IMMUNOLOGY ORIGINAL ARTICLE

Innate profiles of cytokines implicated on oral tolerance correlate

with low- or high-suppression of humoral response

Maria F. Silva,1,2 Alice O. Summary

Kamphorst,3 Elize A. Hayashi,4
Maria Bellio,4 Claudia R. Carvalho,3
Ana M. C. Faria,3 Kátia C. C.
Sabino,5 Marsen G. P. Coelho,5
Alberto Nobrega,4 Daniel Tavares5
and Antonio C. Silva5
1
Universidade Estadual do Norte Fluminense,
2 or antigen-gavaged TR/TS mice. The difference in anti-OVA titres was
Universidade Estadual da Zona Oeste, 3Uni-
versidade Federal de Minas Gerais, 4Universid-
ade do Estado do Rio de Janeiro, and
5
Universidade Federal do Rio de Janeiro, Rio
de Janeiro, Brazil
doi:10.1111/j.1365-2567.2010.03248.x
Received 31 August 2009; revised 15 January
2010; accepted 19 January 2010.
Correspondence: Dr A. C. da Silva,
Laboratório de Imunobiologia, Department
Biol. Cel. e Genética, Instituto de Biologia,
UERJ, Rua S. Francisco Xavier, 524, PHLC,
Maracanã, CEP 20 559-900, Rio de Janeiro,
RJ, Brazil. Email: acdasilva@pq.cnpq.br;
acdasilva.uerj@gmail.com
Senior author: Antonio Carlos da Silva
Oral tolerance (OT) is being studied with great interest because of its
therapeutic potential in allergy and autoimmunity. In the present study,
two mouse strains with extreme phenotypes of OT susceptibility (TS) or
resistance (TR) to ovalbumin (OVA) were used to demonstrate whether
the tr and ts genes, cumulated during 18 generations of bi-directional
genetic selection, influence expression of immunobiological traits in naive
2048-fold between OVA-gavaged TS and TR mice. Tolerance susceptibility
to OVA gavage in individuals from a (TS · TR)F2 population was 24%
high-susceptibility, 62% low-susceptibility and 14% non-tolerant. Differ-
ent antigens, unrelated to OVA, were tested by gavage and TS mice were
generally susceptible while TR mice were resistant. The stability of TS and
TR phenotypes was not affected by the use of strict protocols of intraperi-
toneal immunization or feeding over 30 consecutive days. The levels of
interleukin-2 (IL-2), IL-4, interferon-c and IL-10 cytokines evaluated in
concanavalin A-stimulated spleen cells from naive mice and in OVA-stim-
ulated spleen cells from OVA-gavaged mice were higher in TS mice. Inter-
leukin-10 was up-regulated in OVA-gavaged TS mice and down-regulated
in TR mice. In naive mice, the percentage of CD4+ CD25+ and
CD4+ Foxp3+ spleen cells and IL-10 expression by CD4+ cells was signifi-
cantly higher in TS mice. These results indicate that regulation of IL-10
expression could be an important factor contributing to the mechanisms
controlling OT susceptibility, and that the OT responses of TR and TS
individuals strongly correlate with their innate potential to secrete this
cytokine.
Keywords: antibody responses; cytokines; mice; oral tolerance; regulatory
T cells

munity processes and in alleviating allergic inflamma-

Introduction
Oral tolerance (OT) is a physiological mechanism that
prevents food hypersensitivity and adverse allergic
responses to normal enteric flora.1,2 Several mechanisms
have been proposed to explain OT. Low doses of oral
antigen favour active suppression with the generation of
regulatory cells, whereas high doses favour clonal
anergy/deletion3. Regulatory CD4+ T cells producing the
cytokines interleukin-10 (IL-10) or transforming growth
factor-b controlling both T helper type 1 (Th1) and
Th2 responses4,5 mediate specific or non-specific sup-
pression in antigen-reactive cells.6 The therapeutic
potential of OT has been tested for systemic autoim-
tory diseases.7,8
The OT regulates responses to established gastrointesti-
nal microflora and continuous antigenic challenges from
common food antigens.9 Individuals lacking the ability to
induce OT therefore develop immune responses against
food antigens and commensal flora and this can trigger
food allergies and inflammatory bowel disease.10 Studies
in animal models suggest that there is a genetic predispo-
sition leading to these diseases.11
In isogenic models of mice, it was demonstrated that the
major histocompatibility complex and background genes
control OT.12,13 Our experiments in genetic selection dem-
onstrated polygenic control.14 In the present experiment,

