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Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Division of Pathology, Oslo University Hospital, Rikshospitalet, Norway Induction and function of the mucosal immune system Per Brandtzaeg [email protected]

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Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Division of Pathology, Oslo

University Hospital, Rikshospitalet, Norway

Induction and function of themucosal immune system

Per [email protected]

• The MRKAd5 HIV-1 Gag/Pol/Nef candidate vaccine advanced to a phase 2b test-of-concept trial known as STEP, conducted by Merck & Co., Inc., and the HIV Vaccine Trials Network (HVTN).

• The vaccine neither prevented infection nor had an impact on early plasma virus levels in those who received the vaccine compared with the placebo recipients (2007).

• Additional studies with mucosal and biopsy specimens will be required to explore whether activation of cells at the mucosal sites were different between vaccine and placebo recipients.

Science 2008; 321: 530-2

Sandra Mazzoli et al. Nature Medicine 3:1250-57, 1997

HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals

Most uninfected partners of couples discordant for HIV infection show evidence of HIV-specific secretory IgA at mucosal sites (vaginal wash)

Dimers are a girl’s best friendEditorial: Sarah Rowland-Jones:

The mucosae are an enormous battlefieldMucosal effector sites provide secretory IgA (SIgA) antibodies

Section through skin

Airways and oral cavity

Gastrointestinal mucosa

Hornified layerEpithelial cells

Mucus and cilia

GlandsPlasma cells

Surface epithelium

Glands (crypts)Plasma cells

Epithelial cells

IgAIgGNormal human colon

Plasma cellsH&E

IgA

80% of all plasma cells arelocated in gut mucosa

An adult exports 3 g of SIgA to the gut lumen per day

At the border of hell!

Local formation and export of mucosal immunoglobulins

Dimeric IgA (pIgA)

J chain

Mucus

Gland

IgA+J

SIgM

SIgA

IgM+J

LumenStroma

IgG

pIgR(mSC)

PentamericIgM IgG(±J)

Free SC

IgA+J

Plasma cells

IgA

IgA-coatedbacteria

Brandtzaeg Nature & J. Immunol. 1974; Brandtzaeg & Prydz Nature 1984

Ag‘The IgA pump’

Containment

Inductive site and effector site of gut immune system

Mod

ified

from

: Mac

Don

ald

& M

onte

leon

e S

cien

ce20

05;3

07:1

920-

25

(GALT)

Germinal center

B cell

Homing

Lamina propria

pIgR

Distantsites Normal effector site

SIgA

IgAIgG

Teff

Treg

pIgA

Lymphocyte

differentiation

Barrier function

Gut homing

IgA development

IgA export

Bug controlAntigen uptake

Immunoglobulin A (IgA) is the major antibody class in humans

Plasma cell

Secretory IgA (SIgA) with bound SC (the cleaved ectodomain of pIgR)

SC

Serum IgA(monomeric IgA)

Dimeric IgA (pIgA) with J chain (J)

IgA+J pIgA

J

?

m m m

p p p

IgA

Parotid gland

SC aff. Merge

Brandtzaeg P. Nature New Biol. 1973; 243: 142-43, Nature 1974; 252: 418-20, Mol. Immunol. 1983; 20: 941-66

pIgR

Free SC

Epithelialcell

SC (free or bound) exhibits several anti-microbial properties so the sacrificial pIgR might have originated from the innate defence system to drive the ‘IgA pump’

~40%

Plasma cell

SCIgA+J pIgA

J

? pIgR

Free SC

Epithelialcell

Immunoglobulin A (IgA) is the major antibody class in humansSecretory IgA (SIgA) with bound SC (the cleaved ectodomain of pIgR)

Serum IgA(monomeric IgA)

Dimeric IgA (pIgA) with J chain (J)

m m m

p p p

IgA

Parotid gland

SC aff. Merge

Brandtzaeg P. Nature New Biol. 1973; 243: 142-43, Nature 1974; 252: 418-20, Mol. Immunol. 1983; 20: 941-66

00

PP C-LP I-LP MLN PLN PT

Per

cent

age

J-ch

ain+

()

Per

cent

age

subc

lass

es (

)

100

80

60

40

2020

40

60

80

100

IgA+J

IgA2IgA1

Children

J-LP

Systemic antibodies: Mainly IgGMucosal antibodies: Mainly secretory IgA (SIgA)

MALT(NALT)

MALT(GALT)

Primary lymphoid organs

Secondary lymphoid

organs

Systemic and mucosa-associated lymphoid tissue (MALT)

F

F

FAE

Gut-associated lymphoid tissue (GALT)

GALT structures are strategically situated in relation to the greatest concentration of commensal bacteria (indigenous microbiota)

Peyer’s patches:distal ileum (nos. 100-250)Appendix: cecumIsolated lymphoid follicles (ILFs):distal large bowel (nos. ~ 30 000)

Maintenance of immune homeostasis in the human gut

IgA IgM

Oral tolerance(suppression)

