The Journal of Immunology, 2001, 167: 4187-4195.
Copyright © 2001 by The American Association of Immunologists
Neonatal Exposure to Antigen Primes the Immune System to Develop Responses in Various Lymphoid Organs and Promotes Bystander Regulation of Diverse T Cell Specificities1
Christopher D. Pack2,
Aimee E. Cestra3,
Booki Min4,
Kevin L. Legge5,
Lequn Li,
Jacque C. Caprio-Young,
J. Jeremiah Bell,
Randal K. Gregg and
Habib Zaghouani6
Department of Microbiology, University of Tennessee, Knoxville, TN 37996
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Abstract
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Neonatal exposure to Ag has always been considered suppressive for
immunity. Recent investigations, however, indicated that the neonatal
immune system could be guided to develop immunity. For instance,
delivery of a proteolipid protein (PLP) peptide on Ig boosts the
neonatal immune system to develop responses upon challenge with the PLP
peptide later. Accordingly, mice given Ig-PLP at birth and challenged
with the PLP peptide as adults developed proliferative T cells in the
lymph node that produced IL-4 instead of the usual Th1 cytokines.
However, the spleen was unresponsive unless IL-12 was provided. Herein,
we wished to determine whether such a neonatal response is intrinsic to
the PLP peptide or could develop with an unrelated myelin peptide as
well as whether the T cell deviation is able to confer resistance to
autoimmunity involving diverse T cell specificities. Accordingly, the
amino acid sequence 8799 of myelin basic protein was expressed on the
same Ig backbone, and the resulting Ig-myelin basic protein chimera was
tested for induction of neonatal immunity and protection against
experimental allergic encephalomyelitis. Surprisingly, the results
indicated that immunity developed in the lymph node and spleen, with
deviation of T cells occurring in both organs. More striking, the
splenic T cells produced IL-10 in addition to IL-4, providing an
environment that facilitated bystander deviation of responses to
unrelated epitopes and promoted protection against experimental
allergic encephalomyelitis involving diverse T cell specificities.
Thus, neonatal exposure to Ag can prime responses in various organs and
sustain regulatory functions effective against diverse autoreactive T
cells.
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Introduction
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Medawar
et al. (1) demonstrated almost half a century ago that
rodents injected at birth with splenocytes from a genetically different
donor were able to accept transplants from that donor as an adult.
These landmark experiments suggested that neonatal T cells were somehow
susceptible to the induction of tolerance. Ever since, the neonatal
period has been viewed as a window during which the introduction of Ag
leads to tolerance. Initially, inactivation and/or deletion of T cells
were considered the leading mechanisms for Ag-induced neonatal
tolerance (2, 3). However, recent investigations have
demonstrated that neonatal exposure to Ag can prime, rather than
inactivate or delete, T cells (4, 5, 6, 7), and both Th1 and Th2
cells develop upon this initial Ag encounter. Surprisingly, rechallenge
with the same Ag later in life elicits a predominant Th2 response
(8, 9, 10, 11, 12). Since this type of biased response does not
support inflammatory reactions, neonatal tolerization with Ag has
been applied to deviation of specific naive T cells and has
proven effective in the prevention of autoimmune diseases such as
experimental allergic encephalomyelitis
(EAE)7
(13) and type I diabetes (14). Although the
exact mechanism for the apparent selective maintenance of Th2 cells is
unknown, it has been suggested that primary Th1 cells that arise upon
neonatal exposure to Ag are more vulnerable to apoptosis
(15). Recent investigations have envisioned the
involvement of regulatory T cells to maintain such biased neonatal
immunity (16). Whatever the mechanism might
be, factors such as the dose (17), the form
(18), the adjuvant administered in conjunction with
(12), the APC presenting (5), as well as the
in vivo availability of the Ag (19) were shown to control
the induction of neonatal tolerance.
Previous investigations using Igs as a vehicle for peptide delivery
revealed yet another bias in neonatal-induced immunity
(20, 21, 22). Indeed, Ig-proteolipid protein (Ig-PLP)1
(23, 24), a chimera encompassing the
PLP139151 epitope (25), given to
mice in saline on the day of birth induced an organ-specific regulation
of T cells involving a deviation in the lymph node and a novel form of
anergy in the spleen (20, 21, 22). Specifically, mice given
Ig-PLP1 on the day of birth and challenged with PLP1 peptide at 7 wk of
age developed PLP1-specific T cells in the lymph node that produced
IL-4 instead of IL-2. In the spleen, the cells, although
nonproliferative and unable to produce IFN-
, secreted significant
amounts of IL-2. Furthermore, when supplied with IL-12 or IFN-
,
these cells regained proliferative and IFN-
responsiveness. However,
free PLP1 peptide given to mice on the day of birth in saline had no
effect on the adult response to a challenge with PLP1 in CFA, and the
animals were not protected against EAE (20, 21). This
suggests that the Ig backbone contributed to development of the novel
form of neonatal immunity seen with Ig-PLP1 (20, 21, 22).
Furthermore, a physical link of the peptide to the Ig is required, as
injection of free peptide mixed with the Ig backbone (referred to as
Ig-W) had no effect on the adult response to PLP1 in CFA
(21). Also, PLP1 in IFA on the day of birth, although
protective against EAE, generates a response to immunization with
peptide in CFA characterized by a deviated T cell response in the
spleen, but the lymph node was unresponsive (20, 22).
Consequently, delivery of peptide on Ig provides adjuvanticity and
confers on the peptide the ability to protect against autoimmunity by a
novel mechanism involving lymph node deviation and IFN-
-dependent
splenic anergy (20, 21, 22).
