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Natural Antibodies

Natural Antibodies. Made by B-1 cells*. * Self replenishing B cells that differ in phenotype, location and ontogeny from B-2 cells (the majority B cell population). Natural Antibodies. Present in unimmunized mice Bind to pathogens and cell components A key part of the innate immune system

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Natural Antibodies

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  1. Natural Antibodies Made by B-1 cells* * Self replenishing B cells that differ in phenotype, location and ontogeny from B-2 cells (the majority B cell population).

  2. Natural Antibodies • Present in unimmunized mice • Bind to pathogens and cell components • A key part of the innate immune system • spontaneously secreted (as natural IgM) • first line of defense against invading pathogens

  3. Anti-PtC repertoire • Antibodies expressed by 5-15% of B-1 cells bind PtC • Other (B-2) cells do not make anti-PtC antibodies • Ig heavy chains in anti-PTC antibodies are mainly encoded by 3 VH families; VH11, VH12, VHQ52

  4. PtC-binding B cells are detectable by Hi-D FACS

  5. PtC-binding B cells are detectable by Hi-D FACS IgMa and IgMb are IgM allotypes present respectively in the Balb/C and C.B/17 allotype congenic strains

  6. Is the difference due to enviromental influences (selection) or to genetic differences between the two strains? TRANS VS cis

  7. Genetic Control of the Natural Antibody Repertoire Cis-linked Regulation of Antibody Variable Gene Usage

  8. BALB/c x C.B-17 F1 Mice

  9. BALB/c x C.B-17 F1 Mice

  10. Determining the VH repertoire of anti-PtC antibodies • Single-cell RT-PCS and sequencing of PtC binding cells • FACS analysis of PtC binding cells

  11. Cis-linked regulation of VH12 Predominance

  12. Summary and Conclusions • VH12 predominates the anti-PtC repertoire in C.B-17 mice while VHQ52 predominates in BALB/c • VH predominance is controlled in cis • The element(s) that controls the predominance maps to the VH encoding region.

  13. Summary • Two VH12 Alleles • Differ by a single amino acid in framework 1 (codon 21) • The VH12ala (alanine) is found in BALB/c and C57BL/6 • The VH12thr (thronine) allele is found in the BALB/c Igh bcongenic strain, C.B-17, and in C57BL/10- related strains (including B102a4b from which CH lymphomas were isolated)

  14. Proteins encoded by two VH12 alleles differ by a single amino acid at codon 21 in framework 6 silent mutations also distinguish the VH12 alleles BALB/c and C.B-17

  15. VH12 Predominance Maps to the VH Region

  16. But selection is also important in determining the B-1 repertoire

  17. VH12 has higher avidity for PtC liposomes than VH12ala

  18. Cells expressing VH12thr antibodies that do not bind PtC do not increase in frequency with age

  19. Differential VH expression in PtC-binding cells from C.B-17 x BALB/c heterozygotes

  20. Summary • All VH anti-PtC except VH12thr • Frequency among peritoneal B cells is fixed by 3-4 weeks of age and remains stable at least until 7 months VH12thr • Frequency among peritoneal B cells increases consistently from 3-4 weeks of age at least 7 months

  21. Acknowledgements Lee Herzenberg Len Herzenberg Jennifer Wilshire Nicole Baumgarth Darren Gold The Members of the Herzenberg Laboratory The Stanford Shared FACS Facility

  22. B-1 * Self replenishing B cells that differ in phenotype, location and ontogeny from B-2 cells (the majority population).

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