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-Specific CD8+ T Cell Tolerance1
Département d’Immunologie, Institut Pasteur (Unité de Recherche Associée 161, Centre National de la Recherche Scientifique, et Université Pierre et Marie Curie), Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-positive Ig (Ig) on the C-specific
CD8+ T cell response of knock-out (-/-) pups
resulting from various crosses and foster nursings. These systems
allowed control of physiologic transmission of Ig at defined periods
of ontogeny. Our data show that conventional transfer of maternal Ig
via the placenta plus colostrum/milk or adoptive transfer via only the
colostrum/milk were the most efficient at tolerizing C-specific
CD8+ responses. Surprisingly, tolerance was not detected in
-/- pups born to +/- females obtained by cesarean delivery and
suckled by -/- mothers (transplacental supply only). Tolerance,
which was strong until 5 wk of age, was reversible and waned with the
decrease of Ig serum concentration. Depletion of CD4+ T
cells at the time of C peptide immunization abolished the tolerance
of C-specific CD8+ T cells. These data suggest that an
oral supply of Ig is very efficient at inducing and maintaining
tolerance of C-specific CD8+ T cells, at least for
several weeks after birth, and that suppression rather than deletion is
responsible for this tolerance. In addition, they strengthen the view
that tolerance of CD8+ T cells to a soluble Ag is never
permanently acquired even if it is present in large quantities during
ontogeny. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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More recently, the influence of maternal Ig was investigated in nonimmunized mothers. Comparison of experiments made in F1 scid/+ pups nursed by scid/scid or scid/+ mothers led to the conclusion that milk IgA delay both the development of germinal centers in gut-associated lymphoid tissues and the concentration of serum IgA (12). Conversely, using reciprocal crosses between B cell-deficient and C57BL/6 mice, serum Igs were shown to stimulate B cell development in offspring (13) without modifying the B cell repertoire (14).
Few data are available on the influence of maternal Igs on the T cell
immune responses in offspring. Anti-idiotypic T cells expressing a
V
2315-specific TCR transgene were shown to be deleted in
mice synthesizing 2315 Tg, i.e., in double (TCR x
2315) transgenic mice, but not in mice receiving passive
transfer of maternal transgenic 2315 chain (15). In
contrast, IgG2 ab allotype-specific T cell tolerance was
shown to be strong but reversible in offspring born to
IgG2ab-treated mothers and injected postnatally for several
months with the same allotype (16, 17). In addition, anti-isotypic
CD4+ T cells specific for IgE were shown to be tolerant in
6- to 8-wk-old mice that had received IgE peptide at birth
(18).
To investigate the influence of maternal Ig on the offspring’s T cell
response, we focused our attention on the role of these Ig on the
C-specific CD8+ T cell response of -/- offspring
that were physiologically exposed to high concentrations of Ig.
Indeed, the concentration of light chain
(L)3-bearing Ig reaches 10
mg/ml in serum and is expressed by >90% of B cells in mice. We have
previously shown that spleen cells from +/+ mice are able to elicit,
in 129/Sv (H-2b) C knock-out mice, the emergence of a
diverse repertoire of Kb-restricted C-specific
CD8+ clones recognizing a single peptide of the constant
region (C) of the L (19).
In the present study, we analyzed the cytolytic response of the
following pups: 1) -/- pups born to +/- mothers and suckled
until weaning by +/- mothers (placental plus oral Ig transfer); 2)
-/- pups born to +/- mothers and suckled in the few hours
after birth by -/- mothers (placental plus short term oral Ig
transfer); 3) -/- pups born by cesarean to +/- mothers and
suckled by -/- mothers (placental transfer only); 4) -/- pups
born to -/- crosses and suckled by +/- mothers for various
periods of time (oral transfer only).
