免疫学第三章Bcell

B细胞及其介导的免疫应答B淋巴细胞分化发育在中枢免疫器官中的分化发育1. 祖 B 细胞（pro-B cell）--表达 Igα/Igβ 异源二聚体2. 前 B 细胞（pre-B cell）--表达 μ 链及替代轻链，表达 pre-BCR3. 未成熟 B 细胞（immature B cell）--表达 BCR，为 mIgM4. 成熟 B 细胞（mature B cell）--同时表达 mIgM 和 mIgD——活化后只表达一种在中枢免疫耐受的形成克隆清除未成熟B细胞表达mIgM，若与自身抗原结合，导致B细胞凋亡，发生克隆清除，称为B细胞的阴性选择。

生物学意义：清除自身反应性B细胞克隆，产生自身耐受。

受体编辑部分完成VH、VL基因重排的未成熟的自身反应性B细胞识别自身抗原后，未被克隆清除，而引起VH或VL基因再次重排，改变原来的BCR特异性，增加了BCR的多样性。

若受体编辑不成功，则B细胞发生凋亡。

表面分子BCR-IgαIgβ复合物BCR识别抗原，Igα、Igβ 转导 BCR 接受的抗原刺激信号。

1. 膜表面免疫球蛋白（mIg）B细胞抗原受体（BCR）B细胞特异性表面标志BCR包括 mIgM 和 mIgD2、Igα/Igβ（CD79a/CD79b）胞质区较长，含有ITAM结构，转导抗原与BCR结合所产生的信号。

B细胞共受体促进BCR对抗原的识别及B细胞的活化CD19、CD21 和 CD81 非共价相联，形成 B 细胞的多分子共受体，增强 BCR 与抗原结合的稳定性并与 Igα/Igβ 共同传递 B 细胞活化的第一信号。

共刺激分子CD40分子与活化T细胞表面CD40L结合后传递第二信号，导致B细胞活化、增殖和分化；促进抗体类别转换组成性表达于成熟B细胞；CD80和CD86分子（B7）静息B细胞不表达，活化B细胞高表达；与T细胞表面CD28相互作用，活化T细胞；与T细胞表面CTLA-4相互作用，抑制T细胞活化。

