Immuno Completed Objectives by Lecture

7/28/2019 Immuno Completed Objectives by Lecture

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Lecture 1: Overview What is the immune system and why do we need it?

Immune system is the ability of the body to differentiate self from non self and destroy non self. It enables us tocoexist with microbes in the environment

Difference between self and non self The innate system is made up of cells and molecules that function early in protective response to foreign agents

(antigens) using rectors to distinguish self from non self.

Differentiate between innate and adaptive immunityInnate Immune System Adaptive Immune System

Memory No YesSpecificity diverse Specific. The epitope of each antigen is

recognized by only a few lymphocytesResponse time fast SlowTrigger/regulation No unique antigenic specificity that

triggers responseContingent on action of cytokinesgenerated during innate immunity

Response generated Production of specific cells effector molecules or antibodies

Primary cell types Phagocytes, NKC, inflammatory cells,APC, serum proteins complement

Lymphocyte (T & B cells)

Proliferation Recruitment Binary division, clonal expansion

Barriers to infection first line of defense (innate immunity) Intact skin Mucosal lining (lysozyme) Chemical (stomach pH)

Cells of the immune systemInnate immune system

Receptor FunctionPhagocytes (monocytes,macrophages,neutrophils)

Phagocytose antigens

Antigen presenting cells(dendritic cells,macrophages, B cells)

Class II MHC Present fragments of ingested antigen onsurface that is then recognized by alymphocyte (t-lymphocyte)

NKC Exposure tocytokines

Destruction of virally infected (self) cells

Inflammatory cells (mastcells, basophils,eosinophils)

Receptors. Cells release histamine, leukotrienes, ECF-A, platelet activating factor, major basic

protein, eosinophil cationic protein

Adaptive immune systemReceptor Function

B lymphocytes Antibodies Once b cell recognizes antigen they willclonally expand into plasma cells

T lymphocytes Alpha/betaGamma/delta

CD4 (helper T cells) Recognize class IIMHC

Secrete cytokines

CD8 (cytotoxic T cells) Recognize class IMHC

Cytotoxicity

Define, clone, monoclonal, polyclonal as they apply to B and T cellso Clonal expansion is crucial to generate effective immunity against pathogens during first exposure. In the

presence of an antigen b cells will clonally expand and differentiate into antibody secreting plasma cells.


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o Antibodies secreted by each clone of cells are called monoclonal o Total antibody collection from all different clones are polyclonal

Describe the role of bone marrow and thymuso Hematopoiesis is the renewal of pluripotent stem cells. This process occurs in the bone marrow.o pluripotent stem cells give rise to precursor lymphoid cells some of which mature in the bone marrow. Others

migrate to the thymus.o B cells mature in the bone marrowo T cells mature in the thymus

Role of spleen and lymph nodeso Secondary lymph tissues are major sites of adaptive immune responseo Antigens carried in the blood trigger the adaptive immune response in the spleeno Antigens carried in the lymph trigger the adaptive immune response in the lymph nodeso Lymph nodes

afferent and efferent lymphatics primary follicles have mature b cells secondary follicles contain germinal centers of antigen activated b-cells regions between follicles have t-cells

o spleen secondary lymph organ contains white pulp contains majority of lymphoid cells, t-cells live in PALS contains red pulp

chemical classes of antigenso monoclonal

t-dependant - proteins t-independent

Type I - lipopolysaccharide Type II pneumococcal polysaccharide

o oligoclonal t lymphocytes - superantigens

o polyclonal t lymphocytes

pokeweed mitogen (PWM) concanavalin A (Con A) phytohemagglutinin (PHA)

b lymphocytes pokeweed mitogen (PWM) LPS???

Antigen, antigenic determinant, determinant, epitopeo Antigen molecules that have specific sequence to which adaptive immune responses can be generatedo antigenic determinant a component of an antigen that is recognized by unique lymphocyte receptors

b and t cells only recognize a unique region of the antigen called the epitope t cells only recognize antigens displayed with MHC

o determinant unique portion of an antigeno

epitope unique portion of an antigen antigenicity, immunogenicity

o antigenicity - a property of molecules to which an adaptive immune response can be generatedo immunogenicity refers to a molecule that can serve as the stimulus for an adaptive immune response

hapten & carrier o hapten & carrier small molecule that is not immunogenic unless it binds to a protein; carrier to which a small

molecule has bound permitting an anti-hapten immune response

mitogen


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o mitogen polyclonal activator; naturally occurring molecules that bind to and trigger proliferation of manyclones of lymphocytes. B-cell mitogens include pokeweed mitogen (PWM), t-cell mitogens includeconcanavalin A (Con A)

general structure of Ab, 5 isotypeso general structure

light chain variable and constant regions heavy chain variable and constant regions

o amtibodies are secreted by plasma cellso they are expressed on the cell surface of B cellso they serve as effector molecules for cells functioning in the innate or adaptive immune systemo isotypes (MADGE): IgG, IgD, IgM, IgE, IgA

cytokines and their roleo molecules that are secreted primarily by cells of the immune system. They play an important role in the

activation and in down regulation of immune responses

chemokines, chemoattractant molecules and their roleo molecules produced by activated lymphocytes in the innate immune systemo chemoattractant & chemokines small molecules that attract cells to a particular regiono serve to attact circulating neutrophils and monocytes to site of infection

role of complement and mode of activation, some proteolytic fragment generated during activation of C3b, C3a, C4a,C5a

o activation and regulatory proteins bound to cell membrane or found in circulationo synthesized by liver, macrophages and fibroblastso functions:

facilitate interactiono f phagocytes to induce opsinin-mediated phagocytosis induce osmotic lysis of microbes enhance vascular permeability by inducing degranulation of mast cells/basophils induce chemotaxis of neurtrophils facilitate immune complex elimination

o two pathways: classical and alternativeo both pathways generate C3b fragment in host defense and then converge into a common terminal pathwayo C3a fragment found in classical and alternative pathwayo C4a fragment found in classical pathwayo C5a fragment found in terminal pathway

Lecture 2: Innate Immunity Innate responses to pathogens

Phagocyteso Histology

Phagocytes are cells of innate immunity that include: macrophages, neutrophils Cells are found in the following tissues

Blood MonocyteBone marrow Monoblast

CNS Microglial cellsLiver Kupffer cellsSynovium SynoviocytesLung Alveolar marcrophagesLymph node & spleen macrophages

o Role internalize and destroy antigen, produce molecules that affect immune response

o Receptors Direct: primitive pattern recognition receptors (PPRR) Indirect:


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Fc R (receptor for IgG) CR-1 (complement receptor for C3b) CRP (c-reactive protein for CRP)

o Recognition Direct via primitive pattern recognition receptors on phagocytes that recognize sequences on a

number of microbes or antigens Opsin mediated opsins attach to pathogens.