 2010 Blackwell Publishing Ltd, Immunology 1

M. F. Silva et al.
we used mice selected for extreme phenotypes of suscepti-
bility and resistance to OT over 18 generations of consecu-
tive breeding by assortative mating and avoiding
consanguinity. This strategy favours the selection of coher-
ent sets of multiple genes participating in the desired OT
phenotype. Unlike isogenic strains, in which each mouse
carries a single genome, our tolerance-resistant (TR) and
tolerance-susceptible (TS) mice are genetically homoge-
neous at the relevant loci for the selected character and het-
erogeneous in terms of background genes.
An advantage of using animals with extreme pheno-
types is the possibility of reconstitution of an F2
(TR · TS) segregant population with a normal phenotype
distribution in a widely heterogeneous genetic back-
ground using inter-strain crosses. Another possibility is
the ability to detect phenotypes not found in homoge-
neous strains of mice. Multigenic control of the OT phe-
notype allows different degrees of OT to coexist in a
heterogeneous population.
In the present study, we analysed the influence of the
cumulated tr and ts genes on disparate OT phenotypes in
naive or ovalbumin (OVA) -gavaged TR and TS mice,
further immunized by intraperitoneal injection with the
ingested antigen, using different protocols for tolerance
induction and immunization. We also studied the genetic
inheritance of OT trait, analysing the distribution of OT
antigen-specific phenotypes in F1 and F2 individuals of a
genetically heterogeneous population. The TR and TS
mice were tested for tolerance induction by gavage with
different antigens, unrelated to OVA, measuring their
humoral response after immunization with the respective
antigen. The influence of tr and ts genes on the percent-
age of CD4+ CD25+ and CD4+ Foxp3+ splenic T cells and
production of IL-10 by spleen cells from naive and
gavaged TR and TS mice was also evaluated. Other rele-
vant cytokines such as IL-4, interferon-c (IFN-c) and IL-2
were also quantified. The intracellular IL-10 production
by CD4+ T cells from TS mice was found to be greatly
augmented compared with TR CD4+ T cells, both in per-
centages and in levels of secretion. Percentages of regula-
tory T (Treg) cells, measured by CD25 and Foxp3
markers, were higher in TS mice. These results clearly
indicate that the cumulated tr and ts genes control the
innate profile of cytokine production and Treg cell per-
centages of naive TS and TR animals, before oral treat-
ment with OVA, and strongly correlate with OT
susceptibility or resistance obtained after being fed and
further immunized with antigen.
Materials and methods
Mice
Two- to three-month-old mice of both sexes from an F18
generation of OT-susceptible (TS) or OT-resistant (TR)
2  2010 Blackwell Publishing Ltd, Immunology
F1 hybrids and an F2 segregant population produced by
reciprocal crosses between the parental lines TS and TR
were used in this work. The TS and TR mice were devel-
oped through bi-directional genetic selection, starting
from a highly polymorphic population (F0) derived from
the inter-crossing of eight inbred mouse strains (A,
DBA2, P, SWR, CBA, SJL, BALB/c and C57BL/6). Ani-
mals used throughout the experiments, and their parents,
were maintained on a hen egg OVA-free diet in a produc-
tion colony, separated from the breeding colony where
the best phenotypes of susceptibility and resistance to
OVA OT were mated for the maintenance of the colony.
The TR and TS mice were subjected to experimental pro-
tocols and used as parents to produce F1 and F2 mice
after two to three generations of mating in the produc-
tion colony. The Commission for Care and Use of Labo-
ratory Animals of the Rio de Janeiro State University
(UERJ, Brazil) approved the study protocols.
Antigens
Ovalbumin and human gamma globulin were purchased
from Sigma-Aldrich (St Louis, MO). Egg yolk immuno-
globulin (IgY) was prepared from eggs inoculated with
canine parvovirus according to McLaren15 while peanut
and cashew-nut proteins were extracted as described by
Landry and Moureaux.16 Protein concentrations were
determined using the Lowry method.17 A diet containing
15% casein (Rhoster Indústria e Comércio LTDA, SP Bra-
sil) or purified casein (Sigma-Aldrich) was used. Fresh
sheep red blood cells (SRBC) washed in 015 mM NaCl
were used immediately. Deaggregated OVA was prepared
according to Bruce and Ferguson.18
Tolerance induction and immunization
Mice were gavaged with 02 ml physiological saline con-
taining 5 or 20 mg soluble OVA in a single administra-
tion 7 days before immunization. Control groups were
gavaged with saline alone. Continuous feeding of 20 mg
OVA in the drinking water was given for 1, 3 or 30 con-
secutive days (each mouse ingested 20 mg OVA daily).19
Seven days after the last day of oral treatment, mice
were immunized intraperitoneally (i.p.) with 10 lg
OVA + 1 mg alum and 14 days after with one booster of
10 lg OVA without alum. To evaluate the effect of con-
secutive i.p. immunization on OT maintenance, mice ga-
vaged with OVA 5 mg and i.p. immunized 7 days after
with 10 lg OVA + 1 mg alum were boosted with i.p.
10 lg soluble OVA on days 14, 21 and 28 after the first
immunization. Sera were collected 7 days after the last
immunization. To induce endovenous tolerance, 02 ml
physiological saline containing 2 mg deaggregated OVA
was injected in a single intravenous (i.v.) dose 7 days
before i.p. immunization. To induce oral tolerance to

