Immunology

1. Immunology • Chapter 8

2. Immunity and the Immune Response • Innate immunity • Molecules that pre-exist in the body that recognize common microbial motifs • Adaptive Immunity • Passive • Administration of antibodies specific to a pathogen • Antibodies neutralize pathogen before disease onset • Active • T and B lymphocytes recognize pathogen and specifically target it for destruction

3. Mechanisms of Innate Immunity • Physiologic barriers • Skin • Mucous membranes • Innate immunologic mechanisms • Organs and tissues involved in recognizing foreign substances • Phagocytic cells are strategically located in these organs, adjacent to blood and lymphatic vessels • Cells • Neutrophils - phagocytic and possess inflammatory granules (e.g., histamine) • Macrophages - phagocytic cells that exist in nearly all tissues that monitor for infectious agents • Inflammation • Mediated by cytokines, which are small soluble proteins secreted by many cells • Fever - caused by interleukin-1 that acts upon hypothalamus • Interferons • Natural killer cells - cause lots of collateral damage

4. Mechanisms of Specific Host Defense • Adaptive response • Antibody (humoral) response • Now referred to as Type 2 immunity • Prominent antibody response • But cells are involved, too! • Cell-Mediated response • Now referred to as Type 1 immunity • Prominent cellular response • But antibodies are involved, too! • Cells are lymphocytes • B cells • Restricted to lymphoid tissues (e.g., lymph nodes) • Secrete antibodies specific for the pathogen • T cells - circulate through blood and lymph • Helper T cells (Th) • Direct the actions of other cells by secreting cytokines that signal and coordinate such activities • Cytotoxic T cells (CTL) • Recognize cells infected by viruses and kills them

5. Mechanisms of Specific Host Defense • Antigens (Ag) • Any substance that can elicit an immune response in an animal • The body can distinguish self molecules from nonself molecules • Failure of this system can result in autoimmune diseases • Most antigens are large proteins • Some carbohydrates can be antigenic, but they are generally poor antigens because T cells cannot recognize them • Antigenic determinants (epitopes) are the parts of an antigen that are recognized by lymphocytes • B cells recognize parts of the 3D structure of an antigen • T cells recognize linear peptides of 8-14 amino acids of a protein antigen • Genetic constitution of the host contributes to disease susceptibility • Dosage, route and time of antigen administration influence immune responses • Adjuvants are usually required to elicit an effective immune response to non-living antigens • These are usually irritants that induces localized inflammation

6. Mechanisms of Specific Host Defense • Cellular basis of the immune response • B cells • Develop from stem cells in the bone marrow • The B cell repertoire from the development results in billions of B cells specific for different antigenic determinants • The express an antigen-specific B cell receptor (BCR), which is simply a membrane-bound antibody • T cells • Early development is in the bone marrow • Final development is in the thymus and results in billions of T cells specific for different antigenic determinants • Possess an antigen-specific T cell receptor (TCR) that has substantial sequence similarity to antibodies • Two types of T cells • Helper T cells (Th) • Respond to extracellular protein antigens • Secrete cytokines that mediate local immune responses • Cytotoxic T cells (CTL) • Respond to intracellular protein antigens • Kill cells that are infected

7. Antibodies • Antibodies (Ab) are immunoglobulins that recognize antigens • All antibodies are immunoglobulins, but not all immunoglobulins are antibodies • They are proteins • B cells express BCR specific for an antigen • Each B cell possesses thousands of identical BCRs on their surfaces • When the antigen enters the body, it must find the few B cells that possess a BCR capable of binding to it • This can take several days • When recognition occurs, the B cell, with the help of Th cell cytokines, begins to secrete antibodies in soluble form • The B cell also undergoes clonal expansion; repeated rounds of cell division • The process includes mutations in the antibody genes of the daughter cells that leads to antibodies with greater affinity for antigen; termed affinity maturation • The antibodies also switch to other classes of Ig, each with distinct biological activities • A plasma cell is a B cell that is committed to secreting antibodies for 2 or 3 days, then it dies

8. Antibodies

9. Antibodies • Antibody structure • Composed of two identical heavy chains and two identical light chains • Form a disulfide-linked tetramers • Each antibody molecule includes: • Two variable regions that interact with the antigen • Some antibodies bind to viruses in such a way as to interfere their infectious capacity • These are termed neutralizing antibodies • A constant region that confers biological activities, such as • Binding to receptors on phagocytic cells • Binding to complement proteins

