Mucosal Immunity-I

Sarah Gaffen, Ph.D.Division of Rheumatology & Clinical Immunology

Spring 2009sig65@pitt.edu

Overview

• Organization- what is a mucosal surface?– Development, NALT, GALT etc

• Effectors– IELs– M cells– IgA

• Commensal organisms• Mucosal tolerance & homeostasis• Mucosal diseases: lung, gut, mouth

Mucosal Immunity

• Comprises the “surface exposed” parts of the body (gut, mouth, genital tract, lung, eye, etc)

• Contrasts with “systemic immune system” (spleen, LN)

• Most infections start at mucosal surfaces• Probably the most evolutionarily ancient

Anatomical/Physical Barriers

• Skin - acidic, anti-microbial peptides• Mouth - enzymes, anti-microbial peptides,

directional flow of fluid to stomach• Stomach- low pH, digestive enzymes, fluid flow to

intestine• Large intestine -normal flora compete for niches• Airways and lungs- cilia, mucus, coughing/ sneezing,

macrophages in lung alveoli

Unique features of Mucosal I.S.• Close association of mucosal epithelial layers and

lymphoid structures– Diffuse or organized

• Specialized Ag-uptake mechanisms– Example: Microfold (M) cells in the gut

• Effectors– IgA– Specialized T’s, activated even w/o infection

• Strong immunosuppressive environment– Suppressive responses to commensals

Mucosal-Associated Lymphoid Tissue (MALT)

• Each mucosal surface has immune tissue, more or less organized– GALT (gut): Peyer’s Patches, appendix– NALT (nasopharynx): Adenoids, Tonsils– BALT (bronchial): iBALT– Genital tract? Not as clear, no lymphoid

follicles.

Antimicrobial Defenses of the Lung

• Structural Defenses– Glottis– Cough reflex– Airway secretions– Mucociliary transport

• Innate Defenses– Resident alveolar macrophages– Recruited polymorphonuclear leukocytes– Recruited monocytes

Prototypical mucosal surface: Gut• Largest mucosal surface• Specialized Ag-uptake• Large # of specialized lymphocytes• Villi- large surface area for nutrient exchange• Peyer’s Patch

– secondary lymphoid organs within wall of intestine– Rich in B cell follicles, smaller T cell areas– Surrounded by epithelium interspersed with

“microfold” (M) cells

M (microfold) Cells

• Transport antigens across the mucosal surface• Transport achieved via vesicles; “transcytosis”• Extensively folded – large surface area• Creates a “pocket” for DCs, lymphocytes• DC’s migrate to:

– T cell areas in PP– mesenteric LNs

• Can be exploited by pathogens to gain entry into

Blue=epithelial cellsRed = T cellsGreen = B cells

Luminal antigens are taken up by M cells and presented to T cells by

macrophages

EMBO reports 7, 7, 688–693 (2006)

Lymphocyte circulation in Mucosa

• Naïve lymphocytes that arrive at PP or mLNs enter via HEVs, just like other 2° organs

• If they are activated, they lose expression of CCR7 and L-selectin (which normally directs cells to peripheral LNs)

• They gain expression of the 4:7 integrin, which binds to “MAdCAM-1” on gut (and other mucosal) endothelial cells

• They also express CCR6, CCR9 and CCR10, which directs them to gut

Adhesion molecules in the Integrin family are heterodimers sharing similar

and chains

1 Integrins 2 Integrins 3-7 Integrins

VLA-4

4, 1

LFA-1

L, 2

MAC-1CD11b, CD18M, 2

4, 7

Initial steps in leukocyte migration are mediated by interactions with

endothelium

Selectin dep.Integrin dep

CCR6CCL20

“Common Mucosal Immune System”

• Lymphocytes primed at one mucosal area can recirculate to other mucosal surfaces

• This is because MAdCAM-1, mucosal chemokines are expressed on vasculature of other mucosal sites

• Therefore, priming at one site can provide protection at another– e.g. Immunization in the nose can protect against

infection in lung (flu)