SRBC, we followed the protocol of Mowat.20 Sera were
collected 21 days after immunization.
Evaluation of humoral response
The OVA-specific agglutinin titres of F2 populations were
determined by passive haemagglutination with SRBC-cou-
pled OVA according to Avrameas et al.21 The SRBC-spe-
cific agglutinin titres of TR and TS mice were determined
by direct haemagglutination testing. The titres were
expressed as log2 of the highest serum dilution giving a
positive reaction. Immunoglobulin G antibodies of TR
and TS mice to OVA and other antigens such as casein
(Sigma-Aldrich), fowl gamma globulin (IgY), peanuts and
cashew nuts were assessed by enzyme-linked immunosor-
bent assay (ELISA) as described previously.22 Tetrameth-
ylbenzidine (Zymed Lab., Inc., San Francisco, CA) was
used as the substrate for horseradish peroxidase. The yel-
low colour produced was read at 450-nm wavelength
using a multiscan photometer. Experimental and control
groups were evaluated by comparing the mean ± SD of
the sums of absorbance values read between 1/800 and 1/
25 600 serum dilutions (the most linear part of the absor-
bance curve).22 The mean values were therefore based on
readings of six dilutions of each individual serum.
Cell proliferation and cytokine assays
Spleen cells from individual mice were harvested 14 days
after immunization and cultured in 96-well plates at
106 cells/ml in the presence of anti-CD3 and anti-CD28
antibodies (1 lg/ml each), concanavalin (Con A) (1 lg/
ml), OVA 01, 1 or 5 lg/ml for 72 hr, or Escherichia coli
lipopolysaccharide 125, 25, 5 or 10 lg/ml (LPS; Sigma-
Aldrich). [3H]Thymidine (1 lCi/well) was added for the
last 6 hr of culture and the incorporated thymidine was
measured in a beta liquid scintillation counter. For cyto-
kine assays, culture supernatants were collected at 24 hr
for IL-2 and at 72 hr for IFN-c, IL-4 and IL-10. Levels of
cytokines in supernatants were determined by capture
ELISA as described elsewhere.23 Briefly, 96-well microtitre
plates (NUNC, Naperville, IL), were coated with 100 ll/
well of respective capture antibodies (Pharmingen, San
Diego, CA) at a dilution of 2 lg/ml in coating buffer, and
incubated overnight at 4. The plates were then washed
with phosphate-buffered saline (PBS) –01% Tween-20
and blocked for 60 min at 37 with 10% fetal calf serum
(FCS). Culture supernatants and standards were loaded
onto plates. The plates were thoroughly washed and the
appropriate biotinylated rat anti-mouse IL-2, IL-4, IL-10
or IFN-c monoclonal antibodies (mAbs; Pharmingen)
were added, followed by a 60-min incubation at 37. After
additional washes, horseradish peroxidase-labelled strepta-
vidin (Sigma-Aldrich) was added to each well for 15 min
and each plate was thoroughly washed. Colour reaction
 2010 Blackwell Publishing Ltd, Immunology
Naive mice and OVA oral tolerance
was developed at room temperature with 100 ll/well of
orthophenylenediamine (1 mg/ml), 004% H2O2 substrate
in sodium citrate buffer. Reaction was interrupted by the
addition of 20 ll/well 2 M H2SO4. Absorbance was mea-
sured at 490 nm using an ELISA reader (Bio-Rad Model
450 Microplate Reader, Hercules, CA).
Cell staining and flow cytometry
Freshly isolated spleen cells obtained on the third day
after immunization from naive or previously saline-
gavaged or OVA-gavaged TR and TS mice were stained
with fluorescein isothiocyanate-labelled anti-mouse CD4
(clone RM4-5; Pharmingen) and phycoerythrin-Cy5-
labelled or allophycocyanin-labelled anti-mouse CD25
(clone PC61; eBioscience, San Diego, CA) for analysis, or
with phycoerythrin-Cy7-labelled anti-CD19 (clone
MB19.1; eBioscience) and Alexa 647 IgM (clone b-7-6;
kindly provided by Dr John Cambier, University of Colo-
rado Health Science Center, Denver, CO).
Typically, 1 · 106 to 3 · 106 erythrocyte-depleted
splenocytes in suspension were incubated in fluorescence-
activated cell sorting (FACS) buffer (PBS, 1% FCS, 005%
sodium azide) with mAbs in appropriate dilutions for
20 min on ice, and then washed with FACS buffer. For
FoxP3 staining, cells were further fixed and permeabilized
using the mouse regulatory T-cell staining kit (eBio-
science), following the manufacturer’s instructions. Data
were acquired on a FACSCalibur (BD Biosciences, San
Jose, CA) and analysed using CELLQUEST (BD Bioscienc-
es) or SUMMIT (Dako Cytomation, Glostrup, Denmark)
software.
Con A blast generation and intracellular staining (ICS)
for IL-10 production
Splenocytes from TR and TS mice were cultured for
2 days in complete medium (OptiMEM medium contain-
ing 10% FCS, 100 lg/ml penicillin, 100 U/ml streptomy-
cin, and 50 nM 2b-mercaptoethanol (all from Life
Technologies Inc., Grand Island, NY) in the presence of
Con A (25 lg/ml; Sigma-Aldrich), followed by 3 days of
culture in rIL-2 (20 ng/ml) and rIL-4 (20 ng/ml) (R&D
Systems, Inc., Minneapolis, MN) to generate Con A
blasts. For IL-10 detection, Con A blasts obtained as
described above were further stimulated with phorbol 12-
myristate 13-acetate (PMA; 20 ng/ml), calcium ionophore
A 23187 (200 ng/ml) and monensin (5 lM) for 5 hr. Cells
were then pre-stained for cell surface markers, followed
by fixation in 1% paraformaldehyde for 18 hr at 4 and
permeabilization with 02% saponin in FACS buffer
(PBS-supplemented with 5% FCS and 005% sodium
azide) for 20 min at 4. Cells were then incubated for
20 min with phycoerythrin-conjugated anti-mouse IL-10
mAb or isotype control (eBiosciences) properly diluted in
3
M. F. Silva et al.
02% saponin FACS buffer, on ice. Alternatively, freshly
isolated splenocytes from TR and TS mice were directly
resuspended (25 · 106 cells/ml) in complete medium
with LPS (10 lg/ml, E. coli serotype 0111:B4), PMA
(50 ng/ml), calcium ionophore A 23187 (200 ng/ml) and
monensin (5 lM) (all from Sigma-Aldrich) for 5 hr, in
24-well flat-bottom plates for detection of IL-10 produc-
tion by splenic B lymphocytes, as previously described.24
Cells were pre-stained for B-cell surface markers and pro-
cessed as above for intracellular IL-10 staining.
Genetic analysis
The degree of dominance effect in the F1 (TR · TS)
hybrid was calculated as d/a. The d (dominance devia-
tion) represents the distance from the mid-parental value
caused by dominance and is calculated according the
equation: d = xF1 ) ½(xA + xB), where xA and xB are
the mean response of each parent. The determination of
a (additive effect) might be obtained by measuring
the departure from the F1 hybrid mean to either of
the parents, or halving the parental difference: a = ½
(xA ) xB).25 The F1 mean value would be expected to be
exactly intermediate to both parents if the genetic effects
were additive, departures from this mid-parental value
would indicate the effect of dominance. The value
between zero and one calculated by the ratio d/a signifies
the dominance effect.
Statistical analysis
The chi-square goodness-of-fit test was used to evaluate
whether the observed data were coherent with a normal
curve. Differences between mean antibody levels in differ-
ent mouse groups were determined by Student’s t-test;
groups were considered statistically different if P  005.
Results
Feeding OVA for 30 consecutive days did not inhibit
the IgG response in TR mice
In the present study, the tolerogenic potential of mice
selected for extreme phenotypes of oral tolerance (TR and
TS mice) was evaluated. The mean of specific IgG titres
to antigen (OVA) used for selecting these strains of mice
was significantly different. In the present generation, the
difference was 2048-fold between OVA-gavaged TS and
TR mice and 32-fold between saline-gavaged TS and TR
mice.
We evaluated the phenotypic profiles of TS and TR
mice when submitted to different oral treatment proto-
cols: each mouse received a single dose of 5 mg OVA by
gavage, or daily 5 mg OVA diluted in drinking water
(continuous feeding) in 1 or 3 days (Fig. 1a) or daily
4  2010 Blackwell Publishing Ltd, Immunology
20 mg OVA for 30 days (Fig. 1b). The TR mice showed a
steady resistance after any of these different regimens
whereas TS mice became tolerant on all feeding regimens.
Continuous feeding for 1 or 3 days was more effective
than gavage to down-regulate the in vivo IgG response.
The stability of TS and TR mouse phenotypes was
not changed after continuous i.p. challenge with
OVA + alum or OVA + complete Freund’s adjuvant
To test the robustness of the selection, the TS and TR
mice were evaluated under immunogenically extreme
conditions. Figure 1(c) shows the kinetics of primary and
secondary responses of previously OVA-gavaged mice.
The secondary i.p. challenge did not induce a humoral
response in OVA-gavaged TS mice. The OVA-gavaged or
saline-gavaged TR mice and the saline-gavaged TS mice
responded to the secondary challenge with augmented
specific IgGs. To confirm the stability of susceptibility to
OVA OT, TS and TR mice gavaged with 5 mg OVA were
boosted by i.p. injections of 10 lg OVA in saline on days
14, 21 and 28 after the first i.p. immunization with 10 lg
OVA + complete Freund’s adjuvant (CFA; Fig. 1d). The
IgG response of OVA-gavaged TS mice stayed stable
throughout despite the rigorous immunization protocol.
There was no significant difference in antibody levels
between OVA-gavaged and saline-gavaged TR mice.
TS mice were generally susceptible and TR mice were
resistant to tolerance induction for the majority of
non-related antigens tested
To test the responses to different routes of sensitization,
or to antigens not used during the selection process, TR
and TS mice were gavaged using different and unrelated
soluble [OVA, bovine serum albumin (BSA), IgY, human
gamma globulin, casein, peanut and cashew-nut extracts]
and particulate (SRBC) antigens (Table 1); intravenous
injection with OVA was also evaluated. Following these
treatments, mice were immunized with the homologous
antigens and the sera were obtained to measure levels of
IgG antibodies as described in the Materials and methods.
The results were as follows. (i) The IgG response differ-
ences, between gavaged TS and TR mice (inter-strain dif-
ference), were statistically significant to all tested antigens
except to BSA. (ii) Differences in specific IgG responses
between antigen-gavaged and saline-gavaged TS mice, fol-
lowed by immunization with the same antigen used to
gavage, were statistically significant for casein, peanut,
BSA, SRBC, IgY and OVA. Cashew nuts and human
gamma globulin did not induce OT. The TR mice did
not show differences in antibody titres between experi-
mental and control groups excluding BSA. (iii) Intrave-
nous OVA only induced tolerance in the TS mice
(Table 1).
 Naive mice and OVA oral tolerance

