal centers (TFH help) - Somatic hypermutation (B cells make Ab with increased affinity for the Ag) (5) - B cells differentiate into plasma cells (secrete Ab but loose surface Ab) - Memory B cells made in germinal centers survive for a while (for reinfection) **also make IL-21 = regulatory effects on immune system cells (NK, B cells, T cells) (5) Somatic On B cells after encountering an Ag (often for secondary response) – introducing point Hypermutation = ONLY mutations in V regions (often with CDR) → Ig specificity = better affinity (maturation) FOR B CELLS! **happens during clonal expansion!! T cell Development Process = pluripotent HSC from bone marrow → thymus where they are DOUBLE-NEGATIVE (CD4-CD8-) ○ Go through CD4/CD8 expression moving from cortex → medulla: double(-) → double(+) → single(+) ○ T cell receptor assembly: β, α, γ, δ chains → made two TCRs: TCRαβ and TCRγδ (different T cell clones = diverse antigen-binding specificities) TCRγδ T cells = 1st defense (skin and mucosal surfaces (MHC NOT used for Ag recognition) TCRαβ T cells = recognize and bind antigen-derived peptides on MHC ○ Then do MHC selection (bind self MHC I or II) ○ Then Effect T cell subsets T cells have 4 mechanisms for making a vast repertoire – diversity within TCR repertoire is achieved by: ○ Selection from many mini-gene segments ○ Recombination to join the randomly selected mini-gene segments ○ Addition & deletion of nucleotides during joining of mini-gene segments ○ Association of two different chains to form binding sites ○ NO somatic hypermutations for T cells because after MHC selection, mutations would lose MHC recognition = T cells would not be able to get signals from APC Positive and Negative Selection of T cells = CD4 and CD8 Presentation T-cell precursor development in bone marrow → in the thymus, they aren’t yet T cells. The Notch receptor commits lymphocytes to the T cell lineage. This stage = “double negative” = CD4- or CD8- ○ T cell precursor rearrange TCR β chain = most likely making TCRαβ but if not then TCRγδ ○ If the β chain is expression (CRαβ) → they become “double positive” (CD4+CD8+) and go through the cortex – during this movement, they do positive/negative selection for MHC affinity Recognize self = deleted Cells that survive → medulla to make a single positive T cell (either CD4+or CD8 +) – these go to the periphery to carry out a function. TCRαβ “browse” through cortical thymic epithelial cells which have MHC I and II there → MHC can bind self or non-self peptides. So, as immature T cells are tested on how strongly their affinity is to self antigens in MHC  ○ T cell TCRαβs that bind too strongly to self-peptide/MHC complexes → apoptosis occurs ○ T cell TCRαβs that bind “just right” → they are selected for maturation and enter peripheral tissues Control of auto-reactive cells: ○ Central control = removes cells before T cells are release (95% cells fail positive selection → apoptosis) – T cells released into blood are activated by foreign peptides present on MHC These become T helper cells and cytotoxic T lymphocytes ○ Peripheral control = recognition of self antigens by immature T cells in the thymus → Treg cells Small percentage that see antigens differentiate → CD4+ Treg cells (to inhibit responses) In peripheral tissues, they do Positive and Negative Selection: Positive Selection = FIRST Negative Selection = at medulla CD4+CD8+ DP thymocytes = 80% of thymic Positively selected cells → medulla for more screening. Medullary cells → if these bind MHC-peptide with thymic epithelial cells (mTEC) express and present proteins moderate affinity and it is POSITIVELY from the body, like autoimmune regulator protein (AIRE) = SELECTED and shifts from DP to SP induces them to express, process, and present tissue-specific - TCRαβ can binds MHC II → binds with the proteins CD4 = selecting the CD4+ subset (T helpers) Thymocytes with high-affinity TCR for self are deleted → this - TCRαβ can binds MHC I → it binds CD8 = deletion = NEGATIVE SELECTION to ensure self- tolerance selection to the CD8+ subset (CTL/Tc) (central tolerance = elimination of self-reactive clones) TCRs that can’t bind MHC-peptide (on stromal CD4+ and CD8+ thymocytes that survive → bloodstream and cells) die by neglect complete their maturation in the periphery to recognize foreign antigenic peptides presented to them on the MHC molecules. TYPES OF T CELLS: T cells that recognize LIPID antigens TCRγδ T cells Natural Killer T (NKT) cells TCRγδ cells develop from unsuccessful β chain Heterogenous group of T cells with TCR from invariant α rearrangement into αβTCR and limited number of β (not diverse) Appear early in ontogeny for protection at a young Share properties of T and natural killer cells: age (declines after birth) → protect barrier from - Rapid responders (between innate and adaptive) outside – join innate cells = first of line attack and - Binds foreign and self (lipids and glycolipids) → cellular stress PAMPs on on mycobacteria from tuberculosis with a - High frequency (50-100% T cells) in skin and lot of lipids that NKT cells take care of intestine (mucosal tissue) - Do NOT recognize MHC, instead lipid antigens - Low frequency (5% T cells) in thymus and presented by CD1 (CD1 = non-polymorphic MHC peripheral organs such as lymph nodes I-like APC (PAMPs use CD1 with NKT recognize) DON’T GO THROUGH DP STAGE! → don’t recognize - Make a large amount of cytokines Ag/MHC and instead recognize lipids and head ***Small number of αβTCR commit to NKT*** shock proteins (have helper and CTL activities)  T Helper cells They each have their own cytokines made by that APC → cytokines regulate different transcription factors to activate T cells into their mature state Inhibition of other T helper subsets by competing for resources ○ T helper cells differ in: cytokines, transcription factors, immune reactions, and defense functions ○ Each T cells makes their own cytokine = amplifies itself and inhibits others (polarization) TH1 APC to present IFNγ IFNγ → M1 macrophages to make TH1 stimulates expression (interferon) antigens to naive more IL-12 = positive feedback to of MHC and CD-80/86 for CD4+ T cells make more TH1 (**reciprocal APC, cytokines, NO, and (adhesion molecule induction**) killing microbes binds CD4 receptor) IFNγ stimulates classical Fights intracellular APC