医学免疫学-B细胞Antigen-activated B cells differentiate into centroblasts that undergo clonal expansion in the dark zone of the germinal centre During proliferation the process of somatic hypermutation SHM introduces base-pair changes into the V D J region of the rearranged genes encoding the immunoglobulin variable region IgV of the heavy chain and light chain some of these base-pair mutations lead to a change in the amino-acid sequence Centroblasts then differentiate into centrocytes and move to the light zone where the modified antigen receptor with help from immune helper cells including T cells and follicular dendritic cells FDCs is selected for improved binding to the immunizing antigen Newly generated centrocytes that produce an unfavourable antibody undergo apoptosis and are removed A subset of centrocytes undergoes immunoglobulin class-switch recombination CSR Cycling of centroblasts and centrocytes between dark and light zones seems to be mediated by a chemokine gradient presumably established by stromal cells in the respective zones not shown 14 Antigen-selected centrocyteseventually differentiate into memory B cells or plasma cells d Th cells stimulate B cells to produce Abs of different heavychain classes isotypes Heavy chain class switching is initiated by CD40L-mediated signals and switching to different classes is stimulated by different cytokines Ig heavy chain class isotype switching 蠕虫 Ig heavy chain class isotype switching Mechanisms of Ig heavy chain class switching e Affinity maturation in Ab responses Affinity maturation is the process by which the affinity of Abs produced in response to a protein Ag increases with prolonged 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 mutation Control of Affinity Affinity MaturationFive B cell antigenreceptors -all specificfor but withdifferent affinities due to somatichypermutationof Ig genes inthe germinal centre B B B B B Only this cell that has a high affinity for antigen can express CD40Only this cell can receive signal 2 Only this cell is rescued from apoptosis ie clonally selected The cells with lower affinity receptors die of apoptosis by neglect Affinity maturation in antibody responses - Selection of high affinity B cells The anatomy of humoral immune responses A fraction ofthe activated B cells which are often the progeny of class-switched high-affinity B cells do not differentiate into Ab secretors but instead become memory B cells Germinal Centers Function to generate B cells that produce antibodies with increased affinity for the inducing antigen affinity maturation Germinal Center Reaction Activated B cells give rise to Centroblasts 中心母细胞 - localize in follicle undergo rapid cell division and turn on machinery that causes somatic mutation in V-regions Centroblasts give rise to Centrocytes 生发中心细胞 - migrate to the FDC-rich region of the Germinal Center - survival is dependent on interaction with FDC-bound Ag and presentation of Ag to T cells - centrocytes that successfully compete to bind antigen eg 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 FDC in the form of an immune complexwith C3d and antibodies attached Antigen enters the germinal centre Complement receptor 3 Ig 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 andcomplement receptors on the FDC dendrites The veils ofantigen-bearing dendritic cell surround the beads and the layerof immune complexes is thickened by transfer from the dendriticcell These beads are then released and are then called ICCOSOMES Iccosome formation and release DC veils Iccosomesblack coated particlesbind to and are taken up by B cell surface immunoglobulin YY Y Iccosomes bearingdifferent antigens B Uptake of IccosomesAntigen by B cellsAnti- B cell Surface Ig captures antigen Cross-linking of antigen receptor activates B cell Activated B cell expressesCD40 CD40 Germinal Centers 3 B cell response to TI-Ag TI-1 Bcell mitogen activate B cells - Antigen crosslink to BCR and mitogen receptors Or simpely crosslink mitogen receptors in ahigh dose LPS TI-2 activate mature B cells directly -the repeated epitopes combine with BCR→ BCRcross-linking→produce IgM bacteria capsularpolysaccharides 3 Immune response of B cells to TI-Ag No Th helpno memory early effect T Dependent Antigens TI-1 AntigensTI-2 Antigens Induces response in babies Yes Yes No Induces response in athymia No Yes Yes Primes T cellsYes No No Polyclonally activates B cells No Yes No Requires repeating epitopes No No Yes T Dependent Independent Antigens Examples TD Diptheria toxin influenza heamagglutinin Mycobacterium tuberculosis TI-1 Bacteriallipopolysaccharides Brucella abortis TI-2 Pneumococcalpolysaccharides Salmonella polymerised flagellin TDActivate 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 Primary immune response- longer latent phase- smaller peak response lower Ab titer- remaining in the serum at detectablelevels 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 muchlonger periods - higher average affinity - usually IgG Secondary immune response Features of primary and secondaryantibody responses