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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 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 t
2、o 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,After birth, development continues in the bone marrow,B cell development starts in the foetal l
3、iver,B cell development in the bone marrow,Bone Marrow provides a MATURATION & DIFFERENTIATION MICROENVIRONMENT for B cell development,Bone marrow stromal cells nurture developing B cells,Types of cytokines and cell-cell contacts needed at each stage of differentiation are different,Bone marrow stro
4、mal 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 t
5、he bone marrow are defined by Ig gene rearrangement,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,B cell receptor,Transiently expressed when VHDHJH CHm is productively rearranged,Pre-,B
6、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 heavy-chain,B cell development is coupled with rearrangeme
7、nt of heavy-chain,B cell development is coupled with rearrangement of heavy-chain,Splicing of IgM and IgD RNA,Two types of mRNA can be made simultaneously in the cell by differential usage of alternative polyadenylation sites and splicing of the RNA,B cell development is coupled with rearrangement o
8、f heavy-chain,6000 heavy chains combined with 320 light chains 1.9X106 Abs,Ligation of the pre-B cell receptor,1. Ensures only one specificty of Ab expressed per cell,2. Triggers entry into cell cycle,ALLELIC EXCLUSION Expression of a gene on one chromosome prevents expression of the allele on the s
9、econd chromosome,1. Suppresses further H chain rearrangement,2. Expands only the pre-B cells with in frame VHDHJH joins,Evidence for allelic exclusion,Allotypes can be identified by staining B cell surface Ig with antibodies,AND,ALLOTYPE- polymorphism in the C region of Ig one allotype inherited fro
10、m 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),Suppression of H chain gene rearrangement ensures only one specifi
11、city of Ab expressed per cell.,Allelic exclusion prevents unwanted responses,One Ag receptor per cell,IF there were two Ag receptors per cell,Prevents induction of unwanted responses by pathogens,Allelic exclusion is needed for efficient clonal selection,All daughter cells must express the same Ig s
12、pecificity 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,Antibody,Allelic exclusion is needed to prevent holes in the repertoire,Exclusion o
13、f anti-brain B cells i.e. self tolerance,Anti-brain Ig AND anti-S. aureus Ig,Anti-brain Ig,anti S.aureus B cells will be excluded leaving a “hole in the repertoire”,BUT,1. Suppresses further H chain rearrangement,Ligation of the pre-B cell receptor,1. Ensures only one specificity of Ab expressed per
14、 cell,2. Triggers entry into cell cycle,2. Expands only the pre-B cells with in frame VHDHJH joins,Ligation of the pre-B cell receptor triggers entry into the cell cycle,Many large pre-B cells with identical pre-B receptors,Small pre-B cell No antigen receptor at cell surface Unable to sense Ag envi
15、ronment !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,Physical removal from the repertoire DELETION Paralysis of function
16、 ANERGY Alteration of specificity RECEPTOR EDITING,B cell self tolerance: clonal deletion,Immature B cell recognises MULTIVALENT self Ag,Clonal deletion by apoptosis,Y,B cell self tolerance: anergy,Y,Y,Y,Anergic B cell,IgD normal IgM low,Immature B cell recognises soluble self Ag No cross-linking,Ig
17、M,IgD,IgD,IgD,Receptor editing,A rearrangement encoding a self specific receptor can be replaced,V,V,V,V,V,V,V,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,Immature B cell doesnt recognise any self Ag,B-cell surface markers,1. BCR com
18、plex,BCR (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 receptor-mediated signal transduction in B cells
19、,CD19/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-receptors,The role of the coreceptor in B cell activation,(1)CD40 interacts with CD40L (Th cell) (2)CD80(B7.1), CD86(B7
20、.2) Expressed on activated B cells and other APCs (3)ICAM-1 （CD54）、LFA-1（CD11/CD18)： mediate cell-cell interaction and co-stimulation,3. Co-stimulatory molecules,CD20: function is unclear. It is suspected that it acts as a calcium channel in the cell membrane CD22：Inhibitory receptor with ITIM motif
21、 CD32 (FcRII)： Inhibitory receptor Cytokine receptors Complement Receptors Toll-like receptors MHC,4. Other receptors,3. Subtype of B cells,Conventional B cells (B-2 cells) B-1 cells (expression of CD5),Two B cell lineages,B2 B cells,IgM - no other isotypes,B1 B cells Primitive B cells found in pleu
