Articles

Diversification of memory B cells drives the

continuous adaptation of secretory antibodies
to gut microbiota
Cornelia Lindner1,11, Irene Thomsen1,11, Benjamin Wahl1, Milas Ugur1, Maya K Sethi1, Michaela Friedrichsen1,
Anna Smoczek2, Stephan Ott3, Ulrich Baumann4, Sebastian Suerbaum5, Stefan Schreiber3,6, André Bleich2,
Valérie Gaboriau-Routhiau7, Nadine Cerf-Bensussan7, Helena Hazanov8, Ramit Mehr8, Preben Boysen9,
Philip Rosenstiel6 & Oliver Pabst1,10
© 2015 Nature America, Inc. All rights reserved.

Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis.
However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice
and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the
microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were
clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory
B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how
maternal antibodies are optimized throughout life to protect the newborn.

The gut microbiota affects the host’s susceptibility to infection and
inflammatory diseases1 and shapes the development of the immune
system and metabolism2. Reciprocally, the host immune system
influences the intestinal microbiota through a multilayered system
of innate and adaptive immune responses3. A key component in host-
microbiota interaction is secretory immunoglobulin A (SIgA). Defects
in the SIgA system result in overstimulation of innate immunity by
gut epithelial cells4 and dysbiosis of the intestinal microbiota5,6. Thus,
SIgA production constitutes a hallmark of the intestinal immune
npg
broad agreement that T cell–dependent antibody responses in PPs can
generate affinity-matured IgA, but sites of IgA induction other than
PPs, such as isolated lymphoid follicles and intestinal lamina propria,
have also been proposed. IgA induction at these sites is frequently
linked to T cell–independent pathways of IgA induction10,11 and to
the generation of low-affinity and polyreactive antibodies referred
to as ‘primitive’ or ‘natural’ IgA11–13. However, it is unknown how
T cell–dependent and T cell–independent pathways act in synergy to
generate SIgA responses in response to microbial stimulation.

response and a central element in host-microbiota communication.
However, it is unknown how these beneficial immunoglobulin A
(IgA) responses are regulated.
Induction of SIgA depends on microbial stimulation, and accord-
ingly germ-free mice possess few intestinal plasma cells. However,
plasma cell generation is rapidly triggered after colonization and even
by transient exposure to live microbes7. Notably, different bacterial
species differ in their SIgA-inducing properties and endogenous SIgA
coats in the intestine8,9. The gut microbiota in the intestinal lumen is
constantly being sampled into inductive lymphoid compartments such
as Peyer’s patches (PPs) and triggers the generation of IgA-secreting
plasma cells. Newly generated plasmablasts relocate to the gut lam-
ina propria, differentiate into plasma cells and secrete IgA. There is
To deal with dynamic changes in microbial exposure, such as those
introduced by antibiotic treatment, vaccination or infection, the SIgA
system has to adapt and refine SIgA responses to the microbiota. This
adaptation process might rely on two non–mutually exclusive path-
ways: the recruitment of new clones into the plasma cell repertoire
from the pool of naive B cells after changes in the microbiota and
thus encounters with new antigens, and the development of antibody
specificity to the changed microbiota through somatic hypermutation
and diversification of B cell clones already established in the intestinal
plasma cell pool. Here we introduced changes in human and mouse
microbiota by administering antibiotics and then used defined coloni-
zations and infection models to comprehensively analyze responses in
the plasma cell compartment by means of immunoglobulin repertoire

1Institute of Immunology, Hannover Medical School, Hannover, Germany. 2Laboratory for Animal Science, Hannover Medical School, Hannover, Germany. 3Department
for Internal Medicine I, University Hospital Schleswig Holstein, Kiel, Germany. 4Pediatric Gastroenterology, Hannover Medical School, Hannover, Germany. 5Institute
of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany. 6Institute of Clinical Molecular Biology, University Hospital
Schleswig-Holstein, Kiel, Germany. 7INSERM UMR1163, Laboratory of Intestinal Immunity, Institut Imagine, Paris, France. 8The Mina & Everard Goodman Faculty of
Life Sciences, Bar-Ilan University, Ramat-Gan, Israel. 9Department of Food Safety and Infection Biology, Faculty of Veterinary Medicine and Biosciences, Norwegian
University of Life Sciences, Oslo, Norway. 10Institute of Molecular Medicine, RWTH Aachen, Aachen, Germany. 11These authors contributed equally to this work.
Correspondence should be addressed to O.P. (opabst@ukaachen.de).

Received 26 March; accepted 31 May; published online 29 June 2015; doi:10.1038/ni.3213

880 VOLUME 16  NUMBER 8  AUGUST 2015  nature immunology

 Articles

sequencing. Our data suggest that diversification of memory B cell
responses is a key mechanism that shapes the intestinal antibody arse-
nal in response to changes in the microbiota and offer unbiased insight
into dynamic changes in the intestinal plasma cell repertoire.
RESULTS
A complex microbiota drives diverse IgA repertoires
To study how the microbiota shapes IgA responses, we compared
properties of the IgA sequence repertoire in germ-free mice colonized
with Escherichia coli Nissle (ECN), Lactobacillus GG, a mixture of
two Clostridia strains or a complex microbiota present in specific-
pathogen-free (SPF) mice. Four weeks after colonization, we isolated
RNA from small intestinal tissue and transcribed it into cDNA, and
we amplified IgA heavy-chain variable regions by PCR. Primers were
chosen to flank amino-terminal framework region 1 and a small stretch
of the constant Cα domain and thus contained complementarity-
determining region (CDR) 3, which is particularly important
for antigen binding (Fig. 1a). We characterized amplicons via
high-throughput sequencing and compared a minimum of 4,000
sequences per sample with reference sequences from the IMGT
database (http://www.imgt.org/) 14,15. Sequences that shared
© 2015 Nature America, Inc. All rights reserved.
E. coli, the IgA repertoire was less diverse than that of mice colo-
nized with a complex SPF microbiota (Fig. 1d). Although C3H mice
showed greater overall diversity than C57BL/6 mice (Fig. 1c,d), in
both genetic backgrounds a diverse SPF microbiota was required to
boost IgA repertoire diversity.
We reported previously that in littermate mice colonized by their
mother with a complex SPF microbiota, CDR3 sequences observed in
one mouse are typically not found in any other mouse16. Here we stud-
ied the situation in monocolonized mice. To quantify the similarity in
immunoglobulin repertoires, we calculated the Morisita-Horn index
(MHI)16, which considers the overlap of clonally related sequences
and their respective frequencies. The MHI scores two identical popu-
lations as 1 and two completely nonoverlapping populations as 0. MHI
scores for comparisons of different monocolonized mice were close to
zero (0.006 ± 0.02, mean ± s.d., from an analysis of >40 comparisons).
We therefore concluded that even in monocolonized mice, there is no
apparent convergence of the IgA repertoire that exceeds the common
bias in gene segment use17.
a

CDR1 CDR2 CDR3

identical VH and JH gene segments and 95% identity in the CDR3
nucleotide sequence were assigned to clusters and regarded as FR1 FR2 FR3 Cα

clonotypes thought to derive from the same B cell clone. When we 5′ primer 3′ primer
compared the various colonized mice, we found no apparent bias in
VH, D or JH gene segment use (Fig. 1b). b 80

To quantify how the colonization state affects IgA repertoire diver- 60

sity, we calculated the exponent of the Shannon index, a common
VH use (%)

measure used to describe population diversity. The Shannon index 40

considers species richness and evenness of representation. For our

20
analysis the number of clusters represented richness, whereas even-
ness was represented by cluster size (i.e., how many sequences were 0
assigned to each cluster). We tested the validity of this approach in V H1 VH2 V H3 VH5 VH10 VH14

samples that comprised different numbers of sorted plasma cells from 80

either a single mouse or multiple mice. The exponent of the Shannon

60
index correlated with the number of mice represented in the plasma
D use (%)

cell pool (Supplementary Fig. 1), which indicated that the Shannon 40
index represented a reasonable estimate of IgA repertoire diversity.
The IgA repertoire of SPF-colonized C57BL/6 mice was more diverse 20
npg

than the IgA repertoires of germ-free and monocolonized mice

0
(Fig. 1c). Similarly, in C3H mice colonized for 60 d with segmented D1 D2 D3 D4 D5 D6
filamentous bacteria, a typical mouse commensal, Bacteroides or 80

