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ANTIGEN AND ANTIBODY REACTIONS I II Immunobiology BIOMI 494G 2009 Joe McGillis PhD Sept 16 18 MN 380MN 375 323 6721 jpmcgi01ukyedu Of ce Hours I am here most days from about 930 AM to about 630 PM I will make it point to be in my of ce 30 minutes before and after each lecture However you are welcome and I encourage you to come by any time that you have a question The best thing to do is to call my of ce or lab at 323 67213236689 before you come over to insure that I will be available If not we can easily schedule a time that is convenient If you have questions please come see me as soon as possible After all it is what I get paid for The Ten Most Important Reasons to Pay Attention Ask Questions About Things You Don t Understand Study Immunology All Weekend and Learn All About B Cells and Antibodies By 100 PM there is no edible food left on campus Getting an A in Immunobiology will increase your chances of getting into graduatedentalveterinarymedical school but probably not clown school Life sucks without antibodies Life can also suck with antibodies To understand what Dr McGillis willi a uta you with about complement it will be essential to understand antibodies teach There is generally no good live Jazz to be heard at 100 PM on weekdays UL is going to kick the snot of UK next weekend so there is no reason to go the football game You might as well save 20 bucks stay home and study Immunology Dr Roszman who is right most of the time surprisinglyl and is himselfa pretty hip cat thinks it s important During the last 60 years more fundamental advances in biology and medicine have resulted from research in related to or dependent on Immunology than any other single biomedical discipline Dr McGillis who is always right and is the hippest cat around thinks it s important You are here because you love science and want to learn everything you can before tuition goes up again i III IV ANTIGEN AND ANTIBODY REACTIONS I II Immunobiology BIOMI 494G Sept 16 18 Overall Concepts Review I 2 Clonality Ig Forms a soluble forms b cell membrane bound forms BCR Fc receptor bound Theoretical and practical considerations of antigen antibody interactions 1 2 3 Af nity of antigen antibody binding a non covalent forces b affinity c avidity Precipitin Formation soluble antigens Agglutination particulate antigens Ig Functions 1 2 3 4 5 Opsonization ADCC Sequestration of toxins Prevention of parasite entry or colonization Complement Fixation to be covered in complement lectures Practical applications of antibodies 1 General Considerations a Polyclonal vs Monoclonal b Methods of detection c Direct vs Indirect labeling depending on time we may not cover all topics in the outline For the exam you will only be responsible for topics covered in the lecture Clonality Ep ope Antigenic Determinants BCR Fab Clonal expansion Plasma cell Polyclonal response Agglutination Immune complexes Fc receptors Opsonization Neutralization Sequestration T Cell Signal wick i 593 999 i lt T Cell Signal 34 9999 Noncovalent forces Origin Electrostatic forces Attraction between opposite charges 69 NH3 ooc Hydrogen bonds Hydrogen shared between electronegative atoms NO N H O C 5 5 5 Van der Waals forces Fluctuations in electron clouds around molecules oppositely polarize 5 5 neighboring atoms Hydrophobic groups interact H H6H unfavorany with water and H gt0 5 H Hydrophobic forces tend to pack together to 5 Olt H exclude water molecules 5 The attraction also involves 0 5 van der Waals forces HAH Figure 39 lmmunobiology 6e Garland Science 2005 BindingLAffinitv AbAq Binding 1 Reversible 2 Follows Laws of Mass Action Ab Ka kon Ag koff Kd T association constant Ag Ag dissociation constant at equilibrium AAbAg AbAg At N 3 it M H A kon A t At AbAg t quot76 k Ab39AQ Ka 39 ko 39 AbiliAg 39Wd k AbAg Kd ken m 1Ka For a single Ag binding site Kd s range from 107 to 109 M At equilibrium this means that The concentration of AbzAg complexes is 107 to 109 fold higher than free Ab and Ag Or The on rate for the binding reaction is 107 to 109 times higher than the off rate FIGURE 64 A precipitin curve for a system of one an tibody and its antigen This plot of the amount of anti body precipitated versus increasing antigen concentra tions at constant total anti body revealsthree zones a zone of antibody excess in which precipitation is inhib