B-cell surface markers 1 BCRcomplex BCR mIg VH VL----Ag binding site mature B cellsmIgM and mIgD Function specifically recognizes antigenIgIg CD79aCD79b heterodimer cytoplasmic domains containITAM Function transduce the signals that lead to B cellactivation Antigen receptor-mediated signal transduction in B cells CD19CD21CD81complex CD21 CR2 C3dR EB virusreceptor CD19CD21CD81 interactions with complementassociated with antigen play a role in antigen-induced B-cellactivation 2 Co-receptors The role of the coreceptor in B cell activation 1 CD40 interacts with CD40L Th cell 2 CD80 B71 CD86 B72 Expressed on activated B cellsand other APCs 3 ICAM-1 CD54LFA-1CD11αCD18 mediatecell-cell interaction and co-stimulation 3 Co-stimulatorymolecules CD20 function is unclear It is suspected that it actsas a calcium channel in the cell membrane CD22Inhibitoryreceptor with ITIM motif CD32 FcRII Inhibitory receptorCytokine receptors Complement Receptors Toll-like receptorsMHC 4 Other receptors 3 Subtype of B cells Conventional Bcells B-2 cells B-1 cells expression of CD5 CD5 Two Bcell 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 YY Y Y Y Y Y Y Y Y IgM - no other isotypes B Distinct B cellprecursor B1 B cellsPrimitive B cells found in pleura and peritoneum B-2 cellsconventional B cells Recirculating follicular B cellscirculate between LN follicles and blood mIg IgM IgD ProduceIgG after antigenic stimulation in the presence of T helpercells B1 cells CD5 Many of the first Bcells that appear during ontogeny express CD5 a markeroriginally found on T cells express mIgM no mIgD They respondwell to TI-Ag and may also be involved in the Ag processing and presentation to T cells Functions 1 produceanti-bacterial IgM the first line of defence against microorganisms 2 produce polyreactive Ab clearanceof 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 properties PropertyB-1 cells B-2 cells N regions FewExtensive V region repertoire Restricted Diverse Location Peritoneumpleura Everywhere RenewalSelf renewal in situ Bone marrow Spontaneous Ig production High Low Isotypes IgM IgMGADECarbohydrate 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 typesof antigens are seen by B-1 and B-2 B cells 4 Function of B cells1 Production of antibody Abs prevent microorganism from entry into cells and eliminate microorganisms by opsonization causing phagocytosis complement activation and toxin neutralization2 Ag presentation to T cells3 Immune regulation Secretion of cytokines TNF IFN IL-12 →M DC NK B cell Co-stimulation of T cells→T cell proliferation Y Y Immune effector mechanisms against extracellular pathogens toxins NEUTRALISATION Y Toxin release blocked Prevents toxicityNEUTRALISING ANTIBODIES Adhesion to host cells blocked Prevents invasion Bacterium Y Toxin Fc receptor binding Effector mechanisms against extracellular pathogensOPSONISATION OPSONISATION Phagocytosis Bacteria inextracellular space Ab Effector mechanisms against extracellular pathogens COMPLEMENT Activation Bacteria in plasma Ab COMPLEMENT Phagocytosis binding Complement Fc receptor Lysis Opsonisation ADCC B cell-mediated humoral immune response Humoral immunity is mediated by antibodies andis 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 TI-Ag T cell-independent Response to TD-Ag 1 B cells recognize TD-Ag a BCR directly recognizes B cell epitopes b IgIg transfer the first signal c Signaling pathways d Effect of coreceptors CD21CD19CD81 Transduction of signals by the B cell receptorIga Igb Intracytoplasmicsignalling domains 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[LV]X6-9YXX[LV] BCR Signaling I BCR Signaling II The roleof co-receptors CD19CD21CD81 in B cell activation 2 Role of Th cells in humoral immune response to TD-Ag For a protein Agto 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 helper T cells Th 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 cells B cells 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 cells c Mechanisms of Thcell-mediatedactivation of B cells Th 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 cells Key components of T cell help CD40L triggers CD40 - synergizes with BCR signals to promote mitosis cytokine eg IL4 signals also contribute FasL triggers Fas - BCR signaling protects from apoptosis T cell derived cytokines influence differentiation isotype switchingIL2 IL6 promote differentiation IL4 - IgG1 IgE IFNg - IgG2a IgG3 TGFb and IL5 IgA Many other molecules involved in T-B interactions eg ICAM1LFA1 ICOSICOSL CD30CD30L CD27LCD27 OX40LOX40 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 Response 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 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 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 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 Secreted Factors - CYTOKINES 2Secretion of cytokines by stromal cells B Bone marrow stromal cells nurture developing B cells Types of cytokines andcell-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 Bone marrow stromal cell Maturing B cells B B Stromal cell B lineage