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1、Oral Tolerance,State of immunological unresponsiveness to antigen induced by feeding.It is a feature of the common mucosal immune system.,The mucosal immune system,Consists of the gastro-intestinal tract,respiratory system,genito-urinary system,liver.Common lymphoid circulationEpithelial cells line
2、the mucosaLargest area exposed to the external environmentHeaviest antigenic load,Features of mucosal tolerance?,Normal immune functionTolerance can be local or systemicIt requires a functional immune systemSymbiosis-in the absence of commensals,a poor immune response develops and oral tolerance can
3、not be induced,General properties of mucosal tolerance:,Antigen specific.Often partial(eg.antibodies inhibited,but T cell responses may remain).Not complete(eg.may be a quantitative reduction in antibody levels).Wanes with time.,General properties of mucosal tolerance contd,Easier to abrogate a resp
4、onse than reduce and established response.Good immunogens are better at inducing tolerance!(adjuvant)Dose and route dependent.,Breakdown of oral tolerance,Immune responses to foodleads to food intoleranceeg coeliac diseaseImmune responses to commensal bacterialeads to inflammatory bowel disease(IBD)
5、eg crohns disease,ulcerative colitis,Balance,Respond Dont respondfightand eradicateIgnorePATHOGENS SELFFOOD,Mechanism?,Central tolerance deletion of self-reactive T cells in the thymusPeripheral tolerance an area of very active research!1.deletion 2.immune deviation 3.anergy 4.suppression/regulation
6、,Regulation of self tolerance?,Central tolerance is incompleteTCR bind at low affinity and can potentially recognise a number of MHC/peptide Auto-reactive T cells exist at high frequency in the peripheryAuto-immunity-is it a result of defective T cell regulation?,1.Deletion,Mechanism of central tole
7、rance(negative selection in the thymus)Apoptosis of specific T lymphocytes(eg fas-fasL)Shown to play a role in peripheral tolerance in sites of immune privilege(eg stromal cells in the testes express fasL),Peripheral deletion of antigen-reactive T cells in oral tolerance,REF:Nature 1995 Jul 13;376(6
8、536):177-80 Chen Y,Inobe J,Marks R,Gonnella P,Kuchroo VK,Weiner HL,oral antigen can delete antigen-reactive T cells in Peyers patches,in mice transgenic for the ovalbumin-specific T-cell receptor genes.The deletion was mediated by apoptosis,and was dependent on dosage and frequency of feeding.At low
9、er doses deletion was not observed;instead there was induction of antigen-specific cells that produced transforming growth factor(TGF)-beta and interleukin(IL)-4 and IL-10 cytokines.At higher doses,both Th1 and Th2 cells were deleted following their initial activation,whereas cells which secrete TGF
10、-beta were resistant to deletion.These findings demonstrate that orally administered antigen can induce tolerance not only by active suppression and clonal anergy but by extrathymic deletion of antigen-reactive Th1 and Th2 cells,Deletion summary,Generally observed at high doses of fed antigen:Activa
11、tion induced cell death(AICD)mediated by fas/fasL interactionsGrowth factor deprival,2.Immune Deviation,CD4+T lymphocytes are activated by antigen presenting cells(APC)Th1 cells-important in inflammatory responses(eg delayed type hypersensitivity)Th2 cells-important in helping antibody responses.Sup
12、press Th1 cells(IL-4,IL-10).Therefore Th1 immune responses may be inhibited if Th2 cells are stimulated instead.,Inhibitory cytokines,Transforming growth factor beta(TGF)non-specifically inhibits the growth of lymphocytes(Th3)Specific immune responses can be inhibited by Th2 cytokines(IL-4 and IL-10
13、)Some populations of T lymphocytes(both CD4 and CD8)can consume IL-2,the T cell growth factor.Surrounding cells therefore fail to grow,One example of many,Feeding oral insulin to mice prevents virus induced insulin-dependent diabetes in a mouse model.IL-4 and IL-10 were generated which inhibited a s
14、pecific immune response.REF:Von Herrath et al.,J Clin Invest 98,1324.1996,Bystander suppression,Antigen-specific suppression is induced by feedingSuppression is triggered by re-encounter of antigenRelease of inhibitory cytokines will non-specifically inhibit other cells,3.Anergy,Non-productive antig
15、en presentation,T cells are activated by antigen presenting cells,3 signals are required to activate a T cell,Specific recognition-TCR sees the right MHC-peptide complex.signal 1Costimulation-CD28 binds B7 signal 2Cytokines-local micro-environment will instruct the kind of T cell needed signal 3,Res
16、ponse vs non-response,T lymphocyte activation requires 2 signalsSignal T cell proliferation+Signal(IL-2&IL-2r)Signal alone No proliferation,Signal 2 absence/blockade,Some epithelial cells in the gut and lung normally express class II MHC,but not costimulatory molecules and therefore cannot provide s
17、ignal 2Reagents(eg CTLA4 Ig)have been developed to block the interaction of CD28 with B7 on APC and therefore block signal 2,Anergy,Results in a specific hypresponsivenessAnergic cells do not respond to specific MHC+peptide plus costimulationAnergic cells may then block APC-and inhibit immune respon