The “Common” Mucosal Immune System

M cell

M M M

SIgA

Mucosal inductive site

Organized mucosa-associated lymphoid tissue (MALT)

Antigen

B

BB

B

BB

B

T

TT

T

TT

T

FDC

B

Endothelial gatekeeper function

SIgM (IgG)

Mucosal effector site

HEV

IgG(J)

CD8

APC

CD4

B

pIgR

(SC)

APC

B

DC

Naivecells

Lymphatic vessel

T

Antigen

IgA+J

IgA+JIgM+J

B

Lymph node

Peripheral blood

Peyer's patches

Isolated lymphoid follicles (ILFs)

Appendix

Waldeyer's ring (NALT)

T

IgA

• The dominant class of Ab in the mucosal immune system

• Can exist in multiple forms– Serum- IgA is mostly monomer – Mucosa

• IgA is dimer linked by J chain• After transcytosis, associated with secretory

component

IgA is the most heterogeneous of Ig isotypes: 3 molecular forms

17%

52%

30%

1%

IgM IgG IgA IgD

13%

6%

80%

1%

>6 x 10 10 cells

Ig-producing cells in human tissues

~2.5 x 1010

cells

Mucosal Tissues

Gastrointestinal, respiratory,genito-urinary tracts; eyes;Salivary, lacrimal, mammary glands

Systemic Tissues

Bone marrow,lymph nodes, spleen

Daily production rates (mg) of IgA and IgG in humans (70kg adult)

Tissue/fluid IgA IgG

Circulation 1300 - 2100 2100

Saliva 100 - 200 1 - 2

Tears 1 - 5 ?

Bile 50 - 400 160

Intestine - small

- large

2100 - 5200

1200

600

140

Urine 1 - 3 1 - 3

Nasopharynx 45 15

Genital tract (F)

(M)

?

?

?

?

Total 4800 - 9000 3000

IgA

• Up to 5 GRAMS of IgA produced daily!• Certain pathogens can cleave IgA as a defense

mechanism• How does IgA get across? “Poly-Ig receptor”

– Remains tethered to “secretory component” =sIgA

• Class switching mediated by TGF• IgA-secreting B cells express 47, which binds

to MAdCAM-1 and directs them to mucosal tissues

Biological activities of IgA antibodies

mIgA pIgA S-IgA

Neutralization: Enzymes and toxins

                         Viruses

+

+

++

++

++

++

Inhibition of adherence/uptake at mucosae ++

pIgR-mediated transport across epithelium – ++

Intracellular viral neutralization – +

Comp. activation: Classical pathway

                        Alternative pathway

                        Lectin pathway

–

–

+

–

–

+

–

–

?

Phagocytosis via FcR (CD89) + ++ –

IgA

• Up to 5 GRAMS of IgA produced daily!• Certain pathogens can cleave IgA as a defense

mechanism• How does IgA get across? “Poly-Ig receptor”

– Remains tethered to “secretory component” =sIgA

• Class switching mediated by TGF• IgA-secreting B cells express 47 and CCR6,

which directs them to mucosal tissues

Figure 9-26IgA can prevent pathogen adherence to epithelium

Bacterial IgA Proteases-IgA Hinge Region

O O O O O | | | | |P V P S T P P T P S P S T P P T P S P S

Cl. ramosum

PrevotellaCapnocytophaga

Str. pneumoniaeStr. sanguisStr. oralisStr. mitisGemella haemolysans

H. influenzae 1H. aegyptius

H. influenzae 2H. parahaemolyticusN. gonorrhoeae 2N. meningitidis 2

N. gonorrhoeae 1N. meningitidis 1

IgA

• Up to 5 GRAMS of IgA produced daily!• Certain pathogens can cleave IgA as a defense

mechanism• How does IgA get across? “Poly-Ig receptor”

– Remains tethered to “secretory component” =sIgA

• Class switching mediated by TGF• IgA-secreting B cells express 47 and CCR6,

which directs them to mucosal tissues

IgA

• Up to 5 GRAMS of IgA produced daily!• Certain pathogens can cleave IgA as a defense

mechanism• How does IgA get across? “Poly-Ig receptor”

