(8/29) Woodbury Lecture: Innate and Adaptive Immunity Lymphoid tissues and Organs

- Primary Lymphoid Organs: Locations for B and T cell maturation. [Bone Marrow – B Cell, Thymus – T Cell] - Secondary Lymphoid Organs: Serve to sweep, trap, and concentrate foreign materials, exposing foreigners

o Includes: Lymph nodes, spleen, tonsils, appendix, peyer’s patches (intestines), lymphoid aggregates o Functions as the major site of antibody production

- MALT: Mucosa-associated lymphoid tissue – GI, Respiratory, Genitourinary - GALT: Gut-associated lymphoid tissue- Peyer’s patches, appendix, solitary lymphoid nodules in submucosa - BALT: Bronchus-associated lymphoid tissue - Lymphocyte and Antigen Migration/Recirculation – 3 main routes by which the antigen enters the body

o (1) Bloodstream: Antigen enters the blood, passes through spleen, meeting APC à activate B/T o (2) SkinàLymph Nodes: Antigen penetrates the skin and passes into lymph, meet APC à activate B/T o (3) GI/Respiratory: Antigen meets MALT or BALT, similarly à generate immune response

- Thymus Gland: A pouch-like structure of cortex and medulla. Innervated by adrenergic and cholinergic neurons o Members: Stem cells colonize the thymus, and are termed Thymocytes o Role: Serves as a site of maturation of lymphocytes, particularly T Cells. Macrophages and DC are

present for digesting apoptotic thymocytes (in the medulla) and as APC to induce adaptive immune response

o Age: Thymus stops producing thymocytes as we age and engages in steady atrophy. By the time this happens, however, you have produced a large population of memory cells and stocked the lymphartics

- Spleen: A jelly-like encapsulated organ perfused with blood, abundant in germinal centers o Role: A secondary lymphoid organ that acts as a major blood filter to help generate immune responses to

antigens in the blood. The germinal centers are rich in B cells, the periphery rich in T Cells. Throughout are an abundance of APC. APC generally take up antigen, present to B and T cell à Immune response

- Lymph Nodes: Encapsulated tissues located at the junctions of draining lymphatics o Role: Host B/T cells and APC, such that the node provides great exposure of antigen to necessary

immune system components, similar to the spleen. Jobà Generate immune response. Mechanisms of Innate Immunity

- Participants: Barriers, MonocytesàMacrophages, Dendritic Cells (DC), Mast Cells, Complement, Polymorphonuclear Leukocytes, Natural Killer Cells

- Passive/External Defense: Includes FA on skin (acidic pH+mechanical), gut/skin bacteria (competition), stomach acid (pH), mucus, cilia, Enzymes (lysozymes to hydrolyze), cough reflex, nose hairs

o Hair Follicles: A poorly defended route of invasion. Acne serves as an excellent example - Complement System of Soluble mediators – Also consists of membrane-bound proteins

o Proteins bind to pathogen cell surface (opsonization) and stimulate phagocytic cells to digest the target. Some of these components disrupts the target’s cell membrane (cell lysis)

- Intracellular and Extracellular Killing o Intracellular: Recognition of pathogen-associated molecules à phagocytosis o Extracellular: Secretion of toxic metabolites, pore formation à cell lysis, and induction of apoptosis

§ Natural Killer Cells: Develop in the thymus, related to T Cell precursor. Respond to viral/cancer • Express Killer Inhibitor Receptor (KIR), which binds to MHC I on normal cells, but if

MHC I is not present, then the NK cell attacks via pore-forming agents à Cell lysis, as well as signaling agents to start apoptosis in the target cell

§ Natural Killer T Cells: Develop in the thymus, express TCR! Specific for sphingolipids • Express a T Cell Receptor (TCR) that is specific for membrane-associated sphingolipids,

particularly those found in bacteria. NKT cells secrete IL-4, IFNg, and other cytokines to stimulate other immune cells. Can trigger apoptosis via Fas-FasL interactions

- Pattern Recognition o PAMPs: Pathogen-associated molecular patterns: These are conserved molecules expressed by pathogens

present in large amounts. Such as sugars on bacteria, various lipoproteins, flagellin, etc o PRRs: Pattern recognition receptors: This is different from BCR or TCR, they’re highly expressed on DC

