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1、Lie Wang (汪洌）浙江省杭州市浙江大学紫金港校区医学院科研楼A810-814医学院免疫学研究所B lymphocytes and Humoral Immune ResponseThe discovery of B cell immunity1954 - Bruce Glick, Ohio State UniversityStudies on the function of the bursa of Fabricius, a lymphoid organ in the cloacal region of the chickenBursa was later found to be the
2、 organ in which antibody producing cells developed antibody producing cells were thereafter called B cellsMammals do not have a bursa of FabriciusTransfer marked foetalliver cellsOrigin of B cells and organ of B cellmaturationNo MatureB cellsAfter birth, development continues in the bone marrowNorma
3、l bone marrowDefective bone marrowMature markedB cellsin peripheryB cell development starts in the foetal liverB cell development in the bone marrowBRegulates construction of an antigen receptorBone Marrow provides aMATURATION & DIFFERENTIATION MICROENVIRONMENTfor B cell developmentEnsures each cell
4、 has only one specificityBChecks and disposes of self-reactive B cellsBExports useful cells to the peripheryBProvides a site for antibody productionBSecretedFactors - CYTOKINES2. Secretion of cytokines by stromal cellsBBone marrow stromal cells nurture developing B cellsTypes of cytokines and cell-c
5、ell contacts needed at each stage of differentiation are differentStromal cell1. Specific cell-cell contacts between stromal cells and developing B cellsCell-cell contactBone marrow stromal cellMaturing B cellsBBStromal cellB lineage commitmentHSC (hematopoietic stem cell) MPP ( (淋巴系髓系多能前体细胞淋巴系髓系多能前
6、体细胞) ) ELP (earliest lymphocyte progenitor) ETP (early T-lineage progenitor)CLP (common lymphoid progenitor) HSCHSCHSCHSCDevelopment and migration of B cells (An overview)Stages of differentiation in the bone marrow aredefined by Ig gene rearrangementB CELL STAGEIgH GENECONFIGURATIONStem cellEarly p
7、ro-BLate pro-BLarge pre-BGermlineDH to JHVH to DHJHVHDHJHPre-B cellreceptorexpressedIg light chain gene has not yet rearrangedB cell receptorTransiently expressed when VHDHJH CHm is productively rearrangedIga & Igb signaltransductionmoleculesCHmHeavy chainVHDHJHLight chainVLJLCLVpreBl5Pre-B cell dev
8、elopment is coupled with rearrangement of heavy-chain B cell development is coupled with rearrangement of heavy-chain B cell development is coupled with rearrangement of heavy-chain B cell development is coupled with rearrangement of heavy-chain B cell development is coupled with rearrangement of he
9、avy-chain B cell development is coupled with rearrangement of heavy-chainSplicing of IgM and IgD RNACm1Cm2Cm3Cm4Cd1Cd2Cd3pA1VDJCm1Cm2Cm3Cm4Cd1Cd2Cd3VDJAAA RNA cleaved and polyadenylated at pA2VDJ CmIgM mRNAVDJ CdIgD mRNAC1C3CdCmVDJCm1Cm2Cm3Cm4Cd1Cd2Cd3DNApA1pA2VDJTwo types of mRNA can be made simult
10、aneously in the cell by differential usage of alternative polyadenylation sites and splicing of the RNARNA cleaved and polyadenylated at pA1AAA B cell development is coupled with rearrangement of heavy-chain6000 heavy chains combined with 320 light chains 1.9X106 Abs Ligation of the pre-B cell recep
11、tor1. Ensures only one specificty ofAb expressed per cellLargePre-BStromal cellUnconfirmed ligand of pre-B cell receptor2. Triggers entry into cell cycleALLELIC EXCLUSIONExpression of a gene on one chromosome prevents expression of the allele on the second chromosome1. Suppresses further H chain rea
12、rrangement2. Expands only the pre-Bcells with in frame VHDHJH joinsEvidence for allelic exclusionAllotypes can be identified by staining B cell surface Ig with antibodiesa/ab/ba/bYBbYBaYBbYYBabYBaANDALLOTYPE- polymorphism in the C region of Ig one allotype inherited from each parentSuppression of H
13、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)YYYYSuppression of H chain gene rearrangementensures only one specificity of Ab expressed per cell.