– Remains tethered to “secretory component” =sIgA

• Class switching mediated by TGF• IgA-secreting B cells express 47 and CCR6,

which directs them to mucosal tissues

TGF- is chiefly responsible for directing the IgA ‘switch’

IgA

• Up to 5 GRAMS of IgA produced daily!• Certain pathogens can cleave IgA as a defense

mechanism• How does IgA get across? “Poly-Ig receptor”

– Remains tethered to “secretory component” =sIgA

• Class switching mediated by TGF• IgA-secreting B cells express 47 and CCR6,

which directs them to mucosal tissues

Differential expression of chemokines in tissues explains distribution of T and B cells

between mucosal effector sites

Ontogenic development of mucosal immunity

• Newborn infants have virtually no S-IgA in secretionsbut all the cellular machinery is present:– SC in intestine by gestational day ~40– J chain+ B cells by day ~110– Mucosal T cells by day ~100-120– sIgA+ B cells in intestinal aggregates by day ~120– Peyer’s patches by day ~200

• Ag stimulation required to activate CMIS– colonization by commensal microbiota

NB: germ-free animals remain with poorly developed immune systems

Development of Igs in Infancy100

0

% r

el a

tive t

o a

dul t

level s

Placental transfer of IgG

Mucosal T or T-like cells

• Located in MALT, also scattered throughout• In the gut, found in:

– LAMINA PROPRIA– EPITHELIUM (intraepithelial lymphocytes, IELs)

• LP T cells: cause inflammation in disease– IBD, celiac disease (gluten allergy)– May be involved in tolerance

• IELs: 90% T cells, 80% are CD8+• High proportion of -T cells, IL-17+ cells

Mucosal T cells/IELs• High proportion have memory phenotype

– CD45RO (hu)– Gut homing markers (CCR6, CCR9, 4:7 integrin)

• Constitutively produce IFN, IL-10• IELs

– 80% are CD8+, 50% are form of CD8 (do not see conventional Ags+MHC, but Class Ib MHC)

– Many + T cells, high levels of NKG2D (induced in epithelial cells under stress) – repair? Danger?

– Intracellular granules with perforin, granzymes– Restricted VDJ usage

T cells in PP and lamina propria respond differently to antigens

APC

CD40

AgMHC

B7.2B7.1

T-cell

CD40L

TCRCD4

CTLA4CD28

APC

CD40

AgMHC

B7.2B7.1

T-cellT-cell

CD40L

TCRCD4

CTLA4CD28

proliferation

Circulating memory cells

Peyer’s Patch

APC

CD40

AgMHC

B7.2B7.1

T-cell

CD40L

TCRCD4

CTLA4CD28

APC

CD40

AgMHC

B7.2B7.1

T-cellT-cell

CD40L

TCRCD4

CTLA4CD28

proliferation

Circulating memory cells

Peyer’s Patch

Intestinal lamina propria

APC

CD40

AgMHC

B7.2B7.1

T-cell

CD40L

TCRCD4

CTLA4CD28

APC

CD40

AgMHC

B7.2B7.1

T-cellT-cell

CD40L

TCRCD4

CTLA4CD28

Differentiated CD4 T cell

Naive CD4 T cell

Cytokines

B cell plasma cell

pIgA

Characteristics of Intraepithelial lymphocytes (IELs)

• Large granular lymphocyte morphology• CD3+, CD8+• E, 7 integrin expression• TCR+ more common• Alternative pathways of activation• Produce IL-2, IFN-• Function: cytotoxic, immunoregulatory

