Mucosal Immunity-I
Sarah Gaffen, Ph.D.
Division of Rheumatology & Clinical Immunology
Spring 2009
sig65@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 cells
Red = T cells
Green = 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 a4:b7 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
a and b chains
b1 Integrins
b2 Integrins
VLA-4
LFA-1
a4, b1
aL, b2
MAC-1
CD11b, CD18
aM, b2
b3-7 Integrins
a4, b7
Initial steps in leukocyte migration are
mediated by interactions with
endothelium
Selectin dep.
Integrin dep
CCL20
CCR6
“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
Mucosal effector site
Mucosal inductive site
Antigen
Antigen
M
M
T
B
T
T
B
T
B
CD8
IgG(J)
DC
T
T
T
B
a/b
pIgR
Lymphatic
vessel
(SC)
IgM+J
HEV
(IgG)
APC
APC
B
B
SIgM
g/d a/b
B
FDC
SIgA
Naive
cells
IgA+J
a/b
IgA+J
CD4
B
Organized mucosa-associated
lymphoid tissue (MALT)
B
T
T
B
Lymph node
B
M cell
Peyer's patches
Isolated lymphoid
follicles (ILFs)
Appendix
Waldeyer's ring
(NALT)
Peripheral blood
Endothelial
gatekeeper
function
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
Ig-producing cells in human tissues
Mucosal Tissues
Systemic Tissues
Gastrointestinal, respiratory,
genito-urinary tracts; eyes;
Salivary, lacrimal, mammary glands
Bone marrow,
lymph nodes, spleen
1%
1%
13%
17%
30%
6%
52%
80%
~2.5 x 10
IgM
>6 x 10 10 cells
IgG
10
cells
IgA
IgD
Daily production rates (mg) of IgA and IgG in humans (70kg adult)
Tissue/fluid
Circulation
IgA
1300 - 2100
IgG
2100
Saliva
Tears
Bile
Intestine - small
- large
Urine
Nasopharynx
100 - 200
1-5
50 - 400
2100 - 5200
1200
1-3
45
1-2
?
160
600
140
1-3
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 TGFb
• IgA-secreting B cells express a4b7, which
binds to MAdCAM-1 and directs them to
mucosal tissues
Biological activities of IgA antibodies
Neutralization: Enzymes and toxins
Viruses
mIgA
pIgA
S-IgA
+
+
++
++
++
++
Inhibition of adherence/uptake at mucosae
++
pIgR-mediated transport across epithelium
–
++
Intracellular viral neutralization
–
+
Comp. activation: Classical pathway
Alternative pathway
Lectin pathway
–
–
+
–
–
+
–
–
?
Phagocytosis via FcaR (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 TGFb
• IgA-secreting B cells express a4b7 and CCR6,
which directs them to mucosal tissues
IgA can prevent
pathogen adherence
to epithelium
Figure 9-26
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
Prevotella
Capnocytophaga
H. influenzae 1
H. aegyptius
Str. pneumoniae
Str. sanguis
Str. oralis
Str. mitis
Gemella haemolysans
N. gonorrhoeae 1
N. meningitidis 1
H. influenzae 2
H. parahaemolyticus
N. gonorrhoeae 2
N. meningitidis 2
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 TGFb
• IgA-secreting B cells express a4b7 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 TGFb
• IgA-secreting B cells express a4b7 and CCR6,
which directs them to mucosal tissues
TGF-b 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 TGFb
• IgA-secreting B cells express a4b7 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 secretions
but 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 Infancy
100
Placental transfer of IgG

0
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 gd-T cells, IL-17+ cells
Mucosal T cells/IELs
• High proportion have memory phenotype
– CD45RO (hu)
– Gut homing markers (CCR6, CCR9, a4:b7 integrin)
• Constitutively produce IFNg, IL-10
• IELs
– 80% are CD8+, 50% are a:a form of CD8 (do not
see conventional Ags+MHC, but Class Ib MHC)
– Many gd+ 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
Naive CD4 Peyer’s
T cell
CD40
APC
MHC
Ag
Patch
Circulating
memory cells
CD40L
CD4
TCR
T-cell
proliferation
CD28
CTLA4
B7.1
B7.2
Intestinal lamina propria
Differentiated
CD4 T cell
CD40
APC
MHC
Ag
B7.1
B7.2
pIgA
CD40L
CD4
TCR
T-cell
Cytokines
CD28
CTLA4
B cell
plasma
cell
Characteristics of Intraepithelial
lymphocytes (IELs)
•
•
•
•
•
•
•
Large granular lymphocyte morphology
CD3+, CD8+
aE, b7 integrin expression
gd TCR+ more common
Alternative pathways of activation
Produce IL-2, IFN-g
Function: cytotoxic, immunoregulatory
CD4+ T-cells
IL-4
IL-4
TH2
CD40
APC
MHC
Ag
CD40L
CD4
TCR
B7.1
B7.2
IL-5
IL-10
x
T-cell
IL-2
CD28
CTLA4
IFN-g
TH1
IFN-g
TNF
x
IL-12
IL-17A
TH17
IL-6
TNF
TGF-beta, IL-6,
IL-23
IL-22
Th17 cells in mucosal disease
• IL-17+ T cells can be both ab (classic Th17) or
gd-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
NOD2
ATG16L1
5p13
IRGM
IBD5
NKX2-3
5q33
MHC
3p21
10q21
PTPN2
SBNO2
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 tolerance
The 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 diminished
i.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 be
shown that such T cells have become
anergized - they do not proliferate
in 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 Responses
Antigens:
Food
Commensals
bland, non-viable antigens
killed organisms
viable,
non-aggressive
Pathogens
viable, aggressive
toxins e.g. CT/CTB
repeated
high doses
Responses:
Mucosal
Ab (S-IgA)
Systemic Ab
(IgG>IgM,IgA)
CMI
Little
or none
Moderate, while
stimulus persists
Strong,
persistent
Little
or none
Little
or none
Strong, persistent
Suppressed
‘oral tolerance’
?
Potent mucosal adjuvants e.g. CT
Induced?
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 body
confronts 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.