B Cells and B Cell Development © Dr. Colin R.A. Hewitt crah1@le.ac.uk

The discovery of B cell immunity 1954 - Bruce Glick, Ohio State University Studies on the function of the bursa of Fabricius, a lymphoid organ in the cloacal region of the chicken Bursectomy – no apparent effect None of the bursectomised chickens made anti-Salmonella antibodies Bursectomised chickens were later used in experiments to raise antibodies to Salmonella antigens Bursa was later found to be the organ in which antibody producing cells developed – antibody producing cells were thereafter called B cells Mammals do not have a bursa of Fabricius

Origin of B cells and organ of B cell maturation Transfer marked foetal liver cells Mature marked B cells in periphery Normal bone marrow No Mature B cells Defective bone marrow B cell development starts in the foetal liver After birth, development continues in the bone marrow

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

Bone Marrow M S E M

Scheme of B Cell Development in the Bone Marrow Immature & mature B Central Sinus E n d o os t e u m Progenitors Pre-B X Stromal cells X X Macrophage

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

Maturing B cells Bone marrow stromal cell

B B Stromal cell

Stages of B cell development Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell Peripheral Small pre-B cell Immature B cell Mature B cell Each stage of development is defined by rearrangements of IgH chain genes, IgL chain genes, expression of surface Ig, expression of adhesion molecules and cytokine receptors

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

Cytokines and cell-cell contacts at each stage of differentiation are different Interleukin-7 receptor Interleukin-7 Growth factor Early pro-B Late pro-B Pre-B Stromal cell

Stages of differentiation in the bone marrow are defined by Ig gene rearrangement Pre-B cell receptor expressed B CELL STAGE IgH GENE CONFIGURATION Stem cell Early pro-B Late pro-B Large pre-B Germline DH to JH VH to DHJH VHDHJH Ig light chain gene has not yet rearranged

Transiently expressed when VHDHJH CHm is productively rearranged B cell receptor CHm Heavy chain VHDHJH Light chain VLJLCL VpreB l5 Iga & Igb signal transduction molecules Transiently expressed when VHDHJH CHm is productively rearranged VpreB/l5 - the surrogate light chain, is required for surface expression Ligand for the pre-B cell receptor may be galectin 1, heparan sulphate, other pre-BCR or something as yet unknown

Ligation of the pre-B cell receptor 1. Suppresses further H chain rearrangement 2. Triggers entry into cell cycle Large Pre-B Unconfirmed ligand of pre-B cell receptor 1. Ensures only one specificty of Ab expressed per cell Stromal cell 2. Expands only the pre-B cells with in frame VHDHJH joins ALLELIC EXCLUSION Expression of a gene on one chromosome prevents expression of the allele on the second chromosome

Evidence for allelic exclusion ALLOTYPE- polymorphism in the C region of Ig – one allotype inherited from each parent Allotypes can be identified by staining B cell surface Ig with antibodies a/a b/b a/b Y B a Y B b Y B a Y B b AND Y B a b Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two specificities of antibody per cell (Refer back to Dreyer & Bennet hypothesis in Molecular Genetics of Immunoglobulins lecture topic)

Allelic exclusion prevents unwanted responses One Ag receptor per cell IF there were two Ag receptors per cell Y B Y Self antigen expressed by e.g. brain cells B Y S. aureus S. aureus Y Anti S. aureus Antibodies Y Anti brain Abs Y Anti S. aureus Antibodies Suppression of H chain gene rearrangement ensures only one specificity of Ab expressed per cell. Prevents induction of unwanted responses by pathogens

Allelic exclusion is needed for efficient clonal selection Antibody S. typhi S. typhi All daughter cells must express the same Ig specificity otherwise the efficiency of the response would be compromised Suppression of H chain gene rearrangement helps prevent the emergence of new daughter specificities during proliferation after clonal selection

Allelic exclusion is needed to prevent holes in the repertoire Y B One specificity of Ag receptor per cell Y B IF there were two specificities of Ag receptor per cell Anti-brain Ig Anti-brain Ig AND anti-S. aureus Ig Exclusion of anti-brain B cells i.e. self tolerance BUT anti S.aureus B cells will be excluded leaving a “hole in the repertoire” B Deletion Anergy OR Y B S. aureus

