Ig light chain rearrangement: Rescue pathway

Transcription

1 B Cell Development

2 Ig light chain rearrangement: Rescue pathway There is only a 1:3 chance of the join between the V and J region being in frame Vk Jk Ck Non-productive Rearrangement Light chain has a second chance to make a productive join using new V and J elements Spliced mrna transcript

3 Model to account for allelic exclusion: If one allele arranges nonproductively, a B cell still can rearrange the other allele productively; once a productive rearrangement( 33%) have occurred, the recombination machinery is turned off. ( the protein product acts as a signal to prevent further gene rearrangement)

4 Repeated light chain rearrangements can occur at a single light chain locus Replacement of non-productive VJ junction by rearrangement of upstream V to downstream J High probability of generating a functional light chain

5 Multiple opportunities to generate a functional light chain Two light chain isotypes k and l Pre-B cells usually rearrange k before l A successful rearrangement that can lead to migm expression blocks further light chain rearrangement

6 B cells have several chances to successfully rearrange Ig genes Early Pro B Late Pro B Pre B D H -J H On first chromosome B NO D H -J H On second chromosome NO ES ES NO V H -DJ H On first chromosome NO V H -DJ H On second chromosome NO ES ES k on first chromosome NO k on second chromosome NO l on first chromosome NO l on second chromosome ES ES ES ES Immature B IgMk B IgMl B

7 Gene rearrangements in B cells occur is an orderly fashion

8 Heavy and light chain rearrangement is potentially wasteful V D J C Germline V V V D D J D J C C D H -J H joining V H -D H J H joining Large pre-b With two random joins to generate a heavy chain there is a 1:9 chance of a rearrangement of being in frame V J C Germline V V J C V L -J L joining With one random join to generate a light chain there is a 1:3 chance of a rearrangement being of frame Small pre-b There is, therefore, only a 1:27 chance of an in frame rearrangement Out of frame rearrangements arrest further B cell maturation

9 Consequences of pre-bcr expression Inhibition of further heavy chain rearrangement If the first VDJ H rearrangement is productive then the second allele will not undergo VDJ H rearrangement If the first VDJ H rearrangement is non-productive then the second allele will undergo VDJ H rearrangement This means that a single B cell will only express the product of a single functionally rearranged heavy chain ALLELIC EXCLUSION

10 Consequences of a failure to express pre-bcr If the first VDJ H rearrangement is non-productive then the second allele will undergo VDJ H rearrangement If the second VDJ H rearrangement is non-productive then the cell will die Almost half of the pro-b cell population will die at this stage of development

11 B Cell Development: Introduction B cells are responsible for the production of immunoglobulins B cells are derived from pluripotent hematopoietic stem cells in the liver before birth and from bone marrow thereafter The distinguishing surface characteristics of B cells are; - Surface Immunoglobulins - FC Receptors - Complement Receptors (CR1 and CR2) - MHC Class II Molecules

12 B Cell Types B cells comprise two major cell lineages; B-1 cells and B-2 cells B-1 cells are so named because they are the first to develop and are a self- renewing population that predominates in the peritoneal and pleural cavities Although much of the function of B-1 cells is unknown, it is known that they constitutively secrete IgM antibodies of limited diversity that react strongly with carbohydrates and poorly with proteins A large proportion of serum IgM antibodies found in normal individuals are of B-1 origin

13 In contrast, B-2 cells also called conventional B cells, arise after birth, are continuously replaced from the bone marrow, and are widely distributed through the lymphoid organs and tissues Because of more extensive somatic gene rearrangement, B- 2 cells display a broader repertoire of immunoglobulin specificities and affinities Unlike B-1 cells, resting B-2 cells produce low levels of immunoglobulins that increase only on their activation by helper T cells, causing them to differentiate into antibodysecreting cells

14 Two B cell lineages B cell precursor Mature B cell Plasma cell B B Distinct B cell precursor?? B B CD5 IgM - no other isotypes PC B2 B cells IgG B1 B cells Primitive B cells found in pleura and peritoneum

15 B-1 B Cells IgM uses a distinctive & restricted range of V regions Few non-template encoded (N) regions in the IgM Recognises repeating epitope Ag such as phospholipid phosphatidyl choline & polysaccharides NATURAL ANTIBOD NOT part of adaptive immune response: - No memory induced - Not more efficient on 2nd challenge - Present from birth Can make Ig without T cell help B CD5 IgM

16 Comparison of B-1 and B-2 B cell properties Property B-1 cells B-2 cells N regions Few Extensive V region repertoire Restricted Diverse Location Peritoneum/pleura Everywhere Renewal Self renewal in situ Bone marrow Spontaneous Ig production High Low Isotypes IgM IgM/G/A/D/E Carbohydrate specificity es Rarely Protein specificity Rarely es Need T cell help No es Somatic hypermutation of Ig No High Memory development No es Specificity & requirement for T cell help suggests strikingly different types of antigens are seen by B-1 and B-2 B cells