The data presented in this study show that maternal Ig induces a
strong but reversible state of tolerance of C-specific
CD8+ T cells and emphasize the role of milk-transmitted Ig
in the maintenance of tolerance until at least 6 wk of age. This
tolerance can be readily reversed by injection of anti-CD4 mAb at
the time of C-peptide immunization, suggesting that suppression,
rather than deletion or anergy, is responsible for the tolerance of
C-specific CD8+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-deficient 129 (H-2b -/-)
mice, generated by targeted mutation in the C gene from 129/Sv ES
cells (20), were used in this and our previous study (19). Their
wild-type counterparts, 129/Sv (+/+) mice, were bred in the animal
facilities of the Institut Pasteur (Paris, France). The following
crosses and foster nursing systems were used. +/- heterozygous
females were mated with -/- males. Their -/- offspring were
either nursed by their own mothers, to assess the effect of placenta
plus colostrum/milk-derived maternal Ig, or fostered at birth or at day
19.5 of embryonic age (E19.5; birth at day 20 in 129/Sv mice) by
-/- mothers, to assess the influence of maternal Ig delivered
by placenta only. Pups born to -/- parents and suckled at birth by
+/- foster mothers were analyzed to study the effect of Ig derived
only from colostrum/milk for various periods of time. The -/- pups
were identified by FACS analysis at 3 to 4 wk of age or when required
at the time of in vitro immunization, as described previously (21). PCR
analysis was conducted to identify -/- E19.5 (-0.5)-day-old pups
used in quantification of Ig titers (20).
Quantification of serum Ig titers
ELISA analysis was conducted with the Southern Biotechnology
Associates kit (cat. no. 5300.05; Birmingham, AL). Briefly, Luxlon
microplates were coated with 2.5 µg/ml of affinity-purified goat
anti-mouse Ig in PBS for 18 h at 4°C. After two washes with
PBS/0.5% Tween, plates were saturated for 30 min at room temperature
with PBS/0.5% BSA and incubated for 5 h at room temperature with
serial dilutions of serum samples from individual mice or known
concentrations of 65-1 anti-ß-galactosidase mouse 
1 mAb (E.
Barbier et al., unpublished observations). After three washes, plates
were incubated for 1 h at room temperature with anti-mouse
Ab coupled to horseradish peroxidase (Southern Biotechnology
Associates, Kit No. 5300.05), then washed four times and the presence
of Ig revealed by ATBS (2,2’-azino-di-[3-ethylbenzthizoline]
suffonate diammonium salt crystals) substrate (Sigma Chemical, St.
Louis, MO) diluted at 1.5 mg/ml in citrate buffer and
H2O2 according to the manufacturer’s
recommendations. OD were read at 405 nm. Concentrations of Ig were
calculated on the basis of reference absorbance values of a standard
curve of 65-1 anti-ß-galactosidase mouse 1 mAb and
expressed in mg/ml.
Immunizations and in vivo treatment with anti-CD4 mAbs
Mice (4 to 23 wk old) were injected s.c. at the base of the tail
with 10 µg C(NOREF130–144) peptide (Chiron Mimotopes, Clayton,
Australia and Lyon, France) emulsified in 200 µl of IFA. Ten-day-old
pups received one s.c. injection of 50 µl of IFA containing 2.5 µg
of peptide. For CD4 in vivo depletion, mice received, as described
previously (22), three injections of 300 µg (at days -1, 0, and +1
of immunization) of a Na2SO4 fraction of
GK1.5 rat anti-CD4 isolated from ascitic fluid raised in nude mice
(23). Control mice received the same volume of PBS. At day 8, FACS
analysis, conducted as previously described (21), revealed that spleen
cells from GK1.5-treated mice contained <0.4% CD4+
cells.