60 GF
Figure 1  The complexity of the microbiota determines IgA repertoire SPF
JH use (%)

diversity but not gene segment use. (a) Schematic overview of framework 40 ECN
regions (FRs) and CDRs indicating primer-binding sites for IgA amplicon Lact
generation. (b) Germ-free (GF) mice were left untreated (n = 9, pooled 20 Clost
from three independent experiments) or colonized with an SPF microbiota
(n = 12, pooled from three independent experiments), ECN (n = 10, 0
JH1 JH2 JH3 JH4
pooled from three independent experiments), Lactobacillus GG (Lact;
n = 5, pooled from two independent experiments) or Clostridium sordellii *** ***
plus Clostridium perfringens (Clost; n = 4, pooled from two independent c *** *** d ***
300 * 500 *** **
experiments) for 4 weeks. Use of the main V H, D and JH genes is shown as
)
Diversity (eShannon)

Shannon

a percentage of all identified genes (mean and s.d.). (c,d) IgA repertoire
diversity in C57BL/6 mice colonized as described above (c) and C3H 200
300
Diversity (e

mice colonized for 60 d with E. coli MG1655 (n = 3), Bacteroides

thetaiotamicron (n = 3) or fecal homogenate from SFB monocolonized 100
mice (n = 2) (d). Diversity is displayed as the exponent Shannon index 100

(eShannon), calculated on the basis of 4,000 CDR3 sequences per sample 0 0

and clustered with 95% identity. Each circle represents an individual 4 weeks 4 weeks
F

F
G

mouse; horizontal lines indicate means. One-way analysis of variance

F

es
li
ct

st

co
SP

SP

SP
EC
lo
La

id

(ANOVA) with Tukey’s post-hoc test was used for statistical analysis.
C

E.

ro
te
ac

*P < 0.05, **P < 0.01, ***P < 0.001.

B

nature immunology  VOLUME 16  NUMBER 8  AUGUST 2015 881

 Articles

Figure 2  CDR3 sequences generated in monocolonized mice persist

after complex colonization. (a) Comparisons of IgA repertoire similarity
a *** b c
1.0 1.0 NS ** 300 160
(expressed as the MHI) between biopsy-sized replicate samples

)
)

Shannon
Shannon
0.8 0.8
(Bx versus Bx, n = 3 independent experiments) and between biopsies and 120

IgA (MHI)

IgA (MHI)
200
0.6 0.6
larger tissue samples (Bx versus small intestine (SI), n = 3 independent

Diversity (e
Diversity (e
80
experiments) from the same mouse. Each symbol represents an individual 0.4 0.4
100
40
mouse. To illustrate non-overlapping repertoires between different mice, 0.2 0.2
we also compared samples from two different mice (1 versus 2; one 0 0 0 0
comparison representative of more than 20 mouse-to-mouse comparisons

I
.2

vs F
PF

G GF

N N

F
.S
.B

N SP

SP

SP
EC EC
vs

SP s. S

.S
analyzed). (b,c) IgA diversity in germ-free mice, ECN-monocolonized mice

vs
vs

EC vs.
1
Bx

F
Bx

v
and SPF mice that were biopsied and then inoculated with a complex

F
G
microbiota. (b) Comparison of IgA sequence repertoires between biopsies
and samples of small intestine taken from the same mouse 4 weeks after biopsy (n = 5–6, pooled from three independent experiments). One-way
ANOVA with Bonferroni’s post-hoc test was used for statistical analysis. NS, not significant (>0.05); **P < 0.01, ***P < 0.001. Horizontal lines in a
and b indicate means. (c) IgA repertoire diversity observed in biopsies of small intestine compared to diversity after 4 weeks of SPF colonization. The
exponential Shannon index was calculated on the basis of 4,000 CDR3 sequences per sample clustered with 95% identity. Matched samples (biopsy
and small intestine of the same mouse) are connected by lines in c (n = 4–5, pooled from three independent experiments).

Because individual mice have distinct IgA repertoires, tracking
dynamic microbiota-triggered changes in the IgA system requires
the comparison of serial samples from an individual mouse. To this
end, we obtained biopsies of small intestine from germ-free mice,
ECN-monocolonized mice and SPF-colonized control mice. Then we
© 2015 Nature America, Inc. All rights reserved.
IgA repertoire after colonization with an SPF microbiota. Notably,
4 weeks after the colonization of E. coli-monocolonized mice with SPF
microbiota, γ-proteobacteria, including E. coli, were hardly detect-
able, and the microbiota in the recipients had adopted the donor
composition (Supplementary Fig. 2). This showed that B cell clones

inoculated the mice with cecal content isolated from SPF-colonized
wild-type mice. Whereas the MHI scores observed for the comparison
of repertoire information from different individual mice were close
to zero, comparison of ‘biopsy-sized’ replicate samples showed MHIs
close to 1 (Fig. 2a). Moreover, we observed that the IgA repertoire
of as few as 1,500 sorted plasma cells showed high similarity and
MHI scores when compared with larger samples (data not shown).
This suggested that even in the case of reduced plasma cell densities,
repertoire information based on gut biopsies reliably represented
the overall repertoire. The similarity between the IgA repertoire of a
germ-free or ECN-monocolonized mouse and that of the same mouse
after SPF colonization was consistently higher than the IgA repertoire
similarity between different individuals (Fig. 2b). This observation
suggested that clonotypes present in small intestinal biopsies taken
from germ-free or E. coli–monocolonized mice had persisted in the
persisted in the IgA repertoire even though the bacterial species that
had initially triggered their activation were absent, and colonization
with a complex SPF microbiota resulted in a robust increase in IgA
diversity (Fig. 2c).
The microbiota composition of wild-type mice housed under
SPF conditions in two separate animal facilities (Online Methods)
showed a typical dominance of Firmicutes and Bacteroidetes (Fig. 3
and Supplementary Fig. 2). Still, SPF housing conditions in experi-
mental animal facilities differ fundamentally from a natural environ-
ment and natural microbial exposure. Thus, SPF housing conditions
might affect the architecture of the IgA repertoire and restrict reper-
toire diversity. To evaluate the effect of a semi-natural environment
on IgA repertoire diversity, we housed adult SPF mice together with
wild-caught mice in an enriched environment for 8 weeks. The micro-
biota of wild-caught mice differed from the microbiota of SPF mice

Figure 3  Convergence of microbiota in a semi- 100a Bacteroidetes Firmicutes

natural environment. (a) Adult SPF mice were Bacteroidia Clostridia
npg

housed under SPF conditions or together with 80

Bacteroidiales Unclassified
Unclassified Clostridiales
wild-caught mice in an enriched environment Bacteroidaceae Lachnospiraceae
Frequency (%)

Porphyromonadaceae Ruminococcaceae
for 8 weeks (Online Methods). The composition 60 Proteobacteria
Prevotellaceae
of fecal microbiota was analyzed by 16S rDNA Rikenellaceae Alphaproteobacteria
sequencing. Bars depict bacterial families S24-7 RF32
40 Odoribacteraceae Unclassified
contributing more than 2% of all sequences Paraprevotellaceae Deltaproteobacteria
obtained in individual mice (n = 5–6 mice 20 Cyanobacteria Desulfovibrionales
4C0d-2 Desulfovibrionaceae
per group analyzed in one experiment); colors YS2
Epsilonproteobacteria
Unclassified
match key. Shannon indices based on 1,842 0 Campylobacterales
Deferribacteres
sequences: SPF mice, 6.288 ± 0.432; wild- Deferribacteres Helicobacteraceae
4
5
6
8
9
1
2
3
4
5
6
4
5
6
7
8
30
30
30
30
30
10
10
10
10
10
10
80
80
80
80
80

TM7
caught mice, 6.421 ± 0.307; SPF mice SPF Wild SPF + wild Deferribacterales
TM7-3
Deferribacteraceae
housed with wild-caught mice, 7.460 ± 0.298. CW040
(b) Operational taxonomic units generated b
0.3 300 c Tenericutes
F16 d
100
by Qiime analysis of 16S rDNA sequences 0.2 Mollicutes
Diversity (eShannon)

contribution (%)

RF39
PC2 (15.48%)

shown in a clustered via weighted UniFrac 0.1 80

Component

200 Unclassified
0
principal coordinate (PC) analysis. Each symbol 60
–0.1
represents an individual mouse. (c) Analysis of 100 40
–0.2
IgA repertoires from SPF mice and from SPF –0.3 20 I: low frequent
mice housed with wild-caught mice; repertoire –0.4 0 0 II: expanded
–0.4 –0.2 0 0.2 0.4 4 weeks
diversity is expressed as the exponential
F

PC1 (62.01%)
se

se
SP

SP

Shannon index. Each symbol represents an

F

N
ct

st
ou

ou
SP

EC
lo
La
oh

oh
C

SPF Wild SPF + wild

individual mouse. Horizontal lines indicate
C

means. (d) The distribution of components I (non-expanded clonotypes) and II (expanded clonotypes) as a percentage of all CDR3 sequences in germ-
free mice colonized for 4 weeks with an SPF microbiota (n = 12), Lactobacillus GG (n = 5), Clostridium sordellii plus Clostridium perfringens (n = 4) or
ECN (n = 10) and in mice housed under SPF conditions (n = 5) or housed with wild-caught mice in an enriched environment (n = 5). Data (mean and
s.d.) are pooled from two or three independent experiments as described in Figure 1b.

