Untitled Flashcards Set

The major aspects in more detail: 1. The transcription factor network that regulates B cell identity B cell development is a paradigm of a terminal cell differentiation process. Hematopoietic stem cells generate multipotent progenitors and subsequently lineage-restricted progenitors that further differentiate into specialised effector cell fates. Differentiation of lymphoid-primed progenitors (LMPPs) involves the loss of megakaryocyte-erythroid lineage potential but retention of myeloid and lymphoid potential. Development of T- and B-cells arises from common lymphoid progenitors (CLPs). To make a B-cell two processes are required: 1) B cell specification refers to the induction of B lineage-specific genes such as Pax5 and is coordinated by activity of the transcription factors EBF1, E2A and FoxO1; 2) B cell commitment concerns the repression of alternative lineage choices, exemplified here by Notch1 which initiates the T-cell lineage programme. The transcription factor Pax5 is the B-cell commitment factor and its knock-out in progenitor Bcells leads to reversion of the B-cell lineage fate (with the appropriate cytokines, these cells can differentiate into macrophages, osteoclasts, dendritic cells, granulocytes and natural killer cells). Working together with Pax5, EBF1 also has a role in B-cell commitment. A coordinated transcriptional network specifies the B-cell lineage program, and this involves the combinatorial action of many transcription factors, some of which are not B-cell specific and yet have a crucial role in determining B-cell fate. - Both E2A and FoxO1 are connected with EBF1 in a positive feedback loop stabilizing its expression and thus contributing to the activation of Pax5 - This integrative, genome-wide study demonstrates the intricate relationship between the different transcriptional regulators in promoting B-cell specification and subsequent commitment 2. Classification of stages of B cell development Once lymphoid progenitors are specified into the B-cell lineage, B-cells follow a developmental pathway that can be classified in two different ways 1. Biological definition is based on immunoglobulin gene rearrangement and expression As a rouge guide, B cells undergoing Ig heavy-chain rearrangement catalysed by Rag endonucleases are called progenitor B cells or pro-B cells; once B cells express IgH on the surface together with a surrogate light-chain (as a pre-BCR) they undergo extensive proliferation and downregulate the expression of Rag endonucleases – these are called large pre-B cells; after a few rounds of division, cells arrest and upregulate Rag expression to undergo Ig light-chain rearrangement, they are termed small pre-B cells; once B-cells express IgH and IgL on the surface as the BCR, they are termed immature B cells and exit the bone-marrow. 2. Technical definition is based on cell surface markers used to define the sequential stages of development experimentally. The most widely used classifications are the Hardy nomenclature and the Melchers nomenclature. The Melchers nomenclature is similar to the biological definition just described, with some more granularity. These surface markers can be analysed by flow cytometry and the different populations sorted to the highest level of purity for subsequent analyses. The example illustrates how the use of just 6 cell surface markers allows isolation of 7 different B-cell populations. 3. Regulation of V(D)J recombination at the immunoglobulin heavy chain locus Pro-B cells initiate recombination at the IgH locus in two steps: first one D and one J gene are randomly chosen to carry out a D-to-J rearrangement and then one V gene is randomly chosen to carry out a V-to-DJ rearrangement. Both steps are catalysed by Rag endonucleases which introduce DNA double-strand breaks in specific sequences that flank V, D and J genes. It is the random process of joining V, D and J genes (together with DNA end processing prior DNA repair) that confers diversity to the primary antibody repertoire. Once VDJ recombination occurs, an mRNA encoding for IgH is expressed and this contains the VDJ exon spliced to the first constant exon encoding for IgM. The VDJ exon encodes for the variable region of the IgH, responsible for antigen recognition. The newly expressed IgH pairs with a surrogate light-chain (SLC) on the membrane as a pre-BCR that, amongst other roles, signals for the shut-down of further IgH recombination thus ensuring that only one allele/one VDJ-encoded antigen specificity is expressed in any given cell – a process known as allelic exclusion V(D)J recombination is regulated in two ways: a. Expression of the Rag proteins, Rag1 and Rag2, is tightly restricted to B and T lymphocytes, and to the developmental stages when Immunoglobulin loci or T-cell receptor loci undergo recombination This doesn’t explain how V(D)J recombination is lineage-specific (Ig loci recombine in B-cells, TCR loci recombine in T-cells), ordered within each loci (as shown for IgH, DJ rearrangement occurs first and then VDJ) and within each lineage (IgH rearranges before IgL), and originating only one functional allele. b. Chromatin accessibility. The Accessibility