In vitro restimulation of in vivo-immunized mice and cytotoxicity assay
Eight days after immunization, spleen cells were incubated at
37°C in 5% CO2, for 5 days, at 3 x
106 cells/ml in 10 ml or 2 ml of RPMI containing 10% FCS,
100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 2 mM
pyruvate, and 50 µM 2-ME in 25-cm2 flasks (in an upright
position) or in 24-well microplates, respectively, in the presence of
0.15 µg/ml of peptide. At the end of the incubation period, all
cultures were tested, at serial E:T ratios, on 51Cr-labeled
and peptide-loaded EL4 cells (TIB 64, American Type Culture Collection,
Manassas, VA) or on 2-day-cultured 51Cr-labeled LPS blasts
from +/+ or -/- mice in a classical chromium release assay, as
described in our previous study (19). Data are expressed as percentage
of specific lysis displayed at E:T = 9O:1, except in Figure 1
, in
which all of the E:T ratio values are reported. Student’s t
test was used for statistical analysis, and p values 
0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(NOREF130–144) peptide
Using five independent C-specific CTL clones obtained from 129
-/- mice immunized with 129 +/+ spleen cells, we demonstrated
previously that the L-specific CD8 cytolytic response is restricted
in H-2b mice to a single sequence contained in the
C(NOREF130–144) peptide (19). We asked next whether this peptide was
immunogenic in 129 mice. Figure 1
shows
that a single s.c. injection of 10 µg C(NOREF130–144) peptide
emulsified in IFA induces in 129 -/-, but not in 129 +/+ mice,
CTL that are able to lyse LPS blasts from +/+ mice or
C(NOREF130–144)-loaded EL4 cells but not LPS blasts from -/- mice
or unloaded EL4. Immunization with 100 µg of peptide did not
significantly increase the CTL response of -/- mice and did not
induced a C-specific T cell response in 129 +/+ mice. These data
suggest that 129 +/+ mice are tolerant to this peptide. In addition,
they show that the peptide that we had identified as being recognized
by C-specific T cell clones in vitro is immunogenic in 129 -/-,
strengthening the view that this peptide sequence encompasses the
peptide constitutively expressed on +/+ APC. Accordingly, in the
following experiments the C-specific cytolytic response was tested
only on +/+ and -/- LPS blasts, which do not express the
constitutive C peptide.
C-specific CD8+ T cell tolerance in -/-
pups born to +/- mothers
Bogen et al. (15) previously demonstrated that T cells expressing
a V2315-specific TCR transgene were deleted in
double (TCR x 2315) transgenic mice but not in
single TCR transgenic pups, which received maternal 2315
through both placenta and colostrum/milk routes, suggesting that B
cells must be present in the thymus to induce T cell tolerance. To
determine whether these conclusions should be considered as a general
rule in Ig systems, we assessed the functionality of the C-specific
CD8+ T cells of non-TCR transgenic -/- pups born to
+/- females. In this situation, -/- pups were exposed to large
amounts of Ig from fetal life until weaning. Data reported in Figure 2a show that the cytolytic
activity of in vitro-restimulated spleen cells of -/- pups,
immunized at 5 wk of age with C(NOREF130–144), was markedly reduced when
compared with those of age-matched immunized control -/- mice (in
4 of 10 pups, the response was completely abolished). This tolerance
was specific inasmuch as the response to OVA(NOREF257–264) peptide was not
altered in similar pups (data not shown). These data indicate that in
non-TCR transgenic mice, maternal L is able to induce tolerance in
offspring that are themselves unable to synthesize L.
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-specific CTL tolerance in
-/- pups born to +/- mothers and immunized at different ages.
Figure 2b shows that the CTL activity of 5-wk-old -/-
spleen cells was extremely low (3.5% of specific lysis). As early as 6
wk, the response reached one-half of the control response (28.5 ±
9.9% vs 65.8 ± 7.9%) and returned to normal by 12 wk. In this
kinetic study, -/- adult mice were used as positive controls
because the cytolytic responses of C(NOREF130–144)-immunized -/-
mice remains constant in mice tested between 10 days and 8 mo of age
(data not shown).