882 VOLUME 16  NUMBER 8  AUGUST 2015  nature immunology

 Articles

Figure 4  Adult mice maintain a stable clonal composition of their IgA a Sequences Mutations
repertoire over time. (a) IgA repertoire sequence information from serial
small intestine samples taken from SPF-housed C57BL/6 mice at 5, 9, 1 5 10 40 50 91 0 >15
13 and 17 weeks of age. We clustered 1,000 sequences per time point
separately with 95% CDR3 sequence identity; these are depicted here as
networks. Each node represents a unique full-length sequence. Node sizes
represent the number of identical full-length sequences per node, and the
number of mutations is denoted by a color code. (b) Clustering of 1,000
sequences per time point; node colors in the network represent sampling
time points of 5 (yellow), 9 (red), 13 (turquoise) and 17 (blue) weeks.
Two sequences were considered clonally related if they shared identical
VH and JH gene use and showed 95% identity of their CDR3 nucleotide
sequences. Clusters that remained in the repertoire in mice from 5–17 First biopsy: 211 clusters Second biopsy: 221 clusters
weeks of age are marked by asterisks. Small clusters are not displayed.
(c) MHIs comparing the 7–9-week time points (shaded region) to all other
time points analyzed. Lines represent individual mice (n = 7). Results
showed that 13.3% ± 19.7% (mean ± s.d., n = 6) of expanded clones
were shared between juvenile 4–5-week-old mice and 8–9-week-old
mice, whereas 75.2% ± 9.7% (mean ± s.d., n = 3) of expanded clusters
observed in mice 17 weeks of age or older were already present in 11–13-
week-old mice. Results are pooled from three independent experiments.

Third biopsy: 220 clusters SI (17 weeks): 240 clusters

(Fig. 3a,b), although overall microbial diversity was in a similar range.

© 2015 Nature America, Inc. All rights reserved.

When SPF mice were housed with wild-caught mice, the composition b
of their microbiota shifted to resemble the situation in wild-caught * * * * * *
mice (Fig. 3b), and microbial diversity increased slightly. Consistently,
IgA repertoire diversity increased moderately under these conditions * * * *
compared with diversity in SPF mice housed in isolation (Fig. 3c). *
In wild-type mice housed under SPF conditions, the IgA repertoire * *
comprises expanded and non-expanded clones16. This typical archi-
tecture of the IgA repertoire was observed in SPF mice after they were c 1.0

transferred to a semi-natural environment, as well as in monocolo- 0.8

nized mice (Fig. 3d and Supplementary Fig. 3). We conclude that

IgA (MHI)

0.6
expanded clonotypes are an inherent feature of the IgA plasma cell 0.4
pool and will be observed under a broad range of housing conditions,
0.2
whereas a diverse microbiota is a prerequisite for the generation of a
diverse IgA repertoire. 0
4 6 8 10 12 14 16 18 20 22
Age (weeks)
Antibiotics do not perturb the gut IgA compartment
In mice, the generation of intestinal IgA plasma cells starts after
weaning and coincides with a fundamental shift in diet and intes- that about 13.3% of shared clones were present at both time points.
npg

tinal microbiota18. To monitor the build-up of the intestinal plasma
cell pool during this dynamic phase, we obtained serial biopsies of
small intestine from mice starting when the mice were at weaning
age and continuing until they reached adulthood. To visualize and
analyze IgA repertoire information, we considered each sequence as
a node in a network19. This approach made it possible to display the
clonal architecture of the IgA repertoire along with relevant additional
information such as sample origin, frequency in the overall repertoire
and somatic mutations (Fig. 4a,b). Expanded clonotypes, which
we considered the second component of the IgA repertoire, were
displayed as large clusters of interconnected nodes (sequences).
In contrast, non-expanded clonotypes—those sequences belonging
to the first component of the IgA repertoire—were represented by
single nodes or nodes with few connections. Consistent with our
previous observations 16, the number of somatic mutations in
expanded and non-expanded clonotypes gradually increased as mice
aged (Fig. 4a).
In the graphical network representation, clonotypes present in
the IgA repertoire at several time points appeared as mixed clusters
of clonally related sequences, that is, interconnected nodes in the
network (Fig. 4b). Quantitative comparison of the IgA repertoire in
juvenile 4–5-week-old mice with that in 8–9-week-old mice showed
Consistently, MHIs at these time points were low (Fig. 4c) but still
higher than MHIs typically observed for repertoire information
obtained from different individuals (Figs. 2a and 4c). Notably, some
clones present in 4–5-week-old mice remained in the repertoire and
were still detectable in the guts of 17-week-old mice (Fig. 4b). The
persistence of clonotypes was even more evident when we compared
the repertoires of mice 8–9 weeks of age with those of older mice.
MHIs for these repertoire comparisons always remained substantially
higher than those observed at earlier time points (4–5 weeks of age;
Fig. 4c). In fact, about 75% of expanded clusters observed in mice
17 weeks of age or older were already present in mice 11–13 weeks
old. The slight decline in MHI thus mostly reflected changes in the
frequency of clones, rather than the appearance of new clones or a
loss of old clones from the repertoire. We therefore concluded that
the gut immunoglobulin repertoire is highly dynamic in young mice
and that new B cell clones are constantly recruited. Our observations
also suggested B cell clones introduced into the repertoire early in a
mouse’s life tend to remain in the compartment for a prolonged time
and persist even into late adulthood.
To translate our observations to humans, we analyzed the IgA rep-
ertoire in biopsies of human colon and duodenum. The human IgA
repertoire showed expanded and non-expanded clusters of clonally

nature immunology  VOLUME 16  NUMBER 8  AUGUST 2015 883

 Articles

Figure 5  Dynamics of human and mouse IgA repertoires during antibiotic a 1.0 1.0
b c
treatment. (a) Colon biopsies from healthy human volunteers were
obtained directly before the start of antibiotic treatment (time point A, 0.8 0.8

IgA (MHI)
IgA (MHI)
blue), after 4 d of treatment with paromomycin (time point B, red) and 0.6 0.6
46 d after cessation of antibiotic treatment (time point C, yellow). We 0.4 0.4
clustered 1,000 sequences per sample from one representative individual 0.2 0.2
with 95% CDR3 sequence identity. The 22 most expanded clusters are 0 0
displayed here as a representative network. (b) MHIs for volunteers Abx: + –

.B

.C

.C
vs

vs

vs
(n = 6) comparing CDR3 clusters for time points A–C. Symbols represent

B
different individuals. (c) Biopsies of small intestine were obtained from
7- and 9-week-old C57BL/6 mice housed under SPF conditions. One week after surgery mice received a cocktail of ampicillin and vancomycin in drinking
water, and antibiotic administration continued for 4 weeks. Shown are MHIs for each mouse comparing the IgA repertoire observed in the biopsy before
antibiotic treatment (Abx) to the repertoire after 4 weeks of treatment (+, n = 6). Non-treated control mice (−, n = 6) were kept under SPF conditions
throughout the experiment and are also represented in Figure 2b. Each symbol represents an individual mouse. Horizontal lines in b and c represent
means. Data are pooled from two independent experiments.

related IgA sequences and did not overlap between individuals
(Fig. 5a and data not shown). Thus, the overall architecture of the
IgA repertoire in humans resembled that in mice. The persistence of
B cell clones in the IgA repertoire might reflect only minor changes in
microbial exposure in mice housed under laboratory conditions; thus
we next tracked changes in human and mouse IgA repertoires during
antibiotic treatment, which results in massive changes in microbiota
© 2015 Nature America, Inc. All rights reserved.
microbiota was reduced and profoundly shifted in its composition
relative to that in non-treated control mice (Supplementary Fig. 4).
This finding indicated that in human colon, like in mouse small
intestine, the clonal composition of the IgA repertoire is rather stable
even in the face of extensive changes in the intestinal microbiota that
occur during antibiotic treatment.

