B Cell Development PDF

B cell development Dr Allison Imrie Learning Outcomes At the end of this lecture you should understand: The main stages of B cell development and the anatomical sites where these events occur The structure and function of the B cell receptor The mechanisms whereby B cell receptor diversity is genera...

B cell development Dr Allison Imrie Learning Outcomes At the end of this lecture you should understand: The main stages of B cell development and the anatomical sites where these events occur The structure and function of the B cell receptor The mechanisms whereby B cell receptor diversity is generated – V(D)J recombination; class switching; affinity maturation - and the major enzymes that are involved The major features of central and peripheral tolerance The similarities and differences among B-1 and B-2 B cell lineages – where they arise, how they are activated, and their anatomical distribution How B cells encounter antigen and the anatomical sites where these interactions occur Diseases that may arise as a consequence of mutations in genes that encode proteins involved in B cell development pathways Pluripotent stem cells give rise to distinct B and T lineages. Hematopoietic stem cells (HSCs) give rise to distinct progenitors for various types of blood cells. One of these progenitor populations is the common lymphoid progenitor (CLP). CLPs give rise mainly to B and T cells but may also contribute to NK cells and some dendritic cells (not depicted here). CLP Pro-B cells can eventually differentiate into follicular (FO) B cells, marginal zone (MZ) B cells, and B-1 cells. Commitment to different lineages is driven by various transcription factors (indicated in italics). EBF, E2A and Pax-5 induce expression of genes required for B cell development. These include genes encoding RAG-1 and RAG-2 (recombination-activating gene) proteins and the proteins that contribute to signalling through the pre-B cell receptor and the B cell receptor, Igα and Igβ. Phases of B lymphocyte life history Stages in B (and T) cell development are defined mainly by successive steps in assembly and expression of functional antigen receptor genes Stages of lymphocyte maturation * * * B (and T) cell maturation involves the sequence of changes shown here B cell antigen receptor complex Membrane IgM (and IgD) on the surface of mature B cells is associated with the invariant Igβ and Igα molecules, which contain ITAMs in their cytoplasmic tails that mediate signalling functions. Note the similarity to the TCR complex. B cell receptor complexes in class-switched B cells, including memory B cells, contain membrane immunoglobulins that may be of the IgG, IgA, or IgE classes. Signal initiation by antigens occurs by cross-linking of the BCR and is facilitated by the coreceptors for the BCR, CD21 and C’ proteins Antigen receptor gene rearrangement and expression The rearrangement of antigen receptor genes is the key event in lymphocyte development that is responsible for generation of a diverse repertoire Each lymphocyte clone produces an antigen receptor with a unique antigen-binding structure. In any individual there may be > 107 unique B and T cell clones Functional antigen receptor genes are produced in immature B cells in the bone marrow by a process of gene rearrangement, which is able to generate a large number of variable regionencoding exons using a relatively small fraction of the genome In any given developing B cell, one of many variable region gene segments is randomly selected and joined to a downstream DNA segment. The DNA rearrangement events that lead to production of antigen receptors are not dependent on or influenced by the presence of antigens Clonal selection hypothesis Germline organization of human Ig loci Three separate loci encode, respectively, all of the Ig heavy chains, the Ig κ light chain, and the Ig λ light chain. Each locus is on a different chromosome D (diversity) segments are not found in Ig light chain loci Note variable number of C genes at each Ig locus The germline organization of Ig genetic loci is characterized by spatial segregation of many different sequences that encode variable domains, and relatively few sequences that encode constant domains of receptor proteins; distinct variable region sequences are joined to constant region sequences in different lymphocytes Human Ig protein domains The domains of Ig heavy and light chains are shown. The V and C regions of each polypeptide are encoded by different gene segments. Areas in the dashed boxes are the hypervariable (complementarity-determining) regions. Diversity of antigen receptor genes From the same germline DNA, it is possible to generate recombined DNA sequences and mRNAs that differ in their V-D-J junctions. In the example shown, three distinct antigen receptor mRNAs are produced from the same germline DNA by the use of different gene segments and the addition of nucleotides to the junctions. RSS V(D)J recombination Lymphocyte-specific proteins that mediate V(D)J recombination recognize certain DNA sequences called recombination signal sequences (RSSs), located 3′ of each V gene segment, 5′ of each J segment, and flanking each side of every D segment The RSSs consist of a highly conserved stretch of 7 nucleotides, called the heptamer, located adjacent to the coding sequence, followed by a spacer, followed by the nonamer During V(D)J recombination, double-stranded breaks are generated between the heptamer of the RSS and the adjacent V, D, or J coding sequence The intervening double-stranded DNA, containing signal ends (the ends that contain the heptamer and the rest of the RSS), is removed in the form of a circle, and the V and J coding ends are joined In some V genes, especially in the Ig κ locus, the RSSs are 3’ of a Vκ and 3’ of Jκ, and therefore do not face each other. In these cases, the intervening DNA is inverted and the V and J segments are properly aligned Most Ig and TCR gene rearrangements occur by deletion; inversion is the basis of up to 50% of