1. Basic events during B cell development Stem cells Pre-B “D” Pre-B expansion and antigen selection Immature B cells Mature Peripheral B cells IgH () heavy chain rearrangement VDJ-C() + V L C L IgM VDJ-C() Pro-B “A” Pro-B “C” Pro-B “B” None DJ VDJ Allelic exclusion A,B,C… = Hardy Fractions

2. Bone Marrow

3. But: not all mature B cells are the same

4. Phenotypic differences distinguish four kinds of B cells  B-1a: CD5 +, IgM br, IgD dull, MAC-1 + in PerC  B-1b: like B-1a but CD5 -  B-2 follicular: CD5-, CD23+, IgM dull, IgD br  B-2 marginal zone: CD5-, CD23+, IgM br, IgD dull Phenotype

5.  B-1a: Peritoneal and pleural cavities; gut  B-1b: Peritoneal and pleural cavities  B-2 follicular: spleen, lymph nodes, PerC  B-2 MZ: spleen Location

6. Spleen

7. PerC

8.  B-1a: IgM >> IgG 3 > IgA >IgG 2 > IgG 1  B-1b: IgM > IgE > IgG 1 > IgG 2  B-2 follicular: IgM, IgG1, IgG2…  B-2 marginal zone: IgM, IgG1… Ig Isotype production

9.  Made in response to antigenic stimulation  Usually T dependent  Differentiate to IgG memory cells  Usually made by B-2, but B-1 clearly respond Function: adaptive responses

10.  Made by B-1  Produced and secreted without (known) specific antigenic stimulation  Cytokines increase secretion  IL-9 increases IgE and IgG1 production by B-1b  IL-5 increases secretion by B-1a (?)  Production is T-independent in the ordinary sense  Differentiation to IgG producing cells has been reported in pathologic conditions Function: natural antibodies

11.  B-1a: Arise in fetus and neonate  B-1b: Arise in neonate; adult??  B-2 follicular: Arise around weaning  B-2 MZ: Strains differ but mostly after weaning Ontogeny

12.  B-2 are replenished by de novo development from progenitors in BM throughout life  B-1 cells develop de novo during fetal and neonatal life but persist thereafter as a self- replenishing population Subset maintenance

13. Single lineage model of B cell development Stem cell Pre-B “D” B-1a B-1b B-2 (follicular + MZ) Self-replenishing De novo replacement Normal pre-B expansion and antigen selection Pro-B “A” Pro-B “C” Pro-B “B” Immature B Peripheral B cells DHDJDHDJ Special antigens (self, repetitive, bacterial) Special antigens (self, repetitive, bacterial)

14. Multi-lineage model of B cell development Stem cell Adult BM Fetal liver Feedback loop in mice 3-6 weeks X X B-1a B-1b B-2 (follicular + MZ) Self-replenishing De novo replacement Pre-B expansion Stem cell

15. So how do we determine which hypothesis is valid? Study B cell progenitors activity in mixture-transfer experiments Use Ig allotype expression to mark the mature progeny B cells

16. Basic events during B cell development Stem cells Pre-B “D” Pre-B expansion and antigen selection Immature B cells Mature Peripheral B cells IgH () heavy chain rearrangement VDJ-C() + V L C L IgM VDJ-C() Pro-B “A” Pro-B “C” Pro-B “B” None DJ VDJ Allelic exclusion A,B,C… = Hardy Fractions

17. IgH allelic exclusion in B cells

18. The IgH of the antibody molecules produced by an individual B cell are all encoded by a single VDJ-C rearrangement that occurred on one of the two parental chromosomes “Allelic” exclusion

19. So how do we determine which hypothesis is valid? Study B cell progenitors activity in mixture-transfer experiments Use Ig allotype expression to mark the mature progeny B cells

20. Results of mixture-transfer studies  B-2 are replenished by de novo development from progenitors in BM throughout life  Adult BM readily regenerates the entire B-2 population in adoptive recipients  B-1 cells develop de novo during fetal and neonatal life but persist thereafter as a self- replenishing population  Adult BM regenerates only a few B-1 cells, mainly B-1b  Fetal and neonatal progenitor sources fully regenerate the B-1 population

21. Results of mixture-transfer studies  Mixtures of progenitors (B220 - ) from adult BM and fetal sources fully regenerate the B-1 population  Virtually all B-1 cells are derived from the fetal source Therefore: 1) BM does not contain cells that inhibit B-1 development; and, 2) Fetal sources do not provide support for the development of cryptic progenitors for B-1 cells We conclude that BM (essentially) lacks progenitors for B-1 cells