Immunology

Innate immunity- most primitive- cytokines, phagocytes, and complement; barriers to infection (skin, mucous membranes, pH, temp), inflammation (acute phase proteins, complement, phagocytic cells) Adaptive immunity- LYMPHOCYTES, phagocytes, cytokines, Ab, and complement Active- convalescence and vaccine Passive- transplacental and serum therapy Humoral- Ab and complement Cellular- lymphocytes, cytokines and phagocytes Memory response- if exposed again much greater immune response- controlled by lymphocytes 3 phases of Adaptive immunity Cognitive phase- recognition Activation phase- proliferation and differentiation of lymphocytes Effector phase- memory, elimination of Ag (phagocytes and complement) Acute phase proteins- induced by stress for inflammation and healing (C-reactive protein, serum amyloid) Complement- activation by immune complexes for immune regulation (over 20 proteins) Cytokines- all cells in the body to regulate immune and other responses (interleukins and interferons, TNF Ab- produced by B cells and plasma cells IgM,A,G,E,D Clonal selection- lymphocytes, but not all T and B cells respond to the same Ag, each clone responds to only one Ag determinant Clones against self are deleted or suppressed for the most part Get autoimmunity if those cells are not suppressed Get quicker response when re-exposed by activation of the clonal cells Hematopoiesis Stem cells- blood cells are short lived and continuously renewed from pluripotent stem cells found in the bone marrow; Memory lasts for a long time but a given lymphocyte does not last for yrs and yrs progeny are generated Under influence of cytokines progenitor cells become a megakaryocyte or granulocyte of some kind IL-7 important in B cell development Megakaryocyte makes platelets Colony forming units can produce- rbcs, basophils, eosinophils, neutrophils, or monocytes

T lymphocytes TCR- T cell receptor- binds Ag to T cell o TCRa/b receptor- blood, spleen, lymph nodes, CD4, CD8, o TCR g/d receptor- mucosal surfaces, CD8 Subtypes and surface markers: o Th- CD4 o Tc- CD8 o Ts- CD4 and CD8 o NK- CD16 (no TCR) Surface markers- proteins functioning as receptors/ligands o Common to all T cells- CD3, TCR, CD2, CD28, CD5, and CD7 CD4 has an affinity for MHCII CD8 has an affinity for MHCI B lymphocyte Surface markers- surface immunoglobulin, complement receptors, and Fc receptors Plasma cells- mature B cells, make bulk of Ab and secretes them into blood (lives for about 1 week) Markers: HLA-D, CD22, CD19, CD20, Igb, Iga, sIg, CD40, CR1, CR2, CD72, CD5, Fc_RII, Mononuclear phagocytes- large cells, amoeboid, granular, endosomes, and kidney shaped nuclei Involved in innate immunity (phagocytosis and wound healing) and active immunity (Ag presentation, regulatory cytokines) Markers: MHCII, Fc receptors (Fc_RI, Fc_RII, Fc_RIII), complement receptors (CR1, CR3) Releases many enzymes and prostaglandins Granulocytes- neutrophils, eosinophils, basophils, Blood is 70% neutrophils, 5% eosinophils, and 1% basophils Antigen presenting cells- express MHCII ALL nucleated cells express MHCI Only APC present MHCII Langerhans cells in skin, interdigitating cells in thyroid, follicular dendritic cells in neurons, B cells in blood, macrophages in blood Lymphoid organs Primary organ- thymus, bone marrow- foster maturation of stem cells; cells mature to T or B cells Secondary organ- lymph node, spleen; contain mature cells and foster immune response Thymus- cortex and medulla; macrophages and dendritic cells present self-Ag to differentiating T cells leading to positive and negative selection Positive selection- selecting cells that respond to non-self Negative selection- selecting cells that respond to self (at corticomedullary junction)

