B cell differentiation, maturation and activity

week 4 clinical immunology

B cell maturation

• B cells are central to adaptive humoral immune system

• produce antigen-specific immunoglobulin (Ig) or ABs, directed against invasive pathogens

• early B cell development and commitment to the B cell lineage occurs in the foetal liver prenatally

• B cells differentiate from haemapoietic stem cell (HSC) in bone marrow throughout life

• 1 bill B cells produced each day

• B = bursa

B cell maturation overview

• antigen-independent maturation phase

• antigen-dependent maturation phase

antigen-independent maturation phase

• immunocompetent B cells expressing membrane IgM and IgD are generated in the bone marrow

• 10% of potential B cells reach maturity and exit bone marrow

antigen-dependent maturation phase

• naive B cells in the periphery die within a few days unless they encounter soluble protein antigen and activated Th cells

• activated B cells proliferate within secondary lymphoid organs

• B cells with high-affinity mlg differentiate into plasma ells and memory B cells

• may express different isotypes bc of class switching

progenitor B cells (pro B cell): earliest distinctive B-lineage cell

• expresses a transmembrane tyrosine phosphatase called CD45R

• bone marrow stromal cells are required for maturation of pro-B cells into precursor B cells

• pro-B cells bind to stromal cells via VCAM-1 on the stromal cell and VLA-4 on the pro-B cell

• binding of c-kit (receptor) omn the pro-B cell to stem cell factor (SCF) on the stromal cell, triggers a signal, mediated by tyrosine kinase activity of c-kit → expression of IL-7 receptors

• IL-7 released from the stromal cell then binds to IL-7 receptors, inducing pro-B cell to mature into pre-B cell

stromal cell role

• form specific adhesive contacts with the developing lymphocytes by interactions between cell adhesion molecules and their ligands

• provide soluble and membrane bound cytokines and chemokines that control lymphocyte differentiation and proliferation

Ig-gene rearragement produces immature B cells

• progenitor B cells rearrange their immunoglobulin genes

  • assemble and expression of functional antigen-receptor gene

• recombination activating genes (RAG 1 ) and RAG2 are expressed in developing B cells

• required for rearrangement of antigen receptor genes

allelic exclusion: B cells rearrange one gene locus at a time

• Ig heavy and light chain genes of only one parental chromosome are expressed per cell

• ensures that B cells possess a single antigenic specificity

• allele selected for rearrangement is chosen randomly

• expressed Ig may contain one maternal and one paternal chain or both chains may derive from only one parent

• only B and T cells exhibit allelic exclusion

gene rearrangement in B cell development

• early pro B cells: heavy chain gene rearrangement is inititated with D to JH rearrangements

• no functional μ protein is expressed, although transcription occurs

• late pro-B cells: VH to DJH rearrangement occurs on one chromosome first

  • if no functional H chain produced, VH to DJH rearrangement occurs on second chromosome

• productive heavy chain gene: μ chains are expressed together with 2 other chains: λ5 and Vpre5

• Igα and Igβ signal to halt heavy chain gene rearrangement: drives the transition to the large pre B stage by inducing proliferation

gene rearrangement in B cell development cont.

• progeny of large pre B cells stop dividing and become small pre-B cells, in which light chain gene rearrangements commence

• Vκ-Jκ occurs first, and if unsuccessful, Vλ to Jλ rearrangement occurs next

• successful light chain gene rearrangement results in production of a light chain that binds the μ chain to form complete IgM molecule

• expressed together with Igα and Igβ at the cell surface

pre-B receptor: important checkpoint

• tests for successful production of a complete heavy chain

• test takes place in the absence of light chains

• signals the transition from pro-B cell to the pre B cell stage

pre-B cell receptor initiates signalling through spontaneous dimerization

• surrogate protein chains, VpreB (orange) and λ5 contain unique amino-terminal regions that are not present in other immunoglobulin like domains

• dimerizarion generates signalling from the pre-B cell receptor that is dependent on the presence of the ITAM-containing signalling chains Igα and Igβ