The concentration of Ig in the sera of -0.5 (E19.5)- to 77-day-old
-/- and +/- pups born to +/- mothers was then assessed. As
shown in Figure 2c, the concentration of Ig in both
-0.5-day-old -/- and +/- pups reaches 1.10 mg/ml ±
0.006. Similar concentrations were found in 4-wk-old -/- mice
(1.26 mg/ml ± 0.02), gradually decreasing to 0.005 mg/ml by 6 wk
of age. In +/- pups, the concentration of Ig increases with age,
reaching the adult level in 6 wk.
These data indicate that the tolerance of C-specific T cells under
physiologic conditions is strong but reversible and wanes with the
decrease of serum Ig.
Oral transfer of maternal Ig induces tolerance in C-specific CTL
responses
In the pups examined in the previous section, tolerance could be
induced by a combination of placental and oral/mucosal Ig transfer. To
assess the importance of each of these routes in tolerance induction,
we first examined the C-specific T cell response in 5-wk-old
-/- pups born to -/- mothers but suckled by +/- mothers
from birth (oral transfer) and in -/- pups born to +/- mothers
and fostered in the few hours following birth by -/- mothers
(transplacental plus short term oral transfer).
Data reported in Figure 3 show that the
C-specific CTL response is greatly reduced in -/- pups that
have been suckled by +/- foster mothers, compared with control mice
(group 2 vs group 1, p < 0.001), indicating that
maternal Ig transferred by the oral route are able to induce tolerance
in C-specific CTL. Conversely, the CTL response of -/- pups
born to +/- mothers and fostered at birth by -/- mothers until
weaning was similar to that of age-matched control -/- mice (group
3 vs group 1, p = NS). This first set of experiments
shows that the tolerance observed can be induced and maintained by
colostrum/milk Ig.
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-/- pups born to +/- mothers and
fostered at birth by -/- mothers showed any evidence of tolerance,
CTL responses were examined in similar pups immunized sooner. Indeed,
as shown in Figure 4, these pups
immunized at 10 days displayed a significant reduction of the CTL
response, as compared with 10-day-old -/- pups born to and suckled
by -/- mothers (groups 3 and 2, p < 0.005). These
data indicate that C-specific CTL of -/- pups born to +/-
mothers and fostered at birth by -/- mothers had been tolerized,
but this tolerance was not maintained no more than 6 wk of
age.
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-/- pups born to +/- mothers and fostered at birth by -/-
mothers might be achieved by transfer of colostrum-derived maternal Ig
during the first few hours after birth, i.e., before transfer of pups
to -/- mothers, the experiments were repeated with -0.5
(E19.5)-day-old fetuses obtained by cesarean delivery from +/-
mothers and fostered by -/- mothers. Figure 5 clearly shows that in the total absence
of oral transfer, tolerance was not detectable in 10-day-old immunized
pups (compare group 4 with group 3); the lower response than in
unmanipulated 10-day-old mice (group 2) could easily be explained by
stress induced by the cesarean delivery, because pups from regular
-/- obtained by cesarean (group 3) also have a lower response.
Group 5, which consists of -/- pups born to and suckled by similar
+/- mothers as those of group 4, was used as a positive control of
tolerance.
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-/- pups born to and suckled by +/-
mothers.
Tolerance of C-specific CTL can be reversed by anti-CD4 mAb
treatment
Comparison of the response of -/- pups born to +/-
mothers suckled from birth (Fig. 2a) with those
delivered by cesarean and fostered by -/- mothers (Fig. 5) shows
that oral tolerance plays an important role in the tolerance of
C-specific CTL in -/- mice born to and suckled by +/-
mothers. It is now well established that orally delivered Ag induces
tolerance to the same Ag administered subsequently by the parenteral
route (see Ref. 24 for a recent review). Depending on the concentration
of the tolerogen, clonal deletion or anergy of Ag-specific cells or
induction of regulatory T cells can occur (25, 26). Clonal deletion
could not be approached in our system because of the low frequency of
C-specific CTL in these nontransgenic mice. Clonal anergy elicited
in oral tolerance has been reported to be reversible by high
concentrations of rIL-2 (27). In two sets of experiments, we were
unable to reverse tolerance by incubating spleen cells with various
concentrations of rIL-2 at the time of in vitro restimulation,
suggesting that anergy is not involved (data not shown).