ited and excess antibody can be detected in the superna tant an equivalence zone of maximal precipitation in which antibodyand antigen form large insoluble com plexes purple and neither antibody nor antigen can be detected in the supernatant and a zone of antigen excess in which precipitation is in hibited and excess antigen can be detected in the super natant excess Ab S t upema ants excess Ag Antibody precipitated l Antibody excess re zone i Equivalence Antigencxcess zone zone d a J Antigen added agglutinated a 0 tube 0 000 o o 0 o o o o o o 8 oo 0 well 630 00quot nonagglutinated blood type 0 serum antiA antiA normal serum control serumdilution 12 14 18 116132 164 3amp6SZO ZOE 0i3q r 39 Anl Anllbodx Mglulmmon FIG 2 Passive agglutination Icchniquc ANTIGEN RECOGNITION AND LYMPHOCYTE ACTIVATION 56 SECTION II Macrophage u Bacteria amp L y I W A Fc receptors AnthacterIal IneffICIent antibody phagocyTOSIs 39 2K Enhanced phagocytosis Binding of opsonized bacteria quot to macrophage Fc receptors 58 SECTION II ANTIGEN RECOGNITION AND LYMPHOCYTE ACTIVATION A Antibody dependent cellmediated cytotoxicity ADCC k IgG Lowaffinity FcyR I gt gt TARGET CELL 39 LYSIS Surface Preooated NK CELL antigen TARGET CELL 39 I 4 399 Polyclonal vs Monoclonal Antibodies Polyclonal Antibodies Produced by immunization of an animal followed by preparation of antiserum from blood or isolation of immunoglobulins from serum Antiserum or puri ed polyclonal immunoglobulin contains a collection of distinct antibody proteins that recognize distinct epitopes on the target antigen The antibodies are derived from distinct B cells that respond to the same antigen Most common animal sources are rabbit goat donkey or sheep Polyclonal antibodies have higher avidity and are more ef cient at crosslinking an antigen Polyclonal antibodies are more prone to crossreactivity and overall may be less speci c than monoclonals One reason is that a polyclonal antiserum may contain clones that recognize an epitope that is common to multiple related proteins Monoclonal Antibodies Production Individual antibody producing cells are isolated from and immunized animal and are immortalized in culture Antibodies are isolated from culture media Alternatively the immortalized tumor monoclonal cell line can be injected into the peritoneum of a host mouse The peritoneal uid call ascites can then be collected The ascites uid contains very high levels of the monoclonal antibody Monoclonal antibodies are derived from a single antigen speci c B cell are therefore identical in primary structure amino acid sequence in the variable domains and therefore all recognize a single distinct epitope on an antigen Most common animal sources are mouse and rat Monoclonal antibodies have lower avidities and are less ef cient at crosslinking antigens Monoclonal antibodies are less prone to non speci c artifacts by selection of clones that recognize epitopes that are unique to the target antigen Common Methods of Detection in Antibody Based Assays 1 Fluorescent labels small uorescent molecules can be covalently crosslinked to immunoglobulins Examples Abbrev Chemical Name Color FITC uoresceine isothiocyanate Green PE phyCOerythrin Red APC 7 Blue 2 Enzymes enzymes can be covalently linked to immunoglobulin proteins Examples Horse Radish Peroxidase Glucose Oxidase a colorless substrates converted to colored substrate b Chemiluminescence non luminescent subtrate converted to luminescent substrate that can be detected in a uorimeter or on x ray lm 3 Radioisotopes radioactive moieties can be covalently linked to immunoglobulins Most common is 12 I Can be detected on x ray lm or by coating antibody bound specimen with a photosensitive emulsion Direct and Indirect Methods of Detection 1 Direct the primary antibody is labeled Advantages Disadvantages 0 Can use more colors 0 Less sensitive o More Speci c lower background and non speci c staining 2 Indirect Methods a Second Antibody uses a labeled 239 antibody speci c for the primary antibody Advantages Disadvantages More sensitive 7 ampli cation of primary signal More dif cult to use with multiple colors More prone to high background and non speci c staining b Avidin Biotin Avidin streptavidin SA and biotin bind with a very high af nity Biotin is a small molecule that can be covalently crosslinked to proteins Avidin is a large molecule to which multiple uorescent