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 circulation Epithelial cells line
2、the mucosa Largest area exposed to the external environment Heaviest antigenic loadFeatures of mucosal tolerance?Normal immune function Tolerance can be local or systemic It requires a functional immune system Symbiosis-in the absence of commensals,a poor immune response develops and oral tolerance
3、cannot be inducedGeneral 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 food leads to food intolerance eg coeliac disease Immune responses to commensal bacteria leads to inflammatory bowel disease(I
5、BD)eg crohns disease,ulcerative colitisBalanceRespond Dont respondfight and eradicateIgnorePATHOGENS SELFFOODMechanism?Central tolerance deletion of self-reactive T cells in the thymus Peripheral tolerance an area of very active research!1.deletion 2.immune deviation 3.anergy 4.suppression/regulatio
6、nRegulation of self tolerance?Central tolerance is incomplete TCR bind at low affinity and can potentially recognise a number of MHC/peptide Auto-reactive T cells exist at high frequency in the periphery Auto-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 toleranceREF:Nature 1995 Jul 13;376(653
8、6):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 lower
9、 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-b
10、eta 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 cellsDeletion summary Generally observed at high doses of fed antigen:Activatio
11、n induced cell death(AICD)mediated by fas/fasL interactionsGrowth factor deprival2.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.Suppres
12、s 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)Some
13、 populations of T lymphocytes(both CD4 and CD8)can consume IL-2,the T cell growth factor.Surrounding cells therefore fail to growOne 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 specifi
14、c immune response.REF:Von Herrath et al.,J Clin Invest 98,1324.1996Bystander suppression Antigen-specific suppression is induced by feeding Suppression is triggered by re-encounter of antigen Release of inhibitory cytokines will non-specifically inhibit other cells3.Anergy Non-productive antigen pre
15、sentation T cells are activated by antigen presenting cells3 signals are required to activate a T cellSpecific 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 3Response vs
16、non-response T lymphocyte activation requires 2 signalsSignal T cell proliferation+Signal (IL-2&IL-2r)Signal alone No proliferationSignal 2 absence/blockade Some epithelial cells in the gut and lung normally express class II MHC,but not costimulatory molecules and therefore cannot provide signal 2 R
17、eagents(eg CTLA4 Ig)have been developed to block the interaction of CD28 with B7 on APC and therefore block signal 2Anergy Results in a specific hypresponsiveness Anergic cells do not respond to specific MHC+peptide plus costimulation Anergic cells may then block APC-and inhibit immune responses Ane
18、rgic cells may consume IL-2 Anergic cells are more susceptible to programmed cell death(apoptosis)Ref-Cobbold S&Waldmann H(1998)Infectious Tolerance.Current Opinion inImmunology 10,518-5243.Regulation There has been a great deal of discussion of suppressor cells(especially in the 1980s)Suppressor ce
19、lls have proved difficult to clone and phenotype Many cells exert a suppressive effect A range of regulatory T cells(Treg)have now been describedRegulatory T cells A population of CD4+T cells has been implicated in the suppression of inflammatory immune responses Antigen specific Turn off specific i
20、nflammatory immune responses Mechanism unclearT reg in murine inflammatory bowel disease Pathogenic T cells(Tpath)-can transfer the disease to nave recipients Regulatory T cells(Treg)-inhibit disease&Tpath Treg are a subset of helper T cells(CD4)which express CD25 Major area of investigation in Immu
21、nology ResearchModels of oral tolerance Eat soluble antigen Inject antigen Measure immune response T cell proliferation antibody production cytokine profileMultiple models of oral tolerance have been proposed(Weiner,1997)Animal models Human models Clinical trialsA murine model-Garside Murine model i
22、n which OVA-specific T cells could be tracked with a specific monoclonal antibody Fed OVA Watch 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
23、unable to provide B cell help.J Immunol 2002 May 1;168(9):4318-25Results PRIMING-Ova injection resulted in:specific antibody production proliferation of OVA specific T cells DTH response TOLERANCE-Feeding Ova abrogated these responses demonstrated that priming and tolerance could be induced in this
24、model.Where did the responses take place?PRIMING d3 peak of OVA specific T cells in peripheral lymph nodeTOLERANCE d3 peak of OVA specific T cells in peripheral lymph nodeT cell proliferationPRIMING T cell division in peripheral lymph nodes(pln),mesenteric lymph nodes(mln)and peyers patches(pp)at 2
25、daysTOLERANCE T cell division in peripheral lymph nodes(pln),mesenteric lymph nodes(mln)and peyers patches(pp)at 2 daysT cell phenotypePRIMING Ova specific T cells develop a memory phenotype.Changes detected as early as 6h after feeding.TOLERANCE Ova specific T cells develop a memory phenotype.Chang
26、es detected as early as 6h after feeding.Differences.Early systemic and local immune response in priming and tolerance was very similar However,later immune responses were very different(immunity vs tolerance)Tolerant T cells did not move into B cell area and stimulate their expansionPotential Can o
27、ral tolerance be used therapeutically?Do inbred animal models relate to outbred human populations?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(M
28、S)Myelin Basic Protein(MPB)Rheumatoid Arthritis(RA)Type II collagenType I DiabetesInsulinUveitisS-antigenTransplant RejectionMHC moleculesHuman MS trial 1y double blind study 6/15 MS patients fed MBP had attacks 15/15 control MS patients fed placebo had attacks Those individuals fed myelin had a hig
