IMMUNOLOGY LEARNING OBJECTIVES - Boston ... · Web viewIMMUNOLOGY LEARNING OBJECTIVES CHAPTER 1: ON MICROBES AND HOST DEFENSE Specify the basic characteristics of the four major categories

IMMUNOLOGY LEARNING OBJECTIVES

CHAPTER 1: ON MICROBES AND HOST DEFENSE- Specify the basic characteristics of the four major categories of microbes

o Bacteria Single cell prokaryotes (no nucleus; dsDNA) Gram positive

Retain stain upon treatment with alcohol or acetone Cell wall = thick peptidoglycan layer around cytoplasmic membrane)

Gram negative Loses stain upon treatment Cell wall = thin peptidoglycan layer around cytoplasmic membrane; inside to out: cyto membrane

peptidoglycan lipoprotein layer outer membrane toxic lipopolysaccharide (LPS) LPS = main component of outer membrane

Both gram positive and negative have flagella (protein = flagellin) Some bacteria surrounded by capsule (protective outer layer of polysacc) Bacterial diseases: bacterial pneumonia, gonorrhea, tuberculosis, Legionnaires’ disease, strep throat

o Fungi Eukaryotes (nuclei) Two forms: yeasts (single cell) and molds (multi cell) Cytoplasmic membrane surrounded by multilayered cell wall Cell wall = chitin (glucosamie polymer) and zymosan (complex polysacch) Some yeasts have polysacch capsule Molds have hyphae (branches/tubular filaments with multiple nuclei) Fungal infections: athlete’s foot, jock itch, vaginal yeast infections, thrush

o Parasites Invertebrates that require host Two groups: protozoa (unicellular euk) and metazoa (worms/helminthes; multicellular) Diseases: malaria, sleeping sickness, pinworm, intestinal roundworm

o Viruses Reproduce only inside hosts; therefore are called obligate intracellular parasites Core of nuclei acid surrounded by capsid (protein coat); larger viruses have envelope made of lipids

surrounding capsid Infecting hosts: bind port/carb receptor on host cell via attachment protein on viral surface; host cells with

receptor for viral species are susceptible to that specific infection Virus cycle: virus attaches and penetrates host viral particle uncoated transcription of viral genome

packaging and release Non-enveloped viruses: lyse host cell upon exit Enveloped viruses: exit host via budding (doesn’t kill cell); capsid proteins embed into host cell memb

(envelopment) enveloped viral particles bud out of cell Viral diseases: AIDS, polio, chickenpox, smallpox, measles, hepatitis, herpes infections, mono, flu,

common cold- Identify the components of the immune system

o Self-tolerance: lack of reactivity against self componentso Specificity: discriminating self vs. non-selfo Antigen/immunogen: induces immune responseo Molecular recognition of antigens via noncovalent interactions: electrostatic bonds, hydrogen bonds, aromatic-

aromatic, hydrophobic, van der Waalso Epitope: part of antigen (antigenic determinant) that directly interacts with receptor on immune cells

Repeating epitopes: identical Linear epitopes: contiguous array of subunits Conformational epitopes: 3-D structure that is destroyed when unfolded

- Draw stick diagrams of the structures of antibodies and T cell receptors and specify their secondary structures

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o Antibodies Two heavy chains (V and C) and two light chains (V and C) Noncovalent and covalent intercahin disulfide bonds Variable regions (V): VH-VL responsible for antigen binding; amino acid sequence differences allow

different Ab to bind different antigenic determinants Within one Ab, two VH are identical; two VL are identical = same antigen specificity All constant regions in one type of Ab are identical H and L chains: globular domains Hinge: amino acid stretch connecting CH1 and CH2; allows Ab arms to move CH group: carbohydrates present Ig fold: two antiparallel beta shets connected by intradomain disulfide bond of both variable and constant

domains; conformations fit particular antigen Complementarity determining regions (CDR1, CDR2, CDR3): complementary to parts of antigen

determinant; contact Ag; loops come together to form Ag binding site Framework regions (FR1, FR2, FR3, FR4): interspersed among CDRs and act as scaffold to align

CDR loopso Receptors

Two types of glycoprotein receptors: T cell receptors and antibodies (on B cells) – can make 1011 to 1018

different antigen receptors (diversity) TCR: membrane-bound

All TCRs expressed by one cell are identical Heterodimer: either alpha-beta (95%) or gamma-delta (5%) (but not both) Amino variable region Carboxyl constant region Each chain folded into Ig-like globular domains (one variable and one constant) – each domain

held together by intradomain disulfide bond All alpha (for ex) C regions identical but are different from all delta C regions vs. all V regions

different even if chains are same type Three CDRs interspersed with less variable FRs (4)

Antibodies: membrane-bound or secreted- Distinguish between innate and adaptive immune responses

o Innate: general defense force that first meets foreign antigen; recognition of general features of antigen pattern recognition receptors

Uses pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) on microbes

PAMPs: flagellin, bacterial lipoproteins, LPS, zymosan PRRs: soluble and membrane-bound molecules; Toll-like receptors (TLR1-TLF10); each TLR recognizes

different type of PAMPo Adaptive: generated after 5-10 days after body is familiar with invader

Primary immune response: first specialized response Secondary immune response: greater amplitude and better accuracy; indicates memory

- Explain the interaction between antigens and antibodies in terms of valence, affinity avidity, crossreactivity and crosslinking

o Valence (number of binding sites in molecule) One antigen binding site (eg. Fab, Fv): monovalent Two antigen binding sites (eg. Intact Ab, F(ab)2): bivalent

o Crossreactivity Same antibody combining site can bind (react) with distinct though structurally related epitopes One Ab can crossreact with several Ag determinants

o Affinity Strength of binding for each Ag determinant Measured by ease of association/dissociation (equilibrian association constant = 1/dissociation constant)

o Avidity Measured by Ka; strength of association and ability to not dissociate Two arms of Ab have greater avidity than one arm (Bivalent > monovalent)

o Crosslinking Ab can bring together more than one Ag molecule (when one Ag has multiple epitopes that Ab can bind) Agglutination: crosslinking by Ab on insoluble particle Immunoprecipitation: crosslinking by Ab with soluble antigens

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- Calculate antigen-antibody binding constantso Ka = [AbAg]/[Ab][Ag] = 1/Kd

- Recognize the “big picture of immunology”o Two aims: ID the enemy, destroy enemy without harming bodyo See Fig. 1-21 on page 9o Two main cell type categories

Innate: monocytes/macrophages, granulocytes, NK cells, dendritic cells Adaptive: B and T cells that produce Ab and TCRs respectively

o All cells derived from hematopoietic stem cells in bone marrow migrate thru blood from central lymphoid organs (bone marrow for B cells, thymus for T cells) where they mature exit via blood, circulate thru blood and lymph and stop to inspect peripheral lymphoid organs (spleen, lymph nodes, tonsils, Peyer’s patches); these organs are meeting places for antigens and lymphocytes; where lymphocytes are activated by antigens, divide, and differentiate exit organs via lymph, travel to tissue sites where microbes have entered

CHAPTER 2: THE MAJOR HISTOCOMPATIBILITY COMPLEX AND ANTIGEN PRESENTATION TO T CELLS- Recognize the role of major histocompatibility complex (MHC) molecules in immune responses

o MHC: host molecules that antigen fragments (peptides) associate witho Peptide-MHC complexes that bind complementary antigen receptors on T cells activate T cells to respond (direct

kill or recruitment of killers)o Displayed on host membrane but allows for monitoring what is occurring inside host cello Peptides from both foreign Ag and self Ag are displayed on host cell membrane but immune system is tolerant of

self MHC-self peptide complexes- Distinguish between MHC class I and MHC class II molecules and genetic loci

o Class I MHC Alpha chain – crosses plasma membrane Beta chain – assoc with alpha chain but is not anchored to membrane Peptide binding site – alpha1 and alpha2 domains Bind endogenous antigens Expressed on almost all nucleated cells; therefore, it marks the APC (aka. Target cell) for destruction by T

cells direct killo Class II MHC

Alpha chain and beta chain – both cross membrane Peptide binding site – alpha1 and beta1 domains Bind exogenous antigens Activate T cells to secrete substances that recruit or activate other cells of immune system to eliminate

foreign Ago MHC Loci

Express multiple MHC types because they are encoded by multiple genetic loci MHC – refers to region of genome that contains loci that encode the MHC protein molecules; human MHC

= HLA (human leukocyte antigen on chrom 6) Class I loci – encode alpha chain of class I Class II loci – encode alpha and beta chains of class II Beta-2 of class I encoded on separate chrom (so not in the MHC) Classical HLA loci – class I (B, C, A) and class II (DP, DQ, DR)

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Alpha and beta chains encoded are different but similar (thus different peptide-binding specificities) Many alleles for each loci – polymorphisms (differences within species) found at alleles for one locus;

polymorphic residues concentrated in areas corresponding to peptide binding site (alpha1-alpha 2 class I domain, alpha1-beta1 class II domain)

Codominant MHC expression – inherit different alleles from mom and dad resulting in expression of both maternal ad paternal MHC molecules on surface of one cell

Inherit m haploypte from mom and p haplotype from dad diploid genotype is mp- Compare the peptide-binding characteristics of MHC class I and MHC class II molecules

o Peptide binding site: al-a2 (class I) and a1-b1 (class II); platform of 8-stranded beta sheet (floor of groove) and two alpha-helices (walls)

o Other 2 domains of each class fold up to form globular structure (2 anti-parallel beta sheets connected by disulfide bond)

o Only about 20 MHCs but each one can accommadate many peptideso MHC I – bind endogenous Ag that are 8-10 AA long; peptide ends buried in MHC pockets and anchored at anchor

positions (complementary to peptide side chains, polymorphic)o MHC II – bind exogenous Ag that are 12-20 AA long; peptides allowed to extend outside fo binding site; bind

peptides via hydrogen bonds; bind peptides with key anchor position AAo Upon being bound, each peptide has different part of itself exposed for recognition by T cell R; TCR recognizes

both bound peptide and MHC surface- Draw stick diagrams of the structures of MHC class I and MHC class II molecules and describe their secondary

structures

- Distinguish between the endogenous and exogenous pathways of antigen processing and presentationo Endogenous pathway (cytosolic)

Endogenous Ag derived from host/viral proteins synth in cytosol (hence cyto pathway) Peptides produced as part of normal cell metabolism then degraded by proteosomes meanwhile:

alpha an dbeta-2 class I chains synth with leader sequences that direct their translocation to ER where calnexin (ER transmembrane prot) physically associates with alpha chain and facilitates its dimerization with beta2m to form unstable, incompletely folded class I molecule degraded peptides are transported into ER via TAP (transmemb molecule) peptides bind to incompletely folded class I molecules peptide binding induces conformational change in alpha chain causing it to dissociate from calnexin and to stabilize association with beta2m peptide-MHC molecule packaged into secretory vesicle in Golgi fuses with and displayed on membrane

o Exogenous pathway (endocytic) Ag enter cell via endocytosis (hence endocytic pathway) Ag enters and plasma membrane invaginates it in a vesicle that then fuses with early endosome; some fuse

with late endosome acidic endosome interior causes antigen to unfold and be partially degraded (these endosomal parts are displayed eventually on cell) meanwhile: alpha and beta class II cains translocated into ER and associate with calnexin; invariant chain (transmemb prot; Ii) assembles with alpha and beta and blocks peptide binding site to prevent class II molecules from binding peptides in ER II-Ii complex dissociates from calnexin and goes to Golgi where it is transported to endosomes (not cell surface) peptides and class II meet in endosome whose acidic conditions degrade Ii chain petide loaded onto class II and transported via vesicles to cell surface

- Identify the professional antigen presenting cellso Always express class II molecules and can act as APCs for exogenous antigenso Will also have class I because class I is expressed on all nucleated cells; therefore can present endogenous Ag alsoo Types: B lymphocytes, macrophages/monocytes, dendritic cells, some epithelial cellso Because they express class II MHC, it means they inform T cells, don’t directly kill

CHAPTER 3: T CELL ACTIVATION BY ANTIGEN- Compare and contrast the three categories of T cells and explain how they function

o Cytotoxic (TC) – kill target cells

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Express CD8 (CD8+) Binding of TCRs on TC cell to class I causes death of target Must be activated in order to kill target cells Activate requires T cell-target cell contact and costimulation provided by IL-2; cytokine binding to receptor

induces differentiation to functional cytotoxic T lymphocyte (CTL) Activated CTLs have granules released via exocytosis (perforin: forms channels/pores in membrane that

allows entry of other released granule components like granzymes/proteases); granzymes initate caspase activation (cystein proteases) leads to apoptosis

Activated CTLs have enzymes on cell surface that inactivate perforin (protection) Caspase activation also by Fas (membrane receptor) interaction with Fas ligand (FasL) on CD8+ and CD4+

T cells therefore, CD4+ cells can also be cytotoxico Helper (TH) – enhance immune system

Express CD4 (CD4+) Produce and secrete cytokines: IL-2 (TH cell proliferation via binding IL-2 receptors that have also been

Ag-induced = autocrine stimulation that expands Ag-selected T cell clone) Help activate B cells through contact mediated by adhesion molecules

o Suppressor /Regulatory (TReg) – dampen immune system Prevent activation of other self-reactive lymphocytes that might cause tissue damage Most are CD4+ and express CD25 (IL-2 receptor component) Act via direct contact with cells they suppress or through release of inhibitory cytokines

- Identify the antigen forms recognized by T cellso TCR contacts both the peptide and MHC molecule and binding is specific to both (noncovalent interactions)o Antigen fragments presented via:

MHC Classes I and II Nonclassical (nonpolymorphic MHC class I) – these Ag may be non-peptide (lipids, carbs, etc) CD1 – non MHC protein expressed on many professional APCs; assoc witih beta-2 microglobulin

o Native/intact AG – recognized by gamma-delta T cells; include cell-surface or secreted molecules expressed by microbes

- Specify the molecules and pathways involved ino Antigen recognition by T cells

Antigen recognition (signal 1) –coreceptors adhesion molecule enhanced cell-cell interaction –signal transduction costimulation (signal 2) prolferation and differentation 2 pathways: (1) CTL cell killing and (2) TH or Treg cytokine secretion, contact, help, inhibition

Signal 1 alone is insufficient for full T cell activation Two transmemb proteins that have signaling functions and associate with TCRs

CD3 complex: gamma, delta, epsilon chains that dimerize Disulfide-linked zeta-zeta dimmer TCR + CD3 + zeta-zeta = TCR complex – when TCR engaged, Tyr-sequences of all chains are

phosph (sequences = ITAMs: immunoreceptor tyr-based activation motifs) Coreceptors

CD4 – bind MHC Class II (most TH and Treg are CD4+); therefore, MHC Class II restricted (CD4 T cells bind and are stimulated only by Ag complexed with Class II)

CD8 – bind MHC Class I (most TC are CD8+); class I restricted

98% alpha-beta T cells = CD4+or CD8+ but most gamma-delta T cells lack CD4 and 8 so not MHC-restricted

Adhesion molecules Enhance adherence of T cell to APC/target cell Transmembrane proteins that interact with complementary adneshion molecules (ligands) on other

cell CD45 – transmemb prot tyr phosphatase (PTPase)

Alpha-Beta T Cell APC/TargetTCR Peptide/MHC

Coreceptors CD4 MHC IICD8 MHC I

Adhesion molecules LFA-1 ICAMCD2 LFA-3CD45 CD45L

Signal transduction CD45 CD45LCD3/zeta-zeta

Costimulation CD28 B7 (CD4 & CD8)

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4-1BB 4-1BBL (CD8)

o T cell activation Immunological synapse – area of contact between T cell and APC/target cell; promotes crosslinking to

initiate activation of both cells Signal transduction

CD3 complex + zeta=zeta dimmer + CD4 or 8 + adhesion molecules Singal transmitted to nucleus via phosph and dephosph activates DNA binding prot increase

transcription of specific genes Transient phosph (peak in minutes) transcriptional activation peaks several hours later

Progressive activation T cell/APC target binding upregulation of cell surface proteins Isoform (new form) of CD45 expressed on T cell (shorter extracellular portion)

o CD45RO – interacts with different ligand on APC/target cello CD45RA – isoform on unactivated T cells

B7 costimulatory signalo Aka signal 2 transmitted by CD28 and bound by APC ligand B7o B7 – transmemb protein on APC Class IIo T cell stimulation B7 binds CTLA-4 receptor (with higher affinity than CD28 so out

competes CD28 binding) causes inhibitory signal downregulates T-cell responseo CD4 T cells requires B7 costimulatory signal for full activation; class II restrictedo Cross-presentation – dendritic cells ingest extracellular Ag and yield peptides presented

by class I MHC rather than class II (even though dendritic = professional APC); don’t have to be infected with microbe to display microb peptides

4-1BB on T cell w/4-1BBL on APC interaction = second co-stimulatory signal for full activation of CD8 T cells

Cytokineso Secreted peptides that bind cytokine receptors – paracrine effectso Each cytokine has specific receptoro Autocrine effect – cells that make specific cytokine have cell surface receptors for that

cytokineo Diffuse radially away from cell that produces them and bind target cellso Cytokine concentration (and therefore cytokine receptor occupancy) decreases with

increasing distance therefore mediation is short-rangeo High receptor occupancy needed for signal transduction

o T cell proliferation T cells initially at rest prior to binding peptide-MHC complex (G0) activation induces them to G1

proliferation (peaks after several days from initial cell sitmulation) amplification of antigen-specific T cells

Each activated T cell yields many descendant cells that express same TCR and therefore have same Ag specificity

Clone – cell collection with identical Ag specificy Ag selects T cells with complementary receptors for expansion into clones (clonal selection) – responsible

for generation of Ag-specific T cell responses T cell clonal expansion accompanied by differentation of proliferation cells into T cell blasts CD4+ T cells diff into T helper or suppressor cells CD8+ T cells diff into cytotoxic T cells Polyclonal response – Ag stimulates expansion of many different T cell clones

o Generation of T cell memory Primary immune response – first exposure that generates Ag-specific memory Secondary immune response – quicker, stronger longer-lasting response as a result of re-exposure Memory generated during A-specific activation of resting T cells ot proliferate and differentiate Activated T cells express CD45RO (shorter isoform of CD45) therefore are called CD45RO+ cells Resting T cells = CD45RA+ cells If CD45RO+ memory-type T cells are reexposed to Ag, respond more quickly than the CD45RA+ T cells Memory mediated by two mechanisms