makes IL-12 macrophages (killing) and production microbes via macrophage which activates of IgG to bind their Fab to Fc and IgG production T-bet gene to receptors on phagocytes → (autoimmune diseases and differentiate opsonization (isotype switching, chronic infections) increasing IgG TH2 APC makes IL-10 to IL-4 IL-4 → isotype switch: IgG → IgE **activates alt macrophage* activate GATA3 IL-5 (IgG4 coats parasites for IgE to Fights helminth parasites gene TF IL-13 activate mast cells/eosinophils = SM via activation of mast cells/ (interleukin) contacts and granules kill worms) eosinophils, IgE, and IL-4 + IL-13 → mucus secretion macrophages (peristalsis in intestines to expel (allergic reactions) worms) and M1 macrophages IL-5 → activates eosinophils TH17 APC makes IL-23 to IL-17A IL-17 → leukocytes make pro- Fights extracellular activate RORϒt IL-17F inflammatory cytokines (TNF, IL-1, bacteria and fungi via gene IL-22 IL-6) = recruit neutrophils and neutrophils and monocytes (defensins) monocyte inflammation **loss of IL-17 = autoimmune (organ-specific diseases = increase susceptibility to autoimmunity) mucocutaneous candidiasis = thrush IL-22 → barrier functions in GI tract + fungal/bacterial infection Follicular Subset of CD4+ T IL-21 Use CD40(L) co-stimulatory receptor Help B cell development in Helper T cells in lymph nodes: to engage B cells (isotype switching, germinal center (TFH) IL-6 and IL-21 proliferation, and higher affinity activate TF BCL6 → mutations) TFH *make more IL-21 = positive feedback and self-upregulating  Treg TGFβ (tumor-growth TGF-β TGF-β suppresses T cell activation T suppressor cells = factor β) activates IL-2 (via CTLA-4) = inhibits CD4+ and down regulation to TF FoxP3 (low CD8+ activation maintain peripheral levels) tolerance Memory T After microbe destroyed, most T/B die but small amount remains Low numbers and cells No antigen + IL-7 and IL-15 → memory T cells! Slow proliferation heterogenous (unlike B = Maintenance of memory cells is dependent on cytokines but does high in number and not require antigen recognition express Ab (IgE, IgA, IgG) Classically vs Alternatively Macrophages (M1:M2) both start as monocytes Classical (M1) Alternative (M2) Activated by interferon Activated by interleukins (IL-4, IL-10 and (TH1 activated) IL-13) 1) Increases collagen synthesis and fibrosis = 1) INFLAMMATION! promotes tissue/wound repair 2) Enhance killing of 2) Inhibits inflammation and microbicidal the ingested activity of M1 macrophages = suppress microbe immune response = ANTI-INFLAMMATORY Myeloid Lineage 4 types of common myeloid progenitors 1. Red blood cells (erythrocytes) 2. Megakaryocytes 3. Monocytes/macrophages (phagocytic cells) – migrate into tissues and differentiate into macrophages a. Phagocytic → contain digestive enzymes (lysozymes and acid hydrolase) and make antimicrobial peptides (defensins and cathelicidins) and free radicals = destroy pathogens i. Vessel dilating substances (nitric oxide) = immune cells to rush into the infected area b. Have Fc receptors to present antigens to T cells (MHC) – present antigens to T lymphocytes c. Do tissue (T cell) repair/remodeling d. Differentiate into dendritic cells = present antigens to naive T lymphocytes for initial activation – proliferation / differentiation (contain dendrites = more surface area to interact) i. Plastic = can shape to the environment/homeostatic state 4. Granulocytes (lots of granules) – subtypes differ in granule staining and in protein content and function Neutrophils Eosinophils Basophils / Mast Cells Direct harm (very phagocytic: capture Antiviral / anti-parasite activity Inflammation/allergies → produce and kill them) → make digestive enzymes (uses toxic proteins → ROS) cytokines that modulate adaptive (proteases) and free radicals Vasodilation, basophil responses. Granules contain: (superoxides) Predominant cell at wound degranulation - Histamine - vasodilation, smooth sites Make cytokines for adaptive muscle activation Like macrophages: vasodilation, responses and chemokines - Heparin – ↑ blood flow inflammation, tissue remodeling (activate things downstream) - Leukotrienes – inflammation “PFN” = ?? Have Fcε receptors Multi-looped nuclei with neutral granules Multi-looped nuclei with acidic Multi-looped nuclei with basic (blue in Giemsa stain) granules (red in Giemsa stain) granules (purple in Giemsa stain 60-70% of WBC (highest) 1-4% of WBC (lowest number) Cell Traffic Endothelial cells have intercellular adhesion molecules (ICAM) and produce chemokines The immune cells are activated and roll toward the higher chemoattractant concentration ○ Integrins have higher affinity on the membrane so they stick → leukocyte-endothelium interaction and leukocyte extravasation (roll and attach, then squeeze into the infected area). Cells they use: Macrophages Chemokines: Recruit cells to the region of the infection Adhesion molecules: Selectin, Integrins, ICAM ○ Two types of movement: Margination: immune cells adhere to endothelium by integrins (bind to ICAM) = COMING IN Extravasation: leukocyte move/squeeze between endothelial cells (ICAM for traction) and enters the surrounding tissue = INTO INFECTED SITES Blood vessels dilate to allow the immune cells (and fluid) move into the area they develop inflammation = redness, heat, pain, loss of function ○ Infection Process At the site of infection, innate molecules FIRST encounter the foreign molecule Forgein molecules are funneled into specialized organs where antigen-specific lymphocytes come in (ex. Cells → cells come out of the lymph nodes and go through circulation to the site of infection to fight it) ○ Transition from innate → adaptive immunity Ex. When a foreign antigen enters the body through a splinter into his sole, which of the following portions of the lymphoid system is the antigen MOST likely to reach FIRST? ○ Choices: Spleen (go here is systemic / goes into blood) Liver (for hematopoiesis) Thymus Regional lymph nodes → find these around the feet? Mucosal-associated lymphoid tissue (MALT) Gut (injected and go through mucosa) Lungs (pathogen inhaled into mucosa) Antibodies (Gamma globulin, Immunoglobulins) Antibody Structure In adaptive immunity, antibodies are placed on the surface of B cells → they have two forms: ○ Membrane Bound = signal receptor on B lymphocytes (talked