commitment HSC hematopoietic stem cell MPP淋巴系髓系多能前体细胞 ELP earliest lymphocyte progenitor ETP early T-lineage progenitor CLP common lymphoid progenitor HSC Development and migration of B cells An overview Stages of differentiation in the bone marrow aredefined by Ig gene rearrangement B CELL STAGE IgH GENE CONFIGURATION Stem cell Early pro-B Late pro-B Large pre-BGermline DH to JH VH to DHJH VHDHJH Pre-B cell receptor expressed Ig light chain gene has not yet rearranged B cell receptor Transiently expressed when VHDHJH CHm is productively rearranged Iga Igb signal transduction molecules CHm Heavychain VHDHJH Light chain VLJLCL VpreB l5 Pre- B cell development is coupled with rearrangement of heavy-chain B cell development is coupled with rearrangement of heavy-chain Bcell development is coupled with rearrangement of heavy-chainB cell development is coupled with rearrangement of heavy-chainB cell development is coupled with rearrangement of heavy-chain B cell development is coupled with rearrangement of heavy-chain Splicing of IgM and IgD RNA Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3 pA1 VD J Cm1 Cm2 Cm3 Cm4 Cd1 Cd2 Cd3 V D J AAA RNA cleaved and polyadenylated at pA2 V D J Cm IgM mRNA V D J Cd IgD mRNA 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 B cell development is coupled with rearrangement of heavy-chain 6000 heavy chains combined with 320 light chains 19X106 Abs 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 VHDHJH joins Evidence for allelic exclusion Allotypes canbe identified by staining B cell surface Ig with antibodies aabb ab Y B b Y B a Y B b Y Y B a b Y B a AND ALLOTYPE- polymorphismin the C region of Ig –one allotype inherited from each parentSuppression 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 Y Y Y Y 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 eg brain cells S aureus Y Y Y Y Y B S aureus Y Y Y Y Y Y Y Anti S aureus Antibodies Y Y Y Y Y Y Anti brain Abs One Ag receptor per cell IF 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 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 Allelic exclusion is needed to prevent holes in the repertoire Exclusion of anti-brain B cells ie self tolerance Y Y B B One specificity of Agreceptor per cell S aureus Anti-brain Ig AND anti-S aureus Ig Y Y Y B B IF there were two specificitiesof Ag receptor per cell Anti-brain Ig B B Deletion Anergy ORanti Saureus B cells will be excluded leaving a hole in the repertoire BUT Y Y Y B B 1 Suppresses further H chainrearrangement Ligation of the pre-B cell receptor 1Ensures 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 VHDHJH joins Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B 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 VDJCH chain VL-JL rearranges Proliferation stopsPre-receptor not displayed Small pre-B 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 Physicalremoval from the repertoire DELETION Paralysis of function ANERGY Alteration of specificityRECEPTOR EDITING B cell self tolerance clonal deletionImmature B cell recognises MULTIVALENT self Ag B Clonaldeletion by apoptosis Y Y B Immature B B Small pre-B Small pre-Bcell assembles Ig Y B cell self tolerance anergy B Y Y Y B AnergicB cell IgD normal IgM low Immature B cell recognises solubleself Ag No cross-linking Y Y B Immature B B Small pre-B Smallpre-B cell assembles Ig IgM IgD IgD IgD Receptor editing A rearrangement encoding a self specific receptor can be replacedV C D J V V V Y B B Receptor recognises self antigen B Apoptosisor anergy Y B B Edited receptor now recognises a differentantigen and can be rechecked for specificity C D J V V V V Arrest development And reactivate RAG-1 and RAG-2 Y Y Y Y Y Y MatureB cell exported to the periphery Y Y B cell self tolerance exportof self tolerant B cells IgD and IgM normal IgM IgD IgD IgD IgDIgM IgM IgM Immature B cell doesnt recognise any self Ag Y YB Immature B B Small pre-B Small pre-B cell assembles Ig B Antigen-activated B cells differentiate into centroblasts thatundergo clonal expansion in the dark zone of the germinal centreDuring proliferation the process of somatic hypermutation SHMintroduces base-pair changes into the V D J region of therearranged genes encoding the immunoglobulin variable regionIgV of the heavy chain and light chain some of these base-pairmutations lead to a change in the amino-acid sequenceCentroblasts then differentiate into centrocytes and move tothe light zone where the modified antigen receptor with helpfrom immune helper cells including T cells and folliculardendritic cells FDCs is selected for improved binding to theimmunizing antigen Newly generated centrocytes that produce an unfavourable antibody undergo apoptosis and are removed Asubset of centrocytes undergoes immunoglobulin class-switch recombination CSR Cycling of centroblasts and centrocytesbetween dark and light zones seems to be mediated by a chemokine gradient presumably established by stromal cells in therespective zones not shown 14 Antigen-selected centrocyteseventually differentiate into memory B cells or plasma cells。