Persistence of CD45RO+ activated/memory type T cells – very efficiently activated by Ag Increased frequency of long-lived resting CD45RA+ T cells – expanded during primary immune

response and collective can give rise to secondary immune response of higher magnitude

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CHAPTER 4: B CELL ACTIVATION BY ANTIGEN- Identify the immunoglobulin (Ig) isotypes and draw stick diagrams of their structures

o Functions of Ab parts Fab – antigen binding formed by Vh and VL Fc region – responsible for destruction or removal of bound Ag; recruits other prot and cels to eliminate

antigen (effector functions); also mediates transport functions (binds cell surface receptors that transport Ab)

o Membrane vs. Secreted – carboxyl terminal end present in secreted form is replaed in mIg (memb form) by cyto tail and stretch of hydrophobic amino acids that anchor it to membrane; mIg also known as B cell receptor (BCR)

o Isotypes (classes and subclasses) IgM – H chain = mu IgD – H chain = delta IgG – H chain = gamma; subclasses = G1 thru G4 IgE – H chain = epsilon IgA – H chain = alpha; subclasses = A1 thru A2 Light chains for all isotypes = kappa or lambda (not class and subclass specific like the H chains are)

o Structure Basic structure = Ig monomer (4-chain Y structure H2L2) Cell surface Ab of all isotypes are monomeric Secreted Ab are polymeric for some classes IgG, IgE, IgD – monomers always IgM (secreted) – pentameric or hexameric (5-6 H2L2 monomers present); monomer subunits held together

by interhcian disulfide bonds that connect heavy chain C regions; dominant form (pentameric) has J chain (disulfide-inked polypeptide)

IgA (secreted) – predominantly dimmers (2 H2L2); IgA1 has hinge (IgA2 doesn’t); one disulfide-linked J chain per molecule in dimeric IgA

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- Define Ig allotypes and idiotypeso Allotypes – proteins encoded by inherited differences in C region in same isotype due to presence of different alleles

in individuals within same specieso Idiotypes – different structures specific to V regions of each antibody (IgM kappa Ab specific for flu will have

different idiotype than IgM kappa for pneumococcus)- Specify the molecules and pathways involved in

o Antigen recognition by B cells B cell receptor complex (BCR)

mIg associated with Ig-alpha and Ig-beta heterodimer 2 heterodimers associate with Ig monomer (1 per heavy chain)

cytoplasmic domains of Ig-alpha and Ig-beta have ITAMs (immunoreceptor tyr-based activation motifs) that particiate in signal transduction when BCR is engaged

resting B cells have IgM and IgD BCR complexes but no secreted Ig produced; have samge Ag specificity since mIgM and mIgD on single B cell have identical V regions

Coreceptors in Ag binding by B cells Transmemb proteins CD19, CD21, TAPA-1 strengthen interaction between Ag and B cell CD21 – binds host peptide (C3d) that attaches foreign Ag to clear them (C3d part of complement

system)o B cell activation

Specific binding of IgM and IgD Ab to complementary Ag determinant stimulates B cell and activates it to proliferate and differentiate into Ab secreting cell

Upon Ag binding (since Ig tails aren’t long enough) cyto tails of Ig-alpha and Ig-beta and coreceptor complex interact with protein tyr kinases (PTKs) to start signal transduction kinase cascade leads to change in gene transcription

Signal-initiated phosphorylation increases when Ag that binds has several epitopes and can crosslink BCR complexes enhanced signal transduction

T-dependent antigens Antigens that require T cell help to activate B cells (since Ag binding to mIg may be insufficient –

true for soluble Ag with few identical epitopes so poor crosslinking) T cells help via direct cell-cell contact (contact help) and cytokine secretion

o Contact help – increase the avidity of binding between T helper and B cells via complementary adhesion molecules signal transduction that acts in synergy with signals induced by BCR complex B cell activation

o Cytokine help – activated T cells secrete cytokines that bind to upregulated receptors on contacting B cell cytokines directionally secreted towards contacting B cell max B cell stimulation

Helper T cells via T cell/peptide-MHC bind APC activated T cell interact with and activate B cells to proliferate and differentiate

Increase in number of Ag-specific B cells B cells become major APCs (early in immune reaction, dendritic cells are major APCs) recruit T cell help; B cell is faster in recruitment b/c it endocytoses antigen and displays it on class II molecule B and T cell recognize same Ag (linked recognition)

Haptens

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o Small epitopes that can bind B cell receptors (eg. Drugs)o After being internalized by B cell or APC, can’t recruit T cell help b/c no peptides

produced; therefore, binding of haptens to BCRs does not lead to B cell activation haptens therefore are not immunogenic

o If hapten attached to carrier protein, then hapten-specific B cells can be activated: they bind the hapten-carrier conjugate, form peptides that are presented to T cells on class II

T-independent Ag Ag that can activate B cells without T cell help Large multivalent antigens with many identical epitopes (hence extensive crosslinking) Typical antigens are bacterial cell wall components, flagellin units Extensive crosslinking transduces signal strong enough for full B cell activation Some T-ind Ag (like LPS) at high doses activate many B cells regardless of Ag specificity of

receptors (so may clone selected) – these antigens are polyclonal B cell activators (mitogens)o B cell proliferation

Naïve/resting B cells arrested at G0

Exit G0 upon activation Ag selects only the B cells with complementary receptors for proliferation = clonal selection

o Antibody secretion by B cells Differentiation

Proliferating activated B cells become bigger B cell blasts (plasmablasts) become nondividing plasma cells Ab production and secretion

Sequence of differentiation accompanied by decrease innumber of membrane Ig molecules because plasma cells don’t need Ag stimulation anymore

See class switching and somatic hypermutation below Remember: Ag select the cells in order for immune response to occur so clonal selection is mechanism by

which adaptive immune system operateso Generation of B cell memory

Proliferation of B cells induced by Ag creates 2 cell subsets: plasma cells and memory B cells Memory B cells – nondividng that express mIg but no secreted Ig Most memory B cells underwent somatic hypermutation or class switching (so display IgA, E, G) Encounter Ag memory B cells stimulated to divide differentiate into Ab secreting plasma cells

(quicker and greater magnitude b/c Ag-specific memory B cells were expanded during primary response and are more numerous than Ag-specific naïve cells and b/c somatically mutated and selected Ag receptors on memory B cells have higher affinity than naïve cells)

- Explain how Ig class switching and somatic hypermutation contribute to the maturation of the antibody response Ig class switching

B cell can change H chain isotype of its antibodies In early immune response: main Ig class secreted is IgM and IgD rarely secreted As immune response progresses: B cells can start producing IgG, IgA, IgE – switch occurs both

in membrane and secreted forms Even though Ig isotype has changed, V domains and progeny stay the same and light chain stays

the same Class switching induced by: T cell-released cytokines, binding of CD40L on T cells to CD40 on

B cells Class switching very prevalent in Ab responses to T-dependent antigens Predominant Ig class secreted with T-independent antigens = IgM Somatic hypermutation

Mutations in expressed H and L chain variable regions only in somatic cells with higher rate of mutation than normal somatic mutation

Changes Ag-binding affinity of expressed Ab so can halt signal proliferation if Ab can no longer bind to the correct antigen or can increase affinity for Ag as Ag levels decrease, the differentiated daughter cell with improved binding will beat out the other mutations and continue to proliferate while the others die

Somatic hypermutation occurs at same time as clonal selection: Ag select B cells that produce higher affinity Ab increased average affinity = affinity maturation

Dependent on T cell signals so greater mutation in T-dependent than T-independent Ab responses- Distinguish between T-dependent and T-independent antibody responses

o See B cell activation objective- Distinguish between primary and secondary antibody responses

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o Polyclonal Ab – heterogeneous mixture of Ab molecules all reactive with same antigen; Ab responses to almost all Ag are polyclonal

o Primary Ab response – first encounter with foreign Ag Most secreted Ab produced inearly mmune response to T-dependent Ag = IgM class As immune response progresses, class switching to IgG, A, E, somatic mutations/higher affinity

o Secondary Ab response – subsequent encounter of same Ag with quicker and greater magnitude response Memory B cells stimulated by Ag to proliferate and differentiate into Ab secreting plasma cells Characteristics: faster, higher levels of Ag-specific Ab, longer period of time, higher proportion of non-

IgM isotypes due to class switching, higher affinity for antigen due to somatic hypermutation and affinity maturation

o Most of Ab responses to T-independnet antigens consist mostly of IgM and show little/no memory- Compare B and T cells for antigen binding and activation

CHAPTER 5: GENERATION OF ANTIBODY AND T CELL RECEPTOR DIVERSITY- Distinguish between the germline and somatic configurations of antibody and T cell receptor (TCR) loci

o Germline configuration Antibody Loci

Light chain kappa locus: 5’–V—J—C—3’ Light chain gamma locus has V, J, C genes but in different organization H chain: 5’—V—D—J—constant region C—3’

TCR No switching of C genes nor somatic hypermutation occurs Leader sequences (l exons) Random pairing of alpha and beta or gamma and delta chains (diversity)

- Identify the mechanisms involved ino Rearrangement of antibody and TCR loci

Antibody (somatic) Rearrangement of H and L loci occurs by recombination of V region gene segments One VL gene and one JL gene brough together to generate expressed VL region gene One VH gene, one DH gene and one JH gene brought together to generate expressed VH region Somatic recombination is as above Recomb of VL and JL and VH, DH, and JH occurs by deleting DNA between recombining gene

segments: deleted DNA includes coding and non-coding regions H locus: 2 recomb events hav eto occur to generate VH region

TCR Junctional diversity (diverse joing process) Required activities for recomb of V region

o Alignment of recombining gene segmentso Cleavage of DNA between recombining gene segments

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o Ligation (physical joining) or recombining gene segments Activities carried out by V(D)J recombinase (DNA binding and enzymatic functions); RAG prot

(recombination-activating genes only expressed in B and T cells; components of VDJ recombinase)

o Simultaneous expression of IgM and IgG by the same B cell Naïve B cells express both mu and gamma H chains with identical VH regions due to differential splicing

of RNA transcripts that originate at promoter of recombined VH gene and include VDJ gene, Cmu gene, and Cdelta gene

Primary transcript spliced to generate either mu or gamma mRNA depending on site of cleavage and polyadenylation

o Expression of membrane versus secreted antibodies Leader sequences on L and H chains to direct them to ER; helps to determine which Ig’s are secreted or

remain on membrane sequences cleaved off as chains translocate into ER and then H and L chains associate to form intact Ab

L and H transcription starts at promoters of VJ and VDJ genes respectively on 5’ end of each V gene and transcription is enhanced by enhancers (DNA that activates trancription from any promoter that is a few kilobasepairs away)

In germline: VJ and VDJ recomb brings promoters and enhancers closer Membrane and secreted terminie of C gene of each H chain type are encoded by different exons Each CH gene has several axons (unlike C-kappa and C-gamma genes which have only one exon)

One exon for each CH domain: CH1-CH3 for delta, gamma, alpha; CH1-CH4 for mu and epsilon One exon for hinge region (excludes mu) Two exons for carboxyl terminus of membrane form Carboxyl terminus of secreted form is encoded by 3’ end of last CH exon (CH3 or CH4) Example (C-mu): has six exons: C-mu1—C-mu2—Cmu3—Cmu4 for CH1, CH2, CH3, CH4

domains respectively these genes followed by muM1 and muM2 for exons encoding membrane carboxyl terminus (M1 = transmemb region; M2 = cytoplasmic tail of heavy chain of membrane Ig); if secreted, carboxyl terminus = S and is preceded by intra-exon splice site

o Mu: Cmu1—Cmu2—Cmu3—Cmu4—(intra-exon splice site)S—(ss)muM1(ss)—muM2 Production of secreted or membrane form of H chain RNA depends on competition between RNA splicing

and cleave/polyadenylation PolyA sites – upstream of M1 exon and downstream of M2 If primary mRNA cleaved and polyA-ed upstream of M1 secreted form encoded If first intra-exon splice site used and Cmu4 is ligated to M1 S exon and cleavage site upstream

of M1 eliminated RNA polyA-ed downstream of mu-M2 exon encodes membrane form of H chain

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o Ig class switching B cell may switch H chain isotype to G, E, or A after being induced by Ag Method: differential splicing of long transcripts that include relevant CH gene (this method used by B cells

before lots of secreted Ab have been made) For massive production of IgG, E, A faster more efficient method: produce shorter transcripts

Switch recomb – rearranges H chain locus by bringing VDJ gene close to a G, A, or E C gene by deleted intervening C genes therefore, first C gene downstream of VDJ gene now encodes constant region of expressed heavy chain

Switch regions – long DNA stretches upstream of each CH gene except delta; involved with deletion of intervening C genes; recognized by nuc enzymes and aligned to help the looping-out of intervening DNA (one enzyme = AID) figure below = switch from IgM to IgA:



o Somatic hypermutation After B cell encounters T-dependent Ag, V regions can be changed by somatic hypermutation to increase

affinity of Ab during immune responses Introduces pont mutations AID (activation induced cytidine deaminase) – enzyme expressed only in B cells and mediator of somatic

hypermation: deaminates cytosine to uracil Mutations introduced by repair enzymes that excise and replace uracil with any of the 4 DNA bases or pair

it with A instead of G (so G-C pair not replaced with A-T pair)- Compare the mechanisms for generating diversity in antibodies and TCRs

o Antibody diversity Somatic hypermutation and class switching Heavy chain VDJ recomb and light chain VJ recomb are random processes: can have any one of the

possible VDJ combos for H chain and any one of the possible VJ combos for light chain: 1000 VH regions x 1000 diff VL regions = 1 x 106 total number of H-L pairs to be generated

o TCR diversity Flexible recomb (inexact joining) – differences in length and sequence at joints of recomb gene segments;

results from chaning the ends of cleaved DNA Modifications include:

Deletion (trimming) of coding sequence by exonuclease Adding nt via P nt insertions – insertion of nt that are part of palindromic sequences which are

encoded by ends of recomb gene segments Adding non-coded (non-templated) nt durinig process of recomb through action of terminal

deoxynucleotidyl transferase (TdT) = N region addition Length and sequences differences at joints of recomb gene segments can result from D-D joining (joining

of more than 1 D segment) or from omitting any D segment (skip D joining) Productive rearrangements – unchanged reading frame of J region produces completely functional protein Change J reading frame translation termination codon in J gene or extends into reading frame of C gene

after generation of VJC transcript by RNA splicing Changed C gene reading frame prevents formationof functional protein chains by either changing the

AA sequence or reaching premature translation termination codons

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Non-productive rearrangements – yield non-functional protein chains For VDJ recomb in Ig H, and TCR beta and gamma loci – changing the reading frame of D region during

VD recomb could still yield intact protein chain as long as DJ recomb doesn’t change the J reading frame- Solve antibody and TCR rearrangement problems

CHAPTER 6: MATURATION AND CIRCULATION OF B AND T LYMPHOCYTES- Identify the central and peripheral lymphoid organs and specify their organization and functions

o Central lymphoid organs Bone marrow – for B lymphocytes; B cell matures in bone marrow into IgM and IgD B cells

Trabeculae create maze of interconnected spaces filled with spongy bone marrow Interconnected sinuses empty into larger blood vessel (central sinus) As they mature, B cells migrate from endosteum (lines inner surface of bones) towards central

sinus Mature lymphocytes leave bone marrow through central sinus

Fetal liver – for B lymphocytes Thymus – for T lymphocytes where it matures in to alpha-beta and gamma-delta T cell

Divided by trabeculae into lobules (cortex surrounds medulla) Developing T cells = thymocytes; embedded in stromal cells (epithelial cells, macrophages,

interdigitating dendritic cells, nurse cells As thymocytes mature, they are pushed from cortex towards medulla and once mature, they will

exit thymus from medulla Maturation takes 3 weeks Alpha-beta cells – maturation determined by expression/lack of expression of TCR and CD4/CD8

coreceptorso Double positive cells = express both CD4 and CD8o Double negative = cells lacking both CD4 and CD8o Signle positive = cells expressing either CD4 or CD8o Maturation process: double negative double positive single positive

Gamma-delta cells – develop from same double negative cells but do not express CD4 or CD8 and aren’t MHC-restricted

o Peripheral lymphoid organs Spleen

White pulp and red pulp No afferent lymphatics, only efferent lymphatic vessels (therefore, no lymphatic circulation) Has blood vessels (splenic artery and vein); areterioles surrounded by PALS (T cells); follicles

composed of B cells and are near PALS marginal zone (lymphocytes, macrophages) surrounds PALS

Central arteriole sinusoids in marginal zone and red pulp veins; fraction of blood diverted into splenic cords (not lined by endo cells); this blood reenters sinusoids through small gaps between sinusoidal endo cells splenic vein

Lymph nodes Outer cortex (B cells) organized into lymphoid follicles Central area called paracortex (T cells) Inner medulla (macrophages, B and T cells, plasma cells) Afferent lymphatics enter on convex side and drain into marginal sinus cortex and paracortex

via cortical sinuses medullar sinuses efferent lymphatics Professional APCs (macrophages, dendritic cells) line lymph sinuses and are around follicles and

in paracortex MALT Tonsils, appendix, Peyer’s patches

Composed of follicles (mostly B cells) interspersed with T cells Blood circulation and drained by efferent lymphatics

- Compare the mechanisms of positive and negative selection of B and T cells in the central lymphoid organso Positive selection

T cells Mature T cells activated if TCRs bind to both Ag and MHC molecules – this ability to bind to self-

MHC acquired during maturation in thymus Alpha-beta TCR lineage

o Beta chain gene rearrangement occurs in immature double negative cells in thymic cortex if productive rearrangement functional beta chain produced and expressed in

