7 B Lymphocyte Development.pdf

B Lymphocyte Development ROSS UNIVERSITY SCHOOL OF MEDICINE Raymond F Adebiyi Professor Immunology and Medical Microbiology Learning Objectives 1. Describe the basic functions of B cells. 2. Name the main stages of B cell development. Discuss the major events that characterize each stage of B ce...

B Lymphocyte Development ROSS UNIVERSITY SCHOOL OF MEDICINE Raymond F Adebiyi Professor Immunology and Medical Microbiology Learning Objectives 1. Describe the basic functions of B cells. 2. Name the main stages of B cell development. Discuss the major events that characterize each stage of B cell development. At what stages do the heavy and light chains undergo rearrangement? Discuss the roles of RAG-1, RAG2, and TdT in B cell development. 3. Explain the why allelic exclusion is an important part of B cell rearrangement. 4. Describe central and peripheral tolerance in B cells. What do these processes achieve? 5. Compare and contrast immature B cells, mature B cells, naïve B cells, virgin-B cells, B-1 cells, B-2 cells, activated B cells, plasma cells, and memory B cells. 6. Name the component parts of the B cell receptor complex and list the functions of each component. 7. Describe the process of B cell activation and differentiation. Name the cell-surface interactions and cytokines necessary to drive both B cell activation and differentiation. 8. Describe the outcomes of somatic hypermutation, affinity maturation and isotype switching. What kinds of help do B cells require to undergo these processes? Distinguish between somatic recombination and somatic hypermutation events. 9. Describe how a B cell response to a T-independent antigen differs from the response to a T-dependent antigen. 10. List the surface markers found on B cells and their functions. Recommended Readings: Lauren Sompayrac, 2019, How the Immune System Works, 6th Edition, Wiley-Blackwell, Lecture 3 and Lecture 7 Peter Parham, 2015, The Immune System, 4th Edition, Garland Science, pp 149-173 Any Questions? [email protected] Office Hours https://atge.webex.com/meet/radebiyi Acquired Immune System Development B cell plays critical role in adaptive immune response. Recognize and respond to antigen. Secrete antibodies. Present antigen to T cells. Antibodies – General Structure 5 Different Classes (Isotypes) of Antibodies • Class of antibody determines o Effector function o Tissue distribution Immunoglobulin Genes Adaptive diversity is achieved by gene rearrangements. Mediated by RAG-1 and RAG-2 From “How the Immune System Works” by L. Sompayrac , 2012 p.5 Mutations of the RAG1 and/or RAG2 result in severe combined immune deficiency. Omenn Syndrome with a complete lack of circulating T and B cells due to an early block in lymphoid development. ROSS UNIVERSITY SCHOOL OF MEDICINE | 12 ROSS UNIVERSITY SCHOOL OF MEDICINE B Cell Development Pro-B Cell to Pre-B Cell • If functional heavy chain expressed o Complexes with surrogate light chain to form pre-B receptor. • Surrogate light chain VpreB and 5 o If functional, then progression to Pre-B cell. Elimination of Self-Reactive Clones - Central Tolerance • Negative selection o Heavy and light chain rearrangements are random. o Self-reactive BCRs are often generated. o Self-reactive BCRs (recognize ubiquitous self-antigen) are deleted or functionally inactivated. Elimination of Self-Reactive Clones -Tolerance Induction- Elimination of Self-Reactive Clones • Receptor editing o Immature B cells with self-reactive B cell receptors can attempt to re-arrange light chain. o Some B cells exhaust their attempts to make a BCR that is not self-reactive and get signaled to die by apoptosis (clonal deletion). From Immature to Mature B cell • Immature B cells exit the bone marrow?? • In secondary lymphoid tissue, immature B cells receive signals in form of cytokines to survive and mature. • Exit secondary lymphoid tissue (via efferent lymph) as mature B cells. Mature B cell • Expresses both IgM and IgD. • Considered “naïve” or a “virgin” B cell until it encounters its specific antigen. Receptor Editing of Self-Reactive Clones Peripheral Tolerance • Not all self-antigens are expressed in bone marrow. • When self-reactive B cell recognizes self-antigen in