Immunology TQs - Lecture Notes PDF

Immunology TQs **[Lecture 1]** 1. Innate vs Adaptive (acquired) Immunity Innate Adaptive ------------------ -------------------------------------------------------------------------...

Immunology TQs **[Lecture 1]** 1. Innate vs Adaptive (acquired) Immunity Innate Adaptive ------------------ -------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------- Players involved Neutrophils, macrophages, NK cells, complement proteins B-cells/plasma cells, T-cells (helper and killer), antibodies (IgM, IgG, etc.) Mechanism Phagocytosis, complement, PAMP-recognition = pathogen associated molecular pattern, inflammation Antibodies, cell mediated immunity (T cell) (**intracellular**), humoral immunity (B cell) (**extracellular**), mucosal immunity Onset Fast response (minutes -- hours) Slow response (days) Duration Short-lived, no memory cells Longer-lasting, memory cells made 2. Distinguish between the two branches of the immune system a. Innate immunity i. Non-specific ii. Born with it iii. Always on and **looking for nonself pathogens/Ags** iv. No memory v. It is as effective the first time it is exposed to bacteria as it is on subsequent infections vi. **Kickstarts inflammation** b. Adaptive immunity vii. Specific viii. Must be activated/upregulated by lymphocytes ix. Gets better with each encounter + **memory antibodies** and specific T killers and helpers x. 3 types c. OVERLAP = do not act in isolation xi. **Complement activates B cells** xii. Phagocytes activate/differentiate B and T cells xiii. Antibodies act as **opsonins** that phagocytes can use as markers to opsonize 3. Lymphatic system: a system of vessels that circulate **lymph** which is fluid that comes from **interstitial fluid** (excess fluid between body tissues) d. Lymphatics drain interstitial fluid (fluid between cells in the tissue), Lymph drains from tissues to lymph nodes where it is filtered e. Lymph nodes: filters interstitial fluid/lymph + removes antigens ⇒ antigens are presented to T cells ⇒ provide location for immune response ⇒ allow B and T cells interaction to carry out immune response ⇒ B cells make antibodies f. Lymph cells in blood: T cells (cell mediated), B cells (humoral immunity/ Ab production/ Ag presentation) NK cells ⇒ all come from pluripotent hematopoietic stem cells 4. Immune system roles g. Able to tell self from nonself = most important attribute xiv. PRRs xv. Antigen specific receptors = Abs, BCRs, TCRs, MHCs h. Lymphocytes = B cells, T cells, NK cells 5. Cell mediated vs humoral i. Cell mediated xvi. T cells (CD4⁺, CD8⁺) and activated macrophages. xvii. Critical for **intracellular** pathogens, including **viruses** (infected cells), certain **bacteria** (like Mycobacterium tuberculosis), and **fungi** (e.g., Candida, Pneumocystis). xviii. Also central to **graft rejection** (cytotoxic T cells) and controlling **tumor cells**. j. Humoral xix. B cells produce antibodies (IgG, IgM, IgA, IgE, IgD). 1. IgE triggers anaphylactic reactions xx. Primarily effective against **extracellular** pathogens (e.g., bacteria in the bloodstream, **extracellular viruses**) by mechanisms such as **neutralization**, **opsonization**, and **complement activation**. 2. Autoimmune diseases = autoantibodies Lecture 2 1. Chemical barriers a. Lysozymes: in saliva, **hydrolyzes 1-4 beta linkages between NAG and NAM** in the peptidoglycan wall permeability causing lysis i. More effective against Gram **POSITIVE** b. Lactoferrin and transferrin: sequester iron via **siderophore** 2. Complement protein deficiencies +-----------------------------------+-----------------------------------+ | Complement protein | Effects of deficiency | +===================================+===================================+ | C3 Factor I | Increased susceptibility to | | | encapsulated bacteria; recurrent | | | infections | +-----------------------------------+-----------------------------------+ | **C5-C9** | **Increased susceptibility to | | | Neisseria bacterial species | | | (gonorrhea and meningitis); | | | recurrent infections** | +-----------------------------------+-----------------------------------+ | Factor D | Increased susceptibility to both | | | encapsulated bacteria and | | Properdin (factor P) | Neisseria species | +-----------------------------------+-----------------------------------+ 3. Alternative C3 and C5 convertases c. Microbial lysis: C5b initiates the membrane attack complex (MAC) to lyse pathogens (osmotic lysis) d. Inflammation (chemotaxis): complement pathway produces small fragments **(C3a and C5a)** promotes degranulation of mast cells and basophils**, increase vascular permeability, increase vasodilation (histamine),** recruit neutrophils and monocytes to inflammatory site, granule contents = contraction of visceral smooth muscle ⇒ too much can induce systemic inflammation ii. **= anaphylactic shock** 1. **C3a and C5a are anaphylatoxins** e. Phagocytosis (opsonization) 4. Complement Alternative pathway: **improve phagocytosis, lyse microbes, and drive inflammatory responses (chemotaxis)** f. Step 1: convertase creation iii. Spontaneous conformational change of C3 into ⇒ iC3 2. Or be cleaved to expose thioester bond 3. **nucleophilic attack by H2O = soluble C3b** 4. nucleophilic attack by R-NH2 or R-OH groups of proteins on pathogen's surface = C3b bound to pathogen iv. iC3 binds to factor B ⇒ iC3-B v. **factor D cleaves factor B ⇒ iC3Bb + Ba** 5. **iC3Bb = C3 convertase** vi. **iC3Bb cleaves C3 ⇒ C3b + C3a** vii. **C3b binds to pathogen surface ([complement fixation])**, **C3a released (anaphylatoxin)** 6. **Macrophages express complement receptor 1: CR1 binds to pathogen bound C3b** ⇒ receptor mediated phagocytosis (opsonization) a. C3b is taken up into the phagosome, H+ ions pumped into phagosome ⇒ lysosome fuses with phagosome ⇒ phagolysosome ⇒ low ph activates enzymes to destroy 7. Factor H & I bind surface bound C3b, C3b is cleaved into iC3b which cannot form C3 convertase b. **Factor I deficiency**: unregulated C3bBb complexes ⇒ deplete C3 ⇒ pt more susceptible to encapsulated bacteria (abscess or ear infections) c. **Factor H** can also bind **sialic acid** + recruit factor I to cleave C3b into iC3b ⇒ Strep pyo and Staph aureus cover their cells with sialic acid to inactivate complement g. Step 2: Alt C3 convertase creation viii. Pathogen bound C3b binds factor B ix. C3bB cleaved by factor