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B Cells and B Cell Development © Dr. Colin R.A. Hewitt

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1 B Cells and B Cell Development © Dr. Colin R.A. Hewitt

2 The discovery of B cell immunity 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 Bursectomised chickens were later used in experiments to raise antibodies to Salmonella antigens None of the bursectomised chickens made anti-Salmonella antibodies 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

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

4 B cell development in the bone marrow B Regulates construction of an antigen receptor Bone Marrow provides a MATURATION & DIFFERENTIATION MICROENVIRONMENT for B cell development 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

5 Bone Marrow E M M S

6

7 Immature & mature B Central Sinus Progenitors Pre-B Stromal cells X X X EndoosteumEndoosteum Macrophage Scheme of B Cell Development in the Bone Marrow

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

9 Bone marrow stromal cell Maturing B cells

10 B B Stromal cell

11 Peripheral Stages of B cell development Stem CellEarly pro-B cellLate pro-B cellLarge pre-B cell Small pre-B cellImmature B cellMature 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

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

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

14 Stages of differentiation in the bone marrow are defined by Ig gene rearrangement B CELL STAGE IgH GENE CONFIGURATION Stem cellEarly pro-BLate pro-BLarge pre-B Germline D H to J H V H to D H J H VHDHJHVHDHJH Pre-B cell receptor expressed Ig light chain gene has not yet rearranged

15 B cell receptor Transiently expressed when V H D H J H C H  is productively rearranged VpreB/ 5 - the surrogate light chain, is required for surface expression Ig  & Ig  signal transduction molecules CHCH Heavy chain V H D H J H Light chain V L J L C L VpreB 5 Ligand for the pre-B cell receptor may be galectin 1, heparan sulphate, other pre-BCR or something as yet unknown Pre-

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

17 Evidence for allelic exclusion Allotypes can be identified by staining B cell surface Ig with antibodies a/a b/b a/b Y B b Y B a Y B b Y Y B ab Y B a AND ALLOTYPE- polymorphism in the C region of Ig – one allotype inherited from each parent 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)

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

19 Allelic exclusion is needed for efficient clonal selection 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 S. typhi Antibody S. typhi

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

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

22 Translation in frame 1 MKXLWFFLLLVAAPRWVLSQV HLQESGPGLGKPPELKTPLGD TTHTCPRCPEPKSCDTPPPCP RCPEPKSCDTPPPCPRCPEPK SCDTPPPCXXCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTC VVVDVSHEDXXVQFKWYVDG VEVHNAKTKLREEQYNSTFRV VSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPEE MTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYNTTPPMLD SDGSFFLYSKLTVDKSRWQQG NIFSCSVMHEALHNRYTQKSLS LSPGK* Human IgG3 Heavy Chain nucleotide sequence ATGAAACANCTGTGGTTCTTCCTTCTCCTGG TGGCAGCTCCCAGATGGGTCCTGTCCCAGG TGCACCTGCAGGAGTCGGGCCCAGGACTGG GGAAGCCTCCAGAGCTCAAAACCCCACTTGG TGACACAACTCACACATGCCCACGGTGCCCA GAGCCCAAATCTTGTGACACACCTCCCCCGT GCCCACGGTGCCCAGAGCCCAAATCTTGTG ACACACCTCCCCCATGCCCACGGTGCCCAG AGCCCAAATCTTGTGACACACCTCCCCCGTG CCCNNNGTGCCCAGCACCTGAACTCTTGGG AGGACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGATACCCTTATGATTTCCCGGACCC CTGAGGTCACGTGCGTGGTGGTGGACGTGA GCCACGAAGACCCNNNNGTCCAGTTCAAGT GGTACGTGGACGGCGTGGAGGTGCATAATG CCAAGACAAAGCTGCGGGAGGAGCAGTACA ACAGCACGTTCCGTGTGGTCAGCGTCCTCAC CGTCCTGCACCAGGACTGGCTGAACGGCAA GGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCA AAGCCAAAGGACAGCCCGAGGAGATGACCA AGAACCAAGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTACCCCAGCGACATCGCCGTGGA GTGGGAGAGCAATGGGCAGCCGGAGAACAA CTACAACACCACGCCTCCCATGCTGGACTCC GACGGCTCCTTCTTCCTCTACAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGA ACATCTTCTCATGCTCCGTGATGCATGAGGC TCTGCACAACCGCTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAATGA Large pre-B cells need in frame V H D H J H joins to mature Translation in frame 2 (no protein) * Translation in frame 3 ETXVVLPSPGGSSQMGPVPGA PAGVGPRTGEASRAQNPTW* Large pre-B Development continues Pre-B cell receptor can be activated Development arrests Development arrests

