B cell development Tony DeFranco, 10/29/14

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 cytidine deaminase (AID)

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

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

Overview of B cell development

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

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

Pro-B to pre-B checkpoint requires Pre-BCR signaling Surrogate light chain (VpreB + 5): Expressed only in proB/preB cells Triggers signaling (self aggregation?Ligand present on stromal cells?) Expression turned off by pre-BCR signaling

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

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)

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”)

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

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

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

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

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

A hierarchy of transcription factors specifies B cell fate

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 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

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

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 Ig , Blnk) –It shuts off genes needed to go down other lineages (M-CSF receptor, pre-T , Notch1) or associated with other lineages (myeloperoxidase, perforin, etc.)

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

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

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

pre-B Mature Plasma Cell IgM μ IgM Antigen Independent Antigen Dependent Immature PeripheryBone Marrow IgM IgD T2T1 autoreactive deletion or editing autoreactive deletion or anergy autoreactive anergy (w/o T cell help) Negative Selection Positive Selection Pre BCR antigen encounter & proliferation Theme 4: Fate of self-reactive B cells

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

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

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): -mIg with high affinity for dsDNA results in strong editing and deletion -mIg with lower affinity leads to anergy (Weigert, Erikson)

Characteristics of Anergic B cells Anergic B cells exhibit chronic low grade BCR signaling and attenuated response to further stimulation 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. NOTE: B cell anergy is best thought of as a range of phenotypes from deep anergy to light anergy

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

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)

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

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

Changes to rearranged Ig genes during immune response

Ig Heavy chain class (isotype) switching

Affinity maturation and antibody responses

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

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)

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) 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

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

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 Model for direct actions of AID in somatic mutation and class switch