22、ra and peritoneum,B-2 cells : conventional B cells Recirculating follicular B cells : circulate between LN follicles and blood mIg: IgM, IgD Produce 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
23、 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 presentation to T cells. Functions 1. produce anti-bacterial IgM the first line of defence against microorganisms; 2. produce polyreactive Ab clearance of denatu
24、red 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,Property B-1 cells B-2 cells N regions Few Extensive V region repertoire Restricted Diverse Location Peritoneum/pleura Everywhere Renewal Self r
25、enewal 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,Specificity & requirement for T cell help suggests strikingl
26、y different types of antigens are seen by B-1 and B-2 B cells,4. Function of B cells,1. Production of 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 ce
27、lls 3. Immune regulation Secretion of cytokines (TNF, IFN, IL-12) M, DC, NK, B cell. Co-stimulation of T cellsT cell proliferation.,Immune effector mechanisms against extracellular pathogens & toxins NEUTRALISATION,NEUTRALISING ANTIBODIES,Bacterium,Toxin,Effector mechanisms against extracellular pat
28、hogens OPSONISATION,OPSONISATION,Effector mechanisms against extracellular pathogens COMPLEMENT Activation,Lysis,ADCC,B cell-mediated humoral immune response,Humoral immunity is mediated by antibodies and is the arm of the adaptive immune response that functions to neutralize and eliminate extracell
29、ular 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.
30、 Ig/Ig transfer the first signal c. Signaling pathways d. Effect of coreceptors (CD21/CD19/CD81),Transduction of signals by the B cell receptor,BCR Signaling I,BCR Signaling II,The role of co-receptors (CD19/CD21/CD81) in B cell activation,2) Role of Th cells in humoral immune response to TD-Ag,For
31、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 helper T cells,Th cells that have been activated to differentiate into
32、 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,
33、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 Th cell-mediated activation of B cells,Th cells that recognize Ag presented by B
34、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 (e.g. IL4) signals also
35、 contribute FasL triggers Fas - BCR signaling protects from apoptosis T cell derived cytokines influence differentiation, isotype switching: IL2, IL6 promote differentiation IL4 - IgG1, IgE IFNg - IgG2a, IgG3 TGFb and IL5 IgA Many other molecules involved in T-B interactions e.g. ICAM1/LFA1, ICOS/IC
36、OSL, 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 different cytokines.,Ig heavy chain class (isoty
37、pe) 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
38、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 Ma
39、turation,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,Affinity maturation in antibody responses - Selectio
40、n of high affinity B cells,The anatomy of humoral immune responses,A fraction of the 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 p
41、roduce 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
42、 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 (e.g. by having higher affinity BCR) and to receive T cell help are
43、 selected and may differentiate into long-lived plasma cells or memory B cells,Association of antigen with FDC,in the form of an immune complex with C3d and antibodies attached,Antigen enters the germinal centre,The Immune complexes bind to Fc and complement receptors on the FDC dendrites,The veils
44、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,Iccosomes (black coated particles) bind to and are taken up by B cell su
45、rface immunoglobulin,Iccosomes bearing different antigens,Uptake of Iccosomes/Antigen by B cells,Surface Ig captures antigen,Cross-linking of antigen receptor activates B cell,Activated B cell expresses CD40,CD40,Germinal Centers,3. B cell response to TI-Ag,* TI-1 (B cell mitogen) activate B cells -
46、 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 TI-Ag,No Th he
47、lp，no memory, early effect,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,T Dependent & Independent Antigens,Examples TD: Diptheria toxin, influenza heamagglutinin, M
48、ycobacterium 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,Primary immune response - longer latent phase; - sm
49、aller 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 perio