Opsin is a molecule that binds to antigens and is recognized by receptors on phagocytes Opsins are the products of

o Complement activation (C3b)o B cell activation (IgG)o Cytokine mediated activation of hepatocytes (CRP)

o How it killsLysosomal enzymes NADPH oxidase forms the phagosome

The phagosome is a vesicle that contains the engulfed pathogen Lysosomes in the cytosol fuse with the phagosome forming a

phagolysosome Lysosomal granules contain many enzymes that are cytotoxic

Reactive oxygenintermediates

NADPH oxidase in addition to forming the phagosome also participatesin a reaction forming a reactive oxygen intermediate

O2 + NADPH oxidase superoxide + proton + NADP+ superoxide + proton hydrogen peroxide + O2 superoxide + hydrogen peroxide hydroxyl radical + hydroxyl ion

+ O2Hypochlorite hydrogen peroxide + proton + Cl- hypochlorite + H2OReactive nitrogenintermediates

Nitric Oxide eliminates intracellular organisms resistant to degradation in phagolysosomes by inhibiting ETC which breaks down cellular respiration and DNA synthesis

Nitric oxide reacts with reactive oxygen intermediates and generatesreactive nitrogen intermediates

l-arginine + O2 + nitric oxide synthase l-citrulline + nitric oxide nitric oxide destroys all cells in its vicinity but it has a short half life regulation of nitric oxide synthase

o activation requires two signals 1)priming signal by cytokine,TNF 2)IFN signal

o deactivation occurs in presence of IL-10, IL-4, TGF

Eosinophilso Histology

Derived in bone marrow from myelocyte cell Matures in bone marrow Travels to blood and connective tissues Cell with granules Half life is 8-10 hours

o Role

Immunity to parasites - helminthes (worms) which cannot be destroyed by neutrophils or macrophageso Receptor

FC R

o Recognition Indirect Fab portion of IgE binds to epitope on antigen (helminth) (IgE generated in adaptive immunity!!) IgE binds to FC R

o How it kills Docking of eosinophil with IgE causes degranulation of eosinophil and release of molecules:


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MBP membrane disruption by interacting with anionic lipid membranes Eosinophil cationic protein forms pores in membranes

NKCo Histology

Lymphocyte Derived in bone marrow from lymphoblast Matures in Bone marrow Travels to blood and connective tissues

o Role Attack virus infected cells, transplanted cells and cancer cells

o Receptors Direct - Cell expresses receptors that recognize a variety of viral proteins embedded in the cell

membrane of an infected cell NK activating receptor Killer inhibitory receptor (KIR)

Indirect FC R

o Recognition Direct NK activating receptor recognizes viral antigen receptor Indirect

FC R receptor recognizes IgG Fc

o How it kills Direct

NK activating receptor recognizes viral antigen receptor releases perforin granules thatcause lysis

Indirect host cell becomes infected with virus Viral envelope proteins form on cell surface Antibodies are generated that recognize envelope proteins IgG Fc antibodies enclose viral cell FC R receptor on NKC recognizes IgG Fc leads to Ab dependant cell mediated cytotoxicity (ADCC) NKC releases perforins and granzymes that lyse cell

Cells of Innate immunity (Accessory Cells): Mast Cells & Basophilso Histology

Mast cells Originate from progenitor cells in bone marrow Connective tissue cells with cytoplasm filled with basophilic secretory granules Granules contain histamine and heparin Surface receptor for IgE

Basophils Derived in bone marrow from myelocyte cell Matures in bone marrow Travels to blood and connective tissues Cell granules contain histamine and heparin

o Role release granules in response to certain antigenso Receptors

FC R C5aR C4a and C3a


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o Recognition

FC R receptor recognizes IgE C5aR receptor recognizes C5a C4a and C3a receptors recognize C4a or C3a complement fragments

o How it kills IgE Fc antibodies encase antigen forming cross links

FC R, C5aR, C4aR and C3aR recognize IgE Fc receptors Leads to release of histamine an inflammatory mediator which causes vasodilation of precapillaryarterioles resulting in increased blood flow to capillary beds causing increased vascular permeabilityand EDEMA

Activation of complement receptors release of anaphylatoxins such as histamine and other inflammatory mediators

Lecture 3: Antigen Presenting Molecules

Primary vs secondary immune responseo Primary immune response occurs in secondary lymphoid tissues and not at the site of infection (dendritic cells)o Secondary immune response occurs at the site of infection (macrophages

Types of antigen presenting cells (Professional APC)o Dendritic cells, macrophages, B cells all express MHC class II molecules (and class I molecules)o Antigen presenting cells present their antigen fragment to a CD4+ T cell

Types of target cellso Any nucleated cell all express MHC class I molecules (only)o Target cells present their antigen fragment to a CD8+ T cell

APCs - dendritic cells, macrophages, b cells dirty details

MHC HLA Describe the function of MHC molecules in antigen presentation

o Function of MHC molecules is to present antigenic fragments on the target cell surface so that it can berecognized by a T cell and destroyed

Diagram the genetic organization of the HLA, MHC, for Class I and Class II MHCo MHC is located on chromosome 6 and referred to as HLAo Class I MHC molecules are encoded by 3 loci (A, B, C)o Class II MHC molecules are encoded by a D locus subdivided into three loci (DP, DQ, DR)

Describe the murine system H-2

Dendritic cells Immature cells present in lymphoid and nonlympoid tissue (except brain) including:mucosal epithelium of oral cavity, anus, vagina. Called langerhans cells in skinepidermis

Transporter carry antigens from peripheral tissues to secondary lymph tissues Express Fc R, low concentrations of MHC class II and costimulatory

molecules, CD4 and chemokine receptors (CCR5/CXCR4) Cannot endocytose displays antigen and secretes cytokines

Macrophages Cells present in blood, bone marrow, CNS, liver, synovium, lung, lymph node,spleen

Primitive pattern recognition receptors also express CRI, Fc R, CRP receptors,MHC class II, CD4 and chemokine receptors (CCR5/CXCR4)

Can endocytose displays antigenB cells Recognize antibody specific receptors only (IgM, IgA, IgD, IgG, IgE)

Can endocytose displays antigen


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Explain MCH polymorphism and the likely selective advantageo Polymorphism is the occurrence of different forms of particular MHC proteins with the populationo The advantage of MHC polymorphism is that it can adapt to changing antigens to present fragments to t-cellso Necessary for population survival

Explain codominant expression and its advantage see MHC Class I for more detailso Codominant expression both alleles are expressed

Explain the terms allele and linkage disequilibriumo Allele one of two genes present at a specific locuso Linkage disequilibrium - is a term used in the study of population genetics for the non-random association of

alleles at two or more loci, not necessarily on the same chromosome. Linkage disequilibrium describes asituation in which some combinations of alleles or genetic markers occur more or less frequently in a populationthan would be expected from a random formation of haplotypes from alleles based on their frequencies. It is notthe same as linkage, which describes the association of two or more loci on a chromosome with limitedrecombination between them.