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association with pre-T-alpha (pTa) glycoprotein (beta chain – pTa heterodimer = pre-TCR complex)

o Cells that don’t produce functional beta chain nonproductive rearrangement no pre-TCRs apoptosis

o Pre-TCRs bind intrathymic ligand and receive signals to further proliferate and differentiate signals also lead to alpha chain gene rearrangement and expression of CD4 and 8 on cell surface these CD4+ and CH8+ double positive cells rescued from apoptosis only if they express on alpha-beta TCR tha binds to self MHC molecules on thymic cortical epithelial cells (both class I and class II expressed on thymic cortex) binding signals cells to differentiate

Positive selection – selection of immature thymocytes for survival and maturationo T cells that positively select for reactivity with class II express CD4o T cells that select for reactivity with class I express CD8

B cells B cell progenitors differentiate into pro-B cells rearrange their H chain loci H chain prot

derived from productively rearranged H locus = mu chain which is expressed in cytoplasm and on cell surface in assoc with VpreB and gamma5 proteins (together = surrogate light chain) H chain expressing cells are called pre-B cells two mu H chains and two surrogate L chains assemble into pre-B cell receptor (pre-BCR; analogous to pre-TCR) pre-BCR cells positively selected for proliferation and induced to suppress H chain gene rearrangement and begin light chain rearrangement (no pre-BCR apoptosis)

Productive light chain yields kappa or almbda light chain protein this light hain associates with mu H chain to form membrane IgM expression of surrogate light chain terminated cells stop dividing and are called immature B cells (those that don’t productively rearrange light chain gene apoptosis)

o Negative selection T cells

Immature TC4+CD4+CD8+ T cells die via apoptosis if TCR molecules bind with high avidity to MHC or both MHC and peptide in self MHC-self peptide complexes on thymic cells (esp dendritic cells and macrophages)

Only thymocytes whose TCRs bind weakly to self MHC-self peptide complexes become mature T cells

Due to negative selection, mature T cells that exit thymus are devoid of cells that could mount immune response against peptides in thymus yields self-tolerance to thymic Ag = central tolerance

Small number of strongly auto-reactive T cells is not deleted acquire FoxP3 TF that directs them to develop into TregS

Non-thymic Ag also expressed in thymus produce TF encoded by gene (autoimmune regulator – AIRE) causes expression of tissue-specific antigens allows for negative selection of T cells specific for these Ag

Soluble prot from blood don’t enter thymus b/c of blood-thymus barrier created by epi cells surround B

Remember:o TCR/self-peptide/MHC no binding apoptosiso TCR/self peptide/MHC weak binding maturation (positive selection) activated

when TCR bound strongly to foreign peptide-self MHC complex MHC restriction – T cells can only be activated when foreign peptides

associated with self MHC molecules (CD4+ T cells class II; CD8+ T cells class I)

o TCR/self peptide/MHC strong bindingi apoptosis (negative selection) accounts for 95% of T cells

Thymic education – process by which T cells learn self tolerance and MHC restriction B cells

Immature B cells whose BCRs bind with affinity to Ag on other bone marrow cells die via apoptosis

Immature B cells that bind with affinity to soluble Ag in bone marrow inactivated so that even after maturation they can’t be reactivated = anergy (cells = anergic)

Therefore, emerging B cell population = negatively selected against self antigens in bone marrow establishes B cell central tolerance

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B cells are immature when leaving bone marrow (transitional B cells) and finish development to IgM IgD mature B cells in peripheral lymphoid organs

Mature B cells can be activated if they meet Ag outside bone marrow- Distinguish between central and peripheral tolerance of B and T cells

o Central tolerance – negative selection against self antigens in bone marrow for B cells and thymus for T cellso Peripheral tolerance – tolerance to self antigens present in periphery

T cells Mature T cells that bind self antigens in periphery anergized (inactivated b/c no costimulatory

signals) activation-induced self deatho Anergy: when T cell gets signal 1 via engagement of its TCRs without signal 2 (B7

costimulatory signal)o Suppression of self-reactive T cells mediated by regulatory T cellso If no costim signals Fas and FasL expressed on CD4+ T cells FasL binds Fas

(death receptor) on same cell activation of caspases apoptosis B cells

Mature B cells that bind T-dep self antigens in periphery anergized b/c of lack or anergy of helper T cells

T-ind self Ag in periphery are present in insufficient density to activate B cells- Describe the circulation of mature lymphocytes through the blood, lymph and peripheral lymphoid organs

o High hydrostatic pressure at arterial end of capillaries water, electrolytes filter out into EC space at venous end low hydro P some absorption excess fluid in EC space (lymph) drained through lymph vessels -> left thoracic duct and right lymphatic duct left and right subclavian veins

o LV (lymphatic vessels) everywhere except CNS, cartilage, bone, bone marrow, thymus, placenta, cornea, teetho Lymph nodes along course of larger LVs LV that enter lymph node (afferent) and those that exit (efferent)o Lymphocytes and foreign Ag brought to peripheral lymphoid organs via circulatory systemo Circulation of mature lymphocytes

Exit central lymphoid organs and enter peripheral organs via blood leave peripheral organs via lymph eventually reenter blood to begin another cycle through peripheral organs (recirculation ensures that each foreign Ag will contact few lymphocytes that have complementary receptors)

Entry into peripheral organ: lymphocytes leave BV and pass into tissue by crossing endothelium, basmenet membrane (process of exit = emigration/extravasation): lymphocytes moving with blood flow are slowed down by weak adhesive interactions with tall endo cells of HEVs mature B and T cells have L-selectin adhesion receptor on surface that binds to counter receptors on HEV endo cells (addressins: CD34 and GLyCAM-1) binding causes lymphocytes to slow donw and roll on endo and it also induces signals resulting in lymphocyte activation leading to stronger adhesion between lymphocytes and endo that is mediated by binding of integrins on lymphocytes to ICAM-1 and ICMA-2 on endo cells strengthening adhesion allows lymphocytes to crawl between endo cells into tissue = transendothelial migration/diapedesis

L-selectin and integrins = homing receptors b/c they guide lymphocytes to reach their destination (home) Once in peripheral tissue naïve lymphocytes move through ECM most B cells go to lymphoid

follicles and T cells to paracortex in lymph nodes and PALS in spleen Lymphocytes that don’t encounter foreign Ag exit lymphoid tissue via efferent lymphatics reenter blood

via thoracic duct or right lymphatic duct to recirculate Only few B cells go to follicles where they get survival signals from BAFF cytokine these follicular B

cells enter circulating pool of B cells B cells in marginal zone of spleen = marginal zone (MZ) B cells specific for T-ind antigens and located

to bind and be activated by blood-borne pathogens- Explain how lymphocytes respond to antigen encounter

o Ag binding + costimulatory signaling lymphocyte activationo B and T cells activated even before hey reach follicles or T cell-rich areaso Naïve T cells activated by interacting with APCs (esp dendritic cells)o B cells

Post Ag stimulation germinal centers form in lymphoid follicles at interface between B and T cell areas Lymph nodes with no germinal center = primary follicles (resting cells) Lymph nodes with germinal center = secondary follicle Germinal centers have mostly B cells with few activated TH cells and follicular dendritic cells FDCs – long dendrites/extensions anchored to immune complexes containing intact Ag bound to Ab (after

Ag-specific Ab produced) Ag on FDCs helps activate specific B cells B cell proliferation yields clones that make up germinal center; therefore, all B cells in germinal center may be specific for single Ag

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Ig class switching, somatic hypermutation, affinity maturation, generation of memory all occurs in germinal center

Surviving B cells leave peripheral tissues via lymph (and/or blood in spleen) some B cells diff into plasma clels (in medulla of lymph nodes and splenic red pulp) but most go back to bone marrow where they undergo diff into plasma cells (90% of total Ab produced in blood is produced in bone marrow)

o T cells Activated by Ag on APCs prolif and diff mostly in T cell areas into TH or Tc cells leave tissue via

lymph or bloodo Lymphoblasts (Ag-activated lymphocytes) and memory lymphocytes don’t express L-selectin anymore stop

recirculating change homing pattern and migrate to tissues containing Ago Most circulating lymphocytes are stimulated by antigen within 2 days after that foreign Ag entered body within 5

days, large numbers of activated lymphocytes for that foreign Ag leave periph tissue and deployed back to circulation to fight foreign Ab at site of entry



CHAPTER 7: INNATE AND ANTIBODY-MEDIATED EFFECTOR FUNCTIONS- Classify cell types by the innate and antibody-mediated effector functions they perform

o All cells involved in innate and Ab-mediated functions derived from hematopoietic stem cells and developed in bone marrow

o Monocytes Migrate into peripheral tissues where they become macrophages Membane-enclosed cytoplasmic granules containing toxic substances Circulate in blood

o Granulocytes Include neutrophils, eosinophils, basophils Membrane-enclosed granules Neutrophils and eosinophils move using membrane extensions (pseudopodia)

o Mast cells Cytoplasmic granules similar to basophils Found only in tissues (vs. basophils which are found in blood) Skin, CT, submucosa (gastro, uro, resp, eye)

o NK cells Subset of lymphocytes tha share characteristics esp with T cells Most express neither TCR or BCRs NKT cells – express alpha-beta TCRs of restricted diversity

o Dendritic cells Long membrane extensions Found in lymphoid and non-lymphoid tissues, blood, lymph Different functions based upon locations (eg. IDCs – thymus; Langerhans immune function in skin) Two classes of dendritic cells outside central lymphoid organs

Conventional dendritic cells (cDCs) – from myeloid progenitor; present Ag to and activate naïve T cells)

Plasmacytoid dendritic cells (pDCs) – derived from lymphoid progenitor that produces large amounts of interferons, cytokines needed for innate immunity

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Follicular dendritic cells in germinal centers of pheriph lymph tissues do NOT arise from hematopoietic stem cells

o Abundance: neutrophils > T cells (6:1 T vs. B) > B cells with ratio of CD4+ T cells to CD8+ being 2:1- Specify the molecules and pathways involved in innate and antibody-mediated

o Phagocytosis Cell ingests and degrades large insoluble particles Phagocytic cells/professional phagocytes = monocyte/macrohage; neutrophils Phagocyte binds particle to be phago-ed via molecular recognition of receptors (recognize sugars,

phospholipids, LPS receptor for example, CD14, MD-2, Toll-like receptor 4); pattern-recognition receptors, mannose receptor, scavenger receptors)

Phagocytes also express Fc receptors (Fc-gamma-R and Fc-alpha-R) bind Fc regions of some of IgG and IgA Abs respectively when Ab brought lose together by being bound to large Ag

Binding of Ab and Ag can initiate phagocytosis = opsonization Opsonin – agent that promotes phago of an Ag by binding to and coating that Ag; IgA and IgG = opsonins Phagocyte binds particle transmits signals that activate phago and stimulate it to extend pseudopodia to

engulf particle pseudopodia fuse at tips to create phagosome (vesicle) phagosome fuses with 1+ lysosomes to form phagolysosome ingested particle degraded by lysosomal toxic substances and enzymes

Substances that degrade particles Reactive oxygen species – damages memb of ingested microbes by oxidation of fatty acids;

production referred to as oxidative burst Reactive nitrogen species – NO inhibts iron-containing respiratory enzymes in ingested microbes Defensins – group of cationic peptides that kill ingested microbes by forming channels that embed

in membranes leading to ion leakageo Lysozyme – degrades peptidoglycan layer of bacterial cell wallso Hydrolytic enzymes – degrade proteins and carbs

Some breakdown products are contained in transport vesicles that bud off phagolysosome and fuse with late endosomes in late endosomes of monocytes and macros, peptides form phago-ed Ag associate with class II MHC molecules complexes transported to cell surface (MHC-peptide complex)

Bulk of breakdown products extruded from both mono/macrophages and neutrophils after transport vesicles fuse with plasma memb (exocytosis)

Immature dendritic cells – phagocytic but not considered professional APC b/c that’s not their main function; abilityt o phago microbes allows them to mature into professional APCs that activate naïve T cells

o Cytotoxicity Killing by NK cells

No Ag-specific receptors but can kill tumor cells and host cells virally infected Innate recognition of targets involves activation receptor (aR) (that binds to carb (CHO) ligands

potential targets) as well as inhibitory receptor (iR) (that binds MHC class I molecules) aR + CHO ligand (aR is engaged) killing signal transmitted triggers killing of target iR + class I MHC (iR is engaged) protective signal blocks activation signal and prevents

killing of target Normal host cells with class I MHC bind inhibitory receptor and are protected from killing by the

NK cell If class I expression decreased greater engagement of aR relative to iR protective signal

can’t overcome killing signal results in target cell death Some intracellular pathogens cause MHC class I down-regulated expression (herpes virus, tumors)

els less susceptible to be killed by Tc cells but more susceptible to death by NK cells NK cells express both Fc-gamma-R and FC-alpha-R receptors bind to Ab only when Ab

multimerized (by binding to target) host cells coated with Ab that can recognize specific Ag on surface can be bound by NK cells through Fc receptors results in crosslinking of Fc receptors signal triggers NK cell sot kill AB-coated target cell = Ab-dependent cellular cytotoxicity (ADCC)

NK cell killing mechanism is same as Tc killing mechanism: exocytosis of granules with release of perforin, granzymes, caspase activation, death of target cell by apoptosis

Killing by eosinophils Kill both by innate and by ADCC (med by IgE, G, A) Important in immune response against parasitic works b/c these infections elicit production of high

levels of Ab of IgE isotype

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Ab coat worm by binding work surface Ag Fc receptors oneosinohpils bind Fc regions of Ab multimerized on worm surface transmits signals to eosinohpils that triggers exocytosis of cytoplasmic granules with release of granule contents towards bound worm major granule substance released = cationic proteins (esp major basic protein MBP)) needed to kill worm

Killing by monocytes/macrophages and neutrophils Kill targets too big to phago-ed Exocytose toxic products when receptors for innate recognition and/or Fc receptors on phago cells

are engaged Macrophages can carry-out ADCC through IgG and IgA Ab

o Inflammation Functions triggered by IgE Mast cells and basophils release soluble factors that cause recruitment and local accumulation of leukocytes

(phago, cytotoxic cells, lymphocytes) and accumulation of fluid = inflammation Mediators of inflammation/inflammatory mediators = soluble factors released IgE trigger release fo inflam mediators from mast cells and basophls Both mast cells and basophils express Fc-epsilon-RI receptors – high affinity for Fc regions of IgE

therefore can Fc-epsilon-RI can stably bind IgE even if Ab not first aggregated (multimerized) bound IgE sensitize the cell mediator release occurs only if IgE Ab crosslink Fc-epsilon-RI receptors: in order for crosslinking to occur, IgE monomers must themselves be crosslinked by binding multivalent Ag

Crosslinking of Fc receptors signal transduction activation of mast cells and basophils to exocytose their granules (degranulate) preformed mediators released to set off inflam response (histamine and proteases)

Histamine – vasoactive amine that causes dilation of capillaries which facilitates passage of fluid that contains cells and proteins from blood into tissues

Proteases – released from mast cells and cause partial degradation of basement membrane underlying endo cells to increase vascular permeability

Newly synthesized lipid mediators PAF (platelet activating factor)

o Lipid mediator that recruits monocytes, neutrophils, eosinophils from blood to tissue by binding receptors

o Chemotactic because of its ability to recruit cells (chemotactic substances – cause migration of cells up concentration gradient)

o Aggregates platelets leading to formation of micro-clots and platelet activation to release granule content (including vasoactive amines)

Prostaglandinso Cause increased vascular p ermeabilityo Allows ore fluid containing cells and protein to enter tissue

Thromboxaneso Cause platelet aggregation and vasoconstrictiono Vasoconstriction forces leakage into tissues and brings more cells and soluble molecules

to area Leukotrienes

o Cause contraction of smooth muscles and increased secretion of mucuso Smooth muscle contraction facilitates Ag expulsion from body

Cytokines secreted by activated mast cells and basophils Secrete IL-4: causes B cells to switch to production of IgE Ab that can lead to further mast cell

activation and can mediate clearance of Ag by eosinophils Tumor necrosis factor (TNF) – cytokine produced by activated mast cells and basophils; helps

activate monocytes/macrophages, neutrophils results in increased Ag clearanceo Immune complex clearance

Cytokines are secreted to cause Ag to be decreased in body (see above) RBCs clear out Ag-Ab complexes from classical complement pathway by having CR1 receptors that bind

to C3b and C4b and take them to spleen where they are phago-ed by macrophages- Distinguish the three pathways of complement activation

o Two components: soluble and glycoproteinso Soluble prot – synthesized and secreted by hepatocytes and monocytes, macrophages; circulate in blood o Complement components interact sequentially to eliminate antigenso Three pathways – differ in early reactions but converge into common terminal reaction sequence

Classical pathway of complement activation Activated by Ag-bound IgM and IgG

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Nine complement components (C1-C9) interact sequentially Reaction initiation: C1 binds to Fc regions of IgG and IgM when Ab bound to Ag; C1 can

alternatively bind to carb epitopes on some pathogen surfaceso C1 binds Ab or directly to pathogens via C1q subunit (6 subunits each with globular head

and collagen-like stem; six stems assoc with each other and with 2 protease proenzymes C1r and C1s) each C1q binds to one Fc region or pathogen epitope and at least two C1q heads must be bound for stable interaction betw C1q and antigen this activates classical complement pathway

o IgM and IgG distortion caused by binding to antigen exposes C1q binding sites at least two Cq1 heads bind to Fc regions

o Only one IgM-Ag molecule sufficient for Cq1 bindign because IgM has multiple Fc regions (pentamer)

o IgG has to be in favorable position for Cq1 molecule to bind both Fc regions simultaneously therefore high density of IgG Ab on antigenic surface is needed

o Because of this, IgM more efficient at complement activation than IgG After Cq1 binds Ag-Ab complex Cq1 stems move and induce conformational changes that

cause enzymatic reactions involving C1r, C1s, C4, C2, C3, and C5: one component participates in proteolytic cleavage of the next component cleavages always result in generation of an active enzyme that remains bound to Ag-Ab complex and fragment that diffuses away = cascade effect with amplfication at each step