17 T Independent Antigens (TI-2) TI-2 Antigen Immature B-2 Cell Mature B-1 Immature B cells that bind to multivalent self Ag undergo apoptosis B-2 cell repertoire is purged of cells recognising multivalent antigens during development in the bone marrow IgM Non-bone marrow derived B-1 cells are directly stimulated by antigens containing multivalent epitopes. No T cells are necessary Induces the expression of natural antibodies specific for TI-2 antigens

18 T Independent Antigens (TI-1) LPS LPS binding protein TLR 4 Bacterial Lipopolysaccharides, (TI-1 antigens), bind to host LPS binding protein in plasma LPS/LPSBP is captured by CD14 on the B cell surface CD14 Toll - like receptor 4 (TLR4) interacts with the CD14/LPS/LPSBP complex B Cell Activation of B cell

19 T Independent Antigens (TI-1 e.g. LPS) LPS complexes with CD14, LPSBP & TLR4 B B B B B B Six different B cells will require 6 different antigens to activate them At high dose TI-1 antigens (like LPS) will POLCLONALL ACTIVATE all of the B cells irrespective of their specificity. TI-1 antigens are called MITOGENS

20 Once committed to the B cell pathway, B-2 cell precursors pass through several stages that involve changes in gene activity to provide the appropriate enzymes and structural molecules necessary for mature B-2 cells In many cases, the changes in gene activity are induced by molecules, such as IL-7, produced by the stromal cells of the bone marrow Among the changes defining the developmental progression of the B-cell lineage are those involving the ability to synthesize and express immunoglobulin molecules on the cell surface

21 The B- cell receptor complex

22 B cell development in the bone marrow B Regulates construction of an antigen receptor B B B B Ensures each cell has only one specificity Checks and disposes of self-reactive B cells Exports useful cells to the periphery Provides a site for antibody production Bone Marrow provides a MATURATION & DIFFERENTIATION MICROENVIRONMENT for B cell development

23 B Cell Development is Dependent on Several Factors A.) The presence of bone marrow stromal cells providing cell-mediated contacts. B.) The presence of the cytokine IL-7 secreted mainly by the bone marrow stromal cells. C.) The productive rearrangement of the immunoglobulin genes. In the common lymphoid progenitor both the immunoglobulin heavy and light chains are in their germ-line configuration and must be rearranged to generate an in-frame protein for B cell development to progress.

24 Bone marrow stromal cells nurture developing B cells 1. Specific cell-cell contacts between stromal cells and developing B cells 2. Secretion of cytokines by stromal cells Cell-cell contact B Secreted Factors - CTOKINES Stromal cell Types of cytokines and cell-cell contacts needed at each stage of differentiation are different

25 Cytokines and cell-cell contacts at each stage of differentiation are different VLA-4 (Integrin) Stem Early pro-b Kit Receptor Tyrosine kinase VCAM-1 (Ig superfamily) Cell adhesion molecules Stromal cell Stem cell factor Cell-bound growth factor

26 Bone Marrow Derived IL-7 IL-7 secreted by bone marrow stromal cells binds to the IL-7 receptor expressed on pro- and pre-b cells and stimulates the survival and proliferation of the B cells.

27 Bone marrow microenvironment is required for B cell development Interaction between cell adhesion molecules (CAMs) expressed on lymphoid and stromal cells Interaction between cytokines (SCF) and cytokine receptors (Kit) => Growth or differentiation Cell adhesion molecules include integrins (VLA-4), selectins and other adhesion molecules

28 Bone marrow microenvironment provides soluble and surface bound cytokines Stem cell factor; an example of a cell surface cytokine Stromal cells produce interleukin-7 (IL-7) a soluble cytokine Late pro-b and pre-b cells require IL-7 for growth

29 Pro-B Cells: B Cell Developmental Stages - Initiate the production of enzymes such as Rag-1 and Rag-2, and other molecules necessary for the construction of an immunoglobulin molecule including the chromosomal rearrangement of V, D, and J genes to form the variable V H domain - The first component synthesized is the µ immunoglobulin heavy chain, which remains confined to the cytoplasm

30 Early- Stage Pre-B Cell: - They are characterized by the cytoplasmic presence of a transitional or surrogate light chain - This chain is neither a true λ or k light chain - It serves as a temporary substitute capable of interacting with the µ heavy chains present in the cytoplasm to permit assembly of a monomer-like structure (2 heavy chains and 2 surrogate light chains) that can be transported to the membrane for surface expression