D x. **C3bBb + Ba ⇒ a powerful C3 convertase** 8. **DAF**: binds to C3b and dissociates the Bb to **inactivate C3 convertase** 9. **MCP:** binds to C3b and recruits facto I for cleavage of C3b into iC3b 10. Lack of PIGA ⇒ loss of GPI anchors and little expression of complement inhibitors ⇒ DAF and S protein missing from cell surface ⇒ MAC can form and lyse RBCs ⇒ **paroxysmal nocturnal hemoglobinuria** xi. C3bBb cleaves C3 ⇒ **coats pathogen surface with C3b (amplifie**d) 11. **This is enhanced by Properdin (Factor P) which stabilizes C3bBb and prevents degradation** h. Step 3: C5 convertase xii. **C3b binds to C3bBb ⇒ C3bbBb** 12. **C3bbBb = [alt C5 convertase]** xiii. C3bbBb cleaves C5 into C5a and C5b 13. **C5a = anaphylatoxin, C5b initiates MAC** xiv. C5b binds C6 xv. C6 binds C7 and allows for hydrophobic C7 tail to bind pathogen surface 14. **S protein prevents C7 interaction with microbe membrane** d. **Factor J and clusterin do the same thing** xvi. C8 then binds, poking a hydrophobic piece in the membrane xvii. C9 monomers then assemble the MAC 15. **Protectin (CD59) prevents recruitment of C9 = no MAC** e. **HRF does same thing** i. Step 4: MAC xviii. MAC creates a pore in the pathogen surface leading to osmotic influx and lysis 5. Enhance complement j. Properdin and Factor D: enhances alt C3 convertase activity, stabilizes C3bBb (C3 convertase complex) and prevents degradation by other proteases xix. Deficiency = more susceptible to infection by encapsulated bacteria and Neisseria species k. Factor I deficiency: unregulated C3bBb complexes ⇒ deplete C3 ⇒ pt more susceptible to encapsulated bacteria (abscess or ear infections) 6. Possible Q: Westernized diet and microbiome diversity l. Highest density of bacteria present in the large intestine and lowest O2 conc m. **Westernized diet (high saturated fats, high sugar, low fiber) thought to REDUCE microbio**me diversity while diets including **increased fiber uptake are associated with increased diversity and less inflammation** n. Aging and lack of physical activity decreases microbiome diversity Lecture 3 1. TLRs: toll like receptors are a family of PRRs that have an extracellular domain for PAMP engagement (leucine repeat region = LRR) and the **only PRR with a cytoplasmic signaling domain (TIR)** a. TLR4 i. Chromosome 9, **on surface of plasma membrane, extracellular organisms specifically**, Ligand is lipopolysaccharide, **GRAM -- NEGATIVE,** Cells carrying CD14 are macrophages, DCs, mast cells, eosinophils 1. When a gram-negative bacteria dies, LPS (part of cell wall) is released and binds to CD14 on tissue macrophage a. **CD14 is the coreceptor to TLR 4** 2. **NO CD14 = susceptible to Gram NEGATVIE bacteria** 3. **LBP** (LPS binding protein) and **MD2** (associated with TLR4 and binds LPS-CD14) help also capture LPS and give it to LPS-CD-14 b. **TLR3 = double stranded RNA** c. **TLR7 = single stranded RNA viruses** d. **TLR9 = unmethylated CpG islands** 2. TLR4 e. Binding alters the conformation of the toll interleukin-1 receptor (TIR) and binds to MyD88 f. **MyD88 binds to TLR4 ⇒ activates IRAK4** g. IRAK 4 phosphorylates TRAF6 ⇒ leads to phosphorylation of IKK h. IKK phosphorylates IKB ⇒ **leading to its degradation and release of NFKB which enters the nucleus** i. **NFKB in nucleus ⇒ activates transcription factors of genes for inflammatory cytokines, which are synthesized in the cytoplasm and secreted via ER** ii. **IL-1B, IL-6, TNF-A, IL-12, IL-8** 3. PRRs j. NOD-like receptors = another PRR just like TLRs, recognized degraded peptidoglycan components of bacterial cell walls iii. NOD binds ligand ⇒ **NOD dimerizes and RIPK2 binds via CARD** ⇒ RIPK2 phosphorylates TAK1 ⇒ **NFKB goes to nucleus** 4. **Free NFKB transcribes cytokines** iv. **NOD1** = degraded peptidoglycans from **gram NEGATIVE** v. NOD2 = degraded peptidoglycan from all bacteria 5. Deficiency ⇒ chronic intestinal inflammation ⇒ inc numbers of flora bacteria in the gut k. RIG-1-like receptors = another PRR, IRF3 promotes type 1 IFN (IFNa and IFNb) vi. **Viral RNA binds to RIG-1** & interacts with mito antiviral sign proteins (MAVS) ⇒ **phosphorylation of IRF3 ⇒ transcription of type 1 IFN** vii. **Stops 3 things viruses need:** 6. **Serine/threonine protein kinase (PKR)** ⇒ phosphorylation of EIF2a (translation/elongation initiation factor) ⇒ **protein synthesis inhibited** 7. **2-5 oligoadenylate synthetase** ⇒ activates RNAse which degrades RNA 8. **Mx GTPases** ⇒ interferes with nuclear transport of viral RNA and blocks viral assembly 4. Effector functions l. TNF alpha = capillary endothelial cells ⇒ **chemokine recruitment and inc vascular permeability; leukocyte attachment and cell migration, promotes contraction of endothelial cells** viii. Local effect: inc vasodilation, inc vascular permeability, inc phagocytosis, inc lymphocyte migration into tissue, inc platelet adhesion and blood clots to prevent pathogen ix. **Systemic effect:** decreases blood volume, hypoproteinemia and neutropenia, decreased volume (**[septic shock]**), collapse BV, **DISSEMINATED INTRAVASCULAR COAGULATION (DIC)** m. IL-8 = produced by macrophages to **recruit neutrophils** to the area n. IL-1B = inc prostaglandin E2 (nonspecific myalgia and arthralgia) ⇒ PGE2 binds to glial cells **⇒ inc cAMP ⇒ inducing fever** **⇒ IL-1 and IL-12 activate NK cells** x. Inflammasome needed for amplication of IL-1B production 9. NFKB ⇒ NLRP3 ⇒ pro-caspase 1 ⇒ makes more IL1B b. NLRP3 mutation ⇒ **CAPS** i. Leads to overactivity ⇒ inc inflammasome ⇒ excessive IL-1B ⇒ fever, hives, joint pain 5. Mannose Binding Lectin (MBL) o. **Binds MASP (on PATHOGEN) and kickstarts lectin complement pathway. 