composition. Colon biopsies were obtained from healthy human vol-
unteers directly before antibiotic treatment, after 4 d of treatment and
46 d after the end of treatment. The repertoire observed at the first
examination was as similar to the repertoire directly after antibiotic
treatment as it was to that at the follow-up time point (7 weeks later)
(Fig. 5a,b). Moreover, nearly all expanded clones were shared between
at least two samples and were displayed in the network representation
as mixed clusters (Fig. 5c). Similarly, in mice the IgA repertoires
observed before and after antibiotic treatment were as similar as
those observed in non-treated control mice at the same time points
(Fig. 5e), even though in antibiotic-treated mice the density of colonic
a 70
4–5 weeks
30
7–9 weeks
25
Sequences (%)
Dynamic changes in the pattern of somatic mutations
Despite general clonal stability, we observed ongoing modulation of the
IgA repertoire when we analyzed somatic mutations. The frequency of
somatic mutations observed in the gut of 12–23-week-old mice was
similar to the frequency observed in 1–3-year-old children. In both
mice and humans, the frequency of somatic mutations increased with
age (Fig. 6a,b). Notably, increasing frequencies of somatic mutations in
mice were also evident in clones that persisted in the repertoire in mice
5–17 weeks of age (Fig. 6c). Similarly, B cell clones that remained in the
IgA repertoire of ECN-monocolonized mice after colonization with
SPF microbiota and B cell clones that persisted in the IgA repertoire
of mice during antibiotic treatment differed with respect to somatic
mutation frequency (Fig. 6d). As somatic hypermutation requires cell
12
17–23 weeks
proliferation, collectively these findings indicated that a stable clonal
10 composition of the IgA repertoire can be maintained despite constant

50 20 8 plasma cell generation and dynamic changes in the pattern of somatic

15 6
30 mutations. We therefore hypothesized that in adults, adaptations of
10 4
the gut IgA repertoire might rely largely on the diversification of B cell
npg

10 5 2
0 0 0 clones already fixed in the IgA repertoire.
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
Mutations Mutations Mutations
Memory responses generate extraintestinal plasma cells
b 50 1–3 years 4–10 years 11–16 years To explore this idea further, we compared the immunoglobulin
16 16 16 repertoires of sorted memory B cells and naive B cells from PPs
Sequences (%)

12 12 12

8 8 8 Figure 6  Mutation frequencies increase in mice and humans within

clonally stable IgA repertoires. (a,b) Sequence frequency versus the
4 4 4
number of somatic mutations for mouse (a) and human (b) small intestine
0 0 0 IgA repertoires. Lines represent individual mice or patients (n = 19 or
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 35, respectively); ages of subjects in weeks or years are presented at the
Mutations Mutations Mutations top of each graph. (c) The average mutation frequency in CDR3 clusters
c d *** * *** * in mice at 5, 9, 13 and 17 weeks of age. The diagram shows all clusters
20 12 present at all four time points in one representative mouse out of four
mice analyzed in one experiment. (d) Mice were treated as described
10
16 in Figure 2b (ECN to SPF). All CDR3 clusters that persisted in the IgA
8 repertoire and were represented by at least five sequences were identified.
Mutations
Mutations

12
6 The average number of mutations in these clusters was determined; data
8 shown are for two representative mice out of five mice analyzed. Such
4
clusters were also picked from repertoires of mice treated with antibiotics,
4 2 as described in Figure 5c, to demonstrate the persistence of CDR3
0 0
clusters despite treatment (Before Abx to after Abx). Data shown are for
5 9 13 17
ECN to SPF Before Abx
two representative mice out of six mice analyzed. Paired t-test was used
Time (weeks) to after Abx for statistical analysis; *P < 0.05, ***P < 0.001.

884 VOLUME 16  NUMBER 8  AUGUST 2015  nature immunology

 Articles

Figure 7  Memory B cells recirculate between multiple compartments 2h 24 h 72 h

and give rise to plasma cells in mammary glands. (a) Numbers of
a 105
4
10
CD80+CD73+CD19+CD138−GL-7− B cells expressing Dendra2 in

D-Green
103
photoconverted PPs 2 h, 24 h and 72 h after illumination (representative 99.23 78.65 58.59
of three mice at 2 h and five mice at 24 and 72 h from two experiments). 0

(b) Frequency of D-Red+ cells among Dendra2-expressing CD80+CD73+

and CD80−CD73− B cells in spleen, photoconverted (Photoconv) PPs 0
3 4
10 10 10
5

and non-converted (Non-photoconv) PPs (n = 3 or 5 mice per time D-Red

point). Unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001. (c) IgA
repertoires were compared in mammary glands (MG), small intestine (SI)
b ***
Photoconv PP
***
Non-photoconv PP
* ***
** *** *** *** NS ** * ***
and colon (Co) from lactating mice 2–3 weeks after delivery; similarity is 100 5

D-Red+ cells (%)

expressed as MHI (black squares, n = 4). Additionally, female C57BL/6 80 4
mice were biopsied and mated 1 week later. Two weeks after delivery, 60 3
we collected mammary gland, small intestine and colon tissues from 40 2
four lactating mice and compared IgA repertoires among all tissues,
20 1
including the biopsies taken before mating (red circles, n = 4). Horizontal
0 0
lines represent means. On the left, 1,000 sequences per compartment 2 24 72 2 24 72 2 24 72 2 24 72
that were clustered with 95% CDR3 sequence identity are displayed Time (h) Time (h)
as a network. Small clusters are not displayed. Clones shared between Spleen
mammary gland and small intestinal biopsy are marked by asterisks, and * ***
sample origin is indicated by color according to the key. Data are pooled 5
* * * **
from three independent experiments.

D-Red+ cells (%)

4 Memory
3 Naive
and lamina propria plasma cells. Memory B cells were sorted from
© 2015 Nature America, Inc. All rights reserved.

2
PPs as CD19+ B cells that did not express the plasma cell and ger- 1
minal center markers CD138 and GL-7 but did express CD80 and 0
CD73, characteristic of murine memory B cells20 (Supplementary 2 24 72 2 24 72
Time (h)
Fig. 5a). For comparison, we purified non-memory B cells, mostly
naive B cells (CD19+CD138−GL-7−CD80−CD73−). In contrast to c SI Co MG

plasma cells, naive B cells did not contain expanded sequence clus- * * 1.0
* * 0.8
ters and mostly presented as non-expanded sequences in the net-

lgA (MHI)
0.6
work display (Supplementary Fig. 5b), with few somatic mutations * * *
0.4
(Supplementary Fig. 6). Compared with rates in naive B cells, somatic
* * * 0.2
mutation frequencies were higher in memory B cells for both IgM-
0
encoding and IgA-encoding sequences (Supplementary Fig. 6). MHIs * * SI Co Co SI MG Co
observed for the comparison of non-memory B cells to IgA plasma vs. MG SI Bx
cells were close to zero, which indicated that these populations were
not clonally related. Similarly, IgM sequences obtained from PP mem- Consistently, IgA repertoire analysis showed clonally related mem-
ory B cells were not clonally related to IgA sequences obtained from ory B cells in PPs and spleen (data not shown). This indicated that
intestinal plasma cells. In contrast, the IgA repertoire obtained memory B cells could recirculate between different PPs and spleen.
from PP memory B cells showed substantial similarity to that of To examine the functional relevance of an IgA-switched memory B
IgA-secreting gut plasma cells, and shared clones were evident in the cell population outside of the intestine, we compared IgA repertoires
npg

