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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).
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
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
PtC-binding B cells
are detectable by Hi-D FACS
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
Is the difference due to
enviromental influences
(selection)
or to
genetic differences between
the two strains?
TRANS VS cis
Genetic Control of the Natural
Antibody Repertoire
Cis-linked Regulation of
Antibody Variable Gene Usage
BALB/c x C.B-17 F1 Mice
BALB/c x C.B-17 F1 Mice
Determining the VH repertoire
of anti-PtC antibodies
Single-cell RT-PCS and sequencing of PtC binding cells
FACS analysis of PtC binding cells
Cis-linked regulation of VH12 Predominance
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.
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/10related strains (including B102a4b from which
CH lymphomas were isolated)
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
VH12 Predominance Maps to the VH Region
But selection is also important
in determining the
B-1 repertoire
VH12 has higher avidity for PtC
liposomes than VH12ala
Cells expressing VH12thr antibodies
that do not bind PtC do not increase in
frequency with age
Differential VH expression in PtC-binding
cells from C.B-17 x BALB/c heterozygotes
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
Acknowledgements
Lee Herzenberg
Len Herzenberg
Jennifer Wilshire
Nicole Baumgarth
Darren Gold
The Members of the Herzenberg Laboratory
The Stanford Shared FACS Facility
B-1
* Self replenishing B cells that differ in phenotype, location and ontogeny from B-2 cells
(the majority population).