2^nd^ pathway to initiate** xi. **No MBL = Neisseria susceptibility, no MAC formation, susceptibility to encapsulated bacteria** p. **Acute phase protein induced by IL-6 mainly (IL-1B and TNFa)** xii. **C-reactive protein**, serum amyloid A protein, and mannose binding lectin q. MBL complexed with MASP ⇒ MBL binds to mannose ⇒ autocleavage of MASP2 then cuts second MASP2 ⇒ active MASP2 cleaves C4 and C2 ⇒ C4a (anaphylatoxin), C4b (binds to pathogen), C2a interacts with C4b, C2b released ⇒ **C4bC2a = classical C3 convertase** xiii. 2^nd^ pathway to act r. Deficiency in IL-6 = lower the serum levels of CRP and MBL 6. Classical complement -- prob not a Q s. CRP interact with C1 or C1 interacts with Ab bound to pathogen surface t. C1q binds to CRP ⇒ C1r autocleaves then cuts the other C1r and the two C1s proteases ⇒ cleaved C1s protease is active and cleaves C4 and C2 ⇒ C4a (anaphylatoxin), C4b (binds to pathogen), C2a interacts with C4b, C2b released ⇒ **C4bC2a = classical C3 convertase** xiv. 3^rd^ pathway to act 7. Cellular rolling u. Diapedesis xv. Cytokines **IL-1 and TNFa** cause vascular endothelial cells to express **selectins and ICAMs** 10. **Endothelial cells express selectins and ICAMs** c. Selectins: their ligands roll neutrophils along endothelium to the site of inflammation xvi. Macrophages produce **CXCL8 (IL-8) which recruits neutrophils to the area** 11. **Enhanced by CXCL8** d. **CXCL8 = IL8; neutrophil attachment and migration into tissue** e. **CXCL8 binds to CXCL8R on neutrophil surface ⇒ induces cell signaling to inc the binding affinity of the integrin** xvii. TNFa weakens tight junctions, and neutrophil proteases loosen PECAM-1 interactions; this chemokine gradient drives the cellular movement through the endothelial layer (diapedesis) v. Cell arrest/rolling adhesion xviii. **Integrins ON the immune cells bind to ICAMs (on endothelial cells) which stops immune cells** 12. **LFA-1 (integrin) + ICAM-1 (ligand) = stop** **IL-1 and TNFa inc selectins on ENDOTHELIUM** ⇒ bind to ligands on IMMUNE CELLS **Integrin on NEUTROPHIL** ⇒ bind to receptor on ENDOTHELIUM Lecture 4 1. Phagosome a. O2 rxns (early): **O2 dependent reactions happen first** because they are stronger ones that kill better, **ROS/RNS break DNA** to stop replication **and restrict metabolic pathways** by breaking iron-sulfur enzymes i. **NADPH oxidase: O2 ⇒ O2-** ii. **SOD: O2- ⇒ H2O2** iii. **MPO (myeloperoxidase) : H2O2 ⇒ HOCl (hypochlorous acid)** iv. **RNS formed from INOS** b. **Some microbes can survive the phagosome by blocking the action/function of NADPH oxidase** 2. Phagolysosome fusion c. Most O2 independent killing happens here v. Acid hydrolases (breakdown everything) mixed in from lysosome vi. Lysozyme (destroys bacterial walls) 3. Neutrophil granules: **last** granules to **form** are the **first** granules **released** d. **Secretory and Ficolin-1** vii. **Used to help neutrophil attach to endothelium and extravasation** 1. Necessary for PMNs to enter the tissue site 2. High Ca++ viii. **Band neutrophils DO NOT have secretory and ficolin** 3. **= Immature neutrophils** ix. **Contents: Integrins, CR1, PRR** e. Gelatinase x. Used for neutrophil extravasation into injured tissue xi. Degrades ECM xii. Contents: gelentinase and arginase 4. Arginase helps close tight junctions by decreasing NO levels f. Specific xiii. Secondary. Moderate Ca++ xiv. Makes it harder for pathogen to function xv. **Contents: Lactoferrin, NGAL** 5. Lactoferrin = Hoards iron so bacteria can't use it 6. **Prevents iron acquisition by microbes by binding up siderophores** g. **Azurophilic** xvi. Helps to fuse phagolysosome and release enzymes to destroy pathogens xvii. **Contents: MPO, acid hydrolases, defensins, antimicrobial peptides** 4. NK cell h. Plasmacytoid DCs (PDCs) produce IRNa which activate NK cells xviii. Cytokines can also activate NK cells 7. IL-1b, IL-2, **IL-12**, T1-IFN xix. **Macrophages produce IL-12 ⇒ activate NK cells ⇒ NK cells release IFN gamma ⇒ increases NADPH and iNOS activity in macrophage** 8. System feeds into each other, positive feedback xx. Our cells are tagged with MHC-1 so NK cells are inhibited 9. Cells without MHC-1 (viruses and infecting agents) are targeted by NK cells 5. Neutrophils i. First cells **RECRUITED** from the blood to the tissue in response to pathogen invasion and are the **dominant phagocytic cell** xxi. First cells encountered by a pathogen = macrophages, first cells recruited by a pathogen = neutrophils xxii. 70% of WBCs 10. **More than 70% = neutrophilia** a. Seen **during acute bacterial infections** and **inflammation** 11. **Less than 70% = neutropenia** b. Cases of **chronic or overwhelming** infection by bacteria or fungi j. Circulate in blood until needed k. Live 3-5 days l. Die within hours of tissue entry in response to infection and form pus 6. Mast cells m. 2 ways to trigger xxiii. PRR binds to PAMP 12. Mast cells see Ag and **half** degranulates, later releasing cytokines and chemokines xxiv. Ag binds to IgE 13. IgE is a pre-prepared receptor, triggers **FULL** degranulation Lecture 5 1. Secondary lymphoid organs: site of immune cell storage, activation, and response a. Spleen locations = major site of Ag surveillance. Home to both B and T cells and many other immune cells. i. Red pulp: contains **dendritic cells, plasma cells, RBCs, and macrophages** that **filter blood for pathogens, removes old RBCs, and reserves a store of blood in case of hemorrhage.** 1. Secondary site for hematopoiesis 2. Vascular sinusoids separated by splenic cords contain high numbers of immune cells ii. White pulp: where B and T cells localize 3. **Periarteriolar lymphoid sheath (PALS) aka T cell zone containing DCs and T cells** a. Newly formed B cells leave BM and enter spleen, localizing at T cell zone first 4. **B cells are contained in the follicles** iii. Marginal zone: area between red and white pulp where marginal zone B cells (MZB) live and Ag in blood is captured 5. **The MZB cells provide a rapid IgM response** 2. LN b. LN locations iv. **Follicles**: **naïve B cells and follicular DCs** are localized to the follicles (**activated B cells are concentrated within the germinal centers within the follicles)** v. **Paracortex: T cells** and DCs are localized to the paracortex vi. **Medulla: macrophages and plasma** cells are in the medulla c. Immune cells entry vii. Immune cells enter LN from tissues by afferent lymphatic vessels viii. Immune cells enter LN from **blood circulation by high endothelial venules** 3. Secondary sites d. Liver = largest glandular organ in the body divided into 4 unequal lobes ix. Functions: breaks down fats, converts glucose to glycogen. Makes proteins (including acute phase proteins). Stores vitamins and minerals. x. **Contains Kupffer cells (liver macrophages): KCs line sinusoids and function to capture and remove Ag from blood** e. MALT xi. Sites of immune reactivity in the mucosal tissue that contain lymphocyte aggregations 6. Tonsils: three sets that survey oropharynx for ingested/inhaled pathogens xii. The predominant Ig found in mucosa is IgA f. GALT xiii. **Peyer's patches (lamina propria in your small intestine): contain microfold (M) cells which are the key to GALT responses** 7. **These M cells are phagocytic and direct gut materials to follicles and lymph nodes where they promote IgA production to limit pathogen colonization.