network (Supplementary Fig. 5b). This finding indicated that at least
part of the intestinal IgA repertoire was reflected in IgA-expressing,
but not IgM-expressing, memory B cells in PPs.
Previous work suggests that memory B cells can leave and re-enter
PP germinal centers21. Such recirculation of memory B cells might
enable the coordination of mucosal antibody responses between vari-
ous inductive sites, and it also could explain the stable clonal com-
position of the gut plasma cell compartment. To explore this idea,
we quantified the recirculation rates of naive and memory B cells
between PPs and spleen. To track B cell egress from PPs, we marked
cells in situ in PPs by photoconversion as described22,23. We generated
bone marrow–chimeric mice expressing the photoconvertible protein
Dendra2 in hematopoietic cells (Dendra2 chimera). Dendra2 exhib-
its green fluorescence (D-Green), which changes to red fluorescence
(D-Red) upon exposure to short-wavelength light. We exposed PPs in
Dendra2 chimeras by surgery, illuminated four or five individual PPs
and analyzed the presence of photoconverted cells in converted PPs,
nonconverted PPs and spleen 2, 24 and 72 h after photoconversion.
Within 72 h, almost all D-Red naive B cells had migrated away from
photoconverted PPs, whereas D-Red memory B cells egressed from
PPs at a slower rate (Fig. 7a,b). D-red naive and memory B cells were
readily detectable in non-photoconverted PPs and spleen (Fig. 7b).
of plasma cells from small and large intestine to the de novo–formed
plasma cell pool in mammary glands of lactating mice. The IgA
repertoire in mammary glands largely mirrored the gut IgA repertoire
and comprised highly mutated sequences (data not shown). Moreover,
we frequently noted clones that were shared between gut biopsies
taken from female mice before mating and mammary glands iso-
lated from lactating mice 2 weeks after delivery (Fig. 7c). Notably,
MHIs observed in comparisons between mammary gland and small
intestine were higher than those for comparisons between mammary
gland and colon or between small intestine and colon (Fig. 7c). This
observation indicated that the plasma cell population in mammary
glands might arise from memory B cells originating from previous gut
immune responses and could mechanistically explain the presence of
antibodies in breast milk that recognize gut antigens24.
Infection triggers the recruitment of new B cell clones
We next investigated whether pre-existing B cell clones also contrib-
ute to the response during primary intestinal infection or, alterna-
tively, whether infection-triggered changes in the IgA repertoire rely
on different processes. To this end, we obtained biopsies of mouse
small intestine and subsequently infected mice with an attenuated
strain of Salmonella enterica subsp. enterica serovar Typhimurium

nature immunology  VOLUME 16  NUMBER 8  AUGUST 2015 885

 Articles
Figure 8  S. Typhimurium LPS–binding plasma cells are unrelated to
B cell clones present before infection. (a) IgA+ plasma cells (PC) were
sorted and either enriched (LPS+) or depleted (LPS−) of S. Typhimurium
LPS–binding plasma cells. The IgA repertoire in the sorted plasma cell
population was compared to the repertoire in gut biopsies obtained before
infection (Bx) and total small intestine isolated after infection (SI). Here
1,000 sequences that clustered with 95% CDR3 sequence identity are
displayed as a network. Sample origin is indicated by color according to
the key; small clusters are not shown. (b) For analysis, sequences were
clustered with 95% identity, and 4,000 sequences were considered per
sample. Repertoire similarity was calculated as the MHI for all samples
(n = 4, pooled from two independent experiments). Horizontal lines
represent means. Unpaired t-test, **P < 0.01.
(Salmonella Typhimurium). S. Typhimurium infection did not cause
any major shifts in the overall architecture of the IgA repertoire. In
fact, even though we detected roughly 4% of S. Typhimurium lipopoly­
saccharide (LPS)–specific plasma cells in infected mice but not in
uninfected mice, this was not reflected in discernible changes in the
immunoglobulin repertoire compared with that in uninfected mice
(data not shown).
To investigate how the gut immune system generates Salmonella-
© 2015 Nature America, Inc. All rights reserved.
directed IgA responses, we compared the repertoire of S. Typhimurium
LPS–specific plasma cells to the repertoire present before infection.
Gut plasma cells show surface IgA, which allows for labeling of plasma
cells producing antibodies to a given antigen25,26. We compared the
IgA repertoire observed in gut biopsies obtained before infection to
the overall repertoire 3 weeks after infection and sorted intestinal
plasma cells enriched or depleted in binding of S. Typhimurium LPS.
Sequences generated from sorted LPS-negative cells clustered both
with biopsies before infection and with small intestine after infection
(Fig. 8a). In contrast, sequences obtained from LPS-binding plasma
cells were generally absent from the gut IgA repertoire before
S. Typhimurium infection and appeared in the small intestine
only after infection (Fig. 8b). Sequences derived from plasma cells
enriched in S. Typhimurium LPS binding were found in several
new expanded clusters, which indicated that the S. Typhimurium
LPS–specific IgA response relies on the recruitment of several new
B cell clones into the repertoire.
Collectively, these results reveal complementary mechanisms that
npg
enable adaptation of the intestinal IgA repertoire. In young animals
and during infection, the recruitment of new B cell clones enables
drastic changes in the repertoire that cannot be accommodated on the
basis of pre-existing memory cell specificities. In addition, antibody
specificities generated from memory B cell responses allow for the
establishment of stable host-microbe interactions and the dissemina-
tion of responses generated in the gut immune system to other areas
such as mammary glands.
DISCUSSION
Hosts and microbiota engage in intense crosstalk that in healthy
individuals results in peaceful coexistence. However, how antibody
responses are regulated to achieve sustained host-microbe interac-
tions in spite of changes in diet, antibiotic use, intestinal infections
and other environmental influences is unknown. Here we suggest
that in the intestine, ongoing memory B cell–based diversification
of the immunoglobulin repertoire is a key mechanism for matching
antibody specificities to the intestinal microbiota and fueling IgA
production in mammary glands.
We found that expanded clonotypes were an inherent feature of
both human and mouse IgA repertoires and were preserved under a
broad range of environmental, colonization and infection conditions.
886 VOLUME 16  NUMBER 8  AUGUST 2015  nature immunology
a b **
LPS+ PC LPS– PC Bx SI
0.6
IgA (MHI)
0.4
0.2
0
PC
PC
PC
PC
+
S
S
S
S
LP
LP
LP
LP
vs. Bx SI
These expanded clonotypes were reliably identified in biopsy-sized
tissue samples and can serve as a basis for tracking changes in the IgA
repertoire. IgA repertoires obtained from mouse and human gut biop-
sies before and after antibiotic treatment showed clusters of clonally
related sequences. Moreover, clonotypes present in biopsies of small
intestine taken from E. coli–monocolonized mice persisted in the
IgA repertoire after colonization with an SPF microbiota. This find-
ing indicates that B cell clones, once recruited to the IgA repertoire,
tend to persist over prolonged time periods. A variation from this
scenario was encountered in analyses of S. Typhimurium–infected
mice, which showed that plasma cells producing antibodies to
S. Typhimurium LPS were clonally unrelated to clusters present in the
IgA repertoire before infection. Yet even in infected mice, the overall
repertoire retained striking similarity to the preinfection repertoire.
Collectively, these observations indicate that recruitment of new B
cell clones into the IgA repertoire occurs ‘on demand’ but does not
rapidly result in global remodeling of the IgA repertoire.
Mechanistically, clonal stability despite progressively changing
patterns of somatic mutations and turnover of the plasma cell
population could be achieved via the diversification of memory
B cells. Indeed, hapten-directed IgA responses21 and efficient
vaccine responses27 rely on the repeated entry of memory B cells into
germinal centers. Here we used photoconversion-based cell tracking
to demonstrate the recirculation of PP memory B cells. Consistently,
we found clonally related B cells in memory B cell populations and gut
plasma cells. Recirculation of memory B cells between IgA inductive
compartments such as PPs, isolated lymphoid follicles and mesenteric
lymph nodes might drive ongoing hypermutation, thereby generating
an iterative process by which to adapt IgA specificity to the micro-
biota. But what is the advantage of diversifying existing memory
B cells rather than generating memory from scratch in naive B cells?
We speculate that a confined set of B cell clones dominating the IgA
repertoire might be particularly valuable for sustaining symbiotic
host-microbe interactions. SIgA exerts selective pressure on gut bac-
teria9,28. Thus, the specific composition of the SIgA arsenal might
stabilize an individual’s microbiota and contribute to community
resilience. In addition, diversification of memory B cell clones, as
opposed to recruitment of new B cell clones, might reduce the risk of
introducing specificities to the IgA repertoire that endanger a stable
host-microbe interaction.
Somatic mutations cannot occur in terminally differentiated plasma
cells. Therefore, changes in the pattern of somatic mutations over time
imply constant turnover of the plasma cell pool. Such a scenario is
reminiscent of the situation in cattle and sheep, which show limited
recombination diversity and rely largely on somatic hypermutation