In pilot experiments, we observed that C-specific responses in
immunized -/- mice are not dependent on CD4 T cells, as depletion
of these cells in -/- regular mice did not affect the emergence of
C-specific CTL (data not shown). Therefore, to determine whether
CD4+ regulatory T cells were involved in the tolerance that
we observed, both 5-wk-old -/- pups born to +/- mothers and
-/- pups from regular -/- parents but suckled by +/-
mothers were treated with three injections of 300 µg of depleting
anti-CD4 mAb at days -1, 0, and +1, with peptide given at day 0.
The results are illustrated in Figure 6.
As expected, the CTL response of PBS-treated -/- offspring born to
+/- mothers was absent or strongly reduced as compared with that of
-/- controls from -/- parents. In contrast,
anti-CD4-treated pups from the same litter showed a response
similar to that displayed by control -/- mice (Fig. 6a). Similar data were obtained with
anti-CD4-treated -/- pups from regular -/- mothers but
suckled by +/- mothers (Fig. 6b). Taken together,
these data strongly suggest that CD4+ regulatory T cells
are involved in the tolerance of the C-specific CD8+ CTL
response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-specific CTL,
which has been well characterized for peptide and TCR repertoires (19).
To this aim, we have produced different crosses and various foster
nursings to allow the immune system of knock-out pups to develop in
the presence of large amounts of Ig for defined periods of time:
either during fetal life only, during fetal life and after birth, or
after birth only, for short or prolonged periods of time. The major
findings in our study are: 1) oral supply of Ig via milk/colostrum is
very efficient at inducing and maintaining tolerance of the
C-specific CD8+ CTL response; 2) suppressor CD4 T
cells are involved in, or responsible for, CD8 tolerance induced by
Ig; and 3) tolerance of C-specific CD8+ CTL, although
strong initially, is reversible. Transplacental vs colostrum/milk transfer of maternal Ig
Maternal Abs have long been known to protect mammals against
pathogens during ontogeny (see Ref. 1 for a recent review). However,
their effect on the induction of T cell tolerance has been poorly
documented (15, 16, 17, 18). In mice and rats, maternal Ig can be supplied by
an active process in utero through uptake of IgG via placenta and
after birth by absorption of IgG from colostrum/milk (28). A small but
significant level of maternal Ig has been detected by day 15 of
gestation (29). In both routes, uptake of IgG is ensured by a similar
if not identical FcRn. This receptor consists of an 
-chain homologue
to a class I molecule plus ß2-microglobulin and is
expressed in mice and rats in the fetal yolk sac and in mice in the
small intestinal epithelium until day 16 after birth (30, 31, 32).
Our experiments with -/- offspring born to +/- mothers clearly
show that maternal Ig induces tolerance in C-specific CTL until 5
wk of age. This tolerance progressively wanes with the disappearance of
maternally transmitted Ig. In 5-wk-old pups, inhibition of the
C-specific CTL response is strong, despite a large decrease of
maternal Ig compared with the concentration of Ig in 4-wk-old
pups. This is even more striking in 6-wk-old mice, in which only trace
amounts of circulating Ig are detected, while the cytotoxicity is
still one-half of the control response (see Fig. 2). Therefore, L
peptides on APCs probably maintain tolerance for some time, after which
the peptide concentration must become too low to sustain tolerance and
recovery of the C-specific T cell response occurs. Our data confirm
and extend those of Majlessi et al. (16, 17), in showing that tolerance
of C-specific T cells is not permanently acquired even in the
presence of physiologically high concentrations of tolerogen from early
ontogeny until 4 wk of age (see Fig. 2c), i.e.,
during a critical period for the setting up of the immune system. In
addition, our data strengthen the notion that, except for tolerance of
T cells specific for Ags expressed on the surface of cells or in
certain situations for virus-specific T cells (33, 34, 35, 36, 37, 38), T cell
tolerance induced by soluble Ags is always reversible (17, 36, 39) and
wanes with the decrease of Ag concentration, whatever nature, dose,
route, or ontogenic time at which the Ag is administered.