• signalling inhibits RAG-1 and RAG-2 expression and causes proliferation of the large pre-B cell

large pre-B cells transitions to small pre-B cell

• cell division expands large pre-B cell population with successful in-frame joins

• RAG proteins now cause the rearrangement of the light chain locus

• developing B cells fail to assemble a complete surface immunoglobulin undergo apoptosis in the bone marrow and are eliminated from the B cell pool

immature B cells carry an intact IgM molecule

• IgM: rearranged light chain paired with a μ chain

• antigen receptor is tested for reactivity to self-antigens (autoreactivity)

• elimination of autoreactive B cells ensures that B cell population will be tolerant of self antigens

• central tolerance

• B cells leaving the bone marrow require additional maturation steps that take place in peripheral lymphoid organs

central tolerance: B cell development

1. immature B cells without self reaction migrate from bone marrow to peripheral lymphoid tissues

   they may become mature recirculating B cells bearing both IgM and IgD on their surface

2. if developing BCRs recognise self-molecules, these receptors are deleted from the repertoire

   B cells either undergo receptor editing to eliminate autoreactivity or undergo apoptosis, resulting in clonal deletion

3. immature B cells that bind soluble self-antigens able to cross-link the BCR are rendered unresponsive to the antigen

   bear little surface IgM

   migrate to periphery where they express IgD but remain anergic

4. immature B cells whose antigen is inaccessible to them, or which bind monovalent or soluble self antigens with low affinity don’t receive any signal and mature normally

   potentially self-reactive cells however are clonally ignorant as their ligand is present but unable to activate them

3 major mature B cell populations

• follicular B-2 cells circulate secondary lymphoid organs

• if antigens stimulate a response, B-2 cells migrate to border of B/T cell boundary and interact with stimulated T cells

• B cell activation

plasma cells: terminal differentitation of a B cell

• from germinal centre or the memory B cell population, antigen specific B cells will proliferate (in presence of the antigen) and become plasmablasts and then plasma cells

  • short term plasma cells are in spleen/lymph node

  • long lived plasma cells are in bone marrow and secrete ABs into bloodstream

B cell receptor (BCR)

• antigen binding receptor

• membrane bound immunoglobulin associated with one disulphide-liked Igα-Igβ heterodimer contains the immunoglobulin-fold structure and cytoplasmic tails that are longer than those of mlg signal transduction

B cell co-receptor

• amplifies the activating signal transmitted through the BCR

• complex of 3 cell membrane molecules: TAPA-1 (CD81), CR2 (CD21) and CD19

• binding of the CR2 component to complement-derived C3d that has coated antigen captured by mlg results in the phosphorylation of CD19

• the Src family tyrosine kinase Lyn binds to phosphorylated CD19

• resulting activated Lyn and Fyn can trigger the signal-transduction pathways that begin with phospholipase C

initial stages of B cell activation

• BCR crosslinking causes interaction of immunoreceptor tyrosine-based activation motifs (ITAMs) with Src tyrosine kinases (Fyn, Blk and Lck) and kinase activation

• activated kinases phosphorylate tyrosine residues on the cytoplasmic tails of the Igα/Igβ heterodimer. creating docking sites for Syk kinase which is also activated

B cell activation mechanisms

• B cells recognise soluble or cell-bound epitopes directly through their BCRs

steps in B cell activation by TD antigens

• antigen cross linkage of immunoglobulin induces signal-1 which leads to increased expression of class II MHC and co-stimulatory B7 on B cell

• TH cell recognises antigen-class II MHC on B cell membrane and activates TH cell with co-stimulatory signal

• expresses CD40L, then provides signal-2 with CD40 and CD40L interaction

• B7-CD38 interactions provide co-stimulation to the TH cell

steps in B cell activation by TD antigens (cont)

• B cell expresses receptors for various cytokines including:

  • IL-4

  • IL-5

  • IL-6

  • IFN-γ

  • TGF- α

the humoral response

• activation leads to production of secreted ABs of various isotypes (differ in their ability to mediate specific effector functions)

• naive lymphocytes (primary response) vs memory lympho

AB functions in humoral response

• neutralisation: ABs neutralise bacterial toxins (diphtheria, tetanus) and prevent adherence of microorganisms to their target cells (eg IgA in the gut)

• opsonisation: ABs bind to antogens and enhance phagocytosis

• activation of complement (classical pathway): leads to bacterial lysis

• mediate AB-dependent cellular cytotoxicity: by macrophages and NK cells

• agglutination: ABs clump bacteria leading to phagocytosis

primary response vs. secondary

primary response:

• rapid production of IgM

• slightly delayed IgG response due to class switching

secondary response:

• produces small amounts of IgM initially, larger amounts of IgG with some IgA and IgE

• ABs are made by memory B cells that were generated in the primary response and have already switched from IgM to another isotype

• memory B cells express more MHC class II molecules and the co-stimulatory ligand B7.1 than naive B cells

• helps memory B cells acquire and present antigen more effectively to TFH cells

• characterised by a more vigorous and earlier generation of plasma cells than in the primary response

primary B cells vs. secondary B cells

selective Ig A deficiency

• most common humoral AB deficiency

• 50-80% are asymptomatic

• recurrent sinopulmonary infections arae the most frequent manifestations

• may have severe malabsorption (chronic diarrhea)

• isolated low IgA level

• increased risk of autoimmune disorders

Bruton’s X-linked agammaglobulinaemia

• results from defected BTSK gene (encodes tyrosine kinase)

• no B cells

• child will be clinicallyw ell for first 6 months of life

• recurrent upper/lower respiratory tract infections (RTIs) with encapsulated bacteria

• sepsis, meningitis, skin infections

• paucity of lymphoid tissue (tossils, adenoids)

• markedly decreased IgG, IgA, IgM

• treatment: IVIG, antibiotics

common variable immunodeficiency

• B cells don’t differentiate into plasma cells

• recurrent sinopulmonary infections

• low IgG, IgA, IgM

• treatment: IVIG (IV immunoglobulin G)

• associated with autoimmune disease, lymphoma

lymphocyte recirculation routes

• an individual lymphocyte may make a complete circuit from the blood to the tissues and lymph and back again (as often as 1-2 times a day)

• lymphocytes migrate from the blood into lymph nodes through specialiased areas in postcapillary venules called high-endothelial venules (HEVs)

cell adhesion molcules (CAMs)

• HEVs express variety of cell-adhesion molecules (membrane proteins)

• facilitate lymphocyte extravasation

• CAMs expressed either:

  • constitutively

  • induced by cytokines during inflammation

• vascular addressins (VAs) distributed in a tissue-specific manner and direct the extravasation of different populations or recirculating lymphocytes to a particule lymphoid organ

CAM expression controls lymphocyte movement through tissues

4 families of adhesion molecules:

• selectins: bind to sialic acid residues on mucins

• mucins: heavily glycosylated proteins on cell surface that contain sialic acid residues which bind to selectins

• integrins: heterodimers consisting of a common β chain and a unique α chain

  • integrins bind to ICAMs

• ICAMs (intracellular adhesion molecule): members of immunoglobulin family

extravasation

homing receptors direct trafficking of lymphocytes

• naive lymphocytes attach to HEVs via L-selectin (homing receptor) and adhesion molecules such as GlyCAM-1 and CD34 on HEVs

• antigen-specific lymphocytes undergo rapid proliferation and differentiation

• effector and memory cells that are generated by this process leave the lymphoid tissue and recirculate

effector and memory lymphocytes adopt different trafficking patterns

• effector cells home to regions of infection: by recognising inflamed vascular endothelium and chemoattractant molecules generated during inflammatory response

• memory lymphocytes home selectively to the type of tissue in which they first encountered the antigen

• express different different combinations of cell-adhesion molecules to enter sites of inflammation