about before) ○ Secreted Ig = no cytosolic segment, has hydrophilic segment instead  Sources = made by B lymphocytes and then differentiate into plasma cells (no receptors anymore, only go into circulation to neutralize pathogens → these are highly differentiated with specialized secretion of antibodies) BCR = typical antibody (IgG) that is divalent ○ Globular and symmetrical (2 Fab and 2 binding sites for cross-linking) → heavy and light chains are held together by interchain disulphide bonds 2 identical Light chains (shorter and lighter weight) Kappa (𝛋) or Lambda (𝛌) → λγγλ or κγγκ 2 identical Heavy chains → 5 classes (5 isotypes) → grouped into classes (isotypes) depending on which H Chain is used) Mu (𝛍), Gamma (γ), Alpha (α), Delta (δ), and Epsilon (ε) ○ Contain a hinge region with proline for extension (“Y” shape instead of “i”) Four globular domains (also held by interchain disulphide bonds) ○ VL = variable light, CL = constant light ○ VH = variable heavy, CH = constant heavy 1, 2, 3 ○ Variable region = different clones of B cells (antibody amino acid sequences are made by different clones of B lymphocytes → each B clone has a different sequence for this region Hypervariable regions – within variable domains (3 on light chains and 2 on heavy chains) - this is the tip that comes into contact most with the antigen = “antigen combining sites” or "complementarity-determining regions" Amino acids here determine the shape, charge, and chemistry of an antibody for its antigen = the specificity (what area it binds to!) CDRs: CDR1, CDR2, CDR3 (CDR3 = MOST VARIABLE!!) ○ Constant region = always similar from clone to clone (identical amino acid sequences on matter the B lymphocyte) – pretty much the same except 1-2 amino acid differences among some… Experiment where we removed part of antibody to see what function was deleted: ○ N terminal = Antigen binding sites (Fab) recognition site = this confers antigen specificity (this lost then cannot bind antigens) ○ C terminal = Fc (c = crystallization) to mediate effector activities (this lost then the activity is lost – will crystalize out of solution). It is really the carboxyl end of heavy chain performs biological function Ex. Which hypervariable region has the most differences in amino acid sequence when comparing B cell clones? CDR3 LOCK AND KEY Interaction Antigen binds to Fab fragment (from antibody papain digestion?) → N-terminal binds antigen determinant depending on the 3D shape make by the variable heavy and variable light domain ○ Amino acid residues define the "antigenic determinant" or "epitope" of HA (antigen’s 3D structure) ○ End of Fab = paratop which directs contact with the surface of HA burying the aa residues Fab fragment → anti-HA antibody This interaction is non-covalent (hydrogen, ionic, van der waals) = reversible (can come off from one another!) Interactions was close steric fit → higher affinity = closer fit = tighter Specificity vs. affinity ○ Specificity = binding to one antigen and not the other ○ Affinity = how strong the fit is (strength of reaction between an antigenic determinant and the combining site on the antibody) – strength of an INDIVIDUAL BOND Sum of attractive/repulsive forces between the antigenic determinant and the combining site Interactions may be multivalent → multivalency increases avidity of the interaction Avidity = the combined strength of binding of multiple interactions – high avidity (can be higher than the affinity) – i the sum or more than the sum of the individual parts Secreted Antibodies Antibodies secreted aim to neutralize the toxins that pathogens secreted into circulation A major fraction in sera = key component of Humoral Immunity 5 Classes of Antibodies (conferred by the H chain, on the Fc portion) IgG IgM IgD IgA IgE SIMPLEST monomer and Secreted pentamer (5 Small monomer Dimer Monomer (200,000 very small (150,000 MW) = linked) = BIG/HEAVY (180,000 MW) MW) can cross placenta (900,000 MW) External SURFACE secretions: milk, In serum at the lowest Secreted into blood/tissues In serum (lymph) and BOUND = on sweat, tears, concentrations of all (MOUTH GUMS!) intravascular spaces mature B cells GI,colostrum, other Abs (not secreted/in respiratory, Slow turnover, long half life Shorter half-life (5 days) circulation) urogenital tracts, Shortest half-life (2 (23 days) SALIVA and days) H chain: μ (extra CH4) H chain: δ MUCUS) H chain: γ L chain: k or λ L chain: k or λ Absorbed hough H chain: ε (extra CH4) L chain: k or λ J chain intestinal lumen L chain: k or λ NO subclasses! (when ingested with Subclass: IgG1-4 NO subclasses! milk) NO subclasses! Short half-life (5.5 days) H chain: α L chain: k or λ Subclass: IgA1-2 Functions: - Agglutinates: “glue” – Functions: Functions: bacteria together = can’t - First immunoglobulin - Signal function and dies produced in an immune receptor - Precipitates: soluble in response (1st made by Functions: - Needed for solution, will precipitate out infants = low affinity (not - For parasitic one stage of of solution as much modification → infections differentiation - Opsonizes: phagocytosis t-independent antigens) - Associated with of B (Fc receptor for IgG) – - Agglutinates: VERY allergies or lymphocytes antibody Fab binds to GOOD FOR THIS (so hypersensitivities foreign molecules = many sticky sides!!) → - Reaginic (capacity of “breach” that allows each side has 2 spots so IgE to cause phagocytes to inject the should bine 1- but steric allergies) pathogen easier (antibody hindrance = only bind 5 - Does not agglutinate makes stickiness between - Also good for Functions: nor fix complement the precipitation for the - Agglutinates - Binds to IgE Fc pathogen/macrophages so same reason - Neutralizes receptors on it is easier to grab) - Activates complement → basophils/mast - Mediates ADCC = only need 2 IgM for this! cells, and Antibody-dependent - Poor for neutralization eosinophils cell-mediated cytotoxicity - Induces (Fab binds to target, Fc MOST EFFICIENT degranulation → binds to Fc receptor on NK ANTIBODY FOR triggers granules of cells and stick the NK cells AGGLUTINATION, mast cells and into the target and trigger PRECIPITATION, AND basophils to release them for the lytic activity) COMPLEMENT FIXATION - Activates complement - Neutralize toxins Regulation of Immune Responses Three signals to