Key Contributors: Edward Jenner (1796) demonstrates cowpox vaccine against smallpox Robert Koch ( ) develops kochs postulates

es for disease causing agents Louis Pasteur (1880s) develops vaccines to cholera in chickens, rabies Emil von Behring and Shibasaburo Kitasato (1890s) discovery of antiserum Elie Metchnkoff ( ) macrophages and innate immunity

Immunology Lab Manual Draft 1 Property of Elizabeth Cody

Macarlane Burnet (1950s) clonal selection theory of antibody production James Gowans (1960s) demonstrates lymphocytes as units of clonal selection Ray Owens (1940) demonstrates tolerance in heterogenic twin calves Peter Medawar (1953) demonstrates embryonic exposure as the basis of tolerance Susumu Tonegawa ( Basic Vocabulary: Immunology Pathogens (vs. Commensals, Cancer, or Toxins/Toxoids) Extracellular Parasite Fungi Extracellular bacteria Worms (helminths) Intracellular Bacterial Parasite Virus Immunogenic Vaccine Antibody (Ab) aka Immunoglobulins Humoral immunity Antiserum WBC (White Blood Cells) aka Leukocytes Cytokine Chemokine Plasma surrounds cells in blood Extra Cellular Fluid (ECF) surrounds cells in tissues Types of immune response: ) discovers gene rearrangement as basis for antibody diversity

Nonspecific Immunity Physical barrier Chemical barrier Innate Immunity (Complement + Antibody+ PRR Phagocytosis, Clearance Inflammation) Adaptive Immunity (Cellular immunity + humoral immunity + memory) Primary (Central) Lymphoid Organs of the Immune System: Bone Marrow Hematopoiesis Pluripotent stem cells Common lymphoid and myeloid progenitor cells Common lymphoid progenitor cells lymphocytes + DCs Immature T Cells Immature B Cells mature B cells Bursa of Fabricius NK cells Common myeloid progenitorsLeukocytes + Megakaryocyte + Erythroblast + DCs Myeloid Leukocytes (PMNs + M + MP) Monocyte Mast cell (precursor) Histamine PMNs/Granulocytes (Nps + Eps + Bps) Neutrophil Eosinophil Basophil Megakaryocyteplatelets Erythroblasts erythrocyte (RBCs) Other Cells present: Thymus* *The thymus develops from the fetal gut

Secondary (Peripheral) Lymphoid Organs of the Immune System Lymph flow: arteries capillaries tissue ECF lymphatics lymph nodes thoracic duct L. subclavian vein Superior Vena Cava R. atrium of heart Lymph nodes Afferent vessels Efferent vessels HEV (high endothelial venules) Marginal Sinus Cortical sinus Outer Cortex Primary Follicles Secondary Follicles Germinal center Senescent Deep Cortex (paracortical area/T cell zone) attracts T cells, activated DCs and Mps Medullary chords Medullary sinus Path of nave lymphocytes: artery capillary HEV venule walls cortex/medulla T cells to paracortical area, B cells to follicles activated stay or exit efferent vessel Path of activated APCs or free antigen: Tissue ECF lymphatics afferent vessel marginal & cortical sinus cortex/medulla Activated DCs to paracortical areas, Macrophages to paracortical areas or medullary chords Spleen (filters out old RBCs, blood borne pathogens and free antigen) Red pulp White pulp Central arteriole PALS (periarteriolar lymphoid sheath) mainly T cells Lymphoid follicles mainly B cells (secondary = has germinal center) Germinal center differentiating B cells B cell corona surrounds germinal center Marginal zone - Mps, non-circulating resident MZ B cells, DCs; around corona Perifollicular zone

Path of antigen and lymphocyte: blood splenic artery trabecular artery central arteriole Perifollicular zone (PFZ) follicle Path of APC: macrophage/immature DC follicle marginal zone collect antigen activated PALS (T cell area) Mucosa (MALT, GALT, NALT, BALT - these all have M cells!) Nasal and respiratory mucosa Oral cavity Urogenital tract GALT (tonsils, adenioids, appendix, peyers patches) Peyers patches M cells lack microvilli, mucus, shuttle Ag across gut to waiting DCs Subepithelial dome B-cell follicles (w/ Germinal centers) T cell dependent area Path of antigen: gut M cell waiting DCs in subepithelial zone T cells Path of lymphocytes: blood capillaries HEV subepithelial dome (follicle or T cell dependent area) get activated leave via efferent lymphatic Functions of Mucosa trap antigen, attract APCs, sustain lymphocyte populations until they encounter APCs Non-Canonical Immune Regions with resident lymphocyte populations Liver* *also responsible for stress response, fetal hematopoiesis, filtration of blood etc Gut lamina propria Epidermis* *in mice, not humans Reproductive epithelium (eg uterus) Other Tissues That Mediate Immune Response: Mesothelium influences leukocyte migration, capable of synthesis of cytokines, phagocytosis, antigen presentation Blood (No activated cells, contains Monocytes, nave lymphocytes, plasma molecules) Endothelium

