Oral Combined Therapy with Probiotics and

Alloantigen Induces B Cell −Dependent

Long-Lasting Specific Tolerance

This information is current as
of September 15, 2014.
Downloaded from http://www.jimmunol.org/ at Univ of California-San Diego Serials/Biomed Lib 0699 on September 15, 2014
Ana C. T. Mercadante, Suelen M. Perobelli, Ana P. G.
Alves, Triciana Gonçalves-Silva, Wallace Mello, Ana C.
Gomes-Santos, Anderson Miyoshi, Ana M. C. Faria and
Adriana Bonomo
J Immunol 2014; 192:1928-1937; Prepublished online 22
January 2014;
doi: 10.4049/jimmunol.1301034
http://www.jimmunol.org/content/192/4/1928

Supplementary http://www.jimmunol.org/content/suppl/2014/01/22/content.1301034.
Material DCSupplemental.html
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 The Journal of Immunology

Oral Combined Therapy with Probiotics and Alloantigen

Induces B Cell–Dependent Long-Lasting Specific Tolerance

Ana C. T. Mercadante,* Suelen M. Perobelli,† Ana P. G. Alves,* Triciana Gonçalves-Silva,†

Wallace Mello,‡ Ana C. Gomes-Santos,x Anderson Miyoshi,{ Ana M. C. Faria,x and

Downloaded from http://www.jimmunol.org/ at Univ of California-San Diego Serials/Biomed Lib 0699 on September 15, 2014
Adriana Bonomo*,†,‡
Allogeneic hematopietic stem cell transplantation (aHSCT) is widely used for the treatment of hematologic malignancies. Although
aHSCT provides a good response against the malignant cells (graft-versus-leukemia [GVL]), it also leads to the development of
graft-versus-host disease (GVHD), a severe disease with high mortality and morbidity rates. Therapy for GVHD is commonly based
on nonspecific immunosupression of the transplanted recipient, resulting in the concomitant inhibition of the GVL effect. In this
study, we propose an alternative approach to specifically suppress GVHD while sparing the GVL, based on oral treatment of trans-
plant donors with recipient Ags, associated with the intake of probiotic Lactococcus lactis as tolerogenic adjuvant (combined
therapy). We show that treatment of C57BL/6 donor mice with combined therapy before the transplant protects the recipients
F1 (C57BL/6 3 BAL/c) mice from clinical and pathological manifestations of disease, resulting in 100% survival rate. Importantly,
the animals keep the immunological competence maintaining the GVL response as well as the response to third-party Ags. The
protection is specific, long lasting and dependent on donor IL-10–sufficient B cells activity, which induces regulatory T cells in the
host. These data suggest that combined therapy is a promising strategy for prevention of GVHD with preservation of GVL,
opening new possibilities to treat human patients subjected to transplantation. The Journal of Immunology, 2014, 192: 1928–1937.

T
he main limitation of allogeneic hematopoietic stem cell
transplantation (aHSCT) for the treatment of malignant
and nonmalignant disease is the development of graft
versus host disease (GVHD), a complex multiorgan inflammatory
syndrome that results from recognition of genetically disparate
tissues of the recipient patient by the incoming allogenic donor
T cells (1). GVHD treatment is based on use of immunosupressive
agents that block T cell function in a nonspecific way, leading to
undesired consequences—because these alloreactive T cells also
facilitate engraftment of hematopoietic stem cells, accelerate immune
reconstitution (1–4), and contribute to the graft-versus-leukemia
*Department of Experimental Medicine, Brazilian National Cancer Institute, Rio
de Janeiro 20231-050, Brazil; †Department of Immunology, Microbiology Institute
Prof. Paulo de Góes, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902,
Brazil; ‡Laboratory of Thymus Research, Oswaldo Cruz Institute, Rio de Janeiro
21040-360, Brazil; xDepartment of Biochemistry and Immunology, Biological Sci-
ence Institute, Federal University of Minas Gerais, Belo Horizonte 30161-970,
Brazil; and {Department of Biology, Biological Science Institute, Federal Univer-
sity of Minas Gerais, Belo Horizonte 30161-970, Brazil
Received for publication April 18, 2013. Accepted for publication December 17,
2013.
This work was supported by grants from Conselho Nacional de Pesquisas (57.3806/
2008 and 306624/2010-9), Instituto Nacional de Ciência e Tecnologia (306624/2010-
9), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (E-26/111.423/
2010, E-26/110.949/2008, and E-26/110.323/2010), the Swiss Bridge Foundation
(2301500), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais
(APQ00575-09).
Address correspondence and reprint requests to Prof. Adriana Bonomo, Laboratory of
Thymus Research, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Avenida Brasil
4365, Manguinhos, Rio de Janeiro 21045-900, Brazil. E-mail address: abonomo@
micro.ufrj.br
The online version of this article contains supplemental material.
Abbreviations used in this article: aGVHD, acute graft versus host disease; B6, C57BL6;
BMT, bone marrow transplantation; Breg, regulatory B cell; GVL, graft versus leuke-
mia; HSCT, hematopoietic stem-cell transplantation; LAP, latency-associated polypep-
tide; mLn, mesenteric lymph node; pLN, peripheral lymph node; Treg, regulatory T cell.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
(GVL) effect (4). Because of the close association between GVH
and GVL reactions (4), much effort has been put forth to develop
therapies that reduce the first while maintaining the latter (1).
Oral tolerance is classically defined as the suppression of im-
mune responses to Ags that have been previously administrated by
the oral route (5). Far from being just a matter of hyporesponsive-
ness, oral tolerance is an active immunologic event influenced by
numerous factors such as Ag dose, microbiota, and costimulatory
molecules, resulting from multiple mechanisms of action (6). In-
duction of oral tolerance has been shown to be effective in various
experimental models of immune disorders (7–9) including GVHD
(10–12). In fact, Nagler et al. (12) demonstrated that it is possible
to alleviate acute GVHD (aGVHD) severity by the induction of
oral tolerance in the recipient, although the maintenance of the
GVL effect was not addressed in that study.
Administration of probiotics by the oral route also have been
shown to modulate the immune response, favoring either pro- (13,
14) or anti-inflammatory (15, 16) responses. Recent studies sug-
gest that TLRs and NF-kB signaling pathways play a crucial role
in the outcome of the response (17–21). Also, probiotics can in-
fluence the integrity of the gut epithelial cell barrier through
modulation of mucus, defensins, and IgA production, reduction of
bacterial adhesion, and enhancement of tight junction and cell
survival (22–24).
The relationship between the endogenous microbiota and de-
velopment of aGVHD was proposed ∼40 y ago in experimental
models (25) and confirmed in humans later on (26). As hypothe-
sized by Hill et al. (27), damage of the host intestinal epithelium
by the conditioning regiment results in systemic exposure to mi-
crobial Ags that ultimately lead to an inflammatory milieu that is
adequate for donor T cell activation. With those results in mind,
intestinal decontamination became a common practice in aHSCT
(26, 28). More recently, Jenq et al. (29) showed that composition
of the gut flora of recipient mice that developed GVHD had
a dramatic loss of bacterial diversity and a distinct composition