to generate a diverse antibody repertoire29,30. One might thus specu-
late that diversity of the primary B cell repertoire is less critical than
commonly assumed in human and mouse gut as well; instead
hypermutation-based diversification of pre-existing B cell clones
might have a central role in the adaptation of antibody responses
to the microbiota. Dysbiosis consistently develops in SIgA-sufficient
mice that do not generate somatic mutations6.
Placing memory B cell responses at the core of the intestinal
IgA-inducing machinery also sheds new light on the ways in which
T cell–dependent and T cell–independent pathways integrate to
generate microbe-directed IgA responses10,11,31. Frequent somatic
mutations are a distinguishing feature of unmanipulated intestinal
plasma cells16,32,33, whereas somatic mutations are virtually absent in
plasma cells of germinal center–deficient or T cell–deficient mice16,34.
However, hypermutation can occur independently of T cells and
germinal centers35–37, and in PPs germinal centers can form inde-
pendently of B cell receptor signaling and T cells38–40. Thus, somatic
mutations in the IgA repertoire per se do not establish a strictly
T cell–dependent origin of plasma cells. We propose that some of
this controversy may be reconciled once generation and adaptation
of the intestinal plasma cell repertoire are viewed as parts of a con-
© 2015 Nature America, Inc. All rights reserved.
tinuous process that potentially results from the combined activity of
T cell–dependent and –independent processes.
Collectively, our results suggest that secretory antibodies are shaped
by the ongoing diversification of memory B cells. This process might
be particularly beneficial in the intestine to match transient altera-
tions of the microbiota and provide protective maternal antibodies
secreted into milk. Durable changes in microbiota or infection can
be addressed by the recruitment of new B cell clones into the plasma
cell repertoire. Our findings contribute to the understanding of
host-microbiota interactions and suggest a new concept to explain
the regulation of intestinal antibody responses.
Methods
Methods and any associated references are available in the online
version of the paper.
Accession codes. Immunoglobulin repertoire and 16S microbiota
sequences have been deposited at the European Nucleotide Archive
npg
with study number PRJEB9386.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
We thank U. Kalinke (TWINCORE, Hannover, Germany), A. Krueger, I. Prinz,
O. Schulz, S. Woltemate (Hannover Medical School, Hannover, Germany),
M. Bemark (University of Gothenburg, Gothenburg, Sweden), B. Wabakken
Hognestad and D. Ölsner (both at the Norwegian University of Life Sciences, Oslo,
Norway), and R. Bharti (IKMB, University of Kiel, Kiel, Germany). This work was
supported by the DFG Cluster of Excellence Inflammation at Interfaces (CL VIII
to P.R., S.O. and S. Schreiber), the BMBF (grant SysINFLAME (TP3/4) to P.R.),
the Israel Science Foundation (grant 270/09 to R.M.), the German Centre for
Infection Research (DZIF), partner site Hannover-Braunschweig and Deutsche
Forschungsgemeinschaft (grants PA921/4-1 (to O.P.) and SFB621-Z).
AUTHOR CONTRIBUTIONS
C.L. and I.T. performed experiments and analyzed data; M.U. performed cell-
tracking experiments; B.W. generated tools for data analysis; M.F., M.K.S. and
S. Suerbaum supported sequencing experiments; A.B. and A.S. performed
colonization experiments; V.G.-R. and N.C.-B. provided samples from
monocolonized mice; H.H. and R.M. helped with sequencing data analysis; P.B.
performed cohousing experiments; S.O., U.B., S. Schreiber and P.R. provided
human samples; and O.P. performed mouse surgery, designed the study and wrote
the manuscript.
nature immunology  VOLUME 16  NUMBER 8  AUGUST 2015 887
Articles
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
1. Kranich, J., Maslowski, K.M. & Mackay, C.R. Commensal flora and the regulation
of inflammatory and autoimmune responses. Semin. Immunol. 23, 139–145
(2011).
2. Sommer, F. & Backhed, F. The gut microbiota—masters of host development and
physiology. Nat. Rev. Microbiol. 11, 227–238 (2013).
3. Hooper, L.V., Littman, D.R. & Macpherson, A.J. Interactions between the microbiota
and the immune system. Science 336, 1268–1273 (2012).
4. Shulzhenko, N. et al. Crosstalk between B lymphocytes, microbiota and the
intestinal epithelium governs immunity versus metabolism in the gut. Nat. Med.
17, 1585–1593 (2011).
5. Kawamoto, S. et al. The inhibitory receptor PD-1 regulates IgA selection and
bacterial composition in the gut. Science 336, 485–489 (2012).
6. Wei, M. et al. Mice carrying a knock-in mutation of Aicda resulting in a defect in
somatic hypermutation have impaired gut homeostasis and compromised mucosal
defense. Nat. Immunol. 12, 264–270 (2011).
7. Hapfelmeier, S. et al. Reversible microbial colonization of germ-free mice reveals
the dynamics of IgA immune responses. Science 328, 1705–1709 (2010).
8. Lécuyer, E. et al. Segmented filamentous bacterium uses secondary and tertiary
lymphoid tissues to induce gut IgA and specific T helper 17 cell responses.
Immunity 40, 608–620 (2014).
9. Palm, N.W. et al. Immunoglobulin A coating identifies colitogenic bacteria in
inflammatory bowel disease. Cell 158, 1000–1010 (2014).
10. Suzuki, K., Maruya, M., Kawamoto, S. & Fagarasan, S. Roles of B-1 and B-2 cells
in innate and acquired IgA-mediated immunity. Immunol. Rev. 237, 180–190
(2010).
11. Pabst, O. New concepts in the generation and functions of IgA. Nat. Rev. Immunol.
12, 821–832 (2012).
12. Macpherson, A.J. et al. A primitive T cell-independent mechanism of intestinal
mucosal IgA responses to commensal bacteria. Science 288, 2222–2226
(2000).
13. Slack, E., Balmer, M.L., Fritz, J.H. & Hapfelmeier, S. Functional flexibility of
intestinal IgA—broadening the fine line. Front. Immunol. 3, 100 (2012).
14. Lefranc, M.P. et al. IMGT, the international ImMunoGeneTics information system.
Nucleic Acids Res. 37, D1006–D1012 (2009).
15. Brochet, X., Lefranc, M.P. & Giudicelli, V. IMGT/V-QUEST: the highly customized
and integrated system for IG and TR standardized V-J and V-D-J sequence analysis.
Nucleic Acids Res. 36, W503–W508 (2008).
16. Lindner, C. et al. Age, microbiota, and T cells shape diverse individual IgA repertoires
in the intestine. J. Exp. Med. 209, 365–377 (2012).
17. Larimore, K., McCormick, M.W., Robins, H.S. & Greenberg, P.D. Shaping of human
germline IgH repertoires revealed by deep sequencing. J. Immunol. 189, 3221–
3230 (2012).
18. Schloss, P.D. et al. Stabilization of the murine gut microbiome following weaning.
Gut Microbes 3, 383–393 (2012).
19. Ben-Hamo, R. & Efroni, S. The whole-organism heavy chain B cell repertoire from
Zebrafish self-organizes into distinct network features. BMC Syst. Biol. 5, 27
(2011).
20. Tomayko, M.M., Steinel, N.C., Anderson, S.M. & Shlomchik, M.J. Cutting edge:
hierarchy of maturity of murine memory B cell subsets. J. Immunol. 185,
7146–7150 (2010).
21. Bergqvist, P. et al. Re-utilization of germinal centers in multiple Peyer’s patches
results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses.
Mucosal Immunol. 6, 122–135 (2013).
22. Ugur, M., Schulz, O., Menon, M.B., Krueger, A. & Pabst, O. Resident CD4+ T cells
accumulate in lymphoid organs after prolonged antigen exposure. Nat. Commun.
5, 4821 (2014).
23. Schulz, O. et al. Hypertrophy of infected Peyer’s patches arises from global,
interferon-receptor, and CD69-independent shutdown of lymphocyte egress. Mucosal
Immunol. 7, 892–904 (2014).
24. Brandtzaeg, P. The mucosal immune system and its integration with the mammary
glands. J. Pediatr. 156, S8–S15 (2010).
25. Di Niro, R. et al. High abundance of plasma cells secreting transglutaminase
2–specific IgA autoantibodies with limited somatic hypermutation in celiac disease
intestinal lesions. Nat. Med. 18, 441–445 (2012).
26. Di Niro, R. et al. Rapid generation of rotavirus-specific human monoclonal antibodies
from small-intestinal mucosa. J. Immunol. 185, 5377–5383 (2010).
27. McHeyzer-Williams, L.J., Milpied, P.J., Okitsu, S.L. & McHeyzer-Williams, M.G.