The study presented herein explores whether this new form of tolerance
is intrinsic to the PLP1 epitope or is applicable to other epitopes
presented on Igs. To address this issue, the myelin basic protein (MBP)
8799 sequence, or MBP3 (26), was incorporated into the
same Ig vehicle, and the resulting Ig-MBP3 chimera was tested for
induction of neonatal immunity and protection against EAE.
Surprisingly, the results show that mice given Ig-MBP3 in saline on the
day of birth and challenged with MBP3 in CFA at the age of 7 wk
developed T cell responses in both the lymph node and spleen. T cells
of both lymphoid organs were deviated and produced IL-4 instead of IL-2
or IFN-
. Furthermore, the splenic, but not the lymph node, T cells
produced elevated levels of IL-10 when subjected to prolonged (72-h)
peptide stimulation. Interestingly, this environment enabled unrelated
autoreactive T cells to diverge into the Th2 pathway, leading to the
prevention of EAE involving diverse T cell specificities.
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Materials and Methods
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Mice
SJL/J (H-2s) mice were purchased from
Harlan Sprague Dawley (Frederick, MD) and maintained in our animal
facility for the duration of experiments. For the generation of newborn
mice, breeding sets of one adult male and three females were caged
together. When pregnancy was visible, the females were separated and
caged individually. Offspring were weaned when they reached 3 wk of
age. All experimental procedures were conducted according to the
guidelines of the institutional animal care committee.
Peptides
All peptides used in this study were purchased from Research
Genetics (Huntsville, AL) and purified by HPLC to >90% purity. MBP3
peptide (VHFFKNIVTPRTP) encompasses an encephalitogenic epitope
corresponding to aa 8799 of MBP (26). PLP2 peptide
(NTWTTCQSIAFPSK) encompasses an encephalitogenic sequence corresponding
to aa 178191 of PLP (27). PLP1 peptide (HSLGKWLGHPDKF)
encompasses an encephalitogenic sequence corresponding to aa 139151
of PLP (25). All three peptides are presented to T cells
in association with I-As MHC class II molecules
and induce EAE in SJL/J mice (25, 26, 27).
Ig chimeras
Ig-MBP3 is a chimera expressing MBP3 peptide that corresponds to
aa 8799 of MBP. Construction of Ig-MBP3 used the genes coding for the
L and H chains of the anti-arsonate Ab, 91A3, according to the
procedures described for the construction of Ig-nucleoprotein
(28). In brief, the
91A3VH gene was subcloned
into the EcoRI site of pUC19 plasmid and used as template
DNA in PCR mutagenesis reactions to generate
91A3VH fragments carrying
the MBP3 (91A3VH-MBP3)
sequence in place of CDR3. The 91A3VH-MBP3
fragment was then subcloned into an expression vector (28)
in front of the exons coding for the constant region of a BALB/c
2b.
This plasmid was then cotransfected into the non-Ig-producing SP2/0
myeloma B cell line with an expression vector carrying the parental
91A3 L chain. Transfectants producing Ig-MBP3 were selected in the
presence of geneticin and mycophenolic acid. Ig-PLP2, which encompasses
aa 178191 of PLP, was previously described (21). Ig-W,
the parental Ig not encompassing any foreign peptide, has also been
described (28). Large scale cultures of transfectoma cells
were conducted in DMEM containing 10% iron-enriched calf serum
(BioWhittaker, Walkersville, MD). Purification of Ig-MBP3, Ig-PLP2, and
Ig-W was conducted on separate columns of rat anti-mouse
-chain
mAb coupled to cyanogen bromide-activated Sepharose 4B (Amersham
Pharmacia Biotech, Piscataway, NJ). Large scale culture was conducted
with bovine serum and media that contain minimal amounts of endotoxin.
Also, since the Ig chimeras were purified by affinity chromatography,
the preparations were free of endotoxins.
RIA
Capture RIA was used to assess the secretion of complete Ig-MBP3
constructs from SP2/0 transfectants. Microtiter 96-well plates were
coated with polyclonal rabbit anti-mouse
2b chain-specific Ab
(Zymed, South San Francisco, CA; 2 µg/ml in PBS) overnight at 4°C
and then blocked with 2% BSA in PBS for 1 h at room temperature.
The plates were then washed three times with PBS, and 100 µl/well of
supernatant from SP2/0 cells growing under selective pressure was
incubated for 2 h at room temperature. After three washes with
PBS, captured Ig chimeras were revealed by incubation with 1 x
105 cpm/well 125I-labeled
rat anti-mouse
mAb (American Type Culture Collection, Manassas,
VA) for 2 h at 37°C. The plates were then washed five times with
PBS and counted using a Wallac LKB gamma counter (Rockville,
MD).
Generation of T cell hybridoma
A T cell hybridoma specific for MBP3 was generated by immunizing
SJL/J mice with 200 µg MBP3 peptide in 200 µl PBS/CFA (v/v) s.c. in
the foot pads and at the base of each limb. After 10 days the draining
lymph nodes were removed, and T cells were stimulated in vitro for two
rounds in the presence of irradiated, syngenic splenocytes, 5% T-Stim
supplement (Collaborative Biomedical Products, Bedford, MA), and MBP3
peptide (15 µg/ml). The culture medium used to carry out these
stimulations and other T cell activation assays in this study was DMEM
supplemented with 10% FCS (HyClone, Logan, UT), 0.05 mM 2-ME, 2 mM
glutamine, 1 mM sodium pyruvate, and 50 µg/ml gentamicin sulfate.
