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sciencemag.org SCIENCE
RESEARCH ARTICLE
MUCOSAL IMMUNOLOGY
SCIENCE sciencemag.org
aaf4822-1
R ES E A RC H
R ES E A RC H | R E S EA R C H A R T I C LE
RE S E ARCH | R E S E A R C H A R T I C L E
Ccr6+/+
Peyers patches
Ccr6+/+
Ccr6+/+
25
80
% GC B cells
100
***
60
40
80
7.05
60
1.57
Ccr6-/-
Ccr6-/-
Ccr6-/-
40
15
10
5
10.2
20
**
20
0
-
20
24.7
% IgA+ B cells
***
C
cr
6 +/
+
C
cr
6 -/
100
N
o
MT
BM chimera
measured by FACS from mixed BM chimeras as in (A). (G) Fecal CT-specific IgA
from WT (Igha) and Ccr6+/+ or Ccr6/ (Ighb) mixed BM chimeras orally treated
with CT, measured by allotypic ELISA. (H) Fecal IgA from WT (Igha) and Ccr6+/+
or Ccr6/ (Ighb) B cells cotransferred into either Rag1/ or mMT, or from mixed
BM chimeras as in (A), measured by allotypic ELISA. Each symbol in (A), (B),
(E), (F), (G), and (H) represents an individual mouse, and data are pooled from
at least three independent experiments. Data in (C) and (D) are representative
of at least three independent experiments. **P < 0.01, ***P < 0.005 (unpaired
Students t test).
aaf4822-3
6 -/
W
T
cr
C
W
T
C
cr
6 +/
W
C T (I
cr
6 +/ gA a
+
(Ig )
W A b)
T
(
C
cr IgA a
6 -/
)
(Ig
Ab
)
W
T
cr
6 -/
C
W
T
C
cr
6 +/
SCIENCE sciencemag.org
Rag1-/-
W
T
cr
6 -/
0.0
0.5
20
1.0
W
C T
cr
6 +/
40
***
***
1.5
60
80
IgA
***
2.0
***
IgD
IgG1
4.9
IgG1
W
C T
cr
6 +/
IgA
100
***
2.26
GC Mem
W
T
cr
6 -/
IgG1
Ccr6-/-
IgA
Ccr6+/+ WT
WT
Total gA (OD450)
IgA
Ccr6-/-
WT
tra
ns
fe
r
W
C T
cr
6 +/
Ccr6+/+
WT
IgD
W
C T
cr
6 -/
Total gA (OD450)
IgD
R ES E A RC H | R E S EA R C H A R T I C LE
Pre-GC
0.1
6.9
AID
0.1
23.5
Naive
Naive
GC
Pre-GC
Mem
CD19
Mem
0.01
0.01
Naive
GC
Pre-GC
Mem
Mem
10
GC
0.001
65.8
Pre-GC
0.01
I-C
100
GC
% AID-GFP+ B cells
Pre-GC
Mem
0.1
Aicda (A.U.)
I-C
Naive
10
0.1
GT (A.U.)
GT
GC
Naive
AID-GFP
Fig. 3. Pre-GC B cells in PPs initiate IgA class switching. (A and B) Representative semiquantitative (A) or quantitative (B) RT-PCR on B cells from
PPs sorted according to IgD and CCR6 expression, for the indicated transcripts. (C) Representative FACS staining (left) and frequency (right) of AID-GFP+
PP B cells from reporter mice, according to IgD and CCR6 expression. Each symbol in (B) and (C) represents an individual mouse, and data are pooled from
three independent experiments. Data in (A) are representative of three independent experiments. A.U., arbitrary units.
RE S E ARCH | R E S E A R C H A R T I C L E
2.5
LTbR MFI (WT/Ltbr-/-)
0.8
AU
0.6
0.4
0.2
63
0.11
***
***
29
1.5
Ltbr-/-
0.5
CD11b+
DN
8+
5000
19.6
CD8+
20000
15000
WT
Ltbr-/+ CD11cDtr
Ltbr-/-
IgG1
WT
Ltbr-/+CD11cDtr
IgA
10000
0
CD11b+
32.7
hIgG-Fc
LTR-Fc
60
IgA + (% GC)
IgA
4000
13.8
2000
40
20
LTR-Fc
Fig. 4. LTbR-dependent PP DC support IgA class switching. (A) Quantitative PCR analysis of Ltbr transcript abundance in the indicated DC subsets
from PPs (left) and median fluorescence intensity (MFI) ratio for LTbR on the DC
subsets from PPs of BM chimeras reconstituted with WT or Ltbr/ BM (right).