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Bone marrow- contains all cells and cytokines necessary for B cell development Spleen- filters Ag out of blood and promotes immune response No spleen- more susceptible to blood-borne infections- esp. Strep. pneumo Composed of capsule and reticular fiber and cellular framework Lymphocytes, macrophages, interdigitating cells, follicular dendritic cells, and reticular cells Red pulp surrounds white pulp which surrounds the central artery and contains the PALS w/ T cells and B cell zones (T cells surround the germinal centers of B cells) Lymph node- filters Ag from lymph; Capsule, medulla, cortex; contains lymphocytes and other cells Cortex- outer region B cells inner region T cells Medulla- plasma cells Structure of Ag and Ab Hapten- small moiety such as DNP that induces immunity only when attached to carrier such as bovine serum albumin (BSA) B cells recognize hapten T cells recognize carrier protein Proteins are best immunogens. Epitope- chemical group that confers Ag specificity- proteins have more than 1 to engage a variety of clones to increase chance of stimulation of immune response Need an APC to present non-peptide to T cells Do not need APC to present non-peptide to B cell Lipopolysaccharides: Lipid A- endotoxin- binds to serum proteins and ligates to CD14 which stimulates B cells O antigen- binds to B cell receptors- if receptor is clone w/ affinity for O Ag Core polysaccharide B cells get several signals from a single molecule of LPS- enough to have it develop into plasma cellindependent of T cells Ig Basic structure: 2 Light and 2 Heavy chains both with variable and constant regions Ig Properties 4 IgG subclasses- different heavy chains sIgA- secretory IgA IgG- most common 3 subclasses of IgA

Isotype- gene product that every member of a species has Allotype- gene products that vary among individuals Idiotype- unique gene products for each individual H chain- 3 C regions and 1 V region L chain- 1 C and 1 V region Ag binds to V region- 2 binding sites (bivalent) Fab- Ag binding region (V regions of H and L chains) Fc- terminal piece (most glycosylation sites) J chain- helps link 5 subunits of IgM together to form pentamer IgM can bind 10 Ag. Only 1 IgM pentamer is needed to activate complement since 2 Fc receptors are close to each other. IgA also has a J chain Secretory piece- helps protect IgA from proteolytic digestion in secretion COMPLEMENT: Complement is involved in cytolysis, Opsonization, and inflammation, enhancement of humoral response, and clearance Inappropriate firing of complement system can lead to shock like state. Insufficiency of complement can cause more susceptibility to bacteria and have immune complexes that are not cleared. Classical system- starts with Ab/Ag complex C3b critical to both classical and alternative pathways C5 splitting- entering MAC complex (C5b, C6, C7, C8, C9)- cascading is now creating small holes in bacterial cell walls to cause cell lysis See pg 69 bottom slide C3a, C4a, and C5a o Smooth muscle contraction o Increased vascular permeability o C3a and C5a- degranulation of eosinophils, and platelet aggregation When there is a classical pathways deficiency (C1q, C1r, C1s, C4, C2) cant have cell lysis and also have problems clearing immune complexes leading to autoimmunity, vasculitis, etc Short lasting for fine control Receptors : CR1, CR2, CR3, CR4 Specific factors- regulatory factors that have very specific actions in limiting the complement system o CCP and DAF inhibit C3 convertase o MCP- regulated C4b Missing MAC complex cant undergo Opsonization but can have inflammatory cells brought in