molecules can be attached Primary antibodies can be tagged with biotin and then detected with SA Advantages Disadvantages More sensitive W ampli cation of primary signal More prone to high background and non Can be in multiple color systems speci c staining VI BIOMl 494G Fall 2008 Infection and Immunity Routes of infection 1 Introduction a Mucosal surfaces b External epithelia Ephithelial barriers against infection rst line of defense 1 Mechanical 2 Chemical and antimicrobial peptides 3 Microbiological 4 Innatelike lymphocytes Role of phagocytes macrophage and neutrophil in innate immunity 1 Introduction 2 Macrophages 3 Neutrophils 4 Function of macrophages and neutrophils In amation 1 De nition 2 Role of in ammation in infection 3 Cardinal signs of in ammation a Redness rubor b Swelling tumor 0 Heat calor d Pain dolor 4 Cellular events in the in ammatory response a Vasodilation b Increased capillary permeability c Phagocyte extravasation d Clotting Recruitment of phagocytes of sites of infection 1 Rolling adhesion 2 Tight binding 3 Diapedesis 4 Migration 5 Role of chemokines Kinetics of leukocyte binding to endothelium and in ltration into tissue 1 Endothelial adhesion molecule expression 2 Leukocyte in ltration 39 Fig 22 Pathogens infect the body Routes of infection for pathogens 1hrough a variety of mmes Route of entry I Mode 01 transmission Pathogen Disease Mucosal surfaces inhaled droplet I Influenza virus I I influenza Airway I Neisseria meningitidis II Menmgowxal Spores I Bacillus anthracis T Inhalation anthraxJ Salmonella typhl Typhoid lever Gastrointestinal tract Comam39na39ed I I water 0 100d I Rotavirus I Dianhea I Treponema palidum Syphilis Reproductive tract Physical contact I l HIV AlDS if 1 External epithelia External surface I Physical contact I I Trichophyfon I Athlete39s loot I Minor skin abrasions I Bacillus anthracis Cutaneous anthrax Wounds and abrasions Puncture wounds I Cosi dl um tetani Tetanus Handing mteded animate I Flandsella tutarensis Tuiaremia gggnjegggg Flavivirus Yellow lever Insect bites Deer tick bites Borrelia burgdorien Lyme disease MOW 0 bites Plasmodium s Malaria Anopheles I pp gt 39 39 Fig 24 Many barriers prevent quotMMquot Ephm ia39 barrierstolm mion 39 V 39 pathogens from crossing epithelia 39 i V I and colonizing tissues Surface epithelia provide mechanical chemical and microbiological barriers to infection a semiP cells Mechanical Movement oi Longitudinal tiow oi air or uid mucus by Cilia Low F 39d Chemical any am 5 Antibacterial Microbiological Normal flora microvilli 1 7 n E F r light 5 o junctions intraepithelial lymphocyte ee Paneth cells Figure 35 Paneth cells in the small intestine Paneth cells are specialized cells that secrete a detensins at the bottom of the crypts formed by the infolding of the epithelium of the small intestine Paneth cells synthesize and secrete defensins in response to the presence of bacterial components Figure 37 Mechanism ol delensin killing ol microbes Defensins are highly amphipathic and positively charged molecules They seem to make pores in membranes approximately 20 A in diameter it is thought that their positively charged regions interact with39the negatively charged phospholipids of 3 0 4 39 binding jg 01932 poly lg receptor loss of Smallmolecule I g gradients t 533 39 iimh quot microbial membranes after Which they inserttheir hydrophobic regions into the membrane and oligomerize to form pores disrupling the normal function of the membrane The pores also allow g entry of other effector molecules of immune defense that can contribute to killing microbes Figure 93 Epithelial defens cells in the underlying soft ti a cutaneous barrier Langerhans cell Cy z n epidermis v AV Al3 delensmvs A V V v 4 A v adetensins V v 39 dermal dendritic cell 39 ery rocytes 39 blood vessel 39 l dermis a a i a a b 39 a a l b mucosal barrier microvilli M CE atria lgA Y3 fill ng t t39 39 39 Rafa ightiunctions intraepithelial lymphocyte Paneth cells Ssugabsqganous epithelium showing keratinized outer layer and dendritic o umnar epithelium showing tight junctions and Paneth cells Physical barrier to infection Pepgde anti iotics Killing of microbes L gt39 39 by locally produced 0 antibiotics 13qmw IIntraitapithelial m 00 e Killing of microbes y p yt and infected cells by intraepithelial lymphocytes Figure 121 Epithelial barriers Epithelia at the portals of entry of