periphery. o Cell dies by apoptosis, or o Cell becomes anergic (incapable of future activation). The B Cell Receptor Complex • B cell receptor (antibody molecule) o Surface bound o Complexes with • Ig and Ig which mediate intracellular signaling • CD19, CD20, CD21, CD81 B Cell Subsets B Cell Subsets The BCR and co-receptor cooperate in B cell activation. • CD19 is the signaling chain of the complex. • CD21 (AKA Complement Receptor 2) recognizes C3d fragments deposited on pathogens. • CD81– forms signaling complex with CD19, as well as with other surface molecules. Details of Post-Activation Events Discussed Later Activated B Cells Undergo Differentiation Plasma Cells • Terminally differentiated antibody synthesizing and secreting machines. • IgM is no longer surfacebound, but secreted. • No longer can respond to antigen. o Lose surface IgM (BCR). o Stop MHC class II expression. o Express CD27. Activated B cells undergo differentiation. Memory B Cells • Survive for long periods. • Allow for more rapid and robust secondary antibody response. • Express CD27. Memory cells Activated B cell Differentiation MDBS 1102 B-cell Biology Dr. Raymond F Adebiyi B-Cell Biology LEARNING OBJECTIVES 1. Describe the basic functions of B cells. 2. Describe the stages of B cell development. Discuss the major events that characterize each stage of Bcell development. Understand the stages at which the heavy and light chains undergo rearrangement and discuss the roles of RAG-1, RAG-2 and TdT in B-cell development. 3. Explain the principle of allelic exclusion and the consequence of failure of allelic exclusion. 4. Describe central and peripheral tolerance B-cells and explain the consequence of failure to achieve tolerance. 5. Compare and contrast immature B-cells, mature B-cells, naïve B-cells, activated B-cells, plasma cells and memory B-cells. 6. Discuss the components of the B-cell receptor complex and the functions of each component. 7. Describe the process of B-cell activation and differentiation and the cell-surface interactions and cytokines that are necessary to drive the processes. 8. Discuss the processes and outcomes of somatic hypermutation, affinity maturation and isotype switching. Discuss the role that helper T cells, cytokines and activation-induced cytidine deaminase AID, play in B cell activation, differentiation and function. 9. Describe how a B cell response to a T-independent antigen differs from the response to a T-dependent antigen. 10. List the surface markers found on B-cells and their functions. Recommended Readings: Peter Parham, 2015, The Immune System, 4th Edition, Garland Science, pp 149-173 Lauren Sompayrac, 2019, How the Immune System Works, 6th Edition, Wiley-Blackwell, Lectures 3 and 7 1 MDBS 1102 B-cell Biology Dr. Raymond F Adebiyi B Cell Biology B cells play an essential role in the immune response. These lymphocytes take part in the adaptive immune response against foreign antigens. Their primary role is to synthesize antibodies against antigens but can also serve as antigen presenting cells. By developing into memory B cells, they provide the host with rapid antibody-mediated immunity against previously encountered antigens. The mature B-cell expresses an antigen receptor, the B cell receptor (BCR), which takes the form of surfacebound immunoglobulin (Ig) (for naïve [antigen-inexperienced], mature B cells, IgM and IgD). Surface Ig contains functional rearrangements of the heavy and light chain genes. Thus, each B-cell that exits the bone marrow is committed to a specific antigen and can be activated by that antigen only. B-cell precursors are derived from bone marrow stem cells. Through antigen independent differentiation in the bone marrow, these cells acquire functional competence. These committed cells may then proliferate through antigen dependent activation to form clones of plasma cells for each class of antibody. B-cells possess a vast capacity for diverse development to generate cells that are responsive to the multitude of antigens to which the body could be exposed. B cell development occurs in 4 phases: 2 MDBS 1102 B-cell Biology Dr. Raymond F Adebiyi GENERATION OF B CELLS Stem cells commit to the B-lineage in response to cytokines released by