14、Allelic exclusion prevents unwanted responsesBSelf antigenexpressed bye.g. brain cellsS. aureusYYYYYBS. aureusYYYYYYYAntiS. aureusAntibodiesYYYYYYAntibrainAbsOne Ag receptor per cellIF there were two Ag receptors per cellYYYYYYYAntiS. aureusAntibodiesPrevents induction of unwanted responses by patho
15、gensAllelic exclusion is needed for efficient clonal selectionAll daughter cells must express the same Ig specificityotherwise the efficiency of the response would be compromisedSuppression of H chain gene rearrangement helps prevent the emergence ofnew daughter specificities during proliferation af
16、ter clonal selectionS. typhiAntibodyS. typhiAllelic exclusion is needed to prevent holes in the repertoire Exclusion of anti-brain B cells i.e. self toleranceYYB BOne specificity of Agreceptor per cellS. aureusAnti-brain IgANDanti-S. aureus IgYYYB BIF there were two specificitiesof Ag receptor per c
17、ellAnti-brain IgBBDeletionAnergyORanti S.aureus B cells will be excluded leaving a “hole in the repertoire”BUTYYYB B1. Suppresses further H chain rearrangementLigation of the pre-B cell receptor1. Ensures only one specificity ofAb expressed per cellLargePre-BStromal cellUnconfirmed ligand of pre-B c
18、ell receptor2. Triggers entry into cell cycle2. Expands only the pre-Bcells with in frame VHDHJH joinsLargePre-BLargePre-BLargePre-BLargePre-BLargePre-BLargePre-BLargePre-BLargePre-BLargePre-BLargePre-BProliferationYImmatureB cellLight chain expressedIgM displayed on surfaceIgMLigation of the pre-B
19、cell receptor triggers entry into the cell cycleLargepre-BMany large pre-B cells with identical pre-B receptorsLarge pre-BIntracellular VDJCH chainVL-JL rearrangesProliferation stopsPre-receptor not displayedSmall pre-BBYYYYBSmall pre-B cellNo antigen receptor at cell surfaceUnable to sense Ag envir
20、onment!May be self-reactive!Immature B cellCell surface Ig expressedAble to sense Ag environmentCan now be checked for self-reactivityAcquisition of antigen specificity creates a needto check for recognition of self antigens1.Physical removal from the repertoireDELETION2.Paralysis of functionANERGY3
21、.Alteration of specificityRECEPTOR EDITINGB cell self tolerance: clonal deletionImmature B cell recognisesMULTIVALENTself AgBClonal deletion byapoptosisY YBImmatureBBSmallpre-BSmall pre-B cellassembles IgYB cell self tolerance: anergyBYYYBAnergic B cellIgD normal IgM lowImmature B cell recognisessol
22、uble self AgNo cross-linkingYYBImmatureBBSmallpre-BSmall pre-B cellassembles IgIgMIgDIgDIgDReceptor editingA rearrangement encoding a self specific receptor can be replacedVCD JVVVYBB!Receptorrecognisesself antigen!BApoptosisor anergyYBBEdited receptor now recognisesa different antigen and can berec
23、hecked for specificityCD JVVVVArrest developmentAnd reactivateRAG-1 and RAG-2YYYYYYMature B cellexported to theperipheryYYB cell self tolerance: export of self tolerant B cellsIgD and IgM normalIgMIgDIgDIgDIgDIgMIgMIgMImmature B cell doesnt recognise anyself AgYYBImmatureBBSmallpre-BSmall pre-B cell
24、assembles IgBB-cell surface markers1. BCR complexBCR (mIg): VH, VL-Ag binding site mature B cells: mIgM and mIgD. Function: specifically recognizes antigen. Ig /Ig (CD79a/CD79b): heterodimer cytoplasmic domains contain ITAM. Function: transduce the signals that lead to B cell activation.Antigen rece
25、ptor-mediated signal transduction in B cellsCD19/CD21/CD81complex CD21=CR2, C3dR, EB virus receptor CD19/CD21/CD81 interactions with complement associated with antigen play a role in antigen-induced B-cell activation.2. Co-receptorsThe role of the coreceptor in B cell activation(1)CD40 interacts wit
26、h CD40L (Th cell) (2)CD80(B7.1), CD86(B7.2) Expressed on activated B cells and other APCs (3)ICAM-1 （CD54）、LFA-1（CD11/CD18)： mediate cell-cell interaction and co-stimulation3. Co-stimulatory molecules(1)CD20: function is unclear. It is suspected that it acts as a calcium channel in the cell membrane
27、(2)CD22：Inhibitory receptor with ITIM motif(3)CD32 (FcRII)： Inhibitory receptor(4)Cytokine receptors(5)Complement Receptors(6)Toll-like receptors(7)MHC 4. Other receptors3. Subtype of B cells1.Conventional B cells (B-2 cells) 2.B-1 cells (expression of CD5)CD5Two B cell lineagesBB cell precursorBMat
28、ure B cellB2 B cellsPlasma cellYYYYYYYYYPCIgGBYYYYYYYYYYYYYYYYYYYYIgM - no other isotypesBDistinct B cellprecursor?B1 B cellsPrimitive B cells found in pleura and peritoneumB-2 cells : conventional B cells Recirculating follicular B cells : circulate between LN follicles and bloodmIg: IgM, IgDProduc
