Administrative issues Recommended text GoldsbyKuby Immunology 6 th

Administrative issues: Recommended text: Goldsby/Kuby Immunology, 6 th edition (Note that Innate Immunity is not adequately covered in the 5 th edition. ) Discussion sections start next week. The journal article Akira et al, and the first problem set will be covered. Both are available on the website. Office Hours: Questions about the lecture material are best addressed during office hours (Tues 11 -12). I will be holding extra office hours (date and time TBA) before the first midterm. Email: Please use email only for VERY simple yes/no questions or simple administrative matters. Great questions, keep them coming!

Innate Immunity Innate immune effector mechanisms Physical and biochemical barriers (defensins) Phagocytosis and reactive oxygen species Cell autonomous defenses Apoptosis Interferons and PKR Innate immune recognition discovery of the Toll-like receptors mammalian TLRs and their ligands non-TLR recognition of PAMPs Connections between adaptive and innate immunity

Some microbes hijack cellular machinery to replicate and spread. Intracellular pathogens include viruses (influenza, HIV) and intracellular bacteria (listeria) and intracellular parasites (malaria, toxoplasma). cell-autonomous defense: cell produces an immune response that acts on itself

Apoptosis: Cellular Suicide • Nuclear fragmentation • Proteolysis • Blebbing • Death Remnants undergo phagocytosis

Apoptosis versus Necrosis • Tidy: contents of cells degraded from within, producing small cellular “blebs” • Programmed from inside the cell • Messy: contents of cell released. • Induced by external insult

Cell death by necrosis is more likely to produce inflammation.

Cells can avoid being hijacked by viruses by activating the Protein Kinase R (PKR) pathway. PKR is triggered by ds. RNA and interferon. Interferons are cytokines that are produced in response to viral infection. Produce an “anti-viral state” in target cells. Acts on cell that produces it, as well as neighboring cells. Together with ds. RNA, act to triggering the Protein Kinase R (PKR) pathway. Shuts down protein synthesis machinery of cells, thus preventing viral replication.

Protein Kinase R: Interfering with Infection e. IF 2 a ds. RNA or interferon Kinase domain RNAbinding domain PKR Phosphorylated PKR (active) Phosphorylated e. IF 2 a (inactive)

Innate Immunity (finishing up) Innate immune effector mechanisms Physical and biochemical barriers (defensins) Phagocytosis and reactive oxygen Cell autonomous defenses Apoptosis Interferons and PKR Innate immune recognition discovery of the Toll-like receptors mammalian TLRs and their ligands non-TLR recognition of PAMPs Connections between adaptive and innate immunity

Cell types of innate immunity

Components of the bacterial cell wall, such as lipopolysaccaride (LPS), peptidoglycan, lipoprotein can trigger the innate immune system. Cross section of the cell wall of a gram-negative bacteria.

Comparison of the adaptive and innate immune responses innate adaptive Response time hours days Response to repeat infection identical to primary (no memory) stronger response upon second exposure (memory) Receptors that Mediate pathogen Recognition pattern recognition receptors e. g. Toll-like receptors (TLR) limited diversity, fixed in germline antibodies and T cell antigen receptors (TCR) Ligands Pathogen associated molecular patterns (PAMPs) virtually any component of pathogen unlimited diversity generated by V(D)J recombination

What triggers innate immune responses? ( How do phagocytes know what to eat? ) • Long-known that bacterial cell wall components activate phagocytes • Hypothesis-- microbes contains pathogen associated molecular patterns (PAMPs) which are recognized by pattern recognition receptors (PRRs) • A series of disparate observations in transcriptional regulation and drosophila development lead to the discovery of PRRs

Genetic analysis of early embryonic development in the fruitfly, Drosophila (Nusslein-Volhard and colleagues)

The Dorsal Signaling Pathway controls dorsal/ventral polarity in the Drosophila early embryo Dorsal Toll (a receptor) Cactus Ventral “Toll” is German slang for “weird” Dorsal (a transcription factor)

NF-k. B: a transcription factor which binds to antibody genes (Baltimore and colleagues). Transcription factor Ig k gene enhancer Nuclear factor Igk locus B (as in A, B, C, etc. )

NF-k. B: A critical transcription factor for innate immunity p 65 Ik. B p 50 Inactive, cytoplasmic (cactus) p 50 (dorsal) Active, nuclear Mutant mice lacking NF-k. B subunits have defects in innate immunity. What is the mammalian receptor that leads to NF-k. B activation in response to infection?

Discovery of the mammalian Toll-like receptors (TLR): 1997: Janeway and Medzhitov discovered a human protein with structural similarity to drosophila Toll that could activate immune response genes in human cells (TLR 4). 1998: Beutler discovered that a mouse strain with an altered response to bacterial lipopolysaccharide (called LPS or endotoxin) was due to a mutation in the TLR 4 gene. There are 11 TLR family members in human and 12 in mice. Each responds to a distinct set of microbial products.