bone marrow stromal cells. These early stages occur in the absence of antigen. The B cell differentiation pathway can be subdivided into stages; these stages are defined by the rearrangement status of the immunoglobulin genes and by the expression of specific molecules on the cell surface. The stages are as follows: Early Pro-B cells are the earliest committed B-lineage cells. These cells can be recognized by the expression of surface makers that characterize the B cell lineage, namely CD19. The main event in this stage is the rearrangement of the heavy chain immunoglobulin genes. In early Pro-B cells, the DH and JH gene segments are joined, followed by the late Pro-B cell stage characterized by the joining of the VH segment to the rearranged DJH segment (see below for some details on immunoglobulin gene rearrangements). Rearrangement of the immunoglobulin heavy chain genes requires the expression of the recombinationactivating genes RAG-1 and RAG-2 and terminal deoxynucleotidyl transferase (TdT). If a productive gene rearrangement occurs, then the cell can express a functional heavy chain. Pro-B cells that fail to produce a functional heavy chain die via apoptosis in the bone marrow. Fortunately, because there are two copies of the heavy chain immunoglobulin genes, a cell has 2 attempts to make a functional heavy chain. The heavy chain must demonstrate that it can combine with an immunoglobulin light chain. As the light chain genes have not yet been rearranged, Pro-B cells express a surrogate light chain formed by two proteins, VpreB and 5, that mimic the light chain. The heavy chain, surrogate light chain, Ig and Igβ (transmembrane proteins that associate with the BCR and mediate signal transduction, see membrane-bound IgM figure) form a complex called the pre-B cell receptor that resembles the BCR. The cell then receives signals through the pre-B cell receptor to become a pre-B cell. If a functional pre-B cell receptor complex does not form, the cell dies by apoptosis. Successful assembly of the pre-B cell receptor also signals for transcription of the RAG genes to stop, thus preventing rearrangement of the second immunoglobulin heavy-chain locus. This phenomenon is termed allelic exclusion and is the way the B cell ensures that it only expresses one of its two copies of the heavy chain genes. In pre-B cells, RAG-1 and RAG-2 expression is reactivated, and the immunoglobulin light chain genes can rearrange. Each cell has two  light chains and two  light chains, for a total of 4 attempts to successfully 3 MDBS 1102 B-cell Biology Dr. Raymond F Adebiyi generate a functional light chain. When a functional light chain is formed, it associates with the heavy chain, Ig and Ig β to form the mature B cell receptor (BCR) and transition into immature B cells. Signals through a functional B cell receptor trigger the cell to shut down light chain rearrangement. Immature B cells will undergo a selection process (described below) and then exit the bone marrow and traffic to the secondary lymphoid tissue. These cells express high levels of surface IgM and low levels of surface IgD. Chemokines produced by stromal cells and dendritic cells within the lymphoid tissue (i.e., lymph nodes, spleen, Peyer’s patches) attract the immature B cells and trigger the B cell to mature and survive. The B cell now exits the lymph node as a mature B cell, expressing surface IgM and IgD. Here’s a figure that re-caps these events: ELIMINATION OF SELF-REACTIVE CLONES The heavy and light chain gene rearrangements occur at random and are designed to generate the highest possible number of receptors for antigens yet to be encountered. Obviously, some receptors would be created that are capable of recognizing self-antigen. Cells that recognize ubiquitous self-antigen such as MHC molecules are deleted or functionally inactivated. These cells may undergo receptor editing, so that the self-reactive receptor is deleted or the cell is marked for apoptosis. This process of negative selection occurs in the bone marrow. Further tolerance induction occurs when the B cell enters the periphery. Exposure of a self-reactive, immature B cell to self-antigen triggers the cell to either die by apoptosis or to become anergic, anergy can be induced by down-regulating expression of IgM, which renders the cell incapable of future activation. 