MHC Class I Id tissue distribution of class I MHC

o Any nucleated cell all express MHC class I molecules (only)

List major structural features of Class I MHC

o One transmembrane heavy chain noncovalently bound to 2-microglobulin Has a binding groove for peptide fragment to fit

o 2-microglobulin encoded on chromosome 15 Does not bind antigen Is not a transmembrane protein Assists with folding

o Murine H-2 Class I H2-K, H2-D, H2-L

Co-dominant expressiono MHC class I molecules are HLA-A, HLA-B, HLA-C each of which is codominantly expressed on all nucleated

cellso This means that one copy of each HLA (A, B, C) that is inherited from each parent will be transcribed and

translated into a protein with the potential to express six different HLA class I (MHC) moleculeso HLA molecules are polymorphic with several allelic formso The allelic forms are given number to designate different alleleso Each cell expresses many copies of each of the six HLA (A,B,C) proteins but only a few are loaded with foreign

antigenic fragments during infection

MHC Class II Id tissue distribution of class II MHC

o Dendritic cells, macrophages, B cells all express MHC class II molecules (and class I molecules)

List major structural features of Class II MHCo Heterodimer with two transmembrane proteins noncovalently boundo Heterodimers DQ, DP, DR structurally similar o The invariant chain transiently associates with MHC class II molecule in the ER forming a trimeric complexo This temporarily blocks the active antigen binding site and directs the transport of the MHC class II molecule

through the golgi

Co-dominant expressiono MHC class II molecules are HLA-DP, HLA-DQ, HLA-DR each of which is codominantly expressed on all

antigen presenting cellso This means that one copy of each HLA (DP, DQ, DR) inherited from each parent will be and a minimum of six

different class II molecules will be expressed on the cell surfaceo HLA molecules are polymorphic with several allelic formso The allelic forms are given number to designate different alleles


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Digestion yields two copies of a single Ag binding region F(ab) and one fragment Fc that cannot bindantigen

o Pepsin IgG + pepsin Fab2 + degraded Fc Digestion yields one molecule with two Ag binding sites Fab2. The Fc portion is degraded.

name two different types of light-chain constant regionso o

define allotype, isotype, subclasso allotype antigenic differences w/in a given class of Ig between members of the same specieso isotype - In immunology, the immunoglobulin isotype refers to the type of chain. In humans, there are nine

isotypes: heavy chain

- IgA 1, 2 - IgD - IgG 1, 2, 3, 4 - IgE - IgM

light chain

o subclass subclass within the antibodies (IgG1, IgG2, IgG3)

define Am, Gm, Km allotypeso Am the IgA allotypeso Gm the IgG allotypeso Km the kappa chain allotypes

define affinity and avidityo affinity the force between molecules that impels them to bind to one another (non-covalent)o avidity the sum of affinities

differentiate between monoclonal Ab and polyclonal Abo Monoclonal antibodies are antibodies that are identical because they were produced by one type of immune

cell and are all clones of a single parent cell o Polyclonal antibodies are antibodies that are derived from different B-cell lines. They are a mixture of

immunoglobulin molecules secreted against a specific antigen, each recognising a different epitope.

list Ig isotypes heavy chain

- IgA 1, 2 - IgD - IgG 1, 2, 3, 4 - IgE - IgM

light chain

list structural and biological roles/function of Ig isotypes

IgGmonomer

MW 150kDHighest in serumCrosses placentaPresent in milk and colostrum (IgA primary)Fc/ Fc R: opsonization (phagocytosis) and ADCC (by NKC)


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cells expressing receptors Fc R: NKC, neutrophil, monocyte, B-cellActivates complement: IgG1, IgG2, IgG3 (NOT IgG4)

Neutralization (viruses, toxins)Allotypic form Gm4 subclasses: IgG1, IgG2, IgG3, IgG4Halflife:IgG1, IgG2, IgG4 three weeksIgG3 one week

IgMMonomer &Pentamer formsSecreted form

MW 900kDIntravascular Half life 5-7 daysCovalent attachment: J chainPredominates in primary responseMonomeric form on immature + nave B cellsIsohemagglutinins of the IgM isotype stron agglutinationcapabilityActivates complement (classic pathway)

IgAMonomer, dimer andtrimer Secretory component

MW 320 kDHalf life 5-7 days2 Subclasses: IgA1, IgA2Present in mucosa associated lymph tissue, GI tract, secretions

Present in colostrum and breast milk (PRIMARY)Covalent attachment : J chain (associated with dimer and trimer)Secretory component only in mucosal secretionResistant to proteases and changes in pH

IgEmonomer

MW 195 kDHalf life 2 daysReaginic antibody ( allergic activity)Bound to FC R on mast cells and basophils leading to release of inflammatory and vasoactive mediators from granules in cellsImmunological role in helminth infections Can come on and off

IgDmonomer

MW 180kDExpressed on mature nave B cells along with monomeric IgM

Function unknown difference between dimeric IgA and secretory IgA

o dimeric IgA is in lamina propriao secretory IgA is present in lumeno dimeric IgA is converted to secretory IgAo disulfide bonds differentiate subclasses

describe how dimeric IgA becomes secretory IgAo dimeric IgA is on basolateral side of epithelial cello binds to secretory component and is internalized in epithelial cello secretory components cleaved and released into lumen

ID cells that express receptor for Fc portion of IgG ( Fc R) o cells expressing receptors Fc R:

1)NKC 2)neutrophil 3)monocyte 4)B-cell

explain how a hybridoma is generatedo cell that is the result of fusion of a tumor cell with a normal cello used to manufacture antibodies


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define cross linking and cross reactiveo crossreactive refers to fact that some antibodies will bind to antigens that have similar or same epitopeo cross linking is when two antibodies bind to an antigen epitope

define hypervariable regiono region of antibodies that varies from one antibody to another even within one class

define complementary determining regiono that part of the variable region which is hypervariable between antibodies

explain significance of Rh+ and Rh-o mother is Rh- and baby is Rh+o first pregnancy is ok since no anti Rh antibodies have been madeo second pregnancy will compromise fetus unless treatedo hemolytic disease of newborn

ABO Antigens relate cell surface antigens with blood type and associated isohemagglutinso Blood group polysaccharide antigens

Lecture 5: Complement describe overall function of complement in host defense

o family of ~32 effector and regulatory proteinso found in circulation, lymph, interstitial fluid or bound to the cell membrane

o function as effector molecules of immune system to:1. facilitate interaction of phagocytes to induce opsonin mediated phagocytosis2. induce osmotic lysis3. enhance vascular permeability by inducing degranulation of mast cells/basophils4. induce chemotaxis of neutrophils5. facilitate immune complex elimination

describe source of complement proteins and distribution of soluble components in bodyo synthesized by liver o synthesized by macrophages and fibroblasts

explain CH50 test and rational for using plasma CH50 levels in assessment of disease processo complement hemolytic tests

CH50 tests the classical pathway AH50 tests the overall complement activity

explain why we measure C3, C4 and the conditions that would lead to a decrease in serum levels of these twocomplement proteins

o decrease in C3 and C4 blood serum levels are indicators of an autoimmune disorder o will test C3 and C4 blood serum levels if the CH50 test result is abnormal

compare the alternative and classical pathways: activation , function, components, receptorsAlternative Classical Terminal

Activation C3b deposited on bacterium IgG1, IgG2, IgG3 or IgM (pentamermore efficient at binding complement)

bound to antibodyFunction C3b opsonin for

phagocytes & eliminationof immune complex

C5a chemotaxis for neutrophils (mastcells/basophils)

C3a anaphylatoxin(mast cells/basophils)

C3b opsonin for phagocytes &elimination of immune complex

C5a chemotaxis for neutrophils(mast cells/basophils)