(B) Representative FACS staining (left) and absolute cell number (right) of the
indicated DC subset from PPs of BM chimeras reconstituted with WTor Ltbr/
BM. (C) Frequency of IgA+ and IgG1+ GC B cells in PPs from BM chimeras as in
(B), determined by FACS. (D) Frequency of IgA+ and IgG1+ GC B cells in PP from
mixed chimeras reconstituted with CD11c-DTR and either Ltbr+/+ or Ltbr/ BM,
treated with DT for 3 weeks. (E) Absolute number of the indicated DC subset
60
40
20
c
-F
R
LT
hI
CD8+
gG
-F
0
DN
IgA
SCIENCE sciencemag.org
80
***
hIgG-Fc
6000
LT-tg
IgG1
IgG1
**
CD11b+
WT
LT-tg
IgA
8000
dendritic cells
WT
CD8+
DN
from PPs of WTor LT-transgenic (tg) mice. (F) Frequency of IgA+ and IgG1+ GC B
cells in PPs from WTor LT-tg mice, as determined by FACS. (G) Absolute number
of the indicated DC subset from PPs of mice treated with LTbR-Fc or hIgG-Fc for
7 days. (H) Representative FACS staining of polyclonal B cells in PPs 7 days after
transfer to MD4 recipients that were treated with LTbR-Fc or hIgG-Fc (left) and
frequency of IgA+ B cells and GC B cells in PPs among transferred B cells (right).
Each symbol in (A), (B), (C), (D), (E), (F), (G), and (H) represents an individual
mouse, and data are pooled from at least three independent experiments. In (B), (C),
(D), (E), (F), (G), and (H), *P < 0.05, **P < 0.01, ***P < 0.005 (unpaired Students
t test); in (A), ***P < 0.005 (one-way ANOVA with Bonferronis post-hoc test).
aaf4822-5
IgA
WT
Ltbr-/-
-F
WT
*
20
5000
40
-F
20
10000
20
60
% GC B cells
40
40
dendritic cells
% GC B cells
60
***
80
WT
LT-tg
***
**
LT
**
**
gG
60
**
hI
10000
CD8
CD11c
b+
12.4
11
8+
D
C
b+
11
***
CD11b
0.0
5.8
58.5
0.05
1.0
80
% GC B cells
***
WT
2.0
MHCII
0.0
WT
Ltbr-/-
15000
dendritic cells
Ltbr
1.0
R ES E A RC H | R E S EA R C H A R T I C LE
***
80
***
Lin- Thy1+IL7R+CD25+
% GC B cells
60
85.4
0.2
40
20
IL7R
Thy1
CD25
Lin
LTR-FC
WT
Rorc-/IgA
WT
Rorc-/IgG1
ns
80
ns
***
***
***
***
***
60
% GC B cells
% GC B cells
60
***
40
40
20
20
0
WT
Rorc-/IgA
Rorc-/+Rag1-/-
WT
Rorc-/-
Rorc-/+Rag1-/-
WT
IgG1
IgA
Rorc-/+Lta-/-
WT
Rorc-/+Lta-/-
IgG1
switching may dominate, eliminating the intervening Ig constant regions and thereby limiting
switching to IgG1.
Discussion
These studies identify a network of cellular and
molecular interactions underpinning the induction of IgA responses in PPs (fig. S6). After activation by foreign or commensal-derived antigen
and receipt of CD40-dependent helper signals,
PP B cells up-regulate CCR6 and are attracted
by CCL20 into the SED, where they undergo extensive interactions with CD11b+ DCs. This DC
population is maintained by LTa1b2 provided
locally by ILC3s. CD11b+ DCs express integrin
avb8 and promote TGFb activation during interactions with B cells. After receipt of TGFb and
likely additional SED-derived signals, B cells return
to the follicle by directed migration and participate in the GC response.