Hypothesis (proposed by Yancopoulos and Alt in 1985) postulated that dynamic changes in chromatin that permit or deny access to Rag proteins are the basis of lineage and stage-specific regulation of V(D)J recombination. This proposal was based on the observations that non-coding RNA transcripts (also known as germline trascripts) were generated from Ig domains poised for recombination, indicating an open chromatin conformation. Since then, numerous discoveries of diverse chromatin reorganisation mechanisms support this model for all antigen receptor (AgR) loci (i.e. immunoglobulin loci and T-cell receptor loci). Chromatin accessibility to recombination occurs at three different levels: Local, Regional and global Local regulation VH gene recombination efficiency Despite equal opportunity, the analysis of IgH repertoires at the DNA level revealed that recombination frequency of V genes varies enormously, due to differing local chromatin states at the V genes. Actively recombining V genes are characterised by one of two active states, while non-recombining V genes generally reside in a silent chromatin state. Active states are either characterised by the presence of architectural proteins such as CTCF and Rad21 (also known as cohesin) or lineage transcription factors like Pax5 and IRF4. Regional IgH chromatin opening by non-coding/germline transcription The chromatin of the IgH locus is in a closed state in non-B cells. It opens up into an accessible state for first, D to J recombination, followed by V to DJ recombination. At the locus this activation involves several complementary mechanisms including non-coding RNA transcription and active histone modifications. Global changes in IgH accessibility by locus contraction The large V region (encompassing 195 V genes, grouped in 16 families and spanning 2.5 Mb) presents a conformational challenge – how can all the active V genes have equal access to the DJ recombined gene segment to enable diversity in V to DJ rearrangements? Initial DNA FISH experiments, using fluorescent probes to visualise the spatial conformation of the IgH locus in the 3D nuclear space, showed that the locus contracts in pro-B cells when preparing to undergo recombination - exemplified by the red dot (marking the V region) coming into close proximity to the green dot (marking the constant region) More recently, the development of a technique known as 3C (chromosomal conformation capture) allowed us to appreciate how locus contraction is achieved through highly organised and complex chromatin looping that brings V genes into close enough proximity to the DJ recombined gene segment to enable synapsis and V to DJ recombination. This looping is regulated by several transcription factors including Pax5, YY1 and Ikaros and architectural factors including CTCF and cohesin. These protein-DNA interactions can be captured by fixing of chromatin with paraformaldehyde, restriction enzyme digestion and ligation of two DNA fragments in close spatial proximity and downstream analyses using qPCR or high-throughput sequencing. 3C-based methods allow analysis of chromosome conformation in great detail, and we now know that looping generates a 3D rosette-like conformation of V genes around the DJ segment that confers ‘equal opportunity for all’ V genes to participate in V to DJ recombination. Global changes in IgH accessibility by sub-nuclear re-location The IgH loci are sequestered at the repressive heterochromatic nuclear periphery in non-B cells. In pro-B cells, they relocate to the euchromatic centre of the nucleus. Here, the two IgH alleles are sometimes physically brought together by the Rag1 and Rag2 enzyme complex. They first undergo D to J recombination on both Igh alleles. V to DJ recombination occurs asynchronously, first on one allele. If this produces a product in the correct reading frame, it will be transcribed and translated into an IgH polypeptide. Once a functional Igh polypeptide is expressed on the surface of a pre-B cell, it must signal back to the nucleus to silence the second Igh allele to prevent further IgH VDJ recombination, which might generate a second, competing Igh polypeptide. This process is known as allelic exclusion, which purpose is to generate a monoclonal B cell receptor with a unique antigenic specificity. It remains unclear which cytoplasmic signalling pathways are used for allelic exclusion to occur, but the outcome in the nucleus includes i) downregulation of Rag expression, ii) IgH loci decontraction so that the V region is physically removed from the DJ region (this occurs in both loci); iii) loss of non-coding RNA transcription and active histone modifications. The IgH allele that has not undergone V to DJ recombination (remains DJ) is recruited to pericentromeric heterochromatin (PCH) or the nuclear lamina, repressive chromatin compartments. 