29、e IgG after antigenic stimulation in the presence of T helper cells B1 cells (CD5+): Many of the first B cells that appear during ontogeny express CD5, a marker originally found on T cells. (express mIgM, no mIgD). They respond well to TI-Ag and may also be involved in the Ag processing and presenta
30、tion to T cells. Functions 1. produce anti-bacterial IgM the first line of defence against microorganisms; 2. produce polyreactive Ab clearance of denatured self components; 3. produce auto-Ab, thereby participating in the pathogenesis of some autoimmune diseases. Comparison of B-1 and B-2 B cell pr
31、opertiesPropertyB-1 cellsB-2 cellsN regionsFewExtensiveV region repertoireRestrictedDiverseLocationPeritoneum/pleuraEverywhereRenewalSelf renewal in situBone marrowSpontaneous Ig productionHighLowIsotypesIgMIgM/G/A/D/ECarbohydrate specificityYesRarelyProtein specificityRarelyYesNeed T cell helpNoYes
32、Somatic hypermutation of IgNoHighMemory developmentNoYesYes RarelyRarely YesNo YesCarbohydrate specificityProtein specificityNeed T cell helpSpecificity & requirement for T cell help suggests strikingly different typesof antigens are seen by B-1 and B-2 B cells4. Function of B cells1. Production of
33、antibody Abs prevent microorganism from entry into cells and eliminate microorganisms by opsonization causing phagocytosis, complement activation and toxin neutralization.2. Ag presentation to T cells3. Immune regulation Secretion of cytokines (TNF, IFN, IL-12) M, DC, NK, B cell. Co-stimulation of T
34、 cellsT cell proliferation.YYImmune effector mechanisms against extracellular pathogens & toxinsNEUTRALISATIONYToxin releaseblockedPreventstoxicityNEUTRALISING ANTIBODIESAdhesion tohost cells blockedPreventsinvasionBacteriumYToxinFc receptorbindingEffector mechanisms against extracellular pathogensO
35、PSONISATIONOPSONISATIONPhagocytosisBacteria in extracellular spaceAb+Effector mechanisms against extracellular pathogensCOMPLEMENT ActivationBacteria in plasmaAb & COMPLEMENT+PhagocytosisbindingComplement &Fc receptorLysisOpsonisationADCCB cell-mediated humoral immune responseHumoral immunity is med
36、iated by antibodies and is the arm of the adaptive immune response that functions to neutralize and eliminate extracellular microbes and microbial toxins.It is more important than cellular immunity in defending against microbes with capsules rich in polysaccharides and lipids.TD-Ag： T cell-dependent
37、TI-Ag： T cell-independentResponse to TD-Ag1) B cells recognize TD-Ag a. BCR directly recognizes B cell epitopes b. Ig /Ig transfer the first signalc. Signaling pathwaysd. Effect of coreceptors (CD21/CD19/CD81) Transduction of signals by the B cell receptorIgaIgbIntracytoplasmicsignalling domainsExtr
38、acellular antigenrecognition domainsThe cytoplasmic domains of the Iga and Igb contain Immunoreceptor Tyrosine -based Activation Motifs (ITAMS) - 2 tyrosine residues separated by 9-12 amino acids - YXXL/VX6-9YXXL/VBCR Signaling IBCR Signaling IIThe role of co-receptors (CD19/CD21/CD81) in B cell act
39、ivation2) Role of Th cells in humoral immune response to TD-AgFor a protein Ag to stimulate Ab response, B cells and Th cells specific for that Ag must come together in lymphoid organs and interact in a way that stimulates B cell proliferation and differentiation.a. Activation and migration of helpe
40、r T cellsTh cells that have been activated to differentiate into effector cells interact with antigen-stimulated B cells at the edges of lymphoid follicles in the peripheral lymphoid organs.The interactions of Th cells and B cells in lymphoid tissues.b. Presentation of Ags by B cells to Th cellsB ce
41、lls that bind protein Ags by their BCR endocytose these Ags, process them in endosomal vesicles, display MHC II-peptides for recognition of Th cells. B cells and Th cells recognize different epitopes of the same protein Ag.Ag presentation by B cells to Th cellsc. Mechanisms of Th cell-mediated activ
42、ation of B cellsTh cells that recognize Ag presented by B cells activate B cells by expressing CD40L and by secreting cytokines (IL-2, IFN-, IL-4, IL-5, IL-6, IL-13, etc.).Mechanisms of Th cell-mediated activation of B cellsKey components of T cell helpCD40L triggers CD40 - synergizes with BCR signa
43、ls to promote mitosis; cytokine (e.g. IL4) signals also contributeFasL triggers Fas - BCR signaling protects from apoptosisT cell derived cytokines influence differentiation, isotype switching:IL2, IL6 promote differentiationIL4 - IgG1, IgEIFN - IgG2a, IgG3TGFb and IL5 IgAMany other molecules involv
