Immunology Block 2 Learning Objectives PDF

**[Immunology Block 2 Learning Objectives]** Overview of Adaptive System ***LO 1. State where you would expect to find receptors of the adaptive immune system within the context of the cell and correlate the ligands and receptors of the two types of immune cells with their signaling outcomes*** 1...

**[Immunology Block 2 Learning Objectives]** Overview of Adaptive System ***LO 1. State where you would expect to find receptors of the adaptive immune system within the context of the cell and correlate the ligands and receptors of the two types of immune cells with their signaling outcomes*** 1. **Interferons**: family of cytokines that act specifically to induce cells to resist viral infections a. Rapidly produced when viral PAMPs interact with PRRs b. **IFN-alpha and IFN-beta:** type I interferons that are produced by a variety of cells i. Induce cells to activate antiviral defenses like RNA-dependent protein kinase pathway and apoptotic (programmed cell death) pathway c. **IFN-gamma:** type II interferon that is structurally and functionally related to type I but is produced by activated lymphocytes like CD4 T cells, CD8 T cells, NK cells, and ILCs ii. Acts principally to activate macrophages in innate and adaptive immunity ***LO 2. Compare and contrast NK and NKT cells*** 1. **NK Cells:** kill certain virally infected cells and tumor cells without prior sensitization like some virus-infected cells, cells covered with antibody, and cells that do not express normal levels of MHC I a. Function in innate and adaptive system b. Descended from common lymphoid progenitors c. Don't have variable antigen specific receptors d. Retains memory of its encounters with pathogens e. **Killer Activation Receptors (KAR):** recognize presence of stress-related molecules MICA and MICB expressed by host cells that are unhealthy or infected i. Binding MICA or MICB by NK cell's KAR induces NK cell to attach and destroy target ii. Always the first to bind f. **Killer Inhibition Receptors (KIR**): monitor MHC I molecules displayed on cell surfaces of all nucleated cells in the body iii. Determine normality of host cells iv. Once bound to a target via KAR, KIRs assess expression of MHC I on cell 1. If subnormal MHC I, then target cell is killed (ex: cancer and viruses) 2. If normal MHC I, then target cell is released g. **High Endothelial Venule:** where lymphocytes like NK cells leave the blood and enter the lymphatic system v. Blood vessels are so close to lymph vessels that it's easy for them to cross h. Steps of Function vi. Virus infects cells and triggers interferon response vii. Type I interferon drives proliferation of NK cells viii. Type I interferon drives differentiation of NK cells into cytotoxic effector cells ix. Effector NK cells kill virus infected cells by inducing apoptosis 2. **NKT Cells:** subset of T cells that develop within the thymus and express a rearranged T cell receptor of limited repertoire i. Limited repertoire includes lipids, glycolipids, or hydrophobic peptides j. **CDId:** specialized nonclassical MHC I molecule that presents NKT cells' limited repertoire and secrete large amounts of cytokines, especially IL 4 k. Contain granules that are antiviral and can be secreted without killing the cell x. Granules cause lysis l. No KAR and KIR ***LO 3. Describe how NK cells can work with macrophages and dendritic cells to bring about an immune response*** 1. NK cells and macrophages active each other at sites of infection and results in the induction of NK cell proliferation and differentiation a. Macrophages activated by viral infection secrete cytokines (IL-12) that recruit NK cells b. NK cell and macrophage conjugate at immunological synapse i. Synapse delivers IL-15 that, along with IL-12, activates NK cell c. NK cells proliferate and differentiate into effector NK cells secreting IFN-gamma d. IFN-gamma binds its receptor on the macrophage, increasing phagocytosis and cytokine secretion 2. One interaction is the activation, proliferation, and differentiation of NK cells by dendritic cells e. In virus infected tissue, an immature dendritic cell expressing viral antigens can activate NK cells and drive their differentiation into effector cells to kill 3. The second interaction is the outnumbering of dendritic cells by NK cells, resulting in the death of dendritic cells f. When activated cytotoxic NK cells are abundant and innate immunity is overcoming infection, NK cells kill dendritic cells and prevent their activation of adaptive immunity 4. The third interaction is the outnumbering of NK cells by dendritic cells, resulting in the maturation of dendritic cells and initiation of an adaptive immune response g. When NK cells are scarce and innate immunity cannot control infection, NK cells induce dendritic cells to differentiate into a form that migrates to secondary lymphoid tissue and initiates the adaptive immune response ***LO 4. Outline the movement of lymphocytes throughout the body and key anatomical features*** 1. Movement of lymphocytes: a. Circulating lymphocytes leave in efferent lymph b. Return to blood in left subclavian vein c. Enter lymph node through walls of fine capillaries in secondary lymphoid organs d. Recirculating lymphocytes keep traveling until they encounter pathogens i. Lymphocytes activated by specific antigen stay in lymph node to divide and differentiate into effector cells 1. Results in germinal centers with B cells ii. Lymphocytes not activated by specific antigen leave lymph node in efferent lymph and are carried by lymphatics to thoracic duct and into the blood 2. Naïve Lymphocytes: have not encountered target 3. Mature Lymphocytes: have encountered pathogen; also called active/effector 4. Lymphocytes must go with the flow of blood but can follow a more direct path in lymph e. They can also jump from blood to lymph 5. Movement in draining lymph node: f. Pathogen and antigens (and dendritic cells carrying pathogens and antigens) arrive at a lymph node in afferent lymph draining the infected tissue g. Pathogens and debris are removed by macrophages h. Dendritic cells become residents and move to T cell areas (naïve cells), where they encounter small lymphocytes that have entered lymph nodes from blood i. Dendritic cells orchestrate division and differentiation of small pathogen specific lymphocytes into effector cells (mature cells) iii. Some helper and cytotoxic T cells leave in efferent lymph and travel to infected tissue iv. Other helper T cells stay in lymph node to stimulate division of pathogen specific B cells and their differentiation into plasma cells j. Plasma cells move into medulla of lymph node, where they secrete pathogen specific antibodies v. Other plasma cells leave lymph node and travel to bone marrow where they secrete pathogen specific antibody in quantity k. Antibodies travel to infected tissue by efferent lymph and blood ***LO 5. Describe how adaptive immune system differs from innate and unique features of the adaptive immune system*** 1. Adaptive immune system is initiated in secondary lymphoid tissues 2. Adaptive immune system has two types of cells a. **T lymphocytes** i. **CD4+ Helper T Cells:** assist in B cell activation ii. **CD8+ Cytotoxic T Cells:** attack and destroy virally infected cells b. **B Lymphocytes** iii. Antigen affinity is increased 1. **Antigen Affinity:** as B cells divide, they make receptors more specialized iv. Class switching (between antibodies) can take place 3. Both B and T cells develop into long lived memory cells ***LO 6. Recognize and describe the microenvironments where adaptive immune cells mature, and the adaptive immune system develops*** 1. **Endosteal Niche**: microenvironment of cells that participate in the regulation of hematopoiesis 2. **Vascular Niche:** microenvironment niches in bone marrow required for B cell developed 3. **Follicles:** area in lymph nodes where activation into effector cells occurs a. Located adjacent to T cell areas b. Comprise germinal center, B-cell corona, and marginal zone 4. **Germinal Centers:** where B cells mature by dividing and becoming activated 5. T cell area of maturation is near B cell area because T cells present the antigen to B cells ***LO 7. Identify the primary and secondary immune organs in vertebrates and describe their function in relation to the adaptive immune system*** 1. **Primary Lymphoid Organs:** where immune cells develop a. Bone marrow and thymus 2. **Secondary Lymphoid Organs:** where immune response is initiated, and cells become activated b. **Lymph nodes:** filter and process antigens present in lymphatic fluid as it travels from afferent to efferent vessels i. Site where blood-borne lymphocytes respond to lymph-borne pathogens ii. Swell in infection because active B and T