Ligation of the pre-B cell receptor 1. Suppresses further H chain rearrangement 2. Triggers entry into cell cycle Large Pre-B Unconfirmed ligand of pre-B cell receptor 1. Ensures only one specificity of Ab expressed per cell Stromal cell 2. Expands only the pre-B cells with in frame VHDHJH joins

Large pre-B cells need in frame VHDHJH joins to mature Development continues Pre-B cell receptor can be activated Human IgG3 Heavy Chain nucleotide sequence ATGAAACANCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCACCTGCAGGAGTCGGGCCCAGGACTGGGGAAGCCTCCAGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCNNNGTGCCCAGCACCTGAACTCTTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCNNNNGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCGAGGAGATGACCAAGAACCAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Translation in frame 1 MKXLWFFLLLVAAPRWVLSQVHLQESGPGLGKPPELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCXXCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDXXVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRYTQKSLSLSPGK* Development arrests Translation in frame 2 (no protein) * Translation in frame 3 ETXVVLPSPGGSSQMGPVPGAPAGVGPRTGEASRAQNPTW*

Ligation of the pre-B cell receptor triggers entry into the cell cycle Large pre-B Large Pre-B Many large pre-B cells with identical pre-B receptors Proliferation Large pre-B Y Immature B cell Light chain expressed IgM displayed on surface IgM Proliferation stops Pre-receptor not displayed Small pre-B Intracellular VDJCH chain VL-JL rearranges

Heavy and light chain rearrangement is potentially wasteful Large pre-B Small V D J C Germline V C D J D J DH-JH joining V C D J V VH-DHJH joining 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 C J V VL-JL joining With one “random” join to generate a light chain there is a 1:3 chance of a rearrangement being of frame There is, therefore, only a 1:27 chance of an in frame rearrangement Out of frame rearrangements arrest further B cell maturation

B cells have several chances to successfully rearrange Ig genes Early Pro B Late Pro B Pre B Immature B DH-JH On first chromosome VH-DJH On first chromosome k on first chromosome YES YES YES Y IgMk B YES YES YES NO NO NO k on second chromosome DH-JH On second chromosome VH-DJH On second chromosome NO YES l on first chromosome Y IgMl B YES NO B NO NO l on second chromosome NO

Y B B Acquisition of antigen specificity creates a need to check for recognition of self antigens Y B B Small pre-B cell No antigen receptor at cell surface Unable to sense Ag environment !!May be self-reactive!! Immature B cell Cell surface Ig expressed Able to sense Ag environment Can now be checked for self-reactivity Physical removal from the repertoire DELETION Paralysis of function ANERGY Alteration of specificity RECEPTOR EDITING

B cell self tolerance: clonal deletion Small pre-B Small pre-B cell assembles Ig B Immature Y Immature B cell recognises MULTIVALENT self Ag Clonal deletion by apoptosis

B cell self tolerance: anergy IgD normal IgM low Y Y IgM B Small pre-B Small pre-B cell assembles Ig B Immature B Y Y IgD IgD Y IgD B Immature B cell recognises soluble self Ag No cross-linking Anergic B cell

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

B cell self tolerance: export of self tolerant B cells IgD and IgM normal IgM IgD Y B Small pre-B Small pre-B cell assembles Ig Y B Immature Y Y B Y Y Y Y Y Mature B cell exported to the periphery Immature B cell doesn’t recognise any self Ag

IgM and IgD simultaneously? How can B cells express IgM and IgD simultaneously? Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Cm Cd Cg3 VDJ Cg1 Sg1 Ca1 Cm Cd Cg3 VDJ Cg3 VDJ Ca1 Cg3 VDJ IgG3 produced. Switch from IgM VDJ Ca1 IgA1 produced. Switch from IgG3 VDJ Ca1 IgA1 produced. Switch from IgM N.B. Remember Molecular Genetics of Immunoglobulins lecture – No Cd switch region Consider similarities with mechanism allowing secreted and membrane Ig by the same cell

Splicing of IgM and IgD RNA Cg1 Cg3 Cd Cm V D J Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3 DNA pA1 pA2 V D J Two types of mRNA can be made simultaneously in the cell by differential usage of alternative polyadenylation sites and splicing of the RNA RNA cleaved and polyadenylated at pA1 AAA V D J Cm IgM mRNA Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3 V D J AAA RNA cleaved and polyadenylated at pA2 V D J Cd IgD mRNA Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3 pA1 V D J

Summary B cells develop in the foetal liver and adult bone marrow Stages of B cell differentiation are defined by Ig gene rearrangement Pre-B cell receptor ligation is essential for B cell development Allelic exclusion is essential to the clonal nature of immunity B cells have several opportunities to rearrange their antigen receptors IgM and IgD can be expressed simultaneously due to differential RNA splicing So far, mostly about B cells in the bone marrow - what about mature peripheral B cells?

