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Administrative issues:

Recommended text: Goldsby/Kuby Immunology, 6th edition(Note that Innate Immunity is not adequately covered in the 5th edition.)

Discussion sections start next week. The journal article Akira et al, andthe first problem set will be covered. Both are available on the website.

Office Hours: Questions about the lecture material are best addressedduring office hours (Tues 11-12). I will be holding extra office hours (dateand time TBA) before the first midterm.

Email: Please use email only for VERY simple yes/no questions or simpleadministrative matters.

Great questions, keep them coming!

Innate ImmunityInnate immune effector mechanisms

Physical and biochemical barriers (defensins)Phagocytosis and reactive oxygen speciesCell autonomous defenses

ApoptosisInterferons and PKR

Innate immune recognitiondiscovery of the Toll-like receptorsmammalian TLRs and their ligandsnon-TLR recognition of PAMPs

Connections between adaptive and innate immunity

Some microbes hijack cellular machinery to replicateand spread. Intracellular pathogens include viruses(influenza, HIV) and intracellular bacteria (listeria)and intracellular parasites (malaria, toxoplasma).

cell-autonomous defense:cell produces an immuneresponse that acts on itself

Apoptosis: Cellular Suicide

•Nuclear fragmentation•Proteolysis•Blebbing•Death

Remnantsundergophagocytosis

Apoptosis versus Necrosis•Tidy: contents of cellsdegraded from within,producing small cellular“blebs”

•Programmed from inside thecell

•Messy: contents of cellreleased.

•Induced by external insult

Cell death by necrosis is more likely to produce inflammation.

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Interferons are cytokines that areproduced in response to viral infection.

Produce an “anti-viral state” in targetcells.

Acts on cell that produces it, as well asneighboring cells.

Together with dsRNA, act to triggeringthe Protein Kinase R(PKR) pathway.

Shuts down protein synthesis machineryof cells, thus preventing viral replication.

Cells can avoid being hijacked by viruses by activating the ProteinKinase R (PKR) pathway. PKR is triggered by dsRNA and interferon. Protein Kinase R:

Interfering with Infection

RNA-bindingdomain

Kinasedomain

dsRNAor

interferon

PKR

eIF2a

PhosphorylatedPKR

(active)

PhosphorylatedeIF2a

(inactive)

Innate Immunity (finishing up)Innate immune effector mechanisms

Physical and biochemical barriers (defensins)Phagocytosis and reactive oxygenCell autonomous defenses

ApoptosisInterferons and PKR

Innate immune recognitiondiscovery of the Toll-like receptorsmammalian TLRs and their ligandsnon-TLR recognition of PAMPs

Connections between adaptive and innate immunity

Cell types of innate immunity

Components of the bacterial cell wall, such aslipopolysaccaride (LPS), peptidoglycan, lipoprotein cantrigger the innate immune system.

Cross section of the cellwall of a gram-negativebacteria.

Comparison of the adaptive and innate immune responses

innate adaptive

Response time hours days

Response to identical to primary stronger response uponrepeat infection (no memory) second exposure (memory)

Receptors that pattern recognition receptors antibodies and T cell antigen receptors (TCR)Mediate pathogen e.g. Toll-like receptors (TLR)Recognition limited diversity, unlimited diversity

fixed in germline generated by V(D)J recombination

Ligands Pathogen associated molecular virtually any component of pathogenpatterns (PAMPs)

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What triggers innate immune responses?( How do phagocytes know what to eat?)

• Long-known that bacterial cell wall components activatephagocytes

• Hypothesis-- microbes contains pathogen associatedmolecular patterns (PAMPs) which are recognized bypattern recognition receptors (PRRs)

• A series of disparate observations in transcriptionalregulation and drosophila development lead to thediscovery of PRRs

Genetic analysis ofearly embryonic

development in thefruitfly, Drosophila

(Nusslein-Volhard andcolleagues)

The Dorsal Signaling Pathway controlsdorsal/ventral polarity in the Drosophila early

embryo

Dorsal

Ventral

Toll

Cactus

Dorsal

(a receptor)

(a transcriptionfactor)

“Toll” is German slang for “weird”

NF-κB: a transcription factor whichbinds to antibody genes (Baltimore and

colleagues).

enhancer

Transcriptionfactor

Ig κ gene

Nuclear factor Igκ locus B (as in A, B, C, etc.)