31 Late-Stage Pre-B Cell: - They synthesize either a k or λ light chain that replaces the transitory light chains, resulting in the expression of true IgM molecules on the cell surface - Again, this includes the chromosomal rearrangement necessary for formation of the V L domain - With the formation and expression of IgM, the pre-b cell becomes an Immature B cell - Because the IgM has not yet bound to its antigen, immature B cells are also called naïve B cells

32 Immature B cells: - With their surface antigen receptors now in place, they undergo a selective process to remove any that may be self-reactive - If their surface immunoglobulin finds and binds to epitopes within their bone marrow environment, they undergo apoptosis - Those B cells that survive this test leave the bone marrow and enter the circulation

33 After gene rearrangements and production of a functional molecule, the cell tests whether its specificity is anti-self Maintenance of tolerance requires the persistence of antigen because selfantigens are always present but foreign antigens are transient Alive but not functional Ag binding is too weak to get a response (least important) These are the cells that constitute a normal immune response

34 Before clonal deletion of an antiself B cell, the cell can attempt receptor editing of the light chain Immature B cell edits light chain if it binds antigen (gets negative selection signal). This could rescue the cell from negative selection (i.e., death). Immature B cell continues to rearrange a chain until it gets positive selection signal. (will eventually die if it does not receive positive selection in a few days).

35 Receptor editing A rearrangement encoding a self specific receptor can be replaced V V V V J C B!!Receptor recognises self antigen!! Arrest development And reactivate RAG-1 and RAG-2 B Apoptosis or anergy V V V J C B Edited receptor now recognises a different antigen and can be rechecked for specificity

36 - On leaving the bone marrow, immature B cells have a short half-life and must quickly find their way to a lymph node - Once there, they receive additional signals from the lymph node stromal cells that enable them to continue developing - Among the critical steps that new B cells undertake after entering a lymph node is further modification of their immunoglobulin expression

37 - They begin to synthesize δ heavy chains so that they can simultaneously express both IgD and IgM molecules on their surface - The ability to produce both IgD and IgM with identical antigen-binding regions is accomplished by a mechanism involving alternative splicing of the mrna encoding the heavy chains - With the expression of both IgD and IgM, the immature B cell becomes a mature B cell, even though it may not yet have bound antigen

38 Splicing of IgM and IgD RNA V D J Cm Cd Cg3 Cg1 V D J V D J V D J Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3 DNA pa1 Two types of mrna can be made simultaneously in the cell by differential usage of alternative polyadenylation sites and splicing of the RNA AAA Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3 V D J Cm IgM mrna Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3 pa1 pa2 RNA cleaved and polyadenylated at pa1 RNA cleaved and polyadenylated at pa2 AAA V D J Cd IgD mrna

39 Mature B Cells: - They Co-express IgD and IgM, and they are ready for activation - In the overwhelming majority of cases, activation requires the binding of antigen to surface immunoglobulin and an interaction with T cells

40 Plasma Cells: - They are immunoglobulin producing and secreting cells derived from B cells - These terminally differentiated, oval cells have a relatively short lifespan of less than 30 days and are immunoglobulinproducing factories - They are characterized by a basophilic cytoplasm and large, nonstaining Golgi apparatus. - Although they secrete large quantities of immunoglobulin, very little is displayed on their cell surfaces

41 Ig gene rearrangements occur in an orderly fashion in developing B cells bone marrow periphery

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44 Survival of B and T cells requires that they circulate through lymphoid organs B cell survival signal is probably not received via the antigen receptor. Without antigenic stimulation, most B cells die shortly after leaving the bone marrow; some will survive several weeks (3-8 weeks). B cell will not divide in the periphery without antigenic stimulation. Mature naïve T cells can divide in the periphery (probably get stimulation via weak binding to self peptide and MHC, similar to positive selection in the thymus). Memory T cells can divide without MHC + peptide stimulation.

45 Three types of mature B cells Recirculating follicular B cells (aka conventional B cells, B2 cells): circulate between LN follicles and blood Marginal zone B cells: reside in marginal zone of spleen where they can respond to particulate antigen in blood (bacteria, etc.) 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.

46 Antigen-dependence for B cell maturation Immature B cells that don not experience a strong BCR signal in bone marrow exit to the periphery. These cells have an average lifespan of about 7 days, but about 20% enter the long-lived recirculating B cell population Entry into the long-lived follicular B cell pool is competitive and requires BCR signaling (like positive selection of thymocytes)

47 Recirculating B cells are trapped by foreign antigens in lymphoid organs Antigen enters node in afferent lymphatic B cells proliferate rapidly B cells leave blood & enter lymph node via high endothelial venules GERMINAL CENTRE Transient structure of Intense proliferation Germinal centre releases B cells that differentiate into plasma cells