What are the external signals that activate B cells? Although Fab fragments bind to membrane Ig (mIg), no signal is transduced through the B cell membrane Fab anti-mIg mIg Bridging (or ‘Cross-linking’) of different mIg allows the B cell receptor to transduce a weak signal through the B cell membrane (Fab)2 anti-mIg Anti-(Fab)2 Extensive cross linking of (Fab)2 bound to mIg using an anti-(Fab)2 antibody enhances the signal through the B cell membrane

Ring staining Patching Capping Ig is evenly distributed around the cell surface Patching Ig is aggregated in uneven ‘clumps’ as a result of mild cross-linking of Ig Capping Ig is collected at a pole of the cell in a ‘cap’ as a result of extensive cross-linking of Ig

Transduction of signals by the B cell receptor Extracellular antigen recognition domains The cytoplasmic domains of the Iga and Igb contain Immunoreceptor Tyrosine -based Activation Motifs (ITAMS) - 2 tyrosine residues separated by 9-12 amino acids - YXX[L/V]X6-9YXX[L/V] Iga Igb Intracytoplasmic signalling domains

Phosphorylation by Src kinases Kinase domain Unique region SH3 domain SH2 domain Enzyme domain phosphorylates tyrosines (to give phosphotyrosine) Phosphotyrosine receptor domain Adaptor protein recruitment domain ITAM binding domain Phosphorylation changes the properties of a protein by changing its conformation Changes in conformation may activate or inhibit a biochemical activity or create a binding site for other proteins Phosphorylation is rapid, requires no protein synthesis or degradation to change the biochemical activity of a target protein It is reversible via the action of phosphatases that remove phosphate

Regulation of Src kinases Kinase domain Unique region SH3 domain SH2 domain Activating tyrosine residue Inhibitory tyrosine residue Phosphorylation of ‘Activating Tyrosine’ stimulates kinase activity Kinase domain Unique region SH3 domain SH2 domain Phosphorylation of ‘Inhibitory Tyrosine’ inhibits kinase activity by blocking access to the Activating Tyrosine Residue

Regulation of Src kinases by Csk and CD45 Kinase domain Unique region SH3 domain SH2 domain C terminus Resting cells: Src kinase is inactivated by a constitutively expressed C -terminal Src kinase - (Csk) Activated cells: a phosphatase associated with the Leukocyte Common Antigen - CD45, removes the C terminus phosphate allowing the activating tyrosine to be phosphorylated Kinase domain Unique region SH3 domain SH2 domain The balance between Csk and CD45 phosphatase activity sets the threshold for initiating receptor signalling

P Phosphorylation of ITAMs by Src kinases P P P P ITAM ITAM ITAM ITAM 1. Csk inactived Src interacts with low affinity with the ITAMs of ‘resting’ receptors P ITAM ITAM 2. Antigen clusters B cell receptors with CD45 phosphatases. Src kinases are phosphorylated P ITAM ITAM P P ITAM 3. Src kinases bind to phosphorylated ITAMS and are activated to phosphorylate adjacent ITAMS P and are activated to phosphorylate ITAMS

Syk protein Tyrosine kinases CD45 phosphatase allows activation of Src family kinases Blk, Fyn & Lyn Receptor cross-linking activates Src kinases that phosphorylate ITAMs in the Iga and Igb ITAM P ITAM One Syk binds to Iga, one to Igb - each Syk transphosphorylates the other Syk - 2 x SH2 domains spaced to bind to two phosphotyrosines on an ITAM P P

The B cell co-receptor CD21 (C3d receptor) CD19 CD81 (TAPA-1) CD45 Iga Igb The B cell co-receptor