23 Proliferation Y Immature B cell Light chain expressed IgM displayed on surface IgM Ligation of the pre-B cell receptor triggers entry into the cell cycle Large pre-B Many large pre-B cells with identical pre-B receptors Large pre-B Intracellular VDJC H chain V L -J L rearranges Proliferation stops Pre-receptor not displayed Small pre-B

24 VDJCDJVCDJVCDJ V Germline D H -J H joining V H -D H J H joining VJCVCJ V Germline V L -J L joining Heavy and light chain rearrangement is potentially wasteful Large pre-B Small pre-B With two “random” joins to generate a heavy chain there is a 1:9 chance of a rearrangement of being in frame 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

25 B cells have several chances to successfully rearrange Ig genes Early Pro BLate Pro B Pre B V H -DJ H On first chromosome V H -DJ H On second chromosome Immature B NO YES NO B  on first chromosome  on second chromosome on first chromosome on second chromosome NO YES NO YES Y IgM  B Y IgM B D H -J H On first chromosome D H -J H On second chromosome YES NO

26 B Y Y Y Y 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 Acquisition of antigen specificity creates a need to check for recognition of self antigens 1.Physical removal from the repertoireDELETION 2.Paralysis of functionANERGY 3.Alteration of specificityRECEPTOR EDITING

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

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

29 Receptor editing A rearrangement encoding a self specific receptor can be replaced VCDJ VVV Y B B !!Receptor recognises self antigen!! B Apoptosis or anergy Y B B Edited receptor now recognises a different antigen and can be rechecked for specificity CDJ VVVV Arrest development And reactivate RAG-1 and RAG-2

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

31 How can B cells express IgM and IgD simultaneously? C2C2CC C4C4C2C2C1C1C1C1C3C3CC CC CC CC C3C3 VDJ S3S3 CC CC C3C3 C1C1 S1S1 C1C1 C3C3 C1C1 C3C3 IgG3 produced. Switch from IgM VDJ C1C1 IgA1 produced. Switch from IgG3 VDJ C1C1 IgA1 produced. Switch from IgM N.B. Remember Molecular Genetics of Immunoglobulins lecture – No C  switch region Consider similarities with mechanism allowing secreted and membrane Ig by the same cell

32 Splicing of IgM and IgD RNA C1C1 C2C2C3C3C4C4 C1C1C2C2 C3C3 pA1 V D J C1C1 C2C2C3C3C4C4 C1C1C2C2 C3C3 V D J AAA RNA cleaved and polyadenylated at pA2 V D J CC IgM mRNA V D J CC IgD mRNA C1C1C3C3CC CC V D J C1C1 C2C2C3C3C4C4 C1C1C2C2 C3C3 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

33 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? Summary

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

35 Ring staining 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

36 Transduction of signals by the B cell receptor Ig  Ig  Intracytoplasmic signalling domains Extracellular antigen recognition domains The cytoplasmic domains of the Ig  and Ig  contain Immunoreceptor Tyrosine - based Activation Motifs (ITAMS) - 2 tyrosine residues separated by 9-12 amino acids - YXX[L/V]X 6-9 YXX[L/V]

37 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 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 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 by Src kinases

38 Regulation of Src kinases Kinase domain Unique region SH3 domain SH2 domain Activating tyrosine residueInhibitory 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

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

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

41 One Syk binds to Ig , one to Ig  - each Syk transphosphorylates the other Syk - 2 x SH2 domains spaced to bind to two phosphotyrosines on an ITAM 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 Ig  and Ig  ITAM P P P P P P P P

42 CD21 (C3d receptor) CD19 CD81 (TAPA-1) Ig  Ig  CD45 The B cell co-receptor

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

44 BLNK Cell membrane-associated B cell Linker protein - BLNK - contains many Tyrosine residues Activated Syk phosphorylates BLNK P P P P BLNK binds Tec kinases Tec Tec kinases activate phospholipase C-  (PLC-  ) PLC-  cleaves phosphotidylinositol bisphosphate (PIP 2 ) to yield diacylglycerol (DAG) and inositol trisphosphate (IP 3 ) ITAM P P P P P P 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 Activation of signals that affect gene transcription

