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Innate Immunity (part II) and Antigen Recognition by Adaptive Immunity

Innate Immunity against viruses • Anti-viral immunity has 2 roles

– Blocking infection (antibodies, complement, etc.) – Blocking viral replication (interferon, killing infected cells, antinucleic acid mechanisms)

• Many viruses evolve extremely rapidly, great challenge for innate immunity • This problem is solved in part by a) targeting molecules that viruses have a hard time changing (dsRNA especially), and b) having multiple mechanisms, making it harder for a virus to evade all of them • Viruses are amazingly good at evasion of immune defenses, but often the most lethal viruses are those that are not well adapted to their host (their goal is to grow and be transmitted to others, not to kill)

INTERFERON: cytokine critical for defense against virus infections 1) Virus-infected cell produces interferon to act on neighboring cells infected cell

Interferon-a

2) Uninfected cells respond to become refractory to viral growth (“anti-viral state”) type 1 interferon= interferon a/ type 2 interferon= interferon 

Induction of interferon by dsRNA in cytoplasm and RLRs

RLRs

Mda5

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IRF: interferon regulatory-factor (family of transcription regulators)

Anti-viral effects of interferon a/

Many other responses, less well understood

Note: PKR and OligoA synthetase induced in anti-viral state, but only have enzymatic activity following virus infection QuickTime™ and a decompressor are needed to see this picture.

Second mechanism of interferon production upon virus infection: Toll-like receptors recognize viral nucleic acid in endosomes

IntracellularTLRs recognize pathogen nucleic acids (TLR3, 7, 8, 9)

Induce type 1 interferon production, especially by specialized immune cell (plasmacytoid dendritic cell) Location inside cell aids discrimination from host nucleic acids (Discrimination may fail in SLE)

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Natural Killer (NK) Cells: innate lymphocytes that protect against intracellular infections

Abbas and Lichtman Fig. 2-10

NK cells are regulated by the balance between activating and inhibitory receptors

stress-induced proteins (red)

healthy cell

stressed cell

NK cell has activating receptors and inhibitory receptors: killing believed to occur if activating receptors dominate (relative numbers of ligands on target cell)

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Summary of Innate Immunity to Viruses •Interferon (type 1) is absolutely critical for anti-virus immune defense; it is produced in two ways •by infected cells via RLRs or similar sensors of DNA for DNA viruses •by immune cells via nucleic acid-recognizing TLRs

•Interferon induces anti-viral state, which is fully engaged by recognition of dsRNA in cytoplasm and inhibits virus replication (also promotes adaptive immunity) •Killing of infected cells is also performed by natural killer cells recognizing stress-induced molecules or loss of MHC class I molecule expression and by cytotoxic T cells which recognize virus antigens expressed by infected cells (+MHC I)

Pathogens and Innate Immunity •Particular pathogens often have evolved ways to evade at least some elements of innate immunity •Highly successful pathogens may also have mechanisms for evading adaptive immunity •On the pathogen side, molecules the pathogen uses to evade immunity are among the “virulence factors” of that pathogen

Part II: antigen recognition • Antigen recognition molecules: structure and function • B cell antigen receptors and T cell antigen receptors: Role in clonal selection • Classes of antibodies and their structures • Monoclonal antibodies and their uses in medicine (lab tests, therapies)

2 Types of Antigen Recognition • 1) Antibodies – Defense against extracellular microbes and viruses by soluble binding molecules linked to effector functions – B cells also make a membrane-bound form of immunoglobulin that serves as their antigen receptor

• 2) T cell antigen receptor – Peptide recognition mechanism for detecting antigens inside cells that are displayed on cell surface as peptides bound to MHC molecules

The B cell antigen receptor is membrane Ig + signaling chains •Antigen binding to more than one membrane Ig molecule triggers signaling reactions; tells B cell that antigen is present, induces activation (“clonal selection”) Iga/Ig •Iga/Ig cytoplasmic domains have signaling function (ITAM sequence) •Differential RNA splicing controls secreted vs. transmembrane forms of Ig heavy chain Signal transduction QuickTime™ and a decompressor are needed to see this picture.

The TCR also has ligand-binding chains + signaling chains

Figure 4-1 Abbas & Lichtman

Antibodies • = immunoglobulins • Secreted by B lymphocytes (“plasma cells”) • Great diversity and specificity: >109 different antibodies; can distinguish between very similar molecules • Block infectivity of viruses, action of toxins (“neutralization”) • Tag particles for clearance/destruction (activate complement, “opsonize”for phagocytosis) • Protect against re-infection (vaccines)

Antibody Structure 2 identical heavy chains and 2 identical light chains Both made of repeating structural unit: The Ig domain

Light chain k or l

Ig domain: 110 amino acids; globular domain used in many proteins Heavy chains QuickTime™ and a decompressor are needed to see this picture.

Antibody Structure Domains at ends of heavy chain and light chain are highly variable and responsible for binding antigen

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Fab, variable domains in color QuickTime™ and a decompressor are needed to see this picture.