(a) (b)
9 TS mice 16
TR mice 14
6

lgG titres (log2)

lgG titres (log2)

*** *** 12
3 *** 10
0
8
9
6 **
6 4
3 2
0 0
Gavaged Fed OVA fed 30 days
1d 3d
OVA – + + + – + – +

(c) (d)
20 Primary Secondary
18 10
TS mice
lgG titres (log2)

16

lgG titres (log2)

TR mice 8
14
6
12
10 4
8 2
6
0
14 21 28 14 21 28 14 21 28 35
Days after i.p. immunization Days after i.p. immunization

Figure 1. Consecutive oral tolerance induction and intraperitoneal immunization. (a) Mice were gavaged with a single dose of ovalbumin (OVA;
5 mg), or submitted to continuous OVA feeding for 1 or 3 consecutive days. (b) Mice received 20 mg OVA by continuous feeding daily for 30
consecutive days. In (a) and (b) mice were intraperitoneally (i.p.) immunized with 10 lg OVA + 1 mg alum 7 days after OVA oral treatment.
(c) Mice were gavaged with 5 mg OVA and given a single i.p. immunization with 10 lg OVA + 1 mg alum (primary response) or were i.p.
immunized and boosted with 10 lg OVA (secondary response). (d) Mice were gavaged with 5 mg OVA and i.p. immunized with 10 lg
OVA + CFA and boosted three times, at 7-day intervals, with 10 lg OVA. Arrows indicate booster days. Squares indicate OVA-fed group and
the circles indicate saline-fed group. Results represent mean ± SD of two experiments with eight mice per group. The differences in antibody
titres between OVA-fed TS mice and the other groups were statistically significant at all time-points (P < 001 by Student’s t test).

Table 1. Humoral response to unrelated antigens

Log2 immunoglobulin G titres – intragastric pre-treatment

Tolerance-susceptible (TS) mice Tolerance-resistant (TR) mice

Antigens Saline1 Antigen2 P-value3 Saline4 Antigen5 P-value6 P-value7

Ovalbumin (OVA) 1230 ± 130 600 ± 147 P < 0001 1470 ± 150 1310 ± 270 NS P < 0001
Casein 826 ± 287 418 ± 329 P < 0005 823 ± 285 903 ± 129 NS P < 0001
Peanuts 1150 ± 220 360 ± 180 P < 0001 1320 ± 250 1230 ± 200 NS P < 0001
Cashew nuts 900 ± 120 810 ± 230 NS 1240 ± 220 1200 ± 210 NS P < 0001
Sheep red blood cells (SRBC) 1100 ± 141 800 ± 219 P < 0001 1200 ± 100 1260 ± 151 NS P < 0001
Fowl gamma globulin (IgY) 875 ± 075 770 ± 030 P < 0001 1080 ± 060 1122 ± 017 NS P < 0001
Human gamma globulin (HGG) 870 ± 114 848 ± 123 NS 1117 ± 118 1282 ± 090 NS P < 0001
Bovine serum albumin (BSA) 954 ± 093 743 ± 321 P < 005 857 ± 315 567 ± 296 P < 005 NS
Log2 IgG titres – intravenous pre-treatment
Deaggregated OVA 1156 ± 136 482 ± 219 P < 0001 1300 ± 040 1288 ± 037 NS P < 0001

NS, not significant difference, P > 0.05. Mean value with standard deviation.
1
TS mice saline-gavaged.
2
TS mice antigen-gavaged.
3
Differences between antigen-gavaged and saline-gavaged TS mice.
4
TR mice saline-gavaged.
5
TR mice antigen-gavaged.
6
Differences between antigen-gavaged and saline-gavaged TR mice.
7
Inter-strain differences between antigen-gavaged TR and TS mice.
Results represent means ± SD of 10 mice per group.

