Figures replaced or skipped for Ch 2

1. Figures replaced or skipped for Ch 2 • Figure 2.3 - Enzyme digestion of Ab’s • Flow cytometry - Fig 2.13 • Monoclonal Ab Fig 2.12 was replaced

2. Molecular and structural basis of antibody diversity • How B cells develop and function in the body • How B cells are activated and participate in adaptive immunity Chapter 2 Antibody Structure and the Generation of B-Cell Diversity

3. Location and production of Immunoglobulins Humoral Immunity - immunity dominated by antibodies that can be transferred to another person Antibodies are specific for individual epitopes Membrane bound form is present on a B-cell Ag binding to B cell stimulates it to secrete Ab

4. Antibody structure Fab Fc Heavy (5 classes), Light (2 classes), Constant (C) and Variable (V) Disulfide bonds link heavy chains Fab- fragment antigen binding Fc- fragment crystallizable

5. Immunoglobulin Isotypes or Classes    pentamers N-linked carbohydrate Hinge region Disulfide bonds Monomer as BCR Multimeric forms k l-Light chain Ig domain   Monomers or dimers

6. Figure 2-5 Immunoglobulin (Ig) domain ~100 amino acid domain - very stable Two types Ig domains- variable and constant Antigen Binding site - VH and VL Heavy chain of IgG - four domains - VH, CH1, CH2, CH3

7. Figure 2-6 Anti-parallel -strands - contribute to the structural part Loop region - in the V domain contribute to variability

8. Hypervariable Regions (CDR) of Antibodies HV - hypervariable regions CDR - complementarity determining regions Framework region -  strand region that has reduced variability

9. Amino Acid Sequence Variability in the V domain 110 amino acid light-chain V domain Framework regions can have variability but variability is much higher in the HV regions

10. Physical Properties of Antigens-Antibody Binding • Epitope - part of the antigen bound by Ab • Antigen is usually carbohydrate or protein • Lock and Key concept • Variety of structures and sizes recognized by Ab’s • Affinity vs Avidity - terms describing binding strength of an antibody for its epitope

11. Epitopes • Epitope (antigenic determinant) is the part of the antigen bound by Ab • Most antigens have multiple epitopes (multivalent) • Usually carbohydrate or peptide. Fig. 2.9

12. Mechanisms of Epitope Recognition • Linear and discontinuous epitopes • Multivalent Antigens • Polymeric Antibodies • Affinity vs Avidity - terms describing binding strength of an antibody for its epitope • Epitope binding mechanisms

14. Figure 2-9

15. Figure 2-26 part 1 of 2 First Ab to be made during during an immune response to an antigen Monomers disulfide bonded together via the J-chain (joining chain)

16. Figure 2-30 Monomers disulfide bonded to the J-chain Dimeric in mucosal lymphoid tissue - secreted into the gut to prevent pathogens binding to gut cells and can act as an antitoxin Monomeric made by B-cells in lymph nodes/spleen and is not J-chain dependent

17. Antibody-antigen interaction • Non-covalent binding: • Electrostatic • Hydrogen bonds • Van der Waals force • Hydrophobic forces • Affinity: Strength of interaction between epitope and one antigen-binding site • Avidity: Strength of the sum of interactions between antibody and antigen

18. Figure 2-8 Poliovirus VP1- blue, contains several epitopes (white) that can be recognized by human antibodies

19. Antibodies bind a Range of Structures Grooves Extended surfaces Pockets Molecule DNA Lysozyme

20. Examples of Problematic Ab binding to various structures Pocket: Penicillin - can bind to RBC surface proteins to create a foreign epitope. IgE binds to drug-RBC protein complex and initiates an inflammatory response Groove: DNA - Systemic Lupus Erythematosus (Lupus) - autoimmune disease in which antibodies are made against DNA and other molecules leading to inflammatory reactions in joints, skin and kidney Extended Surface: Lysozyme/Ovalbumin - allergies to hen egg components - more common in children under 5 - “desensitization” protocol can help

21. Haptens Small molecules that are not immunogenic by themselves, but can bind immunoglobulins or TCRs. Haptens can induce an immune response when linked to a larger protein.

22. 3 4 1 2 Polyclonal vs Monoclonal Antibodies 1 Ag 4 2 Isolate B-cells Spleen Myeloma cells Hybridoma Cells 3 1 2 + 4 3 1 2 3 4 Isolate serum Ab-1 Ab-2 Ab-3 Ab-4 Polyclonal antibodies 1 Ag 4 2 Monoclonal antibodies Ab-1, Ab-2 Ab-3, Ab-4 3

23. Crossreactivity Occurs when an Antiserum is raised against antigen A but also reacts with antigen B Antigen A and B share epitopes Antigen A and B have similar (but not identical) epitopes

24. Antibody Structure Summary • Produced by B-cells • Y shape, Four polypeptide chains, Ig domains • Constant and Variable regions • 5 classes - IgG, IgM, IgD, IgA, IgE • CDR, Hypervariable regions • Epitope recognition

25. Examples of Commercial uses for Ab’s • Pregnancy test • Rh disease therapy • Antitoxin serum - Antivenin, rabies, etc • Anti-cancer monoclonals - will be covered later in the course

26. Pregnancy test Pregnancy window Control window Input window Reaction zone Urine Mouse Monoclonal anti-HCG Ab-enzyme conjugate Immobile Polyclonal anti-HCG Ab + dye substrate Immobile Goat Anti-mouse Ab + dye substrate http://mcb.berkeley.edu/courses/mcb150/lecture5/Preg%20Test%20movie.swf