4 MDBS 1102 B-cell Biology Dr. Raymond F Adebiyi ACTIVATION BY ANTIGEN AND DIFFERENTIATION Naïve (antigen-inexperienced), mature B cells become activated when the BCR (surface IgM or IgD) recognizes and binds specific antigen. Binding of the BCR to protein or carbohydrate epitopes on the surface of a microorganism, the BCR molecules become clustered and physically cross-linked to one another. When this occurs, signals are generated from the BCR and Ig and Igβ molecules at the surface to the inside of the cell, leading to changes in gene expression in the nucleus. Binding of BCR with antigen triggers a cascade of events that produce changes in the cell membrane, cytoplasm and nucleus. The metabolic activity of the cell is increased with the production of intercellular messenger molecules. Protein kinases promote the first steps of activation. The expression of MHC-II molecules stimulated (will allow B cell to present antigen to CD4 T cells). Cytokines involved in B-cell activation include IL-1, IL-4, IL-6 and IFN. Cell division results in clonal expansion of activated B-cells. Interactions with activated T cells and cytokines trigger the proliferation of B cells, and trigger isotype or class switching (refer to the end of this handout for details on isotype switching). Some of the activated B cells differentiate immediately into antibody-secreting plasma cells. Specially designed for the synthesis and secretion of immunoglobulin, these cells secrete IgM antibodies, which contribute to the humoral immune response. Plasma cells are no longer capable of responding to antigen or interacting with T cells, as they no longer express surface Ig or MHC Class II. Those activated B cells that do not immediately differentiate into plasma cells migrate to the lymphoid follicles where they undergo several rounds of division. Somatic hypermutation occurs at this point, causing the formation of BCRs with varying affinity for the antigen that stimulated the immune response. This is accomplished by the random addition of single- nucleotide substitutions at a high rate thought the rearranged variable region of the heavy and light- chain immunoglobulin genes. You can imagine that these random nucleotide substitutions sometimes generate antibodies with higher affinity than they started, and some with lower affinity. To address this, the B cells then undergo affinity maturation. Affinity maturation is a round of selection to test the affinity of the BCR that has undergone somatic hypermutation. Those cells with BCRs with high affinity for the antigen are selected to survive (positive selection); likewise, those with low affinity will die by apoptosis. Somatic hypermutation, affinity maturation, and class switching are all very important in the development of an effective humoral immune response. Somatic hypermutation and affinity maturation allow for the generation and selection of antibody molecules that are of very high affinity for the antigen. Class switching enables these antibodies to have varying effector functions, and as you will see in future lectures, gives the body the ability to respond in the most effective way for that particular antigen. 5 MDBS 1102 B-cell Biology 6 Dr. Raymond F Adebiyi MDBS 1102 B-cell Biology Dr. Raymond F Adebiyi Of those activated B cells that undergo isotype switching, somatic hypermutation and affinity maturation, some go on to differentiate into antibody-secreting plasma cells. Others differentiate into memory B cells, which do not secrete antibody, but can rapidly reactivate upon subsequent encounter with specific antigens. These cells can survive in the periphery for long periods and enable the host to produce a secondary antibody response that is stronger and faster than the primary response (anamnestic response). The antibody response to most antigens requires help from CD4 T cells. CD4 T cells that recognize pathogen-derived peptides in the context of MHC Class II on the surface of the B cell become activated and synthesize a surface molecule called CD40 ligand or CD40L. B cells express the corresponding receptor, CD40. Binding of CD40 