Co-receptor phosphorylation C3d opsonised bacterium C3d binds to CD21, the complement receptor 2 (CR2) Antigen recognition Src family kinases then bind the phosphorylated CD19 P P P mIg and CD21 are cross-linked by antigen that has activated complement CD21 is phosphorylated and receptor-associated kinases phosphorylate CD19 Phosphorylated CD19 activates more Src family kinases Ligation of the co-receptor increases B cell receptor signalling 1000 -10,000 fold

Activation of signals that affect gene transcription ITAM P Cell membrane-associated B cell Linker protein - BLNK - contains many Tyrosine residues BLNK Activated Syk phosphorylates BLNK P BLNK binds Tec kinases Tec Tec kinases activate phospholipase C- g (PLC-g) PLC-g cleaves phosphotidylinositol bisphosphate (PIP2) to yield diacylglycerol (DAG) and inositol trisphosphate (IP3) Activated Syk phosphorylates Guanine-nucleotide exchange factors (GEFS) that in turn activate small GTP binding proteins Ras and Rac Ras and Rac activate the MAP kinase cascade

Transmission of signals from the cell surface to the nucleus B cell-specific parts of the signalling cascade are associated with receptors unique to B cells - mIg, CD19 etc. Subsequent signals that transmit signals to the nucleus are common to many different types of cell. The ultimate goal is to activate the transcription of genes, the products of which mediate host defence, proliferation, differentiation etc. Once the B cell-specific parts of the cascade are complete, signalling to the nucleus continues via three common signalling pathways via: The mitogen-activated protein kinase (MAP kinase) pathway Increased in intracellular Ca2+ mediated by IP3 The activation of Protein Kinase C mediated by DAG

Simplified scheme linking antigen recognition with transcription of B cell-specific genes MAP Kinase cascade Small G-protein-activated MAP kinases found in all multicellular animals - activation of MAP kinases ultimately leads to phosphorylation of transcription factors from the AP-1 family such as Fos and Jun. Increases in intracellular calcium via IP3 IP3, produced by PLC-g, binds to calcium channels in the ER and releases intracellular stores of Ca++ into the cytosol. Increased intracellular [Ca++] activate a phospatase, calcineurin, which in turn activates the transcription factor NFAT. Activation of Protein Kinase C family members via DAG DAG stays associated with the membrane and recruits protein kinase C family members. The PKC, serine/threonine protein kinases, ultimately activate the transcription factor NFkB The activated transcription factors AP-1, NFAT and NFkB induce B cell proliferation, differentiation and effector mechanisms

Differentiation in the periphery B B Y B Y Mature peripheral B cell B cell recognises non-self antigen in periphery Ig-secreting plasma cell

B Plasma cells High Yes No Yes Yes Yes Low No Yes No No No Mature B cell Plasma cell Surface Surface High rate Growth Somatic Isotype Ig MHC II Ig secretion hypermut’n switch High Yes No Yes Yes Yes Low No Yes No No No

Summary You should know: Where B cells come from What happens to B cells in the bone marrow How B cell differentiation is linked with Ig gene rearrangement The B cell developmental ‘check points’ that ensure each cell produces a single specificity of antibody that does not react with self How B cells transmit information from the shape and charge of an antigen through the cell membrane to allow the expression of genes in the nucleus What do mature B cells do once activated by an antigen in the periphery?

Recirculating B cells normally pass through lymphoid organs B cells in blood T cell area B cell area Efferent lymph

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

B cells (stained brown) in the Germinal Centre P = Paracortex, Mn = Mantle zone SC = Subcapsular zone

Follicular Dendritic cells (stained blue)in the Germinal Centre

Retention of Antigens on Follicular Dendritic Cells Radiolabelled antigen localises on the surface of Follicular Dendritic cells and persists there, without internalisation, for very long periods

Maturation of Follicular Dendritic cells Club-shaped tips of developing dendrites Filiform dendrites Bead formation on dendrites Bead formation on dendrites

Association of antigen with FDC Antigen enters the germinal centre in the form of an immune complex with C3b and antibodies attached The Immune complexes bind to Fc and complement receptors on the FDC dendrites Complement receptor 3 Ig Fc receptor FDC surface The filiform dendrites of FDC develop beads coated with a thin layer of immune complexes