activate naive T cells: 1. TCR signaling 2. Co-stimulatory interaction 3. Cytokine Signaling TCR signaling Sets the stage for T cell activation via: ○ TCRαβ – Peptide/MHC ○ CD4 or CD8 – MHC (II and I) ○ Adhesion molecules – CD2:LFA3, LFA1:ICAM1 Co-stimulatory interaction Co-stimulator interactions engage clonal expansion = binding between CD80/86 (APC) and CD28 (T cells) are necessary for T cell activation Anergy = non-responsiveness (functional inactivation of cells) via co-inhibitors (CTLA-4 and PD-1) on T cells. ○ No response when: no binding between CD80/86 APC–CD28 OR engagement between CD80/86–CLT-4 or PD-1–PDL1/2 ○ Inhibitor / negative receptors terminate T cell response (turn off their activation) CTLA-4 (CD152) = made 24h after activation, binds to CD80/86 with higher affinity than CD28 PD-1 (“program-death 1”) = on T cells; recognize two ligands (PD-L1/2) and inactivate T cells PD-L1 = on APC and many tissue cells PD-L2 = on mainly APC ○ T cell exhaustions – tumors express inhibitory molecules (PD-L1 or CD80/86) that “exhaust” / turn off T cells to escape elimination. ○ Treg cells down-regulate response: enagne PD-1 and CTLA-4 on T cells via PD-L1 and CD80/86 (forms of suppression) → also make IL-10 = inhibits T cell response and make indoleamine 2,3-dioxygenase (IDO) to deplete tryptophan T cell regulatory pathways = checkpoints ○ Immune checkpoint therapy for cancer: new weapon to decrease immune suppressive mechanisms of tumors (enhance anti-tumor responses): give anti-CTLA-4 antibody or PD-1 inhibitors to prevent T cell inactivation from tumor cells Good for patients who have no clinical signs of cancer for many years (durable, long-term) High risk of immune side-effects (dermatologic, gastrointestinal, endocrine, or autoimmune) SUMMARY of T-cell costimulatory and coinhibitory receptors and their ligands: TCR Ligand Activity Co-stimulatory CD28 CD80 or CD86 – expressed by professional APC Activate naive T cells receptors (and medullary thymic epithelium) Co-inhibitory CTLA-4 CD80 or CD86 – expressed by professional APC NEGATIVE regulation of immune receptors (and medullary thymic epithelium) system (maintain peripheral tolerance, reduce inflammation, etc) PD-1 PD-L1 or PD-L2 – expressed by professional AP, NEGATIVE regulation via regulation some T/B cells, and tumor cells of Treg differentiation Cytokine Signaling  T cells differentiate when interacting with cytokines (T and B cells use signal with cytokine mediators (for both adaptive and innate) → cytokines bind to receptors Different transcription factors are activated in the polarization of subsets → know these subsets and the cytokine signals ○ T helpers (Th1, Th2, Th17) ○ TFH cells ○ T regulatory ***all come from a naive CD4+ T cell*** TGFβ + IL-2 Treg IL-10 Suppresses immune response Inhibits antitumor FoxP3 (periph TGF-β response (suppressive) ) IFN-γ + IL-12 + TH1 IFN-γ Activate macrophages Tissue inflammation IL-18 GM-CSF (regulates) T-Bet TNF-α Activate macrophages TL-3 Activate macrophages TNF Hematopoiesis Inflammatory response IL-4 TH2 IL-4 Activates B cells For allergy GATA3 IL-5 Activates B cells IL-10 Inhibited macrophages IL-3 Hematopoiesis IL-13 TGF-β Inhibits monocyte activation TGFβ + IL-6 + IL-23 TH17 IL-17(F) Autoimmunity tissue RORγt IL-22 inflammation IL-6 + IL-21 TFH IL-4 Regulates affinity maturation for B BCL6 IL-21 cells in germinal centers Cytokines General characteristics Cytokines = small soluble proteins that bind receptors to mediate effector functions. Subset = Chemokines (chemotactic activity for recruiting cells to infected sites → “chemotactic” (tell cells to come to infected areas). Properties: ○ Pleiotropic = different effects dependent on the type of target cell ○ Redundant = mediates similar effects on target cell ○ Synergistic = combines two cytokine activities (greater than additive) ○ Antagonistic = inhibits one cytokine’s effect by another’s action ○ Cascade = effect of one cytokine on one target cell to produce additional cytokine(s) Low MW, short-lived activity (made quickly, do job, and are quickly degraded) Receptors change to have a better affinity (more subunits come together) Make signals by binding to its cell-bound receptor (cytokine = ligand) = increase affinity? Sometimes multiple signals through multiple receptors are needed (integration of signals occurs at the molecular level inside cells  = induces change in transcriptional program of the target cells = cell changes metabolic/proliferative state) Act in three ways: ○ Autocrine action ‒ released but bind to receptors on the cell that made them ○ Paracrine action ‒ released to affect nearby cells ○ Endocrine action ‒ released into the bloodstream to affect distant cells Cytokine functions (unique to a specific T helper subset): Pro-inflammatory: IL-1β, TNFα, and IL-6 Anti-inflammatory (promote tissue repair): TGFβ Regulate hematopoiesis: ○ Erythropoietin (EPO) – RBC production ○ Granulocyte-macrophage colony stimulating factor (GM-CSF) – growth → diverse sets ○ Granulocyte-colony stimulating factor (G-CSF) – stimulates granulocytic colonies ○ Macrophage-colony stimulating factor (M-CSF) – stimulates macrophage colonies ○ Interleukin-3 (IL-3) – myeloid colony formation (granulocyte/macrophage/eosinophils/megak/erythroids) ○ Interleukin-2 (IL-2) – T-cell proliferation (T-cell growth factor) Cytokine Families: Family name Representative members of family Comments Interleukin-1 IL-1α, IL-1β, IL-1Ra, IL-18, IL-33 Inflammatory mediators Class 1 IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-13, IL-15, Small cytokine molecules (hematopoietin) IL-21, IL-23, GM-CSF, G-CSF, GH, prolactin, erythropoietin/hematopoietin Class 2 IFN-α, IFN-β, IFN-γ, IL-10, IL-19, IL-20, IL-22, IFNs = antiviral responses but all are (interferon) IL-24 important for immune responses Tumor necrosis TNF-α, TNF-β, CD40L, Fas (CD95), BAFF, APRIL, Soluble or membrane-bound – immune factor LT-β system development, effector functions, and homeostasis. Interleukin-17 IL-17 (IL-17A), IL-17B, IL-17C, IL-17D, IL-17F Promote neutrophil accumulation and activation, and are proinflammatory. Chemokines IL-8, CCL19, CCL21, RANTES, CCL2 (MCP-1), Chemoattractant function (“chemotactic”) – CCL3 (MIP-1α) induce adhesion molecule to leukocytes / endothelial cells (selectin, selectin ligand, integrin, and