18、sesAnergic cells may consume IL-2Anergic cells are more susceptible to programmed cell death(apoptosis),3.Regulation,There has been a great deal of discussion of suppressor cells(especially in the 1980s)Suppressor cells have proved difficult to clone and phenotypeMany cells exert a suppressive effec
19、tA range of regulatory T cells(Treg)have now been described,Regulatory T cells,A population of CD4+T cells has been implicated in the suppression of inflammatory immune responsesAntigen specificTurn off specific inflammatory immune responsesMechanism unclear,T reg in murine inflammatory bowel diseas
20、e,Pathogenic T cells(Tpath)-can transfer the disease to nave recipientsRegulatory T cells(Treg)-inhibit disease&TpathTreg are a subset of helper T cells(CD4)which express CD25Major area of investigation in Immunology Research,Models of oral tolerance,Eat soluble antigenInject antigenMeasure immune r
21、esponse T cell proliferationantibody productioncytokine profile,Multiple models of oral tolerance have been proposed(Weiner,1997),Animal modelsHuman modelsClinical trials,A murine model-Garside,Murine model in which OVA-specific T cells could be tracked with a specific monoclonal antibodyFed OVAWatc
22、h immune response by tracking OVA-specific T cellsSmith KM,McAskill F,Garside P.Orally tolerized T cells are only able to enter B cell follicles following challenge with antigen in adjuvant,but they remain unable to provide B cell help.J Immunol 2002 May 1;168(9):4318-25,Results,PRIMING-Ova injectio
23、n resulted in:specific antibody productionproliferation of OVA specific T cellsDTH responseTOLERANCE-Feeding Ova abrogated these responses demonstrated that priming and tolerance could be induced in this model.,Where did the responses take place?,PRIMINGd3 peak of OVA specific T cells in peripheral
24、lymph node,TOLERANCEd3 peak of OVA specific T cells in peripheral lymph node,T cell proliferation,PRIMINGT cell division in peripheral lymph nodes(pln),mesenteric lymph nodes(mln)and peyers patches(pp)at 2 days,TOLERANCET cell division in peripheral lymph nodes(pln),mesenteric lymph nodes(mln)and pe
25、yers patches(pp)at 2 days,T cell phenotype,PRIMINGOva specific T cells develop a memory phenotype.Changes detected as early as 6h after feeding.,TOLERANCEOva specific T cells develop a memory phenotype.Changes detected as early as 6h after feeding.,Differences.,Early systemic and local immune respon
26、se in priming and tolerance was very similarHowever,later immune responses were very different(immunity vs tolerance)Tolerant T cells did not move into B cell area and stimulate their expansion,Potential,Can oral tolerance be used therapeutically?Do inbred animal models relate to outbred human popul
27、ations?Can mechanisms of regulation be generated ex vivo or in vivo for clinical treatment?,Clinical trials,A number of clinical trials for auto-immune disease are in progress:DiseaseAntigenMultiple Sclerosis(MS)Myelin Basic Protein(MPB)Rheumatoid Arthritis(RA)Type II collagenType I DiabetesInsulinU
28、veitisS-antigenTransplant RejectionMHC molecules,Human MS trial,1y double blind study6/15 MS patients fed MBP had attacks15/15 control MS patients fed placebo had attacksThose individuals fed myelin had a higher frequency of TGF producing cells(Fukuara et al.,1996.J Clin Invest 98,70).,Human RA tria
29、ls,Several studies have investigated the effect of feeding type II collagen to RA patientsStudy No of centres Dose(mg)TimeResultsGermany50,1,1012 weeks ndUSA6 0.02-2.512 weeks1+Investigators are finding that dose and frequency are important factors,Diabetes,Type I insulin dependent diabetes mellitus
30、(IDDM)Organ specific auto-immune diseaseT cell mediated destruction of pancreatic beta cellsAnti-insulin antibodies developSusceptibility controlled by environmental&genetic factors(particularly MHC genes),Diabetes Prevention Trial(DPT-1),Multi-centre,randomized,controlled,clinical trial designed to
31、 determine if it is possible to prevent or delay the onset of type I diabetes(1994-2002)50%risk injected insulin 25-50%risk oral insulin,Results to date,Injected insulin failed to prevent or delay the onset of diabetes.Skyler JS et al.,(2002)Effects of insulin in relatives of patients with type 1 di
32、abetes mellitus New England Journal of Medicine 346(22):1685-1691,Results to follow,The Oral Insulin Trial completed enrollment on October 31,2002.Study investigators are now collecting final data to determine if oral insulin can delay the onset of type 1 diabetes.They expect to announce trial resul
33、ts in June 2003.,Summary of human clinical trials,Agents that enhance clinical tolerance are under investigation,eg other cytokines,adjuvants(cholera-toxin).However clinical trials have been disappointingPoor study design/suboptimal dose/type of antigen/route?http:/,Diffuse MALT,Lamina propria lymph
