B cell development Tony DeFranco, 10/ 29/14

1. B cell developmentTony DeFranco, 10/29/14

2. 5 Themes in B cell development+ Ig class switch and somatic mutation Theme 1: Checkpoints in B cell development: feedback from Ig gene rearrangements Theme 2: Bone marrow microenvironment Theme 3: Lineage commitment: transcription factors Theme 4: Central and peripheral tolerance of B cells Theme 5: 3 different types of mature B cells Molecular mechanisms of class switch recombination and somatic hypermutation: Activation-induced cytidinedeaminase (AID)

3. Lymphocyte Development Lymphocyte development is designed to generate functional lymphocytes with useful antigen receptors that are not self-reactive Much of what happens during lymphocyte development is designed to improve the efficiency of adaptive immunity

4. B cell Development: Relevance • Immunodeficiencies that affect B cell development • B cell malignancies (pre-B ALL, etc.) • Alterations in B cell tolerance may underlie some autoimmune diseases • B cell development is an especially well understood example of mammalian cell development

5. Overview of B cell development

6. Theme 1: Ig rearrangement checkpoints forB cell development IgH unrearr DJ VDJ VDJ VDJ IgL unrearr unrearr unrearr rearranging VJ (surrogate L chain)

7.  chain expressed in cytoplasm B cell development:distinctive cell surface markers(mouse) B220 - + ++ +++ CD43 (S7) + + - - CD25 - - + - c-kit + + - - Human B cell precursors: see Blom & Spits 2006

8. Pro-B to pre-B checkpoint requires Pre-BCR signaling • Surrogate light chain (VpreB + l5): • Expressed only in proB/preB cells • Triggers signaling (self aggregation?Ligand present on stromal cells?) • Expression turned off by pre-BCR signaling

9. Pre-BCR checkpoint regulates V(D)Jrecombination Controlled by chromatin accessibility; epigenetic marks due to histone modifications match those associated with transcription

10. Theme 2: the bone marrow microenvironment • Role of IL-7 in murine B cell development • Lack of Notch ligands in the bone marrow favor B lineage development (Notch ligands in thymus are important for commitment to the T lineage)

11. Theme 2: the bone marrow microenvironment Based on in vitro culture experiments, the bone marrow microenvironment has several key properties: • Pro-B cells (-) grow indefinitely in vitro only in contact with stromal cell layer from bone marrow • Pre-B cells (+) will grow in vitro for a short period in response to IL-7 and in the absence of stromal cell contact (matches in vivo “large pre-B cell”)

12. Theme 2: the bone marrow microenvironment • Hypothesis: • pro-B cells fill up a niche of sites bound to the appropriate stromal cells • cells that lose contact, stop dividing and can attempt V(D)J recombination of IgH locus • pre-BCR replaces the stromal signal, so combines with IL-7R to induce burst of pre-B proliferation

13. IL-7 and proliferation of B cell precursors Clark, et al. Nat. Rev. Immunol. 14: 69-80, 2014

14. Pre-BCR and IL-7R: cooperation vs. antagonism? Clark, et al. Nat. Rev. Immunol. 14: 69-80, 2014

15. B-ALL have lost pre-BCR signaling through Blnk Rickert Nat. Rev. Immunol. 13: 578-591, 2013

16. Theme 3: B lineage commitment: control by transcription factors E2A-/- EBF-/- Pax5-/- Knockouts of several transcription factors block B cell development at discrete stages

17. A hierarchy of transcription factors specifies B cell fate

18. Pax5 and commitment to the B cell lineage • E2A and EBF are needed to turn on B cell specific genes including Pax5, which turns on additional B cell-specific genes

19. Culture Pax5-/- bone marrow in vitro to get pro-B cell cultures See if the cells can differentiate into other hematopoietic lineages (add various growth factors) Pax5 and Commitment to B cell lineage Nutt et al. Nature 1999

20. Pax5 and Commitment to B cell lineage Nutt et al. Nature 1999

21. Pax5 and commitment to the B cell lineage • Pax5 seems to act in two ways • It promotes progression down the B cell lineage (expression of Iga, Blnk) • It shuts off genes needed to go down other lineages (M-CSF receptor, pre-Ta, Notch1) or associated with other lineages (myeloperoxidase, perforin, etc.)

22. A networkof transcription factors specifies B cell fate Ikaros PU.1 Repression of genes needed to become myeloid cell Repression of genes needed to become T cell or myeloid cell

23. Theme 4: Tolerance of B cells Method for studying the self-reactivity of human B cells Sorted based on phenotype and/or specificity, etc. Test antibodies for properties, self reactivity, etc.

