Immunology

1. Chapter 3 Antigens and Antibodies and T cell receptors Dr. Capers Immunology

2. Hallmark molecules of adaptive immunity • Antibody and T-cell receptor • Antibody is part of the B cell receptor • Innate immunity recognizes patterns, whereas antibodies and T cell receptors have high degree of specificity

3. Antibodies and T cell receptors • Recognize epitopes • Immunologically active regions of immunogen that bind to antigen-specific antibodies or T-cell receptors

4. Antibodies (Abs) • Epitope binding proteins • Membrane bound on B cells OR • Secreted in blood • Humoral immunity • Share structural features, bind to antigen, and participate in number of effector functions • Known collectively as Immunoglobulins (Igs) • Abs don’t kill anything, their job is to plant the “kiss of death” on an invader

5. T cell Receptor • T Cell Receptor • Expressed on surface of T cells • Recognize processed antigen complexed with MHC molecules

6. Immunogenicity • Ability to induce humoral and/or cell-mediated immune response • Immunogen is substance that induces response • Antigenicity • Ability to combine specifically with Abs or T-cell receptor/MHC • Not all antigens are immunogenic • Haptens

7. Haptens • Hapten – too small, lack immunogenicity • If hapten is coupled to carrier protein, immune response can be induced • Hapten-carrier conjugate • Produces 3 types of antigenic determinants • Antibodies to hapten • Antibodies to carrier • Antibodies to hapten-carrier conjugate

11. Properties of Immunogen contribute to Immunogenicity • 4 Properties • Foreignness • Molecular size • Chemical composition and complexity • Ability to be processed and presented on MHC

12. Foreignness • Lymphocytes that do not bind to self antigens are allowed to further develop • Therefore they will later only recognized nonself antigens • For example: • Bovine serum albumin (BSA) is not immunogenic when injected into cow but is when injected into chicken • Some macromolecules are highly conserved throughout evolution and display little immunogenicity • Cytochrome c, collagen

13. Molecular Size • Active (good) immunogens • > 100,000 Daltons • Poor immunogens • < 5,000-10,000 Daltons

14. Chemical Composition • Polymers composed of multiple copies of same amino acid or sugar tend to be poor immunogens • Lipids are haptens and need to be congugated with carrier to produce antibodies • Important for assays for detection of some steroids, vitamins

15. Susceptibility to antigen processing • Large, insoluble macromolecules are more likely to be phagocytized for processing

16. The biological system contributes to immunogenicity • Host Genetic make-up • Manner in which material is presented • Use of agents (adjuvants) to enhance immunogenicity

17. Genotype of recipient animal • Genes of MHC • Genes in coding for specific antibodies

18. Material presentation – immunogen dosage and route of administration • Too low or high of dosage can induce tolerance • Single dose is often not enough – booster is needed • Route • Intravenous (iv) • Intradermal (id) • Subcutaneous (sc) • Intramuscular (im) • Intraperitoneal (ip) • Antigen administered iv would travel to spleen; administered sc would travel to lymph nodes

19. Adjuvants • Enhance immunogenicity • Not exactly sure how they work but are recognized by Toll-like receptors • Water-in-oil adjuvants • Freund’s incomplete adjuvant – antigen in aqueous solution, mineral oil, and emulsifying agent • Antigen is then released very slowly from injection site • Based on Freund’s complete adjuvant - also contained heat –killed Mycobacteria

20. Epitopes • Antigenic determinants recognized by B cells and T cells • B cell epitopes tend to be on the outside of the antigen • For example, the hydrophilic amino acids on a protein’s surface • T cell epitopes from proteins derived from enzymatic digestion of peptide and then association with MHC

22. Multiple noncovalent bonds between antibody and antigen • Hydrogen bonds • Ionic bonds • Van der Waals • Hydrophobic interactions