4. Control of B-cell proliferation versus differentiation V(D)J recombination is inherently imprecise, generating only one third of cells with productive (successful) rearrangements on the first Igh allele. The other two thirds will die, unless they produce a productive Igh on the second allele, which again occurs in 1 in 3 cells. Proliferation is critical for preservation of B cell numbers. As B cells develop, they undergo both basal proliferation, and several rounds of rapid proliferation after each V(D)J recombination event. Proliferation and Ig recombination are tightly regulated and occur at different stages of B-cell development to allow for DNA repair of Rag-induced DNA double-strand breaks, thus ensuring maintenance of genome stability. Proliferation is dependent on a dynamically changing group of ligands and receptors that are characteristic of progressive stages of B cell development. The main players are: The IL-7 cytokine receptor IL7R, the pre-Bcell receptor preBCR and the CXCL12 chemokine receptor CXCR4. Recent studies also support a role for RNA-binding proteins, as B-cell intrinsic regulators of gene expression changes controlling cell proliferation vs differentiation a. Roles of the pre-BCR, the IL7R and CXCR4 IL-7R signalling controls the earliest stages of B-cell development with roles in promoting IgH locus accessibility for recombination, B-cell survival and proliferation. Once a productive IgH rearrangement occurs, a IgH polypeptide is transiently expressed on the cell surface in association with the invariant surrogate light chain (SLC), composed of VpreB and Ig lambda 5, as the preBCR. This marks the first checkpoint in B-cell development. Initially, preBCR signalling cooperates with IL-7R signalling to test for the functionality of the IgH. Together with IL-7R, the pre-BCR promotes cell survival and proliferation leading to downregulation of Rag expression and allelic exclusion; autoreactive B-cells are eliminated, and these are often characterised by long CDR3 sequences, a region formed by the 3’end of V, D and 5’end of J gene segments that is directly involved in antigen recognition. At a later stage, the preBCR signals independently of the IL-7R, by engaging with downstream signalling proteins such as the Burton tyrosine kinase (Btk) and the adaptor protein Blnk (also known as SLP-65). The preBCR is now responsible for inducing B-cell proliferation arrest (through downregulation of IL-7R and SLC expression) and increase IgL accessibility for recombination. Supporting the transition between early and late preBCR signalling is the chemokine receptor CXCR4. The expression of CXCR4 increases as pre-B cells mature and helps diverting B-cells away from IL-7 expressing niches; this supports shut-down of B-cell proliferation (while promoting B-cell survival) and subsequent initiation of IgL recombination. b. A role for RNA-binding proteins? The intricate network of signalling pathways controlling pre-B cell developmental transitions is further supported by rapid changes in gene expression. Recent studies highlight a role for RNAbinding proteins in making this process highly efficient – this is believed to occur through coordination of inter-connected steps in gene expression, a mechanism that’s often referred to as ‘RNA regulons’. The few examples indicated show that RBPs can impact on the expression of IgH germline transcripts, control B-cell proliferation and maintain genome stability. 5. VJ recombination of immunoglobulin light-chain loci The IgL is encoded in two loci – the Ig kappa locus and the Ig lambda locus. Recombination at the Ig kappa locus prevails over the Ig lambda locus and follows the main principles described for the IgH locus, with a few variations. The Ig kappa locus is only composed of V and J gene segments and therefore only one step of Rag-induced recombination is required. However, one important difference is that the Ig kappa locus contains V genes in the opposite orientation, and their recombination leads to the inversion of the intervening region (rather than its deletion). The retention of previously recombined VJ genes can lead to allelic inclusion and this is prevented by deletion of the entire locus, by using a kappa deletion element (KDE) located at the 3’end of the locus. Recombination at the Ig lambda locus is only attempted when Ig kappa recombination has failed; this explains why most B-cells expressing Ig kappa light-chain contain their Ig lambda loci in germline configuration, while B-cells expressing Ig lambda light-chain carry unsuccessfully recombined Ig kappa loci. Similarly to the IgH locus, the Ig kappa locus undergoes chromatin looping mediated by the architectural protein CTCF, thus ensuring usage of both proximal and distal V genes. Studies by us and others have shown that CTCF binds throughout the Ig kappa V region, as well as in the region between V and J gene segments (more specifically the cis regulatory element Sis) and the 3’end of the locus. This sequesters the J-C cluster, containing the Ig kappa enhancers iEk and 3’Ek, in a separate chromatin loop – the configuration needed to orchestrate germline transcription across the V region and upstream of J genes. In the absence of CTCF, Ig kappa enhancers establish increased contacts with proximal V genes and genes outside the Ig kappa locus leading to decreased Ig kappa recombination and a bias towards the usage of proximal V genes. a. Testing BCR specificity and receptor editing Once a IgL polypeptide is expressed, pairing with the IgH polypeptide leads