Iccosome formation and release Iccosomes (black coated particles) bind to and are taken up by B cell surface immunoglobulin DC veils The veils of antigen-bearing dendritic cell surround the beads and the layer of immune complexes is thickened by transfer from the dendritic cell. These beads are then released and are then called ICCOSOMES

Uptake of Iccosomes/Antigen by B cells Anti- B cell Iccosomes bearing different antigens B Y CD40 Surface Ig captures antigen Cross-linking of antigen receptor activates B cell Activated B cell expresses CD40

B B Fate of Antigens Internalised by B cells 1. Capture by antigen specific Ig maximises uptake of a single antigen B 2. Binding and internalisation via Ig induces expression of CD40 3. Antigen enters exogenous antigen processing pathway 4. Peptide fragments of antigen are loaded onto MHC molecules intracellularly. MHC/peptide complexes are expressed at the cell surface

Signal 1 antigen & antigen receptor T cell help to B cells Signal 2 - T cell help Th Th B Y 1. T cell antigen receptor 2. Co-receptor (CD4) Signal 1 antigen & antigen receptor 3.CD40 Ligand

Y Th B T cell help - Signal 2 Cytokines Signal 2 Cytokines Signal 1 IL-4 IL-5 IL-6 IFN-g TGF-b Cytokines B cells are inherently prone to die by apoptosis Signal 1 & 2 upregulate Bcl-XL in the B cell and Bcl-XL prevents apoptosis Signal 1 & 2 thus allow the B cell to survive T cells regulate the survival of B cells and thus control the clonal selection of B cells

Y T cell help - Signal 2 activates hypermutation Th B Receipt of signal 2 by the B cell also activates hypermutation in the CDR - encoding parts of the Ig genes Signal 2, and thus T cells, regulate which B cells are clonally selected. Low affinity Ig takes up and presents Ag to T cells inefficiently. Inefficient presentation to T cells does not induce CD40. With no signal 2 delivered by CD40, low affinity B cells die. Only B cells with high affinity Ig survive - This is affinity maturation Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 CDR1 CDR2 CDR3 Day 6 Day 8 Day 12 Day 18 Deleterious mutation Beneficial mutation Neutral mutation Lower affinity - Not clonally selected Higher affinity - Clonally selected Identical affinity - No influence on clonal selection

Control of Affinity & Affinity Maturation Five B cell antigen receptors - all specific for , but with different affinities due to somatic hypermutation of Ig genes in the germinal centre B Only this cell, that has a high affinity for antigen can express CD40. Only this cell can receive signal 2 Only this cell is rescued from apoptosis i.e. clonally selected The cells with lower affinity receptors die of apoptosis by neglect

Germinal Centre Macrophages (stained brown) Clean Up Apoptotic Cells GC = Germinal Centre, TBM = Tingible Body Macrophages

Y Role of T cell cytokines in T cell help Th B Cytokines Signal 2 PC IL-4 IL-5 IL-6 IFN-g TGF-b B B B B PC B B B B B B B B B Proliferation & Differentiation IgM IgG3 IgG1 IgG2b IgG2a IgE IgA IL4 inhibits inhibits induces inhibits induces IL-5 augments IFN-g inhibits induces inhibits induces inhibits TGF-b inhibits inhibits induces induces

Regulation of specificity - Cognate recognition 1. T cells can only help the B cells that present antigen to them 2. B cells are best at presenting antigens that they take up most efficiently 3. B cells are most efficient at taking up antigens that their B cell antigen receptors bind to 4. T and B cells help each other to amplify immunity specific for the same antigen i.e. Regulates the Characteristics of Adaptive Immunity Sharply focuses specificity - Pathogen specificity Improves specificity & affinity - Better on 2nd exposure Is specific antigen dependent - Learnt by experience Seeds memory in T and B cell pools

Synaptic tethering of a B cell (red) to a T cell (green)

T cell (in centre) surrounded by B cells with cytoskeleton stained green

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

B cells (90%) and T cells (10%) migrate to form a primary follicle 1. Antigen loaded dendritic cells migrate from subcapsular sinus to paracortical area of the lymph node DC B cells (90%) and T cells (10%) migrate to form a primary follicle B T Primary follicle formation T 3. T cells proliferate T 2. T cells migrate through HEV and are trapped by antigen on DC B B 4. B cells migrate through HEV - most pass through the paracortex and primary follicle. HEV Some interact with T cells and proliferate to form a primary focus