cells are dividing and proliferating c. **Spleen:** first line of defense against blood-borne pathogens iii. **Red Pulp:** where old and damaged red blood cells are removed from circulation 1. Most of spleen iv. **White Pulp:** where white blood cells reside v. **Marginal Zone:** specialized region of macrophages and B cells that borders white pulp and separates from red pulp vi. In left upper part of abdomen (largest node) vii. **Periarteriolar Lymphoid Sheath (PALS):** has T lymphocytes near while B cells are situated peripherally viii. **Perifollicular Zone:** erythrocytes, macrophages, T cells, and B cells d. Barrier tissues 3. **Tertiary Lymphoid Organs:** organize and maintain immune response e. Role in innate immune system Cells of the Adaptive Immune System ***LO 1. Compare and contrast how adaptive immune system differs from innate immune system*** 1. **Hematopoietic Stem Cells:** cells in bone marrow that become all cells in blood a. Can do self-renewal b. Differentiation is influenced by array of cytokines and growth factors c. Become common myeloid progenitors from increased PU.1 TF d. Become common lymphoid progenitors from decreased PU.1 TF 2. Common Myeloid Progenitors are innate immune system cells 3. Common Lymphoid Progenitors are adaptive immune system cells ***LO 2. List unique features of the adaptive immune system (specifically immune cells)*** 1. **Common Lymphoid Progenitors:** becomes B cells and common T cell/ILC precursors after stimulation by IL-7 a. Common T cell Precursors becomes CD4 and CD8 i. Common CD4 T cell Precursor becomes Treg, TH17, TH2, and TH1 b. Common ILC Precursor becomes NK, ILC1, ILC2, ILC3, and LTi 2. **Lymphoid Cells:** lymphocytes produced in bone marrow that travel through lymphatic system and further differentiate/proliferate within lymphoid tissue (lymph nodes, thymus, spleen) c. Histologically, B cells and T cells look the same with big purple nucleus inside less seen cytoplasm while plasma cells are two times this size and have nonstaining golgi d. Natural Killer Cells have a clear cytoplasm with an oddly shaped purple nucleus ***LO 3. Describe the function and role of each common lymphoid progenitor descended cell (and various cell types) and be able to identify them based on generalized picture, description of function, or histology*** 1. **Natural Killer Cells:** develop within bone marrow and function in the killing of certain virally infected cells without prior sensitization a. Have a role in both innate and adaptive immunity b. Like ILCs and LTis c. Lack CD3, TCR, and B cell receptor d. Granular appearance from cytoplasmic granules containing perforin and granzyme that can damage membranes of cells they attack e. No antigen-specific receptors and do not undergo clonal selection f. Instead, have receptors that recognize certain microbial molecules, antibody, and class I MHC g. **Killer Activation Receptors (KAR):** recognize presence of stress-related molecules MICA and MICB expressed by host cells that are unhealthy or infected i. Binding MICA or MICB by NK cell's KAR induces NK cell to attach and destroy target ii. Always the first to bind h. **Killer Inhibition Receptors (KIR**): monitor MHC I molecules displayed on cell surfaces of all nucleated cells in the body iii. Determine normality of host cells iv. Once bound to a target via KAR, KIRs assess expression of MHC I on cell 1. If subnormal MHC I, then target cell is killed (ex: cancer and viruses) 2. If normal MHC I, then target cell is released i. Important in early stages of viral infection, before CD8 are selected and expanded 2. **Innate Lymphoid Cells (ILCs):** lymphoid cells found in all tissues, especially mucosal surfaces, that are activated by cytokine signals and participate in innate immune function j. Activated early in immune response to infection and injury, producing cytokines that direct immune response k. Assist with tissue homeostasis and tissue repair l. Do not need antigen to become activated 3. **Lymphoid Tissue Inducer (LTi):** type of ILC that are essential for development of lymph node and Peyer's patches 4. **Lymphocytes:** defined by where they undergo basic training and the type of receptors they display on their cell surfaces m. Includes T cells, B cells, and NK cells n. **TCR:** T cell receptors on T cells and NKT cells o. **BCR/Immunoglobulins:** B cell receptors on B cells p. Two lymphoid cells that remain and develop in bone marrow and are precursors of immunoglobulin producing lymphocytes v. B cells vi. Plasma cells 5. **B Cells:** synthesize immunoglobulin and display it on their surfaces where it functions as their BCR q. There are two distinct lineages for protection r. **B-1 Cells:** first to develop embryologically (have basic education) vii. Self-renewing population that dominates the plural and peritoneal cavities s. **B-2 Cells:** arise during and after neonatal period (have more education) viii. Continuously replaced from bone marrow and widely distributed throughout lymphoid organs and tissues t. Each B cells is specific because it produces immunoglobulin of only one antibody specificity that recognizes only one epitope ix. Generates diversity 6. **Plasma Cells:** derived from differentiated, mature B cells and both synthesize and secrete immunoglobulin u. Increased size and metabolic activity than B cells and are factories that produce large quantities of immunoglobulin during their short life span x. Doesn't make sense to continue producing plasma cells if they're not fighting the infection anymore v. Cease to use immunoglobulin as a membrane receptor and instead secrete it into fluids around cells xi. **Immunoglobulin:** name when bound to cell xii. **Antibody:** name when secreted and released from cell into body w. Characterized by basophilic cytoplasm, nucleus that has starlike pattern within it, and nonstaining Golgi xiii. **Golgi:** packs new immunoglobulins to put them on the surface of plasma cells and cut them off so they're secreted as antibodies 7. **T cells:** participate in immune response by orchestrating and regulating activities of other cells x. Wide diversity in function y. All identified by **cluster of differentiation 3 (CD3)** associated with T cell receptor on T cell surface z. Two main subtypes xiv. Alpha/beta T cells xv. Gamma/delta T cells 8. **Natural Killer T Cell:** develop within thymus and express a rearranged TCR of limited repertoire a. Limited repertoire includes lipids, glycolipids, or hydrophobic peptides b. **CDId:** specialized nonclassical MHC I molecule that presents NKT cells' limited repertoire and secrete large amountsI of cytokines, especially IL 4 c. Contain granules that are antiviral and can be secreted without killing the cell xvi. granules cause lysis d. No KAR and KIR **LO 4. List known inducing cytokine and key effector cytokines** 1. **Type I immunity** a. **IL-12:** inducing cytokine b. **ILC 1:** ILC subset c. **IFN-gamma:** effector cytokine d. Targeted pathogens: **viruses and intracellular bacteria** 2. **Type 2 Immunity** e. **IL-25, IL-33, or TSLP:** inducing cytokine f. **ILC2:** ILC subset g. **IL-4, IL-5, IL-13:** effector cytokines h. Targeted pathogens: **worms** 3. **Type 3 Immunity** i. **IL-1beta, IL-23:** inducing cytokine j. **ILC3:** ILC subset k. **IL-17 and IL-22:** effector cytokines l. Target pathogens: **extracellular bacteria and fungi** ***LO 5. State where you would expect to find receptors of the adaptive immune system within the context of the cell and correlated the ligands and receptors of the two main lymphocytes with their signaling outcomes*** 1. **Immunoglobulin:** diverse group of globular molecules found in blood and tissue fluids a. **IgA:** protects against pathogens i. Found in mucous, saliva, tears, and breast milk ii. One of the shared antibodies that protects baby from mom b. **IgD:** part of the B cell receptor that activates basophils and mast cells iii. Not well known iv. Some help with development c. **IgE:** protect against parasitic worms and responsible for allergic reactions v. Ex: anaphylaxis and hay fever d. **IgG:** secreted by plasma cells in the blood vi. Able to cross placenta into fetus vii. The first e. **IgM:** may be attached to surface of B cell or secreted itno blood viii. Responsible for early stages of immunity 2. **Antibody:** immunoglobulin that specifically binds to a known ligand/epitope 3. **Epitope:** smallest molecular structure recognized by a specific receptor 4. **Antigen:** collection of repeating and/or unique epitopes ***LO 6. Compare and contrast the subtypes of T cells (alpha/beta T cells, CD4+ (and 5 subtypes), CD8+, (Tc and Ts), and gamma/delta cells)*** 1. **Alpha/Beta T Cells:** majority of T cells that express antigen receptor (TCR) composed of alpha chain and beta chain a. Dependent