34、ocytes(primarily B cells)(LP major site of Ig synthesis)Lamina propria:the layer of connective tissue underlying the epithelium of a mucous membraneDerived from O-MALT and represent effector and memory cells from cells stimulated by antigen Intraepithelial lymphocytes(IELs)Plasma cells producing dim
35、eric IgAAntigen-presenting cells(macrophages and dendritic cells),Modes of Antigen Sampling,Stratified,non-keratinized or parakaratinized epithelia(oral cavity,pharynx,esophagus,urethra,vagina)Antigen sampling depends on Dendritic cellsLangerhans cells,phagocytic,antigen-presenting motile“scouts”)De
36、ndritic cells may then transport antigen to local and regional lymphoid follicles.Simple epithelia(bronchiole,intestine,bronchi)Antigen sampling depends on M cells and Transepithelial transportDendritic cells may also participate in antigen transport,Dendritic cells,Capture antigen in tissuesTranspo
37、rt to secondary lymphoid organsProcess and present to T cellsAn essential link between innate and adaptive immunityMay also represent the“Achilles Heel”of the host?(Cutler et al.2001),Maturation of Dendritic Cells,Loss of endocytic and phagocytic receptorsIncreased expression of MHCUp-regulation of
38、co-stimulatory molecules(CD80 and CD86)required for T-cell stimulationUp-regulation of CD40 and adhesion molecules ICAM-1 and LFA-3Fc receptors(endocytosis)decrease,This DC-precursor found in TDL looks very much like a lymphocyte with several important exceptions.Mitochondria were far more numerous
39、in the cytoplasm,and the DC nucleus was convoluted with more delicately distributed heterochromatin and lighter euchromatin than is normally found in lymphocytes.(From A.Anderson),Antigen Sampling across Simple Epithelia,Mucosal surfaces generally lined by a single layer of epithelial cellsBarrier s
40、ealed by tight junctions that exclude peptides and macromoleculesUptake of antigen requires active transepithelial transport(M-cells or Dendritic cells)Sampling is blocked by mechanisms such as local secretions,sIgA,mucins,etc.,Organization of O-MALT,M-Cell,Follicle-associatedepithelium,Dome region,
41、Germinal Center,Parafollicularregion,LUMEN,Lymphoid Follicle,Antigen Adherence to M-Cells,Adherence favors endocytosis and transcytosisAdherent materials tend to evoke strong immune responsesWide variety of pathogens adhere to M-cellsMechanism of adherence is unclearMany commensal microorganisms avo
42、id adherence to M-cells,M-Cells May Serve as Entry sites for Pathogenic Microorganisms,BacteriaVibrio choleraeEscherichia coliSalmonella typhiSalmonella typhimuriumShigella flexneriYersinia enterocoliticaYersinia pseudotuberculosisCampylobacter jejuni,VirusesReoviruspoliovirusHIV,Antigen Recognition
43、,Antigen transport is effected by M-Cells which occur over Organized Mucosa-Associated Lymphoid Tissue(O-MALT)After antigen stimulation,effector B-lymphocytes leave O-MALT and migrate to distant mucosal or glandular sites,Migration and Homing of Lymphocytes,Distribution of Homing Specificities in Mu
44、cosal Tissues Epithelial cells lining postcapillary venules(HEVs)display organ-specific recognition sites called“vascular addressins”Recognized by cell adhesion molecules“homing receptors”,High Endothelial Venules(HEV),Contain specialized endothelial cells lining post capillary venules.Display organ
45、-specific recognition sites called“vascular addressins”that are recognized by specific cell adhesion molecules on lymphocytes.HEV cells are characterized by:Elongated shape and prominent glycocalyx on luminal surfacePolarized,with a domed luminal surface separated from the basolateral surface by adh
46、erent junctions,but not tight junctionsCells rest on a basal lamina that constitutes the rate-limiting barrier to migrating lymphocytes,HEV(continued),In O-MALT,HEVs are present in T-cell areas between B cell folliclesIn D-MALT,venules have flat endothelial cells that share many features with HEVsHE
47、Vs produce sulfated glycolipids and glycoproteins into the vascular lumen(not known whether these products play a role in homing or extravasation),Adhesion molecules cloned so far belong to four main protein families,IntegrinsSelectinsCAMs(cell adhesion molecules)Proteoglycan-link.core proteins,Modu
48、lation of Homing Specificities,Naive lymphocytes prior to antigenic stimulation demonstrate no migration preferenceFollowing antigenic stimulation,lymphocytes acquire homing specificities,Lymphocytes in HEV,Lymphocytes adhering to luminal surfaces of HEV endothelial cells.Note microvilli on surface
49、of lymphocytes.,Cross-section of HEV,Transepithelial Transport in Mucosal Immunity,SamplingSite,Environment,Effector Site,Diffuse MALT,Organized MALT,Mucosal or GlandularTissue,Transepithelial Transport of IgA Antibodies,Polymeric immunoglobulin receptor and its intracellular trafficking poly-Ig rec
50、eptorBinding of IgA to polymeric immunoglobulin receptor,Transport and Distribution of IgA Antibodies,Effector Functions of Mucosal Antibodies,IgA antibodies are not good mediators of inflammatory reactions complement activation neutrophil chemotaxis phagocytosisImmune Exclusion/Serve“escort functio