29、her frequency of TGF producing cells(Fukuara et al.,1996.J Clin Invest 98,70).Human RA trials Several studies have investigated the effect of feeding type II collagen to RA patientsStudy No of centres Dose(mg)Time ResultsGermany 50,1,1012 weeks ndUSA6 0.02-2.512 weeks 1+Investigators are finding tha
30、t dose and frequency are important factorsDiabetes Type I insulin dependent diabetes mellitus(IDDM)Organ specific auto-immune disease T cell mediated destruction of pancreatic beta cells Anti-insulin antibodies develop Susceptibility controlled by environmental&genetic factors(particularly MHC genes
31、)Diabetes Prevention Trial(DPT-1)Multi-centre,randomized,controlled,clinical trial designed to 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 insulinResults to date Injected insulin failed to prevent or delay the onset
32、of diabetes.Skyler JS et al.,(2002)Effects of insulin in relatives of patients with type 1 diabetes mellitus New England Journal of Medicine 346(22):1685-1691Results to follow The Oral Insulin Trial completed enrollment on October 31,2002.Study investigators are now collecting final data to determin
33、e if oral insulin can delay the onset of type 1 diabetes.They expect to announce trial results 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 disappointing Poor
34、 study design/suboptimal dose/type of antigen/route?http:/ MALT Lamina propria lymphocytes(primarily B cells)(LP major site of Ig synthesis)Lamina propria:the layer of connective tissue underlying the epithelium of a mucous membrane Derived from O-MALT and represent effector and memory cells from ce
35、lls stimulated by antigen Intraepithelial lymphocytes(IELs)Plasma cells producing dimeric IgA Antigen-presenting cells(macrophages and dendritic cells)Modes of Antigen Sampling Stratified,non-keratinized or parakaratinized epithelia(oral cavity,pharynx,esophagus,urethra,vagina)Antigen sampling depen
36、ds on Dendritic cells Langerhans cells,phagocytic,antigen-presenting motile“scouts”)Dendritic cells may then transport antigen to local and regional lymphoid follicles.Simple epithelia(bronchiole,intestine,bronchi)Antigen sampling depends on M cells and Transepithelial transport Dendritic cells may
37、also participate in antigen transportDendritic cells Capture antigen in tissues Transport to secondary lymphoid organs Process and present to T cells An essential link between innate and adaptive immunity May also represent the“Achilles Heel”of the host?(Cutler et al.2001)Maturation of Dendritic Cel
38、ls Loss of endocytic and phagocytic receptors Increased expression of MHC Up-regulation of co-stimulatory molecules(CD80 and CD86)required for T-cell stimulation Up-regulation of CD40 and adhesion molecules ICAM-1 and LFA-3 Fc receptors(endocytosis)decreaseAntigen Sampling across Simple Epithelia Mu
39、cosal surfaces generally lined by a single layer of epithelial cells Barrier sealed by tight junctions that exclude peptides and macromolecules Uptake of antigen requires active transepithelial transport(M-cells or Dendritic cells)Sampling is blocked by mechanisms such as local secretions,sIgA,mucin
40、s,etc.Organization of O-MALTM-CellFollicle-associatedepitheliumDome regionGerminal CenterParafollicularregionLUMENLymphoid FollicleAntigen Adherence to M-Cells Adherence favors endocytosis and transcytosis Adherent materials tend to evoke strong immune responses Wide variety of pathogens adhere to M
41、-cells Mechanism of adherence is unclear Many commensal microorganisms avoid adherence to M-cellsM-Cells May Serve as Entry sites for Pathogenic MicroorganismsBacteria Vibrio cholerae Escherichia coli Salmonella typhi Salmonella typhimurium Shigella flexneri Yersinia enterocolitica Yersinia pseudotu
42、berculosis Campylobacter jejuniViruses Reovirus poliovirus HIVAntigen Recognition 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 sit
43、esMigration and Homing of Lymphocytes Distribution of Homing Specificities in Mucosal 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)Con
44、tain specialized endothelial cells lining post capillary venules.Display organ-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 surface Polar
45、ized,with a domed luminal surface separated from the basolateral surface by adherent junctions,but not tight junctions Cells rest on a basal lamina that constitutes the rate-limiting barrier to migrating lymphocytesHEV(continued)In O-MALT,HEVs are present in T-cell areas between B cell follicles In
46、D-MALT,venules have flat endothelial cells that share many features with HEVs HEVs 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 Inte
47、grins Selectins CAMs(cell adhesion molecules)Proteoglycan-link.core proteinsModulation of Homing Specificities Naive lymphocytes prior to antigenic stimulation demonstrate no migration preference Following antigenic stimulation,lymphocytes acquire homing specificitiesLymphocytes in HEVLymphocytes ad
48、hering to luminal surfaces of HEV endothelial cells.Note microvilli on surface of lymphocytes.Cross-section of HEVTransepithelial Transport in Mucosal ImmunitySamplingSiteEnvironmentEffector SiteDiffuse MALTOrganized MALTMucosal or GlandularTissueTransepithelial Transport of IgA Antibodies Polymeric
49、 immunoglobulin receptor and its intracellular trafficking poly-Ig receptor Binding of IgA to polymeric immunoglobulin receptorTransport and Distribution of IgA AntibodiesEffector Functions of Mucosal Antibodies IgA antibodies are not good mediators of inflammatory reactions complement activation ne
50、utrophil chemotaxis phagocytosis Immune Exclusion/Serve“escort function Beneficial not to induce inflammation Intra-epithelial virus neutralization by IgA Excretory function for IgARelationship between Systemic and Mucosal Immunity Oral tolerance(anergy)Oral administration of antigen suppresses syst