NF-κB: A critical transcription factor forinnate immunity

p50

p65

IκB

p50

p65

Inactive,cytoplasmic

Active,nuclear

(cactus)(dorsal)

Mutant mice lacking NF-kB subunits have defects in innate immunity.

What is the mammalian receptor that leads to NF-kB activation inresponse to infection?

Discovery of the mammalian Toll-like receptors(TLR):

1997: Janeway and Medzhitov discovered a human proteinwith structural similarity to drosophila Toll that could activate

immune response genes in human cells (TLR4).

1998: Beutler discovered that a mouse strain with an alteredresponse to bacterial lipopolysaccharide (called LPS or

endotoxin) was due to a mutation in the TLR4 gene.

There are 11 TLR family members in human and 12 in mice.Each responds to a distinct set of microbial products.

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Insert Fig 3-11

Different mammalian Toll-like receptors (TLRs) are specificfor different classes of microbial products

Different mammalian Toll-like receptors (TLRs) are specificfor different classes of microbial products

Toll-like receptors(TLRs)

link microbialproducts (PAMPs) to

transcription factor activation

in a signalingpathway that is

conserved betweenmammals and insects

Toll-mutant drosophila aresusceptible to fungal infections

A more detailed look at the signaling pathway down-stream ofToll-like Receptors (TLRs)

A more detailed look at the signaling pathway down-stream ofToll-like Receptors (TLRs)

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Pathogen recognition receptors (PRR) in the innateimmune system: TLRs are not the whole story

scavenger receptorsNOD proteins (cytoplasmic PRR)

etc, etc

The benefits and hazards of TLR signaling

Septic shock can be caused by systemicinfection with gram negative bacteria

(Salmonella).

Shock is caused by overwhelmingproduction of cytokines and is induced by

presence of LPS in blood stream.

Mice lacking TLR4 are resistant to LPS-induced shock.

Mice lacking TLR4 are also more sensitiveto infection with gram negative bacteria.

(local vs. systemic effects of TLRsignaling?)

Triggering of PRRs on macrophage or dendritic cells can induce aLARGE variety of events including:

Increased phagocytosis

Production of cytokines and inflammatory mediators:Interferons to induce anti-viral stateChemokines to attract migrating cellsEtc, etc.

Increased cell migration

Changes in expression of molecules involved in T cellantigen presenting cell function.

Innate ImmunityInnate immune effector mechanisms

Physical and biochemical barriers (defensins)Phagocytosis and reactive oxygenCell autonomous defenses

ApoptosisInterferons and PKR

Innate immune recognitiondiscovery of the Toll-like receptorsTLRs and their ligandsnon-TLR recognition of PAMPs

Connections between adaptive and innate immunity

When innate immune signaling is insufficient to clear a pathogen, theadaptive immune system kicks in.Innate immune signaling turns on the adaptive immune response.

Macrophage and dendritic cells serve as antigen-presenting cells for adaptiveimmune cells (T cells).

T cell interacting with a macrophage(antigen presenting cell)

TLR-signaling activates innate immune cells (macrophage and dendritic cells).

Innate immune cells that have been activated by TLR-signaling are much moreeffective antigen presenting cells than resting immune cells.

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Dying infected cell(self, no TLR signaling)

Pathogen(non-self, TLR signaling)

Material inphagosome disposedof inside cell-nopresentation to T cells

Material in phagosomeenters antigenpresentation pathway-presentation to T cells

TLR signaling within phagosomes determines fate ofthat phagosome (destruction vs antigen presentation).

Blander and Medzhitov 2006 Nature v440 p808

Comparison of innate and adaptive immune recognition

Receptors that mediateinnate immune recognition: Toll-like receptors (TLR)

Receptors that mediate adaptive immune recognition:Antibody and the T cell receptor (TCR)

The genes encoding the T cell antigen receptor (TCR) are assembledby DNA rearrangement as T cells develop in the thymus

TCR ! locus:structure in germline

TCR ! locus:structure in T cells

DNA rearrangement(rag1, rag2)

transcriptionRNA splicingtranslation

T cell

!"

V segments D J C

The genes encoding the antigen receptors of T and B cells areassembled by DNA rearrangement as these cells develop. As aresult of V(D)J recombination, every B and T cells expresses aunique version of the antigen receptor.