** Lecture 6 1. Definitions a. Immunogens: any substance that can produce an immune response, all [immunogens are antigens but not all antigens are immunogens] i. **Superantigens: polyclonal activation**, meaning it can bind to **ANY B/T cell** receptor, even if the receptor isn't made for it, can cause hyperinflammation ii. Mitogens: antigens that promote B/T cell proliferation, doesn't actually bind to Ag receptor and cause memory for that specific Ag, also polyclonal activation 1. **More potent stimulatory signal than** superantigens iii. Alloantigen: Ags derieved from different individuals from the same species iv. Autoantigens: self-ags, responses produce autoimmunity b. Haptens = **small molecules that are mostly non-immunogenic, unless bound to a large carrier protein**. Contact hypersensitivity. v. Drug induced autoimmune hemolytic anemia = antibodies are generated to bind to penicillin, activate complement and MAC lysis occurs as well as opsonization of red blood cells 2. Characteristics of Immunogens c. Chemical complexity**: complex molecules** are more readily distinguished as foreign rather than self vi. heteropolymers are more immunogenic than homopolymers d. Degradability: **antigens need to be degraded (or processed**) for presentation to T cells. **[If antigen is not easily degraded like medical devices, then these are generally not immunogenic]** e. Chemical properties**: charged molecules** are more immunogenic than neutral vii. Hydrophilic more immunogenic than hydrophobic f. Epitope density**: more epitopes** = more T and B cell responses g. Foreignness: **increased difference from self-antigens =** more immunogenicity viii. Most foreign: a cell that humans don't have h. Size of antigens: **larger antigens** tend to be more immunogenic ix. Proteins \> 10,000 daltons, carbs \> 100,000 daltons 2. Drugs tend to be around 100 daltons, hence why medical therapeutics are not immunogenic i. **BASICALLY: MOST IMMUNOGENIC AG = SUPER COMPLEX, EASILY DEGRADABLE, CHARGED/HYDROPHILLIC, LOTS OF EPITOPES, VERY FOREIGN, VERY LARGE** x. **Proteins = complex in charge, very large, many side chains** xi. **Lipids alone = least immunogenic** 3. **Not immunogenic unless associated with protein or polysaccharide** j. **Strongest immune response: introduced through subQ or intramuscular** 3. Epitopes = small parts of the larger Ag that serves as the component bound by TCR/BCR. Usually present in multiple copies k. B cell epitopes: remember, **B cells can recognized [directly]** so the BCR can bind to the soluble antigen xii. Typically, BCRs recognize conformational (discontinuous epitopes) 4. IgA, IgG, IgD = have three domains (CH1, CH2, CH3) a. Possess a hinge region 5. IgM, IgE = have four domains (CH1, CH2, CH3, CH4) 6. Fab = Ag binding region 7. Fc = mediates ADCC and opsonization, no capacity to bind Ag xiii. **Proteins, lipids, polysaccharides, or nucleic acid** l. T cell epitopes: Ags are processed and epitopes **revealed and displayed on MHC molecules** (anchor residues (amino acids) = interact with residues of MHC to hold peptide in place, or antigenic peptide residues of peptide interact with the TCR residues to drive cell signaling and activation) = recognition of epitope is the essence of T cell specificity xiv. **TCRs bind Ag displayed** and with sufficient residues, it will drive a cell signaling response xv. **Can only recognize peptides (processed proteins, NOT NATIVE PROTEINS)** coming from any source -- intracellular or extracellular 8. **Can recognize linear peptides** m. Immunodominant epitopes: epitopes that elicit a better immune response than others. Preferred for B and T cell activation. 4. Gamma-delta TCR n. Most T cells that are CD4 or CD8 have an alpha-beta chain o. There is a small subset of T cells (5%) that express different chains = gamma delta p. T cells with these gamma delta chains **[DO NOT need to interact with MHC]** xvi. **The gamma delta TCR can recognize Ag without the need for Ag processing and presentation by MHC** 5. Adjuvants q. Adjuvants help antigens become more immunogenic (greater stimulation of Ab production) r. Some inc ½ life of antigen so that there's a continuous release over time xvii. slow release of target antigen over time s. Some inc local inflammatory cytokines t. Others improve antigen processing by making them more easily phagocytosed u. Main target = dendritic cells Lecture 7 1. Haplotype a. **6 total class I genes (3 from mom, 3 from dad) and 6 total class II genes (3 from mom, 3 from dad)** i. **Total of 12 MHC or HLA genes = haplotypes** ii. ** A person expresses MHC class I and MHC class II proteins inherited from both parents.** b. MHC are the most polymorphic loci in the body iii. Since they are so polymorphic, thymocytes must be educated during positive selection to recognize self MHC molecules = MHC restriction c. Demonstrate promiscuity 2. MHC class I: endogenous Ag, alpha chain bind to beta-2 microglobulin (invariable), alpha chain has 3 domains, HLA-A/HLA-B/HLA-C, 8-10 AAs d. Peptide loading in ER e. Endogenous Ags iv. May be host cell peptides or intracellular pathogens inside cytoplasm or nucleus, made inside the cytosol f. **[Endogenous antigens are processed by the proteasome ]** v. When a host becomes infected with an intracellular pathogen, it produced type 2 IFN gamma. Viral pathogens also cause the cell to produce IFNa and IFNb. **All these IFNs cause upregulation of genes in MHC locus, hence more expression of MHC molecules** 1. This can be interrupted or blocked by some pathogens which can potentially be a mistake because NK cells will now be able to recognize their missing MHC I vi. Step 1 = endogenous proteins in the cytosol are chopped up by the proteasome into smaller pieces 2. **Cytokines IFN gamma (Type 2) are released which cause upregulation of super proteasome = immunoproteasome** a. Immunoproteasome preferentially cleaves hydrophobic or basic residues, **increasing likelihood that foreign peptides will have anchor residues that fit into MHC class I** vii. Step 2: Trafficking 3. **TAP1 and TAP2 move peptides that are roughly 8-10 AAs long into the ER to be loaded into