Class-switched memory B cells remodel BCRs within secondary germinal centers.
Nat. Immunol. 16, 296–305 (2015).
28. Peterson, D.A., McNulty, N.P., Guruge, J.L. & Gordon, J.I. IgA response to symbiotic
bacteria as a mediator of gut homeostasis. Cell Host Microbe 2, 328–339
(2007).
29. Wang, F. et al. Reshaping antibody diversity. Cell 153, 1379–1393 (2013).
30. Jenne, C.N., Kennedy, L.J. & Reynolds, J.D. Antibody repertoire development in
the sheep. Dev. Comp. Immunol. 30, 165–174 (2006).
31. Spencer, J., Klavinskis, L.S. & Fraser, L.D. The human intestinal IgA response;
burning questions. Front. Immunol. 3, 108 (2012).
 Articles
32. Dunn-Walters, D.K., Boursier, L. & Spencer, J. Hypermutation, diversity and
dissemination of human intestinal lamina propria plasma cells. Eur. J. Immunol.
27, 2959–2964 (1997).
33. Barone, F. et al. IgA-producing plasma cells originate from germinal centers that
are induced by B-cell receptor engagement in humans. Gastroenterology 140,
947–956 (2011).
34. Bergqvist, P., Stensson, A., Lycke, N.Y. & Bemark, M. T cell-independent IgA class
switch recombination is restricted to the GALT and occurs prior to manifest germinal
center formation. J. Immunol. 184, 3545–3553 (2010).
35. Weller, S. et al. CD40–CD40L independent Ig gene hypermutation suggests a second B cell
diversification pathway in humans. Proc. Natl. Acad. Sci. USA 98, 1166–1170 (2001).
© 2015 Nature America, Inc. All rights reserved.
npg
888 VOLUME 16  NUMBER 8  AUGUST 2015  nature immunology
36. Scheeren, F.A. et al. T cell-independent development and induction of somatic
hypermutation in human IgM+ IgD+ CD27+ B cells. J. Exp. Med. 205, 2033–2042
(2008).
37. Vossenkämper, A. et al. A role for gut-associated lymphoid tissue in shaping the
human B cell repertoire. J. Exp. Med. 210, 1665–1674 (2013).
38. Casola, S. et al. B cell receptor signal strength determines B cell fate. Nat. Immunol.
5, 317–327 (2004).
39. de Vinuesa, C.G. et al. Germinal centers without T cells. J. Exp. Med. 191,
485–494 (2000).
40. Shlomchik, M.J. & Weisel, F. Germinal center selection and the development of
memory B and plasma cells. Immunol. Rev. 247, 52–63 (2012).
 ONLINE METHODS
Human study design. Serial biopsies were collected from healthy volunteers
aged 20–22 years. Inclusion criteria were no history of antibiotic, antifungal or
probiotic therapy and no hospitalization or diarrhea in the preceding 6 months.
All subjects with signs of current infection were excluded, and all subjects with
relative or absolute contraindications to paromomycin were not considered.
All individuals gave written consent as approved by the local ethical committee
of the Medical Faculty of the Christian-Albrechts-University (Kiel, Germany)
under ethics vote A154/06. The antibiotic paromomycin (Pfizer) was admin-
istered at a dose of 4 g/d for a duration of 3 d. A series of sigmoid biopsies was
obtained before treatment (A, day 0) and immediately after cessation of paro-
momycin treatment (B, day 4), as well as 7 weeks after cessation (C, day 46).
Biopsies from children were obtained at Hannover Medical School at the Clinic
for Pediatric Gastroenterology and Hepatology under ethics vote 1016-2011.
Biopsies were stored at −80 °C until use. Total RNA was isolated from snap-
frozen biopsies with RNeasy kits (Qiagen) according to the manufacturer’s
instructions. Reverse transcription was done with random hexamers.
Mice. C57BL/6 and C57BL/6-(Tcrd-H2BEGFP)tm1 mice were bred under SPF
or germ-free conditions at the central animal facility of Hannover Medical
School. A microbially enriched semi-naturalistic model was designed at the
Norwegian University of Life Sciences. Pens (2.0 × 2.5 × 1.25 m) contained
sawdust; soil; compost; twigs; hay; pallets; and fecal content from pigs, cows
© 2015 Nature America, Inc. All rights reserved.
and horses. 15 female C57BL/6N mice (Charles River) were housed for
8 weeks with 10 female Mus musculus mice caught in the wild as described41.
Water and food (wild bird mix containing grains, nuts and sunflower seeds)
was provided ad libitum. Only mice that presented as healthy upon inspec-
tion after cohousing were included in the study. Mice of the same batch were
kept under SPF conditions as controls. For S. Typhimurium infection, mice
were gavaged with 109 bacteria of the attenuated S. Typhimurium ∆aroA
strain. All animal experiments were performed in accordance with institu-
tional guidelines and approved by either the review board of the Hannover
Medical School and the Niedersächsisches Landesamt für Verbraucherschutz
und Lebensmittelsicherheit or the National Animal Research Authority in
Norway (FOTS4788). Capture of wild mice was approved by the Norwegian
Environment Agency (2012/693).
Gnotobiotics. Germ-free and monocolonized C57BL/6 mice were housed at
the central animal facility of the Hannover Medical School. Colonization status
was verified before mice were killed. Mice 6–8 weeks old were colonized with
E. coli Nissle 1917 (2 × 109 bacteria on two consecutive days). C57BL/6-(Tcrd-
H2BEGFP)tm1 mice were colonized with Lactobacillus rhamnosus strain GG
(5 × 107 bacteria) or a mix of Clostridium sordellii and Clostridium perfringens
npg
(1 × 105 bacteria). SPF microbiota was transferred using cecal content. Samples
derived from colonized C3H mice depicted in Figure 1d have been described
previously42.
Bone marrow chimeras. H2B-Dendra2–expressing bone marrow (BM) chi-
meras were prepared as described22. We enriched BM precursor cells from
wild-type mice by depleting lineage-committed cells with an antibody cocktail
and Dynabeads (Invitrogen). Enriched BM precursor cells were cultured with
interleukin 7 (IL-7; 25 ng/ml), IL-6 (20 ng/ml), stem cell factor (50 ng/ml) and
the hematopoietic growth factor Flt3L (23 ng/ml) for 4 d and transduced with
viral particles carrying the H2B-Dendra2 sequence on days 2–4 via the spin-
infection method. On day 5, cells were harvested and 106 cells per recipient
were transferred i.v. to lethally irradiated (10 Gy) wild-type recipients. Mice
were given antibiotics (cotrimoxazole, Ratiopharm) in their drinking water
(0.96 mg/ml) for 2 weeks and analyzed after at least 8 weeks.
Mouse surgery. Mice were anesthetized with ketamine (100 mg/kg, Albrecht)
and xylazine (5 mg/kg, Bayer). To obtain biopsies of small intestine, we opened
each mouse’s abdominal cavity and exposed its small intestine. Approximately
1 × 2 mm2 of intestinal tissue was cut from the antimesenteric site, and the
intestine was closed with suture. Photoconversion was done as described 22.
Each PP was illuminated for 10 s with a low-intensity light from a BlueWave
75 light curing system (Dymax) equipped with a 390/40 bandpass filter. Four
to five PPs were photoconverted per mouse, and their positions were mapped
doi:10.1038/ni.3213
 nature immunology
for analysis. The intestine was reintroduced into the abdominal cavity, and the
peritoneum and the skin were closed with suture.
Immunoglobulin 454 sequencing. Mouse IgA repertoire information
was obtained as described16. Amplicons encoding IgA and IgM from
PP memory and non-memory B cells were generated with semi-nested
PCR. In the first PCR, with 25 cycles, we used primers VH promiscu-
ous 5′-GAGGTGCAGCTGCAGGAGTCTGG-3′ in combination with
Cα(outer) 5′-ATCAGGCAGCCGATTATCAC-3′ and Cµ(outer) 5′-ATGG
TGCTGGGCAGGAAGTC-3′ for IgA and IgM amplicons, respectively. In the
second PCR, also with 25 cycles, we used the same VH primer including the
adaptor sequence (5′-CTATGCGCCTTGCCAGCCCGCTCAGGAGGTGC
AGCTGCAGGAGTCTGG-3′) and Cα 5′-CGTATCGCCTCCCTCGCGCCA
TCAG (MID)GAGCTCGTGGGAGTGTCAGTG-3′ or Cµ 5′-CGTATCGCC
TCCCTCGCGCCATCAG(MID)AGGGGGCTCTCGCAGGAGACGAGG-
3′ for the constant region. Gene-specific sequences are underlined; “MID”
specifies a nucleotide sequence used in ten variations to identify samples;
non-underlined sequences represent adaptor sequences needed for emulsion
PCR and 454 sequencing. Human VH1 variable regions were amplified with
primer binding in the Cµ region and amino-terminal framework region 1
