Genetics in the Generation of Antibody Diversity

1. Genetics in the Generation of Antibody Diversity Folder Title: AbGenesNoTP Updated: October 09, 2014 Chapter Seven, 7th Edition: “Organization and Expression of Lymphocyte Receptor Genes” Includes Immunoglobulin and T-Cell Receptor Genetics

2. Questions About B-Cell Responses • Biochemical Questions: • How can Immunoglobulins recognize so many different epitopes? • What does an antibody protein look like? • How do specific antibodies work as proteins binding so many different specific antigenic determinants?

3. Representation of Sequence Comparisons Among Light Chains from Antibodies with Three Different Antigen Specificities H3N-Ser-Val-Ile-Thr-Gly-Gly-Tyr-Ala... Thr-Glu-Ala-Val-Tyr-Ser-Met-COO- H3N-Ser-Ile-Met-Thr-Arg-Leu-Tyr-Gly..Thr-Glu-Ala-Val-Tyr-Ser-Met-COO- H3N-Thr-Gly-Gly-Thr-Lys-Leu-Tyr-Ile..Thr-Glu-Ala-Val-Tyr-Ser-Met-COO- Variable Amino Terminal HalfConserved Carboxyl Terminal Half (Positions 1 to 107) (Positions 108 - 214) LiteComp

4. See Figure 4-6, Kuby 6th Edition p. 85

6. Questions About B-Cell Responses • Biochemical Questions: • How can Immunoglobulins recognize so many different epitopes? • What does an antibody protein look like? • How do specific antibodies work as proteins binding so many different specific antigenic determinants? • Genetic Question: • How can this diversity of structure leading to enormous diversity of function be coded and controlled by a very limited host genome? • How can we have “one gene - one polypeptide” and make a virtually limitless selection of polypeptides?

7. Problems for Genetics in Generating Antibody Sequence Diversity Vast Sequence Diversity- Encoded by Very Limit Genome Heavy and Light Chain Sequence Variations- Almost Exclusively in only one region Exactly the same V-region sequences- End up on different C-region Isotypes. How is all of this possible?????

8. Differentiated Neoplastic Plasma Cell making a single antibody. Myeloma Cell DNA

9. Myeloma Cell Conclusions Variable and conserved regions of light chain are linked in the differentiated end-product cell line DNA coding for the mRNA for the light chain is all in one Continuous sequence as for any gene for any protein.

10. Embryonic Mouse Cell DNA Variable and conserved regions of light chain are not linked originally in the stem cell lineage!

11. Immunology and Phone Numbers 315-443-1870 212-345-1775 478-367-8903 537-503-2078 409-159-6309 610-970-3970 934-620-8122 909-603-7023 800-620-6021 704-590-5307 703-725-0153 207-502-6671 435-431-0890 412-830-0048 740-592-1954 307-620-4450 490-501-5672 601-909-7002 554-891-7712 335-592-0944

12. 315- 212- 478- 537- 409- 610- 934- 909- 800- 704- 703- 207- 435- 412- 740- 307- 490- 601- 554- 335- Immunology and Phone Numbers -443-1870 -345-1775 -367-8903 -503-2078 -159-6309 -970-3970 -620-8122 -603-7023 -620-6021 -590-5307 -725-0153 -502-6671 -431-0890 -830-0048 -592-1954 -620-4450 -501-5672 -909-7002 -891-7712 -592-0944

13. 315- 212- 478- 537- 409- 610- 934- 909- 800- 704- 703- 207- 435- 412- 740- 307- 490- 601- 554- 335- -443- -345- -367- -503- -159- -970- -620- -603- -620- -590- -725- -502- -431- -830- -592- -620- -501- -909- -891- -592- Immunology and Phone Numbers -1870 -1775 -8903 -2078 -6309 -3970 -8122 -7023 -6021 -5307 -0153 -6671 -0890 -0048 -1954 -4450 -5672 -7002 -7712 -0944

14. Kappa Chain DNA: Vk’s This and the next two slides deal with Kappa Light Chain DNA. Human Kappa DNA was 40 different “V” region genes to work with, and 5 “J” region genes. Lambda light chain DNA works the same way except that the Human Lambda light chain works with 30 “V” regions and 4 “J” region genes.

15. Kappa Chain: K-J- C

16. Complete Kappa Chain

17. Heavy Chain Dna: VH’s, C-Genes This and the next two slides deal with Heavy chain DNA. Human heavy chain DNA has 51 “V” region genes to work with, 27 “D” (diversification) region genes, and 6 “J” (joining) region genes.

18. Coupling a VH option with DH option and a JH option to a Cu gene sequence would give a Mu Heavy chain isotype with a specific antibody recognizing specificity. Coupling that exact same VH-DH-JH genetic information to Cd or Cg or Ce or Ca Heavy chain isotype gene would give the exact same antigen specificity onadifferentisotype. We would have Isotype Switching

19. Complete Heavy Chain

20. Additional sequence diversity estimated at several orders of magnitude (i.e. perhaps 1000-fold) is generated by junctional flexibility, nucleotide addition, and somatic hypermutation. This allows for an estimated 100 million to a billion different possible antibody seuquences in humans. From Kuby Immunology, 4th Ed. Upper Half of Table: Human Data Lower Half (not shown): Mouse Data. 6 Edition V,D,J segment numbers are slightly different

21. B-Cell Differenatiation

22. Please put away all notes and devices other than your XR transmitter. No consulting or reading notes is permitted. No online searches. This are graded response questions.

23. Save Session with Participant List Responses. Close Session. Reset to Anonymous Polling Open New Session for Anonymous Slide on Scope and Pace of the course so far.

24. This question will be set to anonymous so no one will know what any specific student says. For the first 14 classes the pace of the course is: • Too slow. We need to move faster and cover more about Immunology. • About right. I can keep up Ok but it is a challenge. • Way too fast. Slow down! We’ll figure out later what you don’t get to cover in class. Response Counter

25. On a scale of 5 to 1 rate todays AbGenes Presentation5. Too slow. Get moving4. No problem.3. About right. I can figure out what I missed.2. Too fast1. I’m lost!(This is anonymous. We won’t know who you are.)