the binding cleft of MHC I molecules** 4. **Calnexin (chaperone) stabilizes the MHC I molecule and prevents premature exit of the molecule from the ER** 5. **Tapasin** bridges between TAP and MHC I holding them closely together so that peptide entering the ER does not have to go far to be tested in the MHC b. Peptides are entering ER for loading viii. Step 3: once loaded, MHC peptide complex is transported to the cell surface Question that Davis had on the bottom of a slide - How is it that a nuclear pathogen is processed as a cytosolic pathogen and presented on MHC class I? - A nuclear pathogen can be processed as a cytosolic pathogen and presented on MHC Class I because, under certain conditions, nuclear proteins can leak into the cytoplasm where they are then degraded by the proteasome, which is the primary mechanism for generating peptides for MHC Class I presentation - What happens if there is a mutation that prevents MHC I or MHC II from being expressed? 3. MHC Class II: exogenous Ag, **alpha chain (x2) bind to beta chains (x2),** more diversity, HLA-DP/HLA-DQ/HLA-DR, **13-25 Aas (longer)** g. **Peptide loading occurs in vesicles (phagolysosomes)** h. Exogenous Ag ix. Brought in from outside the cell through phagocytosis or peptides that created within the phagosome 6. Intravesicular pathogens: listeria, TB, salmonella x. Phagocytosis: low pH activates the lysosomal enzymes (like **cathepsins) which break down pathogen proteins into peptides which can be loaded onto MHC II** 7. **Cathepsins =  breaks down proteins into peptides for the exogenous pathway** 4. MHC II is **made in the ER** i. It is combined with an **invariant chain** which occupies the binding cleft xi. The invariant chain has 3 functions 8. **Chaperone for proper protein folding of class II proteins** 9. **Prevents binding by endogenous peptides to MHC II in ER** 10. **Traffics the MHC II to the endosomal pathway** 5. MHC II peptide loading j. Step 1: class II invariant chain buds off the golgi complex forming a membrane bound vesicle k. Step 2: **the invariant chain is cleaved by proteases that leave a part of the chain in the binding cleft = CLIP** l. Step 3: vesicle fusion of the MHC II-CLIP complex with the phagolysosome m. Step 4: **HLA-DM removes CLIP and facilitates loading of peptide into MHC II binding cleft** n. Step 5: MHC II-peptide complex is brought to the cell surface to be expressed xii. **HLA-DP** is utilized to present exogenous (endocytosed/phagocytosed) antigen peptides to T cells. 6. Non-peptide Ags o. **MHC I and MHC II present protein peptide Ags** p. **CD1: a nonpolymorphic** receptor that possesses a **hydrophobic** binding cleft xiii. CD1 possesses the ability to present nonclassical Ags xiv. Has a structure resembling MHC I but Ag processing and trafficking similar to MHC II q. **CD1 molecules can be recognized by either iNKT cells for gamma delta T cells** xv. iNKT cells have an invariant alpha chain along w a b chain as part of their TCR 11. the B chain will undergo rearrangement but only a limited number of genes will be selected resulting in the TCR having a **limited repertoire which is chosen to recognize lipid Ags** 12. important for presenting mycobacterial antigens (very hydrophobic cell wall) xvi. gamma delta T cells are mostly found in gut, skin, and lung tissue (mucosal) 13. **Gamma delta T cells can recognize lipid antigens through interactions with CD-1** Lecture 8 1. Hypervariable regions a. **Parts of the V regions that bind to the epitope** are **hypervariable** regions aka **CDRs** b. Bind to epitope c. CDR-1, CDR-2, CDR-3 2. Somatic recombination d. Aka VDJ recombination e. The process of B or T lymphocyte rearranging its DNA in a random but controlled fashion in order to **increase diversity and expand Ag repertoire** i. **Somatic recombination is regulated by recombination signal sequences (RSS)** 3. Allelic exclusion f. Both alleles, one from mom and one from dad, try to rearrange at the same time. Rearrangement occurs on the 1^st^ allele if successful. The 2^nd^ allele is suppressed unless the first fails. **This prevents both maternal and paternal genes from being expressed in a single cell.** ii. **Both rearrange simultaneously but only one will be expressed in the cell** iii. **APPLIES TO BOTH T AND B CELL RECEPTORS** 1. EXCEPTION: TCR alpha chain (rare, but sometimes one T cell can have receptors with different alpha chains but same B chain) 4. Isotypic exclusion g. **ONLY APPLIES TO BCR LIGHT CHAIN** h. **Kappa always rearranges first** i. **Gamma light chain will only rearrange if both kappa genes fail** j. Kappa goes with kappa, lambda goes with lambda iv. The entire chain will be made up of V, J, and C of the same isotype 5. TCRs k. **T cells follow allelic exclusion EXCEPTION: TCR alpha chain (rare, but sometimes one T cell can have receptors with different alpha chains but same B chain)** l. **Beta, delta, and gamma rearrangement occur simultaneously** m. T cells enter the thymus in germline configuration Lecture 9 1. Regulation of gene rearrangement a. RSS flank each V, D, and J gene and come together in specific ways to ensure correct splicing and recombination i. RSS = conserved noncoding DNA sequences ii. **RSS consists of a conserved block of 7 nucleotides (heptamer)** which is always contiguous with the coding sequence followed by a nonconserved region known as the spacer which is either 12 or 23bp long, followed by a second conserved block of nine nucleotides (nonamer) 1. Nonamer -- spacer -- heptamer (gene) heptamer -- spacer -nonamer a. RSS = nonamer spacer heptamer b. **RSSs are recognized by recombination-activating ([RAG1/RAG2)] which facilitate recombination** iii. 1.DNA is looped to align palindromic heptamers iv. **2. DNA of each heptamer is nicked by the endonuclease RAG1/RAG2** v. 3\. Genes are placed adjacent to each other vi. 4\. Intervening DNA is removed and joint repaired 2. = **heptamers are always closest to the gene sequence** 2. Spacer region c. 2 types vii. 12bp = one turn RSS viii. 23bp = two turn RSS d. **A 12bp spacer must always be moved next to a 23bp spacer** ix. **12/23 rule** x. **1 turn/ 2 turn rule** e. This is what keeps the Vs joining with the Js or Ds and not another 3. BCR Diversity f. **Combinatorial diversity** xi. Random joining of VDJ genes xii. **[Occurs at DNA level]** g. **Combinatorial association** xiii. Any L chain can associate