Must generate C3 convertase to cleave C3 into C3b and C3a C3 and C4 b fragments have similar functions (stay bound to Ab-Ag complex) and C3, C4, C5 a

fragments (diffuse away) have similar functions C3b and C4b = opsonins – facilitate phago of Ag-Ab complexes coated by C3b and C4b

o Monocytes/macrophages and neutrophils have surface receptors for C3b and C4b called CR1 receptors that are present also on RBCs and B cells soluble Ag-Ab complexes coated with C3b and C4b bind CR1 receptors on RBCs and are taken to spleen degraded there by macrophages = major route for Ag-Ab complex clearance

o C3b and C4b that bind CR1 receptors on B cells are endocytosed and degraded peptide samples then displayed on surface of mono/macrophages and of B cells assoc with MHC class II

C3a, C4a, C5a brought to sites of inflammation b/c of increased vascular permeability caused by vasoactive amines at sites of inflammation

o C3a/C4a/C5a bind C3a/C4a receptors or to C4a receptors on mast cells, basophils, neutrophils, eosinophils, monocytes/macros, platelets help activate these cells resulting in enhanced effector functions leads to degranulation of cells with release of inflm mediators

o Therefore, C3a thru C5a feed into inflam reaction and amplify it/prolong it in cascade manner

o C3a, C4a, C5a = anaphylotoxins b/c are inflam mediators with C5a being most potent, followed by C3a

Terminal reaction sequence = direct killing of Ag esp Gram negative bacteria and enveloped viruses

o Complement components added to growing complex initiated b bound C5b fragment which in turn binds to C6

o C7, C8 and lots of C9 insert into membranes of cells or enveloped viruses (targets) to generate ring-like channel leads to target lysis

o C5-9 complex = membrane attack complex (MAC)o Complement-dependent cytotoxicity (CDC) = complement-mediated lysis

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Mannan-binding lectin (MBL) pathway Not Ag-bound Ab activated Similar to classical pathway but can’t be activated by Ag-bound Ab Instead, MBL binds mannose residues exposed on bacterial surface MBL similar to C1q structure (glob heads and stems) and is assoc with 2 serine proteases (MASP-

1 and MASP-2) proteases activated when MBL binds mannan proteases then cleave C4 and C2 complement components = cascade

Alternative pathway of complement activation Not Ag-bound Ab activated Alternative path can be initiated directly on Ag surface Initiation dependent on generation of C3b and its binding to Ag (C3b generated via spont cleavage

of C3 into C3a and C3b) Bound C3b interacts with Factor B binding makes Factor B susceptible to cleavage by Factor D

yields C3 convertase stabilized by properdin (prot) C3a fragments diffuse away from site (just as it does in classical and MBL paths) and participates

in inflam reactions C3b fragments ind to nearby Ag surface and act as opsonins or bind to factor B leading to

generation of more C3 convertase molecules and more bound C3b molecules bound C3b leads to MAC formation and target lysis

Complement system regulation Soluble prot circ in blood to prevent spont complement activation while membrane prot present on

host cells protect them from attack by complement systemo Soluble regulators

C1 inhibitor (C1Inh) – prevents pont activation of C1 by binding to C1r and C1s; C1Inh deficiency = hereditary angioneutotic edema

Factor 1 – inactivates bound C3b and bound C4b by cleaving via C4BP or Factor H cofactors (C3b cleaved yields iC3b (inactive C3b = opsonin) or C3d (stays bound to Ag or Ag-Ab; interacts with B cell coreceptor CD21)

S protein (vitronectin) – binds free C5b67 compexes (not embedded in Ag) and prevents their inertion into memb

o Membrane regulators CR1 – binds to C3b and C4b to prevent formation/promote dissoc of C3

convertases DAF (decay accelerating factor) – binds C3 convertases and accelerates their

dissociation Homologous restriction factor (HRF) and CD59 (protectin) – bind C8 and

prevent C5b-8 complex from associating with C9 and preventing MAC formation

Sialic acid on mammalian cell surface protects form complement action by inactivating bound C3b while microbes have low sialic acid and are more sensitive to complement

- Recognize the distribution and functions of the different immunoglobulin isotypeso IgM and IgG

Major Ab in blood Neutralizing Ab; block attachments of pathogens to host cells

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Ag-Ab complexes then eliminated by Ab-mediated effector mech (phago, ADCC, complement) Small amounts in external secretions

o IgG Major Ab in extracellular space and predominant class in blood Ab class transported across placenta to fetus – confer passive immunity to fetus

o IgE Major class in skin, submucosal surfaces of GI, urogenital, respiratory tracts where it is bound to mast cells Very low concentration in blood

o IgA Most abundant Ab in mucosal secretions, cervical/vaginal, resp; predominates in saliva, tears, breast milk Predominant class produced in body – ends up in external secretions more than in blood

o Polymeric IgA and some IgM Made by suepithelial plasma cells in mucosa Transported through epi cells from BL to apical side via transcytosis: Ab first bind pIgR (polymeric Ig

receptor) on epi cells which recognizes J chain in IgA and IgM pIgR-IgA complexes endocytosed transcytotic vesicles transport them to opposite side of cell where they then fuse with memb and IgA attached to extracellular portion of PIgR released into lumen via cleavage of PIgR IgA-boudn piece of pIgR receptor = secretory component which protects hinge region of secretory IgA (sIgA = complex of IgA and SC in mucosal secretions) from degradation by proteases



Neutralizing Ab – mediate immunity by binding Ag surface and preventing their attachment ot mucosal cells so that they don’t penetrate – Ag complexes expulsed

Most Ag’s first encounter is with sIgA (main class in mucosal secretions); therefore IgA = first line of defense

o Main effector functions (summary) IgM

Complement activation (strong) Neutralization

IgG Opsonization ADCC Complement activation Trans-placental transport Neutralization

IgE Sensitization of mast cells and basophils ADCC

IgA Trans-epi transport Neutralization Opsonization ADCC

CHAPTER 8: CYTOKINES AND INFLAMMATION- Identify the four primary signs of inflammation

o Redness – capillary dilation and increased number of RBCso Heat – increased velocity of blood flowo Swelling – accumulation of fluid and cellso Pain – results from pressure on nerves due to the swelling

- Recognize the general features of cytokineso Cytokines – peptides or glycopeptides secreted by host cells that influence other cells via paracrine or autocrine

fashiono Act at short range but some can diffuse to distant sites via circulatory system

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o Bind cell surface receptors initiate signal transduction activate gene transcription; therefore is a way to mediate intercellular communication between cytokine prod cells and cytokine target cells

o Pleiotropic – some cytokines have different effects on different targetso Redundancy – same effect mediated by more than one cytokineo Synergistic – effect of 2 cytokines on target is additiveo Antagonistic – effect of one cytokine blocks that of anothero Cytokine network – cytokines produced as result of cell stimulation by other cytokineso Major cytokine-producing cells = macrophages/monocytes and T cellso Stimulate/enhance innate immune systemo Immunity conferred by T cells either by direct effect (cytotoxicity or contact help) or indirectly (via T cell secreted

cytokines) = mediated immunityo Cytokines can be manufactured from cloned genes (recombinant cytokines) to treat diseaseo Cytokine receptors

Classified into families (eg. TNF-receptor family, chemokine receptor family) Have gammac (common gamma) chain in common in several cytokine receptors: IL-2, IL-4, IL-7

accounts for overlapping bio functiono Cytokine inhibitors

Soluble molecules that antagonize bio action of cytokines downreg immune response Three types:

Cytokines that block (antagonize) action of other cytokines by transmitting conflicting signal after binding receptors (eg. IFN-gamma = antagonist of IL-4)

Soluble cytokine receptors that are truncated forms of intact receptors shed from surface of cytokine receptor-bearing cells and then bind to complementary cytokines and prevent them from binding to cell surface receptors prevents respective cytokines from eliciting response in target (eg. TNF-binding prot (TNF BPs))

Soluble molecules that compete with given cytokine for binding to specific cytokine receptor on target but don’t elicit bio response upon binding the receptor (eg. IL-1 receptor antagonist, IL-1RA) (eg. Anakrina – trts rheumatoid arthritis)

- Classify cytokines by functiono Interleukins

IL-1 Major sources = activated monocytes and macrophages A.k.a. proinflammatory cytokine b/c it promotes process of inflammation Pleiotropic (b/c has different effects in different places) Local effects

o Stimulates mono/macros to make more IL-1 and TNFo Stimulates T cells to make cytokines (incl. IL-2) and to express IL-2 receptors

Made in high concentrations enters bloostream to mediate endcrine effects on nervous system, liver, endo system – what it mediates:

o Fever Temps higher than 37 deg C inhib growth of microbes Synth prostaglandin E (PGE) by endo cells in hypothalamus and smoothmuscle

cells: increased PGE muscle contraction (shivering) and vasoconstriction (heat conservation/production)

Endogenous pyrogens – substances that have the capacity to cause fever (therefore, IL-1 = endogenous pyrogen)

Increased prot synth by liver cells form acute phase proteins partake in host defense against Ag

IL-2 Synth/secreted by activated T cells (mainly CD4+ T helpers) Engage TH TCRs and costimulatory CD28 induces T cells to synth IL-2 and IL-2 receptors

yields autocrine stim of T cell sot prolife and differentiate Neighboring CD4+ or CD8+ T cells, B cells, NK cells that have IL-2 receptors can also be

stimulated to prlife and diff Acts as growth factor for T, B, NK cells Enhances cytotoxicity by NK cells yields lymphokine-activated killer (LAK) cells – cytotoxic

to some cancer IL-4

Pleiotropic Produced by mostly TH, some mast cells and basophils

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Induces B cell prolif Induces Ig class switching in B cells to IgE Down-regulates expression of some T cell and macro-prducing cytokines Therefore, it enhances and dampens immune response

IL-13 – some of the same functiosn as IL-4 = example of cytokine redundancyo Interferons

Interfere with viral infection Type 1 (alpha and beta) IFN

Production induced by almost all cells Induced most efficiently by viruses but can also be induced by gram neg bacteria, some cytkines Virally-infected cells secrete type 1 interferon binds receptors on neighbors induces those

cells into an anti-viral state enzymes in cell activated to inhibit transcription of viral prot and viral replication inhib

IFN alpha and IFN beta bind to same receptors paracrine action Upregulate MHC I/Ag presentation, activate dendritic cells, macros, NK cells Plasmacytoid dendritic cells (pDC) – accum in lymphoid tissues during infection secrete 1000-

fold more type 1 interferon than other cell types; therefore called interferon-producing cells (IPC) Type II (gamma) IFN

Can only be prod/secreted by T cells and NK cells IFN-gamma receptor diff from type I receptor but also found on most host cells Less potent anti-viral activity than alpha/betta A.k.a. immune interferon because has modulatory effects on immune system:

o Up-reg of MHC class I and II; up-reg of expression of all molecules involve din Ag presentation to T cells

o Phago activation (mono/macro, neut)o Stimulation of macros to kill tumor cellso Activation of NK cell cytotoxicityo Effect on Ig class switching in B cells (IFN-gamma inhib switching to IgE antagonizes

action of IL-4)o Tumor Necrosis Factor (TNF)

Initiate inflam immune response to infection and sometimes cancer TNF-alpha

Produced by many cell types: activated mono and macros TNF-beta

Made only by some subsets of activated T and B cells A.k.a. lymphotoxin (LT) b/c only produced by lymphocytes

Both TNF types bind same receptors so induce same responses: Direct killing of tumor cells Mono/macro activation to produce IL-1, cytokines, TNF, PAF Stimulate expression of adhesion molecules on endo cells

Stimulate mono/macro by LPS in gram neg cell wal TNF produced in high amounts enters systemic circulation induces endocrine effects:

Cytokine production by monocytes Fever (TNF = endogenous pyrogen) Increased prot synth in liver

TNF family – both soluble and memb-bound memberso BAFF

“B cell activating factor” elonigng to TNF family A.k.a. BlyS Made by dendritic cells and macros Needed for survival and prolif of follicular B cells

o Colony stimulating factors (CSF) Stimulate hematopoietic stem cells or progenitors to form colonies (collection of cells all descended from

same ancestral cell via cell division) CSF production enhanced during inflammation via inflam stimuli therefore, inflammation leads to

productiono more hematopoietic cells that can partake in inflam reactions Recombinant forms of some CSFs are being used as treatments (Epo – anemia; G-CSF and GM-CSF –

neutropenia (neutrophil defic) and pancytopenias (leukocyte/erythrocyte/platelet defic)) Granulocyte CSF (G-CSF)

Made by T cells and activated mono/macro

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Needed for prolif/diff/activation of neutrophils Stimulates prolife of hemato stem cells

Granulocyte macrophage CSF (GM-CSF) Made by T cells and other cells stimulated by certain cytokines during inflammation Pleiotroopic – promotes prolif/maturation/activation of diff hemato cells

Monocyte/macrophage CSF (M-CSF) Made by variety of cell types Stimulates prolife/diff/activ of mono/macro lineage

Erythropoietin (Epo) Made by kidney cells and liver cells Main factor that stimulates RBC production

IL-3 (multi-CSF) Made mainly by activated T cells Pleiotropic Synergizes (additive effect) with lineage-specific factors to stimulate prolif/diff of hemato stem

cells IL-7

Made by thymic cortical cells and bone marrow stromal cells Stim prolif/diff of T and B cells during maturation on thymus and bone marrow respectiely

o Chemokines Group of cytokines that are chemotactic for leukocytes; induce them to migrate up concentration gradient

toward chemokine source Made by many cell types bind to receptors on target induce cell movt in target cell can induce

granule exocytosis (depending in type of target cell) with release of inflam mediators and up-reg of adhesion molecules

CC chemokines Chemotactic for monocytes and T lymphocytes Includes CCL2 (MCP-1)

CSC subfamily Chemotactic for neutrophils Include CXCL8 (IL-8)

- Explain the molecular and cellular interactions in inflammationo Initiation of inflammatory response

Initiated via recognition of Ag by immune system regardless of whether Ag was previously encountered Immune response can be initiated by innate mech but once Ag-specific Ab and T cells produced, they are

important in inflam response Microbes and products activate macrophages and complement system macros process bacterial prot and

form N-formyl peptides (b/c all bact have N-formylated prot) Activation of complement system (any of the 3 paths) inflammatory C5a, C3a (C4a in classical and

MBL) complement fragments made; C5a and N-formyl peptides (eg. fMLP) are chemotaactic for leukocytes

C5, C3a, C4a (decreasing order of potency) cause mast cell degranulation (release of histamine and PAF) Macrophage activation cytokine production (TNF-alpha, IL-1) and chemokine prod (CXCL8, MCP-1) TNF-alpha and histamine can activate endo cells on PCvs and capillaries to make more MCP-1 as well as

vasodilators (prostacycline (PGI2) and NO) and to express cell adhesion molecules that can interact with leukocytes to facilitate their extravasation capillary dilation with increases endo cell distance and mast cell-released proteases cause degradation of basement memb prot leaks from blood into tissue



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o Cell trafficking in and progression of inflammatory response Capillary dilation causes blood flow through PCVs to slow down allows WBCs attracted by chemotactic

factors to attach loosely to activated endo cells through selectins (cell adhesion molecules) that bind to mucin-like receptors leukocytes start to roll on endo while chemoattractant molecules (C5a, PAF, chemokines) bind to specific receptors on WBCs

This causes conformation change in integrin structure on leukocyte increases affinity of integrins for counter-recptors on endo cells yielding strong adhesion of leukocytes to endo

Leukocytes crawl betw endo cells using adhesive interactions and once in tissue, migrate thru matrix up concentration gradient of chemoattractants to site of Ag

Neutrophils = first cell type recruited and to accumulate (few hours to 3 days) – don’t recirculate – die in tissue phago Ag and degranulate

Eosinophils, baso, NK, mon recruited into inflam site afterward and activated by Ag and/or preexisting cytokines to perform innate functions like phago

Ag-specific T cells also arrive as do Ab that are Ag-specific adaptive immunity now dominates (Ab-mediated effector functions, cytotoxic T cell mediated host clel killing – responses controlled by cytokines secreted by activated T cells, mainly helper T)





o Termination of inflam response When Ag eliminated, inflam response ends Termination aided by cytokine inhibitors (like shed cytokine receptors, IL-4, IL-10), glucocorticoids, some

prostaglandins TGF-beta cytokine inhibits immune functions = anti-inflammatory

- Distinguish between the cytokine profiles of Th1 and Th2 cell subsetso Activated CD4+ T helpers can develop into Th1, Th2, and Th17 based on cytokines they produceo Th17

First subset to be produced during infection Migrate to infected tissues produce IL-17 (proinfam cytokine that induces cytokine production by epi

cells, endo cells, fibroblasts induced cytokines recruit neutrophils to infection sites) Type 1 cytokine profile produced by Th1 cells

Responsible for secretion of cytokine that activate macrophages and cytotoxic T cells and NK Dominant roles in immune response Therefore, Th1 cells predominate in immune responses to intracellular pathogens Activate macrophages (make proinflam factors) therefore, Th1 called inflammatory T cell

subset Secrets IFN-gamma therefore directs Ig class switching in B cells to IgG1 and IgG2

Type 2 cytokine profile produced by Th2 cells Secrete cytokines that increases Ab production and switching to IgE, diff of eosinophils and mast

cells IgE – mediate cytotoxic and inflam responses by eosinophils and mast cells – defend against

worms Therefore, Th2 cells predominate in worm infections Called helper T cell subset b/c better han Th1 at providing help for Ig production Il-4 and IL-10 made by Th2 – suppress T cell and macro functions

Th1 and Th2 inhibit each other’s proif and cytokine production – cytokines from each antagonize the other Th3

CD4 T cells that don’t express CD25 but produce TGF-beta, IL-4, and IL-10 Are induced regulatory T cells found in MALT Suppress excessive immune function