microbes provide physical barriers produce antimicrobial substances and harbor intraepithe lial lymphocytes that are believed to kill microbes and infected cells Make natural antibody protect against infection with Produce cytokines rapidly Streptococcus pneumoniae Produce cytokines rapidly Ligands not MHC associated Cannot be boosted Ligands are lipids bound to 001 d Cannot be boosted Ligands are MHC class IB associated Fig 252 The three main classes of Cannot be boosted innatelike lymphocytes and their properties Activation oi complement and removal ol bacteria Fig 26 Bactericidal agents produced or released by phagocytes on the ingestion of microorganisms Most of these agents are made by both macrophages and neutrophiis Some of them are toxic others such as lactoferrin work by binding essential nutrients and preventing their uptake by the bacteria The same substances can be released by phagocytes interacting with large antibodycoated surfaces such as parasitic worms or host tissues As these agents are also toxic to host cells phagocyte activation can cause extensive tissue damage during an infection Fig 253 8 1 cells might be important in the response to carbohydrate antigens such as bacterial polysaccharides These Tceli independent responses occur rapidly with antibody appearing within 48 hours after infection presumably because there is a high frequency oi precursors of the responding lymphocytes so that little clonal expansion is required in the absence of antigen specific T cett heip only lgM is made and in mice these responses therefore work mainly through the activation oi complement which is most efficient when the antibody is of the lgM lsotype Acidification Toxic oxygen derived products Superoxide O 39 hydrogen peroxide H202 singlet oxygen 10239 hydroxyl radical OH39 hypohalile OCI Toxic nitrogen oxides Nitric oxide NO T Antimicrobial peptides Defensins and cationic proteins 1 Enzymes Lysozy F me dissoives cell walls of some Grampositive bacteria Acid hydrolases iurther digest bacteria Competitors Lactoferrin binds Fe and vitamin Bigbinding protein T Lt The infected tissue becomes 39 2 lnllamed causing redness I heat swelling and pain Vasodilation and Increased vascular permeability allow tluld39proteln and in ammatory cells to leave blood and enter tissue Surface wound introduces bacteria which actlvate resident d Healthy Skln Is net ln ame effector cells to secrete cytoktnes dirt stones etc blood cm x etieclor cell 0 0 Q o Connective tissue K E g EB Figure 16 Innate immune mechanisms establish a state of inflammation at sites of infection Illustrated here are the events following an abrasion of the skin that leads to bacteria invading the underlying connective tissue and stimulating39the innate immune response Tissue damage causes release of Phagocytes and antibacterial vasoactive and chemolactic factors 39 exudate destroy bacteria that trigger a local increase in blood ow and capillary permeability Permeable capillaries allow an in ux of uid exudate and cells L a u A 5393 Major events in the inflammatory response A bacte blood cells including phagocytes and lymphocytes from the blood 39ial infection causes tissue damage with release ofvarious vasoactive into the tissues The serum proteins contained in the exudate have ind chemotactic factors These factors induce increased blood ow antibacterial properties and the phagocytes begin to engulf the bac o the area increased capillary permeability and an in ux ofwhite teria as illustrated in Figure 13 099 0 9099 9999 00 O 9 Fig 28 Infection stimulates macrophages to release monocyfes out of the blood vessel e 39 rt 39 cytoklnes and chemokines that Initiate an inflammatory infected tissue guided by chemokiniesX1 Fife3323 dyatrli lg tiiiigteed response Qytoktnes produced by tissue macrophages at the macrophages The blood vessels also become more permeable Site of infection cause dilation of local small blood vessels and allowing plasma proteins and fluid to leak into the tissues 1 changes in the endothelial cells oi their walls These changes Together these changes cause the characteristic inflammatory lead to the movement of leukocytes such as neutrophils and signs of heat pain redness and swelling at the site of infection CR30LMB2 neutrophil LFA1QLIBQ l 39 ICAM7391Terid lb5iium 39OW Fig 243 Phagocyte adhesion to vascular endothelium is mediated by integrins Vascular