rearrangements in the Ig κ locus V(D)J recombination Double-stranded breaks are enzymatically generated at RSScoding sequence junctions by recombination-activating gene 1 and recombination-activating gene 2 (RAG1 and RAG2) proteins; the Rag-1/Rag-2 complex is also known as the V(D)J recombinase The V(D)J recombinase recognizes the DNA sequence at the junction between a heptamer and a coding segment and cleaves it; makes a nick on one DNA strand; forms a covalent hairpin; generates a blunt end; holds the hairpin ends and blunt ends together before ligation A lymphoid-specific enzyme, terminal deoxynucleotidyl transferase (TdT), adds bases to broken DNA ends to generate junctional diversity. Junctional diversity. Addition of nucleotides at the junctions of the V and D, D and J, or V and J segments at the time these segments are joined, greatly increases diversity New nucleotide sequences, not present in the germline, may be added at junctions Coding segments (e.g., V and J gene segments) that are cleaved by Rag-1 form hairpin loops whose ends are often cleaved asymmetrically by the enzyme Artemis so that one DNA strand is longer than the other. The shorter strand has to be extended with nucleotides complementary to the longer strand before the ligation of the two segments. The longer strand serves as a template for the addition of short lengths of nucleotides called P nucleotides, and this process introduces new sequences at the VD-J junctions Another mechanism of junctional diversity is the random addition of up to 20 non–template-encoded nucleotides called N nucleotides. This addition of new nucleotides is mediated by terminal deoxynucleotidyl transferase (TdT) pre-B cell Complexes of μ heavy chain, surrogate light chains, and signaltransducing proteins called Igα and Igβ form the pre-antigen receptor of the B lineage, known as the pre-B cell receptor (pre-BCR). The μ heavy chain associates with surrogate light chains which are are invariant (i.e., they are identical in all pre-B cells) and are synthesized only in pro-B and pre-B cells Expression of the pre-BCR is the first checkpoint in B cell maturation Bruton’s tyrosine kinase (Btk) is activated downstream of the pre-BCR and is required for delivery of signals from this receptor that mediate survival, proliferation, and maturation at and beyond the pre-B cell stage In humans, mutations in the BTK gene result in the disease called X-linked agammaglobulinemia (XLA), which is characterized by a failure of B cell maturation The pre-B cell receptor The pre-BCR regulates further rearrangement of Ig genes in two ways. if a μ protein is produced from the recombined heavy chain locus on one chromosome and forms a pre-BCR, this receptor signals to irreversibly inhibit rearrangement of the Ig heavy chain locus on the other chromosome An individual B cell can express an Ig heavy chain protein encoded by only one of the two inherited alleles. This phenomenon is called allelic exclusion, and it ensures that every B cell will express a single receptor, thus maintaining clonal specificity If the first rearrangement is non-productive, the heavy chain allele on the other chromosome can complete VDJ rearrangement at the Ig H locus. Thus, in any B cell clone, one heavy chain allele is productively rearranged and expressed, and the other is either retained in the germline configuration or non-productively rearranged. Following the pre-B cell stage, each developing B cell initially rearranges a κ light chain gene. There are no D segments in the light chain loci, and therefore recombination involves only the joining of one V segment to one J segment, forming a VJ exon The assembled IgM molecules are expressed on the cell surface in association with Igα and Igβ, where they function as specific receptors for antigens The IgM-expressing B cell is called an immature B cell Immature B cells do not proliferate and differentiate in response to antigens If they recognize antigens in the bone marrow with high avidity, which may occur if the B cells express receptors for multivalent self antigens that are present in the bone marrow, the B cells may undergo receptor editing or cell death Central B cell Tolerance Immature B lymphocytes that recognize self antigens in the bone marrow with high affinity either change their specificity or are deleted. Receptor editing If immature B cells recognize self antigens that are present at high concentration in the bone marrow and especially if the antigen is displayed in multivalent form (e.g., on cell surfaces), many antigen receptors on each B cell are cross-linked, thus delivering strong signals to the cells The B cells reactivate their RAG1 and RAG2 genes and initiate a new round of VJ recombination in the immunoglobulin (Ig) κ light chain gene locus. A new Ig light chain is expressed, thus creating a B cell receptor with a new specificity Deletion. If editing fails, the immature B cells may die by apoptosis. Central B cell Tolerance Anergy. If developing B cells recognize self antigens weakly (e.g., if the antigen is soluble and does not cross-link many antigen receptors or if the B cell receptors recognize the antigen with low affinity), the cells become functionally unresponsive (anergic) and exit the bone marrow in this unresponsive state Anergy is due to downregulation of antigen receptor expression as well as a block in antigen receptor signaling. If the new receptor is no longer self-reactive, the immature B cell migrates to the periphery and matures Peripheral B cell Tolerance Mature B lymphocytes that recognize self antigens in peripheral tissues in the absence of specific helper T cells may be rendered functionally unresponsive or die by apoptosis Anergy and deletion B cells that have encountered self antigens have a shortened life span and are eliminated more rapidly in lymphoid follicles than cells that have not recognized self antigens The high rate of somatic mutation of Ig genes that occurs in germinal centres has the risk of generating self-reactive B cells. These