with any H cahin to form an antibody xiv. **[Occurs at protein level]** xv. **[UNIQUE TO B CELLS!!! ]** 3. T cells can't do this because only alpha can combine w beta and only delta can combine w gamma h. Junctional diversity xvi. This process involves the addition or subtraction of nucleotides within the DNA during recombination xvii. Happens w/ recombination 4. Recombination begins by bringing the RSS heptamers of the two chosen genes together to join them 5. Step 1: P nucleotide addition -- within hepatmer b. **RAG1/RAG2 (endonucleases)** cut both DNA strands within the heptamers **generating a hairpin loop** c. **Artemis (endonuclease)** then comes in and **opens** those loops leaving uneven strands d. The uneven strands **are filled in by DNA polymerase** (your P nucleotides) 6. Step 2: Junctional flexibility : ONLY AFFECTS CDR3 e. **Exonucleases** in the area can also **remove nucleotides (including P nucleotides that were just added)** f. \*\* more common in H chain \*\* 7. Step 3: N nucleotide addition = only in H chain g. **TdT** **adds 1-10 non-templated nucleotides** of one of the DNA strands h. **DNA polymerase** once again comes in **to fill the gap** on the other strand i. **Ligase seals and joins** the two genes together i. Somatic hypermutation xviii. **Antigen DEPENDENT = only mechanism of generating diversity AFTER antigen exposure = improve affinity** 8. Occurs in lymph follicles following exposure to Ag to improve affinity of the BCR for the target Ag = affinity maturation 9. Occurs during secondary immune responses 10. How it works: j. Activated B cells proliferate, creating many clones called **centroblasts** k. BCRs are endocytosed and **point mutations are introduced in the Variable regions of H and L chains** during clonal proliferation (can occur in HV or FR regions) l. These new BCRs are expressed on the surface and called **centrocytes** m. These BCRs are checked for changes in affinity by FDCs who display the captured Ag n. Only those with better affinity receive survival signals **⇒** GC B cells (and later plasma cells) TCRs: Ag independent only: combinatorial diversity, junctional diversity, lack of allelic exclusion for alpha chain Lecture 10 1. Anatomy of the thymus a. Outer cortex i. **Cortical thymic epithelial cells (cTEC): promote thymocyte development** 1. **Express notch-L and secrete IL-7** ii. First round of selection occurs here (positive and negative) iii. VDJ rearrangement occurs here b. Inner medulla iv. **Medullary thymic epithelial cells (mTEC): present self-peptides during negative selection process** v. The medulla contains Hassall's corpuscles vi. mTECs within Hassall's corpuscles express thymic stromal lymphopoietin (TSLP) vii. TSLP triggers thymic DCs to activate a small subset of thymocytes to become regulatory T cells viii. TSLP activates FOX3P in these thymocytes which allows them to become regulatory T cells 2. Thymic selection c. DN-1 (thymus ⇒ cortex) ix. **Lymphoid progenitor cells enter the cortex and express notch-1** x. **cTECs express notch-L** xi. **Notch + notch-L = commits LPC to T-cell lineage** 2. **The interaction of Notch1 with Notch-L suppresses B cell development** 3. **If either Notch1 or Notch-L are [not expressed, B cells can develop in the thymus]** 4. **Overexpression of Notch1 in the bone marrow can result in T cell development** xii. **The T progenitor express IL-7 R which binds IL-7 from cTEC leading to upregulation of RAG1/RAG2 which plays an important role in VDJ recombination** 5. **If either IL-7 or IL-7R are not expressed, T cell development is inhibited** d. DN-2 (cortex) xiii. T lymphocyte enters the cortex in germline configuration xiv. Beta, gamma, and delta loci rearrange at the same time, unless beta fails then the receptor will be alpha-beta 6. We have more alpha-beta TCR T cells because only one beta chain must rearrange successfully a. To have a gamma delta, both chains must be successful xv. First is D-J recombination e. DN-3 (cortex) xvi. A beta V gene is selected and paired to the D, J, and C that was selected xvii. The primary mRNA is now transcribed, spliced, translated 7. mRNA VβDβJβCβ xviii. TCR beta chain is now expressed xix. The pre-T-alpha chain, CD3, and zeta chains are all expressed after TCR beta chain is expressed 8. **PRE TCR COMPLEX: beta chain, pre T-alpha chain, CD3, and zeta chains** b. **CD3 is a coreceptor** 9. CHECKPOINT 1: passed if pre TCR complex is expressed indicating successful beta chain rearrangement, now this cell can proliferate f. DN-4 (cortex) xx. RAG1/RAG2 expression xxi. Alpha chain undergoes rearrangement xxii. Alpha-beta TCR now expressed 10. FULL TCR COMPLEX: alpha-beta TCR chain, CD3, zeta chains c. CD4 and CD8 are NOT apart of this complex 11. CHECKPOINT 2: passed when alpha chain expressed g. DP (cortex) xxiii. CD4 and CD8 proteins are expressed on surface of the TCR complex xxiv. cTECs express MHC I and MHC II with self peptides 12. **positive selection: thymocytes recognize MHC and binds with low to moderate affinity ⇒ go to medulla** 13. negative selection: thymocytes bind with high affinity ⇒ apoptosis 14. death by neglect: no recognition ⇒ apoptosis h. DP (cortex) xxv. **Positive selection** 15. **if CD4 interacts with MHC II, then CD8 is LOST** 16. **if CD8 interacts with MHC I, then CD4 is LOST** d. cd4 and cd8 are coreceptors that recognize MHC, NOT Ag bound to MHC (that is the job of the TCR) 17. **The thymocytes are now single positive** 18. **This step ensures MHC restriction and T cell effector function is determined** i. SP Negative selection (medulla) xxvi. **mTECs** and **dendrites express MHC I and MHC** **II** for interaction with CD8 and CD4 positive thymocytes, respectively 19. **positive selection: thymocytes binds to MHC bound self-Ag on mTECs weakly ⇒ it will exit the thymus as a mature alpha beta TCR T cell** 20. ** T cells bearing antigen receptors that recognize self-antigens undergo apoptosis, a process known as negative selection.** 21. negative selection: thymocytes binds to MHC bound self-Ag on mTECs strongly ⇒ apoptosis j. gamma delta T cells xxvii. they do not undergo positive or negative xxviii. **they exit the thymus prior to these processes as double negative T cells** Lecture 11 1. 