10. Antibodies • Immunoglobulin classes • IgM • First to be made during a primary (initial) response • Often secreted as a pentamer of 5 IgM antibodies covalently-linked to one another (Fig 8-4) • Low affinity, but high avidity • IgG • Most common serum antibody • Occurs after the IgM response • High affinity • Persist for years in the blood • Four subclasses encoded by different genes • IgG1 • IgG2 • IgG3 • IgG4

11. Antibodies • Immunoglobulin classes (cont.) • IgA • Most common antibody produced by the body • Secreted into mucosal areas • Often found as a dimer of two covalently-linked IgA molecules • IgE • Associated with parasitic and helminth infections, and with hypersensitivities • IgD • Expressed as a BCR only • Immunoglobulin class switching and affinity maturation • All secreted antibodies are initially IgM • As the immune response ensues, the class switches to IgG, IgA or IgE • This phenomenon is also accompanied by affinity maturation • Both of these events require T cell help

12. Cell Surface Receptors for Antigen • B cell receptor for antigen • Naive B cells express IgM or IgD as their surface receptor (sIg) • The antibody receptor possesses transmembrane domains at the C-terminus of the H chains • This domain anchors the H-chain in the membrane of the rough ER when the polypeptides are synthesized • Thus, the antibody receptor protrudes from the B cell’s plasma membrane • This receptor is usually of low affinity because it’s only aclose-match to its antigenic epitope (10-7KD) • This receptor is used to capture the antigen, which is then internalized by receptor-mediated endocytosis • When a B cell is activated (i.e., immune stimulus), transcriptional processing of the antibody mRNA results in a transcript free of the codons for the transmembrane domain, thus the antibody is secreted in a vesicle • Memory B cells have IgG, IgA or IgE sIg • They have undergone class switching • These receptors are high affinity because they have undergone affinity maturation (up to 10-11 KD) • They are extremely efficient at capturing antigen • All B cells have a sIg-associated Igα/Igβ signal transduction molecule

13. Cell Surface Receptors for Antigen

14. Cell Surface Receptors for Antigen • T cell receptor • Transmembrane heterodimer (not secreted) • Structure is similar to the V/H domain of an antibody • Two types (only one type found on a given T cell) • αβ (alpha-beta) • γδ (gamma-delta) • Two cells • Helper T cells (Th) express CD4 on their surfaces • CD4 interacts with MHC class II proteins on antigen presenting cells (APC) • Cytotoxic T cells (CTL) express CD8 on their surfaces • CD8 interacts with MHC class I proteins on APC • All T cells have CD3 and ζ-chain (zeta-chain), signal transduction components

15. Cell Surface Receptors for Antigen

16. Cell Surface Receptors for Antigen • The Major Histocompatibility Complex • Large contiguous region of mammalian genomes that encode about 100 proteins involved in antigen processing and presentation • Name is a misnomer; it was first characterized as controlling tissue rejection in mice in the 1930s-1950s • Two principal classes • Class I molecules (3 loci in humans) • Expressed by all nucleated cells, which act as APC • Responsible for presenting peptides from endogenously-synthesized polypeptides (i.e., made on cellular ribosomes) • At rough ER, viral polypeptides are clipped into 8-10 amino acid peptides by the proteosome and then transported into the lumen of the ER • Class I molecules bind viral peptides at the ER, then traffic to the cell surface • Marks the cell for death by CTL (via CD8 interaction with class I molecule) • Class II molecules (3 loci in humans) • Expressed by professional APC • B cells • Macrophages • Dendritic cells • A few others • For antigens captured by receptor-mediated endocytosis or phagocytosis (i.e., exogenous) • Fusion of phagosome with class II-bearing endosome results phagolysosome • Peptides bind to class II proteins • The class II molecules traffic to the cell surface where they are engaged by helper T cells • CD4 binds to the class II molecule, thus restricting the interaction to Th cells

17. Cell Surface Receptors for Antigen

18. Type II (Antibody-Mediated) Immunity • Principally for exogenous (extracellular) antigens • The primary response • Within days after infection, antigen-specific IgM appears • Within a week, antigen-specific IgG appears • Class switching and affinity maturation improve the efficiency of the antibody response • As antigen becomes limiting (i.e., the immune system clears the infection) the immune response wanes • Most (98%) responding B cells die, but a few remain as memory cells • Unlike naive B cells with IgM, memory cells possess high-affinity sIgG or sIgA • The secondary (anamnestic) response • In the event the pathogen invades again, the immune response will be of greater speed and magnitude because of the memory cells • No need for class switching • No need for affinity maturation • Antibody response is quicker and of higher affinity • There are usually no clinical symptoms of infection

