1. Antibody Diversity

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. Current theory must account for the following known properties of antibodies

4. B lymphocyte development

5. B lymphocyte development (2)

6. 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?

7. 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

8. 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)

9. Genes for immunoglobulin proteins are found on different chromosomes

10. Multigene organization of Ig genes

11. Kappa light chain rearrangement

12. Heavy chain rearrangement

13. 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

14. 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

15. N nucleotide addition at joining segments: the addition of random bases

16. Randomness in joining process helps generate diversity by creating hypervariable of antigen binding site

17. Imprecise joining generates diversity

18. Some rearrangements are productive, others are non-productive: frame shift alterations are non-productive

19. Allelic exclusion: only one chromosome is active in any one lymphocyte

20. 7 means of generating antibody diversity

21. Diversity calculations

22. 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

23. Combination of heavy and light chains adds final diversity of variable region
8262 possible heavy chain combinations
320 light chain combinations
Over 2 million combinations
P and N nucleotide additions and subtractions multiply this by 104
Possible combinations over 1010

24. Location of variability occurs within CDR regions of V domains (antigen binding sites)

25. Class switching among constant regions: generation of IgG, IgA and IgE with same antigenic determinants—idiotypes

26. Synthesis, assembly and secretion of immunoglobulins

27. Regulation of Ig gene transcription
Each lymphocyte rearranged gene has regulatory sequences that control gene expression
Promoters: initiation sites of RNA transcription
Enhancers: upstream of downstream that transcription from the promoter sequence
Silencers: down-regulate transcription in germline cells
Gene rearrangement brings enhancer and promoter regions close together and eliminates silencer regions allowing transcription

28. 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