molecular and cellular basis of immune protection of...

1Slide # Dennis E. Lopatin, Ph.D.

Molecular and Cellular Basis of ImmuneMolecular and Cellular Basis of ImmuneProtection of Mucosal SurfacesProtection of Mucosal Surfaces

Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.

Department of Biologic & Materials SciencesDepartment of Biologic & Materials Sciences

School of DentistrySchool of Dentistry

University of MichiganUniversity of Michigan

Ann Arbor, Michigan 48109-1078Ann Arbor, Michigan 48109-1078

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IntroductionIntroduction

nn Mucosal surfaces represent a vast surface area vulnerableMucosal surfaces represent a vast surface area vulnerableto colonization and invasion by microorganisms.to colonization and invasion by microorganisms.

nn Total amount of sIgA exported on mucosal surfacesTotal amount of sIgA exported on mucosal surfacesexceeds production of circulating IgG.exceeds production of circulating IgG.

nn Antigens on mucosal surfaces are separated from mucosalAntigens on mucosal surfaces are separated from mucosalimmune tissue by epithelial barrier.immune tissue by epithelial barrier.

nn To elicit a mucosal immune response, antigens must beTo elicit a mucosal immune response, antigens must betransported across the epithelium before they can betransported across the epithelium before they can beprocessed and presented to cells of immune system.processed and presented to cells of immune system.


Significance of Mucosal ImmunitySignificance of Mucosal Immunity

nn Protection from microbial colonization (adherence)Protection from microbial colonization (adherence)

nn Prevention of environmental sensitizationPrevention of environmental sensitization

nn Focus of much vaccine workFocus of much vaccine work

nn May have regulatory influence on systemic immunityMay have regulatory influence on systemic immunity

nn May block allergic sensitizationMay block allergic sensitization


Secretory IgASecretory IgA

nn >3 g of sIgA per day>3 g of sIgA per day

nn Structure of IgAStructure of IgA

nn Isotypes (A1 and A2) are tissue-specificIsotypes (A1 and A2) are tissue-specificll A1-A1-

ll A2- mucosal plasma cells (has resistance to IgA1 proteases)A2- mucosal plasma cells (has resistance to IgA1 proteases)

nn J-chainJ-chain

nn Secretory componentSecretory component


Structure of Secretory IgA (sIgA)Structure of Secretory IgA (sIgA)

Secretory Component (five domains)

J chain


J chainJ chain

nn 15,600 kDa15,600 kDa

nn Associated with polymeric IgAssociated with polymeric Ig

nn Synthesized by Plasma cellSynthesized by Plasma cell

nn One J chain per polymer regardless of sizeOne J chain per polymer regardless of size

nn Is probably associated with initiation ofIs probably associated with initiation ofpolymerizationpolymerization

nn Induces confirmation that optimizes binding to SCInduces confirmation that optimizes binding to SC


Secretory ComponentSecretory Component

nn MW 80,000MW 80,000

nn Synthesized by epithelial cells of mucous membranesSynthesized by epithelial cells of mucous membranes

nn IgA dimer binding sites per epithelial cell isIgA dimer binding sites per epithelial cell isapproximately 260-7,000approximately 260-7,000


Organization of Mucosal Lymphoid TissueOrganization of Mucosal Lymphoid Tissue

nn MALT cellular mass exceeds total lymphoid cells inMALT cellular mass exceeds total lymphoid cells inbone marrow, thymus, spleen, and lymph nodesbone marrow, thymus, spleen, and lymph nodes


Organized lymphoid follicles at specificOrganized lymphoid follicles at specificmucosal sites (O-MALT)mucosal sites (O-MALT)

nn Occur in tissues of digestive, respiratory and genitalOccur in tissues of digestive, respiratory and genitalmucosal surfacesmucosal surfaces

nn Light germinal centersLight germinal centers

nn Dark adjacent areas populated by B and T lymphocytesDark adjacent areas populated by B and T lymphocytesand antigen-presenting cellsand antigen-presenting cells

nn Site of antigen sampling and generation of effector andSite of antigen sampling and generation of effector andmemory cellsmemory cells


Diffuse MALTDiffuse MALT

nn Lamina propria lymphocytes (primarily B cells) (LP majorLamina propria lymphocytes (primarily B cells) (LP majorsite of Ig synthesis)site of Ig synthesis)

ll Lamina propria: the layer of connective tissue underlying the epithelium of aLamina propria: the layer of connective tissue underlying the epithelium of amucous membranemucous membrane

nn Derived from O-MALT and represent effector and memoryDerived from O-MALT and represent effector and memorycells from cells stimulated by antigencells from cells stimulated by antigen

nn Intraepithelial lymphocytes (IELs)Intraepithelial lymphocytes (IELs)

nn Plasma cells producing dimeric IgAPlasma cells producing dimeric IgA

nn Antigen-presenting cells (macrophages and dendritic cells)Antigen-presenting cells (macrophages and dendritic cells)