3 signals essential for T cell a. Pathogen invades tissue, innate system fails to eliminate pathogen b. Signal 1: APCs grab peptide and load it onto MHC, present to naïve T cells in peripheral lymphoid organs i. Ag-peptide is the source for signal one and it binds to a TCR c. **Signal 2: expression of [B7 (CD80/86) on maturing dendrite] which will bind to CD28 on the T cell = COSTIMULATION** ii. Source: microbial components can bind PRRs OR cytokines from neutrophils/macrophages d. Signal 3: results in cytokine production that "weaponizes" the T cell by telling it what to differentiate into (helper or cytotoxic) iii. Source: microbial components can bind PRRs OR cytokines from neutrophils/macrophages e. Activated T cells leave the lymph node, go to tissue, must lose CCR7, and upregulate integrin and selectin ligand to enter the tissue 2. T cell signaling cascade f. **Binding of TCR and CD28 elicits cell signaling** g. **CD3 and zeta chains are the signaling complex** h. CD4/CD8 interact with MHC, which causes **Lck to phosphorylate ITAMs of zeta chain** i. **ZAP-70 recruited, phosphorylated, and activated by Lck** j. **ZAP-70 phosphorylates LAT which activates 3 pathways: MAP-kinase, NFKB, and NFAT** +-----------------------+-----------------------+-----------------------+ | LAT activates PKC | LAT activates IP3 | LAT activates RAS-GTP | | | | & RAC-GTP | | ⇒ | ⇒ | | | | | ⇒ | | PKC activates IKB | **IP3 binds to Ca** | | | kinase | channels in the ER | **RASGTP activates | | | | ERK, RACGTP activates | | ⇒ | ⇒ | JNK** | | | | | | **IKB kinase** | Ca released into the | **⇒** | | phosphorylates IKB | cytosol | | | bound to NFKB | | **ERK leads to | | | ⇒ | expression of c-Fos, | | ⇒ | | JNK leads to | | | **Ca ions bind | phosphorylation of | | **NFKB released** | calmodulin** | c-Jun** | | | | | | ⇒ | ⇒ | ⇒ | | | | | | Moves to nucleus and | Calmodulin activates | **AP1** | | induces transcription | **calcineurin** | (transcription factor | | of IL-2 mRNA | | for IL-2mRNA and | | | ⇒ | Fas-L which is an | | **\*Glucocorticoids | | apoptosis surface | | blocks NFKB | Calcineurin removes | signaler) | | activation\*** | inhibitory phosphate | | | | from NFAT | | | | | | | | ⇒ | | | | | | | | **NFAT** moves to | | | | nucleus to promote | | | | transcription of IL-2 | | | | mRNA | | | | | | | | **\*Cyclosporine | | | | inhibits calcineurin | | | | activity\*** | | +-----------------------+-----------------------+-----------------------+ 3. Action of IL-2 k. **IL2 leads to massive clonal expansion** iv. Need to safeguard uncontrolled proliferation 1. **CTLA-4 and PD-1 (programmed death 1) can outcompete CD28 for binding with B7 (CD80/86) ⇒ causing diminished TCR signaling** v. **In CD4 cells, clonal expansion is signaled by a self-produced IL-2 binding to IL-2 receptor** l. Survival vi. **Induces anti-apoptotic protein Bcl** and **upregulates IL2 receptor alpha chain (CD25)** 2. Bcl = prevents Bax and Bak from poking holes and cytochrome C leakage m. **Increases CD40 ligand** and cytokine receptors for **differentiation** vii. When activated, T cells can go to the tissue and bind CD40 on immature DCs to promote maturation 4. Memory T cells maintain IL-7R expression n. **IL-7R is a survival receptor that keeps naïve and memory T cells alive** viii. Want naïve and memory T cells to survive as long as possible o. Effector T cells need to die after they serve their purpose p. Memory and effector T cells are activated in the paracortex and then effector T cells leave the lymph node into the subclavian vessel +-----------------------+-----------------------+-----------------------+ | Naïve T cell (IL-7R+) | Naïve T cell (IL-7R+) | | | | | | | ⇒ | ⇒ | | | | | | | **Effector T cell = | **Memory T cell = | | | loses IL-7R | retains IL-7R | | | (IL-7R-)** | (IL-7R+)** | | | | | | | ⇒ | ⇒ | | | | | | | **Loses CCR7 | | | | (CCR7-)** | | | | | | | | ⇒ | | | | | | | | Effector T cell | | | | leaves the lymph | | | | nodes to infiltrate | | | | tissues (immediate | | | | response) | | | | | | | | ⇒ | | | | | | | | Once done, apoptosis | | | +-----------------------+-----------------------+-----------------------+ | | **Central memory T | **Effector memory T | | | cell (Tcm)** | cell (Tem)** | | | | | | | **⇒** | **⇒** | | | | | | | **CCR7+** | **CCR7-** | | | | | | | ⇒ | ⇒ | | | | | | | Remain in lymph nodes | Leaves the lymph | | | (reservoir) | nodes to infiltrate | | | | tissues (local recall | | | | response) | | | | | | | | most likely to be | | | | activated quickly | | | | within tissues upon a | | | | second encounter with | | | | a pathogen | +-----------------------+-----------------------+-----------------------+ | Tcms can be activated | | | | to generate more Tems | | | | if response is not | | | | sufficient in tissues | | | | (Tcms will lose CCR7) | | | +-----------------------+-----------------------+-----------------------+ q. **CCR7 = is necessary for entry into the lymph node, must be removed for the effector T cell to leave the lymph node and go to the tissue** r. **IL-7R = T cell must be positive for this or else it will undergo apoptosis** Lecture 12 1. Signal 3 cytokines related to specific T cell differentiation +-----------------------+-----------------------+-----------------------+ | Dendritic Cell (or | | | | APC) | | | +-----------------------+-----------------------+-----------------------+ | **IL-12** | **IL-4** | **IL-6, TGF-b** | | | | | | (STAT4) | (STAT6) | (STAT3) | | | | | | ⇒ | ⇒ | ⇒ | | | | | | T-bet | GATA3 | RORyT | | | | | | ⇒ | ⇒ | ⇒ | | | | | | **TH1 cells** | **TH2 cells** | **TH17 cells** | | | | | | **⇒** | **⇒** | **⇒** | | | | | | **IL-2, IFNg, TNFa** | **IL-4, IL-5, IL-13** | **IL-17, IL-22** | +-----------------------+-----------------------+-----------------------+ a. If signal 3 is IL-12: i. This will cause T cell to differentiate into TH1 cell 1. Mainly targets intracellular pathogens, but it is not restricted to just intracellular. Dr. Gregg mentioned in lecture that it can target extracellular because **IFNy can drive IgG production** ii. TH1 releases... 