27. Rh disease Rh disease (Erythroblastosis Fetalis) - Hemolytic disease of a newborn - occurs in Rh- woman carrying Rh+ fetus RhoGAM - human plasma with anti-Rh+ (D antigen) antibodies. Works by binding any fetal RBC’s before the mother is able to produce an immune response and form anti-D IgG

28. Antitoxin serum • Serum can be used in the prophylaxis and/or treatment of rabies, botulism, diphtheria, gas gangrene, snake and spider bites • Antivenin Crotalidae Polyvalent (ACP) - horse serum based antivenom - gold standard for snake bites but can cause “serum sickness” • New types of antitoxins • Fragmented antivenin (CroFab) where only the Fab fragment (sheep-based) is used • Humanized antibodies derived from mouse sources

29. Generation of Ig diversity in B cells Unique organization - Only B cells can express Ig protein - Gene segments k l-Light chain , , , ,  - Heavy Chain present on three chromosomes

30. The Gene Rearrangement Concept • Germline configuration • Gene segments need to be reassembled for expression • Sequentially arrayed • Occurs in the B-cells precursors in the bone marrow (soma) • A source of diversity BEFORE exposure to antigen

31. V-variable, J-joining, D-diversity gene segments; L-leader sequences Light Chain Variable - V, J Constant - C Heavy Chain Variable - V, D, J Constant - C

32. Figure 2-14 Gene rearrangements during B-cell development V-variable, J-joining, D-diversity gene segments; L-leader sequences l - 30 V & 4 pairs J & C (light chain) chs22 k – 40 V & 5 J & 1 C (50% have 2x V) (light chain) chs2 H – 65 V & 27 D & 6 J chs14

33. V-region of light chain consists of one V and one J segment C-region is encoded by one C segment Variability comes from the V segment CDR1 and CDR2 CDR3

34. D-diversity CDR1 and CDR2 CDR3 Two recombination events needed to combine 3 segments to make the Variable region

35. Random Recombination of Gene segments is one factor contributing to diversity + 120*) 10,530 = 3,369,600 Ig molecules (200* X * Only one light chain loci gives rise to one functional polypeptide!

36. Mechanism of Recombination • Recombination signal sequences (RSSs) direct recombination • V and J (L chain) • V D J (H chain) • RSS types consist of: - nonamer (9 base pairs) • - heptamer (7 base pairs) • - Spacer • Two types: [7-12-9], [7-23-9] • RSS features: - recognition sites for recombination enzymes - recombination occurs in the correct order (12/23 rule)

38. Mechanism of Somatic Recombination • V(D)J recombinase: all the protein components that mediate the recombination steps • RAG complex: Recombination Activating Genes (RAG-1 and RAG-2) encode RAG proteins only made in lymphocytes • Recombination only occurs through two different RSS bound by two RAG complexes (12/23 rule) • DNA cleavage occurs to form a single stranded hairpin and a break at the heptamer sequences • Enzymes that cut and repair the break introduce Junctional Diversity

39. Junctional Diversity Recombination + http://www.blink.biz/immunoanimations/#

40. Figure 2-19

41. Figure 2-18 part 2 of 3 Junctional Diversity • Nucleotides introduced at recombination break in the coding joint corresponding to CDR3 of light and heavy chains - V and J of the light chain - (D and J) or (V and DJ) of the heavy chain • P nucleotides generate short palindromic sequences • N nucleotides are added randomly - these are not encoded • Junctional Diversity contributes 3 x107 to overall diversity!

42. Generation of BCR (IgD and IgM) • Rearrangement of VDJ of the heavy chain brings the gene’s promoter closer to C and C • Both IgD and IgM are expressed simultaneously on the the surface of the B cell as BCR - ONLY isotypes to do this • Alternative splicing of the primary transcript RNA generates IgD and IgM • Naïve B cells are early stage B cells that have yet to see antigen and produce IgD and IgM

43. Figure 2-21 Alternative Splicing of Primary Transcript to generate IgM or IgD

44. Summary Biosynthesis of IgM in B cells

45. Figure 2-23 Mature B cell • Long cytoplasmic tails interact with intracellular signaling proteins • Disulfide-linked • Ig and Ig - invariant - Transmembrane proteins - Dual-function • help the assembled Ig reach the cell surface from the ER • signal the B cell to divide and differentiate

46. Principle of Single Antigen Specificity • Each B cell contains two copies of the Ig locus (Maternal and Paternal copies) • Only one is allowed to successfully rearrange - Allelic Exclusion • All Igs on the surface of a single B cell have identical specificity and differ only in their constant region • Result: B cell monospecificity means that a response to a pathogen can be very specific l k H * * 2 14 Chromosome 22

47. DNA hybridization of Ig genes can diagnose B-cell leukemias Peripheral blood from healthy patient is made up of mostly neutrophils Peripheral blood from a leukemia patient has an abnormally high proportion of B-cells. Cancer cells are derived from one clonal line of B-cell which has V and C chains rearranged next to each other

48. Generation of B cell diversity in Ig’s before Antigen Encounter • Random combination of V and J (L chain) and V, D, J (H chain) regions • Junctional diversity caused by the addition of P and N nucleotides • Combinatorial association of Light and Heavy chains (each functional light chain is found associated with a different functional heavy chain and vice versa)

49. Concept of Combinatorial Association

50. Developmental stages of B cells 1. Development before antigen 2. Development after antigen Mature naive B cell (expressing BCR - IgM and IgD) Immature B cell plasma cell (expressing BCR and secretes Antibodies)