endothelium when it is activated by inflammatory mediators expresses two adhesion molecules lCAM1 and lCAM2 These are ligands for integrins expressed by phagocytes aLz g also called LFA 1 or CD11aCD18 and xMz g also called CR3 Mac1 or CD11bCD18 Stable Migration through adhesion endothelium lntegrin activation R ng by chemokines Integrin lowaffinity state Selectin ligand Integrin high M affinity state Integrin O Proteo ligand l can 0 Op Cytokines TNFJLll Macrophage Fibrin and fibronectin W39th micrObes extracellular matrix Figure 12 2 Recruitment of leukocytes At sites of infection macrophages that have encountered microbes produce cytokines such as TNF and ILl that activate the endothelial cells of nearby venules to produce selectins ligands for inte grins and chemokines Selectins mediate weak tethering and rolling of blood leukocytes such as neu trophils on the endothelium integrins mediate firm adhesion of neutrophils and chemokines increase the affinity of neutrophil integrins and stimulate the migration of the cells through the endotheliUm to the site of infection Blood neutrophils monocytes and activated T lymphocytes use essentially the same mechanisms to migrate to sites of infection Fig 29 Monocytes circulating in the blood recognize blood vessel walls near sites of inflammation and leave the L39 to migrate into the Blood vessel lumen tissue toward the site of infection and inflammation The initial interactions are mediated by adhesion molecules that rst capture the monocyte from the bloodstream and cause it to adhere to the vascular endotheiium Chemokines bound to the vascular endotheiium then signal the monocyte to migrate across the endotheiium into the underlying tissue The monocyte now differentiating into a macrophage continues to migrate under the influence of chemokines released during inflammatory responses toward the site of infection Monocyte diiferentiates into a macrophage and migrates to the site of infection Monocyte binds adhesion molecules on vascular endotheiium near sites of infection and gets chemokine signal chemokine receptor migrates Into the surrounding tissue chemokine Tissue Fig 236 Neutrophils leave the blood and migrate to sites of intection in a multistep process mediated through adhesive interactions that are regulated by macrophage derived cytokines and chemokines The first step top panel involves the reversible binding of leukocytes to vascular endotheiium through interactions between selectins induced on the endotheiium and their carbohydrate ligands on the leukocyte shown here for Eselectin and its ligand the sialyl Lewis moiety s Lex This interaction cannot anchor the cells against the shearing force of the ow of blood and instead they roll along the endotheiium continually making and breaking contact The binding does however allow stronger Interactions which occur as a result of the induction of ICAM1 on the endotheiium and the activation of its receptors LFA1 and Mac1 not shown on the leukocyte by contact with a chemokine like ILB Tight binding between these molecules arrests the rolling and allows the leukocyte to squeeze between the endothelial cells forming the wall of the blood vessel to extravasate The leukocyte integrins LFAt and Mac1 are required for extravasation and for migration toward chemoat tractants Adhesion between molecules of CD31 expressed on both the leukocyte and the junction of the endotheliat cells is also thought to contribute to extravasation The leukocyte also needs to traverse the basement membrane it penetrates this with the aid of a matrix metallo proteinase enzyme that it expresses at the cell surface Finally the leukocyte migrates along a concentration gradient of chemokines here shown as IL 8 secreted by cells at the site of infection Endothelial adhesion molecule expression K C 9 g E selectin ICAMA W a VCAM 1 gtlt lt1 5 gt t 0 C 0 l l E 1 I I I I O l 12 224 36 39 48 Hours 7 n J T cells Neutrophils Monocytes 2 Neuwtrophils E I Tcells quot5 Monocytes I n E I 3 Z l I I I I I l I O 12 24 36 48 I HoUrs gt Figure 12 3 Kinetics of leukocyte binding to endothelium and in ltration into tissues In response to cytokines such as tumor necrosis factor TNF endothe lial cells sequentially express different adhesion molecules that prefer entially bind different leukocytes A This activity results in in ltration of the leukocytes into tissues rst neutrophils