on antigen presentation by MHC I and II b. CD4+ helper T cells c. CD8+ cytotoxic T cells 2. **Gamma/Delta T Cells:** minority of circulating T cells that expresses an antigen receptor (TCR) composed of gamma chain and delta chain d. Recognize different types of antigens than alpha/beta cells e. Not dependent on antigen presentation by MHC I and MHC II f. Most do not have CD4+ or CD8+ (small exceptions) 3. **CD4+ T cell:** CD4+ molecules on surfaces of T cells that recognize non-peptide bonding portion of MHC II g. 2/3 of CD3+ T cells h. Restricted to recognition of peptide MHC II **(pMHC II)** complexes i. CD4+ binds MHC II only when it has a peptide bound to it because it needs to have something to show i. Most humoral or cell-mediated immune response require presence of functional CD4+ helper T cells ii. Interference with helper T cell function results in significant immune deficiency j. Cannot recognize antigen in its native state iii. Antigen must first be taken into APC, degraded by that cell and fragmented into peptides, then bound to a suitable antigen presenting molecule (MHC II) k. Have five subsets l. **T Helper Type I (TH1):** activate macrophages and fight intracellular pathogens m. **T Helper Type 2 (TH2):** fight extracellular infections, activate B cells, and involved in allergy n. **T Helper Type 17 (TH17):** protects against infection and maintains mucosal barrier, recruits neutrophils, and possibly autoimmune o. **T Follicular Helper (TFH):** provide help to B cells for forming germinal centers and maturation p. **Regulatory T Cell (TREG):** required to maintain peripheral self-tolerance 4. **CD8+ T Cell:** q. 1/3 of CD3+ T cells r. CD8 molecules displayed on surfaces of T cells recognize nonpeptide-binding portion of MHC I iv. Restricted to recognition of peptide MHC I **(pMHC I)** complexes s. Before acquiring capacity to kill, cytotoxic T cells must usually receive help from **IL-2** from CD4+ t. During immune response, clonal selection operates to select and expand those cytotoxic T cells that can recognize immunogen u. Have two subtypes v. **Tc Cells:** identify body cells infected with intracellular organisms and eliminate cells harboring these organisms vi. **Ts Cells:** downregulate and thus control adaptive immune responses \\ Antigen Presentation I ***LO 1. Identify and explain antigen processing and presentation*** 1. Immune system has system of checks and balances that prevent T-cells from deciding on their own what antigens to destroy to limit the risk of autoimmunity a. Thus, T cell activation is tightly regulated b. It's difficult to slow down cell mediated immune response once it's started 2. It's necessary for other cells to process and present foreign antigens to T cells with additional signals required for recognition and full activation of cell-mediated response c. T cell receptors recognize peptide antigens bound to MHC (pMHC) 3. Antigen Processing and Presentation Pathway: d. Pathogen and its proteins are synthesized by infected human cell or taken up from extracellular environment e. All human cells have housekeeping mechanisms that serve to remove damaged or unwanted proteins f. These mechanisms degrade proteins into small peptides and are used to produce the pathogen derived peptides that are bound by MHC, displayed on cell surface, and presented to T cells ***LO 2. Differentiate professional APCs from atypical/amateur APCs, especially how they present and what types of antigens. What cell types are they found on*** 1. **Professional APCs:** constitutively express high levels of MHC II and antigen processing machinery and express co-stimulatory molecules following activation a. **Dendritic Cells and Macrophages** Characteristics: i. Phagocytic ii. Express receptors for apoptotic cells, DAMPs, and PAMPs iii. Localize to tissues iv. Dendritic cells localize to T cell zone of lymph nodes following activation b. **B cells** Characteristics: v. Internalize antigen via BCRs c. **Professional Antigen Presentation:** high avidity interaction where there's high affinity of T cell receptor, high density of pMHC, co-stimulation, and low net surface charge vi. Results in the production of cytokines and their receptors and proliferation 2. **Atypical APCs:** inducible expression of MHC II and antigen-presentation functions limited to specific immune environments (specifically type 2 immunity) d. Lack of compelling evidence that they can activate naïve CD4+ T cells in an antigen-specific manner e. Includes **mast cells, basophils, eosinophils, and ILC3s** f. **Amateur Antigen Presentation:** low avidity interaction where there's low density pMHC, little co-stimulation, and high net surface charge vii. Results in production of cytokine receptors, but not cytokines and no proliferation without exogeneous cytokines ***LO 3. Identify and explain the locations and functions of MHC molecules*** 1. **Major Histocompatibility Complex (MHC):** membrane bound protein that displays antigen peptides to T cells a. Encoded by genes from both parents to increase diversity b. Each gene has multiple alleles **(polymorphic)** so that each person has unique MHC pattern i. Ability to fight infection varies between people, as every person's adaptive immune system responds slightly differently to microbes c. Genes are found in all mammals d. Immune system uses MHC to recognize cells in body as self 2. Human MHC **(Human Leukocyte Antigens (HLA))** is on chromosome 6 e. **Class I** gene complex contains 3 major loci ii. B, C, A f. **Class II** gene complex contains at least 3 loci iii. DP, DQ, DR g. Human populations all maintain a diversity of HLA I and HLA II iv. Without this diversity, allotypes of humans would not survive 1. Pathogens are constantly changing and can divide easily (quick life span) so they are faster than humans 2. Thus, we need to have diversity too 3. All nucleated cells express MHC I while MHC II is mainly expressed by professional APCs h. Activated T cells have MHC II whereases resting T cells do not i. Most cell types in brain are MHC II negative but microglial cells (like macrophages) are MHC II positive ***LO 4. Compare and contrast MHC I and MHC II molecules including structure, formation, function, type of peptide, cells they present to, etc.*** 1. **MHC I Structure:** composed of membrane bound heavy chain (alpha) and soluble light chain called beta2-microglobulin (beta2m) a. Heavy chain has three extracellular domains, alpha1 and alpha2 domains that form peptide-binding groove b. Alpha3 domain and beta2m are immunoglobulin like domains with similar structure that interact to support peptide binding groove (aka alpha2 and alpha2) 2. **MHC I Molecules:** HLA-A, HLA-B, and HLA-C c. Highly polymorphic with more than 100 alleles at each locus 3. **MHC I Closed Peptide Cleft:** formed from MHC I molecules and beta2m between alpha1 and alpha2 domains to noncovalently bind an 8-9 amino acid sequence d. Presents less information than MHC II e. Slight structural variations in the binding cleft of different allelic forms allows different peptides to preferentially fit into some clefts better than others 4. **"Nonclassical" MHC I:** have limited variability and tissue distribution and may function to present carbohydrate and peptide fragments f. Encoded by HLA-E, HLA-F, HLA-G, HLA-H loci g. Ex: CDId 5. **MHC II Structure:** composed of two structurally similar membrane-bound alpha and beta chains h. Have an amino-terminal domain resembling the alpha1 and alpha2 domains of MHC I i. Have immunoglobulin like domain resembling the beta2m and alpha3 domains of MHC I 6. **MHC II Molecules:** encoded within HLA-DP, HLA-DQ, and HLA-DR regions with alpha and beta loci j. After synthesis, MHC II alpha and beta chains combine with others encoded in same region i. Ex: DPalpha only with DPbeta, not with DQbeta or DRbeta 7. **MHC II Binding Groove:** alpha and beta chain form groove that can accommodate 18-20 amino acid peptide k. Can fit more amino acids so presents larger pieces to T cells than MHC I ***LO 5. Describe the importance of MHC restriction*** 1. **MHC Restriction:** T cells are restricted because TCR cannot bind free antigen in body (need APC with MHC) 2. Two classes of MHC present peptide antigens to two types of T cells 3. **MHC I bind to CD8** a. CD8 co-receptor binds to alpha3 domain of MHC I heavy chain, ensuring MHC I present peptides only to CD8+ T cells 4. **MHC II binds to CD4** b. CD4 co-receptor binds to beta2 domain of MHC II, ensuring peptides bound by MHC II stimulate only CD4+ ***LO 6. Describe the "Sweaty T-Shirt" Study (Your "Also A Scientist")*** 1. Study conducted in 1995 to see how a man's MHC genes could be detected by women through their sense of smell 2. Men were given clean T-shirts to wear for two nights 3. Researchers put