A a result of V(D)J recombination every mature B cell expresses a unique antibody.Encounter with an antigen leads to clonal expansion of B cells with a particularspecificity.

What’s coming up in the next couple of weeks(Innate Immunity)

Antibodies and antigens I (emphasis on antibody structure)

Antibodies and antigens II(emphasis onantigen-antibodybinding interactions)

Techniques based onantibodies

V(D)J recombination

B cell development andfunction

Antigens & Antibodies IDiscovery of antibodies

Basic Antibody Structurebrief review of protein structuredisulfide linked tetramer: 2 heavy and 2 light chainsmyeloma proteins and the primary structure of antibodycrystal structure of antibody: the Ig domain

The antigen binding site of antibodies

Antibody isotypes: IgM, IgG, IgD, IgA, IgEThe advantages of multivalencyeffector functions of antibody isotypes

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Early Observations of Acquired (adaptive)Immunity

•Thucydides, historian: 430 BC noted that those who hadsurvived plague could nurse the sick and not become sickagain.

•1600s, Turks and Chinese practiced “variolation”:intentional exposure to material from smallpox lesions toprovide protection against smallpox.

• Edward Jenner: ~1800 noted that exposure to vacciniavirus (cowpox) protects against smallpox. Tested firstvaccine using cowpox.

•Louis Pasteur: ~1880

•finds that exposureto attenuated bacteria(chicken cholera)protects against livebacteria.

•First vaccine againstrabies

•Immunize rabbits with tetanus bacteria.

•Isolate serum (non-cellular portion of blood) from immunizedrabbits, inject “immune serum” into naïve rabbit

•Challenge treated and untreated rabbits with lethal dose of livetetanus bacteria: only treated mice survived.

•Passive immunization with pooled human immunoglobulin iscurrently used to treat immunodeficiency and to providetemporary resistance to infection (hepatitis, rabies, measles,tetanus).

Von Behring & Kitasato: ~1890 discovered“passive immunization”

•Tiselius & Kabat: 1930s. Discovered that the γ-globulin fraction ofserum contains antibodies.

Immunize rabbits with chicken ovalbumin.Here a purified foreign protein (chicken ovalbumin), rather than a

pathogen, is the “antigen”, or substance that induces an antibody responseresponse. (“model antigen”)

A serum sample from an immunized rabbit contains a substance with the capacityto bind to ovalbumin, called “antibody”.

Serum contains many different proteins. Which serum fraction contains theantibody?

Blue: serumfrom immune rabbit

Black: serum with specificbinding activity depleted

•Tiselius & Kabat: 1930s. Discovered that the γ-globulin fraction ofserum contains antibodies.

Immunize rabbits with chicken ovalbumin. Isolate serum, divide into 2 parts.Separate one serum sample into different protein fractions (based on size, charge) usingelectrophoresis.Mix another serum sample with ovalbumin and remove the insoluble immune complexes(containing ovalbumin plus serum proteins that bind ovalbumin).Re-analyze depleted serum by electrophoresis and compare to the starting serum sample.

The γ-globulin fraction ofserum contains most antibody:Termed “immunoglobulin”

Antigens & Antibodies IDiscovery of antibodies

Basic Antibody Structurebrief review of protein structuredisulfide linked tetramer: 2 heavy and 2 light chainsmyeloma proteins and the primary structure of antibodycrystal structure of antibody: the Ig domain

The antigen binding site of antibodies

Antibody isotypes: IgM, IgG, IgD, IgA, IgEThe advantages of multivalencyeffector functions of antibody isotypes

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Levels of Protein Structure Non-covalent forces that holdantigens and antibodies together

Energy of chemical interactionsinvolved in stability of protein

structure

Bond Length EnergyCovalent 0.15 nm 90 kcal/molIonic 0.25 nm 3 kcal/molHydrogen 0.30 nm 1 kcal/molVan der Waals 0.35 nm 0.1 kcal/molHydrophobic 0.35 nm 0.1 kcal/mol

The Alpha-Helix

The Beta-Sheet Ribbon Diagrams

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Antigens & Antibodies IDiscovery of antibodies

Basic Antibody Structurebrief review of protein structuredisulfide linked tetramer: 2 heavy and 2 light chainsmyeloma proteins, Ig domains, and hypervariable regions

The antigen binding site of antibodies

Antibody isotypes: IgM, IgG, IgD, IgA, IgEThe advantages of multivalencyeffector functions of antibody isotypes

Basic Immunoglobulin (antibody) structure

Biochemical methods tocharacterize IgG structure

(Porter and Edelman 1960s)1972 Nobel Prize for medicine:

partial proteolysis chromatography

Fab: fragment antigen bindingFc: fragment crystalizes

cleavage of disulfide bondschromatography

Heavy chain ~50 KdaLight chain ~25 Kda

Basic Immunoglobulin (antibody) structure

2 identicalantigen bindingsites/molecule

Each antibody molecules consists of twoidentical heavy chains (50 kDa) and two

identical light chains (25 kDa).