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301034
The Journal of Immunology
compared with recipient mice that did not develop GVHD. In ad-
dition, the authors revealed that antibiotic-treated mice showed an
emergence of species that was associated with exacerbated GVHD.
Interestingly, Gerbitz et al. (30) demonstrated that oral adminis-
tration of Lactobacillus rhamnosus GG before and after transplan-
tation not only improved survival and ameliorated aGVHD clinical
scores but also reduced the translocation of enteric bacteria. Both
works suggest that manipulation of the gut microbiota might be as
good as decontamination with antibiotics to prevent GVHD.
Although promising, the clinical application of both protocols,
oral tolerance induction (12) and probiotic treatment (30), cannot
be used in transplanted patients. Once the conditioning regimen
alters the intestinal epithelial barrier, these patients require special
attention regarding their diet. As so, no live or raw food is
permitted during the peritransplant phase, precluding oral ad-
ministration of Ags or probiotics.
In this report, we reasoned if tolerance induction in the donor,
rather than in the recipient (12, 30), could work as a strategy to
dissociate GVH and GVL effects. We propose a new protocol that
combines the induction of oral tolerance in bone marrow trans-
plant donors to Ags from the recipient, with the use of the po-
tential probiotic Lactococcus lactis NCDO2118 as tolerogenic
adjuvant (combined therapy). Using a haploidentical model of
aGVHD, we found that treatment of donor mice with the com-
bined therapy protects recipients from aGVHD while maintaining
the GVL effect. This protection is long lasting, Ag specific, and
dependent on donor B cells, which induce regulatory T cells
(Tregs) in the host.
Materials and Methods
Animals
Male 8- to 10-wk-old C57BL/6 (B6) (H-2b), B6 IL-10 knockout, or F1
(BALB/c 3 B6dXb) mice were used as cell donors for GVHD induction
experiments and female 10- to 12-wk-old F1 (BALB/c 3 B6 2 H-2dXb)
mice were used as recipients. For skin transplants, male 12- to 14-wk-old
BALB/c (H-2d), B10.A (H-2a), or B6 mice were used as donors, and fe-
male B6 mice or F1 (BALB/c 3 B6) mice were used as recipient. All
animals were bred at the Brazilian National Cancer Institute animal facility
(Rio de Janeiro, Brazil), housed in sterilized microisolators, and were
handled according to the institutional guidelines. Animal care and all
animal procedures were previously approved by Ethical Committee for
Animal Experimentation of the Brazilian National Cancer Institute.
Bacterial growth conditions and administration to donor mice
The wild-type L. lactis ssp lactis NCDO2118 strain (obtained from Lab-
oratório de Genética Celular e Molecular from Universidade Federal de
Minas Gerais, MG/Brazil - taxonomy ID 1117941 at the National Center
for Biotechnology Information database). The bacteria were grown in M17
broth (Difco) supplemented with 0.5% glucose at 30˚C without agitation
for 24 h. In the second day, when a 2.0 OD at 600 nm (OD, nm) was
reached, an inoculum with 5 ml was diluted in 25 ml supplemented M17
broth and offered to the animals.
Preparation of protein extract from recipient mice
To prepare the protein extract, F1 (BALB/c 3 B6) spleen cells were
resuspended on ice-cold PBS and filtered through a 40-mm cell strainer,
cells were disrupted by freezing and thawing five times and boiled for
10 min. Soluble fraction was obtained by centrifugation at 14,000 rpm,
30 min, at 4˚C and used as oral treatment. Total protein was quantified
with Bio-Rad protein kit assay, according to the manufacturer’s spec-
ifications.
Treatment of donor mice with combined therapy
Donor B6 mice received 50 mg F1 (BALB/c 3 B6) spleen protein ex-
tract diluted in 100 ml PBS, by gavage, daily for 5 d. During this period,
ad libitum doses of L. lactis NCDO2118 were offered in replacement
of drinking water. Four days later, the animals were immunized with
106 F1 splenocytes i.p. (31), and donor splenic cells were collected 1 wk
after.
1929
Bone marrow transplantation
Bone marrow transplantation (BMT) was modified from described previ-
ously (32). F1 mice were lethally irradiated with 950 cGy total body ir-
radiation (TH780C irradiator with a cobalt 60 [60Co] source), followed by
the i.v. transfer of 5 3 106 bone marrow cells from healthy donors along
with 5 3 106 T cells from total spleen of treated or untreated donors. When
indicated, spleen cells were depleted of CD25+ cells or CD19+ cells. In
GVL experiments, 105 P815 GFP-expressing mastocytoma cells were
injected together with the bone marrow inoculum.
GVHD clinical and histological score
Mice were monitored daily for survival and weekly for GVHD clinical
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score. Clinical evaluation was modified from the literature (33, 34) and was
based on six parameters: weight loss, fur texture, activity, posture, fecal
consistency, and survival rate. For histopathologic examination, samples of
skin, liver, and colon collected at day 21 posttransplant were formalin pre-
served, paraffin embedded, sectioned, and stained with H&E. The pathologic
score system was based on evaluation of individual parameters for each organ
being the final score obtained by the sum of them. For skin sections, in-
flammatory infiltration, fibrosis, loss of appendices, epidermal chances, and
ulceration were evaluated. For liver samples, parenchyma suffering, inflam-
matory infiltration, and portal space destruction were evaluated. For colons,
lamina propria infiltration, deeper layer infiltration, structural changes, and
damage extension were analyzed.
GVL evaluation
At day 25 posttransplant, samples of spleen, liver, bone marrow, total lymph
nodes, and blood were analyzed by flow cytometry for the presence of
P815-GFP+ cell. For determination of tumor total elimination, recipients
were monitored daily for tumor-related morbidity and mortality. Tumor-
related death was determined if mice had paralysis (in that case they were
euthanized immediately and examined) and if autopsy identified hepat-
osplenomegaly and presence of macroscopic tumor nodules on the liver,
spleen, or bone marrow.
Cell depletions
For Treg depletion, spleen cells from donor mice were incubated with anti-
CD25 (PC61.5) rat anti-mouse IgG Ab and positively selected using goat
anti-rat IgG Dynabeads (Invitrogen, Carlsbad, CA). B cell depletion was
done by positive selection using goat anti-mouse IgG Dynabeads (Invi-
trogen). After depletion, cell suspensions presented ,1% of CD25+ and 3%
of CD19+ cells. Both procedures were done accordingly to the manu-
facturer’s recommendations. For in vivo Treg depletion, recipient chimeras
were injected i.p. with PC61 mAb (250 mg/mouse) at day 3 posttransplant.
On day 14 posttransplant another i.p. dose of PC61 mAb were injected
(100 mg/mice).
Purification and adoptive transfer of B cells
CD19+ B cells were isolated from donor spleen by positive selection using
anti-CD19 MACS microbeads (Miltenyi Biotec, Auburn, CA), according
to the manufacturer’s recommendations. For adoptive transfer experi-
ments, 1 3 107 purified CD19+ B cells (purity . 95%) from treated or
untreated donor were injected along with the graft. When indicated, the
spleen used as T cell source was previously depleted of total B cells and
reconstituted with purified CD19+ B cells.
Flow cytometry staining and analyses
For surface staining, cell suspensions were preincubated with 2% normal
mouse serum in PBS (Sigma-Aldrich, St. Louis, MO) on ice, followed by
incubation with appropriate Ab mixture for 15 min. For Foxp3 staining,
cells were labeled as specified in the eBioscience kit standard protocol. All
samples were acquired in a FACSCalibur (BD Biosciences, San Jose, CA)
or in a FACSCanto (BD Biosciences). Acquisitions were performed using
CellQuest (BD Biosciences) software and BD FACSDiva software, re-
spectively. Analyses were performed using FlowJo software (Tree Star,
Ashland, OR). For cell staining the following Abs were used: FITC or
allophycocyanin anti-mouse CD4 (GK1.5); PECy7 CD8a (53-6.7); PE
anti-mouse CD25 (PC61.5, 7D4); allophycocyanin anti-rat/mouse Foxp3
staining kit (FJK-16); FITC or PECy5 anti-mouse CD3e (145-2C11);
biotinylated anti-mouse CD11c (HL3); FITC anti-mouse CD11b (M1/70);
PE, PercP-Cy5.5, allophycocyanin, or FITC anti-mouse CD19 (1D3); PE
anti-mouse B220 (RA3-6B2); FITC anti-mouse IgM (eB121-15F9); allo-
phycocyanin anti-mouse IgD (11-26c [11-26]); FITC anti-mouse CD5 (53-
7.3); PE anti-mouse CD1d (1B1); allophycocyanin-eFluor 780 anti-mouse
CD21 (eBio8D9); PE anti-mouse CD43 (eBioR2/60); allophycocyanin
1930 ORAL THERAPY INHIBITS aGVHD BUT NOT GVL REACTION