T cell motility in the lymph node

Germinal Centre Microanatomy Primary Follicles become secondary follicles when germinal centres develop Germinal Centre Microanatomy 2. B cells (centrocytes) upregulate surface Ig, stop dividing and receive costimulatory signals from T cells and FDC 4. Selected cells leave lymph node as memory cells or plasma cells Dark zone Light zone T Follicular dendritic cells select useful B cells B 3. Apoptosis of self-reactive & unselected cells 1. B cells (centroblasts) downregulate surface Ig, proliferate, somatically hypermutate their Ig genes. AFFINITY MATURATION

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

Few non-template encoded (N) regions in the IgM B-1 B Cells IgM uses a distinctive & restricted range of V regions CD5 B Y IgM Few non-template encoded (N) regions in the IgM Recognises repeating epitope Ag such as phospholipid phosphotidyl choline & polysaccharides NATURAL ANTIBODY 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

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 Yes Rarely Protein specificity Rarely Yes Need T cell help No Yes Somatic hypermutation of Ig No High Memory development No Yes Yes Rarely Rarely Yes No Yes Carbohydrate specificity Protein specificity Need T cell help Specificity & requirement for T cell help suggests strikingly different types of antigens are seen by B-1 and B-2 B cells

T Independent Antigens (TI-2) TI-2 Antigen Y Immature B-2 Cell Y 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 Y Y Y Y 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

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

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

T Dependent & Independent Antigens Induce responses in babies Yes Yes Induce responses in athymics No Yes Yes Prime T cells Yes No No Polyclonally activate B cells No Yes No Require repeating epitopes No No Yes T Dependent & Independent Antigens No

Y Y Y Y Y Y Why are babies unresponsive to TI-2 antigens? In adults: Immature B-2 Cell Y Mature B-1 Adult immature B cells that bind to multivalent self Ag undergo apoptosis Y Y Y Y IgM Adult non-bone marrow derived B-1 cells are directly stimulated by antigens containing multivalent epitopes produce IgM WITHOUT T cell help.

Y Why are babies unresponsive to TI-2 antigens? In babies: All B cells, B-1 & B-2, are immature TI-2 Antigen Immature B cells that bind to multivalent self Ag undergo apoptosis Y Immature B-1 Cell As with adult B cells, immature B cells that bind multivalent self Ag undergo apoptosis Hence babies do not respond to TI-2 antigens. Babies are, therefore susceptible to pathogens with multivalent antigens such as those on pneumococcus

T Dependent & Independent Antigens Induces response in babies Yes Yes No Induces response in athymia No Yes Yes Primes T cells Yes No No Polyclonally activates B cells No Yes No Requires repeating epitopes No No Yes TD: Activate B-1 and B-2 B cells TI-1: Activate B-1 and B-2 B cells TI-2: Activate only B-1 B cells Examples TD: Diptheria toxin, influenza heamagglutinin, Mycobacterium tuberculosis TI-1: Bacterial lipopolysaccharides, Brucella abortis TI-2: Pneumococcal polysaccharides, Salmonella polymerised flagellin

Immune effector mechanisms against extracellular pathogens & toxins NEUTRALISATION Toxin Y ` Y ` Bacterium Y ` Adhesion to host cells blocked Prevents invasion Toxin release blocked Prevents toxicity Y ` NEUTRALISING ANTIBODIES

Effector mechanisms against extracellular pathogens OPSONISATION Bacteria in extracellular space Ab + Fc receptor binding Phagocytosis OPSONISATION

Effector mechanisms against extracellular pathogens COMPLEMENT Activation Bacteria in plasma Lysis Ab & COMPLEMENT + Phagocytosis binding Complement & Fc receptor Opsonisation

Summary B cell tolerance of self is by clonal deletion or anergy of self-reactive cells Receptor editing increases the efficiency of B cell development Follicular dendritic cells acquire antigen and transfer it to B cells T cell help to B cells is via CD40L and cytokines CD40 expression indirectly leads to Ig affinity maturation Germinal centre microanatomy & function There are two lineages of B cells - B1 and B2 B cells The dependency of B cells upon T cells varies