Productive stimulation

Immune exclusion: SIgA/SIgM

IgE DTHIgG

Lymphoidfollicle

CD4

Other secretory effector tissues

Peyer's patch (GALT)

Peripheral blood

APC BT

TB

B

CD8

Thoracicduct

Mesentericlymphnode

TFDC CD4

MMM

M γ/δ

Ag

BpIgR/mSC

CD4

Mucus

Endothelial gatekeeper function

Effector siteInductive site

IgA IgGM-cell area

FAE

GC

GC

Inductive site (GALT) Effector site (normal intestinal mucosa

M-cell area

Crypt

CD4CD8

CD20CD3

Maintenance of immune homeostasis in the human gut

IgA IgM

Oral tolerance(suppression)

Productive stimulation

Immune exclusion: SIgA/SIgM

IgE DTHIgG

Lymphoidfollicle

CD4

Other secretory effector tissues

Peyer's patch (GALT)

Peripheral blood

APC BT

TB

B

CD8

Thoracicduct

Mesentericlymphnode

TFDC CD4

MMM

M γ/δ

Ag

BpIgR/mSC

CD4

Mucus

Endothelialgatekeeper function

Effector siteInductive site

FAE

MAdCAM-1

GCGC

GC

HEV

HEV

TAPC

M M M

B

Antigen

Effector site:normal gut mucosa

BT

IgA

SIgAB

MAdCAM-1

Mucosal leukocyte homing in the normal gut

Peripheral bloodEos.Mo PMN MC

FDC

MφAPC

Endothelial gate-keeper function

α4β7+

Gut associated lymphoid tissue(GALT)

IgA IgG

pIgRPeyer’s patch

CCR10+

CCR9+MECTECK

Microvascular gut endothelium

Human NALT anlagen: prenatal (19 wks).Nasal ILFs in 40% < 2 yrs (inducible?).Rodent NALT: postnatal organogenesis.

Tonsils and adenoids are well designed for antigen trappingInductive sites

TALT

LALT

BALT

Peyer'spatches,appendixand solitary intestinal lymphoid follicles (GALT)

Tonsils &adenoids(NALT)

GC

Crypt

Human palatine tonsil

Scanning electron-microscopy (Owen, 1988)

Reticular cryptepithelium(cytokeratin)

43%

78%

Blood circulation

Mesentericlymph nodes

Inguinallymph nodes

Tonsils &adenoids

Cervicallymph nodes

Lacrimalgland

Bonemarrow

Peyer’spatches

63%

86%

60%

9%

67%43%

28%0%

Nasal &salivaryglands

33%60%

83% Gut

Cervixmucosa

40%

Integration and compartmentalization in the mucosal immune system

Duodenum

Colon

Freqency of various tissue samples with Cμ deletion, reflecting dissemination of sIgD+IgM– effector B cells from Waldeyer’s ring

(tonsils and adenoids) to systemic and mucosal immune compartments

Peyer’s patches

Mesenteric nodes

Inguinal nodes

Bone marrow

Cervical lymph nodes

Normal adenoids

Hyperplastic adenoids

Normal tonsils

Recurrent tonsillitis

IgA def.: tonsils& adenoids

Small bowel

IgA def.: duodenum

Salivary glands

Nasal mucosa

IgA def.:nasal mucosa

Lacrimal glands

IgA def.: lacrimal& salivary glands

IgA def.: colon

0 20 40 60 80 1000 20 40 60 80 100Percentage of positive samples Percentage of positive samples

n = 13

n = 9

n = 10

n = 5

n = 8

n = 6

n = 5

n = 7

n = 7

n = 2

n = 6

n = 8

n = 23

n = 22

n = 3

n = 26

n = 16

n = 3

Uterine endocervix n = 5

Large bowel n = 15

Compartmentalization of the integrated mucosal immune system

Femalegenitaltract

Peyer'spatches(GALT)

Tonsils andadenoids(NALT)

Lacrimal,nasal andsalivaryglands

BALT

Mammaryglands

Indu

ctiv

esi

tes

Effe

ctor

site

s

PNAd/VCAM-1MEC (CCL28)

Appendix and colonic-rectalisolated follicles(GALT)

Bronchi Small bowel

Large bowel

MAdCAM-1TECK (CCL25)

MAdCAM-1MEC (CCL28)

L-sel. (CD62L)CCR7α4β1

CCR10CCR9CCR10

α4β1α4β7

MEC (CCL28)

Shared homingmolecules (CCR10)

Johansen, Baekkevold, Carlsen, Farstad, Soler, Brandtzaeg, Blood 2005; 106:593-600

Systemic homing

The nature of secretory immunity in the female genital tract is obscure

• The most active SIgA system occurs in the gut, but secretory immunity also operates in the female genital tract; there are J chain-producing IgA+ plasma cells and pIgR/SC expression in the cervical mucosa and fallopian tubes