anti-mouse CD40 (1C10); FITC anti-mouse CD86 (GL1); biotinylated
anti-mouse B7-H2 (HK5.3); allophycocyanin anti-mouse I-A/I-E (M5/
114.15.2); biotinylated or allophycocyanin anti-mouse H-2Kb (AF6-
88.5.5.3); PE anti-mouse H-2Kd (SF1-1.1.1) and FITC, PE, PerCP, or
allophycocyanin streptavidins were all purchased from eBioscience (San
Diego, CA). PE anti-mouse latency-associated polypeptide (LAP) (TW7-
16B4) was purchased from BioLegend (San Diego, CA).
Statistical analysis
For grouped analysis, one-way or two-way ANOVA with Bonferroni
posttest were used. Unpaired Student t test was applied for statistical
differences between two groups. Survival data were analyzed with log-rank
(Mantel–Cox) test. Error bars represent SD. Values of p , 0.05, , 0.01,
therapy is used, an elevated survival rate was observed. Also, eval-
uation of clinical parameters reveals an important decrease in dis-
ease severity when compared with all others groups tested (Fig. 1C,
Supplemental Fig. 1). Donor treatment based on gavage of recipient
protein Ags (AgF1) or L. lactis administration ad libitum (Llactis),
used separately, shows only a discrete protection (Fig. 1, Supple-
mental Fig. 1).
To confirm the reduction in aGVHD, we examined histopath-
ologically the skin, colon, and liver of recipient mice 21 d after
BMT. The pathologic scores were significantly lower in recipi-
ents transplanted with spleen cells from treated donors (AgF1/Ll