ICAM-1) → extravasate and go into infected areas Similar Structures for Receptors have Similar Functions !!! Share common peptide sequences Cytokines recognized by receptors with that Common receptor subunit subunit γc IL-2, IL-4, IL-7, IL-9, IL-15, IL-12 βc IL-3, IL-5, GM-CSF gp130 IL-6, IL-11, LIF, OSM, CNTF, IL-27 Therapeutic targets Cytokines can be immunotherapeutic agents: CYTOKINES EFFECTS INDICATIONS IFN-α Antiproliferative and antiviral Treat chronic hepatitis C, AIDS-Kaposi’s sarcoma, and hairy cell leukemia IFN-γ Immunostimulatory and antiviral Control chronic granulomatous disease IL-2 Immunostimulatory Renal cell carcinoma and metastatic melanoma (eliminate cancer) Erythropoietin Erythropoiesis (rejuvenation of RBC) Anemia from chronic renal failure G-CSF Stimulates granulocyte production Reversal of neutropenia after chemotherapy, radiation therapy, or both GM-CSF (monocyte/macrophage production) There can also be therapeutic agent ending in “MAb” = monoclonal antibody for cytokine/receptors MAb EFFECTS INDICATIONS Basiliximab Anti-IL-2 receptor Prevents rejection of organ transplants Daclizumab Cetuximab Anti-epidermal GF Solid organ tumors refractory to treatments receptor Adalimumab Anti-TNF-α Moderate to severe RA refractory to treatment Receptor Decoy → Etanercept (soluble TNF receptor) binds TNF in circulation so it can’t bond receptors on the cell! (for Rheumatoid arthritis?) Ex. Which of the following pairs are antagonistic? IL-4: IFNg * (IL-1 and IL-2?) NOT: IL-2: IFNg,.IL-1: IL-8, IL-4: IL-5, or GM-CSF: IL-17 MHC MHC Molecules What is “MHC”? ○ Major – vigorous and quick reaction (more so than other genetic differences) ○ Histocompatibility – when tissues are transplanted between animals they can be rejected (compatible if accepted = genes are the same between animals, otherwise non-compatible) ○ Complex – genetic locus encoding molecules in a cluster of associated genes (long, continuous stretch of chromosomes – within this CONTAINS THE THREE CLASSES). Also called “human leukocyte antigen complex” (HLA) in humans; located on chromosome 6  MHC are inherited in linked groups = HAPLOtypes (person inherits one haplotype from mom and the other from dad → both alleles are codominantly expressed). Inherit all encoded alleles in the complex as a set! ○ Genetic polymorphism of MHC in human populations: seen in the region that binds the peptide 6 classes (locus) of class I and 12 class II = 6 x 12 x 2 alleles (many of these = lots of variation in humans!) → Each of these molecules is very different between persons – not the same as between clones) These form signature/fingerprint molecules = used for paternity testing! Three classes: ○ Class I/II = cell-surface vessels to hold/display antigen fragments (for antigen presentation) → peptide fragments made inside and placed on the surface to approaching Tαβ cells can engage with these molecules via their T cell receptors. Class I – makes an alpha chain Class II – start with a “D” and make two peptides (alpha and beta peptides) Professional; ad non-professional APC ○ Class III = NOT APC!! Instead, makes complement proteins,tumor necrosis factors and lymphotoxins Antigen Presenting = taking a fragment of the microbe and putting it on the MHC to present to T lymphocytes which will recognize it by their TCR to activate it to a T cell → Class I vs. Class II Class I Class II Alpha peptide with 3 domains: α1, α2, and α3 – 2 peptides each with their own domain: α peptides (α1 these make a groove that faces outward to hold and α2) + transmembrane C-terminal with β peptides the fragment (hold 8-10 aa of only 1 peptide at a (β1 and β2) = with 2 domains (cleabe 10-20 aa) time) - α1 and β1 make a cleft that faces outward → these are - aa in α1 and α2 = very polymorphic/variable, very polymorphic (like class I) same MHC in people but T/B cells variable - α2 and β2 = non-polymorphic → CD4 binds to β2 within 1 person - aa in α3 = non-polymorphic (similar TCD8 on - Also one at a time and have broad specificity! T cells for everyone that binds α3) - Professional APC = constitutively express MHC II C-terminal transmembrane domain: (specialized to present) = macrophages and B cells β2-microglobulin - Macrophage-like cells - Langerhans = in dermal skin epithelium and Broad specificity (different peptides, same mucosal areas (esophagus) → 1st response MHC) - Dendritic cells = in thymus and lymph nodes (poorly phagocytic), use Fc and C3 receptors - These are found in almost every nucleated cell - B lymphocytes = efficient/matured (high affinity to - Classifical transplantation antigens (if MHC I BCR, antibody receptor, better than dendritic) → 2nd not compatible then transplant will be rejected response - Used for Paternity Testing - Occasional expression induced by cytokines from T cells (keratinocytes, endo/epithelial cells) → join “the battle” **on ALL cells for endogenous antigen (viral / tumor) presentation to CD8 **constitutively on B cells and dendritic cells, and induced on non-professional APC, for exogenous MHC I present endogenous antigens to CD8+ antigens expressed to cd4 cells cytotoxic T lymphocytes (CTLs) 1. Cell infected by virus → viral proteins made in MHC II present exogenous antigens to CD4+ T helpers the cell igens to CD-8 cytotoxic T lymphocytes (CTLs) 2. Viral processes sent to proteasomes (here 1. Bacterial proteins endocytosed (internalized)  processed into short peptides) 2. Unfolded and made into fragments in the endosome 3. Short peptides associate with MHC I in the 3. Peptides move to the ER and associate onto MHC II ER 4. Peptide-MHC adopt a new conformation 4. Peptide-MHC adopts a new conformation 5. Recirculate to the surface 5. Circulate to surfaces of infected cells 6. Bind to T cell receptors (on CD4) 6. Peptide-MHC contact TCR (on CD8 cells) and 7. CD4 enhances binding at non-polymorphic, bind to the α3 non-polymorphic region non-peptide binding region (α2 and β2) 7. CD8 enhances binding at α3 region 8. APC and T cells interact → mutual activation and 8. CD8 CTL degranulate and send enzymes cytokine production (perforin = makes holes in target membrane so granzymes can enter = apoptosis) to lyse infected cells Ex. Which of the following is NOT True of MHC class I molecules? a) Present on all nucleated cells b) Present exogenous antigen to CTL → should