to the expression of a B cell receptor (BCR) in immature B cells. Remarkably, around 50-75% of newly formed BCRs recognise self-antigens, highlighting the need for a second checkpoint at which B-cells are checked for autoreactivity. Downregulation of BCR on the membrane and/or distinct BCR signalling trigger a key process that rescues B-cell clones recognising self-antigens, thus preventing autoimmunity – this process is known as receptor editing. If the BCR is self-reactive, the IgL can undergo several rounds of secondary rearrangement, in which Vk and Jk genes flanking the original VkJk join are recombined. This generates a new IgL, which is then paired with the original IgH. If the autoreactivity is resolved the B cell expressing the edited BCR can then progress to become a mature B cell in the spleen. 6. Mechanisms of antibody diversification in mature B-cells Immature B cells migrate from the bone marrow to the spleen, undergoing transitional stages of development, before finally becoming either a mature follicular B cell (FO) (the vast majority) or a marginal zone B cell (MZ). It is believed that MZ B-cells originate from transitional T2 B-cells and FO B-cells may or may not require transition through the T3 stage of development. In fact, T3 Bcells are currently thought to be a subset of splenic B-cells that are locked in a state of known as ‘anergy’, characterised by an overall lack of response to antigenic stimulation. MZ B-cells are non-circulating and are strategically located between the circulation and the white pulp of the spleen. They have an important role in providing a first line of defence against bloodborne pathogens by rapidly producing antibodies. FO B-cells locate in the follicles of secondary lymphoid organs such as the spleen, in close proximity to T-cells, and can circulate throughout the body via the bloodstream and lymphatics. Upon antigen encounter, mature B-cells undergo extensive proliferation and initiate further antibody gene diversification by two distinct but related mechanisms of DNA mutation and recombination. Somatic hypermutation – catalysed by the DNA mutation enzyme AID, involves somatic mutation of the V(D)J exon in Ig heavy and light chain loci. Together with B-cell selection mechanisms occurring in the germinal centre, SHM is required to increase antibody affinity towards antigen. Class Switch Recombination – also catalysed by AID but involving the formation of DNA doublestrand breaks downstream of DNA mutation. CSR is a DNA deletion mechanism that replaces the default IgM-coding, constant exon in the Ig heavy-chain locus for any downstream constant exons (encoding other antibody isotypes – IgG1, IgG2, IgG3, IgE, IgA). This allows B-cells to further tailor their antibody response to the pathogen at hand, with different antibody isotypes controlling antibody location in the body as well as their effector functions (e.g. pathogen neutralization, opsonization, complement or immune cell activation). Recent studies show that CSR occurs infrequently in germinal centres (the prevailing view found in many textbooks) and it is a much earlier process occurring the first 2-3 days after initial antigen encounter and B-cell activation. CSR relies on the targeting of AID to G-rich and repetitive regions located upstream of each recombining constant IgH exon – known as switch regions. Non-coding transcription running through the switch region (and initiating from a promoter located upstream of a non-coding, intervening (I) exon) are known to hybridise to the template DNA due to its G-richness and repetitive nature. Formation of these RNA:DNA hybrid (also called R-loop) structures are wellknown to be required for AID targeting during CSR. More recently, it was shown that non-coding switch transcripts (also called germline transcripts, GLTs) can form another type of nucleic acid structures known as G-quadruplexes. Mutations in the AID protein domain responsible for binding to G-quadruplex structures are found in hyper-IgM syndrome patients (see below). AID binding to G-quadruplexes is believed to also target AID to the IgH locus, a mechanism requiring the RNA unwinding activity of the DEAD-box RNA helicase DDX1. 7. Diseases and dysfunction associated with defective B cell development Immunodeficiency is caused by defects in a wide variety of processes and involve mutations in cytokine receptors, signalling molecules, transcription factors and enzymes inherent to B cell development and Ig gene rearrangement. Some of these defects can cause severe loss of one or more immune cell types and result in severe combined immune deficiencies (SCID). Other are more specific to the B-cell lineage as it is the case for agammaglobulinemias, in large part caused by defects on the signalling molecule Bruton Tyrosine Kinase (Btk). Defects on molecules involved in T:B-cell interactions and BCR signalling lead to defects in SHM and CSR, resulting in decreased levels of IgG production upon infection – these are known as hyper-IgM syndromes. Ig gene diversification mechanisms do pose a risk for B-cell genome stability and can result in characteristic chromosomal translocations characterising B-cell leukemias and lymphomas.