and , 0.001 are referred to in the figure legends with symbols *, **, and
***, respectively.
Results
Treatment of donor mice with combined therapy efficiently
inhibits acute GVHD
Although it has been reported that induction of oral tolerance in the
recipient can ameliorate aGVHD (12), there are no reports on
whether it is possible to induce tolerance to host Ags in the donor
before the transplant is performed. To determine whether donor
T cells can be rendered tolerant to host tissues through the oral route,
donor B6 mice received daily doses of host protein Ags by gavage,
whereas L. lactis was offered ad libitum during 5 d (combined
therapy). These animals were immunized i.p. with recipient spleen
cells after 4 d and 1 wk later used as spleen cell donors along with
normal B6 bone marrow to reconstitute lethally irradiated F1
(BALB/c 3 B6) mice (Fig. 1A). Fig. 1B shows that when combined
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group) when compared with those who received spleen cells from
untreated donors (Ctr+) (Fig. 2), AgF1, or L1actis groups (Sup-
plemental Fig. 2). This reduction might be related to a higher IL-10
production found on affected tissues at that same period (Supple-
mental Fig. 3).
Combined therapy maintains the GVL effect and
immunocompetence in the recipient
The close association between GVHD and the GVL effect reflects
the need for therapies that target the undesired reaction only (1, 4).
To determine whether the combined therapy is able to decrease
GVHD while maintaining the benefic GVL effect, GFP-expressing
P815 mastocytoma cells were injected along with the graft.
Twenty-five days post-BMT, bone marrow, blood, lymph nodes,
spleens, and livers of the recipients were examined for the pres-
ence of GFP+ cells. As expected by the absence of GVHD, GFP+
cells were found in all tissues analyzed of mice in the negative

FIGURE 1. Donor treatment with combined therapy reduces the clinical signs of acute GVHD. Donor mice (AgF1/Ll) received orally by gavage 50 mg
F1 spleen cell protein extract and ad libitum doses of L. lactis daily for 5 d. Four days later, the animals were immunized with 106 F1 splenic cells i.p. After
6–7 d, BMT was done. Lethally irradiated F1 mice (BALB/c 3 B6) were reconstituted with 5 3 106 bone marrow cells from B6 mice along with 5 3 106
splenic T cells from control B6(Ctr+) or B6 mice orally treated with F1 cell extract (AgF1), with L. lactis (Llactis), or both (AgF1/Ll). (A) Schematic
view of combined therapy treatment. (B) Survival rates. (C) Total clinical score. Data are representative of two experiments with five to eight mice per
group. +p , 0.05, +++p , 0.001 for AgF1/Ll versus Latis group. ***p , 0.001 for AgF1/Ll versus Ctr+ group. ###p , 0.001 for AgF1/Ll versus AgF1
group.
The Journal of Immunology 1931

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FIGURE 2. Donor treatment with combined therapy reduces the histopathological manifestations of acute GVHD. Donor mice treatment with combined
therapy and HSCT were performed as described in Fig. 1. Representative micrographs of skin (A), colon (C), and liver (E) from negative control disease
group (left panels), positive control disease group (middle panels), and experimental AgF1/Ll group (right panels) are shown. H&E staining. Total his-
topathological score of skin (B), colon (D), and liver (F). Data were combined from different experiments with 14–18 mice. *p , 0.05, ***p , 0.001.

control disease group. In contrast, in the positive control disease
group, where GVHD developed, no GFP+ cells were observed
(Fig. 3A). However, despite being protected from aGVHD, mice
in the experimental AgF1/Ll group were capable of eliminating
the tumors cells from all tissues. The protected chimeras remained
free of tumor for .15 wk post-BMT (Fig. 3B), whereas chimeras
from negative and positive control disease groups died with clear
signals of tumor growth and GVHD development, respectively.
Because the therapy proposed in this study combines two treat-
ments with immunomodulatory effects, the protection observed
could be a result of a general state of immunosuppression that
would interfere with recipient response to other Ags. To test
whether the immunosuppression induced by combined therapy
was specific for host Ags, recipients protected for .1 y post-BMT
received skin grafts from third-party and from BALB/c donors.
The protected chimeras specifically rejected the unrelated skin
graft but accepted the BALB/c graft (Supplemental Fig. 4A).
However, after 1 y, we cannot exclude the participation of newly
generated T cells because the thymic function of the chimeras, at
that posttransplant period, already had been restored and the newly
emergent T cells are tolerant to the BALB/c Ags. To test the
specificity of the tolerance induced for host Ags, B6 mice treated or
not with the combined therapy received skin grafts from third-
party and from F1 donors. As shown in Supplemental Fig. 4B,
both treated or untreated B6 recipients rejected the skin grafts.
However, when female B6 mice were treated with the combined
therapy using male B6 splenic protein extract on gavage, they did
not reject male skin graft unlike the untreated females (Supple-
mental Fig. 4C). It suggests that combined therapy induces a more
effective tolerance to minor rather than major histocompatibility