be endogenous (she didn't teach us this = cross-representation) c) CD-4 molecules bind to the polymorphic region → CD4 not associated with Class I, this should say CD8 binding to NON-polymorphic mechanism!! d) Beta 2- microglobulin is part of its structure e) Present peptides of protein antigens Peptides bind for presentation by MHC on the surface Any MHC can bind numerous peptides = “promiscuous” (including self) – can exchange in/out of pockets Class I and II = polymorphism in the region that binds the peptides so each individual’s MHC binds different antigenic peptides (people respond to different microbes at different degrees = protect some people better!) ○ Different panel of peptides based on their haplotype (6x12x2 allele) → each person’s ability to activate clones of useful T helper or CTL will be different ○ We all respond to infection/antigens differently!! → idea of “saving the population” Goes along with the idea of population significance (why we need polymorphism) – diversity of what antigens can be presented and what immunity is launched. Some immune systems can better fight a disease = evolutionary survival advantage against mortality from diseases so the population won’t wipe out ○ Advantages: survival of the race, NOT individuals ○ Disadvantages: certain alleles associated with disease Ex. rheumatoid arthritis (DR4) from ankylosing spondylitis (B27): not everything with B27 has ankylosing, but ankylosing will often have B Other Molecules CD1 molecules have lipid-containing antigens (ex. mycolic acid from mycobacterium) → have binding grooves for hydrophobic structures (like an exogenous antigen)  ○ CD1 = structurally like Class I but functionally overlaps with class II! ○ Limited polymorphism compared to MHC class I and III ○ Presents lipid-containing antigens to NKT cells and Tγδ cells Superantigens = bacterial products that stimulate a lot of T lymphocytes specifically (in an uncontrolled way at low concentrations → fever, shock, and death). These bind MHC II and T cell receptors… ○ NOT by antigen-recognition sites but T helper cells clip antigens outside of the antigen-recognition sites to activate many more T helper cells = rigorous response Complement System Complement System = circulating and membrane-fixed proteins Complement System Functions = kill microorganisms, including bacteria, viruses, yeasts and certain cells, directly Induce inflammation ○ Anaphylatoxins = C3a, C4a, C5a → strongly stimulate chemotaxis for more neutrophils and other granulocytes to come in Neutrophils = pro-inflammatory, protease (tissue destruction) Basophils/mast cells = degranulate to release inflammatory mediators including histamine to induce vasodilation (more blood cells come in) ○ Induce expression of adhesion molecules increase extravasation (go into tissues with infected microbes) → enhance production of inflammation mediators ○ Inflammation marker – C-reactive Protein (CRP) = CRP binds to phosphocholine on microbe’s surface → then binds C1q = activates classical pathway Increase phagocytosis by opsonization ○ Complement receptors: CR1, CR3, CR4 expressed on macrophages, natural killer cells, granulocytes ○ Facilitated by opsonization = binding to C3b, iC3b, C4b “i” = inactive → not active and can’t cut/bind but can still help with phagocytosis? IgG receptors in globuline - instead of gamma, complement receptors on phagocytes – CR1 receptor on phagocytes binds C3b on the microbe → binding initiates phagocytosis (better for opsonization) Clearance of insoluble immune complexes ○ Antigen:antibody complexes are very large and get trapped = causes inflammation so they need to be removed (why we don’t get boosters all the time: more antigens → more antibodies → more complexes!) ○ Complement binds Ab and takes in insoluble complexes to digest them Activates complement cascade Complex binds complement receptors (CR3 on phagocytic cells) Enhanced phagocytosis and destruction of Ag:Ab complexes ○ Deficiency in complement → diseases Deficiency in C1, C4, C2 develop systemic lupus erythematosus (SLE) = ipaired ability to clear immune complexes = react to DNA = makes antibodies = make more complexes Deficiency in alternative pathways = increased susceptibility to recurrent infection via pyogenic bacteria (bad phagocytosis!) Three Activation Pathways: Difference: unique proteins & enzymes for the 1st few steps Similarity: results are the same ○ Classical/lectin make the same C3 convertase → after this have identical pathways downstream ○ KEY FOR ALL THREE PATHWAY = C3 → cutting of C3 is the main step (provides amplification) C3 convertase enzyme is very active.  Large amount of C3b deposit form nuclei for forming C5 convertase Large amount of C3a generated to induce inflammation (along with C5a) Large amount of C3b deposit for opsonization After this = formation of membrane attack complex Activation is three-stepped process: 1. Recognition 2. Enzyme activation 3. Expression of biological activity Classical Pathway 9 individual proteins: C1, C4, C2, C3, C5, C6, C7, C8, C9 Initiated by the antigen:antibody complex (C1 binds to the antibody!) 1. Antigen:antibody complex aggregates and binds C1 makes C1 complex = C1q + C1s + C1r a. C1q globular head binds IgG/IgM by the Fc region (binding needs two molecules of IgG and one of IgM = ionic and hydrophobic bonds) 2. Conformation changes activate C1s enzymatic activity (C1 esterase) = cleaves C4 and C2 a. C4 → C4a + C4b b. C2 → C2a + C2b 3. Complex on the membrane from these = C4bC2a (C3 convertase) a. C3 convertase cleaves C3 → C3a + C3b (makes hundreds of these) i. C3b = KEY MOLECULE for the “cascade of reactions” (largest amplification) ii. C3b is also the central step for ALL complement pathways! 