C4a anaphylatoxin (mastcells/basophils) *** not acomponent of the alternativepathway

C3a anaphylatoxin (mastcells/basophils)

Terminal pathway structuregenerated (MAC attack complex)

Receptor C3b CR1 receptor C3b CR1 receptor


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C5a CR5a receptor C3a CR3a/4a receptor

C5a CR5a receptor C4a, C3a CR3a/4a receptor

Components/ protein

Factor BFactor DProperdin (stabilizingmolecule)C3 (C3a, C3b)

C1C2 (C2a, C2b)C3 (C3a, C3b)C4 (C4a, C4b)

C5 (C5b)C6C7C8C9

Regulation

proteins

Factor I

Factor HDAFC3a, C4a, C5a

C1

Factor IC4bpDAFC3a, C4a, C5a

HRF

S-protein (vitronectin)CD59

Alternative Pathway list components of alternative pathway

o Factor Bo Factor Do Properdin (stabilizing molecule)o C3 (C3a, C3b)

explain how pathway is activatedo C3 tickover Spontaneous generation of C3b from circulating C3

Active C3b is inactivated by water or complement regulatory proteins in absence of microbial infectiono C3b binds to microbial surfaceso Factor B binds to microbial surface nearbyo Factor D hydrolyzes Factor B (Ba, Bb)o C3b binds to the Bb fragment to form a C3bBb complex = alternative pathway C3 convertaseo C3 convertase stabilized by properdino C3b binds to the C3bBb convertase to form a C3bBbC3b complex = alternative pathway C5 convertase

list components of the C3 convertase AP, C5 convertase APo C3bBb complex = alternative pathway C3 convertaseo C3bBbC3b complex = alternative pathway C5 convertase

describe components of alternative pathway and role of proteolytic fragments generated during complement activationo Factor B binds to membrane bound C3bo Factor D proteolytically cleaves factor B (Ba, Bb)

Bb assists in formation of C3 convertaseo Properdin (stabilizing molecule)o C3 (C3a, C3b)

C3b Amplifies complement activity Required for both convertase complexes listed above

describe mechanisms that regulate pathway to minimize destruction of self o Factor I inhibitor of C3 convertase (C4b2a)

Cleaves C4b to make complex inactive Needs C1 cofactor

o Factor H Binds with C3b making complex inactive

o DAF inhibitor of C3 convertase (C4b2a) Competes for C3b or C4b binding sites making complex inactive

o C3a, C4a, C5a Anaphylatoxin inhibitor

Classical Pathway list components of classical pathway


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o antibody/antigen complexo C1o C2 (C2a, C2b)o C3 (C3a, C3b)o C4 (C4a, C4b)

explain how pathway is activatedo IgG1, IgG2, IgG3 or IgM (pentamer more efficient at binding complement)bound to antibody IgG

requires 600 molecules close enough for simultaneous binding by C1o Two adjacent regions of Fc regions must be bound by IgM or IgGo C1 bindso C1 cleaves C4 (C4a, C4b)o C4b moves to surface of antigeno C2 binds to C4b and is cleaved by C1 to form C4b2a complex = classical complement pathway C3

convertase o C4b2a complex cleaves C3 (C3a, C3b)o C3b binds to this complex to form C4b2a3b complex = classical complement pathway C5 convertase o C4b2a3b complex cleaves C5 (C5a, C5b)

list components of the C3 convertase CP, C5 convertase CPo C4b2a complex = classical complement pathway C3 convertase o C4b2a3b complex = classical complement pathway C5 convertase

describe components of classical pathway and role of proteolytic fragments generated during complement activationo C3a, C5a anaphylatoxins bind to receptors on mast cells and basophils

inducing degranulation and release of histamine and other inflammatory mediators causing vascular permeability

o C5a is chemotactic for phagocytes recruiting cells to site of infectiono C3a, C5a induce neutrophils to move to site of inflammationo C3b roles

Opsonin Facilitates the elimination of immune complexes Required for both convertase complexes listed above

o C5b deposit on cell surfaces starts the formation of the MAC complex which induces cell lysiso C2b increases vascular permeability, promotes inflammation

describe mechanisms that regulate pathway to minimize destruction of self o C1 inhibitor protein inhibitor of C1o Factor I inhibitor of C3 convertase (C4b2a)

Cleaves C4b to make complex inactive Needs C1 cofactor

o C4bp inhibitor of C3 convertase (C4b2a) Competes for C4b binding site making complex inactive

o DAF inhibitor of C3 convertase (C4b2a) Competes for C3b or C4b binding sites making complex inactive

o C3a, C4a, C5a Anaphylatoxin inhibitor

Terminal Pathway list components of terminal (common) pathway

o C5 (C5b)o C6o C7o C8o C9

describe components of terminal pathwayo C5b activated by C5 convertase, once C5b lands on cell surfaces it initiates MAC (C5b, C6, C7, C8, C9)

inducing terminal lysis


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describe mechanisms that regulate pathway to minimize destruction of self o HRF inactivate MACo S-protein (vitronectin) inactivate MACo CD59 inactivate MAC

Lecture 6: B-cell antigen receptor and antigen independent maturation/differentiation/ontogeny of B cells

B-CELL ANTIGEN RECEPTOR Role of b-lymphocytes

o Interact with Ago Process and present Ag to CD4+ t cells in secondary immune responseo Become Ab factory following differentiation to plasma cellso Differentiation to memory cells

List components of B cell receptor complexo B cells express Antigen recognition receptors on cell surfaceo Receptors are called membrane immunoglobulin ( mIg)o mIg are expressed in association with CD79a/CD79bwhich couples mIg to intracellular signaling pathwayso immunoglobulins are composed of two identical heavy chains and two identical light chains linked together by

disulfide bondso each chain is made of a variable and constant regiono

the light chain variable region is composed of segments V, Jo the heavy chain variable region is composed of segments V, D, Jo constant regions are encoded by same genes in all B-cells

SOMATIC RECOMBINATION Describe and explain molecular genetic mechanisms involved in generation of Ab diversity

GENERAL FEATURESo The clones of the b-cell repertoire make diversity possible because the V,D,J segments which encode variable

regions are present as multiple germ lineso Gene segments are recombined so their sequence differs from that present in the germline DNA somatic

recombination

VARIABLE REGIONSo Somatic recombination requires RAG1/RAG2to activate recombination

Variable heavy chain RAG1/RAG2 randomly select one D and one J to form the DJ segment whichcombines with a V gene to form the variable VDJ region combinatorial diversity

Variable light chain- RAG1/RAG2 randomly select one J and one V to form the VJ segmento During the recombination process the unused VDJ segments are deletedo Additional DNA nucleotides may be inserted at VDJ segment junctions junctional diversity which is

mediated by template independent DNA polymerase terminal deoxynucleotidyl transferase (Tdt)o The successful rearrangement of a heavy chain variable region inhibits the somatic recombination of the heavy

chain variable region on the other chromosome pair allelic exclusion o So all mIg present on the surface of any one B cell will have the same heavy chain variable regiono In nave mature B cells

heavy chain constant regions are and Light chain constant regions are either or

SUMMARY The construction of unique variable regions leading to the construction of different mIgs on Bcell clones is dueto:

o Multiple copies of germline VDJ gene segmentso Random selection and combination of VDJ segmentso Junctional diversity caused by adding and deleting geneso Random assortment of light and heavy chains