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CHAPTER 9: IMMUNITY TO INFECTION- Recognize the general characteristics of host-microbe interactions

o Infection – invasion of host tisues by microbes with microbial multiplicationo Pathogens – microbes that cause disease upon infectiono Immunity – resistance that host puts up against invading microbes (physical, mechanical, chemical, innate, adaptive)o Protective immunity – resistance effect at eliminating/deterring microbe (vs. ineffective immunity)o Commensals – microbes that peacefully coexist with host and colonize them an dform part of host’s normal

microbial florao Flora members – can cause disease if a part of host defense compromised (eg. Cut)o Opportunistic infections – infections by commensals b/c microbes seize opportunity to infecto Mechanisms of pathogens causing disease

Induction of normal host inflammatory response aimed at eliminating invading microbes – clinical responses = fever, fatigue, headache, sore throat, stuffy nose, watery eyes, vomit, diarrhea

Killing of host cells – lysis of cells caused by viruses, toxins made by microbes (endotoxins – toxins attached to microbe, like LPS; exotoxins – toxins secreted by microbes)

Induction of excessive immune responses by host which cause tissue damage – granule release from neutrophils kill microbes but also surrounding cells; excessive inflam response can lead to functional impairment of tissue

o Inflammatory response = innate + adaptive Innate – role in first four days of infection by microbes which are encountered for the first time (primary

infection) Size of inoculum – number of infecting organisms Adaptive – comes in if innate cannot eliminate infection; B and T cell-mediated; develops within 4 days

post-first invasion (B cells/Ab; T cells/cytokines/kill/help B cells make Ab) Primary immune response – B and T cell mediated immunity; peaks at one week post-infection

and wanes after 2-3 weeks Possible outcomes of first encounter

o Acute infection outcome: invading microbe eradicated immune system winso Acute infection outcome: host killed by invading microbo Chronic infection outcome: host and invading microbe learn to coexist

o Acute infections – yield memory B and T cells to elicit stronger secondary immune response upon reinfection

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o Chronic infections – can also elicit secondary immune response but it would immediately follow primary response (no demarcation)

o Microbes can evade (undermine) or subvert (corrupt) immune mechanisms- Identify the microbial mechanisms of infection and survival

o Crossing epithelial barriers Adhesins – microbe surface molecules that mediate adhesion to skin or mucosa Pili – bacterial adhesins After adhesion, can cross epil layer when it is damaged using attachment prot (glycoprot) on microbe)

microbes internalized by epi cells and then released on subepi side M cells – cross bacteria and viruses over when they enter small int mucosa; deliver antigens from gut to

Peyer’s patches but some microbes use M cells as port of entry to establish infection Macrophages also help microbes enter body – phago them; microbes that survive the phago are carried to

inner body tissueso Multiplication and spread

After crossing epi barrier, microbes can be destroyed by innate or adaptive immune responses but if they evade the responses they can multiply and spread

Microbes taken by local lymphatics to local lymph nodes where microbes are trapped by dendritic cells and macros when number of microbes is high or when phago cells not effective at destroying microbes, they continue to spread through lymphatics and enter blood

Microbes not cleared from blood infect tissues either by infecting endo cells via receptors or damaging endo layer

Microbes passively spread thru body being carried by body fluids, movement Some microbes like HIV can invade neighbor cells by causing cell fusion

o Invasion of host cells Viruses enter host cells via receptor-mediated endocytois Bacteria, fungi, parasites can survive in phagocytc or non-phago host cells Microbes use attachment prot to bind to specific receptors on hosts and are then phago-ed or endo-ed Known host receptors

HIV – CD4 (on T helper)influenza – sialic acid on hosts

Rhinovirus – ICAM-1 or ICAM-2- Specify the first and second barriers to infection

o First barriers Physical, mechanical, chemical, biochemical

Physicalo Epi cells have tight junctions to prevent free passageo Whirling bone system, twisted nasal passageso Short hairs in nasal passages (filter)

Mechanicalo Discharge and flow of fluids: sweat, saliva, tears, mucuso Desquamation – continuous shedding of skin to prevent excess microbial build upo Peristalsis – push microbes out of GI tracto Ciliary movement – cilia synchronous beating to propel microbes out of bodyo Coughing – air/thick mucus containing microbes expelled from airwayso Sneezing – air containing microbes expelled; provoked by irritation

Chemical/biochemicalo Enzymes – damage cell walls (lysozyme in saliva, sweat, tears; pepsin in stomach)o Lactoferrinn – Fe binding prot in mucosal secretions that decreases ferric ion

concentration to prevent bacterial growtho Mucin – confers viscous consistency and helps trap microbes and expel themo HCl – gastric secretion that redues pH in stomach to inhib microbe growtho Fatty acids – in sebaceous and sweat glands that inhiit bacterial/fungal growth on skino Defensins – prod by intestinal cells; kill bacteria, fungi, enveloped viruses by forming

membrane channels Normal flora

Parts of skin colonized by flora: skin, nose, throat, mouth, large int, outer part of urinary tract, vagina

Interferes with pathogen growth by preventing other microbes from attaching to epi and by secreting Ab or bacteriocins (antimicrobial toxins)

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Defends against infectious disease by being constant source of immune stimulation Ag from microbial flora go to secondary lymphoid organs via diffusion betw epi cells or via M

cells Ag activate B and T cells plasma cell (esp IgA plasma cell) generation IgA

sIgA (secretory) in external secretions bind to and neutralize microbes by preventing their attachment to host cells

Microbial toxins can slightly be neutralized by sIgA Immune complexes/microb toxins with sIgA excreted from body

Intraepithelial lymphocytes In mucosal surfaces and skin to destroy invading microbes Majority of IELs in intestine and skin are T cells, and most are CD8+ Tcyto cells derived from self-

renewing lymphocytes specific for Ag on common pathogens Eliminate infected host cells and enhance innate functions via cytokine or Ab secretion

o Second barriers Skin, supporting CT of mucosa, submucosa Macrophages (phago), subepi lymphocytes, IgE-sensitized or unsensitized mast cells, Langerhans, dendritic

cells (endocytose viruses) Local containment of infection and initiation of inlflam response which recruits other cells and factors Macrophages and dendritic cells activated by general features of microbes: LPS on gram neg (LPS

interacts with TLR4 Toll signaling pathway via TLR2); activated macros/dendritics act as APCs to activate Ag-specific cells that then kill host cells infected and activate helper T’s to release cytokines that further Tc cells to kill and macros to secrete proinflam cytokines

Mast cells degranulate upon stimulation by C5a ro C3a complement fragments IgE specific for previously encountered microbial antigens may be present on mast cells; crosslinking of

Ab with Ag leads to degranulation Langerhans migrate to pheriph lymph organs to stimulate Ag-specific B and T cells Inflammatory stimulus (microbe) triggers local immune response and release of inflam mediators tha then

initiate inflam response and bring recruit reinforcements recruitments = adaptive and innate; recruited forces perpetuate inflam response

Local inflam occurs in places where microbes have spread Both innate and adaptive immune forces take time to induce – innate induced earlier (within few hours);

rate of induction for adaptive varies (primary vs. secondary response) First adaptive response – arrive after day 4; memory B and T cells made recirculate intissue they were

initially activated in reinforces second line of defense, increases sIgA level so that microbe less likely to get past sIgA and IELs in the future

Second adaptive response – quicker and more effective so little inflammation; usually asymptomatic



- Identify the innate and adaptive mechanisms of defense which operate against different types of microbeso Innate

Pathogen recognition Microbes bind PRRs and activate innate system; PRRs recognize PAMPs TLR family

o Membrane-bound receptors expressed on dendritic cells and macroso Bind ligands via leucin-rich repeat domaino Most TLRs on cell surface (TLR 3, 7, 9 on endo and phago vesicles)o CD14/MD2/TLR4 – LPS receptor on gram neg bacteria

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o TLR1/TLR2 – binds peptidoglycan and lipoprot on gram poso TLR2/TLR6 – bind yeast zymosano TLR9 – bind viral and bacterial unmethylated CpG-containing DNAo TLR engagement signal activates NF-kappaB (key inflam transcription factor) celll

activation and expression of cytokines/costimulators NOD family

o Nucleotide oligomerization domaino In cytosol and recognize pathogens that replicate in cytosol (eg. Bacterial proteoglycans)o Ligand binding domain = like TLRo Nucleotide-binding domain that mediates oligomerization of NOD upon ligand-binding

signal trasnduction NF-kappaB activation RNA helices

o Cytoplasmic and bind to viral dsRNA produced during viral replication signal to activate production of type 1 interferon

Other PRRso Macrophage mannose receptor (binds mannose residues on bacteria) and scavenger

receptors (bind microbial anionic polymers and lipoprot) are membrane-bound PRRso Formyl peptide receptor (FPR) – on neutrophils; binds bacterial-derived fMLP (N-formyl

peptide) neutrophil migration towards bacteria (chasing effect) phago Soluble prot PRRs

o LPS-binding prot (LBP) – exchanges LPS monomers for lipids and delivers them to CD14 on mono/macrophage surface CD14 interacts with MD2 and TLR4 signal transduction

o Collectins – lectins (sugar-binding prot) that bind sugar residues on microbes while interacting with host prot and collectin receptor on phagos collectins act as opsonins to enhance phago

Innate effector functions Phago (mono/maco/neutro), cytotoxicity (NK, eosinophils), complement-dep cytotoxicity (CDC) Regulated b cytokines, vasocactive amines, PAF, PG thromboxanes, leukotrienes, C5a, C3a, C4a Interferons directly inhibit replication in host cels

Products released from activated phagocytes Lysosomal granule products (mono/macro/neutro) – reactive oxygen and nitrogen species with

some hydrolytic enzymes that damage both microbes and host cells Lysozyme, lactoferrin, defensins – granule-released products with Ab activity that specifically

attack microbes Acute phase proteins (APR)

Local inflam induces APR – generalized response characterized by fever, changed vasc permeability, biosynth metabo and catab changes

Acute phase proteins (APPs) or acute phase reactants (APRs) involved in defense against microbes and in control of inflam response

Most APPs made by liver cells and activated by binding of cytokines (TNF-alpha, IL-1, IL-6, LIF, IFN-gamma) from macrophages and NK cells to cytokine receptors on hepatocytes yields signal transduction regulation of APP transcription

IL-1 and TNF-alpha act on hypothal to induce fever inhib microbial growth Major APRs – massively induced (1000 fold) = serum amyloid A (SAA) and C-reactive protein

(CRP) CRP – clears nuclear material released from illed microbes and illed host cells during inflam; can

bind bacteria, parasites, immune complexes, act as opsonin by interacting with Cq1, enhance NK killing activity

SAA – control inflam by inhib platelet actiation and oxidative burst in neutrophils Other prot induced during APR – complement prot, coagulation prot, protease inhibitors, metal-

binding prot (bind iron), LPS-binding prot Abscess – large numbers of dead neutrophils can overwhelm phago capacity and form this

localized collection of pus (dead leukocytes and live/dead microbes) – can cause tissue damage (con of inflam response)

o Adaptive Humoral immunity – soluble Ab; most effective against extracellular microbes and extracellular stages of

microbes that have an intracellular stage Cell-med immunity – T cells and cytokines; ;most effective against intracell stages of infections Adaptive immunity to bacteria

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Extracellular bacteriao Mostly humoral immunityo Polysacch in cell walls and bacterial capsules are most immunogenic components of

microbes (immunity-inducing); polysacch act as T-ind Ag that elicit mostly Igmo Bacterial prot – act as T-dep Ag – macros that phago bacteria and B cells that bind to and

internalize microb prot display peptide/class II complex act as APCs for T helpers T helpers then provide contact help and cytokine to B cell to produce Ab

o Anti-bact Ab effector functions lead to microbe deatho Classical complement path activation (esp by IgM) bacteria lysiso Opsonization by Ab and C3b phago of bacteriao C3 complement deficiency extremely susceptible to bacterial infectionso Ab neutralize bacterial toxins (anti-toxin Ab = antitoxins prevent toxin binding to

host cell receptor)o Neutralizing Ab/toxin complexes bind C3b, C4b, iC3b AgAb-C complexes then

eliminated by opsonization via binding to Fc or CR1 receptors on phago or to CR1 receptors on RBC

Intracellular bacteriao Mostly CMI (cell-mediated immunity)o Major players = CD4+ T cells of Th1 subset (inflam T cell subset) – produce IFN-gamma

that activates killing mech of infected phago leads to enhanced microbial killingo Hard for immune system to get rid of intracellular bacteria (like the ones that cause TB

and leprosy) b/c they persist in macros for long time leading to chronic inflammationo Gamma delta T cells also involvedo TB – T cell mediated chronic inflammation manifests as granulomas containg infection

and preventing bacteria from spreading; granulomas contain infected macros that become giant cells, CD4+ Th1 cells (stimulate macro killing) and fibroblasts

Fibroblasts – secrete collagen fibrosis impaired lung function blood supply obstructed so necrosis of macrophages in center of granuloma

Therefore, Th1 immune response effective in containing infection If granulomas rupture, some bacteria re-establish function

o Leprosy – two forms of CMI responses Tuberculoid leprosy – Th1 cells stimulated yields protective immune

response with formation of granulomas containing bacteria; minimal disfigurement and not infectious

Lepromatous leprosy – Th2 cells stimulated to produce IL-4 and IL-10 that inhib cytokine prdo by Th1 cells as well as killing mech of macros; ineffective immune response resulting in progressive destructive lesions filled with bacteria; swollen disfiguring nodules

o The type of CD4+ T cell (Th1 or Th2) determines severity of leprosy Adaptive – fungi

CMI and humoral T cells cytokines activate phago of macro AIDS – decrease in CD4+ T cells increased occurrence of fungal infections

Adaptive – parasites Most parasites can’t be completely eradicated by either innate or adaptive usually establish

chronic infections in host Protozoa

o Both humoral (IgG – opsonize protozoa for phago and killing) and cellular immunityo Protozoa peptides/classII displayed by phago cells activate mostly CD3+ T cells of Th1

subset Th1 make/secrete IFN-gamma and TNF which activates killing of phagos Worms

o Mostly humoral immunity with IgE Ab that mediate ADCC by eosinophilso Shed Ag from worms and displayed by class II phago cells activate CD4+ T cells of

Th2 subset Th2 makes IL-4 induces B cells to switch to produce IgE; IL-5 stimulates prolife of eosinophils

o Worm-specific IgE and IgG Ab coat worm and eosinophils bind to aAb activates eosinophils to kill worm by exocytosis

o High levels of IgE Ab and eosinophils in blood = sign of worm infectiono Worms too large to be phago-ed which is why eosinophils are involved

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o Worm Ag bind mast cell or basophil bound anti-worm Ab triggers mast cell or basophil degranulation with release of inflam mediators smooth muscle contraction and mucus secretion helps to expel worm

Adaptive – viruses Both humoral and cell-mediated Humoral – predominates early in viral infections before virus enters host

o Ab bind viral attachment receptors or for enveloped viruses components of fusion neutralize viruses (prevent them from entering host)

o Effector mech: ID Ab-coated virus particles and eliminate themo Classical complement path activated cytotoicity of enveloped viruseso sIgA virus-specific Ab in secretions

CMI – main defense against viruses that have already infected host cellso CD8+ T cyto cells bind viral peptides in assoc with MHC class I and kill infected cellso CD4+ T cells provide contact and cytokine help to virus specific B cells and CD8+ T

cellso ADCC by NK cells elimin virus-infected host cells

- Recognize the microbial mechanisms of evasion from innate and adaptive immunityo Microbes like to interfere with the host’s effector mechanisms (phago, complement system, Ab action)o Antigenic variation – microbes vary their Ag to escape recognition by BCRs and TCRs and soluble Abo Hide inside host cells – viruses do this; bacteria, fungi and parasites can survive and replicate inside ephago or non-

phago cells and survival inside phago cells is dependent on ability of these microbes to evade killing mecho Microbes hiding inside hosts are still visible to immune system b/c T and NK cells ID and destroy infected host cellso Latency period – way for microbes to become invisible to immune system; virsues like herpes and HIV can be

reactivated at later times and exit latency to continue infectiono Evasion by (extracellular) bacteria

Antigenic variation – evades adaptive immune response Gonococci – vary structure of pilin (pili prot) at genetic level by gene conversion (replacement of 1+ out fo

6 segments/minicassettes of expressed pilin gene) – 1 million diff possible combos Ag distinct strains can develop prevents immuno memory

o Evasion by parasites Variation of surface antigens due to different developmental stages of parasite Antigenic variation – African trypanosomes have VSG coat that can change via gene conversion, rapidly

multiply and cause waves of parisitemia (parasites in blood) with each wave dominated by a different VSG variant

Shedding of surface Ag – that bind to Ab – divert the Ab from binding to parasiteso Evasion by viruses

Main mech = antigenic variation HIV, influenza, rhinovirus (common cold) Influenza Ag variation

2 virus surface glycoproteins (hemagglutinin (H) and neuraminidase (N)) that are responsible for ability to cause disease (virulence)

Point mutations slowly accumulate in H and N gradual accumulation of mutations = antigenic drift (RNA reassortment) yields new influenza strains and new epidemics

New strains due to reassortment of RNA segments (antigenic drift) from human influenza strain and animal strain – can cause pandemics

Down-reg of MHC I expression on host cell surface; herpes viruses interfere with class I assembly Establishment of latency (Herpes, HIV)

- Explain how superantigens and microbial products can cause septic shocko Superantigens

Bacterial and viral prot that bind both MHC class II and alpha-beta TCRs regardless of antigen specificy can stimulate so many T cells

SuperAg to MHC II binding is in non-polymorphic region Binding to TCRs involves V-beta region at site encoded by V-beta gene Bacterial superantigens are often toxins: staph, enterotoxins, toxic shock syndrome toxin, TSST-1, strep

pyrogenic exotoxins Superantigen-bidning CD4+ T cel activation large amounts of cytokines produced systemic

toxicity mediated by TNF-alpha and beta, immunosupression mediated by IL-4 contribute to pathogenicity (ability to cause disease) of organisms that produce superantigens organisms have advantage because immunosuppression allows them to multiply and excretion allows them to spread

