Natural Antibodies

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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).
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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

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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

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PtC-binding B cells are detectable by Hi-D FACS
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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
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Is the difference due to enviromental influences
(selection) or to genetic differences between
the two strains? TRANS VS cis
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Genetic Control of the Natural Antibody Repertoire
Cis-linked Regulation of Antibody Variable Gene
Usage
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BALB/c x C.B-17 F1 Mice
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BALB/c x C.B-17 F1 Mice
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Determining the VH repertoire of anti-PtC
antibodies

• Single-cell RT-PCS and sequencing of PtC binding
  cells
• FACS analysis of PtC binding cells

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Cis-linked regulation of VH12 Predominance
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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.

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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)

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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
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VH12 Predominance Maps to the VH Region
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But selection is also important in determining
the B-1 repertoire
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VH12 has higher avidity for PtC liposomes than
VH12ala
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Cells expressing VH12thr antibodies that do not
bind PtC do not increase in frequency with age
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Differential VH expression in PtC-binding cells
from C.B-17 x BALB/c heterozygotes
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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

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Acknowledgements
Lee Herzenberg Len Herzenberg Jennifer
Wilshire Nicole Baumgarth Darren Gold The
Members of the Herzenberg Laboratory The Stanford
Shared FACS Facility
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B-1
Self replenishing B cells that differ in
phenotype, location and ontogeny from B-2 cells
(the majority population).