 2010 Blackwell Publishing Ltd, Immunology 5

M. F. Silva et al.

(a) (b) (c) (d)

35 1·0 8
30 TS mice 7 0·3

IFN-γ (ng/ml)
c.p.m. (× 103)
TR mice

IL-10 (ng/ml)
0·8 6

IL-2 (ng/ml)
25
20 0·6 5 0·2
15 * ** 4
0·4 3
10 2 0·1
5 0·2
1
0·0 0 0·0
35
30 1·0 8
c.p.m. (× 103)

25 7 0·3

IFN-γ (ng/ml)
0·8

IL-10 (ng/ml)
6

IL-2 (ng/ml)
20
0·6 5
15 4 0·2
** **
10 0·4 ** 3
0·1
5 2
0·2 **
0 1
i ii iii i ii iii i ii iii 0·0 0 0·0
Saline Gavage Feeding Saline Gavage Feeding Saline Gavage Feeding Saline Gavage Feeding

Figure 2. Specific proliferative responses and cytokine production. Mice received 5 mg ovalbumin (OVA) by either gavage or continuous feeding
for 24 hr and were immunized subcutaneously with 10 lg OVA + complete Freund’s adjuvant (CFA) 7 days later. Control mice received oral sal-
ine and were immunized. Spleen cells were collected 14 days after immunization and 106 cells/well were cultured in 96-well plates in the presence
of anti-CD3 and anti-CD28 antibodies in medium containing 01, 1 or 5 lg/ml OVA for measuring the proliferative response (a column i, a col-
umn ii and a column iii, respectively) and 1 lg/ml OVA for cytokine production (b–d). Bars represent the mean ± SD of three experiments with
eight mice per group. *P < 005, **P < 001 comparing experimental with control groups.

Oral treatment with OVA affects the in vitro specific T-cell and B-cell proliferative responses and cytokine
proliferation of spleen cells from TS and TR mice production differed in naive TS and TR mice
The specific proliferative response of spleen cells from TS To rule out the possibility that TS mice were simply low
mice submitted to gavage or continuous feeding (Fig. 2a) responders, the polyclonal proliferative response and cyto-
was inhibited significantly when compared with saline- kine production were studied in naive TS and TR mice
gavaged TS mice. Although OT was more effective in TS after in vitro stimulation of spleen cells. To induce B-cell
mice, suppression of specific proliferation was induced in cell proliferation splenocytes were stimulated in medium con-
cultures from OVA-gavaged TR mice stimulated with taining LPS. B-cell proliferation was lower in TS mice
01 lg/ml OVA (Fig. 2a, column i). The cells from TR mice when 125, 25 or 5 lg/ml LPS were used (P < 0001)
submitted to OVA continuous feeding showed increased (Fig. 3a). To investigate T-cell proliferation and cytokine
proliferation when stimulated with 1 lg/ml OVA if com- production, splenocytes were cultured in medium con-
pared with the saline-gavaged TR group, suggesting oral taining Con A or anti-CD3/CD28 (Fig. 3b–f). T-cell pro-
priming (Fig. 2a,b). Proliferation of spleen cells from saline- liferation was higher in TS mice when anti-CD3/CD28
gavaged mice was higher in TS than TR mice, after 1 or 5 lg/ stimulated (P = 0002) (Fig. 3ab). The production of
ml OVA stimulation (Fig. 2a, columns ii, iii). However, IL-2, IFN-c, IL-4 and IL-10 cytokines by T cells were
OVA 01 lg/ml did not stimulate TS spleen cells (Fig. 2a). significantly higher in TS than TR mice (Fig. 3c–f).

IL-2, IL-10 and IFN-c levels were reduced in OVA- Inter-strain differences in CD25+ and Foxp3+ CD4+
gavaged TR mice T-cell frequency
We investigated the effect of OVA administration by either Naive or saline-gavaged TR mice showed a higher CD4+
gavage or continuous feeding on cytokine secretion by T-cell count (Fig. 4a) but a lower relative CD25+ percent-
spleen cells from TR and TS mice. Gavage led to decreased age than naive or saline-gavaged TS mice (Fig. 4b). These
IL-2 production in both strains of mice (Fig. 2b). High lev- inter-strain differences disappeared after OVA-gavage as
els of IFN-c and IL-10 were observed in TS mice result of a reduction in CD4+ cells in TR mice (Fig. 4b,c).
(Fig. 2c,d). On the other hand, IFN-c and IL-10 levels were Despite differences in the proportion of CD25+ within
reduced in OVA-gavaged TR mice (Fig. 2c,d). Interleukin- CD4+ T cells, no significant difference between TR and
10 was also reduced in OVA-fed TR mice (Fig. 2D). Reduc- TS mice was observed in the proportion of CD25+ cells
tion in IFN-c and IL-10 production (Fig. 2c,d) may be among total splenocytes (Fig. 4c). Similar to our observa-
mediating the TR mouse resistance to OT, as observed by tions with CD25, we also found that the Foxp3+ cell pro-
increased proliferation of spleen cells harvested from OVA- portion within the CD4+ T-cell population from naive TS
fed TR mice (Fig. 2a, column ii). mice was significantly higher than in TR mice (Fig. 4d).