Our studies indicate that sustained CCR6 upregulation in PP B cells occurs in a CD40-, and
thus most likely T celldependent, manner, and
CCR6 deficiency strongly affected T-dependent
IgA responses. How CD40 signaling promotes
sustained CCR6 expression is not yet clear. Because
BCR engagement is sufficient to promote CCR6
sciencemag.org SCIENCE
80
RE S E ARCH | R E S E A R C H A R T I C L E
Velocity (mm/min)
20
15
10
5
0
FO
SED
35
FO
30
SED
25
20
15
10
5
0
0
50m
20 m
20
10 m
1:30
12:00
23:00
25:00
co
nt
ac
an
0:00
Pa
u
se
Fig. 6. PP B cell migration dynamics and interaction with SED DCs. (A)
Representative TPLSM of PP for transferred CFP+ B cells (green) in CD11cYFP mice. YFP+ cells (red) are shown by volume rendering. White dotted line
indicates location of the SED. (B) Median velocity and (C) displacement
versus square root of time of B cells in follicle (black) and in the SED (blue).
(D) Percentage of B cells in the SED pausing (Pause), scanning (Scan), or not
contacting (No contact) CD11c-YFP+ cells. (E) Representative time-lapse
images of CFP+ B cell interaction with CD11c-YFP+ cell in the SED. Arrowheads
highlight a single B cellDC interaction. (Yellow color is due to bleed-through
aaf4822-7
40
Sc
60
R ES E A RC H | R E S EA R C H A R T I C LE
***
40
40
20
IgG1
IgA
an
ti-
D
C
IgA
Cre+
an
ti-
Cre-
Is
Cre+
Is
o
0
Cre-
8+
IgG1
ns
10
Itgb8 (MFI)
Nil
antiLTR
RA
antiLTR
+
RA
an
G
+T
8+
ti -
2000
1000
8
tian
an
ti-
LA
F
TG
iu
tian
mLN DCs
Cre+
ed
PP DCs
Cre-
Cre+
10
0
Cre-
15
20
3000
b+
4000
20
30
Itgb8-/-
11
10
25
15
IgA+ (%B cells)
***
***
40
***
***
20
WT
***
fl/fl
Itgb8
Fig. 7. DC integrin-b8 promotes TGFb-dependent IgA switching. (A) Quantitative PCR analysis of Itgb8 (left) and Itgav (right) transcript abundance in
the indicated DC subsets from PPs. (B) Median fluorescence intensity (MFI)
ratio for b8 on the indicated DC subsets from PPs of either CD11c-Cre (WT) or
CD11c-Cre+ Itgb8fl/fl mice. (C) Frequency of IgA+ and IgG1+ GC B cells in PPs of
either CD11c-Cre or CD11c-Cre+ Itgb8fl/fl mice as determined by FACS. (D)
Frequency of IgA+ and IgG1+ GC B cells in PPs 7 days after adoptive transfer
in MD4 mice and treatment with either isotype control or anti-b8. (E) Frequency
of IgA+ splenic B cells upon 5 days of culture with DCs sorted from PPs or
mLNs of CD11c-Cre or CD11c-Cre+ Itgb8fl/fl mice. (F) Frequency of IgA+ splenic
exclude dead cells then stained for surface antigens, treated with BD Cytofix Buffer and Perm/
Wash reagent (BD Biosciences), and stained with
anti-IgA.
Immunohistochemistry and
immunofluorescence microscopy
For immunohistochemistry, cryosections of 7 mm
were acetone fixed and stained as described (45)
with combinations of the following antibodies:
anti-IgD (11-26c.2a, BD Biosciences), anti-IgDa
(AMS9.1, BD Biosciences), anti-IgDb (217-170, BD
Biosciences), and anti-IgMa (DS-1, BD Biosciences).
In some case, the slides were counterstained with
hematoxylin. For immunofluorescence, tissues
were fixed in 4% paraformaldehyde in PBS for
2 hours at 4C, washed three times for 10 min in
PBS, then moved to 30% sucrose in PBS overnight.