This MBP3-specific T cell line was then fused using polyethylene glycol
4000 (Sigma, St. Louis, MO) with the 
TCR-negative thymoma BW1100
(American Type Culture Collection). Hybrids were selected by
supplementing the culture medium with hypoxanthine-azaserine (Sigma).
The resulting hybridomas were screened for reactivity to MBP3 peptide
by testing for production of IL-2 and IFN-
in the supernatant
following stimulation with irradiated (3000 rad) splenocytes in the
presence of 15 µg/ml MBP3 peptide. Positive hybridomas were then
cloned by limiting dilution and used to assess the presentation of MBP3
peptide from the Ig-MBP3 chimera.
Neonatal injections of tolerogen and adult immunizations with
peptide
Neonatal injections of Ig chimera were performed i.p. in 100
µl saline within 24 h after birth. When the mice reached 7 wk of
age they were subjected to immunization with peptide to analyze their
proliferative and cytokine responses. The immunization of adult mice
with either 200 µg MBP3 or a combination of 200 µg MBP3 and 100
µg PLP2 in 200 µl PBS/CFA (v/v) was conducted s.c. in the food pads
and at the base of the limbs. After 10 days the mice were sacrificed to
examine the elicited immune response.
Induction of EAE
EAE was induced by s.c. injection in the foot pads and at the
base of the limbs with 200 µl IFA/PBS (v/v) solution containing 200
µg Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI)
and MBP3 peptide (200 µg), a mixture of MBP3 (200 µg) and PLP2 (100
µg), or MBP3 (200 µg) and PLP1 (100 µg) peptides. Six hours later
200 ng Bordetella pertussis toxin (List, Campbell, CA) in
100 µl PBS was given i.v.. A second injection of B.
pertussis toxin was given to the mice after 48 h. Mice were
then scored daily for clinical signs of EAE as follows: 0, no clinical
sign; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb
paralysis; 4, forelimb paralysis; and 5, moribund or dead.
Proliferation assays
Lymph node (axillary, lateral axillary, and popliteal) and
spleen cells were incubated in 96-well plates at 4 x
105 and 10 x 105
cells/100 µl/well, respectively, with 100 µl stimulator for 3 days.
MBP3 was used at the optimal dose of 30 µg/ml. Therefore, the control
peptides PLP1 and PLP2 were also used at 30 µg/ml. Subsequently, 1
µCi [3H]thymidine (ICN Pharmaceuticals, Costa
Mesa, CA) was added per well, and culture was continued for an
additional 14.5 h. The cells were then harvested and incorporated
onto glass-fiber filters. [3H]Thymidine was
measured using the Trace 96 program and an Inotech beta counter
(Wohlen, Switzerland). A control of medium without stimulator was
included for each mouse and used as background. All results presented
in the figures represent counts per minute of test samples from which
the background was deducted.
ELISA
Cytokine production by spleen cells was measured as previously
described (20). Briefly, 10 x
105 cells/100 µl/well were incubated with 100
µl stimulator for 24 h, and cytokine production, excluding
TGF-
, was measured by ELISA according to the instructions provided
by the manufacturer (PharMingen, San Diego, CA). TGF-
was measured
according to the Genzyme protocol (Cambridge, MA). Some spleen cytokine
measurements were conducted at 72 h. OD405
was measured on a SpectraMAX 340 counter (Molecular Devices, Sunnyvale,
CA) using SoftMAX PRO 1.2.0 software. Graded amounts of recombinant
mouse IL-2, IL-4, IFN-
, IL-10 (PharMingen), and TGF-
(Genzyme)
were included in all experiments to construct standard curves. The
concentration of cytokines in culture supernatant was estimated by
extrapolation from the linear portion of the standard curve. All
anti-cytokine Abs used in these studies were purchased from
PharMingen, except for the pair used for detection of TGF-
, which
was obtained from Genzyme. Capture Abs were rat anti-mouse IL-2,
JES6-1A12; rat anti-mouse IL-4, 11B11; rat anti-mouse IFN-
,
R4-6A2; and rat anti-mouse IL-10, JES5-2A5. Biotinylated
anti-cytokine Abs were rat anti-mouse IL-2, JES5-5H4; rat
anti-mouse IL-4, BVD6-24G2; rat anti-mouse IFN-
, XMG1.2; and
rat anti-mouse IL-10, JES5-16E3.
ELISPOT assay
ELISPOT assay was used to measure cytokines produced by lymph
node T cells during Ag stimulation as previously described
(20). Briefly, 5 x 105
cells/100 µl/well along with 100 µl stimulator were added to
multiscreen-HA plates (Millipore, Bedford, MA) that had been previously
coated with capture Ab. After 24 h of incubation the plates were
washed and subsequently incubated with biotinylated anti-cytokine
Ab overnight at 4°C. Following incubation with avidin-peroxidase
(Sigma) for 1 h at 37°C, spots were visualized by adding
substrate (3-amino-9-ethylcarbazole; Sigma) and then counted under a
dissecting microscope. The capture and biotinylated anti-cytokine
Abs used for ELISPOT were the same as those used for ELISA.