FIGURE 3. Combined therapy preserves the graft versus tumor effect. Donor mice treatment with combined therapy and HSCT were performed as
described previously. Along with hematopoietic stem cells, 105 mastocitoma tumor cells (P815) expressing GFP were injected. (A) Twenty-five days
posttransplant, spleen, liver, bone marrow (BM), total lymph nodes (tLn), and blood from the chimeras were analyzed by flow cytometry for the presence of
GFP+ cells. (B) Survival rate of chimeras that also received tumor cells. Data are representative of two experiments, with five mice individually analyzed per
group. ***p , 0.001 for AgF1/Ll (+P815) versus Ctr2 (+P815) group. ###p , 0.001 for AgF1/Ll (+P815) versus Ctr+ (+P815) group.
1932 ORAL THERAPY INHIBITS aGVHD BUT NOT GVL REACTION

Ags, which may explain how it is able to protect from GVHD pre-
serving the GVL effect.
Altogether, the data show that treatment of donor mice with com-
bined therapy efficiently reduces clinical and pathological aGVHD
development on a specific and long-lasting fashion, without immuno-
suppressing the recipient and preserving the GVL effect.
Protection against aGVHD by the combined therapy is not
mediated by donor Foxp3+ Tregs
The phenomenon of oral tolerance can occur by different mech-
anisms being the expansion of regulatory-suppressive T cells gen-
combined therapy protocol. Spleen cells from combined therapy–
treated donors were depleted of B lymphocytes before transplan-
tation. As indicated in Fig. 5A, elimination of B cells completely
abrogated the protection from aGVHD, leading to a higher total
clinical score and an elevated mortality rate in this group (AgF1/
Ll [-LØB]) when compared with non-B cell–depleted one (AgF1/
Ll). Because the role of B cells in GVHD pathophysiology is still
controversial (37), one may argue that loss of protection could be
related not only to the absence of B cells tolerized by treatment
but also being exacerbated by the absence of B lymphocytes at all.
To overcome this issue, spleen cells from combined therapy–

erally related to repeated lower doses of Ag (5, 6). To investigate the
mechanism by which the combined therapy exerts the protection
against aGVHD, spleen cells from treated and untreated donors
were analyzed. No significant differences were observed in absolute
number or frequency of splenic cell subpopulations, including
Foxp3+ T cells (Fig. 4A, 4B). To further exclude the participation of
donor Tregs, CD25+ T cells were depleted from spleen cells of
treated donors before transplantation. Depletion of donor Tregs did
not abrogate the protection, and the elevated survival rate was
maintained (Fig. 4C, 4D), confirming that these cells are not nec-
essary for the protective effect.
Donor B cells tolerized by the combined therapy are
responsible for GVHD protection
Several recent studies in both mice and humans have showed the
participation of B cell on immunoregulation and protection against
acquired inflammatory syndromes (35). In models of oral tolerance
induction, B cells seem to play an important role in tolerance
induced with low doses of Ag (36). Because the combined therapy
is based on induction of oral tolerance with small amounts of
recipient Ag, we evaluated the participation of donor B cells in the
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treated donors depleted of B cells were reconstituted with purified
splenic CD19+ B cells from untreated donors. The reconstitution
had no effect, and the recipients still developed aGVHD. However,
when CD19+ B cells from a treated donor were used to recon-
stitute the combined therapy B-depleted spleen, protection was
completely restored (Fig. 5A).
We next tested whether tolerized B cells were sufficient to inhibit
aGVHD by transferring CD19+ B cells from a treated donor along
with spleen cells (and bone marrow) from a control untreated B6
donor (Fig. 5B). Of interest, depletion of B lymphocytes from
untreated donor spleen cells ameliorated the GVHD, suggesting
that B cells from untreated mice, at least in this model, have a
pathogenic role in GVHD development. Taken together, the data
show that protection of recipient mice against aGVHD is mediated
by B lymphocytes with regulatory functions generated by the
combined therapy treatment of donor.
Oral administration of recipient protein Ag with L. lactis
induces an increase in splenic B cells compatible with Bregs
To better characterize the B cells involved in the regulatory activity
over aGVHD development, donor mice were treated or not with the

FIGURE 4. Depletion of donor CD25+ T cells before transplantation does not affect the protective effect. Donor mice were treated with combined
therapy as described previously. (A and B) Evaluation of leukocyte populations in treated donor spleens. Splenic cells were analyzed 6 d after the end of the
oral therapy. Nontreated mice were used as controls. (A) Frequency of spleen cell subpopulations. (B) Absolute number of spleen cell subpopulations.
(C and D) Analysis of GVHD development in absence of donor Tregs. Before transplantation, CD25+ T cells were depleted from combined therapy–treated
donor splenocytes. (C) Total clinical score. (D) Survival rates. Data are representative of five pools of three mice per group in (A) and (B), and a total of
10 mice was individually analyzed per group in (C) and (D). ***p , 0.001 for AgF1/Ll (2CD25) versus Ctr+. #p , 0.05, ##p , 0.01, ###p , 0.001 for
AgF1/Ll versus Ctr+.
The Journal of Immunology 1933