6  2010 Blackwell Publishing Ltd, Immunology

 Naive mice and OVA oral tolerance

(a) 6 (b) (c) 8

TR mice 160
6
4 TS mice 120
4
80
2 *** *** *** 40
2
c.p.m. (× 103)

c.p.m. (× 103)

IL-2 (ng/ml)
0 0 0

160 8
6
120 6
4
80 4
***
2
40
** 2 **
0 0 0
0 1·25 2·5 5 10 Con A α-CD3/CD28 Con A α-CD3/CD28
LPS concentration (µgml)

(f)
(d) (e)
20
30 0·4
16

20 12
8 0·2
10
IFN-γ (ng/ml)

4
IL-10 (ng/ml)

IL-4 (ng/ml)
0 0 0·0
20
30 0·4
16

20 12
8 * 0·2
10 *** *** 4 ***
**
0 0 0·0
Con A α-CD3/CD28 Con A α-CD3/CD28 Con A

Figure 3. Proliferative response to concanavalin A (Con A), anti-CD3/CD28 antibody and lipopolysaccharide (LPS), and cytokine production
from naive mice. For proliferative response, 106 spleen cells/well were cultured (a) in medium containing LPS 125, 25, 5 or 10 lg/ml, or (b–f)
in medium containing anti-CD3/CD28 antibody or Con A. (c–e) interleukin-2 (IL-2), interferon-c (IFN-c) and IL-10 were stimulated by Con A
or anti-CD3/CD28 antibody. (f) IL-4 was stimulated by Con A. Bars represent the mean ± SD of three experiments with six mice per group.
*P < 005, **P < 001, ***P < 0001 comparing experimental tolerance-susceptible (TS) and tolerance-resistant (TR) groups.

(a) (b) (c) (d)

CD25+ in the spleen (%)

40 P = 0·03
CD25+ T cells (%)
CD4+ T cells (%)

P < 0·01
CD4+ Foxp3+ (%)

P < 0·01 50 P < 0·01 P < 0·01 14

8
30 40 12
6
30 10
20 4
20 8
2 6
10 10
TR TS TR TS TR TS TR TS TR TS TR TS TR TS TR TS TR TS TR TS
Naive Saline OVA Naive Saline OVA Naive Saline OVA Naive

Figure 4. Frequency distribution of CD4+, CD25+ and Foxp3+ T cells measured by flow cytometry. Three experimental groups are shown: naive
(non-immunized) and saline or ovalbumin (OVA) -gavaged [both intraperitoneally (i.p.) OVA-immunized]; (a) CD4+ cells as a percentage of
gated splenic lymphocytes, (b) CD25+ cells as a percentage of gated CD4+ cells. (c) CD25+ cells as a percentage of spleen cells. (d) CD4+ Foxp3+
T-cell frequency. A representative evaluation with six mice of each group is shown.

(CD19+ IL-10+) (Fig. 5a). However, a greater proportion

Higher IL-10 levels were produced by CD4+ T cells
of IL-10+ non-B cells was detected in TS mouse spleno-
from TS mice
cytes (Fig. 5b). We aimed to evaluate whether such an
No difference between naive TS and TR mice was inter-strain difference was the result of an intrinsic defect
observed in the proportion of IL-10-producing B cells of CD4+ T cells in the response to IL-10-inducing stimuli,

 2010 Blackwell Publishing Ltd, Immunology 7

M. F. Silva et al.

(a) 6 (b) 4
P = 0·004

% IL-10 (non-B cells)

% IL-10 (B cells)

3
4

3
2
2

1 1 Figure 5. Frequency distribution of CD4+ IL-

TR TS TR TS 10+ and CD19+ IL-10+ cells and interleukin-10
(IL-10) mean fluorescence intensity (MFI)
(c) P < 0·0001 P < 0·0001 from tolerance-susceptible (TS) and tolerance-
(d)
20 100 resistant (TR) spleen cells analysed by flow
cytometry. (a,b) Interline comparative propor-
MFI CD4+ IL-10+ (%)

tion of CD19+ IL-10+ and CD19- IL-10+ cells

CD4+ IL-10+ (%)

15
in freshly isolated spleen between naive TS and
80 TR mice. (c,d) Inter-strain difference between
10 TS and TR mice on proportion of CD4+ IL-
10+ T cells and intracellular IL-10 levels by
5 CD4+ T cells after in vitro stimulation for gen-
60 eration of IL-10-producing blasts. A represen-
0 tative evaluation with six mice of each group is
TR TS CtrTR CtrTS TR TS shown.