Of all the data obtained in our various foster nursing systems, the
most surprising are those showing that tolerance is not detectable in
10-day-old -/- pups that have been delivered by cesarean from
+/- mothers and transferred to suckling -/- mothers (Fig. 5, group 4). This is in contrast with moderate but highly significant
levels of tolerance displayed by -/- pups born to similar +/-
mothers and transferred to -/- foster mothers in the 10 h
following birth, then immunized at 10 days of age (Fig. 4, group 3).
The concentrations of Ig in the late term (E19.5 = -0.5 day
old) -/- fetus sera and those of +0.5-day-old newborns born to
similar mothers were 1.10 ± 0.006 mg/ml and 1.13 ± 0.07
mg/ml, respectively. Inasmuch as these concentrations were nearly
identical, two nonexclusive possibilities can explain these data: 1)
the difference in the duration in which the fetal (E19.5) and
newborn immune systems see Ig (4.5 days in utero in E19.5 pups and 5
days in utero + 0.5 days ex utero in newborns); and 2) the
efficiency of the oral route through the mucosal system at inducing
tolerance or in maintaining tolerance induced during in utero
development.
We observed that the transfer of maternal Ig for only 10 to 18 h
to -/- pups born to -/- parents via colostrum could not, by
itself, ensure tolerance of C-specific T cells (data not shown).
These data suggest that maternal Ig transferred through the placenta
might contribute to the tolerance observed in -/- pups born to
+/- mothers and fostered at birth by -/- mothers and that
colostrum-derived Ig maintain tolerance in these pups (Fig. 4, group 3).
Our data clearly show that CD8+ T cells in 5-wk-old
-/- pups born to -/- parents and suckled until weaning by
+/- mothers are tolerant to Ig (Fig. 3). These data confirm in a
new antigenic system that mucosal delivery of Ag is an efficient
mechanism of induction of Ag-specific tolerance (See Refs. 24 and 40
for recent reviews). Although this mechanism has been well established
in several antigenic systems in adult mice and rats, controversy
remains regarding the efficiency of this tolerizing method in newborn
or young animals (41, 42, 43, 44, 45). Our data are in line with Peppard’s data,
which demonstrated that the feeding of young rats with IgG
anti-horseradish peroxidase mAb induced a profound suppression of
the Ab response to this Ag (41). They are, however, in contrast with
those of Ryelandt et al., who did not detect T cell tolerance to
deaggregated human gamma globulin (dHGG) through lactation (42). This
discrepancy could be explained by different dose of "tolerogen,"
which in our system consists of Ig expressed in the mother’s serum
at 10 mg/ml and which persists at constant level in the offspring sera
from at least day 19.5 of gestation until 4 wk of age. In the Ryelandt
et al. study, the tolerogen consisted of 20 mg of human gamma globulin
(HGG) given only twice to mothers of suckling mice, immediately after
delivery and 3 days later.
Our data are also in apparent contrast with those of Bogen et al. (15).
In their models, anti-idiotypic T cells expressing a
V2315-specific TCR transgene, which are deleted in
double transgenic mice, could not be deleted by passive transfer of
maternal transgenic 2315 chain through the placenta and
milk. However, it is important to stress that the frequency of cells to
be tolerized in transgenic mice is extremely high in comparison with
the frequency of C-specific CD8 cells. Moreover these authors did
not check the functional capabilities of the
V2315-specific cells in such mice.
