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.

SummaryTwo 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)

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

SummaryAll 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).