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o Septic shock Excessive systemic inflammatory response to infection that causes extensive tissue damage organ failure

and shock (BP drop) Initiated esp by LPS endotoxin (gram neg) that stimulates release of pro-inflam cytokines (TNF-alpha and

IL-1) from macrophages cytokines enter blood and stimulate production of cytokines by monocytes and endo cells up-reg of adhesion molecules generalized inflammation and endo damage vasodilation with fluid leaking drop in BP (systemic hypotension) shock

Also caused by gram positive exotoxins with superantigen properties stimulate lots of T cells ot secrete cytokines activate macros and monos to secrete high levels of TNF-alpha and IL-1 sepsis

- Identify the host and exogenous factors that influence immune responses to microbeso Immune system factors that influence immune response

Dependence on MHC alleles Cytokine regulation of immune system Immune responses in immunodeficient people

o Immune-neuroendocrine interactions Through hormone actions steroid hormones have suppressive actions on immune system (cortisol) Neuroendocrine system affected by macro and T cell-produced cytokines (IL-1 and TNF-alpha)

reciprocal interaction between neuroendo and immune systems b/c hormone receptors are expressed by immune cells whereas cytokine receptors are expressed by endo and nervous cells

Severe stress diminished immune responseo Nutrition

Impaired immunity Malnutrition is most common cause of immunodeficiency in world Circle cycle = malnutrition impaired immunity infection causes malnutrition itself (fever + loss of

appetite/anorexia to decrease nutrients available to microbes and fever increases energy requirement) Rest and drink plenty of fluids – preserve body energy and prevent dehydration Cycle of malnutrition broken if living in poor sanitation, poverty because that would prevent nutritional

recovery between infectionso Age

Fetus – no immunological competence; dependent on passive immunity from IgG Infants – highly susceptible to infections; Ab passed via milk Childhood – immune response ability increases with age b/c of exposure to microbes which leads to

immuno experience memory T and B bigger quicker adaptive responses Aged people decreased ability for immune response 85+ age significant immune function reduction Immunosenescence = aging of the immune system

o Pregnancy Mild immunosuppression because of increased level of steroid hormones Fetus is in part immunologically foreign b/c of paternal histocompatibility molecules being expressed

o Drug abuse – various degrees of immunosuppression increased susceptibilityo Preexisting infections

Positive or negative effects on immune response Gram neg – enhance immune response because of LPS stimulation of macrophages Viral and parasitic infections immunosuppression (eg. HIV decreases CD4+ count loos of

cytokines and contact help) Microbial infections predisposition t secondary infections because host response to initial infection

(primary) produces god environment for growth of other microbes resp secretions, fluid accumulation in response to viral resp tract infections (influenza) = good environment for bacterial growth

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CHAPTER 10: IMMUNOLOGICAL METHODS- Recognize the relevance of immunological methods and immunobiologics (substances that provide, enhance, stimulate

immunity when administered to host) too Diagnose

Determine the presence and/or concentration of specific substances in biological samples such as blood, urine, tissue samples, CSF

o Therapy Treatment approaches for many diseases (infections, autoimmune, immunodeficiency, cancer)

o Prophylaxis Protective/preventive measures against disease Vaccination – prophylaxis against infections diseases

o Advancement of medical knowledge

- Recognize the sources and uses of antibodieso Uses

Individuals exposed to particular Ag are “immunized” by that Ag Circulating Ab – if developed and reactive with specific Ag, then person is immune to Ag Intentional exposure (immunization) to immunogen (Ab) Oral immunization, parenteral (topical application) Intratracheal immunization – through trachea Via injection: intravenously (blood), intradermally (skin), subcutaneously (under skin), intramuscularly

(into muscle tissue), intraperitoneally (into ab cavity) Anti-Ig Ab

Ab to Ab Anti-Ig from one species can be produced by immunization of another species) (can mount Ab

response to Igs from other species) Rabbit antihuman IgG – anti-Ig made in rabbits: use human IgG to immunize the rabbit and the

rabbit develops anti-human IgG then combine the human IgG with anti-human IgG and you have an immune reaction (Ab attack Ab)

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Detection (serological methods) – rely on Ag-Ab interaction; use known Ab prep to detect presence of complementary Ag or use known Ag prep to detect presence of complementary Ab = serotyping

o Sources Humans, other animals exposed to specific microbes, other antigens in the environment Antiserum – serum containing Ag-specific Ab; used directly as Ab source

Polyclonal (heterogeneous population of Ab even against one Ag) Polyantigen – consists of many Ag and antiserum is heterogenous Finite supply of Ab Ab concentration depends on immunogenicity (ability to induce immune response),

structure/form/dose of Ag, and host Specific Ab – Ab to specific Ag or polyAg Ab can be detected 5-7 days post primary immunization (priming) Ab concentration peaks at

day 12 and then drops (= primary Ab response) during this period, Ag-specific memory B cells generated – on secondary immunization with same Ag, memory B cells reactivated (secondary response) secondary immunization = booster b/c boosts the response

Myelomas B cell cancers of Ab-secreting plasma cells Develops from plasma cell that has become cancerous Clonal origin so all cells in myeloma from a certain host produce/secrete same antibody

(monoclonal) Ab found in serum of patients with myeloma Ag-binding specificity is unknown because specificity of plasma cell that happened to become

cancerous is unknown Hybridoma technology – used to make unlimited supplies of monoclonal Ab with known antigen-

binding specificity: hybrid cells (hybridomas) are created by fusing normal (noncancerous) B cells with immunized host non-producing myeloma cells (don’t make any Ab anymore) hybridomas retain immortality and Ab secretion of myeloma and Ag-binding specificity of normal B cell

Genetically engineered Ab Generate collections of vector molecules (libraries) eoncding different H chain/L chain pairs (Ab

libraries)- Specify the applications for and identify the steps and reagents involved in

o Immunoprecipitation Interaction of multivalent Ab with multivalent soluble Ag that leads to aggregates that come out of solution Centrifuge sample to separate Ab-Ag complex from precipitates Amount of ppt formed depends on relative concentration of Ag for a constant concentration of Ab = bell-

shaped curve (amt ppt vs. Ag concentration) which = precipitin curve Low Ag concentration relative to Ab concentration = zone of antibody excess of precipitin curve mostly

small (soluble) immune complexes formed b/c Ab molecules are around so most Ag have their own Ab rather than have to share Ab and aggregate

High Ag concentration relative to Ab concentration = zone for Ag excess mostly small soluble immune complexes formed b/c enough Ag around so that each Ab can have own Ag and not share with other Ab

Ag and Ab concentrations similar = zone of equivalence extensive cross-linking and large precipitates formed



Can inhibit Ag-Ab ppt with monovalent antigens (eg. Haptens; monoval Ag can be used to determine/verify Ab specificity) and monovalent Ab (Fab, Fv)

Nephelometry – measure concentration of Ag in biological samples Measure Ag concentration in sample by adding known cncentration of Ab – rxn carried out in

zone of Ab excess to form small ppt forming turbidity meas turbidity using nephelometer Concentration of Ag in test sample determined by using standard curve

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Conventional neph – Ag-Ab is first allowed to reach equilibriumo Agglutination

Same principle as immunoprecipitation except the antigen is Insoluble Hemagglutination – agglutinated particles are RBCs; performed as either a direct test or indirect test (assay)

Direct – agglutinating Ab specific for one or more epitopes on cells; IgM good at direct agglut b/c of multivalent capabilities that facilitate cross-linking; IgG poor agglutinins (b/c only bivalent so can’t form as many links)

Can use Anti-Ig Ab to enhance agglutination of cells coated with Ag-specific IgG Ab; many anti-Ag Ab even if bivalent can bind IgG on diff cells crosslinking = indirect agglutination (b/c agglut Ab don’t bind directly to cells)

Agglutination reaction Detects presence of Ag-specific Ab in samples by determining titer of agglutination Determine hemagglutination titer by adding serial dilutions of Ab-containing sample to wells

followed by consant number of RBCs – each well contains dilution and last well gets no Ab (negative control)

Observe agglutination after an hour visually – see clumps of RBCs inwell Hemagglutination titer – reciprocal of the Ab dilution in the last well that shows agglutination Prozone – lack of agglutination resulting from too much Ab; similar principle to zone of Ab

excess in precipitin curveo Enzyme linked immunosorbent assays (ELISA)

Label for immunoassay (labels can detect substances present in concentrations as low as picograms/mL) is enzyme

Label is attached to Ab or Ag to allow visualization or quantification of Ab-Ag reaction Enzymes: alkaline phosphatase, horseradish peroxidase – enzyme causes conversion of added substrate to

colored product that is either sol or insoluble Commonly used in clinical and exp labs or sold OTC EMIT (enzyme-multiplied immunoassay technique) – ELISA variation that doesn’t require separating free

from bound ligand; competitive binding assay in solution: free ligand competes with enzyme-labeled ligand for binding an Ab this binding inactivates enzyme prevents cleavage of substrate and release of colored product; if free ligand present binding of enzyme-labeled ligand to Ab more inhib more colored product formed when substrate added

Color change proportional to concentration of ligand in test sample: more free ligand more color Pregnancy tests – ligand to be detected is captured by solid phase Ab and then detected with labeled Ab

o Radioimmunoassay (RIA) Label is radioactive isotope Iodine-125, sulfer-35, carbon-14, tritium Labels detected by autoradiography (x-ray exposed to radioactive surface to generate image) Can also detect label by counters that count number of disintegrations per minute Can use phosphorescence plate imager – expose sample to plate image scanned and generated Quantitative but require special instruments and facilities; therefore have been replaced by ELISA

o Immunofluorescence assays (IFA) Label is fluorescent Fluorescein isothiocyanate 9FITC) and rhodamine Electrons can be excited to higher state by absorption of light of certain wavelength Fluorescence = light emitted by excited electrons when they drop back down to ground sate

o Immunoblot (Western blot) analysis SDS-page (detergent that coats macromolecules with negative charge) is used on Ag (unlabeled) mix After electorphoresis, Ag on gel transferred to paper or nylon membrane via electric current prot adhere

to memb through hydrophobic interactiosn and create imprint of gel on membrane Band corresponding to Ag of interest can be visualized alone by treating memb (whose remaining prot

binding sites are blocked) with specific primary Ab Memb can be treated with complementary labeled Ab or can use unlabeled primary antibody, wash it away,

and then an anti-Ig antibody If label is radioactive isotope visualize band via autoradiography F label is enzyme Ag band visualized by adding soluble substrate that is converted into insoluble

colored product insoluble product deposits where Ab-attached enzyme has been immobilized Biotin – labels primary or secondary Ab; vitamin with high affinity for avidin or streptavidin: wash awyay

unbound biotin enzyme labeled streptavidin or avidin added substrate added Ag bands to which strept or enzyme labeled Ab are bound visualized by chemiluminescence

o Flow cytometry / fluorescence activated cell sorting (FACS)

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Use of flow cytometer to obtain quantitative info on single cells in population Number of cells expressing one or more surface markers can be determined if cells are tagged with

fluorescent Ab tha recognize those markers Create droplets with single cells that flow past laser; florescent dye on Ab is excited by laser Amount of fluorescencen on cell indicates level of expression of cell surface molecules to which the

fluorescent Ab are directed Data plotted as histogram of cell number vs. fluorescence intensity

o Immunohistochemistry (IHC) Same as immunofluorescence except Ab labeled with enzyme (peroxidase, alk phosphatase) instead of

fluorescent label Enzyme can convert colorless, soluble substrate into an insoluble product that deposits locally where Ab-

attached enzyme has been immobilized Advantage – areas where labeled Ab has bound can be visualized with light microscope instead of

fluorescence microscope Good for IDing types of macro-molecules synth by tumor cells in surgically removed tissues

- Distinguish between direct and indirect immunoassayso Direct – primary antibody binding to Ag of interest causes precipitation or agglutination or is labeledo Indirect – secondary Ab, which binds to primary Ab, causes ppt or agglutination or is labeled; are more sensitive

than direct- Interpret results of immunobiologic assays

o Immunobiologics – substances that provide, enhance, or stimulate immunity when administered to a hosto Clinical trials determine efficacy of an immunobiologic

Phase 1 – involve few subjects; intended to assay safety and potential efficacy of vaccine Ab levels in response to an experimental vaccine

Phase 2 – conducted with high-risk groups or with patients suffering from relevant disease and are inteded to determine efficacy of vaccine

Phase 3 – divide volunteers into 2 groups (blind): experimental that gets vaccine and control that gets placebo

- Recognize different types of immunobiologicso Passive type

Pooled human Ig (IG) and intravenous Ig (IVIG) – intramuscular or intravenous admin to people with Ab deficiencies; IG used for passive immune against measles and hep A

Specific IG – hep B immune globulin, varicella zoster, rabies, tetanus (pooled from individuals selected for high Ab titers against Ag); antitoxi and antivenin from animals

o Active type Attenuated vaccines – live viral or bacterial preps that have been weakened by growth in nonhuman hosts;

administered where natural infection would occur; produces mild disease; elicit humoral and T cell responses; can’t be admin to immunodeficient individuals; problem with reversion to virulence

Whole microbe inactivated vaccines – heat treatment or chemicals inactivate vaccines; more heat stable and unless inactivation is incomplete, don’t have problem of possible reversion to virulence BUT have disadvantages:

Require higher doses because no replication occurs in host Require boosters to achieve sufficient level of immunity Don’t induce CD8+ cytotoxic T’s required for destroying virus-infected cells that express only

MHC I b/c no virus particles replicate inside host to produce peptides for presentation Subunit vaccines – comprise only parts o fmicrobe that are criticule for inducing effective immunity against

microbe; avoid exposure to whole microbes that could carry disease risk Bacterial capsular polysacch – may/may not be conjugated to prot carrier (carrier used to enable

generation of T helps for Ab production against poly and for switching from IgM to IgG or IgA production

Viral surface Ag – large amounts of these Ag are difficult to produce in pure form, so recombinant vaccines made by cloning the surface antigen in bacteria or yeast

Toxoids – microbial toxins that were inactivated by formaldehyde; modify toxin so that it can’t bind receptor on host but epitopes aren’t changed so antibodies will cross-react with toxin if encountered later during infection; don’t induce immunity to microbe that produces toxin but rather to disease that induces toxin

- Distinguish between active and passive immunizationo Passive immunization

External source of immunity; admin Ab that provides temporary protection until Ab are catabolized (3-8 wks)

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Given to imunodeficient individuals and immuno normal individuals who were exposed to microbes Admin of immunobiologics that stimulate host’s own immune system – active immunization/vaccination Provides rapid immunity but no immune memory

o Active immunization Involves administration of vaccine (fools system into thinking that a real infection is taking place) Generates memory T and B cells Most cost-effective way of preventing disease

- Recall the recommended vaccines for children in the United States



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CHAPTER 11: HYPERSENSITIVITY- Compare the four types of hypersensitivity for the

o Immunologic mechanism involved Type I – Anaphylaxis, atopy, or allergy

Caused by allergens Innocuous antigens Initiated by interaction of allergen with pre-formed complementary Ab of IgE bound to mast an

basophils activates inflammatory functions yields symptoms within minutes (therefore known as immediate-type hypersensitivity)

On second encounter of allergen for which there are mast cell-bound IgE Ab in submucosa or skin: allergen interacts with mast/IgE complex if interaction is multivalent, IgE and FC-epsilon-RI receptors to which Ab are bound become crosslinked and will aggregate mast cell activation and degranulation pre-formed and new mediators released from mast cells cause inflammation

Local reactions: localized to site of entry of allergeno Hay fever (allergic rhinitis) – allergen/mast cell IgE interaction in nasal submucosa,

conjunctival tissueso Asthma – allergens inhaled; interaction in airway submucosao Reaction to insect bites – allergens enter via skin; inflammation yields edema and rim of

redness (wheal and flare/wheal and erythema reaction; wheal = swollen area; flare = rim)o Food allergies – eggs, milk, strawberries, lima beans, peanuts, shellfish – interaction in

intestinal submucosa; symptoms include hives (urticaria), eczema, asthma System reactions: triggered by interaction of allergen with igE Ab on mast and basophils with

subsequent degranulation of cells generalized inflam leads to capillary dilation and smooth muscle contraction all over body swelling of lips, tongue, larynx, airway constriction, fall in blood pressure (anaphylactic shock)

o In response to allergens directly injected into blood or can diffuse through blood from entry site

Sensitizationo T cells

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What makes these allergens (Ag) special is that they have capacity to activate T helpers of Th2 subset make high levels of IL-4, IL-5, IL-10, IL-13 (IL-4 and IL-13 induce class switching to IgE)

Allergens mostly soluble, enter mucosa so only small amounts of ree allergen reach local lymph nodes

Some allergen prob internalized by mucosal dendritic cells, Langerhans cells, migrate to lymph nodes and present Ag in class II complexes to T cells

Penicillin becomes allergen by acting like hapten complexes with class IIo Allergen specific B cells

Activated by interaction with allergen and by T cell help to proliferate and differentiate and to switch to making IgE

o Both B and T cells and IgE enter circulation submucosa skin IgE bind and sensitize mast cells

o Recurrent allergen exposure causes intensified reactions because repeated entry further activates and expands T and B allergen-specific cells in nodes



Stages of hypersensitivityo Immediate reaction (in minutes of encounter) – caused by release of inflam mediators

from mast cells and basophils capillary dilation, increased vasc permo Late phase reaction (several hours) – due to cytokines that lead to recruitment and accum

of eosinophils, neutro, baso, lymphocytes to intensify inflammation; Th2 and B cells come; Th2 secrete IL-4, 10, 3 (to promote degran of mast cells, and bring basos), 5 (activate eosino); continues as long as allergen present can result in long lasting chronic inflammation (like asthma lungs)



Genetic predisposition – controlled by genetic factors that influence Th2 and therefore IgE development or the HLA alleles at DR loci

Environmental predisposition – air pollutants increase permeability of epi and facilitate allergen entry