Altogether, our data confirm in a new antigenic system and extend to CD8+ cells the view that the nature, dosage, and age at which Ag is orally delivered are important parameters in the resulting immune response (43, 44, 45).
Mechanism of induction of C-specific CTL tolerance
Our data show that suppression of induction of C-specific CTL
in -/- pups born to and suckled by +/- mothers and in -/-
pups born to regular -/- crosses but suckled by +/- mothers
could be abrogated by concomitant administration of depleting
anti-CD4 mAb. Accordingly, CD4+ T cells are
involved in the tolerance observed. In the absence of C-specific TCR
transgenic mice, we cannot exclude the deletion of some C-specific
CD8+ T cells. However, the recovery of a C-specific CTL
response, which is high in most and total in some pups within 8 days
following anti-CD4 mAb treatment, strongly suggests that
suppression is the dominant mechanism underlying tolerance in our
system. Deletion of the majority of C-specific CD8 clones would most
likely require the presence in the thymus of B cells synthesizing the
appropriate chain, as postulated by Bogen et al. (15).
Suppression of CD4+ T cell function has been reported to be mediated by CD4+ regulatory T cells in several antigenic systems and in some autoimmune diseases following oral administration of Ag (40, 46, 47). Both CD4+ and CD8+ T cell activities could also be suppressed by CD8+ regulatory T cells (48, 49). Conversely, inhibition of CD8+ T cell activity by CD4+ T cells in adult mice has been reported following oral administration of Ag (50, 51), but to our knowledge, this is the first report of such regulatory T cells in newborns.
There is evidence that oral administration of high doses of Ag in adult
animals favors clonal deletion or anergy, while low doses stimulate
regulatory T cells producing inhibitory cytokines such as TGF, IL-4,
and IL-10 (24, 26, 27). In our study, Ig were delivered actively and
daily through FcRn from day E15 until gut closure at day 16 postpartum
(30, 31, 32). The concentration of maternal Ig found from -0.5 day to 4 wk
of age shows that high amounts of Ig have crossed the placental
barrier and the gut epithelium. Therefore, even in the presence of a
high concentration of Ag, tolerance can result from suppressor
mechanisms. Consequently, we suggest that not only the dosage of orally
administred Ag but also its nature and probably the means by which it
is taken up by the mucosal system (FcRn or phase fluid pinocytosis) are
determining factors in the mechanisms leading to oral tolerance.
We have been unable to identify the cytokines synthesized by
CD4+ regulatory T cells or to observe proliferative
responses in lymph node cells from tolerant -/- mice immunized
with L or C(NOREF130–144) peptide. The reason for this failure is
probably threefold. 1) The peptide specificity of these cells is
unknown. CD4+ regulatory T cells might recognize a
L-derived peptide inasmuch as the only difference in +/- and
-/- is the presence or the absence of the L chain. However, we
cannot exclude the recognition of peptides derived from other proteins
(see below). 2) Regulatory CD4+ T cells have been shown to
belong to the Th2, Th3, or T regulatory cells (Tr1) subtypes and to
have low proliferative capacity (26, 52). 3) These cells are likely
present in very low frequency, as previously suggested for Ig-specific
CD4+ T cells (21, 53, 54).
How can CD4+ T cells suppress C-specific
CD8+ CTL responses?