each T-shirt in a box equipped with a smelling hole and invited the women to sniff the boxes, describe the odor, and give each box a score for intensity, pleasantness, and sexiness 4. Women preferred the scents of T-shirts worn by men with MHC genes different from their own a. We are more attracted to people genetically different from ourselves b. Useful to the species because more diversity in genetics increases chances of survival and resistance to change Antigen Presentation II ***LO 1. Compare and contrast phagocytosis and macropinocytosis*** 1. **Phagocytosis:** receptor-mediated endocytosis a. Cells, particles and molecules are captured by PRRs associated with clathrin coated pits b. Clathrin-associated membrane invaginates and pinches off to form a phagosome c. Clathrin is recycled back to cell membrane to form new coated pits (to bring in more) 2. **Macropinocytosis:** actin-mediated endocytosis d. Cytoplasmic protrusions or ruffles engulf and surround microbes, particles, or molecules e. Forms a cytoplasmic vesicle f. Cytoplasmic vesicle fuses with lysosome to become phagolysosome i. Lysosome cuts down microbe, so it fits in MHC g. Phagolysosomes containing enzymatically degraded material fuse with vesicles containing MHC h. Empty phagolysosomes are recycled back to the cell membrane ***LO 2. Identify and explain the purpose, function, and steps of antigen processing and presentation. Elaborate on steps of MHC I and II processing/presentation and compare*** 1. **Antigen Presentation:** process by which protein antigen is presented to lymphocytes in form of short peptide fragments a. When ingested, proteins are enzymatically degraded and resulting peptide fragments are loaded into MHC class molecules, forming pMHC I or pMHC II 2. Antigens must first be processed and presented to T cells via APCs b. Phagocytosis or macropinocytosis ***LO 3. Identify proteins of peptide-loading complex that aid assembly and peptide loading with MHC*** 1. **MHC I Peptide-Loading Complex** a. MHC I heavy chain is stabilized by calnexin until beta2m binds i. **Calnexin:** holds up leaning MHC I to stabilize it b. Calnexin is released c. **Peptide-loading Complex:** TAP, tapasin, ERp57, calreticulin, MHC I (heavy chain and beta2m) ii. **Tapsin, ERp57, and Calreticulin:** chaperones that structurally stabilize MHC I iii. Interactions of chaperones with TAP and MHC I occur in ER iv. Tapasin and ERp57 form heterodimer linked by a disulfide bond v. Tapasin contacts MHC I which stabilizes empty conformation of peptide-binding groove 1. MHC I bind to low affinity peptide 2. Tapasin binds MHC I 3. Alpha 1 and 2 domains widen the groove 4. Peptide is released 5. Tapasin stabilizes empty MHC I 6. MHC I bind high affinity peptide 7. Groove narrows, forcing release of tapasin vi. Calreticulin binds to monoglucosylated N linked glycan at Asp 86 of MHC I heavy chain vii. Transmembrane region of tapasin connects peptide-loading complex with TAP, bringing empty MHC I into proximity with peptides transported from cytosol into ER 8. **TAP:** transporter that connects the endoplasmic reticulum to the cytosol d. Peptide delivered by TAP binds to MHC I heavy chain, forming mature MHC I e. MHC I dissociate from peptide-loading complex and is exported from endoplasmic reticulum to be presented ***LO 4. Compare and contrast MHC I and II processing and presentation*** 1. MHC I and II bind peptides in different intracellular compartments 2. **MHC I Processing:** peptides produced in cytosol are transported to endoplasmic reticulum for binding MHC I a. In nucleated cells, proteasomes degrade cellular proteins in the cytosol that are poorly folded, damaged, or unwanted into peptides b. These peptides are transported from cytosol into lumen of endoplasmic reticulum by TAP (embedded in ER membrane) c. MHC I is loaded with a peptide d. If peptide is too long at the N terminal end, ERAP is needed i. **Endoplasmic Reticulum Aminopeptidase (ERAP):** enzyme that removes amino acid residues in sequence from amino terminus to make peptide fit in MHC I e. MHC I with peptide is taken by exocytic vesicles through golgi to plasma membrane 3. **MHC II Processing:** Pathogens taken up from extracellular fluid are transported via endocytic vesicles to lysosomes f. In lysosomes, proteins are degraded to peptides g. Peptides are transported via endocytic vesicles to fuse with vesicles containing MHC II h. Peptides are loaded onto class II and taken to plasma membrane ***LO 5. Understand the significance of invariant chain*** 1. **Invariant Chain:** prevents MHC II from binding peptides in endoplasmic reticulum a. Antigen is taken up from extracellular space into intracellular vesicles b. In early endosomes of neutral pH, endosomal proteases are inactive c. Acidification of vesicles activates proteases to degrade antigen into peptide fragments d. Vesicles containing peptides fuse with vesicles containing MHC II e. In vesicles, variant chain is cleaved, leaving CLIP fragment bound i. **CLIP:** class II-associated variant protein f. CLIP blocks binding of peptides to MHC II in vesicles g. **HLA-DM** (membrane protein) releases CLIP allowing peptides to bind h. Vesicle containing MHC and peptide travel to surface to be recognized ***LO 6. Describe steps of dendritic cell maturation and its importance in antigen presentation*** 1. Dendritic cells take up antigens at an infected skin wound and carry them to the draining lymph node for presentation to naïve T cells 2. Maturation of dendritic cells changes their form and function i. In peripheral tissues, have arms sticking out ii. In lymphatic circulation, cluster to be compact for travelling iii. In lymphoid tissues, change shape to interact with T cells 3. Dendritic cells use several pathways to process and present protein antigens iv. Because they are nucleated and APC, they have both MHC I and II v. Can present bacteria and viruses at the same time and activate CD4 and CD8 at the same time vi. Very adept and versatile 4. Dendritic Cells do antigen processing for CD4 and present on MHC II when: vii. Receptor-mediated endocytosis of bacteria viii. Macropinocytosis of bacteria or virus 5. Dendritic Cells do antigen processing for CD8 and present on MHC I when: ix. Viral infection x. Cross presentation of exogeneous viral antigens 6. When an immature dendritic cell senses an invasive threat, it rapidly begins to mature a. Threats are detected by PRRs directly or indirectly xi. **Direct Sensing:** engagement of PRRs that recognize PAMPs on viruses, bacteria, fungi, and protozoa xii. **Indirect Sensing:** engagement of other receptors is responsible for perceived threats b. Threat sensing causes dendritic cells to migrate to nearby lymph nodes, decrease their phagocytic and macropinocytic activity, and increase MHC II synthetic activity c. MHC II make no distinction between peptides of self and non-self origin xiii. Self-antigens displayed on phagocytic surface usually go unrecognized because most self-reactive CD4+ T cells have been eliminated during development d. Naïve T cells encounter antigen presented by dendritic cells in secondary lymphoid tissues and respond (three ways) xiv. CD8 T cell recognizes viral antigens presented by MHC I on virus-infected cell 1. CD8 cell kills virus-infected cell xv. CD4 T cell recognizes bacterial antigens presented by MHC II on macrophage 2. CD4 secretes cytokines that activate macrophage, increasing its capacity to kill bacteria xvi. CD4 T cell recognizes bacterial antigens presented by MHC II on B cell 3. CD4 secretes cytokines that drive differentiation of B cell into plasma cell making bacteria specific antibodies ***LO 7. How can a peptide that is derived from an intracellular pathogen that circumvents phagolysosome vesicle load into class II molecules? (cross-penetration)*** 1. Some intracellular antigens are broken down and their peptide fragments are loaded into MHC I and form pMHC I 2. To avoid detection by adaptive system, some pathogens employ a stealth mechanism a. Circumvent phagolysosome vesicles together or enter phagosomes and then leave them by going into cytoplasm b. This ruse is not perfect because as some infected cells die dendritic cells take up dead cells and cellular debris by phagocytosis or macropinocytosis i. Proteolytic peptides are then displayed by MHC II 3. **Cross Presentation:** ability of certain professional APCs (mostly dendritic cells) to take up, process, and present extracellular antigens with MHC I to CD8 cells c. Enables extracellular antigens to be presented by MHC I d. One route could involve translocation of ingested proteins from phagolysosome into cytosol, where they undergo degradation by proteasome, enter endoplasmic reticulum via TAP, and be loaded onto nascent MHC I e. Another route could involve transport of antigens directly from phagolysosome into vesicular compartment (without passage through cytosol) where peptides are allowed to bind to mature MHC Receptors ***LO 1. State where you expect to find antigen receptors of the adaptive immune system*** 1. Specialized receptors of B and T cells are created in lymphocytes of everyone through random somatic chromosomal rearrangements and mutations a. Results in a varied array of receptors specific for precise molecular details found in unique epitopes that may be encountered in the future 2. **Receptors of the Innate Immune System:** limited in number and diversity and are consistent from one normal individual to another b. Include PRRs and complement receptors 3. **Somatically Generated Receptors of Adaptive System:** use random combinations of genes to assemble many different receptors ***LO 2. Describe the structure and organization of the immunoglobulins of the adaptive immune system (BCR, antibodies, TCR, etc.)