Explain autoreactiveo The random selection of VDJ gene segments generates b-cell receptors that may be autoreactiveo Autoreactive b-cells are deleted or inactivated


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Describe role of the products of the following in somatic recombination

o recombinase activating genes ( RAG-1, RAG-2) recombination process requires activation of recombinases (RAG-1, RAG-2)

o enzyme terminal deoxynucleotidyl transferase (Tdt) incorporation of nucleotides at junctions is mediated by template independent DNA polymerase called

Tdto CD79a/CD79b- couples mIg to intracellular signaling pathwayso Pseudo light chain sequence common to all b-cells needed to express pre-BCR o CD19 expressed in pro b-cell stage

Define the term B cell repertoireo A person can generate 10 9-10 11 different b-cell clones each bearing mIg whose variable regions differ from

those on other cloneso Collectively these clones are called the b-cell repertoireo Diversity is made possible because of the V,D,J segments which encode variable regions are present as multiple

germ lines

Define somatic recombination, combinatorial diversity, multiple germline genes, allelic exclusiono somatic recombination during differentiation of b cell the DNA in the locus is cut and recombined to make

an intact gene for the variable regions in the light chain and havey chain. This gene can be transcribed to mRNA

and translated into the light and heavy chain occurs in bone marrow during b cell developmento combinatorial diversity multitude of gene segments that comprise variable regions of BCR and TCR o multiple germline genes diversity in a b cell is made possible because the V, D, J gene segments which

encode the variable regions are present as multiple germline genes gene segments are recombined so their sequence differs from that present in germline DNA

o junctional diversity heterogeneity that results from either the removal or addition of bases at the junction of DNA segments: V, D, J

o allelic exclusion rearrangement of a heavy chain variable region from one chromosome inhibits the somaticrecombination of the heavy chain variable region on the other chromosome pair

B-CELL MATURATION/DEVELOPMENT/DIFFERENTIATION

Describe sequence of events in b cell developmental pathway from a stem cell to a mature nave cello Self renewing stem cello Pluripotent stem cello Lymphoid progenitor o B-cell progenitor o Pro-b cello Pre-b cello Immature b cello Nave mature b cell

Describe the steps of b-cell differentiationo Occurs in bone marrow Characterized by expression and silencing of genes at discrete stages of development

o Produces a nonautoreactive cell repoirtoire

Pro b-cell stage Transcription of multiple genes RAG-1/RAG-2

o CD19o Tdto CD79a/CD79bo Pseudo light chain -

Pre b-cell stage DNA gene rearrangement of heavy chain Allelic exclusion Initial transcription of heavy chain associates with light chains to form pre -BCR


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pre-BCR is expressed on the cell surface with CD79a/CD79bheterodimer composed of

o two heavy chains o two pseudo light chains

DNA gene rearrangement of light chains or CD 19 still expressed CD 20 expressed

Immature b-cell stage Initial transcription of light chains or Association of light chains or with heavy chain to form BCR Shift in receptor expression from pre-BCR to BCR BCR is expressed on the cell surface with CD79a/CD79b heterodimer

complexed witho two rearranged heavy chains synthesized for BCR

expression associated with two light chains or Downregulation of pseudo light chains Downregulation of pre-BCR Tolerance induction apoptosis CD 19 still expressed CD 20 still expressed

Mature b-cell stage Alternative splicing (RNA) heavy chain constant region change to makeIgD from IgM default

Surface expression of both IgD and IgM Surface expression of adhesion molecules Surface expression of CD40, CD21, CD81, CD45 CD 19 still expressed CD 20 still expressed

Explain how CD19 and CD20 can be used to play a role in clinical practiceo A lymphoma that expresses CD19 would be classified as a B-cell tumor o Measurement of CD19 levels helps to classify the stage of cell developmento When we know the stage of cell development of the tumor it aids in determine how to treat it

Explain allelic exclusion with respect to immunoglobulin gene expressiono Allelic exclusion is the successful rearrangement of a heavy chain variable region from one chromosomeinhibits somatic recombination of the heavy chain variable region on the other member of the chromosome pair

What is meant by autoreactiveo One of the events that can happen during recombination is to make a receptor that recognizes self o These are destroyed or degraded before reaching maturityo The event of a b-cell maturation is the generation of a b-cell repertoire that is nonreactive

Explain tolerance induction during b cell development, apoptosis, anergyo Specific inactivation of b cells expression autoreactive mIg tolerance induction is accomplished by apoptosis or

induction of anergy which makes them unresponsive

Explain alternative splicing in B cell development, when does it occur and where?o

Default B-cells are IgMo Alternative splicing occurs on some cells to make IgDo The RNA of the constant region of the heavy chain is altered not the DNAo This occurs in the bone marrow after tolerance induction in the mature b-cell stage

Lecture 7: When a B-cell encounters an antigen (B-cell antigen induced differentiation) General features

o Antigen encounter leads to isotype switching after affinity maturationo Differentiation to antibody secreting plasma cells and memory b-cellso Primary encounter with antigen occurs in secondary lymphoid tissue producing the primary immune response


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Activation of nave b cellso Secondary lymph tissues contain regions with T and B cells

B cells live in follicles T cells live in regions around follicles

o Initiating stimulus is antigen induced crosslinking of the b-cell receptor complexo Followed by endocytosis in a cytosolic endosomeo Lysosomes fuse with the endosome , release enzymes and degrade antigeno Class II MHC fuse with antigen and moves to B cell surfaceo The complex is recognized by activated CD4+T cells and leads to T-cell/B-cell conjugate formation stabilized by

ICAM I LFA-1 LFA-3 CD2

o Conjugate formation facilitates several membrane bound ligand interactions that encompass b-cell signals B7/CD28 CD40/CD40

o Proliferation occurs in secondary lymph tissueso Activated B and T cells migrate to primary follicles where enhanced proliferation of B cells leads to formation

of germinal centers

Fate of activated B cells in germinal centerso Germinal centers are associated with

B cell clonal expansion Isotype switching Affinity maturation Differentiation to plasma cells Differentiation to memory cells

o Differentiation to IgM secreting plasma cells - pentamer B cells express antigen specific antibodies Plasma cells secrete antibodies (IgM, IgG) Antibodies are not expressed on cell surface Antibodies have same specificity Differentiation to plasma cell stage occurs 4 days after immunization and is marked by secretion of

IgM antibodies Nave b cells undergo isotype switching

o Isotype switching switch recombination Depends on prior CD40-CD40 ligand interaction Cells expressing IgM and IgD are modified at the genomic level and produce antibodies of different

isotypes (IgA, IgE, IgG) IgE needs cytokine 4 Changes in the heavy chain constant region determines the antibody isotype

DNA in and regions is exised resulting in , , Specificity is maintained only because the constant region of the Ig heavy chain is altered B cells expressing new isotype will differentiate to plasma cells or memory b cells

o Affinity maturation/somatic mutation Point mutations that occur in the hypervariable region within both light and heavy chain variable

regions

Leads to production of antibodies with higher specificity Occurs in germinal centers 7-10 days following b cell activation during a primary immune responseo Generation of memory b cells