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Results
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Expression of MBP3 peptide on an Ig molecule drives efficient
presentation to T cells
Recent studies revealed that neonatal exposure to an Ig expressing
the PLP sequence 139151 induced, rather than suppressed, an immune
response (20, 21, 22). This response was organ specific and
involved Th2 deviation in the lymph node and an IFN-
-mediated
splenic T cell anergy in the spleen. This investigation addresses
whether this organ-specific response is intrinsic to the
PLP139151 sequence or whether delivery by an Ig
is the key factor, thus allowing a similar response to develop with
other peptides. In addition, we explored whether any bystander effect
of the Th2 response on the response to other myelin protein epitopes
might occur. To address these issues, an Ig chimera was constructed to
include MBP3 within the H chain CDR3. DNA sequence analysis confirmed
insertion of the nucleotide sequence for MBP3 in the correct reading
frame (Fig. 1
a). The H chain
gene incorporating the MBP3 sequence was then cotransfected with the
parental L chain gene into the non-Ig-secreting myeloma B cell line
SP2/0 to generate cells producing complete Ig molecules. As depicted in
Fig. 1
b, supernatant from an Ig-MBP3 transfectant incubated
on plates coated with anti-
2b Ab bound a rat anti-mouse
L chain mAb, indicating that the mutated H chain paired with the
parental L chain and formed a complete Ig molecule. Ig-W, the parental
91A3 Ab with an intact CDR3 domain, paired as well. The better binding
observed with Ig-MBP3 could be related to the site of DNA integration
as well as the number of copies incorporated into the chromosomal DNA.
Alternatively, peptide insertion may have influenced protein folding in
such a way that the isotypic determinants are better accessible
for Abs.
To test Ig-MBP3 for peptide delivery and presentation to T cells, the
chimera was purified by affinity chromatography from the supernatant of
large scale cultures of transfectant and assayed for presentation using
an MBP3-specific T cell hybridoma, designated TCH-MBP3-A7. This
hybridoma line was generated by fusing MBP3-specific short term T cell
lines with the 
TCR-negative thymoma BW1100. As is evident in Fig. 2
, the T cell hybridoma TCH-MBP3-A7
produced IL-2 and IFN-
upon stimulation with irradiated APCs loaded
with MBP3 peptide. The negative control PLP2 peptide, corresponding to
aa 178191 of PLP and presented by I-As
(27), like MBP3 peptide, did not stimulate the T cells.
Similarly, Ig-MBP3 was able to induce both IL-2 and IFN-
, while
Ig-PLP2, a chimera encompassing PLP2 peptide (21), did
not. In addition, immunization of SJL/J mice with Ig-MBP3 in CFA
induced MBP3-specific T cell responses in both the lymph node and
spleen that were predominantly Th1 in nature, exhibiting MBP3-specific
production of both IL-2 and IFN-
(data not shown). These results
indicate that MBP3 peptide is cleaved from the Ig and presented to T
cells as other peptides expressed on Igs (23, 29, 30, 31, 32).

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FIGURE 2. Presentation of the Ig-MBP3 chimera to specific T cells. Irradiated
(3000 rad) SJL/J splenocytes (5 x 105 cells/100
µl/well) were incubated with graded amounts of Ag, and 1 h later
the culture was supplemented with 5 x 104
MBP3-specific T cell hybridoma, A7. After 24 h the supernatant was
harvested, and 100 µl was used for detection of IL-2
(a) and IFN- (b). PLP2 and Ig-PLP2,
Ags presented by I-As, such as MBP3 and Ig-MBP3, were used
as negative controls. Each point represents the mean ± SD of
triplicate determinations.
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Neonatal injection of Ig-MBP3 confers resistance to induction of
EAE by free MBP3 peptide
Neonatal tolerance was examined by injecting Ig-MBP3 in saline
into SJL/J pups within 24 h after birth, challenging these mice as
adults with a disease-inducing regimen of free MBP3 peptide, and then
scoring daily for paralysis. As Fig. 3
illustrates, the group of mice that received no Ig at birth (Nil group)
exhibited a disease course typical for MBP3, which generally manifests
a mild, monophasic, nonrelapsing/remitting disease pattern
(26). In contrast, mice that were injected with Ig-MBP3 as
neonates showed virtually no clinical manifestations of EAE. The
control mice that received Ig-W, the parental wild-type not containing
the MBP3 epitope, developed a pattern of disease resembling the Nil
group.

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FIGURE 3. Ig-MBP3 neonatally tolerized mice resist induction of EAE by MBP3
peptide. Newborn mice (four to seven per group) were injected with 100
µg affinity-purified Ig-MBP3 (Ig-MBP3 Tol) or Ig-W (Ig-W Tol) in
saline within 24 h of birth and were induced for EAE at 7 wk of
age with free MBP3 peptide as described in Materials and
Methods. Mice were then scored daily for signs of paralysis.
For comparison purposes a group of mice that did not receive any
injection (Nil) was included. The maximal SD for the groups of animals
tested did not exceed 0.6.