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FIGURE 5. Protection of recipient mice against GVHD is dependent on donor B cells generated by combined therapy. Donor mice were treated with the
combined therapy as described previously. Before transplantation, B cells were depleted from combined therapy treated or untreated donor spleen. (A) Total
clinical score and survival rates of mice reconstituted with untouched splenocytes from treated donors (AgF1/Ll group), splenocytes depleted of B cells
(AgF1/Ll[2LøB] group), splenocytes depleted of B cells, and reconstituted with B cells from treated (AgF1/Ll[2LøB]+[LøBAg/Ll] group) or untreated
donors (AgF1/Ll[2LøB]+[LøB B6] group). (B) Total clinical score and survival rates of mice reconstituted with untouched splenocytes from untreated
donors added (Ctr+[2LøB]+[LøBAg/Ll]) or not with B cells from treated donors (Ctr+) and spleen depleted of B cells (Ctr+[2LøB]). Data are repre-
sentative of two experiments with 6–10 mice/group. *p , 0.05, ***p , 0.001 for (AgF1/Ll) versus (AgF1/Ll[2LøB]). #p , 0.05, ##p , 0.01, ###p , 0.001
for (AgF1/Ll) versus (AgF1/Ll[2LøB] + [LøB B6]) in (A). *p , 0.05, **p , 0.01, ***p , 0.001 for (Ctr+) versus (Ctr+[2LøB] + [LøBAg/Ll]). #p , 0.05,
###
p , 0.001 (Ctr+) versus (Ctr+[2LøB]) in (B).

combined therapy and their splenic B cells analyzed by flow
cytometry.
When the B lymphocytes were grouped according to the ex-
pression of IgM and IgD, we found a higher frequency of transitional
B cells (CD19+IgM+IgDlo) in treated animals. No differences were
observed among the immature (CD19+IgMhiIgD2) and mature
(CD19+IgMloIgD+) B cells subpopulations (Fig. 6A), suggesting that
combined therapy induced an increase in transitional Bregs. How-
ever, several different phenotypes have been attributed to B cells
with regulatory functions and at least two subpopulations were de-
scribed: transitional Breg, mentioned above, and the B10 cells (37).
In common, both express CD21 and CD1d molecules, being that the
B10 is also positive for CD5 (38). Despite the increase in overall
CD21 expression in splenic B cells from treated donors (Fig. 6B),
we observed that CD19+CD21+CD1d+ and CD19+CD21+CD1d+
CD5+ subpopulations were reduced on these animals (Fig. 6E, 6F).
Although there is not a unique marker, or set of markers, that
exclusively identifies regulatory B cells (Bregs), expression of
molecules such as CD86 (39) and class II MHC (40) are necessary
for most of their regulatory functions, regardless of the specific
B cell immunophenotype. In fact, splenic B lymphocytes from
combined therapy–treated donor express high amounts of both
molecules (Fig. 6C, 6D).
Recently, B cells with strong suppressive functions in vitro and
in vivo were generated by the incubation of naive B cells with
a relevant Ag conjugated to cholera toxin B subunit. In this sce-
nario, tolerance was shown to be at least, partially dependent on
B cell expressing the LAP/TGF-b (41). On the basis of these data,
we looked at LAP expression in CD19+ cells and found a higher
expression of this molecule in B cells from treated when com-
pared with untreated mice (Fig. 6G, 6H).
Convincing data have attributed the suppressive capacity of
Bregs to their IL-10 production (41–43). To check whether the
protection observed in this study is due to B cell–derived IL-10,
CD19+ B cells from B6 IL-10 knockout mice treated with the
combined therapy were transferred to GVHD developing mice
(irradiated F1 receiving spleen and bone marrow cells from control
B6 mice). The results showed that B cells from IL-10–deficient
mice were not able to protect the recipient from GVHD devel-
opment, unlike B cells from IL-10–sufficient donors pretreated
with the combined therapy (Fig. 7A, 7B).
Altogether, the above results indicate that the protection ob-
served after oral combined therapy administration depends on IL-
10–producing Bregs.
Splenic B cells from combined therapy–treated donor mice
induce Foxp3+ Tregs on recipient mice
A growing body of evidence has demonstrated the involvement of
Bregs in the generation or recruitment of regulatory T lymphocytes
(41, 44). To test whether B cells from combined therapy–treated
donors were inducing Tregs, chimeras transplanted with control
spleen cells or spleens depleted of B lymphocytes from both,
treated or untreated donors, were evaluated for the presence of
donor regulatory Foxp3+ cells in spleens and mesenteric and pe-
ripheral lymph nodes 4 d post-BMT. As shown in Fig. 8A and 8B,
recipients reconstituted with spleen cells from treated donors had
higher frequencies and absolute numbers of Foxp3+ cells in the
spleen and lymph nodes. In addition, depletion of B lymphocytes
from the graft completely abrogated this increase. Of note, the
increase of donor Foxp3+ cells happens in the recipient mice,
indicating that the regulatory phenotype is induced during the
alloresponse in the host and not before because spleen cells from
1934 ORAL THERAPY INHIBITS aGVHD BUT NOT GVL REACTION

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FIGURE 6. B cells generated by combined therapy in the donors are phenotypically compatible with Bregs. Donor mice were treated with combined
therapy as previously described, and splenocytes were analyzed at the end of treatment. (A) Frequency of immature (CD19+IgMhiIgD2), transitional
(CD19+IgM+IgDlo), and mature (CD19+IgMloIgD+) B cells. (B) CD21 expression on CD19+ B cells (mean fluorescence intensity [MFI]). (C) CD86
expression on CD19+ B cells (MFI). (D) Class II MHC expression on CD19+ B cells (MFI). (E) Frequency of splenic CD19+CD21+CD1d+ subpopulation.
(F) Frequency of splenic CD19+CD21+CD1d+CD5+ subpopulation. (G) Frequency of LAP+ B cells. (H) Quantification of (G). Data are representative of
10 mice individually analyzed per group. *p , 0.05, **p , 0.01, ***p , 0.001.