Tissues were flash frozen in Tissue-Tek Cryomold
(VWR) the next day, and 7-mm sections were cut
and then dried for 1 hour before staining. Sections
were rehydrated in PBS with 1% bovine serum
albumin (BSA) for 10 min and then stained in
aaf4822-8
B cells upon 5 days of culture with DCs sorted from PPs and incubated with the
indicated antibodies. (G) Frequency of IgA+ splenic B cells upon 5 days of
culture with the indicated DC subset sorted from PPs. (H) Median fluorescence
intensity (MFI) for b8 on the BMDCs generated from CD11c-Cre or CD11c-Cre+
Itgb8fl/fl BM upon 7 days of culture and incubation with the indicated reagents.
Each symbol in (B), (C), (D), (E), (F), (G), and (H) represents an individual
mouse, and data are pooled from at least three independent experiments. Data
in (A) are pooled from three independent experiments. In (B), (F), and (G), *P <
0.05, **P < 0.01, ***P < 0.005 (one-way ANOVA with Bonferronis post-hoc
test); in (C), (D), and (E), **P < 0.01, ***P < 0.005 (unpaired Students t test).
b+
11
**
20
0
8+
N
C
60
***
0.5
C
D
11
b
C
D
8+
C
D
11
b
1.0
0.0
0.0
***
60
0.0
fl/fl
IgA + (% GC)
0.2
1.5
0.1
80
0.4
0.2
Itgb8
2.0
A.U.
0.6
A.U.
0.3
Itgav
% GC B cells
Itgb8
RE S E ARCH | R E S E A R C H A R T I C L E
Sorting
For DC sorting, PPs and mLNs were digested
and stained as described above and sorted on a
FACSAira III with a 70-mm nozzle. In same cases,
DCs were isolated from PPs of 8- to 10-week-old
mice that had been injected subcutaneously in
the flank with 5 106 B16 murine Flt3L-secreting
tumor cells 7 to 10 days earlier. This treatment led
to a ~10-fold expansion in total PP DC numbers.
Bone marrow chimeras, retroviral
transduction, and DT treatment
Ly5.2 congenic B6 mice were lethally irradiated
with either 1100 or 1300 rad in split doses and
reconstituted with (1 to 3) 106 BM cells from
the indicated donors. Mice were analyzed 10 to
14 weeks later. For retroviral transduction, PlatE
cells were transfected with murine stem cell virus
(MSCV) retroviral constructs encoding full-length
mouse Ccr6 with Lipofectamine 2000 (Invitrogen) following the manufacturers protocol. For
transduction of BM-derived cells, BM cells were
harvested 4 days after 5-flurouracil (Sigma) injection and cultured in the presence of recombinant
IL-3, IL-6, and mouse stem cell factor (SCF) (100 ng/
ml, Peprotech). BM cells were spin-infected twice
with a retroviral construct expressing Ccr6 and
an internal ribosomal entry site (IRES)Thy1.1
cassette as a reporter. One day after the last spin
infection, the cells were injected into lethally irradiated C57BL/6 recipients. Eight to 12 weeks later,
splenic B cells were isolated and then injected in
C57BL/6 recipients, and their positioning in PP
was assessed 3 days later. For DT treatments, BM
chimeras received 4 ng of DT (EMD Bioscience)
per gram of body weight every 72 hours for 3 weeks.
Rag1/ and mMT cells transfer
Splenic B cells were sorted on a FACSAira III with
a 70-mm nozzle as CD3, CD43, CD4, CD8, CD11c,
Ly6C, Ly6G, CD90 neg, and 106 cells from each
genotype were transferred i.v. Recipients were
analyzed 4 weeks later.
