Aims

1. Aims • Gene rearrangement and class switching of B-cell Igs. • T cell receptors- What are they and where do they fit into immunology? • Gene rearrangement to generate diversity • Readings: Abbas & Lichtman, Chapters 4 & 7

2. Human Immunoglobulin Loci Heavy Chain Light Chain Abbas & Lichtman’s Basic Immunology 4-9

3. Heavy Chain Synthesis J1-n D1-n 5’ 3’ Cm Cd Cg Ca Ce V1 V2 • Variable (VH) segments • Diversity (DH) segments • Joining (JH) segments • Conserved (CH) segments Vn Germline DNA Similar to Abbas & Lichtman’s Basic Immunology 4-10

4. Heavy Chain Synthesis J1-n D1-n Germline DNA 5’ 3’ Cm Cd Cg Ca Ce V1 V2 • Intervening DNA between the D and J segments is looped and cut out. Vn Rearranged B cell DNA 5’ 3’ Cm Cd Cg Ca V1 V2 Ce Vn Similar to Abbas & Lichtman’s Basic Immunology 4-10

5. Heavy Chain Synthesis Rearranged B cell DNA • Intervening DNA between the V and D segments is looped and cut out. 5’ 3’ Cm Cd Cg Ca V1 V2 Ce Vn Rearranged B cell DNA 5’ 3’ Cm Cd Cg V2 Ca Ce V1 Similar to Abbas & Lichtman’s Basic Immunology 4-10

6. Heavy Chain Synthesis Rearranged B cell DNA 5’ • The gene is transcribed resulting in a primary RNA containing the VHDHJH segment, the remaining J segments and only the Cm and Cd segments. 3’ Cm Cd Cg V2 Ca Ce V1 Primary RNA transcript 3’ 5’ Cm Cd V2 Similar to Abbas & Lichtman’s Basic Immunology 4-10

7. Heavy Chain Synthesis Primary RNA transcript 3’ 5’ Cm • The transcript is processed and the introns and Cd sequences are removed. The resulting mRNA contains the Cm segment and will code for an IgM. Cd V2 mRNA transcript 3’ 5’ Cm V2 Similar to Abbas & Lichtman’s Basic Immunology 4-10

8. Heavy Chain Synthesis mRNA transcript 3’ 5’ Cm V2 • The mRNA is translated by ribosomes into m chain. m heavy chain polypeptide NH2 Cm COOH V2 Adapted from Abbas & Lichtman’s Basic Immunology 4-12

9. Light Chain Synthesis J1-n • Variable (Vk) segments • Joining (Jk) segments • Conserved (Ck) segment • 2 isotypes l (40%) and k (60%) 3’ 5’ Ck V1 V2 V3 Vn Germline DNA

10. J1-n 3’ 5’ Ck V1 V2 V3 Vn Light Chain Synthesis Germline DNA • Intervening DNA between the V and J segments is looped and cut out. Rearranged B cell DNA 3’ 5’ Ck V3 V1 V2 Similar to Abbas & Lichtman’s Basic Immunology 4-10

11. Light Chain Synthesis • The B cell transcribes a segment of DNA into a primary RNA transcript that contains a long intervening sequence of J segments and introns. Rearranged B cell DNA 3’ 5’ Ck V3 V1 V2 Primary RNA transcript 3’ 5’ Ck V3 Similar to Abbas & Lichtman’s Basic Immunology 4-10

12. Primary RNA transcript 3’ 5’ Ck V3 Light Chain Synthesis • This RNA transcript is processed into mRNA by splicing the exons together. mRNA transcript 3’ 5’ V3 Ck Similar to Abbas & Lichtman’s Basic Immunology 4-10

13. Light Chain Synthesis mRNA transcript • The mRNA is translated by ribosomes into k chains. • Combines with heavy chain to make IgM. 3’ 5’ V3 Ck k chain polypeptide NH2 COOH Ck V3

14. Light Chain Synthesis m heavy chain polypeptide NH2 Cm COOH V2 k chain polypeptide NH2 COOH Ck V3 IgM Abbas & Lichtman’s Basic Immunology 4-12

15. mRNA Splicing IgD mRNA 3’ 5’ V2 Cd Primary heavy chain RNA transcript 3’ 5’ Cm Cd V2 IgM mRNA 5’ 3’ Cm V2 Abbas & Lichtman’s Basic Immunology 4-12

16. Ig Class Change • All classes of immunoglobulin use the same set of variable region genes (ie V-D-J). • All that changes is the constant region of the heavy chain. Abbas & Lichtman’s Basic Immunology 7-10

17. Ig Class Change • Involves a switch sequence upstream of the constant region gene segments. • Recombination occurs between the Sμ recombination region and a downstream switch region (Sg). • The intervening region containing genes for IgM and IgD in this instance is looped out and then cut, with deletion of the intervening regions and joining of the two switch regions. Adapted from Abbas & Lichtman’s Basic Immunology 7-10

18. Ig Class Change • Class change occurs upon B cell activation and maturation. • Requires signals from ____________________________ (CD3+, CD4+, CD8-). • Binding of receptors (CD40 ligand) • Cytokines Abbas & Lichtman’s Basic Immunology 7-9