Modes of Antigen SamplingModes of Antigen Sampling

nn Dendritic cells in stratified and pseudo stratifiedDendritic cells in stratified and pseudo stratifiedepitheliaepithelia

ll (Langerhans cells, phagocytic, antigen-presenting motile(Langerhans cells, phagocytic, antigen-presenting motile“scouts”)“scouts”)

nn M cells in simple epithelia with tight junctionsM cells in simple epithelia with tight junctionsll Transepithelial transportTransepithelial transport


Antigen Sampling across Simple EpitheliaAntigen Sampling across Simple Epithelia

nn Mucosal surfaces generally lined by a single layer ofMucosal surfaces generally lined by a single layer ofepithelial cellsepithelial cells

nn Barrier sealed by tight junctions that exclude peptidesBarrier sealed by tight junctions that exclude peptidesand macromoleculesand macromolecules

nn Uptake of antigen requires active transepithelialUptake of antigen requires active transepithelialtransporttransport

nn Sampling is blocked by mechanisms such as localSampling is blocked by mechanisms such as localsecretions, sIgA, mucins, etc.secretions, sIgA, mucins, etc.


Antigen Adherence to M-CellsAntigen Adherence to M-Cells

nn Adherence favors endocytosis and transcytosisAdherence favors endocytosis and transcytosis

nn Adherent materials tend to evoke strong immuneAdherent materials tend to evoke strong immuneresponsesresponses

nn Wide variety of pathogens adhere to M-cellsWide variety of pathogens adhere to M-cells

nn Mechanism of adherence is unclearMechanism of adherence is unclear

nn Many commensal microorganisms avoid adherence to M-Many commensal microorganisms avoid adherence to M-cellscells


M-Cells May Serve as Entry sites forM-Cells May Serve as Entry sites forPathogenic MicroorganismsPathogenic Microorganisms

nn Polio, reovirusPolio, reovirus

nn SalmonellaSalmonella


Antigen RecognitionAntigen Recognition

nn Antigen transport is effected by M-Cells which occur overAntigen transport is effected by M-Cells which occur overOrganized Mucosa-Associated Lymphoid Tissue (O-Organized Mucosa-Associated Lymphoid Tissue (O-MALT)MALT)

nn After antigen stimulation, effector B-lymphocytes leaveAfter antigen stimulation, effector B-lymphocytes leaveO-MALT and migrate to distant mucosal or glandularO-MALT and migrate to distant mucosal or glandularsitessites


Organization of O-MALTOrganization of O-MALT

M-Cell Follicle-associatedepithelium

Dome region

Germinal Center

Parafollicularregion

LUMEN

Lymphoid Follicle


Migration and Homing of LymphocytesMigration and Homing of Lymphocytes

nn Distribution of Homing Specificities in MucosalDistribution of Homing Specificities in MucosalTissuesTissues

ll Epithelial cells lining postcapillary venules (Epithelial cells lining postcapillary venules (HEV’sHEV’s))display organ-specific recognition sites calleddisplay organ-specific recognition sites called“vascular addressins”“vascular addressins”

ll Recognized by cell adhesion molecules “homing Recognized by cell adhesion molecules “homingreceptors”receptors”


High Endothelial Venules (HEV)High Endothelial Venules (HEV)

nn Contain specialized endothelial cells lining post capillaryContain specialized endothelial cells lining post capillaryvenules.venules.

nn Display organ-specific recognition sites called “vascularDisplay organ-specific recognition sites called “vascularaddressins” that are recognized by specific cell adhesionaddressins” that are recognized by specific cell adhesionmolecules on lymphocytes.molecules on lymphocytes.

nn HEV cells are characterized by:HEV cells are characterized by:ll Elongated shape and prominent Elongated shape and prominent glycocalyx glycocalyx on on luminal luminal surfacesurfacell Polarized, with a domed Polarized, with a domed luminal luminal surface separated from thesurface separated from the

basolateral basolateral surface by adherent junctions, but not tight junctionssurface by adherent junctions, but not tight junctionsll Cells rest on a basal lamina that constitutes the rate-limiting barrierCells rest on a basal lamina that constitutes the rate-limiting barrier

to migrating lymphocytesto migrating lymphocytes


HEV (continued)HEV (continued)

nn In O-MALT, In O-MALT, HEV’s HEV’s are present in T-cell areas between Bare present in T-cell areas between Bcell folliclescell follicles

nn In D-MALT, venules have flat endothelial cells that shareIn D-MALT, venules have flat endothelial cells that sharemany features with many features with HEV’sHEV’s

nn HEV’s HEV’s produce sulfated produce sulfated glycolipids glycolipids and and glycoproteinsglycoproteinsinto the vascular lumen (not known whether theseinto the vascular lumen (not known whether theseproducts play a role in homing or products play a role in homing or extravasationextravasation))


Migration and Homing PathwaysMigration and Homing Pathways

HEV (High endothelialvenules in O-MALT) SV (small venules in

D-MALT))