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Alternative system- no Ab needed C3b can start alternative system on bacterial cells C3b can enter the self-amplification loop Tickover- C3 hydrolyzed into C3a and C3b Complement Deficiencies: Early components- absent o Immune complex disease - increased incidence o Pyogenic infections when missing C1, C4, C2 increased incidence Late components o Neisseria- chronic infections Regulatory factors missing o C1H- most common autosomal problem (controls C1) HANE- hereditary angioneurotic edema when deficiency in C1Hcomplement system always firing leading to inflammation Complement receptors o LAD- leukocyte adhesion deficiencyinflammatory cells do not enter area since they are missing complement receptor Ag/Ab Interactions and Functions: CDR- complementary determining region- has many substitutions- diff aa sequences in CDR determine Ag specificity Overall affinity for Ag is higher after proliferation of Ab Each clone has a different specificity for each Ag. Carboxy end motifs of Ig to activate complement: 3 aa in 2nd region of IgG found to activate C1q 3 aa in 3rd region of IgM activates C1q IgE Fc_RI- can bind between 2nd and 3rd region for immune complex removal IgD- helps B cell respond to Ag (w/ IgM) Fc receptors are involved in binding Fc regions of Ab and leading to phagocytosis of the Ab/Ag complex, also for degranulation and allergy response Transport of IgA- Ab produced on abluminal surface of mucosa and are transported to luminal surface by poly Ig receptor Ab-based lab testing: Primary interaction- bivalent Ab and univalent Ag Secondary interaction- multivalent Ag- lattice formation Tertiary- involves secondary interaction leading to activation of 3rd component such as complement Ab tests- ppt, aggultination, complement fixation, EIA, RIA, ELISA, flow cytometry Cant measure Ag/Ab union kinetically- must measure at equilibrium

Primary union- direct and indirect Secondary union- ppt- reaches max then decreases (Ab>Ag, Ab=Ag, Ab<Ag) Immunodiffusion- secondary union- where lattice formation occurs size of ring formation is proportional to [Ag] Agglutination- secondary union- add Ab and see if agglutination takes place (blood typing) Tertiary union- complement fixation Ag + Ab and just Ag Add complement to both Add rbc to both If complement still active then get lysis of rbc Sandwich ELISA: Coat plate w/ Ab Add specimen containing Ag Add enzyme linked Ab Add substrate Measure color change Flow cytometry- used to analyze cells in suspension Generation of Ab diversity and Ig genes: Clonal selection- individual lymphocyte express membrane receptors specific for distinct Ag. Receptor specificity determined prior to Ag exposure. Somatic mutation- method by which point mutations are elicited; responsible for affinity maturation L chain- kappa and lambda genes- constant regions plus V and J regions V chain- V, D, and J regions and a number of different constant regions RSS- sequence that allows for cutting and recombining sequences RAG1 and 2- important in enhancing or making recombinase enzymes Class switching Occurs only in heavy chains Changes effector function of Ig Cannot reverse switching Cytokines can control Does not change specificity Removes introns rather than exons Accompanied by somatic mutations in Ig hypervariable region caused by point mutation in DNA Seeing Ag again can lead to switching from IgM to other Ig via Th cell cytokines Hyper IgM immunodeficiency- cant switch from IgM to IgG- X-linked; susceptible to infection; treat with IV IgG

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T Cell Receptors and MHC MHC- in humans found on short arm of chromosome 6; HLA Class I Co-dominant inheritance ALL nucleated cells 2 chains (alpha and beta) that are not covalently linked; Ag binds to looped domains alpha1 and alpha2. Beta chain does not bind Ag recognize self vs. non self (transplants, autoimmunity) recognize self + foreign Ag (viral infections, malignancies) CD8 Class II- found on APC- alpha and beta chains coded for by D gene regions binding zone made by alpha 1 and beta 1 recognize processed Ag in context of self CD4 TCR Near CD3 Binds Ag and CD3 sends signal CD8 recognizes alpha 3 domain on class I CD4 recognizes alpha 2 domain of class II

T cell cytokines- IFNg, GMCSF, IL-4, TNFb Redundancy in cytokines so if one not made proper course still occurs. CYTOKINES: Cytokines are soluble mediators involved in many cellular processes. Generalities: Low molecular weight Glycosylated protein Surface receptor Mode of action- autocrine, paracrine, endocrine Function and Properties: Lots of overlap in function and tasks Produced in novo Made when contact with Ag is made Non-specific mediators of immunity Regulatory of lymphocyte activation, growth, and differentiation Activators of nonspecific cells Stimulators of immature leukocytes Major cytokines of inflammation: IFN, TNF, IL-1, IL-6 TNFa leads to necrosis and inflammation- why cancer pts with bacterial infections do better. TNF can also result in shock and death Can make Ab to cytokines. TNF more problematic at higher concentrations. IL-1: Co-stimulator of inflammation and T cell function; produced by macro, epithelial, and endothelial cells; increases cAMP, nuclear factors, other cytokines, and prostaglandins, can slow cells down with adhesive properties (low concentrations) High concentrations- fever, APR, cachexia, but NO TISSUE DAMAGE OR NO TUMOR NECROSIS LPS causes increases in TNF, IL1 and IL6