Differentiation of activated b cells to memory cells occurs in germinal centers one week after theantigen encounter

Most memory b cells are in the pool of recirculating b cells that circulate in the blood, lymph andtissues that were primary sites of previous antigen encounter

Memory b cells survive for weeks and months

B cell regulationo Fc R turns off antibody production


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Primary vs secondary immune response

o Primary exposure leads to antigen dependant b-cell differentiationo Secondary exposure to the same antigen induces the activation and differentiation of memory b cells generating

a secondary immune response No lag phase All steps happen faster Memory b cells are activated

Antibody secretion occurs sooner Magnitude of the response is greater Predominantly antibodies of new isotype

Lecture 8a: T-cell antigen receptor and antigen independent maturation/differentiation/ontogeny of T cells

T-CELL ANTIGEN RECEPTOR Role of T cells

o CD4+T cells secrete cytokineso CD8+T cells induce osmotic lysis of infected cells

What is the Tcell receptor complexo T-cells express unique receptors that recognize Ag fragments only when the fragments are displayed with a

Class I or Class II MHC molecule.o T-cells express several different TCR due to somatic recombination of the TCR variable geneso Recombination occurs in the thymus

Describe / and / recepto r complexeso TCR are composed of two transmembrane polypeptide chains classified as / or / o The / receptor heterodimer complex is most prevalent o The variable region has V and J segments o The variable region has V, J and D segments o The / receptor heterodimer complex is joined by the CD3 complexo These heterodimers are expressed on CD8+T cells and CD4+T cells

CD4+T cells secrete cytokines that modulate the activity of NK cells, macrophages, B cells, CD8+Tcells and CD4+T cells

CD8+T cells induce osmotic lysis of infected cells

Compare T-cell receptor complex with B-cell receptor complexo T-cell receptor complex - / receptor heterodimer complex is joined by the CD3 complexo B-cell receptor complex - membrane immunoglobulin (mIg) is joined by CD79a/CD79b heterodimer complex

SOMATIC RECOMBINATION Describe and explain molecular genetic mechanisms involved in generation of T cell receptor diversity

o There can be as many as 25,000 identical TCRs on any given cello Clones expressing these receptors represent the t-cell repertoireo Somatic recombination of the TCR variable region occurs prior to TCR

The variable region has V and J segments The variable region has V, J and D segments

o Diversity is made possible because of somatic recombination of the V, D, J segments that encode the variableregions, specifically it occurs due to: 1) Multiple germline genes

2) Combinatorial association 3) Junctional diversity from Tdt 4) Random selection of / chains

Describe role of the products of the following in somatic recombinationo recombinase activating genes (RAG-1, RAG-2)

recombination process requires activation of recombinases (RAG-1, RAG-2)o enzyme terminal deoxynucleotidyl transferase


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incorporation of nucleotides at junctions is mediated by template independent DNA polymerase calledTdt

Explain allelic exclusiono Inhibition of somatic recombination on the other alleleo Prevents two different chains being expressed with a chain which would result in two different antigenic

specificities bad

Define the term T cell repertoireo There can be as many as 25,000 identical TCRs on any given cello Clones expressing these receptors represent the t-cell repertoire

T-CELL MATURATION/DEVELOPMENT/DIFFERENTIATION Describe the steps of T-cell developmental pathway from stem cell (bone marrow) to mature CD4+, CD8+ T cell

(thymus)o Self renewing stem cello Pluripotent stem cello Lymphoid progenitor o T-cell progenitor o Thymus migrationo Growth only occurs in the thymus with presence of cytokine IL-7

Describe the steps of T-cell developmental pathway within thymusPhase I expressionof pre-TCR

Phase II-doublepositive thymocytesand expression of /TCR

Phase III-thymocyteselection

Phase IV negativeselection

Thymiclocation

Cortex Cortex cortex o Cortex (double positive selection)

o medulla (single positive selection)

Genesexpressed

RAG1/RAG2CD2 panmarker TCR Pre- T CD3

RAG1/RAG2CD2 panmarker CD4CD8TCR Pre- T CD3

CD4 or CD8CD2

CD4 or CD8CD2

Somaticrearrangement

TCR TCR ---- ----

Genesdownregulated

--- Pre- T CD4 or CD8 ---

Cell surfaceexpression

CD2Pre PCR complex

CD2CD4CD8TCR complex (CD3 +TCR /)

CD4 or CD8CD2TCR complex (CD3 +TCR /)

CD4 or CD8CD2TCR complex (CD3 +TCR /)

Other Allelic exclusion ---- Positive selection Negative selectionDeath by neglect(double positive)

Lineage selection

Negative selection(single positive)

Comment Need thymicepitheliumCell proliferation

Need thymicepitheliumCell proliferation

Need thymicepithelium

Need APCs that are bone marrow derived(MHC/antigen)

Explain process of positive and negative selection in thymus and death by neglect


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o Maturation of MHC complexes in the thymus is dependant on how a t-cell receptor recognizes an MHCcomplex. This recognition is called avidity. This differentiation occurs on double positive t-cells

o Positive selection(cortex) When the t-cell receptor recognizes an MHC Proliferation occurs appropriate interactive avidity

o Negative selection for double positive(cortex/medulla) the t-cell receptor recognizes an MHC but the avidity is too high apoptosis very high interactive avidity

o death by neglect (cortex) When the t-cell receptor does not recognize an MHC Death negligible interactive ability

o There is still one more phase a cell has to go through negative selectionfor single positive thymocytes(medulla)

High interactive avidity with bone marrow derived APC CD4 or CD8 interaction with MHC also occurs

Explain terms lineage selection, double positive thymocytes, single positive thymocyteso lineage selection

refers to the transition of a double positive thymocyte (CD4+,CD8+) to a single positive thymocyteexpressing either CD4 or CD8 but not both

silencing of either the CD4 or CD8 gene occurs in each individual T cell

t-cell lineages are classified by the expression of CD4 or CD8 reflective of subsequent biologicalactivity (cytokine secretion or killing)o double positive thymocytes

cell surface expression of both CD4 and CD8 on the developing thymocyte occurs early in phase IIo single positive thymocytes

transition of a double positive thymocyte (CD4+,CD8+) to a single positive thymocyte expressingeither CD4 or CD8 but not both

Explain term interactive avidity and list four factors on which interactive avidity dependso Interactive avidity determines fate of double positive T-cellbased on

# of TCR on T-cell # of MHC molecules on APC amount of intrinsic affinity of TCR for MHC peptide # adhesion molecules # antagonistic molecules There are approximately 25,000 t-cell receptors on a cell

NAVE LYMPHOCYTE RECIRCULATION

List the selectins and integrins expressed on a nave lymphocyteo Mature nave t cells exit the thymus and populate other lymph tissues or enter the lympho Mature nave t cells travel in blood and lympho Bloodo lymph

migration to lymph occurs at postcapillary venules called high endothelial venules (HEV) glycocalyxof endothelial cell traps molecules t-cells move through lipid bilayer by extravasation by the mechanism below:

l-selectin - grabs t-cell integrins (LFA-1) makes t-cell sticky matrix metalloproteinases degrade collagen to make channels in basement membrane for

t-cells transendothelial migration of lymph into tissue

In the absence of antigen recognition lymphocytes exit via efferent lymphatics and enter mainlymphatic circulation