4. New complex made = C4bC2aC3b (C5 convertase) a. C5 convertase cleaves C35→ C5a + C5b 5. C5b initiates formation of the Membrane attack complex (MAC) (C5b = nucleus for MAC formation) a. C6 binds to C5b b. C7 binds to C5bC6 c. C8 binds to C5bC6C7, which is a complex that serves as a receptor for C9 6. C9 polymerizes into poly-C9 tube = inserts into the membrane for ions escape, water enters, and cell to lyse → this all ends in killing the microorganism (cells, bacteria, virus, and yeasts DIRECTLY) Lectin Pathway Initiation by lectins that bind mannose (MBL) on microbes (mannose = PAMP → fast and innate!) ○ Lectins = proteins that bind carbohydrates ○ MBL (“mannose-binding lectin) = binds carbohydrates on surface of microbes 1. MBL proteins bind like Cq1 (globular proteins) → releases associated proteins (MASPS) 2. MASPS (“MBL-associated serine proteases”) cleave C4 and C2 a. C4 → C4a + C4b b. C2 → C2a + C2b 3. Fragments associate together making C4bC2a (C3 convertase) complex on the membrane 4. AFTER THIS = CLASSICAL PATHWAY (identical downstream!) a. Same thing there C3b = kep molecule to make C5 convertase for C5b to initiate the formation of the MAC → then C6/C7/C8 form a complex for C9 to make the poly-C9 tube Alternative (Properdine) Pathway Activated by microbe cell wall (this is more complex) 1. Start from C3 → bind to surface microbes and is hydrolyzed by H2O a. Now C3 → C3a + C3b and C3b can bind to microbe surface forming a nucleus and activating the complement cascade b. After C3b binds to the microbe, it binds Factor B and form C3bFactor B complex 2. Factor D cleaves Factor B bound to the C3b, into Bb which now makes C3bBb (C3 convertase)  a. C3bBb makes more C3b = positive feedback to perpetuate the cycle b. C3bBb = not stable so Properdine binds making a C3bBbP that is more stable (this cleaves more C3 = makes more C3b which can bind Factor B = amplification loop) → go back to first step and amplify itself! 3. 3bBbC3b cleaves C5 (like C4bC2aC3b in classical pathway) a. C5 → C5a + C5b b. Again, C5b initiates the formation of Membrane attack complex (MAC) – same as other pathways Regulation of the Complement System Can make high levels of tissue destruction/inflammation so need to be tightly controlled!! C1 inhibitor (C1 INH) = inhibits 1st step of classical pathway ○ C1 INH stops the C1r2s2 from becomes active/proteolytic ○ C1 INH deficiency → angioedema (hereditary and acquired) = uncontrolled activation of C Decay Accelerating Factor (DAF) = protein that binds C4 (dissociates C4bC2a) = C3 convertase so blocks formation of convertase = can’t make C3b or C3a) Factor H = binds C3b and displaces Bb from C3bBb (alternative pathway C3 convertase not formed) Membrane cofactor protein for Factor I = cleaves C3b making it inactive (iC3b) ○ This still does opsonization because can bind the complement receptor but does NOT cut/function Serum carboxypeptidase Inactivate Anaphylatoxins C3a, C5a, C4a ○ C59 binds C9 to prevent the formation of MHC = turn down destruction ***Factor H and I = InHibitory!! Ex. Phagocytic removal of microbes and immune complexes from our tissues is helped by which of the following? → All of the above (IgG, C3bi, C3b) – think about complement = all working together for opsonization Ex. Although complements are considered part of the innate defense, which of the three pathways may require time and acts with antigen specificity? a. Classical – antigen specificity = specific reaction to a specific antigen = ADAPTIVE IMMUNITY (takes a while for antibody to come in) b. Lectin – innate / non-specific bc mannose is on all microbes c. Alternate – innate / non-specific d. C reactive protein Hypersensitivity 4 types of Hypersensitivity TYPE I TYPE II TYPE III TYPE IV (Delayed = DTH) Mediated by IgE (IgE bind Soluble IgG or IgM IgG and antigen T-cell mediated (Th1 to Fc receptor on mast (cytotoxic) (Ab binds (cell-mediated) (chronic secrete cytokines = cells/basophils → allergen antigen = complement exposure → make Ab:Ag activate macrophages binds IgE = cross-linking = activation and lysis) complex = activates complement and CTLs) degranulation) → inflammatory mediators, neutrophils release enzymes Rxn in in minutes Rxn in hours Rxn 24-48 h after Rxn in days-weeks contact Ex. anaphylaxis, allergies Ex. hemolytic anemia **Wheal-flare (MINUTES) (RBC destruction from Ex. serum sickness, rheumatoid Ex. Rash, MS, 1 diabetes mismatches arthritis **diffused redness from **mediated by T cells = transfusion) complex → complement cascade long time for lesio to form (4-6 HOURS) (DAYS) Type I Events: 1. Sensitization Phase = activation of Th2 makes IL-4 for isotype switching (B lymphocytes → B cells → IgE) a. Generates memory (B cells have memory for next year/next reaction) 2. Activation Phase = IgE activates basophils and mast cells (cell-triggered) a. Cells located around BV, in VT, in gut lining/lungs/nasal passage, and conjunctiva b. IgE binds to Fc receptors on FcγR1 → FcγR1 get signals from allergies for crosslinking (allergen binding in a few min) to which activates mast cells to degranulate and release things 3. Effector Phase = activation of basophils/mast cells to release vasoactive amines and lipid mediators (prostaglandins and leukotrienes) a. Activation of Mast cells/Basophils mechanism: membrane reaction like lipase i. Calcium influx → decrease in cAMP = increase in cGMP (cyclic GMP) ii. cGMP activates of phospholipases (metabolize arachidonic acids) which make lipid mediators = prostaglandins & Leukotrienes iii. Tyrosine kinase is activated = makes cytokines (IL-5) = inflammation escalation → make/release lipid-mediators like neutrophils which degranulate and release histamine = increase eosinophil infiltration b. Activities of mediators released → functions to modulate adaptive immunity: i. Inflammation (cytokines) ii. Tissue damage (proteases) iii. Vasodilation (prostaglandin) iv. Smooth muscle contraction (Leukotrienes and amines) 4. Late Phase = next day (6-24 hours) cytokines make = recruits mast cells = make eosinophil chemotactic factor that recruits eosinophils a. Eosinophils make their own cytokines/chemokines so the reaction lasts for days b. IgE binds FcεRII = activates eosinophils = ribonuclease (antiviral and antiparasitic activity) i. Examples of Type I Reactions ○ Respiratory tract allergies – hay fever, rhinitis, asthma (from pollens, dust, mold sports, dander) ○ GI tract allergies – vomiting/diarrhea (from peanuts, eggs, etc.) ○ Skin reactions – hives, urticaria (from insect bites) Wheal and Flare skin reaction allergy test = antigen intradermal injection of the antigen (immediate rxn = ~15 min), more IgE = larger response Flare = erythema (Redness) from dilation of blood vessels Wheal = raised area (edema/swelling) from the release of serum into tissue, makes a well-defined edge ○ Anaphylaxis = toxin/allergy moves through blood and contacts IgE on mast cells in lung → mast