• The origin of these local IgA+ plasma cells remains partly undefined because organized inductive mucosa-associated lymphoid tissue (MALT) is generally absent from the genital tract

Role of the endometrium in secretory immunity

• Like cervical glandular epithelium, the endometrium can also perform pIgR-mediated external translocation of dimeric IgA

• These dimers appear to be mainly derived from serum, partly under hormonal regulation

• Paracellular diffusion of IgG and monomeric IgA through epithelia is also an important part of humoral immunity in the female genital tract

There is considerable local production as well as leakage of IgG into the female genital tract

• Human cervical mucosa often contains a substantial number of IgG-producing plasma cells

• It is possible that these plasma cells are derived from NALT or perhaps from the draining cervical lymph nodes

• Intranasal vaccination also appears to be quite efficient for systemic immunization, providing high-affinity serum IgG antibodies

Class distribution and J-chain expression of IgA- and IgG-producing plasma cells in human cervical mucosa

Imm

unoc

yte

clas

s di

strib

utio

n (%

)

0

IgA IgG IgM

20

40

60

80

100

J-ch

ain

expr

essi

on (%

)

IgA IgG0

20

40

60

80

100

Brandtzaeg and Johansen (unpublished)

IgA/IgG IgA + J chain IgG + J chain

IgA- and IgG-producing plasma cells with J-chain expression in cervical mucosa

IgA- and IgG-producing plasma cells with J-chain expression in duodenal mucosa

IgA + J chain IgG + J chain

Components of humoral immunity in the human female genital tract

IgA- and IgG-producing cells in adenoid specimen

CryptLymphoid follicle

GC

IgA IgG

Plasmacells

Normal IgD:IgM:IgA:IgG:

82%62%51%2%

79%54%19%1%

Recurrent tonsillitis

82%55%29%36%

IgD:IgM:IgA:IgG:

50%63%2%

45%

IgA+J chain

Cryptepithelium

GC

IgA J chain

GC

GC

Tonsillar germinal centre (GC) and extrafollicular Ig-producing cells express J chain (% of total isotype)

Brandtzaeg & Korsrud Clin. Exp. Immunol. 1984; 58: 709-18

Tonsillar germinal centre contains IgG-producing plasmablasts which often show J-chain expression

IgG+J+

IgG J chain

IgAIgG

sIgM sIgDsIgM sIgA IgA+J

sIgD

sIgGsIgA

Extra-follicular compart-

ments

IgD

IgG

Tonsillargerminal centre

Founder cellMigration to distant secretory effector

sites

Mantle zone

J

J

J

J

JJJ

IgM+J

IgA + J chain

Lymphoidfollicle

GC

IgA J chain

Brandtzaeg Mol. Immunol. 1983; 20: 941-66

B-cell differentiation in germinal centres

The nasal route is attractive for mucosal vaccine administration

But be aware of direct communication between the olfactory bulb and CNS

Adenoids

Middle turbinate

Oral cavity

Tounge

Epiglottis

Inferior turbinate

Olfactory regionTubal tonsil

Palatine tonsil

Lingual tonsil

Skin

Waldeyer’s ringOrganized lymphoid tissue with M cells

Nasal anatomy and location of regional lymphoid tissue

Olfactory region

Ciliated mucosa

Nasal mucosaExtremely rich in dendritic cells

150-200 cm2

Jahnsen FL, Gran E, Haye R, Brandtzaeg P. Am J Respir Cell Mol Biol, 2004; 30:31-37

HLA-DRRFD-7

Human nasal mucosa

Adapted from Hordnes et al. Vaccine 1996; 64:1643-52

Effects of parenteral immunization with albumin on con-current penetration (2h) of human serum albumin (HSA) and transferrin (Trf) through rabbit sublingual mucosa

Bas

al c

onc.

x10

5 /api

cal

conc

. (30

mg/

ml)

Brandtzaeg & Tolo Nature 1977; 266: 262-63

HSA (69 kDa)Trf (90 kDa)

Naïve animals Immunized (IgG anti-HSA)

2

4

6

8

HSA

TrfHSA

Trf

IgG antibodies may increase mucosal permeability for bystander molecules

Confirmed in vivo (mice) that this complication is caused by IgG but not SIgA antibodiesLim & Rowley, 1982

• Mucosal vaccination offers several distinct advantages for protection of the female genital tract (SIgA balancing IgG antibodies)

• Nasal spray might be more acceptable than an intravaginal or rectal vaccine

• A combined nasal/vaginal/rectal approach would probably be most effective but perhaps socially less acceptable

Conclusions

AcknowledgementsLaboratory for Immunohistochemistry and Immunopathology

(LIIPAT) is part of Centre for Vaccinology and Immunotherapy (CEVI, 2001) and Centre of Excellence for Immune Regulation (CIR, 2007), funded by the Research Council of Norway, University of Oslo and Rikshospitalet

University Hospital

Marie Curie Training Network: CROSS-TALK(EC, 2008)