1. Immunoglobulin Genetics

2. Problem…the immune system makes over one billion different antibody proteins In 1950’s: central dogma stated DNA—to RNA—to protein One gene for each protein Required millions of genes just for the immune system Does not seem possible, but most scientists thought it might be Today we know the human genome is less than 30,000 genes So, what is really going on???

3. B lymphocyte development

4. B lymphocyte development (2)

5. Immunoglobulin Genetics History Same C region could associate with many V regions –IgG Ab with different specificities Same V region could associate with many C regions –Same idiotype on IgG and IgM Ab V and C regions coded for by separate genes

6. Genes for immunoglobulin proteins are found on different chromosomes

7. Light Chain Gene Families Germ line gene organization J1J1 C1C1 E J2J2 C2C2 E J3J3 C3C3 E J4J4 C4C4 E P L V1V1 P L VnVn P L V2V2 Lambda light chain genes; n=30 P L V  1 P L V  n P L V  2 J2J2J3J3J5J5 CC E J1J1J4J4 Kappa light chain genes; n=300

8. Light Chain Gene Families Gene rearrangement and expression L V 1V 1 L V nV n L V 2V 2 J2J2J3J3J5J5 CC J1J1J4J4 DNA PPP E P P E L V 1V 1 L J5J5 CC V 2V 2J4J4 DNA Rearrangement VC J V C Protein Transport to ER Protein Translation C L V J E L V CJ RNA Transcription Primary transcript C L V J RNA Processing RNA mRNA

9. Heavy Chain Gene Family Germ line gene organization

10. Heavy Chain Gene Family Gene rearrangement and expression P L V1 P L Vn P L V2 D2D2 D3D3 D1D1DnDn CC CC J2J2J3J3J5J5J1J1J4J4 E DJ rearrangement P L Vn P L V1 P L V2 D2D2 D1D1 CC CC J5J5 J4J4 E DNA VDJ rearrangement D2D2 CC CC J5J5 J4J4 E P L V1V1 P L V2V2 DNA Transcription D2D2 CC CC J5J5 J4J4 E L V2V2 RNA Primary transcript

11. Heavy Chain Gene Family Gene rearrangement and expression E Primary transcript E J4J4D2D2 V2V2 CC CC J5J5 L CC CC J5J5 L D2D2J4J4 V2V2 RNA Processing AnAn mRNA for C  CC L DJV CC L DJV AnAn mRNA for C  Translation C  heavy chain CC L DJVCC L DJV C  heavy chain Transport to ER C  heavy chain V C CC DJVCC DJV C  heavy chain V C

12. Mechanism of variable region rearrangements Each V, D and J segments of DNA are flanked by special sequences (RSS—recombination signal sequences) of two sizes Single turn and double turn sequences (each turn of DNA is 10 base pairs long) Only single turn can combine with a double turn sequence Joining rule ensures that V segment joins only with a J segment in the proper order Recombinases join segments together

13. Mechanism of DNA Rearrangements Recombination signal sequences (RSS) –Nonmer –Heptamer –1 or 2 turn signals Rag-1 and Rag-2 L V 2 J C 1 Lambda light chains L V 1 J C 2 Kappa light chains Heavy chains L V 2 J C 2 1 1 D Heptamer CACAGTG GTGTCAC Nonamer ACAAAAACC TGTTTTTGG 23 bp Two turn RSS 2 Nonamer GGTTTTTGT CCAAAAACA Heptamer CACTGTG GTGACAC 12 bp One turn RSS 1

14. Order of Ig Gene Expression + - Heavy chain gene VDJ recombination Productive rearrangement ? Yes No Transcribe/translate 1st 2nd or Apoptosis Progenitor B cell Pre B cell    (  ) Heavy chain Paternal Maternal

15. Order of Ig Gene Expression + - Light chain gene VJ recombination Productive rearrangement ? Yes No Transcribe/translate Light chain 4 th Apoptosis 1st -  2nd -  3rd -  4th -  or Pre B cell Mature B cell     + 

16. Order of Ig Gene Expression Explains: Why an individual B cell only produces one kind of Ig with one kind of heavy and one kind of light chain Why an individual B cell makes only makes antibodies of one specificity Why there is allelic exclusion

17. Origin of Antibody Diversity History Definition – Diversity (repitoire) is the total of all the Ab specificities that an organism is capable of expressing Theories –Germ line theory –Somatic mutation theory

18. Germ-line vs somatic-variation theories Germ-line: stated that each antibody had its own gene….nothing special, but required billions of genes to account for numbers of antibodies Somatic-variation: some mutation and recombination created vast number of genes for antibody formation This introduced a new concept: targeted mutation or recombination of DNA: is it possible?? Paradox: how could stability be maintained in C region and diversity exist in V region?