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Neonatal injection of Ig-MBP3 leads to normal lymph node
proliferation, reduced responsiveness in the spleen, and Th2 deviation
in both organs
Investigation of the proliferation and cytokine profiles of the
lymph node and spleen of mice injected at birth with Ig-MBP3 and
challenged at 7 wk of age with MBP3 peptide in CFA yielded results
similar to those obtained from neonatal studies conducted with Ig-PLP1
(20, 21, 22). Lymph node proliferation remained unaltered in
Ig-MBP3 tolerized mice vs Ig-W-tolerized mice (Fig. 4
a). In contrast, the lymph
node cytokine production of mice tolerized with Ig-MBP3 revealed a
strong deviation from a Th1 to a Th2 response. Ig-MBP3-tolerized mice
secreted high levels of IL-4 in response to stimulation with MBP3
peptide, while mice injected with Ig-W at birth secreted relatively
none (Fig. 4
b). In addition, while the Ig-W-tolerized mice
showed weak IFN-
production in response to stimulation with MBP3
peptide, the Ig-MBP3 group did not show IFN-
production (Fig. 4
c). These responses were specific for MBP3 peptide, since
the negative control, PLP2 peptide, generated no significant response
in either group. In the spleen, the Ig-MBP3-recipient group
demonstrated significantly reduced proliferation
(p < 0.05) in response to stimulation with
MBP3 compared with mice that had been neonatally injected with Ig-W
(Fig. 4
d). Surprisingly, however, the cytokine profile of
Ig-MBP3 mice revealed deviation to a Th2 phenotype and production of
IL-4 rather than IFN-
upon stimulation with MBP3 (Fig. 4
, e and f). These responses were also specific, as
there was no detectable response to PLP2.

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FIGURE 4. Exposure to Ig-MBP3 on the day of birth drives T cell deviation in both
lymph node and spleen. Newborn mice (eight per group) were injected
i.p. within 24 h after birth with either 100 µg Ig-MBP3 or Ig-W
in saline. When the mice reached 7 wk of age they were immunized with
200 µg free MBP3 peptide in 200 µl CFA/PBS (v/v) s.c. in the foot
pads and at the base of the limbs. Ten days later the mice were
sacrificed, and the draining lymph node (LN; 0.4 x
106 cells/well) and spleen (SP; 1 x 106
cells/well) cells were stimulated with 30 µg/ml free MBP3 or PLP2.
The lymph node (a) and spleen (d)
proliferation was measured by [3H]thymidine incorporation
after 3 days of stimulation. Cytokine production was analyzed in the LN
by ELISPOT (b and c) and in the SP by
ELISA (e and f) after 24 h of
stimulation. The indicated values represent the mean ± SD of
eight individually tested mice. Statistical analysis (Students
t test) was performed. *, Difference between the test
group (Ig-MBP3) and the control (Ig-W) group is significant
(p < 0.05).
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Neonatal exposure to Ig-MBP3 confers resistance to the induction of
EAE with multiple epitopes
Since Ig-MBP3 neonatally tolerized mice exhibited Th2 deviation in
both lymph node and spleen, we hypothesized that such an environment
might be able to modulate the response to a separate myelin epitope
associated with EAE. To address this issue, mice were injected with
either Ig-MBP3 or Ig-W at birth and at the age of 7 wk were assayed for
resistance to EAE induction with multiple epitope regimens. As shown in
Fig. 5
, Ig-MBP3-tolerized mice showed a
significant reduction in clinical paralytic severity when they were
induced for disease with a mixture of MBP3 and PLP2 peptides. Mice that
had received no Ig molecule (Nil group) during the neonatal period and
those injected with the control Ig-W molecule had a normal disease
pattern (Fig. 5
a). In addition, Ig-MBP3 neonatal
tolerization conferred resistance against a regimen including MBP3 and
PLP1 peptides. Although Ig-MBP3-tolerized mice exhibited only a
slightly reduced initial disease peak compared with both the
Ig-W-injected and Nil control groups, they were completely protected
from relapses (Fig. 5
b). In contrast, both control groups
suffered a severe relapse that resulted in a significant mortality rate
(see Table I
).

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FIGURE 5. Ig-MBP3 neonatally tolerized (Tol) mice show resistance to EAE induced
with two epitopes. Newborn mice (510/group) were injected with 100
µg affinity-purified Ig-MBP3 or Ig-W in saline within 24 h of
birth and were induced for EAE at 7 wk of age with a mixture of MBP3
and PLP2 (a) or MBP3 and PLP1 (b)
peptides or with PLP2 alone (c) as described in
Materials and Methods. Mice were then scored daily for
signs of paralysis. A group of mice that did not receive any injection
on the day of birth (Nil) was included for control purposes. The
maximal SD did not exceed 0.6 for groups shown in A and
C and 1 for the groups shown in B.
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To show that the deviated MBP3-reactive T cells must be activated
at the same time as the naive PLP1- or PLP2-specific pathogenic T cells
to achieve protection, EAE was induced in Ig-MBP3- or Ig-W-tolerized
mice with PLP2 peptide, and the signs of paralysis were assessed. As
illustrated in Fig. 5
c, the two groups showed no significant
difference in disease pattern. Therefore, it appears that neonatally
induced MBP3-specific T cells had to be activated by the Ag during
induction of disease with PLP2 to support deviation and/or modulation
of PLP2-specific T cells and prevent the development of EAE.
Organ-specific regulation of IL-10 production in neonatally
tolerized mice
Although both the lymph node and splenic T cells of
Ig-MBP3-tolerized mice were deviated and produced IL-4, the
proliferative response of the splenic cells was significantly reduced
relative to that of their Ig-W counterparts. This suggested that the
splenic T cells might be producing anti-proliferative cytokines
such as IL-10 or TGF-
(34, 35). To test this hypothesis
the MBP3-specific T cells from both lymphoid organs were assayed for
production of IL-10 and or TGF-
. The results indicated that although
after a 24-h stimulation neither TGF-
nor IL-10 was detectable (not
shown), at 72 h a high level of IL-10 was observed in the spleen
(Fig. 6
a). The lymph node T
cells did not produce any detectable IL-10 in either group (Fig. 6
b). Cells from the control mice tolerized with Ig-W did not
secrete a detectable level of IL-10. No TGF-
was observed in either
group (data not shown). This IL-10 production in Ig-MBP3-tolerized mice
was specific for MBP3 peptide, since in vitro stimulation with PLP2
peptide yielded no significant IL-10 production.