donor mice do not have higher numbers of Tregs nor do the spleen
cells depend on the Tregs to exert their inhibitory effect (Fig. 4).
Reinforcing these data, in vivo depletion of CD25+cells from re-
cipient reconstituted with combined therapy–treated donors totally
abrogated the protection (Fig. 9A, 9B).
Taken together, the results show that tolerized B cells from the
donors mediate the protection against GVHD by inducing Foxp3+
cells in the transplant recipients.
Discussion
HSCT is an immunotherapy for treatment of malignant and non-
malignant diseases that is seriously limited by GVHD, an immune
syndrome with high morbidity and mortality rates (1–3). Pro-
phylaxis and treatment of GVHD is based on nonspecific immu-
nosupressive agents that, among other complications, eliminate
GVLr, which elevates the risk of tumor relapse (4). Education of
the immune system by induction of oral tolerance (12) and ma-
nipulation of gut microbiota by ingestion of probiotic bacteria (30)
represent rationally viable and noninvasive therapies to reduce the
development of GVHD. Nonetheless, their clinical application has
been limited because the HSCT recipients are subjected to con-
ditioning regimens that require a strict diet in which live or raw
food is precluded.
In this study, we demonstrate that treatment of donor mice with
combined therapy reduces clinical and pathological manifestations
of aGVHD, improving the survival rates to 100%. This phenom-
enon is specific and long lasting, and it is not a result of a non-
specific immunosuppressive milieu. Maintenance of beneficial
GVL effect is as important as the reduction of GVHD when HSCT
is used to treat a malignant disease (45–47). In humans, although it
has been suggested that GVL and GVHD are temporally separate
(48) and that the severity of GVHD does not implicate in im-

FIGURE 7. Protection of recipient mice against GVHD is dependent on IL-10 produced by tolerized donor B cells. Lethally irradiated F1 mice (BALB/c 3
B6) were reconstituted with 5 3 106 bone marrow cells from B6 mice along with 5 3 106 splenic T cells from control B6 (Ctr+). Splenic CD19+ cells (LøB)
from untreated mice (Ctr+LøB B6), from combined therapy treated wild-type (Ctr+LøB AgF1/Ll) or IL-10–deficient (Ctr+LøB IL10KO) donors were trans-
ferred along with the bone marrow and spleen cells to assess the role of IL-10 in GVHD protection. (A) Total clinical score. (B) Survival rates. A total of
10 mice was individually analyzed per group. *p , 0.05, **p , 0.01, ***p , 0.001. *(Ctr+) versus (Ctr+LøB AgF1/Ll).
The Journal of Immunology 1935

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FIGURE 8. B cells generated by the combined therapy increase the frequency of donor Foxp3+ cells in the recipient mice. Donor mice were treated with
the combined therapy as described previously. Before transplantation, B cells were depleted from combined therapy treated or untreated donor spleen. Four
days posttransplant, the recipients were euthanized, and the presence of H-2Kb+H-2Kd2Foxp3+ was analyzed in the spleen and mesenteric and peripheral
lymph nodes. Representative data of Foxp3+ cells on peripheral and mesenterical lynph nodes (A) and spleen (C) gated on H-2Kb+H-2Kd2 cells. Frequency
and absolute number of H-2Kb+H-2Kd2Foxp3+ in peripheral or mesenteric lymph nodes (B) and in the spleen (D). Data are representative of the individual
analysis of six to eight mice per group for the spleen and analysis of three to four pools of five animals for lymph nodes. *p , 0.05, **p , 0.01, ***p , 0.001.

proved GVL effect (49), both reactions are clinically associated
(4), and there is no specific therapeutic approach to target the
undesired reaction only. In our approach, recipients reconstituted
with spleen cells from combined therapy–treated donors develop
less severe aGVHD and are still capable of eliminating tumor cells
injected along with the graft, showing that the GVLr is main-
tained. This dual capacity may be related with the fact that
combined therapy induces a more effective tolerance to minor
histocompatibility Ags. Actually, it is expected because the Ags
are administered in the form of soluble proteins that will be
captured and processed, losing the entire MHC peptide com-
plexes.