Adhesion

De-Adhesion

AdhesionPrimaryLymphoid Tissue

SecondaryLymphoid TissueO-MALT

EffectorSiteD-MALT


Adhesion molecules cloned so far belongAdhesion molecules cloned so far belongto four main protein familiesto four main protein families

nn IntegrinsIntegrins

nn SelectinsSelectins

nn CAMs (cell adhesion molecules)CAMs (cell adhesion molecules)

nn Proteoglycan-link.core proteinsProteoglycan-link.core proteins


Modulation of Homing SpecificitiesModulation of Homing Specificities

nn Naive lymphocytes prior to antigenic stimulationNaive lymphocytes prior to antigenic stimulationdemonstrate not migration preferencedemonstrate not migration preference

nn Following antigenic stimulation, lymphocytesFollowing antigenic stimulation, lymphocytesacquire homing specificitiesacquire homing specificities


Lymphocytes in HEVLymphocytes in HEV

Lymphocytes adhereing to luminalsurfaces of HEV endothelial cells. Notemicrovilli on surface of lymphocytes.

Cross-section of HEV


TransepithelialTransepithelialTransport inTransport in

MucosalMucosalImmunityImmunity

SamplingSite

EnvironmentEffector Site

Diffuse MALT

Organized MALT

Mucosal orGlandularTissue


Transepithelial Transport of IgATransepithelial Transport of IgAAntibodiesAntibodies

nn Polymeric immunoglobulin receptor and itsPolymeric immunoglobulin receptor and itsintracellular traffickingintracellular trafficking

ll poly-Ig receptor poly-Ig receptor

nn Binding of IgA to polymeric immunoglobulinBinding of IgA to polymeric immunoglobulinreceptorreceptor


Transport and Distribution of IgATransport and Distribution of IgAAntibodiesAntibodies


Effector Functions of Mucosal AntibodiesEffector Functions of Mucosal Antibodies

nn IgA antibodies are not good mediators ofIgA antibodies are not good mediators ofinflammatory reactionsinflammatory reactions

ll complement activation complement activation

ll neutrophil chemotaxis neutrophil chemotaxis

ll phagocytosis phagocytosis

nn Immune Exclusion/Serve “escort" functionImmune Exclusion/Serve “escort" function

nn Beneficial not to induce inflammationBeneficial not to induce inflammation

nn Intra-epithelial virus neutralization by IgAIntra-epithelial virus neutralization by IgA

nn Excretory function for IgAExcretory function for IgA


Rational Strategies for Mucosal ImmunizationRational Strategies for Mucosal Immunization

nn RequirementsRequirements

ll Safe taken orally Safe taken orally

ll Long-lasting due to continued maintenance of memory Long-lasting due to continued maintenance of memory

ll Survive in gastric and intestinal environments Survive in gastric and intestinal environments

ll Must escape normal clearance mechanisms Must escape normal clearance mechanisms

ll Must compete for inclusion within M-Cell transport Must compete for inclusion within M-Cell transport

ll Must arrive intact to antigen-processing cells Must arrive intact to antigen-processing cells

ll Must induce dimeric sIgA reactive with cell surface Must induce dimeric sIgA reactive with cell surface


Rational Strategies for MucosalRational Strategies for MucosalImmunization (continued)Immunization (continued)

nn Strategies for Delivery of Vaccine Into O-MALTStrategies for Delivery of Vaccine Into O-MALT

ll Inert particulate carriers Inert particulate carriers

ll Biodegradable copolymers Biodegradable copolymers

ll Immune-stimulating complexes (ISCOMs) Immune-stimulating complexes (ISCOMs)

ll Hydroxyapatite crystals Hydroxyapatite crystals

ll Live vaccine vectors (recombinant) Live vaccine vectors (recombinant)

llVaccinia virusVaccinia virus

ll SalmonellaSalmonella

ll Mycobacterium bovis Mycobacterium bovis


Rational Strategies for MucosalRational Strategies for MucosalImmunization (continued)Immunization (continued)

nn Strategies for Enhancing Mucosal Immune ResponseStrategies for Enhancing Mucosal Immune Response

ll Co-delivery with cytokines Co-delivery with cytokines

ll Co-immunogens (Cholera toxin) Co-immunogens (Cholera toxin)

ll Peptides presented with potent T-cell epitopes Peptides presented with potent T-cell epitopes


ReferencesReferences1. Brown, T. A. Immunity at mucosal surfaces. Adv Dent Res.1996; 10(1):62-5.

2. Kiyono, H; Ogra, Pearay L, and McGhee, Jerry R. Mucosalvaccines. San Diego: Academic Press; 1996. xix, 479 p .

3. Kraehenbuhl, J. P. and Neutra, M. R. Molecular and cellularbasis of immune protection of mucosal surfaces. Physiol Rev.1992; 72(4):853-79.

4. Neutra, M. R.; Frey, A., and Kraehenbuhl, J. P. Epithelial Mcells: gateways for mucosal infection and immunization. Cell.1996; 86(3):345-8.

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