Basic Effector Mechanisms of Innate Immunity: Effector mechanisms processes by which pathogens are destroyed and cleared from the body. Innate and adaptive immune responses use most of the same effector mechanisms to eliminate pathogens. (Janeway 8th Ed.) Complement* Opsonization Proteolytic reactions Antibodies* Structure: Short chain Heavy chain Variable region Antigen binding site Gene rearrangement Junctional diversity Constant region Isotypes (IgA, IgE, IgG, IgM, IgD)* *differ in location and response triggered Effector Functions: Neutralization Opsonization Complement activation PAMPS (Pathogen associated molecular patterns) & MAMPS (molecular) PRRs *(Pattern recognition receptors) Mannose rich oligosaccharides Peptidoglycan LPS (Lipopolysaccharides) Unmethylated CpG DNA *All 3 methods trigger early responses occurring rapidly after (re)exposure and targeting only exposed molecules or microorganisms Antigen Presentation: Antigen Epitope (antigenic determinant) Peptide Professional Antigen Presenting Cells* (Mainly DCs, also Mp + B cells)

*also some activated epithelial cells Non-professional APCs (e.g fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, vascular endothelial cells, mesothelium) Cells of Innate Immunity (Myeloid Leukocytes + DCs) Dendritic Cells Macrophages Macropinocytosis Neutrophils Eosinophils Basophils Mast Cells Degranulation Inflammatory cells (Mp + Np) Inflammation (rubor, tumor, dolor, calor) Phagocytes (DCs + PMNs + MP) Phagocytosis Basic Concepts of Adaptive Immunity MHC (Major Histocompatibility Complex) MHC Molecules MHC Class I (display internal peptides) on most cells MHC Class II (display processed external peptides) mostly on APCs mhc genes TCR (T cell receptor) TCR Co-receptors CD4 CD8 BCR (B cell receptor) aka mIg Activation Costimulatory signal Lymphoblast Clonal expansion Anergy Clonal Selection Theory Self antigens

Apoptosis Tolerance Memory Memory B and T cells Primary Immunization and response Secondary Immunization and response Effector Lymphocytes Plasma cells - generate optimized antibodies to exposed/free antigen Development: immature B cell in bone marrow nave mature B cell circulates blood and 2ry lymphoid organs encounters antigen + costimulatory signals from Th cells activated B cell becomes lymphoblast clonal expansion differentiation into plasma cells in peripheral tissues or in bone marrow Effector function: Generate antibodies sIg mIg Affinity maturation Effector T Cells recognize internally processed peptides displayed on a cells surface in the context of an MHC molecule Development: immature T cell in bone marrow nave mature T cell in thymus circulation in blood and 2ry lymphoid organs APC present antigen & MHC + Costimulatory signal activated T cell becomes lymphoblast clonal expansion cytokines influence differentiation effector T cells migrate to tissues or stay in 2ry lymphoid structures and activate B cells Cytotoxic T cells (CD8) (MHC II) recognize viral peptides Target cell killing Helper T cells (CD4) Responses are mediated by cytokine profile Th1: activate macrophages, Ab stimulation, target intracellular bacteria Th2: stimulate Ig E Ab production, recruit Eosinophils, target extracellular parasites Th17: produce Il-17, recruit neutrophils, target extracellular bacteria, fungi Other Th subsets: Tfh cells, Th3 Regulatory T cells

Cell

Immature

Mature Nave

Activated Lymphocyte

Effector Lymphocyte B cells = Plasma cells T cells =

Memory Lymphocyte

Lymphocyte Lymphocyte

Location

Bone Marrow (B or NK cells) or Thymus (most T cells)

Located in blood and travels to 2ry lymph organs (LN, spln, mucosa etc), circulates until it finds antigen or dies

Activated in 2ry lymph organs (stops migrating)

T cells stay in 2ry lymph organ to activate B cells, or migrate to sites of infection B cells stay in peripheral lymph organs or migrate to bone marrow to release Ab

Features

Lacks

Express many different BCRs/TCRs Looks inactive little cytoplasm, few organelles, condensed nuclear chromatin

Nucleus, cytoplasm volume increses, can split 2-4x per 24h for 3-5 days. Only B cells undergo affinity maturation during clonal expansion

Diverse.

Reactivate more quickly than nave cells

Response Mechanisms

B cells: Antigen + costimulatory molecule T cells: MHCpresented Antigen + costimulatory molecule activation No costim Anergy

Clinical Conditions: Immunodeficiency disease Acquired immune deficiency syndrome (AIDs) Allergy Asthma Autoimmune disease Graft rejection H2 Locus Polymorphism Immunosuppressants Vaccines Diptheria Polio Measles Adjuvent Extracellular Pathogens (disease) Streptococcus pneumoniae (Pneumonia) Clostridium tetani (Tetanus) Trypanosoma brucei (Sleeping sickness) Pneumocystis jirovecii (Pneumocystis pneumonia) Ascaris (ascariasis) Schistosoma (schisotosomiasis) Intracellular pathogens (diseases) Mycobacterium leprae (leprosy) Leishmania donovani (Leishmaniasis) Plasmodium falciparum (malaria) Variola (smallpox) Influenza (flu) Varicella (chickenpox)