(5′-CGTATCGCCTCCCTCGCGCCATCAG(MID)GAATTCGAGTGGCTC
CTGGGGGAAGA-3′ and 5′-CTATGCGCCTTGCCAGCCCGCTCAGGGCC
TCAGTGAAGGTCTCCTGCAAG-3′). PCR conditions for mouse and human
IgA and IgM amplicons were as follows: 95 °C, 4 min; 25 rounds of (94 °C,
30 s; 62 °C, 30 s; 72 °C, 35 s); and 72 °C, 10 min. Amplicons were purified by
gel electrophoresis followed by gel extraction (QIAquick Gel Extraction kit,
Qiagen), and DNA concentration was quantified with a Quant-iT dsDNA HS
Assay kit (Invitrogen) and measured with a Qubit fluorometer (Invitrogen).
Amplicons were prepared with the GS FLX Titanium SV emPCR kit (Lib-A)
for 454 pyrosequencing on the Genome Sequencer FLX system (Roche) per
the manufacturer’s instructions.
Immunoglobulin sequence analysis was done as described16, with some
modifications. Sequences longer than 320 bp and containing both primers
were sorted according to their MID. Sequences were further analyzed with
ImMunoGeneTics (IMGT) HighV-QUEST (http://www.imgt.org/)14,15. All
sequences were compared with reference sequences from the IMGT database.
Results obtained from IMGT were further analyzed with in-house-generated
Excel and VBA scripts, and only productive sequences were used for down-
stream analysis (Supplementary Note). Mutation frequencies were calculated
as the number of mutations divided by the number of all nucleotides of the
given framework regions and CDRs. Clonally related sequences were identified
by pairwise sequence alignment with USEARCH 5.0.1 (ref. 43). Networks were
displayed with Cytoscape 2.8.2 (refs. 44,45). In the figures, nodes represent
unique full-length sequences; clonally related CDR3 sequences are connected
by lines. Repertoire similarity was expressed as the MHI, calculated as46
2Σ(ani × bni ) Σani2 Σbni2
MHI = da = 2 db =
(da + db) × aN × bN aN bN 2
xN represents the total number of clusters in a given sample x, and xni equals
the number of clusters of type i present in xN. The Shannon index, used
to measure immunoglobulin repertoire diversity, was calculated with
R studio (version 0.94.110; http://www.r-project.org/, http://rstudio.org/)
and the library “vegan” (command: diversity). The Shannon index is equal
to −Σpi × ln pi; pi = ni/N, where N represents the number of clusters present
in the repertoire and ni equals the number of a given cluster i in the same
repertoire, making pi the ratio of cluster i within the overall sequence set.
We expressed repertoire diversity as the exponential Shannon index, which
corresponds to a theoretical number of equally abundant species that would
have the same Shannon index as the actual observed data set.
16S rDNA 454 sequencing. Fresh fecal pellets or 1 cm of proximal ileum was
frozen on dry ice and stored at −80 °C. Samples were homogenized in 1.6 ml
ASL buffer (QIAamp DNA Stool Mini Kit, Qiagen) with a tissue lyser (Qiagen)
for 5 min at 30 Hz using a 5-mm steel bead. Homogenized samples were trans-
ferred into fresh tubes containing 200 µl of 0.1-mm glass beads (Roth) and
 shaken with the tissue lyser two times for 5 min each time at 30 Hz. Subsequent
DNA isolation steps were carried out according to the manufacturer’s instruc-
tions, and samples were incubated at 95 °C for 5 min. 16S rDNA amplicon
libraries were generated with a primer set covering the first three variable
regions of the 16S rRNA gene (8F, 5′-CGTATCGCCTCCCTCGCGCCATCAG
TCAGAGTTTGATCCTGGCTCAG-3′; 541R, 5′-CTATGCGCCTTGCCAGC
CCGCTCAG(MID)ACWTTACCGCGGCTGCTGG-3′). PCR conditions were
as follows: 94 °C, 3 min; 25 (feces) or 35 (small intestine) rounds of (94 °C, 15 s;
61 °C, 45 s; 72 °C, 60 s); and 72 °C, 8 min. 16S rDNA amplicons were purified
by gel electrophoresis and sequenced as described for immunoglobulin
variable regions. 16S rDNA sequences were analyzed with the Qiime software
package47. Sequences with an average quality score of <20, missing the forward
primer or shorter than 300 nucleotides were removed. Chimera sequences
were identified with Usearch6.1 and the Uchime reference database “gold.”
Chimera filtered sequences were clustered into operational taxonomic units
(OTUs) using Uclust with a sequence-similarity threshold of 0.97. One repre-
sentative sequence from each OTU was aligned with Pynast, and the taxonomy
was assigned with the RDP classifier48 with a confidence of 0.8.
Cell isolation and cell sorting. Lamina propria cells were obtained as
described49. Dead cells were excluded by staining with the DNA-binding dye
DAPI, and plasma cells were sorted as IgA+CD138+ single cells. Cells were
isolated from PPs, spleen and BM, strained through nylon mesh and stained
© 2015 Nature America, Inc. All rights reserved.
with the antibodies listed in Supplementary Table 1. Memory B cells from PPs
were sorted as DAPI-negative CD4−CD8−GL7−CD138−CD19+CD80+CD73+
single cells, and non-memory B cells were sorted as DAPI-negative CD4−C
D8−GL7−CD138−CD19+CD80−CD73− single cells. To sort S. Typhimurium
LPS–positive IgA plasma cells, we labeled S. Typhimurium (Sigma-Aldrich)
LPS according to the manufacturer’s instructions with Hydrazide-Biotin rea-
gent (Pierce Biotechnology) and dialyzed it against PBS (Slide-Lyzer, Mini
Dialysis Unit, 10 K MWCO, Pierce). Cells were stained with 30 µg/ml bioti-
nylated LPS, IgA–fluorescein isothiocyanate and CD138-phycoerythrin at
4 °C for 45 min, after which they were stained with streptavidin–Alexa Fluor
647 (SA) and DAPI. LPS-binding and non-binding plasma cells were sorted
as DAPI-negative, SA-positive IgA+CD138+ and DAPI-negative, SA-negative
IgA+CD138+, respectively. RNA was extracted for sequencing with an
miRNeasy Micro Kit (Qiagen).
npg
nature immunology
Immunoglobulin sequence analysis (component analysis). For component
analysis, clusters of clonally related CDR3 sequences were sorted by frequency,
and clusters with the same frequency were grouped into ranks ranging from
single clusters with rank 1 to highly abundant clusters given a rank number
according to their frequency. Log transformation of rank versus frequency
separated the repertoire into two components divided at a critical point, which
was calculated as described50.
Statistical analysis. Statistical analysis was done with Prism software
(GraphPad Software) and R studio (version 0.94.110). For comparison of two
groups, P values were determined by unpaired two-tailed Student’s t-test, and
paired data were analyzed by paired Student’s t-test. For comparison of more
than two groups, significant values were calculated via one-way ANOVA with
Bonferroni’s post-hoc test for fewer than five groups and with Tukey’s post-hoc
test for five or more groups.
41. Boysen, P., Eide, D.M. & Storset, A.K. Natural killer cells in free-living Mus musculus
have a primed phenotype. Mol. Ecol. 20, 5103–5110 (2011).
42. Gaboriau-Routhiau, V. et al. The key role of segmented filamentous bacteria in the
coordinated maturation of gut helper T cell responses. Immunity 31, 677–689
(2009).
43. Edgar, R.C. Search and clustering orders of magnitude faster than BLAST.
Bioinformatics 26, 2460–2461 (2010).
44. Shannon, P. et al. Cytoscape: a software environment for integrated models of
biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).
45. Cline, M.S. et al. Integration of biological networks and gene expression data using
Cytoscape. Nat. Protoc. 2, 2366–2382 (2007).
46. Magurran, A.E. Ecological Diversity and Its Measurement. (Princeton University
Press, 1988).
47. Caporaso, J.G. et al. QIIME allows analysis of high-throughput community sequencing
data. Nat. Methods 7, 335–336 (2010).
48. Wang, Q., Garrity, G.M., Tiedje, J.M. & Cole, J.R. Naive Bayesian classifier for rapid
assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ.
Microbiol. 73, 5261–5267 (2007).
49. Schulz, O. et al. Intestinal CD103+, but not CX3CR1+, antigen sampling cells
migrate in lymph and serve classical dendritic cell functions. J. Exp. Med. 206,
3101–3114 (2009).
50. Naumov, Y.N., Naumova, E.N., Hogan, K.T., Selin, L.K. & Gorski, J. A fractal
clonotype distribution in the CD8+ memory T cell repertoire could optimize potential
for immune responses. J. Immunol. 170, 3994–4001 (2003).
doi:10.1038/ni.3213