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; V H 11, V H 12, V H Q52

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

IgM a and IgM b 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

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 V H 12 Predominance

Summary and Conclusions  V H 12 predominates the anti-PtC repertoire in C.B-17 mice while V H Q52 predominates in BALB/c  V H predominance is controlled in cis  The element(s) that controls the predominance maps to the V H encoding region.

Summary Two VH12 Alleles  Differ by a single amino acid in framework 1 (codon 21)  The V H 12 ala (alanine) is found in BALB/c and C57BL/6  The V H 12 thr (thronine) allele is found in the BALB/c Igh b congenic 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 V H Region

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

V H 12 has higher avidity for PtC liposomes than V H 12 ala

Cells expressing V H 12 thr 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 V H anti-PtC except V H 12 thr  Frequency among peritoneal B cells is fixed by 3-4 weeks of age and remains stable at least until 7 months V H 12 thr  Frequency among peritoneal B cells increases consistently from 3-4 weeks of age at least 7 months

Acknowledgements Lee HerzenbergLen Herzenberg Jennifer WilshireNicole 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).