Presentation on theme: "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."— Presentation transcript:
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).