*** 1. Immunoglobulins and TCRs are antigen receptors of adaptive immunity 2. Immunoglobulins are made up of four structures a. **Antigen Binding Site:** where antigens bind b. **Variable Region:** different between antibodies, allowing for specific recognition of an antigen c. **Constant Region:** same between antibodies d. **Transmembrane Region:** anchors immunoglobulin **LO 3. Compare and contrast BCR and TCR** 1. **B Cell Receptors:** cell surface bound monomeric immunoglobulin associated with disulfide linked heterodimers (Igalpha and Igbeta) a. Bind epitopes b. **Igalpha and Igbeta:** specialized cytoplasmic tails that support BCR and initiate intracellular signaling cascade that may lead to B cell activation c. Some activated B cells terminally differentiate into plasma cells i. **Plasma Cells:** secrete immunoglobulins that have same epitope binding specificity as their BCR 2. **T Cell Receptors:** heterodimers consisting of either alpha/beta or gamma/delta chain pair d. Always membrane bound and recognize antigen combined with MHC molecules e. Associated with cluster of differentiation 3 (CD3) complex of transmembrane surface molecules ii. CD3 functions like Igalpha/Igbeta of BCRs (links TCR with intracellular signaling molecules) f. Additional molecule (CD4 or CD8) present to serve as a type of coreceptor for TCR (most delta/gamma cells don't have CD8 and CD4 with some exceptions) g. Composed of an alpha chain of 40-50 kDa and a beta chain of 35-46 kDa h. Extracellular portion of each chain consists of two immunoglobulin like domains iii. **Constant Domain:** nearest to membrane iv. **Variable Domain:** farthest from membrane i. Alpha and beta chains both span cell membrane and have very short cytoplasmic tails (reason for CD3) j. TCR 3D structure formed by domain resembles membrane associated Fab fragment of immunoglobulin v. **Fragment with Antigen Binding (Fab):** proteolytic fragment of IgG that consists of light chain and amino terminal half of heavy chain held together by disulfide bond between chains k. **Complementarity Determining Regions (CDR):** loops that are highly variable and bind pMHC molecule ligand vi. Arrayed across relatively flat top surface of TCR vii. Short region with high diversity in amino acid sequences within variable region of immunoglobulins and TCR chains viii. Three CDRs (CDR 1, CDR 2, CDR 3) in each variable region that contribute to antigen binding site and determine antigenic specificity ix. Called **hypervariable regions** because most variable parts of domains ***LO 4. List the components and their functions of the TCR complex*** 1. TCR core formed by alpha and beta chains binding antigen a. Core heterodimer associates with 1 copy each of CD3 gamma and CD3 delta and two copies each of CD3 epsilon and zeda chain 2. This is necessary for transport of newly synthesized TCR to cell surface and for transduction of signals to cell's interior after TCR has bound antigen (antigen presentation) 3. Transmembrane domains of alpha and beta chains contain positively charged amino acids which form strong electrostatic interactions with negatively charged amino acids in transmembrane regions of CD3 gamma, delta, and epsilon chains b. Electrostatic interactions explain how it stays in certain area because of polarity ***LO 5. Describe the organization and rearrangement of the TCR genes to create a final TCR product*** 1. TCR alpha and beta chain loci have four gene segments in germline arrangement a. Variable b. Diversity c. Joining d. Constant 2. During T cell development, V region sequence for each chain is assembled by DNA recombination e. For alpha chain, rearrangement of Valpha gene segment and Jalpha gene segment creates a functional axon encoding the V domain f. For beta chain, rearrangement of Vbeta, Dbeta, and Jbeta gene segments creates functional V domain exon 3. Assembled genes are transcribed and spliced to produce mRNA 4. Expression of TCR on T cell surface requires association with additional proteins ***LO 6. Compare and contrast alpha/beta and gamma/delta TCRs*** 1. A distinctive population of T cells expresses a second class of TCR with gamma and delta chain a. Although gamma/delta T cells are mainly double negative (neither CD4 nor CD8) for the classical T cell marker, CD4 and CD8, small fractions express CD4 or CD8 2. Alpha/beta TCRs and gamma/delta TCRs have similar structures, but they are encoded by different sets of rearranging gene segments and have different functions ***LO 7. Define and use vocab words associated with this lecture*** 1. **Antibodies:** soluble (secreted) immunoglobulins a. Immunoglobulins are the same thing as BCR and TCR ***LO 8. Know the key chromosomes and segments*** 1. **Chromosome 14:** houses delta locus within alpha chain between clusters of Valpha and Jalpha gene segments 2. **Chromosome 7:** houses gamma chain locus 3. There are at least 3 Vdelta gene segments, 3 Ddelta gene segments, 3 Jdelta gene segments, and 1 Cdelta gene segment a. Vdelta segments are interspersed among Valpha and other gene segments b. This arrangement means that DNA rearrangement within alpha-chain locus results in deletion of delta chain locus c. The gamma locus (chromosome 7) resembles beta locus with 1 set of V segments and 2 C gene segments each with its own set of J segments 4. Alpha/Beta TCR d. **Alpha Chain:** chromosome 14, has V and J segments, and 1 constant segment e. **Beta Chain:** chromosome 7, has V, D, and J segments, and 2 potential constant segments 5. Gamma/Delta TCR f. **Gamma chain:** chromosome 7, has V and J segments, and 2 constant segments g. **Delta Chain:** chromosome 14 (within alpha locus), has V, D, and J segments, and one constant segment 6. Gamma is like Alpha with the V and J 7. Gamma is like Beta with the two constants 8. Delta is like Beta with the VDJ 9. Delta is like Gamma with the one constant T Cell Development (From Slides Called "T Cell Development" ONLY) ***LO 1. Outline the major events that transform a hematopoietic stem cell into a mature, naïve T cell*** 1. **Prothymocytes:** T cell precursors (immature) that migrate from bone marrow to thymus 2. Thymocytes run selective tests as they migrate from thymic cortex to medulla (making sure they don't attack self) a. Selection process is demanding; only 1-5% graduate as T cells while the rest leave the thymus before undergoing selection or die apoptotic death after failing 3. Identify proteins and receptors that aid in the development and drive of hematopoietic stem cells b. **CD34 and CD44:** stem cell surface and adhesion markers, respectively, that are seen in hematopoietic stem cells c. **CD2 and CD5:** function in adhesion and signaling and are present in thymus i. If seen, then lymphocyte is now T cell d. **CD4 and CD8:** coreceptors that are inactive in hematopoietic stem cell and thymocytes committed to T cell lineage e. **RAG:** helps with gene rearrangement and is expressed in thymocytes committed to T cell lineage, but not hematopoietic stem cells 4. Describe the steps and importance of Notch signaling f. **Notch1:** membrane associated receptor on surface of thymocytes that binds to its ligand (Notch1 Ligand) on thymic epithelium g. Notch1 induces protease to cleave intracellular domain of Notch1, releasing it from plasma membrane h. Soluble intracellular domain is translocated to nucleus of thymocyte i. Removes repressive transcription factors and recruits coactivating transcription factors j. Turns on expression of genes essential for T cell development 5. Without Notch1, there is no T cell maturation ***LO 2. Describe the microenvironments of the thymus where each state of T cell development takes place*** 1. T cells are created in the bone marrow 2. Immature T cells travel through the blood into the thymus 3. T cells develop in the thymus a. **Thymic