2. **IL-2** = proliferation of T cells (clonal expansion), activation of NK cells (inc proliferation + mobilization of cytotoxic granules) 3. **IFNy = activates [M1] macrophages (inflammatory) increasing phagocytic oxidative killing mechanisms** a. **Polarization of Macrophages (M1) = (IL-1, IL-6, IL-12, TNFa)** i. Inc NADPH oxidase (ROS) and iNOS (RNS) 1. Forces lysosome to fuse with phagosome = more phagolysosomes ii. If process of enhancing phagocytosis **fails** then: 2. Upregulate MHC-1+peptide and induce B7 on macrophage a. Activates CD8+ T cell to kill failed macrophage i. M1 with phagocytosed microbe better target for cytotoxicity b. **Positive feedback loop**: when macrophages encounter a pathogen, they release IL-12. IL-12 acts on NK cells stimulating them to produce IFNy. The IFNy acts on the macrophage to produce even more IL-12, promoting the differentiation of CD4 T cells into TH1 cells. c. Th1 cells activate macrophages via **IFNy and CD40** **ligand** signaling iii. Th1 cells interact w macrophages in the tissue iv. T cells bind through TCR-peptide-MHC v. Triggers release of IFNy vi. **CD40L binds to CD40 on macrophage** vii. Together they promote microbe digestion (NADPH oxidase and iNOS activity) d. Basically, SECOND engagement with Ag is required to get effector cells to produce cytokines viii. **Cytokines only released upon [re-engagement] of TCR** 4. **TNF-a** = activation of endothelia, takes over the inflammatory process e. inc vascular permeability, inc endothelial cell adhesion (upregulates selectins and ICAMs) ix. Acute phase response f. Induce apoptosis g. Dr. Gregg states in lecture: Inflammation starts with macrophages, then mast cells, then Th1 cells take over b. If signal 3 is IL-4: iii. This will cause T cell to differentiate into TH2 cell 5. Mainly targets parasitic infections (parasitic worms; helminths) iv. TH2 releases... 6. **IL-4 = causes B cells to produce IgE which binds to FceR1 on mast cells ⇒ Ag binds to IgE ⇒ degranulation ⇒ release histamine, heparin ⇒ allergic response** h. Leads to inc inflammation and **responses that** **expel** **the Ag source** x. Ag can be microbial, self, environmental, or an allergen i. IgE is the primary product of plasma cells when they bind to IL-4 7. **IL-5 = stimulates and recruits eosinophils (parasitic infections)** j. IgE covers the parasite and eosinophil will use its Fc receptor to find IgE ⇒ degranulation ⇒ inflammation 8. **IL-13 = works with IL-4 to activate M2 macrophages** which promote wound healing and suppression of inflammation k. **M2 macrophages (IL-10, VEGF, TGFb)** = apoptotic cell clearance, high phagocytic capacity, and wound healing l. IL-4 and IL-13 can **inc mucous production** and peristalsis to remove Ag xi. Eosinophils also produce IL-4 and IL-13 9. Can also produce IL-10 m. IL-10 ⇒ B cells ⇒ plasma cells ⇒ inc IgG and IgM Ab production and supports germinal center survival n. IL-10 ⇒ dec macrophage activation and proliferation ⇒ dec NK cell IFNy ⇒ inhibits Th1 indirectly o. IL-10 ⇒ dec IL-12 ⇒ dec Ag presentation (dec CD80/86, dec IL-12) ⇒ suppresses MHC II ⇒ dec activated CD4 T cell proliferation (dec IFNy) ⇒ inhibits Th1 10. [IL-10 suppresses IL-12, IL-12 suppresses IL-10] 11. [IFNy suppresses IL-4, IL-4 suppresses IFNy] c. If signal 3 is IL-6 and TGFb: v. This will cause T cell to differentiate into TH17 cell 12. Drives responses to extracellular pathogens (BACTERIA) vi. TH17 releases 13. **IL-17** = drives inflammatory response**, recruits neutrophils** (its effects are through other cytokines, not directly) p. Promotes inflammation xii. Promotes IL-6 & IL-8 (inflammation) in epithelial and endothelial cells xiii. G-CSF = inc neutrophil numbers and monocyte output from bone marrow (epithelial cells) xiv. GM-CSF = aids in macrophage and DC differentiation of monocytes (epithelial cells) xv. Tissue factor = thrombosis (endothelial cells) xvi. Fibroblasts: MMP = modify ECM q. Promotes cellular activation xvii. **(G-CSF, IL-8) recruits and activates neutrophils** 3. **MAJOR RECRUITER AND ACTIVATOR OF NEUTROPHILS** b. Related to inflammatory bowel disease xviii. (GM-CSF) recruits monocytes and aids in differentiation 14. IL-22 = drives inflammatory response, helps create antimicrobial response in mucosa, and wound healing r. Paneth cells and keratinocytes = antimicrobial peptides xix. A deficiency in the production of defensins by Paneth cells would best correlate with an increase in microbiota.   s. Hepatocytes = acute phase response proteins (C3) t. Enterocytes = weak junctions permit water efflux for flushing of Ag, hemopexin (sequesters iron), IL-18 (inflammation) u. Epithelial cells, fibroblasts, keratinocytes = proliferation and ECM production (wound healing) 2. Activated CD8 T cells d. Kill via 2 methods vii. Release of perforin (perforates membrane) and granzymes (stimulates apoptosis) 15. Granzymes activate caspases, caspases activated induce DNA fragmentation (nucleus and mitochondria) and apoptotic body formation (cell blebbing) viii. **Fas-L on CD8 binds Fas on infected cell ⇒ DISC ⇒ DISC binds caspase-8 ⇒ caspase-8 activated (upon cleavage) ⇒ caspase-8 initiates effector caspases ⇒ effector caspases** **induce DNA fragmentation apoptosis induced** 16. This can be blocked by cytokines (IL-7) or NFKB inducing c-FLIP which outcompetes caspase 8 binding to FADD ⇒ blocks apoptosis v. c-FLIP also blocks TNFa apoptosis 17. This process needs TCR engagement 3. How to get rid of T cell army e. 2 mechanisms for this ix. **AICD (activation induced cell death)** 18. Upon activation, T cells express Fas (for eventual apoptosis) and Fas-L (for inducing apoptosis on infected cells) w. IL-2 and TCR expression can drive expression of Fas and FasL expression on activated T cells xx. FasL expression is first for killing xxi. As they keep getting activated, Fas catches up 19. **When IL-2 levels decrease, cFLIP levels drop, now CTLs can have Fas -- Fas-L interaction** between each other that will not be blocked by cFLIP x. = CTLs will undergo apoptosis = T cell contraction x. **MOMP (mitochondrial outer membrane permeability)** 20. During T cell proliferation and active response, IL-2 is stimulating Bcl-2 production to block Bax and Bak from creating pores in the mitochondrial membrane and inducing apoptosis 21. **After the pathogen has been eliminated, IL-2 levels drop and the protective Bcl-2 is no longer stimulated** y. Bak and Bax are now able to **create pores in outer membrane of mitochondria releasing cytochrome C which activates caspases and induces apoptosis** xi. CTLA-4 stops T cell proliferation 22. CTLA-4 outcompetes CD28 for B7 (CD80/86) binding ⇒ T cell diminishes TCR signaling (action of phosphatases) ⇒ cell cycle arrest z. Plateau stage on graph Dr. Davis Qs from lectures Lecture 1 - What happens If we fail to recognize non-self Ags? - What happens if we react to self Ags?