Immune System Recognition and Antibody Function

1. Chapter 4 and 5 Ig study questions: • How does the immune system recognize diverse possible antigens? • How do antibodies simultaneously recognize a huge variety of antigens and carry out a limited number of effector functions? • What is V(D)J recombination? What do the letters (and parentheses) signify? • What is the 12/23 rule? • What are RAG-1/2 and TdT? What do they recognize? What do they do? • What changes at the Ig gene loci are required to switch from membrane-bound to secreted Ig? Which Ig genes are involved?

2. You can skip the non-mammalian stuff from the textbook: • On the midterm there will be no chickens. • On the midterm there will be no sharks. • On the midterm there will be no lampreys. • On the midterm there will be no Drosophila. • You can also skip the single-chain camelid antibodies on page 151-152. • This slide not in your slide deck. If you are attending class and taking notes, good for you.

3. Ig Structure and Function Lecture – Sept. 3, 2019 Dean Tantin, PhD, Department of Pathology Division of Microbiology & Immunology University of Utah School of Medicine dean.tantin@path.utah.edu

4. N C N N C N C C

5. (B cell receptor, BCR) Abcam (an Ab company)

6. N C N C N N • Epitope: the portion of the target antigen (Ag) bound • Never before seen epitopes can be recognized • Too many epitopes to allow direct encoding in the genome C C

7. Fab = “Fragment antigen binding” Fc = “Fragment crystallizable” Why name it F(ab’)2? VH CH1 VL CL CH2 CH3 Figure 4.4

8. VL CL heavy light

9. Ig domain is a recurring protein structure in immunology • Two beta sheets in a sandwich configuration stabilized by a disulfide • Immunoglobulin superfamily • This is how the textbook will denote: heavy light Figure 4.3

10. A quick preview of the T cell receptor (TCR). • Structurally related to immunoglobulin. • Does not recognize free antigen. Instead usually recognizes peptide antigens in the context of presentation by major histocompatibility complex (MHC) molecules. Figure 4.13

11. One difference between Ig and TCR: Ig recognizes Ag in native form, whereas TCR typically recognizes Ag as peptide presented by MHC molecules.

12. These three regions vary. • Known as “hypervariable regions” or “complementarity-determining regions”. Figure 4.3

13. Hypervariable regions= complementary determining regions (CDRs) amino acid amino acid Figure 4.6

14. Figure 4.7

15. Another difference between Ig and TCR: TCR solely functions as an antigen-specific receptor, whereas Ig encodes both antigen receptor and the major effector molecule (antibody). Effector functions are mostly mediated by the IgH constant regions.

16. Antigen (Ag) recognition HERE Effector function end of the molecule is HERE, encoded by heavy chain (IgH) C regions. IgH C regions specify the isotype: IgM, IgD, IgG, IgE, or IgA

17. Review. So far… • IgG is example • 4 polypeptide chains • Modular construction • Ig superfamily • Flexibility in hinge region • Heavy chain constant region defines the isotype Figure 4.1

18. Your first look at the immunoglobulin genes • This is just one of your two copies of heavy chain. There is also k and l light chain. • Modular construction, just like Ig proteins! • Here mainly concerned with the 5’ (V(D)J) part of the locus, and at the end of the lecture (and Thurs) with the 3’ C regions. VH CH1 CH2 CH3 CH4 Related to Fig.5.5, but more complex

19. Your first look at the immunoglobulin genes • V=variable • (D)=diversity • (heavy chain only) • J=joining • This is just one of your two copies of heavy chain. There is also k and l light chain. • Modular construction, just like Ig proteins! • Here mainly concerned with the 5’ (V(D)J) part of the locus, and at the end of the lecture (and Thurs) with the 3’ C regions. VH CH1 CH2 CH3 CH4 Related to Fig.5.5, but more complex