Epithelial Cells:** form a nurturing network around the thymocytes and make the structure of the thymus b. **Hassall's Corpuscles:** sites of cell destruction (more pronounced in thymic evolution) c. Dendritic cells and macrophages are present as antigen presenters of self d. Thymic involution has a direct relationship with age 4. After selection tests, migrate from thymus to secondary lymphoid tissue to be activated 5. Immature T cells that don't pass the selection tests undergo apoptosis and are ingested by macrophages in the thymic cortex ***LO 3. Describe the changes in expression of CD4, CD8, and TCR that occur during T cell development*** 1. Thymocytes commit to the T cell lineage before rearranging their TCR genes a. Commitment to T cell lineage involves changes in expression of various cell surface and intracellular proteins 2. The two lineages of T cells arise from a common double-negative (no CD4 or CD8) thymocyte progenitor 3. T cell development is driven by receptor Notch I 4. **RAG:** essential for gene rearrangement and selectively expressed at two stages where beta and alpha gene rearrangements are made 5. Other enzymes involved in somatic recombination: b. **TdT:** inserts N nucleotides c. **pTalpha:** defining component of pre-T cell receptor that's expressed when rearrangements are made so beta chains can immediately be tested for their capacity to assemble a pre TCR i. if successful, cell stops recombination and initiates cell division and clonal expansion d. Signals from pre TCR depend on presence of CD4, CD8, CD3 signaling complex, tyrosine kinases ZAP70, and Lck e. **CD2:** adhesion molecule on T cells interacts with CD58 on other cells f. **Th-Pok:** transcription factor expressed late in development and necessary for single positive CD4 T cells to develop from DP thymocytes ***LO 4. Compare and contrast alpha/beta and delta/gamma cells*** 1. Alpha/Beta TCR a. **Alpha Chain:** chromosome 14, has V and J segments, and 1 constant segment b. **Beta Chain:** chromosome 7, has V, D, and J segments, and 2 potential constant segments 2. Gamma/Delta TCR c. **Gamma chain:** chromosome 7, has V and J segments, and 2 constant segments d. **Delta Chain:** chromosome 14 (within alpha locus), has V, D, and J segments, and one constant segment 3. Gamma is like Alpha with the V and J 4. Gamma is like Beta with the two constants 5. Delta is like Beta with the VDJ 6. Delta is like Gamma with the one constant ***LO 5. Define and describe the importance of, and the basis for, positive and negative selection*** 1. Epitope-specific TCRs and BCRs are randomly generated within individual thymus and bone marrow derived lymphocytes by gene arrangement a. Consequences is that some lymphocytes develop receptors that react with self b. Adaptive immune system carefully regulates development and differentiation of lymphocytes to prevent maturation of self-reactive T and B cells 2. Positive selection and MHC restriction 3. Negative selection and central tolerance c. Negative selection is a mechanism that removes self-reactive lymphocytes before they become fully functional and attack the body's own tissues ***LO 6. Recognize the variety of T cells that are generated in the thymus and articulate a basic understand of the events that lead to the development of each lineage*** 1. T cell precursors that enter the thymus express hematopoietic stem cell marker CD34, but no characteristic marker of mature T cells 2. Common progenitors proliferate into prothymocyte, then rearrange delta, gamma, and beta genes by picking which way to go a. Cells that productively rearrange both a gamma and delta chain gene (but not beta chain) commit to gamma/delta T cell lineage i. Once gamma/delta T cell receptor appears on cell surface, cells exit thymus and travel in blood to other tissues b. Rearrangement of a beta chain gene shuts down recombination machinery ii. May already have a rearranged delta or gamma chain gene or be cells in which their first rearrangement was a beta chain iii. In absence of recombination, beta chain is made and quality tested (by alpha as pTalpha to make sure it's structurally sound) 1. If the test is passed, cell proliferates to form clone of beta chain positive cells iv. If successful beta chain gene rearrangement before gamma and delta genes has both made successful rearrangement, a pre-T cell receptor (pTalpha) assembles and signals cell to proliferate, express CD4 and CD8, and become a pre-T cell 3. Recombination machinery is reactivated and targeted at alpha, gamma, and delta chain genes c. Can productively rearrange their gamma and delta chain genes to give rise to additional gamma/delta T cells d. In majority of cells, productive alpha chain gene rearrangement is made, followed by assembly of an alpha/beta receptor and commitment to alpha/beta lineage (CD4 + CD8 + alpha/beta cells) e. In minority of cells, alpha chain gene failed to be rearranged so the test failed and there's no successful alpha chain v. Cells die f. When alpha rearrangement begins, delta cannot be made because delta is within the alpha chain g. Rearrangement of alpha chain gene occurs only in pre-T cells because must test beta chain 4. pTalpha becomes TCR in four steps h. Two heterodimers form a beta chain and pTalpha chain form pre-T cell receptor i. If beta chain has capacity to form a function TCR, the two heterodimers can form a superdimer j. Interaction of superdimer with CD3 complex and zeta chain forms a functional pre-T cell receptor vi. Generates signals that initiate rearrangement of alpha chain genes and stops synthesis of pTalpha k. When a functional alpha chain is made, it associates with beta chain to form TCR 5. Successful rearrangements can rescue an initial non-productive beta chain gene rearrangement but only if that rearrangement involves D and J gene segments associated with Cbeta1 gene segment l. a second rearrangement is then possible where a second Vbeta gene segment rearranges to a DJ segment associated with Cbeta2 gene segment m. Cbeta1 and nonproductively rearranged gene segments are deleted 6. Having multiple V and J gene segments in TCR alpha chain genes allows successive rearrangement events to jump over nonproductively rearranged VJ segments, deleting intervening gene segments 7. Process continues until either a productive rearrangement occurs, or supply of V and J gene segments is exhausted n. If V and J segments are exhausted, the cell dies 8. The delta chain locus lies between V and J gene segments of alpha chain locus 9. During a recombination event that joins Valpha segment to Jalpha segment, intervening region, which contains a delta chain locus, is inevitably deleted as a small circular DNA that is disconnected from genome T Cell Development (From Beginning of Slide Show Called "T Cell Development and Activation) ***LO 1. Describe the microenvironments of the thymus where each stage of T cell development takes place*** 1. In the cortex, during the first part of early development, progenitor cells that commit to T lineage are double negative, so they do not have CD4 or CD8 2. In the subcapsular region: a. Checkpoint for pre-TCR must be passed b. Proliferating T cells are still double negative 3. Later in the cortex: c. Checkpoint for TCR must be passed d. Mature double positive cells express both CD4 and CD8 ***LO 2. Describe the changes in expression of CD4, CD8, and TCR that occur during T cell development*** 1. Double negative thymocytes have no cell markers 2. Double positive thymocytes can have a. Alpha/beta TCR, CD3+, CD4+ and CD8+ markers b. Gamma/delta TCR and CD3+ c. Alpha/beta TCR and CD3+ (NKT cells) 3. Single positive thymocytes have alpha/beta TCR, CD3+ and either CD4+ or CD8+ markers 4. Mature T cells can have: d. Alpha/beta TCR, CD3+, and CD4+ markers e. Alpha/beta TCR, CD3+, and CD4+ markers f. Gamma/delta TCR, CD3+, and sometimes CD4+ and/or CD8+ g. Alpha/beta TCR, CD3+, CD4+, or CD4+CD8+ 5. Possible restriction elements: h. MHC I or II i. CD1d ***LO 3. Compare and contrast alpha/beta and delta/gamma*** 1. Transitional B and T cell Lineages a. Cell type: gamma/delta and b-1 b. Ontogeny: develop earlier c. Repertoire: limited d. Receptor selection: none or extremely limited e. No education f. Location of mature cells: gamma/delta in integument, respiratory, and peritoneal while b-1 is in peritoneum and lungs g. Response time to initial antigen is quick h. Cell to cell cooperation for gamma/delta is not needed but might be for b-1 i. No memory 2. Adaptive B and T cell Lineages j. Cell type: alpha/beta and b-2 k. Ontogeny: develop later l. Repertoire: vast m. Receptor selection: alpha/beta is within thymus and b-2 is within the bone marrow n. Education: alpha/beta cells in thymus, and in b-2 nowhere o. Location of mature cells: both are throughout the body