T cell activation Most Ag are monomeric Capping of B cells leads to their activation. IL-2 induces the release of other cytokines. ANTIGENS ARE PROCESSED BEFORE THEY ARE PRESENTED TO T CELLS!!!! Class I- endogenous protein processing Class II- exogenous protein processing TAP- transporter necessary for moving peptides into RER for MHC I. Invariant chain- MHCII binding domain that prevents the binding of endogenous peptides. Co-stimulation reaction between B7 on APC and CD28 on T cell that leads to T cell activation for proliferation. Binding proteins: T cell- LFA-1, CD4, CD3(zeta chain), TCR, CD2, CD28 APC- ICAM-1, MHCII, LFA-3, B7 APC make IL-1 which causes Th cells to make IL-2 receptor and APC cells to make more MHCII receptors. IL-2 helps Tc cells in killing target cells- does not require costimulatory event. APC cytokines- IL-1, IL-6, TNFa, IL-12, IL-15

Interferon- (alpha [mac] and beta [fibroblasts])- inhibits viral replication, increases MHC class I, decreases MHCII, activates NK cells Chemokines- small cytokines Produced by leukocytes, endothelial cells, and fibroblasts Stimulate neutrophils, basophils, eosinophils, and lymphocytes Interferon (Type II (gamma))- produced by T cells Function- MAF, increased class I expression, activates T and B cells, neutrophils, NK cells etc

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IL-2 Produced by T cells (CD4) Major autocrine growth factor for T cells Leads to synthesis of other cytokines Acts on B, T, and NK cells Can live w/out IL-2

Inflammatory mediators: His, 5-HT, PAF, NCF, IL-8 (chemokines), C3a, C5a, bradykinin, fibropeptides, PG, LT, (mast cells and macrophages produce many of these) TNF causes PMN to be more adhesive. Macrophages release IL-12, and TNFa NK- IFNgamma Adhesion and Migration ICAM- intracellular adhesion molecule- allows cell to move through endothelium CD15 E selectin- slows movement of phagocytic cells in blood ADCC- Ab dependent cellular cytotoxicity- killer cell w/ Fc_ receptor (IgG) becomes active if it senses an immune complex to kill target cell Th0- makes IL12, IFN gamma, and IL4; proliferate into Th1 and Th2 Th1- controls classical side of immunity- activates macrophages- secretes IFNgamma, TNFa, and IL2 Th2- controls allergy side of immunity- activates mast cells, B cells and eosinophils (makes Ab)- produces IL4,5,6,10 MHC restricted- as T cells develop recognize self from nonself Perforin- in cytoplasmic granules Tc and NK cells induces lysis by making channels in target cell surfaces Tc and NK lytic mechanisms- lytic granules store and release perforin and granzymes- to form pores and induce apoptosis (respectively) When natural DNA repair is inhibited by TNF and granzyme can lead to apoptosis. Macrophages- phagocytize and present Ag to T cells and activate immune cytokines to release more cytokines, kill microbes and tumors Macrophage products Inflammation and fever- IL-1, IL-6, and PG Lymphocyte activation- processed Ag and IL-1 Tissue rebuild- angiogenesis factor, collagenase Microbicidal- reactive O2 and NO, lysozyme Tissue damage- H2O2 and TNF VACCINES: Vaccination- infection w/ agent (Ag) that induces an immune response- doesnt protect against infection, just the disease Passive immunity- immediate but transient; administration of preformed Ab; can result in serum sickness Adoptive immunity- T cells transferred into animal to impart immunity Active immunity- delayed but more permanent (vaccines)