Different mammalian Toll-like receptors (TLRs) are specific for different classes of microbial products Insert Fig 3 -11

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

Toll-like receptors (TLRs) link microbial products (PAMPs) to transcription factor activation in a signaling pathway that is conserved between mammals and insects

Toll-mutant drosophila are susceptible to fungal infections

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

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

Pathogen recognition receptors (PRR) in the innate immune system: TLRs are not the whole story scavenger receptors NOD proteins (cytoplasmic PRR) etc, etc

The benefits and hazards of TLR signaling Septic shock can be caused by systemic infection with gram negative bacteria (Salmonella). Shock is caused by overwhelming production of cytokines and is induced by presence of LPS in blood stream. Mice lacking TLR 4 are resistant to LPSinduced shock. Mice lacking TLR 4 are also more sensitive to infection with gram negative bacteria. (local vs. systemic effects of TLR signaling? )

Triggering of PRRs on macrophage or dendritic cells can induce a LARGE variety of events including: Increased phagocytosis Production of cytokines and inflammatory mediators: Interferons to induce anti-viral state Chemokines to attract migrating cells Etc, etc. Increased cell migration Changes in expression of molecules involved in T cell antigen presenting cell function.

Innate Immunity Innate immune effector mechanisms Physical and biochemical barriers (defensins) Phagocytosis and reactive oxygen Cell autonomous defenses Apoptosis Interferons and PKR Innate immune recognition discovery of the Toll-like receptors TLRs and their ligands non-TLR recognition of PAMPs Connections between adaptive and innate immunity

When innate immune signaling is insufficient to clear a pathogen, the adaptive immune system kicks in. Innate immune signaling turns on the adaptive immune response. Macrophage and dendritic cells serve as antigen-presenting cells for adaptive immune 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 more effective antigen presenting cells than resting immune cells.

TLR signaling within phagosomes determines fate of that phagosome (destruction vs antigen presentation). Pathogen (non-self, TLR signaling) Material in phagosome enters antigen presentation pathwaypresentation to T cells Dying infected cell (self, no TLR signaling) Material in phagosome disposed of inside cell -no presentation to T cells Blander and Medzhitov 2006 Nature v 440 p 808

Comparison of innate and adaptive immune recognition Receptors that mediate innate immune recognition: Tolllike receptors (TLR) Receptors that mediate adaptive immune recognition: Antibody and the T cell receptor (TCR)

The genes encoding the antigen receptors of T and B cells are assembled by DNA rearrangement as these cells develop. As a result of V(D)J recombination, every B and T cells expresses a unique 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 particular specificity.

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 on antigen -antibody binding interactions) Techniques based on antibodies V(D)J recombination B cell development and function

Antigens & Antibodies I Discovery of antibodies Basic Antibody Structure brief review of protein structure disulfide linked tetramer: 2 heavy and 2 light chains myeloma proteins and the primary structure of antibody crystal structure of antibody: the Ig domain The antigen binding site of antibodies Antibody isotypes: Ig. M, Ig. G, Ig. D, Ig. A, Ig. E The advantages of multivalency effector functions of antibody isotypes

Early Observations of Acquired (adaptive) Immunity • Thucydides, historian: 430 BC noted that those who had survived plague could nurse the sick and not become sick again. • 1600 s, Turks and Chinese practiced “variolation”: intentional exposure to material from smallpox lesions to provide protection against smallpox. • Edward Jenner: ~1800 noted that exposure to vaccinia virus (cowpox) protects against smallpox. Tested first vaccine using cowpox.

• Louis Pasteur: ~1880 • finds that exposure to attenuated bacteria (chicken cholera) protects against live bacteria. • First vaccine against rabies

Von Behring & Kitasato: ~1890 discovered “passive immunization” • Immunize rabbits with tetanus bacteria. • Isolate serum (non-cellular portion of blood) from immunized rabbits, inject “immune serum” into naïve rabbit • Challenge treated and untreated rabbits with lethal dose of live tetanus bacteria: only treated mice survived. • Passive immunization with pooled human immunoglobulin is currently used to treat immunodeficiency and to provide temporary resistance to infection (hepatitis, rabies, measles, tetanus).

• Tiselius & Kabat: 1930 s. Discovered that the g-globulin fraction of serum 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 response. (“model antigen”) A serum sample from an immunized rabbit contains a substance with the capacity to bind to ovalbumin, called “antibody”. Serum contains many different proteins. Which serum fraction contains the antibody?