45 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: 1.The mitogen-activated protein kinase (MAP kinase) pathway 2. Increased in intracellular Ca2+ mediated by IP 3 3.The activation of Protein Kinase C mediated by DAG

46 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 IP 3 IP 3, produced by PLC- , 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 NF  B The activated transcription factors AP-1, NFAT and NF  B induce B cell proliferation, differentiation and effector mechanisms Simplified scheme linking antigen recognition with transcription of B cell-specific genes

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

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

49 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? Summary

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

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

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

53 Follicular Dendritic cells (stained blue)in the Germinal Centre

54 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

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

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

57 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 Iccosome formation and release DC veils Iccosomes (black coated particles) bind to and are taken up by B cell surface immunoglobulin

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

59 B 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 Fate of Antigens Internalised by B cells 1. Capture by antigen specific Ig maximises uptake of a single antigen

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

61 T cell help - Signal 2 Y Y Y B Th Signal 2 Signal 1 B cells are inherently prone to die by apoptosis Signal 1 & 2 upregulate Bcl-X L in the B cell and Bcl-X L 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 Cytokines IL-4 IL-5 IL-6 IFN-  TGF- 

62 T cell help - Signal 2 activates hypermutation Y Y Y B Th Signal 2 Signal 1 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 CDR1CDR2CDR3 Day 6 CDR1CDR2CDR3CDR1CDR2CDR3CDR1CDR2CDR3 Day 8Day 12Day 18 Deleterious mutation Beneficial mutation Neutral mutation Lower affinity - Not clonally selected Higher affinity - Clonally selected Identical affinity - No influence on clonal selection

63 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 B BB 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

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

65 Role of T cell cytokines in T cell help Th Signal 2 Y Y Y B Signal 1 Cytokines IL-4 IL-5 IL-6 IFN-  TGF-  IgMIgG3IgG1IgG2b IgG2aIgEIgA IL4inhibitsinhibitsinducesinhibitsinduces IL-5augments IFN-  inhibitsinduces inhibitsinduces inhibits TGF-  inhibitsinhibitsinduces induces PC BBBBBBBBBBBBBB Proliferation & Differentiation

66 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

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

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

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

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

71 T cell motility in the lymph node

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

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

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

75 Comparison of B-1 and B-2 B cell properties PropertyB-1 cellsB-2 cells N regionsFewExtensive V region repertoireRestrictedDiverse LocationPeritoneum/pleuraEverywhere RenewalSelf renewal in situBone marrow Spontaneous Ig productionHighLow IsotypesIgMIgM/G/A/D/E Carbohydrate specificityYesRarely Protein specificityRarelyYes Need T cell helpNoYes Somatic hypermutation of IgNoHigh Memory developmentNoYes 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

76 T Independent Antigens (TI-2) 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 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y IgM Y Y Y Y Y Mature B-1 Y Y Immature B-2 Cell 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 TI-2 Antigen

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

78 YYYYYY BBBBBB YYYYYY YYYYYY YYYYYY YYYYYY YYYYYY YYYYYY T Independent Antigens (TI-1 e.g. LPS) 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 LPS complexes with CD14, LPSBP & TLR4

79 T Dependent Antigens TI-1 Antigens TI-2 Antigens Induce responses in babiesYesYes Induce responses in athymicsNoYesYes Prime T cellsYesNoNo Polyclonally activate B cellsNoYesNo Require repeating epitopesNoNoYes T Dependent & Independent Antigens No

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

81 Y Y Immature B-1 Cell TI-2 Antigen Why are babies unresponsive to TI-2 antigens? Immature B cells that bind to multivalent self Ag undergo apoptosis 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 In babies: All B cells, B-1 & B-2, are immature

82 T Dependent Antigens TI-1 Antigens TI-2 Antigens Induces response in babiesYesYesNo Induces response in athymiaNoYesYes Primes T cellsYesNoNo Polyclonally activates B cellsNoYesNo Requires repeating epitopesNoNoYes T Dependent & Independent Antigens Examples TD:Diptheria toxin, influenza heamagglutinin, Mycobacterium tuberculosis TI-1: Bacterial lipopolysaccharides, Brucella abortis TI-2:Pneumococcal polysaccharides, Salmonella polymerised flagellin 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

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

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

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

86 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


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