Type II – Ab-dependent cytotoxic hypersensitivity Mechanisms Destruction or alteration of cells by immune system Affected cells or connective tissue are of same species and often the host Reaction involves cell killing Initiated by interaction of insoluble antigens with preformed IgG or IgM activates classical

complement system (esp by IgM) complement-dependent cytotoxicity opsonization by IgG, C3b, C4b, iC3b Ab-mediated cellular cytotoxicity (ADCC), agglutination of hosts by IgM Ab to interfere with their functions

Involves destruction of foreign or host RBCs RBC antigens that elicit reactions = blood group Ag – products of polymorphic genetic loci so

different individuals of same species have diff alleles at same locus

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Types of hypersensitivityo Transfusion reactions – if blood donor and recipient differ at blood group loci –

destruction of transfused RBCs ABO blood locus – can encode glycosyl transferase that adds sugar residues to

H substance A allele – encodes gly transf that adds GalNac to H substance yeidls A Ag

expression B allele – encodes gly transf that adds Gal to H B Ag expression O allele – encodes no enzyme to act on H unmodif H expression





Isohemagluttinins – anti-A and anti-B Ab directed against Ag in individual of same species; re almost always of IgM type

AB – universal recipients / O – universal donoro Hemolytic disease of the newborn

Mom makes Ab against RBCs of fetus – fetal RBCs destroyed Erythroblastosis fetalis (Rh); RhD Ag (express it – RhD+); RhD- woman will

make Ab against RhD+ child (anti-RhD Ab) for first kid; admin Rhogam (anti-RhD Ab) so that mom doesn’t make Ab (form of passive immune = prophylactic trt)

o Autoimmune diseases Autoimmune hemolytic anemia – Ab to RBCs Thrombocytopenic purpura – Ab to platelets Hashimoto’s – Ab to thyroid cells Grave’s – Ab to TSH receptor Goodpasture syndrome – Ab to alveolar and glomerular basement memb Myasthenia gravis – Ab to ACh receptor Rheumatic fever – Ab to heart muscle; induced by crossreacting strep Ag Pempigus vulgaris – Ab to epidermal cadherin (prot in skin jxns)

Type III – immune complex hypersensitivity Results from interaction of preexisting IgG and/or IgM Ab with soluble Ag yields Ag-Ab

complexes that aren’t easily cleared by immune system Local type III rxns

o Can occur in people with circulating Ab to specific Ag Ab diffuse into tissues If Ag enters tissue, Ag-Ab complexes form

o Rxns occur wihen complexes form in zone of Ag-Ab equivalence complexes ppt and bind to C1q activate classical complement path (vs complexes that form in Ab axis with low Ag doses – small and easily cleared by phago) activation of complement system yields complement fragments including inflam mediators C5a, C3a, C4a trigger degranulation edema eventual clearance of immune complexes via opsonization (Fc and C3b receptors, CR1, on neutro and macro and by binding CR1 receptors on RBCs) Ag eliminated

o With continuous or repeat exposure to Ag, inflam response can become chronic tissue damage; chronic occur:

In response to repeatedly inhaled Ag persistent inflam in lungs Farmer’s lung – Ag from fungal spores in moldy hay Pigeon fancier’s disease – serum prot present in dust of dried pigeon feces Allergic bronchopulm aspergillosis – Ag from fungus

o Arthus reaction – skin reaction that resembles late phase type 1 reactiono Arthus-like reaction – local type III rxns at sites other than skin

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Systemic type IIIo In response to large doses of Ag if Ag’s Ab present in circulationo Free antigen present or can diffuse into blood form Ag-Ab complexes in zone of Ag

excess small complexes that activate complement inefficiently b/c lack close Fc for Cq1 binding not readily cleared by phago accum on basement memb of capillaries local inflam of tissue

Glomerulonephritis – filtration memb that is inflamed is glom BM; impairs kidney fnction blood in urine

Arthritis – complexes accum in BM of capillaries that infiltrate joint synovium; inflam of joints

Complexes in choroid plexus – inflam in brain Vasculitis – complexes accum on vascular wall (arteritis for arteries) – skin

rashes Lymphadenopathy – inflam in lympathics, obstruction of lymph flow Serum sickness – injection of animal serum or large doses of foreign prot into

human; type III reaction is self limiting if Ag eliminated before permanent tissue damage occurs; in this case, foreign prot induce productionof specific Ab within 5-8 days; b/c of high amount of prot, it is still present once Ab produced so Ab bind to Ag, make circulating complexes and cause serum sickness; fever, rashes, arthritis, lymphadenopathy, glomerulonephritis; recovery within 3 wks with increasing Ab amounts



o Formation of complexes with microbial Ag Post-strep glomerulonephritis – after strep Polyarteritis nodosa – yields severe inflam of arllow hep B infectiosn due to

complexes with hep B surface Ago Systemic lupus erythematosus – due to Ag against cell components; immune complexes

deposit everywhere Type IV – delayed type hypersensitivity (DTH)

Inflam reaction resulting from engagement of TCRs on memory Ag-specific T cells Takes 24-72 hrs to devlop Manifestations: edema, erythema, induration due to heavy infiltration by WBCs Part of response to intracell microbes, viruses (chickenpox, measles, skin lesions from herpes),

parasites, fungi, bacteria (bacteria tha cause TB and leprosy) Contact dermatitis – eczema, skin rashes; referred to as allergic reactiosn like type I

hypersensitivity; caused by neomycin Ab, cosmetics, poison ivy, poison oak, metal ions (nickel, chromate)

Autoimmune diseases: Rheumatoid arthritis, inflam bowel disease Sensitization

o Occurs in peripheral lymphoid organs where Ag samples brought in from tissueso Major Ag transporters = dendritic cells and Langerhans cells – serve as APCs with

peptide/MHC complexes to T cells Peptides from endogenously synth prot are on class I MHC Peptides form exo are on class II Since endo expressed on CD8+ and exo on CD4+, both cell types can be

sensitizedo Ag stimulate T helpers of Th1 subset these cells = TDTH cells – produce IL-2 to help

activate CD8+ and CD4+ cells; activate cell prolif these Ag-sensitive cells enter circulation and stay there or recruited when Ag originate in tissues some activated T cells become memory cells

o Hypersensitivity by TDHT cells41

Activated Th1 (TDHT) secrete cytokines recruit monocytes become macros that phago Ag and in process release reactive O2 species and enzymes some leak out of cells and damage surrounding tissues activated macros also secrete IL-1, TNF-alpha that activate endo cells to make vasodilators and express adhesion molecules promotes entry of RBCs, NK cells, lymphocytes, more Ag-sensitized T cells NK cells (stim by IL-2) have cytotoxic rxns against target cells

o Hypersensitivity by CTLs Kill those host cells via apoptosis extensive tissue damage Sometimes, CTL caused tissue damage more detrimental to host than vial

infection (hep B: CTLs cause liver damage)o Methods of detection

Type I RAST (radioallergosorbent test)

o Measures IgE levelo Immunoassay – ELISA with enzyme-linked secondary Abo Paper discs coated with allergen immersed in patient’s serum to allow allergen Ab to bind

(primary Ab) wash away unbound Ab amount of allergen-specific IgE Ab bound to disk determined by treatment with anti-human IgE (2ndary Ab)

Skin testo Determines ability of allergens to elicit wheal and flare reactiono Prick several antigens onto skin if mast-cell bound IgE Ab specific for one of the

allergens are present, get rxn in 30 minutes Type II

Coombs testo For rxns involving IgG anti-RBC Abo Hemagglutination assay – patient’s RBCs treated with goat anti-human IgG ab; if IgG Ab

(primary) already bound to RBCs, addition of anti-IgG Ab causes aggutinationo Immunofluorescence fo Ab to tissues

Type III Immunofluorescence

o On tissue sections from biopsieso Granular, lumpy bumpy

Type IV Skin tests – with Ag

o Tuberculin test – determ if person exposed to PPD (TB bacterium); intradermal injection that is monitored for redness in 24-72 hrs

o Patch test – low dose of suspected Ag placed on patietn’s skin; if eczema develops 24-72 hrs laer, have hypersensitivity

o Therapies Type I

Avoidance of allergen Hyposensitization – accomplished via repeated injection of increasing doses of allergens (allergy

shots) or high doses of synthetic peptides that represent immunodominant T cell epitopes in allergen

o Allergen injected in high doses activate Th1 (not 2) cytokines induce B cells to make IgG not IgE Ab diffuse into tissues and bind allergen phago of allergen-IgG prevents allergen from binding to complementary igE Ab on mast cells blocks mast cell degranulation; therefore, IgG Ab called blocking Ab ORinjected peptide interacts with a llergen specific Th2 cells anergizes the cells makes them tolerant to allergen and unable to provide help to allergen-specific B cells

Antihistamine s- bind histamine receptors and prevent His binding Mast cell and basophil stabilizing drugs – inibit mast cell and basophil degran by increasing

cAMP (inhibitor of degranulation); epinephrine, theophylline, sodium cromolyn General anti-inflam agents – corticosteroids

Type II Diuretics – treat transfusion rxns to prevent kidney damage from Hb accumulation Trt hemolytic disease in newborns (HDN) via exposure to UV light to break down bilirubin and

prevent brain damage or by repeated blood transfusions with RhD- RBCs before and after birth Corticosteroids treat autoimmune diseases

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Type III Avoidance of inhaled antigens Treat serum sickness with drugs that prevent release of inflam mediators form mast eclls (sodium

cremolyn) and platelets (heparin) Corticosteroids – treat SLE (systemic lupus)

Type IV Reactiosn subside when Ag eliminated Bacterial infections – Ab Viral – gancyclovir (for herpes) to reduce infection Contact dermatitis – hydrocortisone ointments (immunosuppresants) Rheumatoid arthritis – soluble CTLA-4 to inhibit T cell activation or Ab to TNF-alpha or soluble

TNF receptor fused to Ig Fc- Classify immune disorders by the type of hypersensitivity involved

o See above objective- Distinguish between types II and III hypersensitivity by immunofluorescence of tissue sections

o Type II – pattern of fluorescence is smooth and described as linear b/c Ag on tissue (BMemb)o Type III – pattern is granular and described as lumpy bumpy b/c Ag is in complex so complexes lodge in filtration

memb

CHAPTER 12: TRANSPLANTATION- Identify the mechanisms and stages of allograft rejection

o Solid organ allografts (transplant non-identical of same species) Donor cells that can leave the graft are MHC class II-expressing cells, dendritic, mono, B cells

(professional APCs) can migrate through lymphatic channels to draining lymph nodes of recipient = passenger cells

Passenger cells sensitize B and T recipient cells in nodes B cells recognize surface Ag on passenger cells activated to prolif/diff into Ab-secreting cells

Alpha-beta T cells can recognize class I/II activate CD8 and CD4 respectively Strong reactivity between T cells and allo-MHC molecules

Lots of T cells recognize all-MHC molecules because of the weak reactivity they already have (due to positive selection) with self MHC molecules; therefore, allogeneic MHC molecules are seen by some T cells as if they were peptide-self MHC complexes

High avidity between T cell and donor cell: lots of allo-MHC on donor cell Donor specific CD4+ T cells act as helps – help B cells with Ab production and CD8 with toxicity; some

can act cytotoxic Donor-specific Ab and donor-specific activated T cells reach graft via vasculature bind endo cells of BV

in graft inflam hypersensitivity reaction RBC and platelet aggregation (clotting) in BV of grafted organ loss of blood supply causes organ death



Stages of rejection Hyperacute rejection

o Within 24 hrs of transplant or within minutes

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o Type II hypersensitivityo Caused by graft-specific Ab that preexist in recipient due to blood transfusion, pregnancy

– directed to blood group or MHC antigenso Ab bind graft tissue activate complement cascade aggregation of RBCs and

platelets in capillaries necrosis of graft Acute rejection

o Acute cellular rejection – T cell mediated component 10 days to a few weeks post transplantation Type IV hypersensitivity rxn Cause: activated CD4/CD8+ T cells recognize MHCs on grafted organ T

cells mediate delayed type hypersensitivity (DTH) and cytotoxic (CTL) responses infiltrate organ death

T cells were activated after transplantation Accelerated rejection – T cells may already exist in patients getting second

transplant (memory T) – T cells rapidly activatedo Acute vascular rejection – involves Ab

Days to months post transplant Type II hypersensitivity Caused by graft-specific Ab that develop post transplant Ab bind grafted

organ cells in BV tissue damage via complement activation and ADCC platelet aggregation

o Chronic rejection Months to years post transplant Type III and IV hypersensitivity

III caused by Ab to soluble Ag that are shed from graft immune Ag-Ab deposits on BM of vessel

IV caused by T cells cellular infiltration of organ inflam organ death

Dendritic cells – most potent stimulators of allogenic reactionso Bone marrow or hematopoietic stem cell transplantation

Allografts of lymphoid tissue are immunogenic b/c of high content of class II-expressing cells allografts rapidly rejected by immunocompetent people

Bone marrow transplants treat immunodeficient people Other patients have to be immunoablated (treated with radiation or drugs to destroy immune system) to

prepare for bone marrow transplantation – therefore, upon transplantation, donor’s immune cells replace those of recipient

Since bone marrow contains hemato stem cells and lymphocytes at various stages, immune cells (esp T cells) will react against host tissue in grat versus host (GvH) reaction as opposed to host versus graft (HvG) reaction that occurs in organ transplant

GvHD – graft versus host disease – acute (mononuc infiltration of skin) vs. chronic (collagen and fibrosis deposits); skin rash, fever, anemia, weight loss, fatality

G-CSF mobilized blood replaced bone marrow transplant – inject G-CSF in donor to increases hemato stem cell production; stem cells spill out of marrow into blood and then that blood is taken from donor and processed ot obtain stem cell rich leukocyte prep used for transplantation

Autologus HSC (hemato stem cell) transplantation – used when HLA-matched donors not available; get bone marrow from patient before radiation, treat sample to kill tumor cells, then return cells ot patient post-radiation

- Recognize the features of graft versus host disease (GvHD)o See above

- Interpret results of tissue typing assayso Tissue typing – process of determining which HLA alleles are expressed by prospective donors and recipients

Serological testing Ab-mediated CDC (complement dep cytotoxicity) assay Treat samples of person’s WBCs with panel of anti-HLA anitsera and complement If test WBCs express certain HLA specificity AB binding and complement activation yields

cell death that can be detected with dyes Takes a few hours so WBCs from cadavers can be typed Used more for MHC class I than II b/c 1 are more immunogenic than II and therefore better

antisera available for I

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One way mixed leukocyte reaction (one way MLR) B cell line homo for certain HLA specificity (typing B cell) mixed with WBCs from person to be

typed (test leukocytes) Typing B cell tested for ability to stimulate cell prolif of test WBC population (potential responder

cells) Detect prolif of test cells by incorp 3H-thymidine (indicates DNA synth)



Oligomer typing assays Involve PCR and detect differences between HLA alleles at DNA level Take hours

Cross matching Immunoassay used to determ whether serum of recipient has Ab that react with WBC of donor;

positive reaction prevents transplantation from donor Match at HLA loci (3 on each parent) – six Ag match Always match ABO and Rh blood group Ag Cross matching performed to exclude possibility of preexisting graft-specific Ab in recipient HLA matching more important for bone marrow transplants than solid organ (HLA perfectly

matched for marrow)- Distinguish between generalized and antigen-specific immunosuppression and recognize examples of each

o Generalized (blanket) Corticosteroids (ex. Prednisone) – anti-inflamm: decrease immune response by decreasing number of

lymphocytes in circulation, decreasing phago and cyto ability, downreg MHC and cytokines Anti-mitotic drugs (ex. Azathiprine, methotrexate, cyclophosphamide) – inhib nuc acid synth inhib

division of immune cells Xenobiotics – drugs derived from microorganisms or chem. Synth molecules

Cyclosporine A (CsA), FK506, rapamycin – form complexes with immunophilins drug-immunophilin complexes interfere with signal transduction needed for T cell activation

Mycophenolate mofetil (MMF) – chem. Synth molecule converted to active metabolite MPA in body which inhib DNA synth and B/T cell prolif

More selective: Lymphocyte-specific Ab – bind Ag on T and B cells, mark cells for destruction Anti-lymphocyte globulin (ALG) and anti-thymocyte serum (ATS) – polyclonal Ab from animals

just immunized with human lympocytes or just T cells OKT3 – mouse monoclonal Ab directed to human CD3; targets T cells Monoclonal Ab to subunit of IL-2 receptor – prevents T cell activation b/c IL-2 receptor only

expressed on activated T cell

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Ab induce strong immune response, lead to serum sickness (type III hypersens) and eliminate therapeutic agent therefore, patients given just one treatment

Chimeric Ab – human constant regions and heterologous variable regions used to reduce immunoreactivity to heterologous Ab

Fusion proteins between IL-2 and toxins – target activated T cells by binding to IL-2 receptor; internalization of fusion prot causes target death by toxin

o Ag-specific Multiple blood transfusions from prospective donor to prospective recipient – if transfusions don’t induce

strong anti-HLA response in recipient, recipient becomes more tolerant to donor Ag upon transplant Pretreatment of graft with anti-lymphocyte Ab or anti-HLA ab to eliminate MHC class II passenger cells Soluble CTLA-4 – used with recipient WBCs to pretreat bone marrow grafts

CTLA-4 binds By on recipient APCs blocks B7 costim signal needed for donor T cell activation when interacting with recipient Ag on APCs engagement of TCR in absence of B7 signal yields anergy (negative selection) of recipient specific donor T cells



CHAPTER 13: AUTOIMMUNITY- Recognize the names of autoimmune diseases and their target specificities

o Ankylosing spondylitis – immune complexes of Ab to vertebral Ago Autoimmune hemolytic anemia – Ab to Rh and I blood group Ago Grave’s disease – more common in women than meno Hashimoto’s thyroiditis – women > meno Insulin-dependent diabetes mellitus (IDDM) – CD4+ T cells to pancreatic beta cells preceded by dept of Ab to beta-

cell antigens, insulin, and other pancreatic Ago Multiple sclerosis – women > men; CD4/8+ T cells and Ab to myelin basic prot and proteiolipid prot (PLP) in

insulating myelin sheath of nerve fibers in CNSo Rheumatoid arthritis – women > meno Systemic lupus erythematosus – women > men