44、ed in T-B interactions e.g. ICAM1/LFA1, ICOS/ICOSL, CD30/CD30L, CD27L/CD27, OX40L/OX40, .d. Th cells stimulate B cells to produce Abs of different heavy chain classes (isotypes)Heavy chain class switching is initiated by CD40L-mediated signals, and switching to different classes is stimulated by dif
45、ferent cytokines.Ig heavy chain class (isotype) switching（蠕虫）Ig heavy chain class (isotype) switchingMechanisms of Ig heavy chain class switchinge. Affinity maturation in Ab responsesAffinity maturation is the process by which the affinity of Abs produced in response to a protein Ag increases with p
46、rolonged and repeated exposure to that Ag.The increase in affinity is due to point mutations in the V regions, and particularly in the Ag-binding HVR, of the Abs produced.Affinity maturation occurs in the germinal centers of lymphoid follicles.Affinity maturation in antibody responses somatic mutati
47、onControl of Affinity & Affinity MaturationFive B cell antigenreceptors - all specificfor , but withdifferent affinitiesdue to somatichypermutationof Ig genes in the germinal centreBBBBBOnly this cell, that has a high affinity for antigen can express CD40.Only this cell can receive signal 2Only this
48、 cell is rescued from apoptosis i.e. clonally selectedThe cells with lower affinity receptors die of apoptosis by neglect Affinity maturation in antibody responses - Selection of high affinity B cellsThe anatomy of humoral immune responsesA fraction of the activated B cells, which are often the prog
49、eny of class-switched high-affinity B cells, do not differentiate into Ab secretors but instead become memory B cells.Germinal CentersFunction: to generate B cells that produce antibodies with increased affinity for the inducing antigen = affinity maturationGerminal Center Reaction:Activated B cells
50、 give rise to Centroblasts （中心母（中心母细胞）胞）- localize in follicle, undergo rapid cell division and turn on machinery that causes somatic mutation in V-regionsCentroblasts give rise to Centrocytes （生（生发中心中心细胞）胞）- migrate to the FDC-rich region of the Germinal Center- survival is dependent on interaction
51、 with FDC-bound Ag and presentation of Ag to T cells- centrocytes that successfully compete to bind antigen (e.g. by having higher affinity BCR) and to receive T cell help are selected and may differentiate into long-lived plasma cells or memory B cellsAssociation of antigen with FDCin the form of a
52、n immune complex with C3d and antibodies attachedAntigen enters the germinal centreComplement receptor 3Ig Fc receptorFDC surfaceThe filiform dendrites of FDC develop beads coated with a thin layer of immune complexesThe Immune complexes bind to Fc and complement receptors on the FDC dendritesThe ve
53、ils 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 ICCOSOMESIccosome formation and releaseDC veilsIccosomes (black coated particles) bind to and are taken up by
54、 B cellsurface immunoglobulinYYYIccosomes bearing different antigensBUptake of Iccosomes/Antigen by B cellsAnti- B cellSurface Ig captures antigenCross-linking of antigen receptor activates B cellActivated B cell expresses CD40CD40Germinal Centers3. B cell response to TI-Ag* TI-1 (B cell mitogen) ac
55、tivate B cells- Antigen crosslink to BCR and mitogen receptors-Or simpely crosslink mitogen receptors in a high dose (LPS) * TI-2 activate mature B cells directly -the repeated epitopes combine with BCR BCR cross-linkingproduce IgM (bacteria capsular polysaccharides)3. Immune response of B cells to
56、TI-AgNo Th help，no memory, early effectTDependentAntigensTI-1AntigensTI-2AntigensInduces response in babiesYesYesNoInduces response in athymiaNoYesYesPrimes T cellsYesNoNoPolyclonally activates B cellsNoYesNoRequires repeating epitopesNoNoYesT Dependent & Independent AntigensExamplesTD: Diptheria to
57、xin, influenza heamagglutinin, Mycobacterium tuberculosisTI-1: Bacterial lipopolysaccharides, Brucella abortisTI-2: Pneumococcal polysaccharides, Salmonella polymerised flagellinTD: Activate B-1 and B-2 B cellsTI-1: Activate B-1 and B-2 B cellsTI-2: Activate only B-1 B cellsPrimary immune response -
58、 longer latent phase; - smaller peak response (lower Ab titer); - remaining in the serum at detectable levels for much shorter periods; - lower average affinity; - usually IgM;4. General features of Ab responses in vivo(The immune response followed by secondary antigenic challenge) - shorter latent phase; - bigger peak response (higher Ab titer); - remaining in the serum at detectable levels for much longer periods; - higher average affinity; - usually IgG.Secondary immune responseFeatures of primary and secondary antibody responses
