1. Chapter 4 and 5 Ig study questions (Th): How does the immune system recognize a diverse universe of possible antigens? How do antibodies simultaneously recognize a wide variety of antigens and carry out a small number of effector functions? What is V(D)J recombination? What do the letters (and parentheses) signify? What are RAG-1/2 and TdT? What do they recognize/do? What changes are required to switch from membrane-bound to secreted Ig?

2. Ig Structure and Function Lecture – Sept. 10 & 15, 2015 Dean Tantin, PhD, Department of Pathology Division of Microbiology & Immunology University of Utah School of Medicine JMRB 5200B, 7-3035 dean.tantin@path.utah.edu

3. 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 N N N N C C C C

4. Figure 4.3 Fab = Fragment antigen binding Fc = Fragment crystallizable Why name it F(ab’) 2 ? VLVL CLCL VHVH CH1CH1 CH2CH2 CH3CH3

6. Figure 4.5 Ig domain is a recurring protein structure in immunology Immunoglobulin superfamily This is how the textbook will denote: heavy light

7. A quick preview of the T cell receptor (TCR). Structurally related to immunoglobulin. Does not recognize free antigen. Instead recognizes peptide antigens in the context of presentation by the major histocompatibility complex (MHC) molecules. Figure 4.11

8.  One difference between Ig and TCR: Ig recognizes Ag in native form while TCR recognizes Ag as peptide presented by MHC molecules.

9. Figure 4.5 These three regions vary. “They are know as hypervariable regions” or “complementarity- determining regions”.

10. Figure 4.6 Hypervariable regions=CDRs, complementary determining regions AA

11. Figure 4.7

12.  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. Effector functions are mediated by the IgH constant regions.

13. Business 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 Antigen (Ag) recognition here

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

15. VHCH1CH2CH3CH4VHCH1CH2CH3CH4 Figure 5.3 + 5.16 + 5.18 Your first look at the immunoglobulin genes This is just heavy chain. There is also  and light chain. Modular construction, just like Ig proteins! Today will mainly be concerned with the 5’ end of the locus, and Tuesday with the 3’ end.

16. VHCH1CH2CH3CH4VHCH1CH2CH3CH4 Figure 5.3 + 5.16 + 5.18 Your first look at the immunoglobulin genes This is just heavy chain. There is also  and light chain. Modular construction, just like Ig proteins! Today will mainly be concerned with the 5’ end of the locus, and Tuesday with the 3’ end. V=variable (D)=diversity (heavy chain only) J=joining

17. Figure 5.3 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 B cells, either  or locus is used, not both Heavy chain defines isotype: C  =IgM

18. Figure 5.2, 5.3 No need to memorize this chart. Know that there are many V segments, several J segments; D segments restricted to IgH/TCR  Not to scale Figure above only shows protein- coding regions. Regulatory elements in DNA provide control of e.g. recombination

19. Figure 4.1

20. Figure 5.1 Basically Ig recombination only in the B lineage

21. Figure 4.6, 4.7

22. Figure 5.3, 4.7 CDR 1 and 2 encoded in V segment CDR3 made up of the junction between gene segments Questions?

23. Figure 5.3 **** ********* ****************** ** ****** ************** Recombination signal sequences (RSSs, asterisks) mark potential sites of V(D)J recombination. Recognized by the RAG-1 and RAG-2 proteins.

24. Figure 5.4 Recombination signal sequences (RSSs) are recognized by RAG (recombination activating gene) proteins-1 and -2 12mers/23mers do not recombine with themselves, only with their pair (“12/23 rule”) ∴ V segments never recombine with other V segments 5’

25. Figure 5.5 ✔✔ -or-and-or- ✔ or… You are looking at an Ig light chain, either  or …

26. Figure 5.5 “Direct repeat” RSSs leads to joining and deletion of the intervening DNA “Inverted repeat” RSSs leads to joining and inversion of the intervening sequence. You will have to study the topology of this figure to see it.

27. Figure 5.6 This is the basis of the 12/23 rule

28. Figure 5.6 Variability in the cut generates “P-nucleotide varariability” TdT=terminal deoxy- ribonucleotidyl transferase TdT is lymphocyte specific TdT introduces additional nucleotides at the coding junctions, and increasing diversity “N-nucleotide variability” Lack of RAG proteins, or DNA-PK, or Artemis  SCID (C=combined, meaning B and T cells affected)

29. Figure 5.11 junctional diversity + somatic hypermutation In B CELLS ONLY (next lecture) combinatorial diversity

30.  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. Effector functions are mediated by the IgH constant regions.

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

32. i.e., gene expression from the Ig loci is increased many fold. In plasma cells (effector B cells), Ig accounts for 50% of all gene expression. Ig is secreted as antibodies.

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

34. i.e., there are specific molecular mechanisms that change immunoglobulin from membrane-bound to secreted forms

35. B cells secrete antibody upon stimulation B cells become activated by signaling through B cell Ag receptor (BCR) complex, plus costimulatory signals. Same thing at other heavy chain regions. SC: secretion coding; MC: membrane coding Figure 5.18 cell surface

36. B cells secrete antibody upon stimulation B cells become activated by signaling through B cell Ag receptor (BCR) complex, plus costimulatory signals. Same thing at other heavy chain regions. SC: secretion coding; MC: membrane coding Figure 5.18 cell surface

37. i.e., there are further recombination events to generate antibodies with specific effector functions (Ig isotype switching), and site-specific mutagenesis that allows the selection of high- affinity antibodies (affinity maturation)

39. Chapter 4 and 5 Ig study questions (Th): How does the immune system recognize a diverse universe of possible antigens? How do antibodies simultaneously recognize a wide variety of antigens and carry out a small number of effector functions? What is V(D)J recombination? What do the letters (and parentheses) signify? What are RAG-1/2 and TdT? What do they recognize/do? What changes are required to switch from membrane-bound to secreted Ig?