§ Toll-like Receptors (TLR): Extracellular, Recognize various bacterial components, gram +/- • TLR4: First to be characterized, often responsible for toxic shock syndrome, recognize

LPS endotoxin • TLR3: Recognize CPG sequences without methylation

§ C-type lectin Receptors (CLR): Extracellular, Specific for polysaccharides/carbohydrates from fungi, depend on calcium

§ NOD-like Receptors (NLR): Intracellular, recognizing cytoplasmic PAMPs. Specific for nucleic acids. May generate an inflammasome, releasing certain cytokines related to inflammation

§ RIG-I-like receptors (RLR): Intracellular receptors, sensing cytoplasmic RNA helicases, signal production via interferons.

§ Formylated-methionine receptors: Found in many bacterial peptides - Inflammation- characterized by pain, redness, localized heat, and swelling

o Systemic response mediated by increased blood flow, cell metabolism, vasodilation, cytokines, and hypothalamus raises internal body temperature. This is usually in response to a big cut or wound

o First responders are the PMN leukocytes, WBC, and other macrophages Acute Phase Response of Innate Immunity

- First: Response to PAMPs, whose recognition by innate immune cells stimulates release of pro-inflammatory cytokines (IL-1, IL-6, TNFa).

- Thus, fever is induced, causing release of CRP, complement, fibrinogen (clot), and other mediators - Denoted by the inflammatory cytokines, leukocytes are directed to migrate to the source of the immune response - Phagocytosis and destruction of pathogens

(9/1) Woodbury Lecture: Antigens Characterizing the Antigen (Ag)- Non-self

- General: Complex non-self molecule that is usually polymeric (protein, carb, nucleic acid) susceptible to being broken down (Ag processing) thus enhancing immunogenicity. Proteins have the greatest diversity in sequence and therefore generally have the most epitopes on a single protein – good for provoking response

- Antigenic Determinants = The parts related to eliciting an immune response, equitable to Epitopes/Haptens. They are the parts of antigens that interact with antibodies and T Cell antigen receptors.

o The more antigenic determinants carried = more likely and more diverse the immune response Characterizing the Antibody/Immunoglobulin (Ig)

- Structure: Composed of 4 peptide chains, 2 short/light identical chains, 2 longer/heavier variable chains. Chains are held together by disulfide bonds. The disulfide linkages vary by antibody type.

o Ag-binding region/Complementary Determining Region (CDR): Location between heavy and light chain where the antibody binds

o Fc Domain: The constant stem region, which is recognized by the complement system (C1Q). Sugar linkages sometimes exist in the Fc region, often for glucosamine. The Fc stem region can be cut by plasmin/trypsin to create bioactive peptides

o Fab Domain: The variable arm region, which serves as the Ag-binding region. Have a characteristic beta-pleated sheet pattern. The CDR is at the tip of the Fab, which has “hypervariability” in its AA to determine specificity

o Hinge Region: Confers flexibility of the antibody, allowing for free rotation- which is biologically important for the binding of both arms to surface with more than one hapten. Helps coat a single bacterium/virus + enables crosslinking

§ **Solubility of Ab-Ag is related to the hinge region reactivity** - Interaction with Ag [Ab+Agßà AbAg]: Form a tight complex at the CDR of Fab region, held together by

numerous noncovalent interaction, including Hydrophobic (entropic), Van der Waals, Ionic, and Hydrogen bonding

o High Affinity: Equilibrium lies to the right. It is a good fit with low steric clash and low repulsion. o Low Affinity: Equilibrium lies to the left. Poor fit, with steric clash and thus repulsion. Prefer free Ab Ag o Cross-reactivity: Conceptually – you do not have 0% specificity or 100%, there’s a range. This introduces

the concept of polyclonal antibodies, multiple lines of Ab to one Ag (though probably multiple epitopes) - Reactions of Ab-Ag- Below is archaic terminology, just refers to methods to create better detection of Ag

o Precipitation: Soluble Ag binds with Ab, forming a crosslinked latticework of alternating Ab, Ag. Ppt! o Agglutination: Insoluble particulate binds with Ab, crosslinking leads to clumping of particles o How to determine the difference in vivo? Pull that shit out & shove it in a centrifuge and check solubility o Univalent antigens (1 hapten per particle) prove very difficult to cross-link. Need 2 or more epitopes per

antigen in order to get cross-linking