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FIGURE 6. Production of IL-10 in the spleen, but not the lymph node, of
Ig-MBP3-tolerized and peptide-immunized mice. Newborn mice (eight per
group) were injected within 24 h after birth with either 100 µg
Ig-MBP3 or Ig-W in saline. When the mice reached 7 wk of age they were
immunized with 200 µg free MBP3 peptide in 200 µl CFA/PBS (v/v)
s.c. in the foot pads and at the base of the limbs. Ten days later the
mice were sacrificed, and the lymph node (0.4 x 106
cells/well) and spleen (1 x 106 cells/well) cells
were stimulated with 30 µg/ml free MBP3 or PLP2 for 72 h. IL-10
production was measured by ELISA in the spleen (SP) and by ELISPOT in
the lymph node (LN). The indicated values represent the mean ± SD
of eight individually tested mice. *, Difference in the amount of
cytokine produced by the Ig-MBP3 group vs that produced by the
corresponding Ig-W group is significantly different
(p < 0.05).
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Since recent studies have revealed that APCs could, under certain
circumstances, produce IL-10 (36), we used purified T
cells and mitomycin C-treated APCs to ascertain whether the IL-10 seen
in the culture was produced by MBP3-specific T cells. To this aim,
purified T cells from Ig-MBP3- and Ig-W-tolerized mice were stimulated
with MBP3 peptide-loaded, mitomycin C-treated splenocytes from naive
SJL/J mice. After 6 days of culture the cytokine production of both
groups was analyzed. Adult splenic T cells derived from
Ig-MBP3-tolerized mice demonstrated a polarized Th2 response
characterized by elevated levels of both IL-4 and IL-10 (Fig. 7
, b and c) in
response to free MBP3 peptide as well as a significant decrease in the
level of IFN-
secretion (Fig. 7
a). Mice that received
Ig-W as neonates mounted only a characteristic Th1 response against
MBP3 peptide typified by IFN-
release (Fig. 7
a). These
responses were specific to MBP3 in each case, as stimulation with the
control peptide, PLP2, yielded negligible cytokine production.

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FIGURE 7. T cells, not APCs, produce the splenic IL-10 in Ig-MBP3-tolerized mice.
Newborn mice (four per group) were injected i.p. within 24 h after
birth with either 100 µg Ig-MBP3 or Ig-W in saline. When the mice
reached 7 wk of age they were immunized with 200 µg free MBP3 peptide
in 200 µl CFA/PBS (v/v) s.c. in the foot pads and at the base of the
limbs. Ten days later the mice were sacrificed, and the spleen was
homogenized to a single-cell suspension and passed over a nylon wool
column to enrich for T cells. Purified T cells (1 x
105 cells/well) were then stimulated for 6 days with APCs
that were previously pulsed with either 30 µg/ml free MBP3 or PLP2
peptide and treated with mitomycin C (50 µg/ml for 30 min).
Subsequently, the production of IFN- (a), IL-4
(b), and IL-10 (c) in the supernatant was
analyzed by ELISA. The indicated values represent the mean ± SD
of four individually tested mice. APCs or T cells alone did not produce
any significant cytokine. *, The amount of cytokine produced by the
corresponding group is statistically significant (p
< 0.05).
|
|
IL-10 produced by neonatally induced MBP3-specific T cells displays
bystander function and suppresses the responsiveness of diverse T cells
In an effort to delineate the mechanism underlying the resistance
to EAE induction with multiple epitopes and to assess the bystander
function of MBP3-specific T cells, mice that received either Ig-MBP3 or
Ig-W at birth were immunized as adults with a combination of MBP3 and
PLP2 peptides in CFA. The T cell response to both peptides was then
analyzed. The results indicate that the spleen proliferation of
Ig-MBP3-tolerized mice was significantly reduced in response to both
MBP3 and PLP2 peptide (Fig. 8
a) compared with that of
Ig-W-tolerized mice. Even more surprising, PLP2-specific T cells
produced significant amounts of IL-4, and their IFN-
levels were
reduced (Fig. 8
, b and c). Further examination of
IL-10 production indicated that these cells were producing elevated
levels (Fig. 8
d). The mice tolerized with Ig-W instead of
Ig-MBP3 had significant proliferation and IFN-
production in
response to both peptides, with neither IL-4 nor IL-10 observed.

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FIGURE 8. Neonatal injection of Ig-MBP3 induces bystander suppression of diverse
T cell specificities. Newborn mice (eight per group) were injected i.p.
within 24 h after birth with either 100 µg Ig-MBP3 or Ig-W in
saline. When the mice reached 7 wk of age they were immunized with a
combination of 200 µg MBP3 and 100 µg PLP2 peptides in 200 µl
CFA/PBS (v/v) s.c. in the foot pads and at the base of the limbs. Ten
days later the mice were sacrificed, and the spleen (1 x
106 cells/well) cells were stimulated with 30 µg/ml MBP3,
PLP2, or PLP1. Proliferation (a) was measured after 3
days of stimulation, the cytokines IL-4 (b) and IFN-
(c) were measured after 24 h of stimulation, and
IL-10 (d) was measured after 72 h of stimulation.