p. Response time to initial antigen takes time to develop q. Cell to cell cooperation is required r. Have memory ***LO 4. Define and describe the importance of, and the basis for, positive and negative selection (articulate relationship between positive selection and MHC restriction, as well as the relationship between negative selection and central tolerance)*** 1. For mature, antigen-recognizing T cells to develop without being self-reactive, T cells must go through positive and negative selection 2. **Positive Selection:** thymocytes that bind self-MHC/peptides in the cortex with intermediate affinity are selected to mature, migrating to the medulla for negative selection a. This happens first in the cortex b. Thymocyte with TCR that binds self-MHC I or II on a cortical epithelial cell, macrophage, or other cell in the thymic cortex is signaled to survive and proceed to negative selection c. If a thymocyte with a TCR does not bind self MHC, it is signaled to die d. Interaction of TCR with peptide presented by MHC I or II determines whether cell commits to CD4 of CD8 lineage i. TCR that interacts with pMHC I on a thymic endothelial cell with higher affinity becomes a single positive CD8 thymocyte ii. TCR that interacts with pMHC II on a thymic endothelial cell with higher affinity becomes a single positive CD4 thymocyte 3. **Negative Selection:** thymocytes in medulla whose TCRs bind self MHC/peptide complexes with too high affinity are induced to die (apoptosis) e. Less than 2% graduate f. TCR that binds too tightly to self MHC I or self MHC II on antigen presenting dendritic cells and other APCs in the thymus are signaled to die g. TCR that binds moderately to self MHC I or II on dendritic cells, macrophages, and other cells in the thymus are signaled to survive, mature, and enter peripheral circulation ***LO 5. Recognize the variety of T cells that are generated in the thymus and articulate a basic understanding of the events that leads to the development of each lineage, particularly the CD4+, CD8+, and delta/gamma*** 1. Progenitor T cells enter at high endothelial venule (HEV) from blood into thymus 2. Progenitor cells proliferate in medulla 3. Double negative T cells commit to T lineage in cortex f. Lack all surface proteins that characterize T cells, including CD4, CD8, and CD3 4. First Phase: Progenitor T cells proliferate and rearrange their TCR genes, leading to development of mature gamma/delta cells and immature alpha/beta cells expressing beta chain and pre-TCR g. Rearrangement of beta genes begins in cortex, followed by checkpoint for pre-TCR in the subcapsular region h. Pre-TCR induces expression of CD4, CD8, CD3, making T cells immature double positive thymocytes i. By end of first phase, cells have matured into double positive thymocytes with rearranged alpha chain genes and have both an alpha/beta TCR and CD3 5. Second Phase: Positive Selection j. Matching receptor specificity for MHC with appropriate coreceptor eventually leads to single positive CD4 and CD8 T cells after third phase 6. Third Phase: Negative Selection k. Occurs at corticomedullary junction where maturing thymocytes are tested by densely packed dendritic cells l. Any self-reactive T cells that bind too strongly are eliminated in the medulla 7. Thymocytes surviving positive and negative selection leave thymus as mature, single-positive CD4 or CD8 (and CD3+) T cells and enter circulation (mature, but naïve) T Cell Activation and Effector Function (Combined Wed/Fri Slides) ***LO 1. Review dendritic cell maturation and antigen presentation*** 1. Dendritic cells carry antigens from sites of infections to secondary lymphoid tissues 2. Dendritic cells take up antigens at an infected skin wound and carry them to the draining lymph node for presentation to naïve T cells a. Naïve T cells first encounter antigen presented by dendritic cells in secondary lymphoid tissues ***LO 2. Describe the "homing" of T cells to secondary lymphoid tissue and lymphocyte trafficking*** 1. Naïve T cells recirculate through secondary lymphoid organs and enter lymph node two ways a. \(1) Naïve T cells leave blood at high endothelial venule (HEV) and enter lymph-node cortex, where they mingle with professional APCs i. **High Endothelial Venule:** specialized post-capillary blood vessels especially adapted for lymphocyte trafficking in secondary lymphoid organs like lymph nodes b. \(2) In afferent lymph that comes from an upstream lymph node ii. Two lymph nodes are connected by a lymphatic iii. Downstream lymph node is draining an infection in the skin and upstream lymph node drains only healthy tissue iv. Naïve T cells specific for infecting pathogen can only be activated in downstream lymph node 1. Naïve T cells enter **downstream lymph node** directly from blood 2. Naïve T cells enter **upstream lymph node** in blood, transfer into lymphoid tissues via HEV, and leave upstream node in lymphatic that connects the two nodes, which delivers the naïve T cells to downstream lymph nodes v. Once there, pathogen specific T cells are activated by dendritic cells arriving in lymph that drains the infected tissue 2. T cells that encounter specific antigen APCs are activated to proliferate and differentiate into effector cells c. These effector T cells also leave lymph node in efferent lymph and enter circulation 3. **Homing** of naïve T cells to secondary lymphoid tissues is determined by chemokines and cell adhesion molecules d. Circulating naïve T cell enters high endothelial venule in the lymph node e. L-selectin binding to GlyCAM-1 and CD34 attaches the T cell to the endothelium f. LFA-1 is activated by chemokine and binds tightly to ICAM-1 g. In extravasation, lymphocyte leaves the blood and enters lymph node 4. T cells that fail to find specific antigen leave lymph node in the efferent lymph and reenter bloodstream 5. Naïve T cell binds dendritic cell with low affinity LFA-1-ICAM1 interactions h. Subsequent binding of T cell receptors sends a signal to LFA-1 vi. When a T cell binds to its specific ligand on APC, intracellular signaling through TCR induces a conformational change in LFA-1 i. Conformational change in LFA-1 increases affinity to ICAM on dendritic cell and prolongs cell-cell contact 6. Activation of naïve T cell requires antigen specific-signal and co-stimulatory signal j. **Antigen-Specific Signal (1):** delivered when TCR and CD4/CD8 co-receptor recognize pMHC II/pMHC I on dendritic cell k. **Costimulatory Signal (2):** delivered when T cell's CD28 co-stimulatory receptor binds B7 co-stimulator on dendritic cell vii. **CD28 and B7:** members of immunoglobulin superfamily 3. **B7.1 (CD80) and B7.2 (CD86)** are two forms of B7 that are co-stimulatory l. Activation of clonal expansion of antigen-specific naïve T cell occurs only if antigen-specific and co-stimulatory signals are delivered together viii. First signal only results in tolerance and anergy (only knows something is there) 4. **Anergy:** state of non-responsiveness to an antigen ix. First and second signal results in activation m. CD4+ Activation: signal 1 is TCR and CD4+ coreceptor recognizing pMHC II and signal 2 is interaction between CD40 and CD40L (stimulation by CD28 interaction with CD86) to fully activate n. CD8+ Activation: signal 1 is TCR and CD8+ coreceptor recognizing pMHC I and signal 2 is interaction between 4-1BB and 4-1BBL (stimulated by CD28 interaction with CD86) to fully activate ***LO 3. Define the immunological synapse and state its purpose*** 1. **Immunological Synapse:** structure formed when two immune system cells (naïve T cell and dendritic cell) bind to each other with multiple receptors and cell-adhesion molecules where signals can be exchanged and effector molecules secreted a. Divided into two complexes b. Supramolecular Activation Complex (c-SMAC): initiates activation c. Peripheral Supramolecular Activation Complex (p-SMAC) ***LO 4. Describe the T cell signal transduction and what signals are needed (which activate, which trigger, and which trigger effector function)*** 1. Clustering of T cell receptors and co-receptors initiates signaling within the T cell a. In a resting T cell, **ITAMs** of CD3 are not phosphorylated (inactive T cell) co-receptor has bound, inactive Lck in cell b. Binding of pMHC to TCR leads to CD4/8 coreceptor binding to MHC and incorporating into the cluster i. ITAM phosphorylation by Lck ii. **Lck:** tyrosine kinase associated with complex that phosphorylates ITAM c. **ZAP70** (tyrosine kinase) binds phosphorylated zeta chain ITAMs and is phosphorylated by Lck d. Activated ZAP70 transmits signal onward along T cell signaling pathway iii. Series of reactions leads to activation of phospholipase C -- gamma ***LO 5. What are ITAMs and what are their roles in the immunological synapse and signal transduction?