using an in vitro treatment that favours the generation of males are, respectively, used for mating. No significant
IL-10-producing blasts from splenic CD4+ T cells. A difference was observed between OVA-gavaged and sal-
higher proportion of CD4+ IL-10+ T cells and intracellu- ine-gavaged F1 mice.
lar IL-10 production by CD4+ T cells were observed in The difference between OVA-gavaged and saline-
naive TS mice (Fig. 5c,d). gavaged F2 populations (Fig. 6d,h) was highly significant.
To differentiate tolerogenic or non-tolerogenic mice in an
OVA-gavaged F2 population we used one standard devia-
Genetic inheritance of OT susceptibility in TR, TS, F1
tion of the feeding TR and TS means as values to estab-
and F2 mice and the dominance effect observed in F1
lish these distinct groups. The high-tolerance individuals
hybrids gavaged with OVA
had values lower than 80 log2 (24%), the high-resistance
TS and TR mice, and their F1 and F2 populations result- mice had values over 130 log2 (14%) and the low-toler-
ing from interbreeding TR · TS, underwent tolerance ance (62%) comprised a normal F2 population (Fig. 6d).
induction and immunization protocols, and the titres of The IgG titres were distributed according to a Gauss nor-
serum IgG anti-OVA obtained were evaluated. Compari- mal distribution.
sons of the frequency distributions of IgG titres between The genetic significance of the overlap between the
TR and TS OVA-gavaged mice and with their respective phenotypic frequencies of F1 and TR (Fig. 6b,c) was esti-
saline-gavaged control groups, and between OVA-gavaged mated by calculating the degree of dominance effect using
F1 and F2 populations with their saline-gavaged control the mean of the responses of TS, TR and F1 hybrids trea-
groups, are shown in Fig. 6. We observed a left bias in ted by gavage with OVA. Using the equation d = xF1 ) ½
the gavaged TS IgG titre distribution, suggesting a critical (xA + xB), as described in Material and methods, demon-
value for maximal OT induction in the population of strates an additive effect of a = 5015, a dominance devia-
nearly 5 log2 (Fig. 6a). tion of d = 2725, and a dominance effect of d/a = 054.
To evaluate if maternal effects influenced the immuno- This value ensures the dominance effect of OT-resistance
logical expression of naive or antigen-stimulated TR and over the OT-susceptible phenotype measured by IgG level
TS mice, the F1 hybrids produced by reciprocal crosses (Fig. 6b,c).
were gavaged with OVA or saline. No significant differ-
ence (P = 050) was observed between the means of F1
Discussion
offspring from TR or TS mothers (1454 ± 166 and
1420 ± 152, respectively), indicating non-maternal influ- Models of quantitative variation in immune responses
ence. Therefore, offspring of TS females can express have explained phenotypic attributes successfully for
OT-resistant or OT-susceptible phenotypes if TR or TS many biological systems25 and OT phenotypes can be

8  2010 Blackwell Publishing Ltd, Immunology

 Naive mice and OVA oral tolerance

60 TS mice
(a) 30 TS mice (e)
40 20
20 10
0 0
5 10 15 20 5 10 15 20
60 TR mice

Frequency distribution
(b) 30 (f)

Frequency distribution
40 TR mice
20
20 10
0 0
5 10 15 20 5 10 15 20
10 (c) 8 (g)
F1 mice 6 F1 mice
5 4
2
0 0
5 10 15 20 5 10 15 20
20 F 2 mice L = 62% (d) 20 (h)
15 15 F2 mice
10 S = 24% R = 14% 10
5 5
0 0
5 10 15 20 5 10 15 20
Agglutinin titres (Log2) Agglutinin titres (Log2)
OVA-gavaged mice Saline-gavaged mice

Figure 6. Frequency distribution of agglutinin titres from tolerance-susceptible (TS) and tolerance-resistant (TR) mice, F1 hybrid and F2 popula-
tion. Mice were previously ovalbumin (OVA) -gavaged (a–d) or saline-gavaged (e–h) and presented the following immunoglobulin G (IgG) titres
mean ± SD. (a) OVA-gavaged TS = 662 ± 187 (81 mice); (b) OVA-gavaged TR = 1665 ± 228 (102 mice); (e) saline-gavaged
TS = 1042 ± 174 (52 mice); (f) saline-gavaged TR = 1613 ± 165 (66 mice). In F1 populations, the values were: (c) OVA gavaged
F1 = 1436 ± 157 (28 mice) and (g) saline-gavaged F1 = 1419 ± 204 (18 mice). In F2 populations, the values were: (d) OVA-gavaged
F2 = 1008 ± 249 (89 mice) and (h) saline-gavaged F2 = 1142 ± 204 (47 mice). The mean differences between OVA-gavaged TS and OVA-
gavaged TR were statistically significant (P < 0001) by t-test as were those between OVA-gavaged TS and its respective saline-gavaged control
(P < 0001), and between OVA-gavaged F2 and saline-gavaged F2 (P < 001). There was no significant difference between OVA-gavaged and sal-
ine-gavaged F1 or between F2 groups so as between OVA-gavaged F1 and OVA-gavaged TR mice. H, high susceptible; L, low susceptible; R, resis-
tant.

understood through them. The goal of a trait-related for the induction of OT.27 None of these regimens was
genetic selection is to create mouse strains with extreme able to abrogate resistance to OT in the TR mice. This
and easily differentiated phenotypes. Although we selected indicates that TR mice are strongly resistant, and TS mice
TS and TR mice in response to feeding with OVA, it is are susceptible, not only to protocols used during the
clear from results that susceptibility or resistance to OT selective process but to others that are apparently more
are not restricted to OVA, but is a general property of efficacious (Fig. 1; Table 1).
the TS and TR strains (Table 1). Tolerance susceptibility Because the physiology of antigen absorption and pre-
for multiple immunogens suggests that the outcome of sentation in the intestinal mucosa is peculiar, we investi-
the genetic selection based on a single antigen could not gated whether TS and TR mice would maintain their
be dissociated from a more profound alteration of the tolerance phenotypes for intravenous OVA. The suscepti-
immune response. ble and resistant phenotypes were unchanged (Table 1).
To investigate if OT susceptibility could be maintained Therefore, oral and intravenous tolerance could share
by using different regimens of immunization, TS mice fundamental mechanisms, involving organs such as the
were treated with sequential immunogenic boosters with liver and processing and presentation by specialized
OVA (Fig. 1c,d). In these immunization protocols, we cells.28
used alum or CFA as adjuvant in accordance with the Oral tolerance is a mechanism that probably arose to
observation by Tobagus et al.26 that the nature of inflam- protect the organism against inflammatory diseases such
matory co-stimulation by adjuvant used to express spe- as allergies and autoimmune disorders,2,7,29 so absolute
cific responses after feeding antigens determines the resistance to OT would not be compatible with life
qualitative aspects of the tolerance process. The tolerogen- under normal conditions and we are unlikely to find
ic capacity of TS mice was not modified by boosters or animals resistant for all immunological parameters in
by use of different adjuvant. The TR mice treated either natural populations. We observed that in special condi-
by gavage with low or high doses of OVA, or submitted tions the TR mice were primed or suppressed depend-
to continuous feeding for short and long periods were ing on whether high or low doses of OVA were given
not tolerized (Fig. 1a,b). Continuous feeding resembles orally. We found both down-regulation and up-regula-
natural feeding and is more effective than gavage to sup- tion in antigen-specific cellular proliferation (Fig. 3a), as
press antibody production. Continuous uptake of small in previous results for delayed-type hypersensitivity reac-
amounts of antigen in physiological conditions can signal tion and IgE responses.22,29 This dose-dependent