Enzyme-linked immunosorbent
assay (ELISA)
Ninety-sixwell plates (Thermo Fisher Scientific)
were coated with purified anti-IgA (RMA-1, BD)
or 0.5 nmol/ml monosialotetrahexosylganglioside
(GM1) (Sigma-Aldrich) followed by 0.5 mg/ml CT
overnight at 4C (47). The plates were washed and
clocked with PBS5% BSA before diluted fecal
samples were added and twofold serial dilution
was made. Samples were incubated overnight at
4C, followed by biotinylated anti-mouse antibodies: anti-IgA (C10-1, BD), anti-IgAa, and antiIgAb (Hy16 and HISM2, UCSF Hybridoma Core)
at 1 mg/ml in PBS0.1% BSA. Detection antibodies
were labeled by streptavidin-conjugated horseradish
peroxidase and visualized by the addition of Substrate Reagent Pack (R&D). Color development
was stopped with 3 M H2SO4. Purified mouse IgA
(Southern Biotech) served as standard. Absorbances at 450 nm were measured on a tunable
SCIENCE sciencemag.org
lated with 10 mg/ml anti-IgM and 20 mg/ml antiCD40 (clone FGK4.5, UCSF Hybridoma Core) in
the presence of TGFb (2 ng/ml) and RA (100 nM)
for 5 days.
For BMDCs, 5 106 BM cells were cultured in
10-cm tissue culture dishes in 10 ml of medium
supplemented with supernatants from 3T3 cells
transfected with the gene-encoding murine GMCSF (granulocyte-macrophage colony-stimulating
factor) for 7 days. Cells were treated with 1 mg/ml
LTbR agonistic antibody (clone 3C8) and 100 nM
RA every 3.5 days for 7 days.
Intravital two-photon laser-scanning
microscopy (TPLSM) of PPs
Mice were anesthetized by intraperitoneal injection of 10 ml kg1 saline containing xylazine (1 mg
ml1) and ketamine (5 mg ml1). Maintenance
doses of intramuscular injections of 4 ml kg1 of
xylazine (1 mg ml1) and ketamine (5 mg ml1)
were given approximately every 30 min. An incision was made in the abdominal wall, and the
small intestine was gently stretched and scanned
by eye to identify PP structures. Only small areas
(1 to 2 cm long) were exposed at any time. Once a
PP was located, the area was embedded in warm
saline and stabilized by placing a spring-loaded
platform over the mouse and screwed down until
the cover glass made contact with the PP. The
tissue was placed with the interface between the
intestinal lumen and PP facing upwards in an
orientation that allowed maximal viewing of the
SED. The mouse was placed on a Biotherm stage
warmer at 37C (Biogenics) for the duration of the
imaging. Images were acquired with ZEN2009
(Carl Zeiss) with a 7MP two-photon microscope (Carl
Zeiss) equipped with a Chameleon laser (Coherent).
For video acquisition, a series of planes of 2- or
3-mm z-spacing spanning a depth of 30 to 69 mm
were collected every 15 to 20 s. Excitation wavelengths were 850 to 890 nm. Because most of the
transferred CFP+ B cells occupied the follicular
compartment, we used automated tracks (generated by Imaris 7.4.2 64, Bitplane) to highlight the
follicular region, as previously described (36). The
SED was identified by the presence of CD11c-YFP
DCs and by its typical shape and location above
the follicles. The interfollicular regions, which are
also rich with CD11c-YFP DCs, were identified on
the basis of their distinct positioning and were
excluded from analysis. Videos were made and
analyzed with Imaris 7.4.2 64 (Bitplane). To
track cells, surface seed points were created and
tracked over time. Tracks were manually examined
and verified. Data from cells that could be tracked
for at least 15 min were used for analysis. Data
presented in Fig. 6 were collected from four independent movies, some of which were split
into 2 by 30 min segments and analyzed with
Imaris (Bitplane AG), MATLAB (MathWorks),
and MetaMorph software. In Fig. 6D, the contact
time between B cells and DCs in the SED was measured manually (n = 150 B cells, derived from four
independent movies). The behavior of a B cell
engaged in prolonged interactions was defined
as Pause (5 to 25 min of contact time), Scan
(2 to 5 min of contact time), or No contact when
13 MAY 2016 VOL 352 ISSUE 6287
aaf4822-9
aaf4822-10
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
www.sciencemag.org/content/352/6287/aaf4822/suppl/DC1
Figs. S1 to S5
Movies S1 to S4
15 February 2016; accepted 30 March 2016
10.1126/science.aaf4822
sciencemag.org SCIENCE
R ES E A RC H | R E S EA R C H A R T I C LE
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