and then monocytes and T cells B ICAM intercellular adhesion molecule VCAM vascular cell adhesion molecule Humoral Immunity Thymus Dependent TD Response 3 32 cells predominant lgM39 lgDhiCDS39 b Requires T helper cells No response in athymic mice nude mice c Typically elicits a germinal center response Somatic hypermutation Af nity maturation Class switching Memory B cell Thymus Independent T I Response a Responses observed in nude mice b Tl type 1 response 31 cells lgM lgD39 CD5 c Tltype 2 response Marginal zone B cells lgM lgD39 CD5 Humoral immunity Primary Response Pnaurytcsooasc k A WW 5 I 1 I P E U l toe 5 I 1C g I I I I V g l I z I I T E 1 l 5 f 39 Time after immunization Duration of 39 Attainment of Antigen Lag Phase Peak Response Erythrocytes 3 4 days 4 5 days Soluble pmtcins 5 7 days 910 days Bacteria 1014 days 2 3 weeks Toxoid gt3 weeks lt3 months Secondary Response I 4 Privacy csoonsc Scmnduyrcwonsc c 39 3 t E l 3 g I g g Tvmc Cf hmuniutb Caplllary endothetium CCRS Chemokines orchestrate the trafficking of dendritic cells T cells and B cells needed to generate an immune response By activating TLRs LPS on the surface of bacterial pathogens stimulates the local release of chemolu nes such as MlP Ia MlP t MlP aa and IP10 Immature dendritic cells are attracted to this site through activation of chemokine receptors such as CCRl CCR5 and CCR6 that they constitutiver express Immature dendritic cells are ef cient at pickingup antigen but must mature and differentiate into cells that can activate naive T cells The local milieu into which the immature dendritic cell is attracted contains pathogenassociated molecular patterns eg LPS that are recognized by PRRs such as 0014 and TLR5 which induce the differentiation and maturation of dendritic cells into potent antigen presenting cells During this process the dendritic cell downregulates expression of CCRI CCR5 and CCR6 and upregulates expression of CCR39I causing its migration into the afferent lymphatic system The CCR7 ligand SLC which is expressed on the endothelium of the afferent lymphatic system plays an important role in directing the migration of the antigenloaded mature dendritic cells Chemokines are Amwai aiigw dendritiooellr Endothelium lymphatics Afferent 39 lymph mirtw m s Efferenl lymph Current Opinion In Immunology also involved in bringing naive T cells and B cells from blood across high endothelial venules HEVs into the lymph nodes and into contact with the activated dendritic celL The molecular details remain to be elucidated but it is likely that chemokines such as DCCKI and ELC are important in juxtapositioning these cells in the lymph node SLC and ELC are produced from stromal cells in the Tcell zone and BLC is expressed from stromal cells in the Bcell follicle helping to guide cells to T and 3ch areas respectively Some activated T cells downregulale expression of CCR7 but upregulate CXCR5 and so become directed toward the follicle to deliver help to B cells whereas other activated T cells upregulate CXCR3 and are attracted into inflamed tissue T cells that are activated in regional lymph nodes alter encountean antigenloaded dendritic cells subsequently return to sites of inflammation by sensing chernokine gradients established at these local sites Chemokines such as lPto which is induced by LPS and lFNy and a ligand for CXCRS which is highly expressed on activated T cells are believed to be important in this process in immune responses mediated by Th1 cells Phasequot 39T39B prepc msmace Day 1 Day 7 Fig l Initiating adaptive immunity and dcvebpment of effector function Following protein immtmiution in adjuvant antigen experi enced mature dendritic cells migrate to the T Cell zona of sec ondary lymphoid follicles Phase I signi es the cognate interaction of these pMHCl39lexpressing mature 06 and pMHClI speci c naive T helper Th cells Naive B cells must also spcd mlly recognize the protein antigen to initiate the T cell dcpendent Ball response lt is rusonable to consider that these B cells contact cellassociated antigen for priming during this early initiation phase of adaptive immunity Costimulation 4 5 m 7 MHC ll pep ide g i v I Ag retention lsotype switch signals Naive Th cells expand differentiate into effector Th cells and migrate to the TB borders to contact pMHClLuprasing antigenprimed B cells Phase II signi es the many pairings of thse