*** 1. **ITAM:** immunoreceptor tyrosine-based activation motif that's on zeta chain in cytoplasmic domain of TCR a. Gets phosphorylated by Lck ***LO 6. What is the importance and significance of IL-2?*** 1. **IL-2:** drive proliferation and differentiation of activated naïve T cells a. Naïve T cell and activated T cells express different forms of the IL-2 receptor i. Naïve T cell has gamma and beta chains bound with low affinity IL-2 ii. Activated T cell has gamma, beta, and alpha chains bound with high affinity IL-2 2. Process of proliferation and differentiation with IL-2 b. Naïve T cell expresses low affinity IL-2 receptor c. Presentation of specific antigen activate naïve T cell d. T cells make IL-2 and the high affinity IL-2 receptor e. Proliferation of the antigen specific T-cells (clonal expansion) ***LO 7. Compare and contrast CD4+ and CD8+ maturation*** 1. Naïve CD8 T cells require stronger activation than that for naïve CD4 T cells a. Because if CD8 cells don't have something telling them what do, they just kill all 2. Naïve CD8 cell activated two ways b. Activated directly by virus-infected dendritic cell i. Activated CD8 makes IL-2 that drives its division and development to form a clone of cytotoxic T cells c. Dendritic cell (or other virus-infected cell expressing MHC II) that induces insufficient co-stimulation can be helped by CD4 effector T cells to activate naïve virus-specific CD8 T cell ii. IL-2 secreted by CD4 acts directly on naïve CD8 T cell interacting with same dendritic cell and provides necessary boost to activate CD8 3. Costimulatory signals are required for activating naïve T cells but not for activating effector T cells 4. Effector CD8 T cells recognize and kill virus-infected epithelial targets without more co-stimulation d. Functions are mediated by cytokines and cytotoxins e. Acts as selective and serial killers of target cells at sites of infection by inducing apoptosis iii. CD8+ recognizes virus-infected cell iv. Programs first target cell to die v. Moves on to second target cell vi. First target cell is dying, second is dying, and third is being attacked ***LO 8. Identify the different subtypes of CD4+ cells, the roles of the five different subtypes we learned, and activation pathways (cytokines) that trigger the different responses*** 1. The cytokine environment determines which differentiation pathway (effector function) a naïve T cell will take 2. **TH1:** activate macrophages to become highly microbicidal (better kill function) a. Cytokines that induce differentiation: IL-12 and IFN-gamma b. Cytokines secreted by TH1: IL-2 and IFN-gamma 3. **TH2:** activate cellular and antibody response to parasites (anti-inflammatory phenotype) c. Cytokines that induce differentiation: IL-4 d. Cytokines secreted by TH2: IL-4, IL-5, IL-10, TGF-beta 4. **TH17:** enhance neutrophil response e. Cytokines that induce differentiation: IL-6, TGF-beta, and IL-23 f. Cytokines secreted by TH17: IL-17 and IL-22 5. **TFH:** activate B cells to refine the antibody response g. Cytokines that induce differentiation: IL-6 and IL-21 h. Cytokines secreted by TFH: IL-21, IL-4, IFN-gamma i. Naïve B cells and their helper TFH cells recognize different epitopes of the same antigen j. Activation of naïve B cell by TFH: i. Naïve B cell binds antigen with BCR ii. Antigen is internalized by receptor-mediated endocytosis (phagocytosis), processed, and presented via MHC II iii. Specific TFH cell forms cognate pair with B cell iv. Cognate interaction leads to expression of CD40L on TFH v. CD40L on T cell interacts with CD40 on B cell and secretes IL-4, IL-5, and IL-6 by TFH 6. **TREG:** suppress other effector T cells (limits the activities of effector CD4 and CD8) k. Cytokines that induce differentiation: TGF-beta l. Cytokines secreted by TREG: TGF-beta and IL-10 m. TREGs (autoreactive regulatory CD4 T cells) prevent the proliferation of autoreactive helper CD4 T cells vi. Suppression depends on both T cells interacting with same APC, allowing cell-cell interactions and cytokines to affect in paracrine fashion (released to effect nearby cells) 7. Cytotoxic CD8 T cells and TH1, TH2, and TH17 cells work at sites of infection (often sites of inflammation) n. In naïve cells, LFA-1 binds ICAMs and is expressed by all leukocytes vii. **LFA-1:** integrin adhesion molecule that contributes to T cell interactions with a variety of types of target cells o. In effector T cells, VLA-4 binds VCAM-1 adhesion molecule viii. **VCAM-1:** integrin vascular adhesion molecule that is selectively expressed on endothelium of blood vessels in inflamed tissue and recruits effector T cells from blood into infected tissue ***LO 9. Define the different co-stimulatory molecules and signals (and back-ups)*** 1. **CD58:** backup for CD8 T cells that lack CD28 a. CD28 binds CD80/86 which is the second signal important for activation 2. For cytotoxic CD8 cells: b. Cytotoxins: perforin and granzymes c. Cytokines: IFN-gamma and IL-2 3. For CD4 effector cells, cytokines are listed above d. **IL-21:** key role in B cell differentiation to plasma cells and in the development of TFH, promoting functional germinal centers and immunoglobulin production ***LO 10. What is a memory T cell?*** 1. **Memory CD4+ T cells:** express CD28, increase their expression of some adhesion molecules, and decrease their surface expression of L-selectin a. By increasing CD28 expression, more likely to respond rapidly to CD80/86 displayed by APC b. By decreasing L-selectin expression, they are home to sites of inflammation because of increased expression of other adhesion molecules c. Can switch from CD45RA expression to CD45RO expression 2. CD152:CD86 engagement inactivates CD4+ T cell (suppression) 3. CD8+ T cells can also form memory cells d. CD58 is increased in memory connections to active CD8 in future CAR-T Cell ***LO 1. Describe the typical interaction between T cells and APCs*** ***LO 2. Explain the domains of CARs and their respective functions*** 1. Made up of three components a. **scFvs:** single-chain variable fragments that are small sized artificial constructs composed of immunoglobulin heavy and light chain variable regions connected by a peptide linker i. Heavy and light chain region of anti-CD19 immunoglobulin (antibody targeted against CD19) b. Transmembrane portion of CD28 (stabilizes and holds CAR to surface) c. CD3 zeta proteins (with ITAMs) make up cytoplasmic domain 2. Co-stimulation signaling domains have been added to newer generations of CAR-T cells to improve their ability to produce more T cells after infusion and survive longer in circulation d. More signals are better because they give a stronger response ***LO 3. Explain how CARs allow T cells to interact with and attack both cancer cells and normal cells*** 1. **CAR-T Cell Therapy:** a type of treatment in which a patient's T cells are changed in the laboratory so they will attack cancer cells a. T cells are taken from a patient's blood and a gene for CAR will be added to the T cells in the laboratory i. **Leukapheresis:** separates T cells from blood and the rest of blood is returned to body ii. **CAR:** chimeric antigen receptor that binds to a certain protein in patient's cancer cells b. Large numbers of CAR-T cells are grown in lab and given to patient by infusion iii. T cells are reprogrammed by introducing a genetic sequence through a lentiviral vector, so T cells produce CARs c. CAR-T cells can recognize and attach to specific marker proteins on cancer cell surface, signaling its destruction d. CAR-T cells are expanded in lab e. Before CAR-T are reintroduced, patient undergoes conditioning chemotherapy (don't want the body to kill the CAR-T cells) f. CAR-T cells are infused back into patient 2. Used to treat certain blood cancers and is being studied in treatment of other types of cancer 3. Memory CAR-T cells allow the therapy to be lifelong with renewal when there is relapse 4. In this case study, CAR-T cells are targeting CD19 on B cells g. **CD19:** part of B cell co-receptor complex h. Involved in intrinsic B cell signaling thresholds through modulating both B cell receptor dependent and independent signaling i. Target for chronic lymphocytic leukemias iv. Most often arises from malignant clone of B cells 5. CD19 is a marker of normal and neoplastic (cancer) B cells j. So, CAR-T can kill normal B cells as well, since all B cells express CD19 k. Patients need to be given synthetic antibodies to counteract the effects on normal cells l. Patients become more susceptible to other pathogens and are considered immunocompromised ***LO 4. Apply knowledge of immune system to current applications of cancer immunotherapies*** ***LO 5. Understand the side effects of immunotherapy treatment and compare this treatment with typical chemotherapy***