24. The primary repertoire of B cells includes many self-reactive cells Meffre and Wardemann, COI 20:632, 2008

25. Theme 4: Fate of self-reactive B cells Bone Marrow Periphery Antigen Independent Antigen Dependent Pre BCR IgMμ IgM IgM IgD IgM Plasma Cell pre-B Immature T1 T2 Mature antigen encounter & proliferation autoreactive autoreactive autoreactive deletion or editing deletion or anergy anergy (w/o T cell help) Positive Selection Negative Selection

26. Receptor Editing Mechanisms 1. Upstream V can rearrange to downstream J Vk Jk Ck 2. Upstream V can rearrange to KDE (deleting element) deleting C; this would be followed by a rearrangement of another light chain allele KDE

27. Receptor editing vs. clonal deletion • Contact with antigen in bone marrow leads to maturational arrest (no exit from bone marrow) and receptor editing • Contact with antigen in periphery leads to deletion • Bone marrow stromal cells promote survival to allow editing to occur

28. Clonal deletion vs. clonal anergy • Anti-lysozyme transgenic mice with high affinity antibody: -presence of soluble lysozyme either as transgenic or injected leads to anergy (Goodnow et al.) -membrane-bound form of lysozyme induces deletion • Anti-DNA transgenics (autoantigen of lupus): -mIgwith high affinity for dsDNA results in strong editingand deletion -mIgwith lower affinity leads to anergy(Weigert, Erikson)

29. Characteristics of Anergic B cells • Anergic B cells exhibit chronic low grade BCR signalingand attenuated response to further stimulation • AnergicB cells localize to the edge of the T cell zone next to B cell follicles, same as acutely stimulated naïve B cells. • AnergicB cells have decreased survival in vivo due to decreased ability to respond to the survival factor BAFF (BLyS). • Anergyin the presence of competent helper T cells is enforced by Fas killing. NOTE: B cell anergy is best thought of as a range of phenotypes from deep anergy to light anergy

30. Theme 5: 3 types of mature B cells B1, marginal zone, and follicular B cells

31. Three types of mature B cells • Recirculating follicular B cells (aka “conventional B cells”, B2 cells): circulate between LN follicles and blood: size of population determined by BAFF levels • Marginal zone B cells: reside in marginal zone of spleen where they can respond to particulate antigen in blood (bacteria, etc.); also dependent on BAFF for survival. Also dependent on Notch signaling • B1 B cells: prominent in peritoneal and pleural cavities, present in spleen, absent in lymph node. Produce “natural antibody” and also respond to T-independent antigens. (less dependent on BAFF)

32. Biological roles of three types of B cells follicular MZ + FO MZ + B1 B1

33. BCR signaling and B cell fate (maturation B1) (follicular>MZ?)

34. Changes to rearranged Ig genes during immune response

35. Ig Heavy chain class (isotype) switching

36. Affinity maturation and antibody responses

37. Ig mutations are localized near transcription start site from Longacre and Storb Cell 102: 541, 2000.

38. Activation-induced cytidinedeaminase (AID) • Discovered as an induced gene in a cell line with inducible class-switch recombination (subtractive hybridization) • Transfection into B cell lines induces class switch recombination • AID KO mice have strong defect in class switch recombination AND in somatic hypermutation • Hyper-IgM syndrome type 2 (autosomal) is due to mutation in AID; very similar phenotype to mice (no IgG, IgA, IgE; very much reduced somatic mutation)

39. AID: How does it work? • AID is highly related to APOBEC-1, a cytidinedeaminase that edits mRNA for Apolipoprotein B (via a targeting subunit) and APOBEC-3, which mutates retroviral genomes • indirect action or direct action in class switch and hypermutation? AID could edit mRNAs for factors that act in class switch and factors that act in class switch OR it could act directly in both processes

40. AID as a mutator of DNA • AID is mutagenic in bacteria and mutations are increased by deficiency in Uracil-DNA glycosylase (enzyme that removes U from DNA and triggers DNA repair) • Class switch is inhibited and hypermutation perturbed in UNG-deficient mice • These results favor the hypothesis that AID directly acts on C residues in DNA to promote class switch and hypermutation

41. Model for direct actions of AID in somatic mutation and class switch In hypermutation: U in DNA could lead to direct mutations and secondary mutations via mismatch repair and/or error-prone DNA polymerases In class switch recombination:U in DNA could lead to nick formation by repair enzymes: nicks on both strands-->ds breaks-->recombination