to CD40L, in combination with IL-4 secreted by the T cell, triggers the B cell to divide. T cells also secrete IL-5 and IL-6, which drive differentiation of the activated B cell to plasma cells. The cytokine secretion profile also induces class switching and different cytokines promote switching to different isotypes (see discussion on class switching below). Some antigens can activate B cells in such a way that T cell help is not required. These antigens are appropriately named thymus-independent antigens. These antigens can include repeating polymers (i.e., bacterial polysaccharides) or bacterial cell wall components (i.e., lipopolysaccharide). While the B cell becomes activated and secretes antibodies, it cannot undergo affinity maturation and class switching, and therefore only produces IgM antibodies. 7 MDBS 1102 B-cell Biology Dr. Raymond F Adebiyi B-CELL SUBSETS Development Stage MainEvents Stemcell Pro-Bcell Pre-Bcell H-chain rearrangement,D-J, then V-DJ; Allelicexclusion ExpressionofPreBCR; Downregulation of H-chain rearrangement; Initiation of L-chain rearrangement; Allelicexclusion ImmatureBcell MatureBcell Plasmacellor MemoryBcell Expression of BCR; Negative selection Antigenbinding toBCRpluscostimulatory signalbyT-cell (CD40/CD40L) CD19 BCR(IgMIgD) CD19 (plasma cells do notexpressBCR) - - Surface Markers CD34 CD19 CD19 CD19 BCR(IgMIgD) Expressionof RAG genes - ++ ++ - Most peripheral B-cells are ‘conventional’ B-cells that express the usual B-cell surface markers. These are also called B-2 cells. B-2 cells express more sIgD than sIgM. A subset exists that may express the CD5+ marker normally found on immature cells in addition to expressing more sIgM than sIgD. These are called B1 cells. B-1 cells are found in the fetus and neonate but most mature into B-2 cells in adults. Antibodies secreted by B-1 cells tend to be of low affinity and can bind to many different antigens. B-1 cells appear to preferentially respond to T-independent antigens and are responsible for the production of isoantibody to the ABO substance. B cell development can be summarized by these events: 8 MDBS 1102 B-cell Biology GENERAL CHARACTERISTICS OF B - LYMPHOCYTES 1. 20% of peripheral blood lymphocytes (PBLs) 2. development occurs in bone marrow 3. possess sIg (IgM, IgD) 4. possess FcR 5. high rER 6. receptor for Epstein-Barr Virus – C3dR, CR2, CD21 7. precursors of plasma cells 8. B-1 sub set a. CD5+, CD19+, CD21b. Non-conventional B cells – reactivity to T-independent antigens 9. B-2 sub set a. CD5-, CD19+, CD21+ b. Conventional B cells – reactivity to T-dependent antigens 10. present in lymph node germinal centers 11. CD40, B7.1/B7.2 (CD80/CD86) 9 Dr. Raymond F Adebiyi MDBS 1102 B-cell Biology Dr. Raymond F Adebiyi OPTIONAL READING Variable region sequences are constructed by the rearrangement of gene segments: The heavy chain locus is on chromosome 14 and contains about 60 Variable (V) regions, about 25 Diversity (D) regions and 6 Joining (J) regions. The light chain loci are on chromosomes 2 and 22. The -chain on chromosome 2 contains about 40 V regions and 5 J regions. The -chain on chromosome 22 contains about 30 V regions and 4 J regions. Together, the random combination of heavy and light chain could make at least 1.6 million different antibodies! Isotype or class switching is a process that B cells use to further recombine the antibody heavy chain genes such that the rearranged Vregion can be used with other constant (C)genes than  ( gene encodes the constant region for the IgM isotype). You will recall that antibodies with different C regions have different functions and traffic to different anatomical sites. Helper T cells secrete cytokines that program the activated B cell to undergo class switching (as mentioned above, CD40-CD40L interactions are also required for this to occur). Th2 cells secrete IL-4, IL-5 and TGF-β, which promotes the differentiation into plasma cells that secrete IgM, and promote class switching to IgG2, IgG4, IgA and IgE. Th1 cells produce IFN, which promotes switching to IgG1, IgG2a, and IgG3. Each isotype has different implications for effector function. http://www.vatlieu.us/aboutvietnam.html?view=mediawiki&article=Immunoglobulin_class_switching 10