The indicated values represent the mean ± SD of eight
individually tested mice. *, Data are statistically significant
(p < 0.05).
|
|
To evaluate the contribution of IL-10 to the resistance against
multiple epitope-induced EAE, mice neonatally tolerized with Ig-MBP3
were subjected to induction of EAE with a mixture of MBP3 and PLP2
peptides and given anti-IL-10 mAb. As shown in Fig. 9
, in vivo neutralization of IL-10 by
anti-IL-10 Ab restored the severity of EAE to a level comparable to
that obtained in the susceptible mice neonatally injected with Ig-W.
The control group injected with rat IgG instead of anti-IL-10 mAb,
like the Ig-MBP3 neonatally tolerized mice not given anti-IL-10
during disease induction, failed to restore the severity of paralysis.
These results indicate that splenic IL-10 produced by MBP3-specific T
cells probably contributes to bystander suppression of newly migrant,
MBP3-reactive, as well as unrelated T cells and prevents EAE even when
diverse T cell specificities are involved.

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FIGURE 9. Requirement of splenic IL-10 for bystander suppression of EAE involving
diverse T cell specificities. Newborn mice were injected within 24
h of birth with 100 µg Ig-MBP3 in saline. When they reached 7 wk of
age, a group of four mice was injected i.p. with 1 mg/mouse
affinity-purified JES5-2A5 anti-IL-10 Ab in 1 ml PBS. A second
group of four mice was injected with 1 mg/mouse of rat IgG in 1 ml PBS
to serve as a control. On the next day EAE was induced in all mice with
a combination of free MBP3 and PLP2 peptides as described in
Materials and Methods. Five days after disease induction
the mice were given a second injection of 1 mg/mouse of JES5-2A5 or rat
IgG. The mice were scored daily for signs of paralysis. For comparison
purposes the clinical scores of a group of mice that was injected with
either Ig-MBP3 or Ig-W at birth, but did not receive any Ab treatment,
were included. The maximal SD for the groups of animals did not exceed
0.5. Tol, Tolerized.
|
|
 |
Discussion
|
|---|
In recent years it has become clear that neonatal exposure to Ag
primes, rather than ablates, immunity. However, neonatal-induced
responses in most cases evolve to tolerate the Ag and do not mediate
the inflammatory reactions that usually develop upon immunization of
animals that have not been exposed to the Ag during the neonatal stage
(8, 9, 13, 14). Moreover, in addition to these qualitative
differences, neonatal exposure to Ag seems to drive an organ-specific
regulation of T cells that supports the development of responses in the
spleen, but leaves the lymph nodes unresponsive (12).
However, if the antigenic peptide is given to newborns on an Ig, T cell
responses attempt to develop in the spleen upon rechallenge with Ag
later in life (20, 21, 22). Indeed, SJL/J mice given Ig-PLP1
on the day of birth and challenged with PLP1 peptide as adults
generated lymph node T cells, but the development of splenic response
required exogenous IL-12 (20). Specifically, the lymph
node T cells were deviated and produced IL-4 instead of IL-2, and the
splenic cells, although nonproliferative and unable to produce IFN-
,
still secreted significant amounts of IL-2 (20). Exogenous
IL-12 or IFN-
restored splenic proliferation both in vitro and in
vivo. In the present studies using an Ig chimera incorporating the
encephalitogenic MBP8799 amino acid sequence
(referred to as MBP3), we found that neonatal exposure to Ig-MBP3
promoted immune responses in both the lymph node and spleen. However,
splenic T cells, unlike the Ig-PLP1 system, were proliferative and
produced IL-4, thereby extending deviation to both lymphoid organs. As
the deviation was broadened to both the lymph nodes and spleen, we
sought to test the T cells for bystander modulation of neighboring
cells and suppression of disease involving diverse T cell
specificities. Indeed, mice tolerized with Ig-MBP3 at birth exhibited
significantly reduced disease severity when they were induced for EAE
with a combination of MBP3 and PLP2 peptides or MBP3 and PLP1 peptides
(Fig. 5
, a and b, and Table I
). Also, as
resistance to disease was not present when Ig-MBP3 neonatally tolerized
mice were induced for EAE with PLP2 peptide, we concluded that
neonatally deviated MBP3-specific T cells had to be stimulated during
disease induction for bystander T cell modulation to occur (Fig. 5
c). Furthermore, to determine how Ig-MBP3-induced Th2 cells
were capable of regulating unrelated pathogenic cells and modulating
the disease, two types of analyses were performed. On the one hand, we
tested the neonatally induced MBP3-specific Th2 cells for production of
IL-10, a cytokine that has previously been shown to mediate bystander
suppression (37, 38) and exert pleiotropic,
down-regulatory effects on ongoing Th1 immune responses (for review,
see Ref. 34). On the other hand, we analyzed the
proliferative and cytokine responses to PLP2 peptide upon immunization
of Ig-MBP3 neonatally tolerized mice with the MBP3 and PLP2 peptide
mixture. Splenic MBP3-specific T cells that arise in Ig-MBP3 neonatally
tolerized mice upon stimulation with MBP3 peptide produced, along with
IL-4, copious amounts of IL-10 when cultured in vitro for 72 rather
than 24 h (Fig. 6
a). IL-10 production was Ag specific
and occurred only when the stimulator was MBP3 peptide. Neutralization
of the splenic IL-10 during disease induction restored the severity of
EAE, indicating that IL-10 plays a major role in the resistance to
disease induction (Fig. 9
). IL-10 and IL-4 produced by MBP3-specific T
cells provide an environment suitable for the development of Th2 cells,
but unfavorable for the generation of Th1 cells. This statement is
supported by the observation that immunization of Ig-MBP3
neonatally tolerized mice with a combination of MBP3 and PLP2 generates
weakly proliferating, deviated T cells to both peptides (Fig. 8
). More
striking, the PLP2-specific T cells also produce significant amounts of
IL-10 (Fig. 8
d). In addition, since neutralization of IL-10
restores disease, it is possible that during adult immunization IL-10
produced by MBP3-specific T cells exerts bystander suppression on both
newly migrant, MBP3-reactive T cells that were not exposed to Ig-MBP3
during the neonatal period and PLP2-specific cells, thereby limiting
Th1 development. Moreover, the simultaneous presence of IL-4 might
drive these cells to differentiate into noninflammatory Th2 cells.