CD4+ T-cells

APC

CD40

AgMHC

B7.2B7.1

T-cell

CD40L

TCRCD4

CTLA4CD28

APC

CD40

AgMHC

B7.2B7.1

T-cellT-cell

CD40L

TCRCD4

CTLA4CD28

TH2IL-4

IL-5

IL-10

TH1

IL-2

IFN-

TNF

IL-12IL-12

IFN-IFN-

IL-4IL-4

x

x

TGF-beta, IL-6, TGF-beta, IL-6, IL-23IL-23

TH17

IL-17A

IL-6

TNF

IL-22

x

Th17 cells in mucosal disease

• IL-17+ T cells can be both (classic Th17) or -T or NKT cells

• Th17 cells express CCR6 and CCL20• IL-17 and IL-22 also stimulate expression of

CCL20 in the epithelium, which amplifies recruitment to mucosal tissues

• Th17 cells protect from pathogens– Selectively depleted in HIV infection in gut

• Th17 cells cause inflammation in IBD, Crohn’s

IL23R

ATG16L1

3p21

5p13

IBD5

IRGM 10q21 NKX2-3

NOD2

PTPN2MHC

SBNO2

5q33

You are only 10% human!

• Humans = 1012 to 1013 cells• Flora: skin, gut, other mucosal sites: 1013 to

1014 bacteria

Oral Tolerance

• Oral tolerance is the generation of systemic immune unresponsiveness by feeding of antigen

• Oral tolerance is likely a mechanisms for prevention of harmful immune responses to harmless antigens such as foods

• Necessary to prevent excessive response to normal flora and food antigens

“Oral Tolerance” = mucosally induced systemic toleranceThe classic experiment:

1. Immunize a mouse i.p. with ovalbumin (OVA) in adjuvant develops CMI revealed by DTH reaction i.e. ear-swelling at 24-48h after test injection of OVA

2. First give OVA orally, then immunize i.p. DTH reaction to OVA is diminished

i.e., the mouse has been tolerized to OVA

3. First give bovine serum albumin (BSA) orally, then immunize i.p. with OVA

DTH reaction to OVA is NOT diminishedi.e., tolerance is Ag-specific!

NB: “oral tolerance” is most easily demonstrated in terms of T cell responses or DTH; systemic Ab responses are not so easily tolerized in this way.

A modern version of the same experiment:

Mice are first given naïve CD4+ T cells that transgenically express TCR specific for OVA peptide epitope.

After tolerance induction, it can beshown that such T cells have becomeanergized - they do not proliferatein response to OVA in vitro.

Smaller numbers of OVA-specific T cells also suggest some clonal deletion of these cells (especially at very high, nonphysiological Ag doses).

Limitations to Oral Tolerance

• Can be overcome with mucosal adjuvant (e.g. Cholera toxin)

• Alter physical characteristics of antigen: antigen in micro-spheres that target PP

• Feeding of attenuated enteric pathogen expressing the antigen (Salmonella)

Mucosal tolerance and mucosal antibody responses can coexist!

Spectrum of Mucosal Immune Spectrum of Mucosal Immune ResponsesResponsesSpectrum of Mucosal Immune Spectrum of Mucosal Immune ResponsesResponses

viable, aggressive toxins e.g. CT/CTB

bland, non-viable antigens

Food

killed organismsviable,

non-aggressive

Commensals Pathogens

Potent mucosal adjuvants e.g. CT

Mucosal Ab (S-IgA)

CMI

Systemic Ab(IgG>IgM,IgA)

Littleor noneLittle

or none

Suppressed‘oral tolerance’

Suppressed‘oral tolerance’

Moderate, while stimulus persists Moderate, while stimulus persists

??

Strong, persistentStrong,

persistent

Induced?Induced?

Strong, persistent

Strong, persistent

Littleor noneLittle

or noneLittle

or noneLittle

or none

Antigens:

Responses:

repeated high doses

The immune response is not something that happens only on those infrequent occasions when you inject yourself, or injure your skin.

It is constantly happening, mostly unregarded, as the bodyconfronts the commensal microbiota, and the mass of ‘foreign’ material consumed as food, or inhaled as dust.

Most of the time, the response seems to be: “Leave it alone, it is not harmful or threatening”;

or maybe:“Keep an eye on that, but don’t over-react”.

Yet, when necessary, the mucosal immune system can respond as vigorously as the circulatory immune system.