23. Receptor-ligand interactions induce signal transduction pathways • Translated to biochemical change within affected cell • Ligand binding can • Induce conformational changes in receptor • Alter receptor location within membrane • Phosphorylation is an early step in signaling pathways • Phosphorylation of certain amino acids on enzymes can activate or deactivate them • Phosphorylation of tyrosine on some molecules is seen early, serine and threonine later • PIP2 in cell membrane phosphorylated to PIP3, serves as binding site for other proteins in membrane • PIP2 also hydrolyzed by other enzyme to IP3 and DAG • IP3 interacts with endoplasmic reticulum vesicles, release of stored calcium, altering activity of other proteins • For example – in lymphocytes, calcium ions bind calmodulin altering its conformation allowing dephosphorylation of NFAT (nuclear factor of Activated T cells)

24. Immunoglobulin Superfamily • All have similar structures • Examples: • Antibodies • T-cell receptors • Class I and II MHC molecules • Part of B cell receptor • Most members of immunoglobulin superfamily cannot bind antigen

26. Antibodies and B cell Receptor • B cell epitopes have characteristic properties • Located on surface of immunogen – accessible to antibody • When talking about proteins, the epitopes can be sequential or nonsequential (referring to amino acid sequence) depending on protein folding

27. Basic Structure of Antibodies • Known since late 19th century that antibodies are in serum • Serum is fluid phase that remains after plasma is allowed to clot • Antibodies are also found in other secretions

28. Antibodies are heterodimers • 2 light chains • ~ 22, 000 daltons each • 2 heavy chains • ~ 55,000 daltons each • First 110 aa of amino-terminal end of heavy and light chain vary depending on antibody specificity

29. Different digestion procedures reveal different fragments • F(ab’)2 still shows antigen binding capability

31. Light Chains • When aa sequences of light chains from several individuals were sequenced, pattern emerged: • Amino-terminal end (110 aa) varied • Other part remained constant • Were found to be either kappa (κ) OR • Lambda (λ) • In mice and humans, different lambda subtypes have been found

32. Heavy Chains • Amino-terminal end also shows variability • 5 different heavy chain constant regions (isotypes) • IgM – μ • IgG – γ • IgA – α • IgD – δ • IgE – ε Some subisotypes have been discovered in some species Each antibody has 2 identical heavy chains, 2 identical light chains

34. Overall structure of immunoglobulin • Primary – sequence of amino acids • Secondary – folding into series of β pleated sheets • Tertiary – compact globular domains • Quarternary – adjacent light and heavy chains interact

36. Hypervariable regions = complimentarity-determining regions (CDRs) • Complimentary to epitopes that they will bind

37. Hinge Region • γ (gamma), δ (delta), and α (alpha) heavy chains have extended peptide sequence • Rich in proline and cysteine • Gives flexibility • Immunoglobulins can be secreted or membrane-bound • Membrane-bound differ in the carboxyl-terminal end: • Extracellular “spacer” of 26 aa • Hydrophobic transmembrane sequence • Cytoplasmic tail

38. B Cell Receptor (BCR) • Heavy chain portion of membrane-bound antibody does not extend far enough through the cell membrane for signaling • Membrane bound antibody is accompanied by Igα and Igβ

39. Antibody-mediated Effector Functions • Remember, they plant “kiss of death” on an invader • In addition to binding antigen, Abs can: • Promote phagocytosis (opsonization) • Activate complement • Antibody dependent cell mediated cytotoxicity (ADCC) • Natural killer cells have receptor for Fc portion of antibody • Some can cross epithelial layers to be excreted through mucous or across placenta

42. Monomeric IgM expressed on B cells Secreted is pentameric 1st class produced in primary response Activates complement Very good at agglutination

43. Membrane bound on B cells

44. Most abundant 4 human subclasses Crosses placenta Involved in complement

46. Involved in allergic reactions Involvement in parasitic infections

48. Predominant class in secretions J chain and secretory component helps with transport across intestinal wall J chain makes IgA more resistant to acids and enzymes found in digestive tract IgA and macrophages restrict commensal bacteria that occasionally enter the tissues from the intestines Better for IgA to interact than IgG – this is because the Fc portion of IgG has high affinity for receptors of immune cells and would constantly trigger inflammatory responses Can cross-link large antigens Exists as dimer

50. Immunoglobulins when injected into another species can be immunogenic Isotypic – differences in constant region from one species to another Allotypic – differences (alleles) that occur in some individuals Idiotypic – differences in variable regions; will differ even on Abs of same isotype