cognate interactions between a spectrum ofeffector T11 cells and primed B cclLs that emerge after initial priming There is a major bifurcation in B cell development at this juncture B cell Clonal expansion in the T cell zones produces preplasma cells as precursors to the shortlived plasma cell pathway Expansion in the B cell zones is the aregerminal center precursor to the GC fraction and production of B cll memory Fig 8 A model for the interaction of dendritic cells T cells and B cells in the initiation ofantibody synthesis z ewe processing recognition by 391 h mIg antigen intcmalizaiion peptide class ii MHC FIG 15 Antigen presentation by B cells B cells that bind a particular antigen via the BCR can efficiently internalize this antigenThe internalized protein antigen is then processed by proteofysls in either late endosomes or lysosomes Appropriate antigenderived peptides can combine with class II MHC molecules which are routed to late endosomes before traf ddng to the cell surface Once formed the peptideMHC complex can come to the cell surtace where it serves as a recognition structure for antigenspeci c helper T cells Cell adhesion molecule LFA l B7 1 f B72 G 16 Multiple roles for the BCR in antigen presentation to helper bi In addition to antigen uptake as depicted in Fig 15 antigen tea quot9 to the BCR also triggers signal tramduction reactions These m 011008 induce class if MHC gene expression which provides 59 39 Glass ll MHC molecules to the late endosomes where they an the anugendenved peptides lnaddition BCFt signaling modulates af nity of cellcell adhesion molecules on the Bceli surface and in West the expression of 872 and thereby promotes effective emotion with helper T cells FIG 20 Cell surface molecules involved in B cell helper T cell inter actions A wide variety of cell surtace molecules on B cells have the ability to interact with cell sudace molecules on helperT cells These include antigenpresenting and antigen recognition stmctures MHC class it and the TCR CD4 molecules involved in activating heller T cells 872 and 0028 molecules involved in activating B cells CD40 and CD40L and cell cell adhesion molecules 0045 moie cules are postulated to have interactions with molecules on other cells such as 0022 on the B eel or laterally with other cell surfaCe molecules on the same cell Intracellular Cellular activation K 1 WW n Possble roles for coreceptor molecules 0 the B lymphocyte in membrane imrmmoglobdii mlg signafmg a Ligation ol 0022 by tes 0n neighbwrbg eels may remove the negative regulator SHP I a tyrosine phosphatase from the signaling complex b 0043936011 of the 001900210081 complex with mlg recruits positive signal transduction effectors shown in itafccs which deorease the threshold or cellular activatlon CD23 also can serve as a ligand for 0021 as indicated by dashed arrow 47 c Fc coligation with mlg by antigen lgG comple39xes exerts an inhbitory effect on mlg signals through its association with SHPt BCR wmplex CR2CD19CDBIU39APA1 wrnplex Fclelb n A Ll 1141 l V l W Signalling motif TAM TAM ITIM Primary signal transducers PTKs Lyn Fyn Lyn SHIP dcElk and Syk Vav SHP2 Pl3K Slgnals delivered by An T or 39 Opsontzed Ag T w T Ops lgMoontnking K T of T lquot lgGoontahlng K T or 1 Ops lgGoontalrhg C T o T l Figure l Signalling precast induced by ligation of Bocll receptor BCR complement receptor type 2 CR2 and FcyRIl with cpsomzed antigen lad immune complexes Ag lC immune complexes 36 hmnuaogiobulh G 13M ixnmnnoglobulin M ITAM immunomocptor tyrosinebased lotivation motif ITIM hnmunoreoeptor tyrosinebased inhibition motif Ops opsonizod Pl 3K phosphatidylinositol3 kinase PTK protein tyrosine Rinasc SHIP inositol phosphate 5 phosphatase SHPZ protein tyrosine phosphatase SHP Z Vav gumidine nucleotide exchange factor Vav I Imitation 1 inhibition no e oct or 0r nonspeci c B cells respectively Activmion CIoh39al proliferation39 EN H 2H0 a n 3M P0 Q 3 OQQQmW Q t The GC reaction This ligure illustrates the microenvironmental organization at the GC reaction in the top panel and the corresponding cellular and molecular processes that underpin the development ol 8 cell memory in the lower panel a The recruitment of antigenactivated Bcells into the GC reaction is helperT cell dependent and thought to be initiated in the Tcellrich regions ol the secondary lymphoid organs b Rapid clonal expansion first rCSults in the