20. This is how your Ig loci look while you are a developing embryo, and in every cell of your body except B cells • “Gene segments” • In any given B cell, either k or l locus is used, not both • Heavy chain defines isotype: Cm=IgM Figure 5.5

21. No need to memorize this chart. Know that there are many V segments, several J segments, and D segments restricted to IgH/TCRb. • Not to scale. • Figure above only shows protein-coding regions. Regulatory elements in DNA provide control of e.g. recombination. Figure 5.5, 5.4

22. Figure 4.1

23. Basically Ig recombination only in the B lineage Figure 5.3

24. Figure 4.6, 4.7

25. CDR 1 and 2 encoded in V segment, which vary significantly in this region from segment to segment (positive selection). • CDR3 made up of the junction between gene segments. • Questions? Figure 5.5, 4.7

26. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * • Recombination signal sequences (RSSs, asterisks) mark potential sites of V(D)J recombination. • Recognized by the RAG-1 and RAG-2 proteins. Figure 5.5

27. RSSs are recognized by RAG (recombination activating gene) proteins-1 and -2 5’ 5’ 5’ 5’ • 12mers/23mers only recombine with the other spacer, not themselves (“12/23 rule”) • ∴ V segments never recombine with other V segments Figure 5.6

28. You are looking at an Ig light chain, either k or l… -or- -or- and or… ✔ ✔ ✔ Figure 5.7

29. “Direct repeat” RSSs leads to joining and deletion of the intervening DNA • “Inverted repeat” RSSs leads to joining and inversion of the intervening sequence. • Study the topology of this figure to see it. Figure 5.7

30. The RAG-1/2 heterotetramer binds one 12mer and one 23mer. • This this the basis of the 12/23 rule. Figure 5.8

31. Imprecision in position of cut generates “P-nucleotide varariability”. • TdT=terminal deoxy-ribonucleotidyl transferase. • TdT is lymphocyte specific. • TdT introduces random additional nucleotides at the coding junctions, increasing diversity (“N-nucleotide variability”). • Lack of RAG proteins, or DNA-PK, orArtemisSCID (C=combined, meaning B and T cells affected). Figure 5.8

32. No need to memorize this chart. combinatorial diversity junctional diversity + somatic hypermutation In B CELLS ONLY (next lecture)

33. A difference between Ig and TCR: TCR solely functions as an antigen-specific receptor, whereas Ig encodes both antigen receptor and the major effector molecule (antibody). Effector functions are mostly mediated by the IgH constant regions.

34. Consequently, the molecular biology of the immunoglobulin (BCR/antibody) gene loci following activation is going to be much more complex

35. i.e., gene expression from the Ig loci is increased by orders of magnitude. In plasma cells (effector B cells), Ig accounts for 50% of all gene expression. This does not happen with T cell receptor.

36. Conversion to Ab secretion changes cellular moprhology Resting B cell: Antibody secreting (plasma) cell: Zucker, Atlas of Blood Cells

37. i.e., specific molecular mechanisms change immunoglobulin from membrane-bound to secreted forms. This does not happen with T cell receptor.

38. B cells secrete antibody upon stimulation cell surface • B cells become activated by signaling through B cell Ag receptor (BCR) complex, plus costimulatory signals. • Same thing at other heavy chain regions, i.e. all classes can be expressed at surface. • SC: secretion coding; MC: membrane coding Figure 5.22

39. B cells secrete antibody upon stimulation cell surface • B cells become activated by signaling through B cell Ag receptor (BCR) complex, plus costimulatory signals. • Same thing at other heavy chain regions, i.e. all classes can be expressed at surface. • SC: secretion coding; MC: membrane coding Figure 5.22

40. i.e., further recombination events generate antibodies with specific effector functions (Ig isotype switching), and site-specific mutagenesis events allow for the selection of high-affinity antibodies (affinity maturation).

41. If you have not already, read the book chapters before Thursday I will relying less on figures from your textbook in the next lectures. Textbook therefore becomes an additional rather than redundant resource.