Overall, these observations raise two significant points. The first
relates to how unrelated pathogenic T cells are deviated during
immunization of Ig-MBP3 neonatally tolerized mice with a mix of
peptides, and the second concerns the source of IL-10. One likely
explanation for the deviation of PLP2 responses is that IL-4 and/or
IL-10 from MBP3 cells guided the development of PLP2-specific cells
into the Th2 pathway (39). This would assume that the
cells producing IL-4/IL-10 cytokine differentiate at the same site as
stimulated memory MBP3-specific T cells. As for the production of
IL-10, two sources may be considered. The deviated MBP3-specific T
cells secreting IL-4 could be the producer of IL-10 seen in the
culture. However, the delay in such production relative to IL-4 (72 vs
24 h) suggests that the two cytokines are produced by different
cells. It has been previously shown that regulatory T cells can arise
in an IL-10-rich environment (40) and secrete significant
levels of IL-10 (41, 42). Therefore, one could envision
that the splenic cell population includes regulatory cells that
function as a source of IL-10 and regulates autoimmunity
(41, 42, 43, 44). This statement may garner support from our
recent findings showing that exposure to Ig peptide during the neonatal
stage prevents up-regulation of CD40 ligand (CD40L) expression
(45), a phenomenon that could result in suboptimal
expression of B7 molecules on APCs and favor the development of
regulatory T cells (44). If this is the case, a question
arises as to whether regulatory cells are Ag specific. If not, what is
the underlying mechanism that triggers their expansion and
colocalization with pathogenic T cells to modulate the disease?
The other issues that arise from these observations relate to the
factors driving neonatal T cells to deviate to Th2 and home either to
the lymph node only in Ig-PLP1-induced neonatal immunity or to both the
lymph node and the spleen in the Ig-MBP3 system. Neonatal T cells have
been shown to express quantitatively reduced levels of CD40L
(46). In addition, when the exposure uses Ig-peptide
instead of free peptide, CD40L expression remains at background levels
(45). Consequently, upon Ag recognition on APCs,
CD40-CD40L interactions would be limited, resulting in little or no
production of IL-12 and a biased T cell differentiation. In recent
studies we have shown that the splenic T cells in Ig-PLP1-tolerized
mice lack CD40L expression and could not progress in the
differentiation pathway (47). However, since the same SJL
mouse strain and Ig backbone are used in the Ig-MBP3 and Ig-PLP1
systems, minimal expression of CD40L may not account for the
differential pattern among T cells induced by Ig-PLP1 vs Ig-MBP3.
Therefore, it is possible that a discrepancy in peptide affinity among
PLP1 and MBP3 influences T cell-APC interactions, contributes to
differential regulation of CD40L, and leads to an unbalanced Th2 bias
between the two systems. Alternatively, as the autoimmune T cell
repertoire in the SJL mouse is dominated by PLP1-specific T cells
(48), it is possible that higher T cell frequency in the
PLP1 system is responsible for the emergence of anergic T cells in the
spleen (45).
Overall, the neonatal immune system can be guided to develop responses
in both the spleen and lymph node and protect animals against
autoimmunity involving diverse T cell specificities.
 |
Footnotes
|
|---|
1 This work was supported by startup funds (to H.Z.) from the University of Tennessee (Knoxville, TN). 
2 Current address: Laboratory of Viral Immunology, Department of Microbiology, University of Tennessee, Knoxville, TN 37996. 
3 Current address: Antigenics, Inc., Woburn, MA 01801. 
4 Current address: Laboratory of Immunology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Building 10, Room 11N314, Bethesda, MD 20892-1892. 
5 Current address: Beirne B. Carter Center for Immunology Research, University of Virginia, 400 Lane Road, MR-4 Building, P.O. Box 801386, Charlottesville, VA 22908-1386. 
6 Address correspondence and reprint requests to Dr. Habib Zaghouani at the current address: Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine. M616 Medical Sciences Building, Columbia, MO 65212. E-mail address: zaghouanih{at}health.missouri.edu 
7 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; CD40L, CD40 ligand; MBP, myelin basic protein; PLP, proteolipid protein. 
Received for publication May 1, 2001.
Accepted for publication August 8, 2001.
 |
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J. J. Bell, B. Min, R. K. Gregg, H.-H. Lee, and H. Zaghouani
Break of Neonatal Th1 Tolerance and Exacerbation of Experimental Allergic Encephalomyelitis by Interference with B7 Costimulation
J. Immunol.,
August 15, 2003;
171(4):
1801 - 1808.
[Abstract]
[Full Text]
[PDF]
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A.-C. Field, L. Caccavelli, M.-F. Bloch, and B. Bellon
Regulatory CD8+ T Cells Control Neonatal Tolerance to a Th2-Mediated Autoimmunity
J. Immunol.,
March 1, 2003; |