 2010 Blackwell Publishing Ltd, Immunology 9

M. F. Silva et al.
suppressor or enhancer capacity of TR mice was also
observed as bystander OT for Leishmania amazonensis
infection.30
The cytokines IFN-c, IL-4, IL-10 and transforming
growth factor-b have been implicated as active mediators
of oral tolerance. They are associated with the develop-
ment of Th1, Th2 or Th3 responses and generation of
regulatory T cells.31–33 The OVA-gavaged and OVA-fed
TS mice maintained high levels of IL-10, IL-4 and IFN-c
and OVA-gavaged TR mice showed reduced IFN-c and
IL-10 secretion levels concomitant with the decreased
IL-2 levels (Fig. 2b,d). The TS naive mice also produced
higher in vitro IL-4, IL-10 and IFN-c levels as along with
T-cell proliferation than did TR mice (Fig. 3b–f), but
lower B-cell proliferation (Fig. 3a) in response to LPS.
We found that TS mice produce IL-10 after intravenous
LPS (unpublished work). Results of other authors show
the relationship of OT and bacterial products of intestinal
flora and that LPS administered at the time of OVA
ingestion increases OT.34 Indeed, these responses in
naive animals may represent the background response
to environmental antigens that come into contact with
the immune system via the intestinal and respiratory
mucosas.
Naive CD4+ T cells can differentiate into either IFN-c-
producing Th1 cells or IL-4-producing Th2 cells, and that
reciprocal regulation of Th1 and Th2 responses is medi-
ated by IL-4 and IFN-c,35 both of which antagonize Th17
differentiation.36 This subset of CD4+ Th cells, called
Th17, was implicated in the protection of the host against
extracellular pathogens encountered at mucosal surfaces,
and plays a detrimental role in autoimmune disorders, as
well as in human inflammatory bowel disease and psoria-
sis.37 The information that IL-4 and IL-10 inhibit the Th1
responses, and that IL-4, IL-10 and IFN-c are required to
control the Th17 responses,38 suggest the participation of
these cytokines in establishing phenotypes of specific OT
susceptibility/resistance and in the diverse inflammatory
responses of TR and TS mice.39
Regulatory T lymphocytes (Treg cells) are important
regulators of the immune response that contribute to
the maintenance of intestinal immune homeostasis by
actively suppressing reactive responses to food antigens
and commensal flora. Naive TS mice have higher CD25+
and Foxp3+ mean frequencies among CD4+ lymphocytes
than TR mice (Fig. 4). However, in TS and TR mice fed
with OVA the CD25+ frequencies were similar. Instead,
the significantly higher IL-10 level produced by T cells
in both naive and OVA feed TS mice is suggestive of
Treg cell participation in the discrimination of TS and
TR immune responses. Treg cells are indispensable for
homeostasis and regulation of the immune system, and
there is also a suggestion that multiple signals could
confer resistance to Treg cell suppression.40 TR mice
produce higher prostaglandin E2, intercellular adhesion
10
moelcule, IL-1 and IL-12 levels and acute inflammatory
responses39, probably as a consequence of low IL-10 lev-
els, and these molecules can signal resistance to suppres-
sion.
The quantitative character of OT was demonstrated by
the normal curve of the heterogeneous population used
to start the selection, and by successful production of
mice with extreme phenotypes of OT susceptibility and
resistance (TS and TR strains) selected during 18 genera-
tions of assortative mating (Fig. 5a–c,e).14 The reconstitu-
tion of a segregant F2 population with a normal
phenotype distribution in a widely heterogeneous genetic
background and its use in OT studies allows determina-
tion of the frequency of well-defined phenotypic groups
and can be useful for therapeutic studies in human popu-
lations. (Fig. 5d,h). As TR and TS phenotypes were
selected to OVA-specific OT, we produced F1 hybrids by
reciprocal crosses to test if maternal antibodies could
interfere on the immunological expressions of naive or
antigen-stimulated mice. Both TR and TS mothers pro-
duced OT-resistant offspring (Fig. 5c,g) in these crosses.
The OT-resistant offspring from mating TS females with
TR males, and OT-susceptible offspring from TS females
with TS males, therefore indicate no maternal interference
in the studied mice.
The F1 hybrid response closer to that of the TR mice
(dominance effect), suggests epistatic deviation of OT
genes in the direction of the TR mouse phenotype
(Fig. 5a–c). Recent results show that the TS mice are low-
inflammatory mice while TR and the F1 mice are fivefold
more inflammatory than TS mice39. This is suggestive of
an association between OT and inflammation. Hence, the
opposite capacity of the genetically modified mice may be
involved in co-adaptive mechanisms, reflecting a dynamic
relation between gene frequencies in a natural popula-
tion.29
Our results reinforce the importance of regulatory T
cells producing IL-10 as key players in OT. We suggest
that the disparate immunological profiles of naive or fed
TS and TR mice are the result of OT-selected immuno-
regulatory genes, and that the IL-10 up-modulation in TS
mouse CD4+ T cells and the IL-10 down-modulation in
TR mouse CD4+ T cells are coherent mechanisms to
explain the naive and post-antigen feeding profiles of
both strains of mice.
Acknowledgements
We thank Dr Claudia G. F. Matta (North Fluminense
State University) for kindly supplying IgY, Dr Gerlinde A.
P. B. Teixeira (Federal Fluminense University) for the nut
extracts, and Drs Cid Couto Chaves and Marcia Giesta
(Rio de Janeiro State University) for care of the animal
colonies. The research was supported by CAPES, CNPq,
FAPERJ, FENORTE and FUJB (Brazil).
 2010 Blackwell Publishing Ltd, Immunology

Disclosures
There is no conflict of interest.
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11