Chains are held together by inter-chaindisulfide bonds.

Structure made up of repeating structuralunits of ~110 amino acids called Ig

domain (2/light chain and 4/heavy chain).

N-terminal Ig domain is variable (antigenbinding domain). The C-terminal Ig

domains are constant (effector functions).

Basic Immunoglobulin (antibody) structure Antibodies are bivalent (2 identical antigen binding sites).

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Membrane-associated vs.secreted immunoglobulin

Distinct carboxy-termini

Basic Immunoglobulin (antibody) structure

2 identicalantigen bindingsites/molecule

Each antibody molecules consists of twoidentical heavy chains (50 kDa) and two

identical light chains (25 kDa).

Chains are held together by inter-chaindisufide bonds.

Structure made up of repeating structuralunits of ~110 amino acids called Ig

domain (2/light chain and 4/heavy chain).

N-terminal Ig domain is variable (antigenbinding domain). The C-terminal Ig

domains are constant (effector functions).

Variations in the heavy chain lead todifferent antibody isotypes, and membrane

vs secreted forms of antibody.

Antigens & Antibodies IDiscovery of antibodies

Basic Antibody Structurebrief review of protein structuredisulfide linked tetramer: 2 heavy and 2 light chainsmyeloma proteins, Ig domains, and hypervariable regions

The antigen binding site of antibodies

Antibody isotypes: IgM, IgG, IgD, IgA, IgEThe advantages of multivalencyeffector functions of antibody isotypes

In a normal individual, antibodies are extremely heterogeneous.

Myeloma protein: key to determining Igstructure

• Heterogeneity of antibodies makes sequencingimpossible (each B cell clone produces a unique versionof antibody).

• Multiple myeloma: cancer derived from an antibodyproducing cells (plasma B cell).

• Myeloma patients have large amounts of one particularIg molecule in their serum (and urine)

• Many patients produce a large amount of one light chain,known as “Bence-Jones” proteins.

Antibodymolecules arecomposed ofrepeats of a singlestructural unitknown as the“immunoglobulindomain”

When the amino acid sequences of several differentBence-Jones proteins were compared, they were found toconsist of two repeating units of ~110 amino acids: onevariable and one constant.

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Protein homology• Identity or similarity between domains in two or more

proteins• Most easy to see at the level of primary amino acid

sequence (computer programs find it)• Sometimes no obvious primary sequence homology but

striking structural homology• Homology can sometimes predict structure and function

All Ig domains have asimilar 3D structure

known as an“Immunoglobulin Fold”.

2 β-pleated sheets cometogether to form a

sandwich, held togetherby disulfide bond and

hydrophobic interactions.

3 flexible loops at end:correspond to

hypervariable regions ofprimary sequence (HV).

The ImmunoglobulinFold is a very commonlyused structural motifamongst cell surface proteins

Ig domain: Genome Project Champion!

The variability of antibodies occurs within 3 discrete regionsof the primary sequence: hypervariable regions HV1-3

The hypervariable regions (HV1-3) are separated in primary structure,but come together in the tertiary structure where they form the antigenbinding site. Alias Complementary Determining Regions or CDR1-3.

The HV regions formloops at the end of the Ig

domain.

The intervening frame-work regions (FR1-4)

make up the rest of thestructure.

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The quaternary structure of immunoglobulin

Associations between Ig domains.

Interchain disulfide bonds

Hinge region allows flexible movementof Fc regions

6 CDR (3 from HC, 3 from LC) combineto make up antigen binding site

Because the CDR are highly variable, each antibody moleculehas a unique antigen binding site with its own dimensions and

complementarity.

Antibodies thatbind to large

proteins antigens

Antibodies thatbind to small

molecules