19. Type II (Antibody-Mediated) Immunity • The role of helper T cells in Type II immunity • Exogenous antigens (such as free virus) are captured by professional APC • B cell uses its sIg receptor • Macrophages are phagocytic • Dendritic cells are pinocytotic • The antigen is imported into a lysosome (phagolysosome) that has more than 60 types of molecules, including digestive enzymes • Proteins are digested into peptides of varying length • The phagolysosome is fused with another endosome containing nascent MHC class II proteins • Packaged with the class II endosome is the invarient chain, which occludes the peptide binding cleft to prevent premature peptide binding • The peptides bind to the class II proteins and additional protease activity occurs until the peptides are about 11-15 amino acids • The vesicle traffics to the cell surface where it fuses with the plasma membrane (i.e., class II/peptide complex is outside of the cell) • The Th cell must recognize both the class II protein and peptide before the T cell can become activated (proliferation and cytokine synthesis)

21. B cell Secreted antibodies Helper T cell Cytokines that modulate the immune response (e.g., class switching, affinity maturation, antibody production)

22. Type I (Cell Mediated) Immunity • Principally for containment of endogenously-produced (intracellular) antigens • Viruses rely upon cellular ribosomes for the biosynthesis of their polypeptides • A small proportion of all polypeptides (self and non-self) synthesized in a cell are re-directed to the proteosome complex in the cytoplasm • The proteosome is a tubular protein structure that has protease activity • Polypeptides enter the proteosome, which can hold about 8-10 amino acids, then its protease activity results in clipping into peptides • The peptides are ejected from the other end of the proteosome • The transporter associated with antigen processing (TAP) translocates the peptides into the lumen of the ER • This event requires ATP • Inside the ER, nascent MHC class I molecules bind the peptides • After budding from the ER, the class I/peptide traffic to the Golgi, then the cell surface • The class I/peptide complex is displayed on the outside of the cells for scanning by the CTL repertoire • The CTL kills the cell by sending it a death signal (apoptosis) or by secreting perforin, a protein that polymerizes to form channels in the membrane of the target cell

24. The Complement System • The complement system is composed of several pre-existing blood proteins that puncture membranes • There are three pathways, one of which is important to viral infections, the classical pathway • The classical pathway is antibody-dependent • When IgM or IgG bind to envelope viruses, the complement cascade ensues • This cascade is a series of enzymatic reactions carried out by the complement proteins • Two final outcomes occur • Complement proteins attack the envelope, thus compromising it • The complement-bound viruses are opsonized, that is, they are more efficiently phagocytosed

25. Cytokines • Cytokines are hormone-like proteins • There are more than 80 known • Interleukins - secreted by leukocytes • Interferons - potent antivirals • Chemokines - mediate chemotaxis • Inflammation/immunosuppression • Potent, often active at picomolar concentrations • Bind to receptors on target cells and modulate gene expression • Some are used therapeutically (recombinant) • Some are associated with T cell subsets • Th1 cells secrete... • IFNγ • TNF • LT • Th2 cells secrete... • IL-4 • IL-5

26. Overly Aggressive Immune Responses • Often results in immunopathology • There are no good clinical strategies for managing pathologic immune responses

27. Immunologic Diagnostic Tests • The use of blood to detect antigen-specific antibodies in testing is termed serology • Relies upon the fact that serum antibodies to specific pathogens cannot be detected until after infection (or immunization) • Provides diagnostic indicator of infection • IgM - current/recent infection • IgG - current or past infection (decades) • Common serological tests • Enzyme-linked immunosorbant assay (ELISA) • Rapid • High throughput • Inexpensive • Immunofluorescent antibody (IFA) • Immunoblotting

28. Immunologic Diagnostic Tests

29. Immunologic Diagnostic Tests • Immunoblotting • More specific and sensitive than ELISA • Also more work and more expensive • Most common is western blot • First step is electrophoresis of antigen (SDS-PAGE) • Second is transfer of antigen from gel to solid phase, such as nitrocellulose membrane • Steps for western blot • Block membrane with irrelevant protein (nonfat powdered milk solution) • Incubate serum sample • Incubate anti-antibody, enzyme conjugate • Incubate substrate • Look for bands appearing on membrane

30. Immunologic Diagnostic Tests