Several hypotheses can be put forward. 1) CD4+
regulatory T cells may act directly on CD8+ T cells by
recognizing a L-derived peptide on CD8+ T cells. This
hypothesis is very unlikely in our system because the L-derived
peptide would have to be presented by class II molecules, which have
not been detected on mouse CD8+ T cells. 2) CD4 suppressor
T cells may act directly on CD8+ T cells by recognizing a
V- or Vß-derived peptide expressed by C-specific CD8 T cell
TCRs. This mechanism was reported in a reversed situation by Jiang et
al. in which regulatory CD8+ T cells recognized a
Vß-derived peptide presented by Qa-1 molecules on autologous
CD4+ T cells (55). This hypothesis should not apply to our
system because the CD8+ response induced toward the
C-peptide we used is diverse (19) and the intensity of suppression
of the response we observed strongly suggests that the majority of the
CD8+ clones become tolerant to L during the neonatal
period. 3) CD4+ regulatory T cells may belong to the
NK1.1+, V 14-J 281+ T cell subpopulation
that recognizes the CD1 molecule (56). This is a possible explanation
for our results because, in addition to lipoglycan and mycolic acid,
CD1 binds hydrophobic peptides (57), which are found in the C
sequence, including the C(NOREF>130–144) peptide. In this hypothesis,
CD4+ T cells should act through a bystander effect. A
population using this particular V rearrangement has already been
shown to suppress a keyhole limpet hemocyanin (KLH)-specific Ab
response (58). 4) CD4+ suppressor T cells may act via a
bystander effect on CD8+ T cells, each cell recognizing the
same or a different L-derived peptide presented on the same APC by
class II and class I molecules, respectively. This mechanism of
suppression has been shown to occur in vivo in several autoimmune
situations (24, 47). Different strategies are currently used in our
laboratory to test these hypotheses.
In conclusion, we have described a system that allows us to dissociate
the influence of placental vs oral delivery of maternal Ig in the
induction of tolerance of C-specific CTL. Our data strongly suggest
that oral transfer of maternal Ig is more efficient at inducing and
maintaining tolerance than placental transfer and that regulatory T
cells with suppressor activity are involved in this process.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dominique Rueff-Juy, Département d’Immunologie, Institut Pasteur (Unité de Recherche Associée 161, Centre National de la Recherche Scientifique and Université Pierre et Marie Curie) 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. E-mail address:
3 Abbreviations used in this paper: L, light chain; C, L-constant region; -/-, knock-out mice; E, embryonic age in days; FcRn, neonator Fc Receptor.
Received for publication January 8, 1998. Accepted for publication March 23, 1998.
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1–3 dextran and phosphorylcholine by early administration of monoclonal antiidiotype antibody. H. Köhler, and J. Urbain, and P.-A. Cazenave, eds. Idiotypy in Biology and Medicine 187. Academic Press, San Diego.
knock out mice of a diverse cytotoxic T lymphocyte repertoire that recognizes a single peptide from the immunoglobulin constant light chain region. Eur. J. Immunol. 25:2752.[Medline]
subtype expression in the B cell repertoire of mice with a disrupted or normal C gene segment. Int. Immunol. 6:711.): identification of a naturally processed self-C peptidic region by CD4+ T cell hybridomas obtained in L-deficient mice. Cell. Immunol. 180:84.[Medline]
-chain gene in a functional, antigen-specific suppressor-T cell hybridoma. Proc. Natl. Acad. Sci. USA 83:8708.This article has been cited by other articles:
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  C. M. Snyder, K. Aviszus, R. A. Heiser, D. R. Tonkin, A. M. Guth, and L. J. Wysocki Activation and Tolerance in CD4+ T Cells Reactive to an Immunoglobulin Variable Region J. Exp. Med., November 8, 2004; (2004) jem.20031234. [Abstract] [Full Text] [PDF]
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 O. Annacker, R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa, A. Cumano, and A. Bandeira CD25+ CD4+ T Cells Regulate the Expansion of Peripheral CD4 T Cells Through the Production of IL-10 J. Immunol., March 1, 2001; 166(5): 3008 - 3018. [Abstract] [Full Text] [PDF]
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M. Faure, S. Calbo, J. Kanellopoulos, A.-M. Drapier, P.-A. Cazenave, and D. Rueff-Juy Tolerance to Maternal Immunoglobulins: Resilience of the Specific T Cell Repertoire in Spite of Long-Lasting Perturbations J. Immunol., December 15, 1999; 163(12): 6511 - 6519. [Abstract] [Full Text] [PDF]
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