B cells may be actively eliminated by the interaction of FasL on helper T cells with Fas on the activated B cells Signaling by inhibitory receptors Inhibitory receptors set a threshold for B cell activation, which allows responses to foreign antigens with T cell help but does not allow responses to self antigens – eg. CD22 inhibitory receptor B lymphocyte subsets Immature B cells that are not strongly self-reactive leave the bone marrow and complete their maturation in the spleen before migrating to other peripheral lymphoid organs. Mature B cells cells are IgM+IgD+ Most immature B cells leaving the bone marrow will not survive to become fully mature B cells - >50% die every 3 days Selection occurs at the lymphoid follicle – limited number of cells can enter A. Most B cells that develop from fetal liver-derived stem cells differentiate into the B-1 lineage. They are found in peritoneal and pleural cavity fluid B. B lymphocytes that arise from bone marrow precursors after birth give rise to the B-2 lineage. Two major subsets are derived from B-2 B cell precursors: (1) follicular B cells are recirculating lymphocytes (2) marginal zone B cells are found in in spleen and lymph nodes Distinct subsets of B cells respond preferentially to different types of antigens B-2 cells Follicular recirculating B cells populate follicles in the spleen and lymph nodes Static marginal-zone (MZ) B cells are enriched in the MZ of the spleen B1 cells recirculate between the blood and the body cavities Antigen may be delivered to naive B cells in lymphoid organs in different forms and by multiple routes Antigen that is presented to B cells is generally in its intact, native conformation and is not processed by antigenpresenting cells Most antigens from tissue sites are transported to lymph nodes by afferent lymphatic vessels that drain into the subcapsular sinus of the nodes Subcapsular sinus macrophages capture large microbes and antigen-antibody complexes and deliver these to follicles, which lie under the sinus Large antigens may be captured in the medullary region by resident dendritic cells and transported into follicles, where they can activate B cells Antigens in immune complexes may bind to complement receptor CR2 on marginal zone B cells, and these cells can transfer the immune complex–containing antigens to follicular B cells. Blood-borne pathogens may be captured by plasmacytoid dendritic cells in the blood and transported to the spleen, where they may be delivered to marginal zone B cells. Protein antigens are recognized by specific B and T lymphocytes in peripheral lymphoid organs, and the activated cell populations come together in these organs to initiate humoral immune responses Extrafollicular plasma cells are short-lived (~3 days) Follicular/Germinal Centre plasma cells are long-lived, migrate to bone marrow, MALT; memory cells Naive CD4+ T cells are activated in the T cell zones by antigen processed and presented by dendritic cells, and differentiate into helper T cells. Naive B cells are activated in the follicles by the same antigen (in its native conformation) transported there. The helper T cells and activated B cells migrate toward one another and interact at the edges of the follicles, where the initial antibody response develops. Some of the cells migrate back into follicles to form germinal centres, where the more specialized antibody responses are induced. On activation, helper T cells express CD40 ligand (CD40L), which engages its receptor, CD40, on antigenstimulated B cells and induces B cell proliferation and differentiation, initially in extrafollicular foci and later in germinal centers Mutations in the CD40L gene result in a disease called the X-linked hyper-IgM syndrome, which is characterized by defects in antibody production, isotype switching, affinity maturation, and memory B cell generation in response to protein antigens, as well as deficient cell-mediated immunity The Germinal Centre Reaction The characteristic events of helper T cell–dependent antibody responses, including affinity maturation, isotype switching, and generation of long-lived plasma cells and memory B cells, occur primarily in organized structures called germinal centres that are created within lymphoid follicles during T-dependent immune responses. FDCs are found only in lymphoid follicles and express complement receptors (CR1, CR2, and CR3) and Fc receptors. These molecules are involved in displaying antigens for the selection of germinal centre B cells. FDCs do not express class II MHC molecules Ig heavy chain isotype switching In T-dependent responses, some of the progeny of activated IgM- and IgD-expressing B cells undergo heavy chain isotype (class) switching and produce antibodies with heavy chains of different classes, such as γ, α, and ε The Ig heavy chain DNA in B cells is cut and recombined such that a previously formed VDJ exon that encodes the V domain is placed adjacent to a downstream C region, and the intervening DNA is deleted Activation-induced cytidine deaminase (AID) is the initiator of mutations in somatic hypermutation and class switching Affinity maturation leads to increased affinity of antibodies for a particular antigen as a T-dependent humoral response progresses, and it is the result of somatic hypermutation of Ig genes followed by selective survival of the B cells producing the antibodies with the highest affinities Somatic hypermutation is the phenomenon in which a high frequency of point mutations are generated within a 1–2-kb segment in the variable region of expressed immunoglobulin genes in response to the presence of an antigen. These processes occur in the germinal centres of secondary lymphoid organs B cell development occurs in distinct regions Molecular events: Cellular events: Antigenindependent phase (bone marrow, foetal liver) Antigendependent phase (spleen, lymph node) V(D)J rearrangement Class switch, somatic hypermutation proB > preB > mature B cell B cell activation, memory and plasma B cell differentiation