- Explain the mechanisms that guard against autoimmunityo T and B cells with significant avidity for self Ag are eliminated/anergized (negatively selected) during maturation in

central organso But some auto-reactive mature lymphocytes can be directed to tisusue-specific Ag that aren’t encountered in central

organs or can be directed to epitopes on cell surface Ag or peptide-MHC complexes inc entral organs epitopes yield low avidity interactions with B and T developing cells that express complementary Ag receptors lymphocytes not activated after maturation when Ag encountered in periphery if avidity still low

o If Ag density higher in periphery, auto-reactive T cells are anergized via activate-induced cell death (AICD) via Fas-Fas ligand interactions for lack of CD28-B7 costimulatory signals

o Even if B7 present on professional APCs displaying self peptides, auto-reactive T cells prevented from activation via CD4+CD25+ T reg cells (make TGF-beta and IL-10 block l-cyte activation)

o Suppressive function of Treg depends on its interaction with self Ag via TCR and costim via CTLA-4 binding B7 on same APC

o Therefore, under normal circumstanaces, self reactive T and B cells either deleted, anergized or ignorant ot self Ag (see ppt slide)

- Explain the mechanisms and factors associated with development of autoimmunityo Molecular mimicry (microbial infections)

Cross-reaction of foreign and self epitopes with same Ag receptors Autoimmune diseases post strep infectiosn (eg. Rheumatic fever) Cross-reacting foreign epitopes provide higher avidity interactions to self-reactive T cells than do

complementary self Ag

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o Polyclonal B or T cell activation (microbial infections) Microbial products can activate many B or T cells regardless of their Ag specificity Staph and strep toxins = superAg and thus polyclonal T cell activators that by chance can result in

activation of self-reactive l-cyteso Preferential activation of Th1 or Th2 cells resulting in cytokine imbalance (microbial infections)

Th1 and Th2 inhib each other’s effects and thus prevent excessive immune responses One Th subset can be favored over the other = unblanaced cytokine production Th1 – make inflam cytokines (TNF, IFN-gamma, IL-2) promote inflam and upregulate MHC and B7 on

tissue macrophages push self reactive T cells over threshold necessary for activation Preferential activation of Th1 yields excessive cell-med immunity (eg. Multiple sclerosis, ins-dependent

diabetes)o Epitope spreading (microbial infections)

Activation of T or B cells specific for different epitope than that which originally induced adaptive immune response

Epitope spreading can be caused by up-reg of B7 on APCs after microbes interact with PRRs of innate system APCs that may also be displaying self Ag can now provide costim signals for activation of bystander self-reactive lymphocytes bystander responses can be maintained by sustaining inflam process and by generation of higher affinity self-reactive Ab (in case of B cells) via somatic hypermutation

o Injury May cause previously sequestered tissue-specific Ag to enter circulation and activate T or B cells with

complementary Ag receptors Brain Ag can cross BBB upon head injury and enter circulation can lead to multiple sclerosis

o Predispositon Women > men Expression of some HLA alleles – higher relative risk Genetic defects causing autoimmunity

APD – autoimmune polyglandular diseaseo Autosomal recessiveo Can’t use marrow transplanto Occurs in AIRE gene – defect leads to poor T cell negative selectiono Affects mainly endo gland and other tissues

IPEXo X-linked so mostly men afflictedo Defect in gene that encodes FoxP3 (essential for Treg development)o Affects esp endo glands and gut

- Identify immunological methods that can be used in the diagnosis of autoimmunityo Diagnosed by clinical symptoms and presence of Ab or T cellso Immunoassays for Abo Immunofluorescence for anti-nuc Ab in systemic lupus (SLE)o Prolif or cytotoxicity assays to detect autoimmune T cells

- Describe therapeutic approaches for autoimmunityo Generalized immunosuppression to reduce immune responses – methotrexate, azathioprine, cyclophosphamide,

corticosteroids, cyclosporine Ao Plasmapheresis – replace plasma with plasma substitute so that immune complexes or just Ab can be removed from

bloodo Anti-TNF Ab or soluble TNF receptor – rheumatoid arthritiso IFN-beta or Ab to integrin subunit for multiple sclerosiso Ab to B cell surface molecule CD20 for B cell-involving diseaseso Bone marrow transplantation for IPEX

CHAPTER 14: CANCER IMMUNOLOGY AND IMMUNOTHERAPY- Recognize the general characteristics and types of cancer

o Transformed/neoplastic – host cells that have lost ability to respond to normal growth; give rise to tumorso Benign tumors – limited growth capacity; localized to tissue of origin; don’t kill host unless in location where blood

or lymph flow can be blockedo Malignant tumors – cancers; invade adjacent tissues; metastasize (migrate via blood/lymph to tissues to form new

tumors); if not treated, can kill hosto 3 agents that cause malignant alterations

Chemical carcinogens – tobacco smoke, chemical carcinogens; cause local changes in DNA

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Ionizing radiation (UV and x-rays) – cause chrom breaks and translocations Oncogenic viruses – insert DNA or cDNA copies of genome into host; disrupt host genes

o Alterations in cell’s genome leading to malignancy involve 3 categories of genes Genes whose products cause cell prolif Genes whose products inhibit cell proliferation Genes whose products regulate programmed cell death

o Cancer types Sarcomas – cancer of mesenchymal origin (bone, CT, fat, muscle) Cancers of hematopoietic origin

Lymphomas – from lymphocytes (Hodgkin’s and non-Hodgkins) Myelomas – from plasma cells; most secrete Ab or part of Ab that can be detected in serum/urine

= M-component (of any Ig class and may consist of only light chain) Leukemias – single cells that circulate through blood and lymph; acute (grow faster and arise from

less mature cells) and chronic (grow slower and arise from more mature cells); AML, ALL, CML, CLL

Carcinomas – cancers of epithelial origins- Distinguish between tumor specific and tumor associated antigens

o TSA (tumor specific) Expressed only in tumor cells, not normal cells Glycoprot and glycolipids that differ in cancer cells due to diff orders of actia by glycosyl transferases;

include blood group Ag, mucin CA125 (on ovarian carcinomas)o TAA (tumor associated)

Upregulated in tumor cells compared to normal cells Cell surface Ag or intracellular Ag (expressed on MHC) HER-2 growth factor receptor – overexpressed breast cancers Carcinoemryonic Ag (CEA) – on colorectal cancers

- Describe the immune responses against cancer cellso Immune responses are weak b/c most Ag on cancer cells are normal host products to which host is toleranto Immune responses that do occur = adaptive and innate

Adaptive CD8 CTLs (directed to peptide/MHC I complexes) Ab to TSAs (directed to cell surface Ag) CD4 T helper cells – react to TSA-derived MHC II complexes on APCs that may have

endocytosed fragments of dead cancer cells Recognition of overexpressed TAAs

Natural Killers Botha adaptive (via ADCC) and innate (recognize carb epitopes and MHC I on tumor cells) IFN-gamma and IL-2 secreted to activate NKs

Macrophages – kill tumor cels via ADCC, TNF NKT cells

Exress alpha-betta TCRs directed to glycolipid alpha-gal-cer on tumor cells Gamma-delta T cells – TCRs for phospho-Ag or stress-induced prot Tumor infiltrating lymphocytes (TILs) – inflamm response Immune surveillance against cancer

- Identify methods of detection and diagnosis of cancero Clinical symptoms, e.g., pain, bleeding, weight loss, lumpso Imaging, e.g., X-rays (for lung cancer), mammography, colonoscopy, CT, MRI, ultrasoundo Antibody radioimaging with antibodies or antibody fragments to TSAs, TAAs or tissue-specific Agso Immunoassays, usually ELISA, for blood levels of TAAs or TSAs, e.g., CEA, CA125o Histological and immunohistochemical analysis of biopsies, blood smears, Pap smears

- Describe immunotherapeutic approaches to cancer and identify specific immunotherapeutic agents and their clinical use

o Conventional cancer therapy (and in this order) – always tried first Surgery Radiation – destroy remaining cancer cells Chemotherapy – interfere with growth of certain cancer types Solid tumors that can’t be removed by surgery are treated only with radiotherapy and chemo Toxic side effects of radiation and chemo

o Immunotherapy Strategies to provide passive or active immunity

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Given when cancers become resistant to conventional treatments Passive

Admin of anti-cancer Abost are mouse monoclonal Ab against TSAs, TAAso Anti-idiotypic Ab directed to specific surface Ig on B cell lymphomas; target only

lymphoma cells and not other B cellso Ab to prostate-specific Ago Trt with Ab relies on killing cancer cells (ADCC and CDC)o Chimeric Ab with mouse V and human C regions

Allogeneic hematopoietic stem cell transplantation after bone marrow ablation with chemoradiotherapy; effect against tumors but also causes GVHD

Adoptive transfer immunotherapy with in vitro expanded or modified leukocytes (autoloous leukocytes – patient’s own)

Active General activation

o Inject IFN-alpha activates cytotoxic NK functions and inhibits tumor cell growtho Intratumor injection of adjuvants (TLR agonists) or Ab to CD40 t activate dendritic cells

and macroso Cancer vaccines – activate T and or B cells which will then react against patient’s own

cancer cellso Enhance immunogeneicity of tumor Ag by activating dendritic cells (DCs)

Isolate patient DCs and introduce Ag in vitro before returning the Ag-pulsed DCs to patient

Immunize with inactivated tumor cells from patient that had been modified to secrete GM-CSF which promotes local accum of DCs

Immunize with inactivated DC-tumor cell fusions Inject TLR ligands together with the Ag to stimulate DCs

o Immunize with inactivated tumor ells from patient that were modified to express B7 to activate anti-tumor T cells

CHAPTER 15: AIDS & OTHER IMMUNODEFICIENCY DISEASES- Recognize immunodeficiency diseases and understand the consequences of general types of immunodeficiency diseases

o Either acquired or inheritedo Most common cause in developing countries is malnutritiono Inherited – due to genetic defecto Types

B cell deficiencies – extracellular bacteria and fungi T cell deficiencies – viral infectiosn and infectiosn with other intracellular microbes Combined B and T cell defic – general susceptibility to variety of microbial infections Phagocytic defic – extracell and intracell bacteria, fungi and parasites Complement deficiencies – extracell bacteria X-linked or autosomal

o Diseases to know XLA

B cell deficiency X-linked Block in maturation of pre-B cells few mature B cells, no plasma cells, no circulating Ig

Hyper-IgM syndrome B cell deficiency X-linked and autosomal Defect in CD40L expression Reduced IgG and IgA, normal IgM

DiGeorge syndrome T-cell deficiency Block in T-cell maturation due to lack of thymus development Reduced number of T cells, normal/reduced circulating Ig

X-linked SCID Combined B and T cell deficiency Block in T cell maturation due to defect in gamma chain of several ILs encoded on X chromosome Reduced number of T cells, normal/increased B cells, reduced circulating Ig

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Combined B and T cell deficiency ADA enzyme deficiency so toxins accumulate and affect T cells more than B cells Reduced number of both B and T, reduced circulating Ig, no thymus, tonsils, lymph nodes

detectable Zap-70 deficiency

Combined B and T cell deficiency Autosomal recessive defect in Zap-70 involved with TCR signal transduction Absence of CD8+ T cells, normal or high CD4+ T cells that don’t respond to Ag, reduced

circulating Ag CGD

Phagocyte deficiency X-linked and autosomal defects in neutrophil nad macrophage pathways Impaired killing of phagocytosed microbes, formation of granulomas in response to infection

LAD Phagocyte deficiency Defect in synthesis of integrin Beta chain No extravasation by neutorphils and monocytes, impaired CTL function, impaired B cell

activation by T helpers- Identify general methods for diagnosis and treatment of immunodeficiency diseases

o Diagnosis Clinical symptoms Flow cytometry for number of each leukocyte cell type Functional assays for different leukocyte types Immunoassays for serum concentrations of different Ig isotypes and of complement components DNA analysis for characterized genetic defects

o Treatment IVIG (intravenous Ig) – both B cell deficiencies

XLA Hyer-IgM

Bone Marrow transplant DiGeorge syndrome (T) X-linked SCID (B and T) ADA (B and T) Zap-70 (B and T)

Prophylactic Ab and IFN-gamma CGD (phago)

- Recognize the structure, genetic organzation, and infectious cycle of HIVo Structure and genetic organization

HIV = retrovirus RNA copies into DNA via reverse transcriptase cDNA copy incorp into host genome via integrase (viral

enzyme); RT does 10 mutations per round of cDNA synthesis (virus has no editing mechanism – this contributes to viral variation)

HIV virion = 2 copies of RNA genome packaged Structural proteins

Core protein capsid has core proteins – p24, reverse transcriptase, integrase, protease Two viral envelope glycoprot – gp41 (on lipid bilayer), gp120 (viral attachment glycoprot)

Genes Gag - proteins encoded: p24 – core protein Env – proteins: gp120 (attachment protein), gp41 (fusogenic protein) Pro – enzymes: RT, protease, integrase

Regulatory proteins Rev – regulator of viral expression Tat – transactivator that can increase transcription of HIV gene 1000 fold

Chemokine receptors that act as coreceptor for HIV entry = CXCR4 and CCR5 CD4 = HIV receptor; CD4+ T cells = main target for HIV infection Other cell types with low CD4 expression can be targetsed: macro, mono, dendritic cells

o Infectious cycle Gp120 binds receptor and coreceptor on host cells conformation change exposes gp41 domain

domain induces fusion of viral envelope with plasma memb of host cell HIV core released into host cell’s cytoplasm HIV genetic material uncoated and exposed viral reverse transcriptase assoc with

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HIV RNA copies viral RNA and gnerates circular cDNA cDNA, integrase, and viral coat prot translocated to host nucleus in nucleus, HIV cDNA integrated into host cell DNA at random locations via viral integrase integrated HIV genome = provirus (Fig. 15-3)



Provirus becomes part of genetic material of host lays dormant (latency – no transcription of provirus)) even though it is transmitted to daughter cells rate of proviral transcription increases in response to activation of host cel via Ag stimulation of T cells, cytokine stim of T cells, macrophages or infection by other viruses this activates NF-kappa-B increased transcription of provirus leads to production of new virions = productive stage

Newly formed free virions can infect neighbor host cels via fusion of infected and non-infected cells to form multinucleated cells (syncytia) syncytia formation due to binding of gp120 on memb of infected cells to receptors on niehgbor cels follwed by gp41-mediated fusion of infected and target cells

- Correlate the clinical course of HIV disease with the immune response to HIVo Clinical response

Initial infection asymptomatic but individuals experience “mono” type of illness (fever, headaches, muscle aches, sore throat, swollen lymph nodes, lethargy, rashes) up to 2 weeks after infection

Acute phase – clinical latency – asymptomatic for up to 12 years or longer; persistent lymphadenopathy, occasional night sweats, diarrhea

Progression to AIDS via one or more of the following: Constitutional disease – fever persisting for more than one month, involuntary weight loss,

diarrhea for more than one month Neurologic disease – dementia, PNS disorders Opportunistic infections – with microbes, pneumonia, diarrhea, skin and mucous memb infectiosn,

CNS infections Cancers – Kaposi’s sarcoma (rare cancer, skin nodules), herpes, viruses, lymphomas

o Immune response High titer viremia (virus particles in blood) Rapid viral multiplication mostly in infected CD4+ T cells and less in macrophages yields both humoral

and cell mediated responses aginst HIV Humoral – Ab against viral components, gp120, p24 CMI – HIV-specific CTLs directed towards viral components

Ab get rid of bulk of virions HIV leads to eventual death of T cells but not macrophages – just impairs macrophages – viruses and CTLs

kill these CD4+ cells Immune response = recovery from HIV acute illness with return to almost normal CD4+ count but virus

continue sto multiply in peripheral lympho organs, in activated CD4+ cells and in macros = chronic patho during clinical latency

Immune system can’t eradicate virus b/c HIV can hide from immune system by being dorman and because immune response contributes to invasiveness of HIV (Ab-coated virions bind Fc receptors on effectors = another way for infection of macros and monos)

Clinical latency – immune system keeps viral multiplication in check: low viremia and very gradual CD4+ T cell decline because new CD4+ cells are produced at very high rate while 5-10% infected at given time

Some of the naïve and memory cellsa re in latent phase while activated cells are in productive phase productively-infected CD4+ cells are impaired so they die therefore, NKs, CTLs and B cells that depend on the cytokines that were to be released from the CD4 cells are affected and yield slow and deteriorating immune function

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Further immune deterioration as latently-infected CD4+ memory T cells are activated and enter productive cycle leads to depletion of memory cells and impaired ability to fight infection

At this point, remaining CD4+ T cell population consists of new naïve T cells that are targets for HIV infection and dependent on peripheral organs for survival (organ affected by HIV as it progresses b/c lymphoid follicles disappear and macrophages are long-term reservoirs of virus since they aren’t killed by HIV and they travel to other parts of the body causing infection)

CD4+ T cell count drops from 1000-12000 mm3 to 200 mm3 at advanced AIDS and ratio fo CD4+ to CD8+ T cells in blood decreases from 2 to less than 0.5 immune system unable to contain HIV increased spread of HIV multiplication and spike in viremia

After acute illness, level of HIV-specific Ab and CTLs stays constant but declines at end stage of AIDS Fig. 15-6



- Specify the methods fo detection of HIV infectiono ELISA – to detect anti-HIV Abs; serum dilutions are added to wells of a multi-well plate coated with HIV

components; bound antibodies are detected with enzyme labeled anti-human Ig 2o Abso PCR or RT-PCR – to detect proviral DNA in blood cells or viral RNA in plasmao Immunoblot (Western blot) – to detect anti-HIV Abs

- Describe therapeutic approaches against HIVo Nucleoside analogs that inhibit the viral RT, e.g., AZT (azidothymidine)o Cocktails of RT inhibitors and protease inhibitors and of the gp41 fusion protein

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