List their counter molecules present on the post capillary venules in peripheral lymph nodes and in mucosal lymph nodeso high endothelial venules (HEV) -migration to lymph occurs at postcapillary venuleso glycocalyxof endothelial cell traps molecules


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o l-selectin- grabs t-cello integrins (LFA-1) makes t-cell stickyo matrix metalloproteinases degrade collagen to make channels in basement membrane for t-cells

Lecture 8b: Primary Immune Response CD4 +T cells and CD8 +T cells

PRIMARY IMMUNE RESPONSECD4+T CELLS

T cells that leave the thymus areo CD4+ nave cell (Thp)o Precytotoxic CD8+ T cells (pCTL)

general features of CD4+T cellso CD4+ T cells are a subset of T cells whose primary biological function is the secretion of cytokineso CD4 +T cells that survive the screening process in the thymus are nave because they are unable to perform

biological function without further differentiationo This differentiation stage takes place outside the thymus in secondary lymph tissues differentiation takes

place where? Blood spleen Mucosal tissue MALT lamina propria Lymph lymph node white pulp

o Differentiation is induced by interaction with peptide/class II MHCo Differentiation produces T-cell subsets Th1, Th2 that secrete unique cytokines

o T-cells require that the antigen be displayed on the surface of the APC (dendrite) in association with class IIMHC

o T-cell interacts with peptide/MHC and co-stimulatory molecules on dendrite (or other APC) antigenrecognition - how?

Dendrite (APC) with antigen/peptide class II MHC on surface Nave T- cell has CD3 complex with / receptor

Cell surface interaction for nave T-cell (Thp) activation Relevant molecular interactions to activate Thp o CD4+ T cell activation only occurs with all of the following interactions

APC T-cell Class II MHC TCR Class II MHC CD4ICAM (1,2,3) LFA-1 (CD11a/CD18)CD40 CD40L (CD154)LFA-3 CD2CD80 CD28CD86 CD28

Progression from Thp to Th1 or Th2 subseto An active Thp now expresses

Expression of IL-2R and secretion of IL-2 induces expansion of Th0 clones from antigenstimulated T-cells

Secretion of IL-12 from dendrite activates NKC to secrete IFN o Th0 clones secrete cytokines

IFN Th1 cells (type 1 cytokines) IL-2 makes more Th0 clones IL-4 Th2 cells (type 2 cytokines)

o Concentration of cytokine secretion determine pathway and development of Th1 or Th2o Th1 and Th2 cells secrete cytokines

Some cells become memory T cells (memory Th1, memory Th2) Most cells die by apoptosis

CD4 Thp cell activation (antigen dependant) Key Points Thp cells differentiate to Th0 cells Th0 cells secrete IL-2, IFN , IL-4 IL-12 stimulates NK cells to secrete IFN as well


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Mast cells trigger secretion of IL-4 Whichever cytokine predominates [ IFN , IL-4] determines the cell that is made Th1 or Th2

Activation of Thp to Th1 cells: Secondary Immune ResponseType 1 Secretes

IL-2IFN TNF

o Supports immune responses in which macrophages, NK and CTL are effector cells

o Responsible for DTH delayed hypersensitivity responseo Support isotype switching to IgG2ao Required for immunity against viruses, parasites, fungi,

and intracellular bacteriaType 2 Secretes

IL-4IL-10IL-13TGF

o Supports B cell induced activationo Supports antigen induced differentiation of B cells to

plasma cellso Supports isotype switching to IgG1 and IgEo Required for immunity against extracellular bacteria and

parasites

T-Cell regulation occurs viao CD152 with CD80/CD86 for Th1

CD80/CD86 CD152 (CTLA-4) T-cell downregulation

CD80/CD86 CD28 Tcell activationo Reciprocal regulation

IFN Th1 cytokine turns off Th2 IL-10, IL-4 Th2 cytokines turn off Th1

o Mode IL-10 mediated regulation for Type 2 IL-10 inhibits secretion of IL-12 by APC IL-10 also downstream inhibits Th1 and prevents NKC from producing IFN

PRIMARY IMMUNE RESPONSECD8+T CELLS

T cells that leave the thymus areo CD4+ nave cell (Thp)

o Precytotoxic CD8+ T cells (pCTL)

Activation of pCTLo pCTL needs to interact with

CD4+T cell derived IL-2 cytokine Target cell expressing antigen (APC (dendrite, macrophage) expressing antigen with Class I MHC) CD2 LFA3, LFA1 ICAM (1-3) CD8 needs to bind to class I MHC at a site separate from where it binds to the TCR

o pCTL does not need a special environment to induce differentiation (CD4+Tcell needs secondary lymph tissue),it can develop at site of infection

progression from pCTL to CTLo IL-2 expressiono preCTL detatches from target cell expressing antigeno differentiation takes about a week

delivery of lethal hito mature CTL (CD8+) conjugates with target cello conjugate formation triggers locatization of lytic granules to membrane where two cells are attachedo granules are released outward toward target cello polarized release of granules ensures specificity of killing target cell and not self


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o death caused by osmotic lysis or apoptosiso CTL detaches and continues its function of immunological surveillance

delivery of lethal hito as elimination of virally infected cells occurs the antigenic stimulus for specific CD8+Tcells decrease.o CTL clones that are generated with TCRs specific for the antigen will die others will become dormant (memory

cells)o Memory cells will become activated immediately in the event that the antigen reappearso These cells require less costimulation than for the primary response

superantigens explain mechanism of superantigen recognition of t-cell compare with nominal antigen recognition why do superantigens activate so many t-cells?

E rosettes explain e rosettes and role in clinical lab

Lecture 9: InflammationInflammationo

5 hallmarks of inflammationo Redness (rubor) increased blood flow, extravasation of RBC to areao Heat (calor) due to increased blood flowo Edema (tumor) increased vascular permeability, intersitital fluid o Pain (dolor) bradykinin stimulates nerve endingso Pus necrotic tissue from dead neutrophils

o Relevant cells and proteins in tissues and in circulation prior to inflammationo

o Tissue damage, microbial infections, toxic agents induce inflammation activating:o Complement systemo Mast cell degranulationo Blood coagulation contact system producing bradykinino Macrophages, dendriteso T-calls/b-cells

Activation of intrinsic coagulation pathway and consequenceso Role of kallikrein in production of C5a, bradykinin, neutrophil activation

o Kallikrein hydrolysis of C5 generates C5a cleaves kininogen to release bradykinin

o Stimulus for histamine release from mast cellso Mast cells express FC R o FC R binds to IgE antibodies with active Fc regions that have a bound antigeno Antigens bind to FAB region of IgE via crosslinks to the FC R o triggering mast cells to release histamine

o Role of histamine on expression of p-selectin on endothelial cellso P-selectin is moved from the cytoplasm to the plasma membrane in the presence of histamine

o Role of p-selectin in acute inflammationo P-selectin is expressed on plasma membranes not on circulating cellso P-selectin induces rolling of the leukocytes on the endothelium

o Effects of histamine on endothelium and role it plays in acute inflammationo Histamine causes increased vascular permeability