FIGURE 9. Posttransplant in vivo depletion of CD25+ T cells abrogates the protection. Donor mice treatment with combined therapy and HSCT was
performed as described previously. On third day posttransplant, chimeras reconstituted with combined therapy treated donor spleen received an i.p. dose of
PC61 mAb (250 mg/mice) (54) for depletion of CD25+ cells. On day 14 posttransplant, a reinforced i.p. dose of PC61 mAb was injected in recipient
chimeras (100 mg/mice). (A) Total clinical score. (B) Survival rates. A total of 10 mice was individually analyzed per group. ***p , 0.001 for AgF1/Ll
versus AgF1/Ll (2CD25). ##p , 0.01, ###p , 0.001 for AgF1/Ll versus Ctr+. ++p , 0.01 for AgF1/Ll (2CD25+) versus Ctr+.
1936
Recently, B lymphocytes subsets able to modulate inflammatory
responses were described previously (35). These Bregs seem to
play an essential role in oral tolerance induced with low amounts
of Ags (36). In accordance to that, protection obtained with com-
bined therapy is totally abrogated in the absence of B lymphocytes.
Moreover, CD19+ cells from treated donors, but not from untreated
ones, are sufficient to inhibit aGVHD in recipients reconstituted with
control spleen cells, showing that combined therapy induces the
generation of a B cell subpopulation with regulatory functions.
Generation of specific Foxp3+ Tregs by allogeneic primary
B cells (50) and by LAP/TGF-b+ B cells (41) have been reported
previously. Indeed, our data show that combined therapy not only
increases the CD19+LAP+ population but also induces donor
Foxp3+ Tregs. Interestingly, in our model, the expansion of Tregs
did not occur in treated donors because we did not observe dif-
ferences in frequency or absolute number of donor splenic Foxp3+
cells. Moreover, elimination of CD25+ population in the graft
before the transplant does not abrogate the protective effect from
combined therapy. The induction of donor Foxp3+ cells, possibly
from effector alloreactive T cells, happens on recipient after the
transplant and is dependent on those B cells generated by the
treatment, indicating that Ag-specific presentation and recognition
are important steps for acquisition of a regulatory phenotype by
allo-T cells and for their following activation. The abrogation of
protection after in vivo depletion of CD25+ cells on recipient
supports this hypothesis. On the basis of these data, we propose
a model in which B cells from orally treated mice present cognate
Ags to allospecific responder T cells responsible for the GVH
reaction, explaining the Ag-specific suppression toward aGVHD
only. These results are in accordance with previous reports
showing that the generation of specific Tregs after allostimulation
in vitro inhibits aGVHD and spare GVL/GVT responses in some
experimental models (51, 52).
The heterogeneity among Bregs and the absence of a master
transcriptional factor associated with this subpopulation strongly
suggest that, like their T cell counterpart, they can be divided into
different regulatory subsets originated from ontogenically distinct
subsets (53). However, some features such as the production of IL-
10 (35) and expression of molecules like CD86 (39) and class II
MHC (40) along with an immature phenotype are common among
the different subsets. In fact, we observed increased IL-10 in
protected recipients. Moreover, combined therapy induces a high
frequency of transitional B lymphocytes, with high expression of
CD21, CD86, and class II MHC, a phenotype compatible with
Bregs. In addition, we demonstrated that IL-10 production by
splenic B cells from treated donors is necessary for the protection
observed after combined therapy, indicating the B10 cells, the IL-
10–producing Breg (42), is in fact the key player in this setting.
The contribution of a distinct cell type in the immediate induction
of B cell with regulatory function cannot be excluded, although to
our knowledge there is no report showing the dependence of other
cells, such as dendritic cells, on Breg activation.
Finally, B cell differentiation into a regulatory subtype was
described by Lampropoulou et al. (43) as being dependent on two
signals. A first signal delivered by TLR initiates IL-10 production
by B cells. In a second phase, engagement of receptors classically
involved with B cell survival and expansion, such as the BCR,
amplifies the initial population of IL-10–producing B cells, allow-
ing the effective suppression. Altogether, these data might support
the idea that the ingestion of L. lactis gives the innate stimulus
necessary to the acquisition of a regulatory phenotype by the
B cells, which is dependent on IL-10 in our model. The recipient
Ags in the protein extract would then provide BCR stimuli that
would enable specific B cells to present the cognate Ags to T cells,
ORAL THERAPY INHIBITS aGVHD BUT NOT GVL REACTION
generating Tregs. Activity of these alloreactive Tregs would dampen
the response to recipient Ags but preserve the response to Ags de-
rived from the tumor cells (51, 52).
Our results suggest that oral tolerance induction by the use of
a probiotic bacteria and the recipient protein extract, on HSCT
donors before transplantation, might prove safe, simple, and ef-
fective for preventing GVHD in human patients.
Acknowledgments
We thank Ligia Peçanha for critical reading of the manuscript and sugges-
Downloaded from http://www.jimmunol.org/ at Univ of California-San Diego Serials/Biomed Lib 0699 on September 15, 2014
tions, João P. Monteiro for discussions and critical reading and Jane Littrell
for English review.
Disclosures
The authors have no financial conflicts of interest.
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 The Journal of Immunology

Corrections
Mercadante, A. C. T., S. M. Perobelli, A. P. G. Alves, T. Gonçalves-Silva, W. Mello, A. C. Gomes-Santos, A. Miyoshi, A. M. C. Faria, and
A. Bonomo. 2014. Oral combined therapy with probiotics and alloantigen induces B cell–dependent long-lasting specific tolerance.
J. Immunol. 192: 1928–1937.

The eighth author’s name was omitted from the article. The corrected author and affiliation lines are shown below.

Ana C. T. Mercadante*, Suelen M. Perobelli†, Ana P. G. Alves*, Triciana Gonçalves-Silva†, Wallace Mello‡, Ana C. Gomes-Santosx,
Anderson Miyoshi{, Vasco Azevedo{, Ana M. C. Fariax, and Adriana Bonomo*,†,‡

*Department of Experimental Medicine, Brazilian National Cancer Institute, Rio de Janeiro 20231-050, Brazil; †Department of Im-
munology, Microbiology Institute Prof. Paulo de Góes, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil;
‡
Laboratory of Thymus Research, Oswaldo Cruz Institute, Rio de Janeiro 21040-360, Brazil; xDepartment of Biochemistry and Im-
munology, Biological Science Institute, Federal University of Minas Gerais, Belo Horizonte 30161-970, Brazil; and {Department of
Biology, Biological Science Institute, Federal University of Minas Gerais, Belo Horizonte 30161-970, Brazil

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