4 Themes in B cell development + Ig class switch and somatic mutation Tony DeFranco, 10/28/13 Checkpoints in B cell development: feedback from Ig

Transcription

1 4 Themes in B cell development + Ig class switch and somatic mutation Tony DeFranco, 10/28/13 Checkpoints in B cell development: feedback from Ig gene rearrangements Lineage commitment: transcription factors and microenvironment Central and peripheral tolerance of B cells 3 different types of mature B cells: choice of cell fate and relative roles Molecular mechanisms of class switch recombination and somatic hypermutation: Activation-induced cytidine deaminase (AID)

2 Overview of B cell development

3 Theme 1: Ig rearrangement checkpoints for B cell development IgH unrearr DJ VDJ VDJ VDJ IgL unrearr unrearr unrearr rearranging VJ (surrogate L chain)

4 B cell development:distinctive cell surface markers (mouse) µ chain expressed in cytoplasm B CD43 (S7) CD c-kit Human B cell precursors: see Blom & Spits 2006

5 Pro-B to pre-b checkpoint requires Pre-BCR signaling Pre-B cell transition is blocked by: Deletion of J H or µm exon Knockout of Rag1, Rag2 or Scid (DNA-PKcs) Knockout of signaling components: Igα, Igβ, Syk, triple Src family KO (lyn-fyn-blk-), Btk (human, X- linked agammaglobulinemia), or Blnk (adapter molecule) Conclusion: Need pre-bcr and signaling from pre-bcr

6 Pre-BCR checkpoint regulates V(D)J recombination Cell proliferation (IL-7-dependent) Change in cell surface markers Turn off expression of SLCs Change in targeting of Rag1/2

7 Theme 2: B lineage commitment: the bone marrow microenvironment The Bone marrow microenvironment has several key properties: Pro-B cells (µ-) grow indefinitely in vitro only in contact with stromal cell layer from bone marrow Pre-B cells (µ+) will grow in vitro for a short period in response to IL-7 and in the absence of stromal cell contact Hypothesis: pro-b cells fill up a niche of sites bound to the appropriate stromal cells and only if express the pre-bcr can they proliferate further and proceed down developmental pathway In addition: this microenvironment does not express Notch ligands (in contrast to the thymus), which aid in choice of commitment to the T cell lineage

8 Theme 2: B lineage commitment: control by transcription factors E2A-/- EBF-/- Pax5-/- Knockouts of several transcription factors block B cell development at discrete stages

9 A hierarchy of transcription factors specifies B cell fate

10 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

11 Pax5 and Commitment to B cell lineage 1. Culture Pax5 -/- bone marrow in vitro to get pro-b cell cultures 2. See if the cells can differentiate into other hematopoietic lineages (add various growth factors) Nutt et al. Nature 1999

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

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

14 Ikaros" Repression of genes needed to become myeloid cell" Repression of genes needed to become T cell or myeloid cell"

15 Putting it all together: growth factors + transcription factors in B cell development Harinder Singh et al Solid lines: more solid data Dashed lines: less well established

16 Theme 3: 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.

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

18 Theme 3: Fate of self-reactive B cells Bone Marrow! Periphery! Antigen! Independent! Pre BCR IgM µ Antigen Dependent! IgM IgM IgD IgM Plasma Cell pre-b Immature T1 T2 Mature autoreactive!! autoreactive!! autoreactive! antigen encounter &! proliferation! deletion! or editing! deletion! or anergy! anergy! (w/o T cell help)! Negative Selection Positive Selection

19 Receptor Editing Mechanisms 1. Upstream Vκ can rearrange to downstream Jκ Vκ Jκ Cκ 2. Upstream Vκ can rearrange to KDE (κ deleting element) deleting Cκ; this would be followed by a rearrangement of another light chain allele KDE

20 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 The difference appears to be that bone marrow stromal cells promote survival to allow editing to occur. If apoptosis is blocked with caspase inhibitors, editing can occur in vitro. Also in vitro culture of immature B cells + bone marrow stromal cells can allow editing

21 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.) In contrast, membrane-bound form of lysozyme induces deletion Anti-DNA transgenics (autoantigen of lupus): mig with high affinity for dsdna results in strong editing and deletion but mig with lower affinity leads to anergy (Weigert, Erikson)

22 Characteristics of Anergic B cells Anergic B cells exhibit chronic low grade BCR signaling, further stimulation of BCR gives weak response (contrast to acute stimulation of BCR of naïve B cells). Attenuation of the PI-3kinase-Akt signaling pathway seems important Anergic B cells localize to the edge of the T cell zone next to B cell follicles, same as acutely stimulated naïve B cells. Anergic B cells have decreased survival in vivo due to decreased ability to respond to the survival factor BAFF (BLyS). Anergy in the presence of competent helper T cells is enforced by Fas killing. Autoantibody production in MRL/lpr mice (Fasdeficient) results at least partly from defect here. NOTE: B cell anergy is best thought of as a range of phenotypes from deep anergy to light anergy

23 Mechanisms of B cell tolerance (summary) Immature B cells in bone marrow are very sensitive to antigen: receptor editing and deletion Immature B cells in periphery ( transitional B cells ) can be deleted by antigen (strong signal) Immature or mature B cells in periphery + moderate BCR signal move to T cell zone; in absence of T cell help they become anergic. Anergic B cells can receive T cell help normally, but are killed by Fas unless the BCR can signal adequately to protect (anergic B cells have weak BCR signaling induced by the anergizing form of the antigen)

24 Theme 4: 3 types of mature B cells B1, marginal zone, and follicular B cells

25 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)

26 Marginal zone of the spleen The spleen filters the blood, marginal zone B cells are exposed to particles in the blood

27

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

29 SM610 transgenic B cells develop and produce autoantibody only in animals expressing Thy1: suggests positive selection by antigen

30 BCR signaling and B cell fate (maturation B1) (follicular>mz?)

31 Ig Heavy chain class (isotype) switching VDJ µ δ γ3 γ1 γ2b γ2a ε α 55 kb T cell help (IL-4) antigen IgG1+ memory cell IgM/D+ naive B cell IgG1 secreting plasma cell

32 Affinity maturation and antibody responses"

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

34 Comparison of VDJ recombination, class switch recombination and somatic hypermutation Process Type of change Recognition sequence Mechanism Factors involved VDJ recomb. recomb. + mutation heptamer + nonamer dsdna breaks RAG1 RAG2 Class switch recomb. S regions (repetitive) dsdna breaks Hypermutation mutation None (enhancer directs) dsdna ssdna breaks? nicks? AID

35 Activation-induced cytidine deaminase (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)

36 AID: How does it work? AID is highly related to APOBEC-1, a cytidine deaminase that edits mrna for Apolipoprotein B (via a targeting subunit) 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

37 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

38 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