19. Dreyer and Bennett In 1965 proposed radical theory to account for diversity of antibodies Each antibody was coded for by two separate genes One for the variable region One for the constant region Combined at the DNA level and expressed single mRNA Suggested 1000’s of variable region genes and only one constant region gene Close, but no cigar Most biomedical scientists did not like this idea and rejected it

20. Tonegawa’s demonstration 1976—used restriction enzymes and DNA probes to show that germ cell DNA contained several smaller DNA segments compared to DNA taken from developed lymphocytes (myeloma cells)

21. conclusion Susumu Tonegawa "Nobel prize winner" discovered that antibody genes can move and rearrange themselves within the genome of a differentiating B cell. The same applies for TCR. Many different V region genes can be linked up to a single constant "C" region gene.

22. Origin of Antibody Diversity Current concepts Multiple V genes V-J and V-D-J joining Junctional diversity N region insertions – Amino acid sequences not encoded in the germ line D region TGGCCC Pro Trp CCCCCC V gene TGG D region CCCCCC V gene TGG CGGCCC Pro Arg CCCCCC V gene TGG D region CGCCCC Pro

23. Junctional and Insertional Diversity The absence of precision in joining during DNA rearrangement leads to deletions or changes of amino acids "junctional diversity" that affect Fab region as it occurs in parts of the hypervariable region. Small sets of nucleotides may be inserted "insertional diversity" at the V-D and D-J junctions. It is mediated by the enzyme terminal deoxynucleotidyltransferase "TdT". The additional diversity generated is termed N region diversity.

24. P and N region nucleotide alteration adds to diversity of V region During recombination some nucleotide bases are cut from or add to the coding regions (p nucleotides) Up to 15 or so randomly inserted nucleotide bases are added at the cut sites of the V, D and J regions (n nucleotides_ TdT (terminal deoxynucleotidyl transferase) a unique enzyme found only in lymphocytes Since these bases are random, the amino acid sequence generated by these bases will also be random

25. - GTCAATG - GTCAATG -+ Break DNA TdT adds nucleotides Ligate N region insertionIntron JD J J J D D D Generation of N region insertions

26. Origin of Antibody Diversity Current concepts Multiple V genes V-J and V-D-J joining Junctional diversity N region insertions Somatic mutation Combinatorial association Multispecificity

27. Somatic hypermutation adds even more variability B cell multiplication introduces additional opportunities for alterations to rearranged VJ or VDJ segments These regions are extremely susceptible to mutation compared to “regular” DNA, about one base in 600 is altered per two generations of dividing (expanding) lymphocyte population

28. N.B All these mechanisms contribute to the formation of a huge library. Or repertoire of B lymphocytes that contain all the specificities required to deal with the multitude of diverse epitopes that antibodies could encounter. The number of total Ig specificities that can be generated in an individual are on the order of 1015 which is increased even more by somatic hypermutation.

29. Regulation of Ig Gene Expression Each B cell uses only one set of VDJ genes and one type of light chain. As a result, a single B cell produces an Ig of only one antigenic specificity. Ig chains are coded for by only one set of genes, either from the maternal or the paternal chromosome, e.g. H chain may be coded for by genes on the paternal chromosome and the L chain "  or " by genes on the maternal chromosome. This phenomenon of using genes from only one parental chromosome is known as allelic exclusion. If the process of rearrangement fails in one chromosome, the second parental chromosome will continue rearrangement. Gene exclusion ensures that every B cell and the Ab it synthesizes are monospecific e.g. specific for one epitope.

30. Generation of Diversity Ag independent process Clonal selection

33. Copyright © 2007 by W. H. Freeman and Company ANTIBODY GENE REARRANGEMENTS CLASS SWITCHING : Selection of CH Genes Mechanism Unclear, but Involves Switch Region Switch Recombinase Isotype Specific Cytokine Signals

34. Class or Isotype Switching One B cell makes Ab of a single specificity that is fixed by the nature of VJ "L chain" and VDJ "H chain" rearrangements. These rearrangements occur in the absence of Ag in the early stages of B cell differentiation. B cells can synthesize IgM and IgD with the same antigenic specificity. An individual B cell have the ability to switch to make a different class of Ab... such as IgG, IgE, IgA. This phenomenon is known as class or isotypic switching. Class switching changes the effector function of the B cell but does not change the cell's antigenic specificity.

35. Class Switching among Constant-Region Genes

36. At the 5` end of every H chain C region "CH" gene apart from C delta, is a stretch of repeating base sequences called a switch "S" region. This S region permits any of the C H genes "other than C delta" to associate with the VDJ unit. Under the stimulating influence of Ag & T cell- derived cytokines, a B cell with a VDJ unit linked to Cu and C delta further rearranges its DNA to link the VDJ to an S region in front of another C H region gene " γ1 in previous fig".

37. Cytokines play a key role in C H gene selection during isotype switching. e.g. in the presence of IFN- γ, B cell can switches to IgG2 synthesis. In the presence of IL-4, a B cell can switches to IgG4 or IgE synthesis respectively. Each cytokine is thought to activate an enzyme known as a "switch recombinase" to recognize DNA coding for specific C regions.

38. Understanding of immunoglobulin structure and formation has opened up a new world of possibilities Monoclonal antibodies Engineering mice with human immune systems Generating chimeric and hybrid antibodies for clinical use Abzymes: antibodies with enzyme capability