• Tiselius & Kabat: 1930 s. Discovered that the g-globulin fraction of serum 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) using electrophoresis. 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. Blue: serum from immune rabbit Black: serum with specific binding activity depleted The g-globulin fraction of serum contains most antibody: Termed “immunoglobulin”

Antigens & Antibodies I Discovery of antibodies Basic Antibody Structure brief review of protein structure disulfide linked tetramer: 2 heavy and 2 light chains myeloma proteins and the primary structure of antibody crystal structure of antibody: the Ig domain The antigen binding site of antibodies Antibody isotypes: Ig. M, Ig. G, Ig. D, Ig. A, Ig. E The advantages of multivalency effector functions of antibody isotypes

Levels of Protein Structure

Non-covalent forces that hold antigens and antibodies together

Energy of chemical interactions involved in stability of protein structure Covalent Ionic Hydrogen Van der Waals Hydrophobic Bond Length 0. 15 nm 0. 25 nm 0. 30 nm 0. 35 nm Energy 90 kcal/mol 3 kcal/mol 1 kcal/mol 0. 1 kcal/mol

The Alpha-Helix

The Beta-Sheet

Ribbon Diagrams

Antigens & Antibodies I Discovery of antibodies Basic Antibody Structure brief review of protein structure disulfide linked tetramer: 2 heavy and 2 light chains myeloma proteins, Ig domains, and hypervariable regions The antigen binding site of antibodies Antibody isotypes: Ig. M, Ig. G, Ig. D, Ig. A, Ig. E The advantages of multivalency effector functions of antibody isotypes

Basic Immunoglobulin (antibody) structure

Biochemical methods to characterize Ig. G structure (Porter and Edelman 1960 s) 1972 Nobel Prize for medicine: partial proteolysis chromatography Fab: fragment antigen binding Fc: fragment crystalizes cleavage of disulfide bonds chromatography Heavy chain ~50 Kda Light chain ~25 Kda

Basic Immunoglobulin (antibody) structure 2 identical antigen binding sites/molecule Each antibody molecules consists of two identical heavy chains (50 k. Da) and two identical light chains (25 k. Da). Chains are held together by inter-chain disulfide bonds. Structure made up of repeating structural units of ~110 amino acids called Ig domain (2/light chain and 4/heavy chain). N-terminal Ig domain is variable (antigen binding domain). The C-terminal Ig domains are constant (effector functions).

Basic Immunoglobulin (antibody) structure

Antibodies are bivalent (2 identical antigen binding sites).

Membrane-associated vs. secreted immunoglobulin Distinct carboxy-termini

Basic Immunoglobulin (antibody) structure 2 identical antigen binding sites/molecule Each antibody molecules consists of two identical heavy chains (50 k. Da) and two identical light chains (25 k. Da). Chains are held together by inter-chain disufide bonds. Structure made up of repeating structural units of ~110 amino acids called Ig domain (2/light chain and 4/heavy chain). N-terminal Ig domain is variable (antigen binding domain). The C-terminal Ig domains are constant (effector functions). Variations in the heavy chain lead to different antibody isotypes, and membrane vs secreted forms of antibody.

Antigens & Antibodies I Discovery of antibodies Basic Antibody Structure brief review of protein structure disulfide linked tetramer: 2 heavy and 2 light chains myeloma proteins, Ig domains, and hypervariable regions The antigen binding site of antibodies Antibody isotypes: Ig. M, Ig. G, Ig. D, Ig. A, Ig. E The advantages of multivalency effector functions of antibody isotypes

In a normal individual, antibodies are extremely heterogeneous.

Myeloma protein: key to determining Ig structure • Heterogeneity of antibodies makes sequencing impossible (each B cell clone produces a unique version of antibody). • Multiple myeloma: cancer derived from an antibody producing cells (plasma B cell). • Myeloma patients have large amounts of one particular Ig molecule in their serum (and urine) • Many patients produce a large amount of one light chain, known as “Bence-Jones” proteins.

When the amino acid sequences of several different Bence. Jones proteins were compared, they were found to consist of two repeating units of ~110 amino acids: one variable and one constant. Antibody molecules are composed of repeats of a single structural unit known as the “immunoglobulin domain”

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 a similar 3 D structure known as an “Immunoglobulin Fold”. 2 b-pleated sheets come together to form a sandwich, held together by disulfide bond and hydrophobic interactions. 3 flexible loops at end: correspond to hypervariable regions of primary sequence (HV).

The Immunoglobulin Fold is a very commonly used structural motif amongst cell surface proteins

Ig domain: Genome Project Champion!

The variability of antibodies occurs within 3 discrete regions of the primary sequence: hypervariable regions HV 1 -3

The hypervariable regions (HV 1 -3) are separated in primary structure, but come together in the tertiary structure where they form the antigen binding site. Alias Complementary Determining Regions or CDR 1 -3. The HV regions form loops at the end of the Ig domain. The intervening framework regions (FR 1 -4) make up the rest of the structure.

The quaternary structure of immunoglobulin Associations between Ig domains. Interchain disulfide bonds Hinge region allows flexible movement of Fc regions 6 CDR (3 from HC, 3 from LC) combine to make up antigen binding site

Because the CDR are highly variable, each antibody molecule has a unique antigen binding site with its own dimensions and complementarity. Antibodies that bind to large proteins antigens Antibodies that bind to small molecules