cells degranulate = release histamine (during effector phase) = stimulates bronchoconstriction (constriction of airways = wheezing and breathing difficulties) Drop in pressure when you can’t breath → heart into arrhythmia = shock (suffocation) GI affected (nausea, abdominal pain) Skin reactions (hives over the body) Brain = dizzy or unconscious  Complement system induces inflammation from anaphylatoxins (C3a, C4a, C5a) → stimulate chemotaxis = attracts neutrophils, basophils, and mast cell (cells increase inflammation through release of histamine and induce vasodilation) To reduce inflammation, use antihistamines, glucocorticoids, and epinephrine Epinephrine can be administered subcutaneously through en epipen which will contract smooth muscle, increase cardiac output, and relax muscular airways Reaction from: atrophy (inhaling allergens) or anaphylaxis (systemic reaction) Interventions ○ Environmental – avoid exposure (use air filter to remove pollen ○ Pharmacological – people know they are allergic to carry epipen (with epinephrine) = smooth muscle is still controlled even with the loss of control of blood pressure Or use corticosteroids or leukotriene antagonists to relax SM, or phisphodie to prevent formation of lipid mediators ○ Immunological – modified allergens given in a different route (subcutaneously won’t activate T helper cells = IgE is not made and instead makes IgG = hyposensitization = ↓Th2 so ↑ Th1 ↑ IgG Type II Mechanism: soluble antibody (IgG/IgM) directed to surface of target cell and bind (allergens on the surface) → this activates the complement system via the LECTIN PATHWAY ○ Generation of anaphylatoxins (C3a, C5a, C4a) ○ Induce inflammation: infiltration of inflammatory cells (neutrophils, macrophages, etc) = activation of inflammation and tissue destructive mediators (leukotrienes, prostaglandins, free radicals and proteases) Examples: ○ Blood transfusion (mediated by IgM) ABO polysaccharides only need IgM! Carbohydrates induce IgM production = T-independent (IgM binds blood antigens on donated mismatched blood = agglutination/destruction of donor cells = inflammation symptoms: fever, nausea/vomiting, back/chest pain, RBC debris) Type A = A antigens only = make anti-B antibodies (Ab for B antigen) ○ Hemolytic disease (erythroblastosis fetalis) of the newborn - mediated by IgG Rh- mom has baby with Rh+ dad → Rh+ baby → when baby is born, mom is exposed to Rh antigen (sensitization). During next pregnancy, Rh+ fetus causes mom Rh antigen to make IgG → IgG crosses placenta to destroy fetus’s RBC = kills second baby Also enlargement of liver and spleen, hemorrhages, and elevated bilirubin Diagnosis through indirect Coombs test (find it mom has anti-Rh antibody, IgG) Test sample (mom’s serum) added to reagent (Rh+ RBC) → RBC washed, and then add Coombs reagent (anti=human IgG) If there is agglutination (clumping) = human IgG attach to RBC Prophylactic treatment: Rh immune globulin (RhIG Rhogam) after delivery to prevent sensitization (remove fetal RBC before mom can make an immune response) Rh goes into mom – give mom Rhogam which binds fetal RBC and remove them from mom so they don’t make antibodies = prevention tactic Type III Mechanism: persistent infection (chronic exposure) from extrinsic antigens (mold) → antigen and IgG antibody complexes which go to tissues (failed to clear = activate complement cascade) – immune complexes  ○ Anaphylatoxins released, inflammation induced, basophils/mast cells/platelets activated and release vasoactive substances and inflammation mediators Vasodilation occurs → extravasation increases expression of adhesion molecules Macrophages and neutrophils make cytokines (TNF and IL-1) = increase inflammatory mediators production = escalate inflammation Examples: ○ Arthus reaction (localized) from related injection/immunizations of antigens (too many complexes formed). Person has high [Ab] levels → Ab binds Ag during shot and causes redness on the arm (if it goes worse then it can lead to necrosis) = LOCALIZED Continuously makes complement = make neutrophils (makes enzymes = destruction) and make mast cells (release vessel-dilation things) → also platelets activated ○ Serum Sickness (systemic) = injection of xenogeneic antigen (anti-toxin – snake venom) → high levels of free antigen that stay for a while (~ 5 days) = induce antibodies (anti-horse IgG) = SYSTEMIC Ab:Ag complexes made → activate complement system (at first complexes are soluble and small, but get bigger and deposit onto basement membranes causing nephritis and arteritis (feel inflammation) → when complexes are large and insoluble they are cleared = symptoms subside (complement inactive and now unbound/free antibody in circulation) Immune complexes made and → tissue destruction/feel sick Complexes large = removed → damage/fever subsides and person feels okay Type IV (DTH) Mechanism: antigen activates APC (like macrophage) which takes up the antigen and presents it → activates Th1 (CD4) → Th1 makes cytokines/chemokines (IFNγ, TNFβ, IL-3/GM-CSF) ○ CD8+ (CTLs) for all cell-mediated destruction ○ Cytokines recruit cells (T cells/phagocytes) to antigen exposure → develop lesions 3 types of DTH: Contact dermatitis Tuberculin reactions Granulomatous reactions Antigens contact skin (cutaneous level) Skin test to see previous When things are hard to destroy → from poison ivy, metals, and chemicals exposure to Mycobacterium tuberculosis, leishmaniasis (live in (detergents) → or infection with viruses (intradermal/ subcutaneous macrophage/Kupffer cells), leprosy (chickenpox blisters: contact actually injection of soluble (Hansen disease) makes the blister) antigens) → appearance < 24 hours from DTH reaction Macrophage infiltration → bacteria not Antigens enter dermally and bind to digested into lysosome so host proteins → presented by Patient looks at next day and macrophages fuse into Giant Cells = Langerhan cells and keratinocytes determines if there was a recruit Th1 = lymphocytes activate to activate T-helper 1 (Th1) cells to reaction fibroblasts to encase the whole make cytokines and interferons. thing and make a granuloma Antigen presented by (pathogen confined within) Cytokines activate macrophages dendritic cells and (phagocytes) with protease and other macrophages → Th1 Fibrosis of the organ = pathogen dies things to lyse