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Generation of Antibody Diversity

Generation of Antibody Diversity. The big question:how do you get all those different antibodies? Two other big questions- getting different classes with the same variable regions, and getting secreted and membrane-bound forms of the same class

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Generation of Antibody Diversity

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  1. Generation of Antibody Diversity • The big question:how do you get all those different antibodies? • Two other big questions- getting different classes with the same variable regions, and getting secreted and membrane-bound forms of the same class • WARNING: This is hard stuff- and I'll cover more than you need to know- partly to impress you on the capabilities of what's happening to you daily.

  2. Overall picture • Overall picture- diversity produced by combining a number of variable regions with at least one joining region, plus the constant regions. Diversity is increased by the total number of possible rearrangements. The regions in between are deleted. • Both light and heavy chains variable regions involved. We have multiples of multiples resulting in millions of combinations from ~245 gene segments.

  3. Spend some time on this one!

  4. The story in mice 3 possibilities Gene segments Chromosome 16 85X 4 possible combinations=340 Chromosome 6 134X 13X 4 possible combinations =6968 Chr. 12 Total = 343X6968= 2,390,024- a lot, but not gazillions.

  5. How we know this- Southern blot, using J region as a probe. In a germ line culture, the probe only finds the J region. In a B cell line, it finds the rearranged V region as well. Further cloning and sequencing verified this result. Fig. 22-9 for more information

  6. ~85 V regions

  7. http://www.bio.davidson.edu/COURSES/Immunology/Flash/somaticrecomb.htmlhttp://www.bio.davidson.edu/COURSES/Immunology/Flash/somaticrecomb.html http://www.uni-greifswald.de/~immuteach/basics/somrec/somrec.html

  8. RSS= Recombination signal sequence

  9. This shows the details of the next three slides.

  10. Joining flexibility adds more diversity, and also contributes to nonproductive rearrangements These are the ends of the J and V regions after any P-nucleotide addition has taken place, and also N-nucleotide addition (in H chain)

  11. Allelic exclusion: successful rearrangement of 1 allele excludes rearrangement of the other allele. The cell “knows” when it’s done an unproductive rearrangement!

  12. + Error!

  13. MORE Diversity- Somatic Hypermutation!! • This occurs AFTER B cell formation, and in response to antigen in the germinal centers. • Rate of mutation ~ 10-3 per bp per cell generation- 105 X higher than normal, and just in the CDR 1,2 &3 regions. • The process is random, and there’s selection for those that are better at binding antigen.

  14. Immunize mice w/ antigen (a hapten) Periodically remove cells and make monoclonals- after primary immunization, 2o, 3o immunizations Sequence H and V chain mRNA from these monoclonals Compare sequences and antibody affinity

  15. Quiz on Wed: • Ab classes • Somatic recombination: sample calculation • Definitions: • “sloppy”- DJ v-DJ, or VJ joining produces a variety of outcomes • Non-productive rearrangement • Allelic exclusion • Somatic hypermutation

  16. Class switching (no- not what happens between 9:50 and 10:00!) • Antibodies come in classes • In the maturation process, there is class switching- initially IgM is made, this switches to IgG or A or E due to cytokine influences- e.g. IL-4 induces switch to IgG and sometimes also to IgE. • The switching come from deleting previous heavy classes. • ?consequences?

  17. May be sequential, may be Su recombines directly with SE. IL-4 induced Switch recombinase, signaled by cytokine Consequences? Can’t go backwards! These have been found

  18. Membrane vs secreted • Apparently all antibody classes can be membrane-bound or secreted, but this mainly gets involved in the initial change of a B cell w/ mIgM and mIgD to a plasma cell that’s secreting IgM. • The switch involves alternative splicing.

  19. Note that the sequences are different!

  20. In eukaryotes, we make a primary transcript, that can then be cut- where it is cut is where we add a “polyA tail” Mu really consists of six exons Delta really conists of 5 exons

  21. Antibodies for health and wealth • Monoclonals are quite useful, but typically of mouse origin. • For use in humans, we want to minimize the antibody response to the mouse-specific Ag determinants. • There are a variety of ways to put mouse variable regions onto human antibodies, but we’re not going to talk about them, just the results.

  22. “humanized” antibody

  23. Things to know from this chapter- lots of stories!! • Be able to tell the generation of Ab diversity story- with some numbers and estimates. • Be able to define the processes that add to diversity: p and n-nucleotide addition, joining flexibility, somatic hypermutation. • RSS’s, RAGs, nonproductive rearrangement, allelic exclusion, class switching. • Be able to tell the story of how class switching and the switch from membrane to secreted Ig occurs.

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