formation of the secondary follicle in the B cellrich regions The secondary follicle subsequently polarizes into the light zonedarkzone regions of the typical GC reaction 0 Centroblasts continue to clonally expand and diversity their BCRs by somatic hypermutation and receptor editing d The centroblasts express their diversified BCRs and exit the cell cycle as centrocytes that now emerge into the light zone of the GC reaction Anitigen is present in the light zone as immune complexes on FDC processes 8 Diminished binding for antigen leads to programmed cell death and rapid clearance by resident tingiblebody macrophages Mo 0 improved binding for antigen results in positive selection 9 Positive selection may lead to reentry into the dark zone and retention in the GC reaction for another cycle of expansion diversification and selection or h exit lrom the GC microenvironment and the ability to recirculate throughout the body as a long lived memory 8 cell quotT BCR d Centrocyte 2 l lb e 2 Selection X7 I h MemOry 1 70a il Light zone Dark zone h g Retention 391 a K Recruitment 1 Conant Opinion in Immunong CDZiL J a locosome 39 TRENDS in lmmmology L Fi g 1 Model illustrating the important receptors and ligands used by Tcells and follicular dendritic ca 3915 FDCs in signaling to Bcelis The requirement for Bcell MHC class II to present antigen AL derived peptides as ligands for the T cell receptor TCR is well known as is the involvement of 0040 on the B cell interacting with T cell CD40 ligand CDdOL The FDC8 cell interaction delivers a primary signal through FDC AE H cell receptor BCR interaction and a msignal through 8 cell C021 interacting with FDC CD2 ligand C021 L The events Ola recall response are sdmmarized as follows 1 FDCs trap Agantibody Ab complexes and provide intact A9 for interaction with BCRs on germinal center GC 8 cells this Ag BCR interaction provides a positive signal for Bitequot activation and quot 39 l2 FDCs pmvmo r39 A 39 n4 CDZlLfochellCDZl itsinleraction withthe CDZl CDlS CDBl complex delivers a positive cosignal for Bcell activation and differentiation Coligation of BCR and CD21 as illustrated here with a single molecule olAgl facilitates association of the two receptors and the cytoplasmic tail of C019 is phosphorylated by a tyrosine kinase associated with the BCR complex The arrow pointing from the BCR to 0019 indicates this known interaction 3 A high density of Fcyreceptor IlB Fcy llBlon FDCs binds lg Fe in theAg Ab complex and the signal quot quot d hythe39 r 39 based inhibition motiflTlM in the B cells might be blodted This inhibitory signal is initiated by Ag Ab complexes crosslinking BCR and Fcy llB on B cells Note that BCR is not crosslinked with B cell FcleIB in the model and thus a high concentration of Fcy llB on FDCs minimizes a negative signal to the B cell 4 In addition FDCs provide 39 p L quot which are readilvtaken up by B cells The iocosome membrane is derived from FDC membranes that have Ag CDZlL and lg Fc attached locosomes bind tightly to 8 cells and are rapidly endocytosed We reason that binding of BCR complement receptor 2 CR2 and possibly 8 celch receptor FcR to the icoosomal AgCDzlL lg Fc complex is crucial to the process olendocytosis1he B cells process this FDC derived Ag present it and thus obtain Tcell help Processed Ag reactivation 0 memory 8 cell protection by longlived plasma coils activation memory 8 cells speci c serum antibody cormntratlon time Fig 7 Humoral memory adapts to the environment antigen concentratlon A K I 39 6V5 C 0 0 0 4 4h4h4h dhdhdhdhdh PolyclonaI Bcell activation nonspecific antibody response Figure 916 lmmunobiology 6e CD Garland Science 2005 Tl1 antigenspecific antibody response mama 726 m 7 ii KEELIISEITTEF355 Muml m m Figure 917 lmmunobiology6e 0 Garland Science 2005 L uf 39 Antibody response in infants Antibody production in congenitally athymic individual Antibodyquot response in absence of all T cells Primes T cells Polyclonal Bcell NO activation Requires 39 repeating epitopes N0 N0 Examples of Diphtheria toxin Bacterial lipopoly Pneumococcalpoly antigen Viral hemagglutinin saccharlde saccharide39 39 puri ed protein Braceua abort Salmonella polymerized derivativePPD agellm of Mycobacterium Dextran tuberculosis HaptenconiUgated Ficollpolysucrose Figure 918 lmmunobiology 6e C Garland Science 2005
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