19. Mechanisms of Antibody Diversity • Combinatorial diversity • Multiple germ line V genes recombining with J and D segments. • Junctional diversity • Imprecision in V-J and V-D-J recombinations (N-region diversity) • N-nucleotide additions (N-region diversity) • Note addition is done without a template. • Somatic point mutation • Assorted heavy and light chains

20. Combinatorial Diversity • Possible combinations of V-D-J and V-J germ line gene segments. Abbas & Lichtman’s Basic Immunology 4-11

21. G C A C T G G G T G T A • Recombined DNA Ser Arg Trp Tyr • Protein Junctional Diversity (N- region diversity) • J segment • V segment • Changes in nucleotide sequence at junctions of V, D, and J segments. • Two types of changes • Exonuclease removal of nucleotides. • Random addition of nucleotides by terminal deoxyribonucleotidyl transferase (TdT). A C T G T G C G G A T G • Germ line DNA

22. G G C T G G T T G A C • Recombined DNA Val Arg Trp • Protein Junctional Diversity (N- region diversity) • J segment • V segment • Changes in nucleotide sequence at junctions of V, D, and J segments. • Two types of changes • Exonuclease removal of nucleotides. • Random addition of nucleotides by terminal deoxyribonucleotidyl transferase (TdT). A C T G T G C G G A T G • Germ line DNA

23. G T C C A G G C T T G G A T • Recombined DNA Leu Arg Trp Val • Protein Junctional Diversity (N- region diversity) T C • J segment • V segment • Changes in nucleotide sequence at junctions of V, D, and J segments. • Two types of changes • Exonuclease removal of nucleotides. • Random addition of nucleotides by terminal deoxyribonucleotidyl transferase (TdT). A C T G T G C G G A T G • Germ line DNA

24. sIgM Surface vs. Secreted Soluble IgM mRNA 3’ 5’ Primary RNA transcript Cm Ts V2 • Which poly A site is transcribed determines whether the protein is surface or secreted. 3’ 5’ Cm Ts Tm V2 mIgM • Poly A • Poly A Membrane IgM mRNA 3’ 5’ Cm Ts Tm V2

25. Membrane Bound Ig B cell receptor (BCR) complex • IgM or IgD • Associated with a heterodimer of Iga and Igb. • Activation of the BCR results in activation of Iga and Igb leading to cytoplasmic signaling and B cell activation • Iga • Iga • Igb • Igb

26. Antibody Kinetics • __________________________ is the tendency of antibody to form stable complexes with an antigen • + • Ag

27. Antibody Kinetics • _________________________ is the strength in which a multivalent antibody binds a multivalent antigen. • Ag • Y • Y • Y • Y • Y • Y • IgG • IgM

28. T Cell Receptor • Present on a majority of T cells both CD4+ and CD8+. • Heterodimeric molecule. • Linked by a disulfide bond. • Generated by 4 sets of genes • a and b genes are expressed in a majority of peripheral T cells. • g and d are expressed on a minority of thymic and peripheral T cells (10%). • Binds to the peptide-MHC complex.

29. T Cell Receptor Variable domain • 2 a chains • 2 b chains • Associated with CD3 • 2 e chains • 1 d chain • 1 g chain • 2 z chains Conserved domain CD3 Adapted from Roitt’s Immunology 4-6

30. T-Cell Receptor Loci Adapted from Abbas & Lichtman’s Basic Immunology 4-9

31. T Cell Receptor Diversification J1-n 3’ 5’ Ca V1 V2 Vn Germline a DNA J21-n J11-n D2 D1 3’ 5’ Cb2 Cb1 V1 V2 Vn Germline b DNA • Variable (VH) segments • Diversity (DH) segments • Joining (JH) segments • Conserved (CH) segments

32. T Cell Receptor Diversification J1-n 3’ 5’ Ca V1 V2 Vn Germline a DNA J21-n J11-n D2 D1 3’ 5’ Cb2 Cb1 V1 V2 Vn Germline b DNA • Recombination between V, D, and J gene segments. • a and g chains contain V and J gene segments. • b and d chains contain V, D, and J gene segments. • Similar to Ig.

33. Comparison of TCR and Ig • Ig binds to proteins, polysaccharides, Lipids, and nucleic acids, while TCR only binds peptides in MHC. Forms of Antigen Recognized Diversity Antigen Recognition Mediated by Signaling functions Mediated by Effector functions Mediated by Abbas & Lichtman’s Basic Immunology 4-1

34. Antigen Recognition • Ig binds antigen all by itself, while TCR needs CD4 or CD8 to simultaneously bind. Antigen Binding Region Structure of antigen recognized Affinity of antigen binding On-rate and Off-rate Accessory molecules Involved in binding Abbas & Lichtman’s Basic Immunology 4-7

35. Next Time • T cell maturation • Cell-mediated immune responses (CMI) • Humoral immune responses • B cell activation • Readings: Abbas & Lichtman, Chapters 5 & 6

36. Objectives • Describe the structure and synthesis of the various immunoglobulin Heavy and Light chains, and T-cell receptors. • V, D, J, C Segments • Describe the process of immunoglobulin class change. • Describe the mechanisms of antibody diversity. • Combinitorial vs Junctional • Surface vs Secreted • Know the difference between affinity and avidity. • Compare and contrast immunoglobulins and T-cell receptors.