Variability in antibodies is clustered in the loops in the variable domains of the heavy and light chains (green); these regions are responsible for binding to antigen (“hypervariable regions”; “complementaritydetermining regions”)

Similarities between Ig and TCR • Both are composed of Ig domains • Both have two variable subunits (H + L for Ig; TCR a and TCR ; there are also “d T cells”) • Both have great diversity and exquisite specificity • Both recognize antigen via hypervariable loops at the ends of the variable domains • Both couple antigen recognition to lymphocyte activation (“clonal selection”) via signaling chains with very similar signaling mechanisms (ITAM sequences) • (Only Ig is secreted and only Ig has class switching to change the constant domains)

5 Different Antibody Classes

Heavy chains are named by the greek letter corresponding to the type of antibody (m, , d, a, e) All classes can have k or l light chains

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Antibody Classes II: Distinctive features • IgM – First antibody produced in immune response: multimeric structure adds to “avidity” for antigen – Antigen receptor of naïve B cells • IgD – Used primarily as antigen receptor, little secreted • IgG – Major antibody of serum and extravascular tissues – Crosses placenta to provide protection in utero • IgA – Major antibody of secretions (mucus, saliva, milk, etc.) • IgE – Important for immune defense against helminths; allergy and asthma

Affinity and Avidity •Affinity: the strength of binding between a single binding site and a single ligand •Avidity: the strength of binding between a molecule and a complex ligand. If there are multiple binding sites then the avidity may be increased by increasing the number of binding sites or by increasing the affinity of those binding sites

Affinity and Avidity II •IgM is produced early in an immune response when the affinity for antigen often is low; as an immune response continues, antibody affinity is improved, this is combined by “class switching” to the use of smaller molecules (IgG, IgE and IgA). The increased affinity compensates for the decrease in number of binding sites in maintaining the overall avidity for antigen

Antibody Classes II: Distinctive features • IgM – First antibody produced in immune response: multimeric structure adds to “avidity” for antigen – Antigen receptor of naïve B cells • IgD – Used primarily as antigen receptor, little secreted • IgG – Major antibody of serum and lymph – Crosses placenta to provide protection in utero • IgA – Major antibody of secretions (mucus, saliva, milk, etc.) • IgE – Important for immune defense against helminths; allergy and asthma

Fc receptors: •FcR have two functions: directing antibodies to correct locations in body and promoting effector function •Polymeric Ig receptor is a transport receptor for polymeric IgA and transports it across epithelial cells •FcRn is a transport receptor for IgG: -Transports IgG across placenta; -Responsible for long half-life of IgG in the blood

Antibodies and medicine • Vaccination works by inducing production of protective antibodies • Antibodies from immunized or pooled donors (“IVIG”) can provide “passive immunity” (used for tetanus,snake bites, etc.; to treat immunodeficiencies) • Antibodies are often used to diagnose infectious diseases (e.g., presence of antibodies in patient’s blood), determine bloodtype, diagnose type of cancer, etc. • Increasingly, antibodies are used to treat diseases (cancer, autoimmune disease, etc.): advantage of monoclonal antibodies

Monoclonal Antibodies • Normal antibodies are “polyclonal”, as they are mixtures of antibodies made by several different clones of B cells • Monoclonal antibodies: Single antibody (all same H and L chains): more reliable, consistent; can be produced in unlimited quantities • Made by fusion of B cells to a transformed cell line of the plasma cell type and selection for “hybridomas” that produce antibody with the desired properties

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Use of antibodies in lab tests: immunoprecipitate-based tests

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(Polyclonal antibodies)

Use of antibodies in lab tests: RIA and hemagglutination

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Hemagglutination assay: test blood type, etc.

Radioimmunoassay (RIA): measure hormone levels by displacement of binding of a radioactive hormone standard with hormone in a blood sample (etc.)

Use of antibodies in lab tests: ELISA

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Enyzyme-linked immunosorbant assay (ELISA): measure levels of antibody or antigen, depending on assay design (“sandwich ELISA is shown)

Use of antibodies in lab tests: flow cytometry

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Flow cytometry: measure the amount of a protein on the surface (or inside) individual cells; measure the numbers of particular types of cells in blood (etc.)

Monoclonal antibodies used in medicine Standardized, unlimited reagents for diagnosis or therapy (human antibodies or “humanized” antibodies can be made)

The list of monoclonal antibodies FDA-approved for therapy is increasing by several per year Names: fully human: -mumab humanized: -zumab chimeric: -ximab murine: -omab

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Anti-TNF therapeutics

TNFR2

Adalimumab (Humira) etc.

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Infliximab, etc.: anti-TNF monoclonal antibodies (more coming) Etanercept: fusion between TNF receptor extracellular domain and Fc part of IgG1, which greatly extends half-life in serum These agents are used to treat Rheumatoid arthritis, Crohn’s disease, etc.