MICR 304 Immunology & Serology

1. MICR 304 Immunology & Serology Lecture 8A Antibodies Part II Chapter 4.2- 4.5, 4.12 – 4.20, 9.14 – 9.20

2. Overview of Today’s Lecture • Generation of antibodies • Secondary antibody modifications • Effector functions of antibodies

3. Key Players in Immunology

4. Variable Regions Are Constructed From Gene Segments • Variable regions of light chain • V (variable) gene segments • J (joining) gene segments • Variable regions of heavy chain • V (variable) gene segments • D (diversity) gene segments • J (joining) gene segments • From multiple genes, randomly one each of V and J gene segments for the light chain and one each of V, D and J gene segments for the heavy chain are recombined.

5. Construction of the Antibody V Regions L: Leader sequence; directs protein to cell’s secretory pathway : Hinge region

6. Recombination of a Limited Number of Gene Segments Results in Millions of Variations ~

7. Germline Organization of the Light and Heavy Chain on chromosome 22 on chromosome 2 on chromosome 14 Includes a significant number of non-functional segments (pseudogenes)

8. Gene Rearrangement and Recombination in Variable Regions • Somatic gene recombination • Lead to unique antibodies • Conserved regions flank gene segments (blue, orange) • Recombination signal sequences (RSS) • Aid in rearrangement • Performed by lymphocyte specificrecombinases and ubiquitous DNA modifying enzymes • RAG1 and RAG2 • Rearrangement followed by looping out and joining by recombination • Irreversible changes!

9. Recombination Signal Sequences • Consist of • Conserved heptamer • Spacer (12 OR 23 base pairs) • Conserved nonamer • 12/23 rule In heavy chain, V cannot be directly joined with J.

10. Antibodies Can be Membrane Bound Or Secreted • 2 exons located at the end of constant regions from heavy chain • Code for hydrophobic amino acid stretch for membrane spanning domain • Code for more for hydrophilic amino acids for secretion • Both are initially transcribed • Either membrane coding or the secretion coding sequences are removed by splicing prior to translation

11. Transmembrane and Secreted Forms of Antibodies

12. Secondary Modifications of Antibodies • In activated B cells (after first antigen contact) • Driven by antigen • Initiated by activation-induced cytidine deaminase (AID) • Somatic hypermutation • Gene conversion • Class switching Variable region Constant region

13. Diversification of the Antibody Repertoire by Three Major Processes Involves heavy chain only Involves variable region on heavy and light chain

14. Somatic Hypermutation • In mice and humans in germinal centers • Requires also signals from activated T cells • Random point mutations in V regions • Alter affinity of the antibody for its antigen • Reduced antigen binding leads to negative selection and cell death • Improved antigen binding leads to positive selection, proliferation and finally plasma cell development

15. Successful Somatic Hypermutations Occur Predominantly in CDR Regions Mutations in the framework tend to disrupt the antibody structure and are selected against.

16. Gene Conversion • Modification of re-arranged variable region • Introduction of sequences derived from V gene segement pseudogenes • Creates additional antibody specificities • Occurs in some species • Birds, rabbits, cows, pigs, sheep, horses • Little or no germ line diversity

17. Immunoglobulin Class Switch • Same as isotype switch • Initial m heavy chain is replaced by heavy chain regions of another isotype • d, g, a, e • Aided by switch regions • Induced by cytokines (T cell derived) or mitogenic signals from pathogen • Modification of antibody effector function • Does not change antigen specificity • B cell can undergo multiple rounds of switching

18. Germline Organization of Constant Region of the Heavy Chain • Naïve but mature B cells co-express IgM and IgD • IgM is always secreted first • IgM indicates acute infection/antigen challenge Carbohydrate group Pseudogene

19. IgM and IgA Can Form Multimers IgM Pentamer (5mer) Excellent agglutination ~960 kDa Monomers are cross-linked by disulfide bridges that connect each other and J-chain IgA Dimer (2mer) Epithelial transcytosis ~320 kDa

20. IL4 induces IgE IL5 augments IgA IFNg induces IgG TNFb induces IgG and IgA Cytokine-Induced Switching of Isotypes

21. Distribution of Antibodies in the Body

22. Basic Functions and Distribution of Antibodies

23. Neutralization (IgG and IgA) Toxin Virus

24. Antibodies Can Block Bacterial Adherence to Host Cells

25. Opsonization Antibody only In conjunction with complement

26. Antigen:Antibody complexes Antibody conformation change Activate classical pathway of complement Binding of C1q to the Fc piece Pentameric IgM is more efficient Complement Activation

27. Complement Receptors are Important in the Removal of Antigen:Antibody Complexes • Immune complexes activate C1q • Deposition of C3b • Binding to CR1 on erythrocytes via bound C3b • Transport to liver and spleen • Removal from circulation by phagocytes

28. Anti-Parasitic Function of IgE • Helminths are too bnig to be phagocytosed • IgE binds to helminths • Eosinophils have Fce receptors • After crosslinking degranulation • Granules contain anti-helminthic proteins

29. IgG Mediated Sensitization for Killing by NK Cells Antibody Dependent Cell Mediated Cytotoxicity

30. Today’s Take Home Message • The variable regions of an antibody molecule are coded by V and J gene segments in the light chain and by V, J, and D gene segments in the heavy chain. • There are a limited number of V, J, and D gene segments. • Gene rearrangement, looping out and gene recombination lead to the antibody diversity. • Isotype switch is greatly influenced cytokines and dictates the functions of antibodies • The major functions of antibodies include: neutralization, opsonization, complement activation, NK cell activation.