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Activation of tissue macrophageso List six actions of activated macrophages

o Secrete cytokines (IL-1, IL-6, IL-12, TNF)o Secrete chemokines (MCP-1, IL-8)o Secrete matrix metalloprotieinases (degradation)o Secrete tissue inhibitors of matrix metalloproteinases (regulation)

o List local effects of TNFo Enhance cytotoxicity of macrophages

o List systemic effects of IL-1 and TNFo TNF and IL-1 stimulate endothelial cells to express adhesive molecules

E-selectin VCAM-1 Chemokines IL-8 MCP-1 Produce vasodilation Induce fever

o Explain how IL-6 secretion is induced and the source of IL-6o

Cytokines affect IL-6o IL-6 induces the secretion of acute phase proteins (CRP)

o Why do levels of c-reactive protein increase in pt with heart diseaseo IL-6 induces secretion of acute phase proteins from the liver called CRPo CRP serves as an opsonin for enhanced phagocytosis of bacteriao People with heart failure will have higher levels of CRP

Activated endotheliumo Role and stimulus for cell surface expression of e-selectin/p-selectin

o E-selectin Induced by TNF and IL-1 Lead to leukocyte rolling on endothelium

o P-selectin Induced by histamine Lead to leukocyte rolling on endothelium

o Effects of IL-1 and TNF on vascular endotheliumo Produces vasodilation contributes to low blood pressureo Induce fever modifies hypothalamus

o List integrins present on both neutrophils and monocytes and integrin counter molecules and which ones areupgregulated in acute inflammation

o PSGL-1 p-selectino Ligand:e-selectin eselectino MAC-1 ICAM-1o LFA-1 ICAM 1 or ICAM 2

o Name counter molecule for VLA-4 and the effect of IL-1 and TNF on its expressiono VLA-4 VCAM 1o IL-1 and TNF will promote expression of VCAM-1 on endothelial cell surface

o What is CD18?o CD18 activation epitopes induced by leukocyte activation

o Role of IL-1 and TNF to induce cells to express MCP-1 and IL-8 and why circulating leukocytes move toward region inwhich these molecules are present in high concentration

o IL-1 and TNF stimulate endothelial cells to secrete chemokines IL-8 and MCP-1


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IL-8 induces adhesiveness of 2 integrins (LFA-1 and MAC-1) shedding of L-selectin chemotactic for neutrophils neutrophils will move to a region with a high concentration

of IL-8 MCP-1

chemotactic for monocytesand lymphocytes monocytes and lymphocytes will move to aregion with a high concentration of MCP-1

o Explain what is meant by IL-1 and TNF act synergisticallyo They have a pleiotropic effect to stimulate the endothelial cell to increase adhesiveness

Rolling, margination, diapedesiso List integrins present on both activated or memory lymphocytesand integrin counter molecules

o PSGL-like p-selectino Ligand:e-selectin eselectino VLA-4 VCAM-1o LFA-1 ICAM 1 or ICAM 2o LPAM VCAM-1

o Role of p-selectin, e-selectin, eLFA-1, MAC-1, VLA-4, PE-CAM with respect to Rolling, margination, diapedesiso What cells are these molecules expressedo What cell is the ligand expressd

Lecture 10: Cytokines

Cytokine Mac Den NK Mast Thp Th0 Th1 Th2 T-cellsIL-1IL-2IL-3IL-4IL-5IL-6IL-10

IL-12IFN Type 2interferonTNFTGF CSFGM-CSFIFN / Type 1interferonIL-7

Cytokine Sources Target Effect and role in immune responsesIL-1 macrophages 1-Activate macrophages

2-Triggers inflammation3-Activates endothelial cells to express

o Denovo - ICAM-1, VCAM-1, e-selectino Enhance ICAM-2o Secrete chemokines IL-8, MCP-1

4-Systemic effectso Low blood pressure/shock o Fever (hypothalamus)

TNF Macrophages 1-Activate macrophages


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Th-1 2-Triggers inflammation3-Activates endothelial cells to express

o Denovo - ICAM-1, VCAM-1, e-selectino Secrete chemokines IL-8, MCP-1

4-activate iNOS (along with IFN enhancing cytotoxicity of macrophagesREMEMBER that TGF **, IL-10, IL-4 downregulate iNOS)

IL-2 ThpTh0Th1

o Growth factor for T-cells (activated with low concentration of IL-2)o Enhances NK cell cytotoxicity (activated with high concentration of IL-2)o Plays role in pCTL to CTL differentiation (cytotoxic t-lymphocyte)

IL-3 T-cells Hematopoesis (growth factor)IL-4 Mast

Th0, Th21)Required to isotype switch to IgE2)Down regulation of Th1 cytokines3)Down regulation of iNOS (along with IL-10 and TGF **)4)Shifts Th0 response to Th2 phenotype

IL-5 MastTh2

o Hematopoesis (growth factor) differentiation of eosinophilso Chemotactic for eosinophilso Activation of eosinophils

IL-6 MacTh2

o Inflammatory response (IL-12, IL-1, TNF) stimulus for secretion of CRP from hepatocytes assist with opsonization of bacteria

o Can test blood for levels of IL-6 to determine CHDIL-7 Mac

Den NK MastThp, Th0,Th1, Th2T cells

o Hematophoesis bone marrow and thymuso Amplifies amount of B & T cells

IL-10 Th2 Th1 o Downregulates IL-12 secretion by dendritic cells and macrophages (leadsto downregulation of Th1 cytokines)

o Downregulates iNOS but not as effective as TGF IL-12 Mac

Den NK Activate NK cells to secrete IFN which leads to development of Th1 (CD4+)

cellsIFN / Type 1interferon

All cellsinfected withviruses that

producedoublestranded RNS

Triggers production of enzymes (2,5, oligoadenylate synthetase) that interferewith replication of some viruses

IFN Type 2interferon

NK Th0Th1

1)Activates iNOS2)Enhances activation of NADPH oxidase3)Downregulates production of Th2 cytokines4)Role in Th0 differentiation to Th1 phenotype5)Along with IL-2, promotes pCTL to CTL differentiation

TGF Th2 Downregulates iNOS (along with IL-10, IL-4)CSF Mac o Hematophoesis granulocyte

GM-CSF Mac

T cells

o Hematophoesis granulocyte or monocyte

Chemokine Sources Target Effect and role in immune responsesIL-8(CXCL8)

MacEndothelialcellsTh2

Neutrophils Chemotactic for neutrophilsInflammation induces adhesiveness of 2 integrins making the endothelialsurface sticky for neutrophils (during rolling and diapedesis process)

MCP-1(CCL2)

MacEndothelialcellsTh2

Monocytes Chemotactic for monocytes


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Eotaxin(CCL11)

Epithelial cellsFibroblasts

Eosinophils Chemotactic for eosinophils

Chemoattractant Sources Target Effect and role in immune responsesC5a Chemotactic for neutrophils

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Immuno Completed Objectives by Lecture