IL-4: produced by T cells (CD4) and acts on B cells IL-10: Produced by T cells, decreases IFNg, increases B cells Hematopoiesis: CSF- colony stimulating factor IL-3- early progenitor GM-CSF: granulocyte/monocytes colony stimulating factor M-CSF, G-CSF Can be used by chemo pts and bone marrow transplant patients B CELL ACTIVATION Major APC: Interdigitating dendritic cells- induces T cell proliferation most effectively Macrophages- produce proliferation of T cells and helper functions B cells- most effective APC when Ag conc. is lowhigh affinity receptor on cell surface IgM binds complement the best Ab mediation is very important need B cell control. Plasma cells are terminally differentiated. Memory B cells have a long _ life Cross-linking is important w/ monomeric Ag and receptors on B cells. C3d- degradation product of C3b, binds CR2 on B cells to lower B cell activation threshold. CD40- important for B cell activation and class switching. No CD40 ligand on T cell when hyper IgM immunodeficient. CELL MEDIATED IMMUNITY: Innate CMI; Chemotaxis- activate macrophages or PMN directly or through C5a Phagocytosis- enhanced by C3b deposition on microbes and binding to complement receptors on phagocytes. Acute phase cytokines- binding and uptake of microbes by phagocytes also induces them to synthesize and release cytokines.

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Types of Vaccines: Th1- not susceptible to IFN gamma Killed organism- inactivated or killed vaccine Ag part of disease causing organism Attenuated or weakened preparation Toxoid vaccines- toxins treated w/ or absorbed w/ aluminum salts; usually must add adjuvant to increase immunogenicity Organisms similar to virulent organisms but that does not cause human disease Subunit vaccines- utilizes techniques of genetic engineering DNA plasmid vaccines- circular DNA plasmids expressing specific proteins are injected w/ presentation of the protein to the immune system CR2- augment immune response by binding to complement receptor Anti-Ids- produced to either non-antigen binding idiotyopes or to Ag binding idiotyopes Anti-Ids can either increase or decrease immune response Changes in the alpha chains can lead to changes in the binding cleft of MHCI and II. Polymorphism of MHC allows us to respond to some idiotopes and not others can respond to only a certain set of Ag due to genetic coding IMMUNITY TO TUMORS Tumors- breakdown of normal growth regulatory mechanisms; changes that occur in surface Ag of malignant cells can sometimes be recognized by the immune system Often a number of subclinical tumors. Tumor specific antigens- unique to cancerous cells and are not found on normal counterparts Tumor associated antigens- expression is greatly increased on tumors

Ways to give vaccines: Subcutaneous Intramuscular Intradermal Oral Live attenuated vaccines: Polio, measles, mumps, rubella, yellow fever, varicella zoster, hep A, TB Killed vaccines: polio, rabies, typhoid, cholera, influenza, plague, and pertussis Toxin Based Vaccines: Clostridium tetani (inactivated toxin), Corynebacterium diptheria (inactivated toxin), vibrio cholerae (toxin B subunit), Clostridium perfringes (inactivated toxin) Vaccines based on subcellular microbial fragments: Nisseria meningitis, Strep pneumoniae, H. influenzae B, hep B virus Safety problems with vaccines: Attenuated vaccines- reversion to wild type, severe disease in immunocompromised Killed vaccines- vaccines not killed, yeast, animal viruses, or endotoxin contaminant Organisms that have no vaccine: HIV, staphylococcus, Candida, malaria, schistomlasis Adjunvants: inorganic salts, delivery systems, bacterial products, natural products DNA plasmid based vaccines- live attenuated vaccines, make Ab and cellular immunity but risk infection; DNA seen as endogenous Ag REGULATION AND TOLERANCE: Regulation by antigens Proteins are best Too much or too little can lead to immune unresponsiveness or tolerance Subcutaneous or intradermal induce immunity Adjuvant augments immunity

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