By
From the Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996
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Abstract |
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Induction of neonatal T cell tolerance to soluble antigens requires the use of incomplete
Freund's adjuvant (IFA). The side effects that could be associated with IFA and the ill-defined mechanism underlying neonatal tolerance are setbacks for this otherwise attractive strategy for
prevention of T cell-mediated autoimmune diseases. Presumably, IFA contributes a slow antigen release and induction of cytokines influential in T cell differentiation. Immunoglobulins
(Igs) have long half-lives and could induce cytokine secretion by binding to Fc receptors on
target cells. Our hypothesis was that peptide delivery by Igs may circumvent the use of IFA and
induce neonatal tolerance that could confer resistance to autoimmunity. To address this issue
we used the proteolipid protein (PLP) sequence 139-151 (hereafter referred to as PLP1),
which is encephalitogenic and induces experimental autoimmune encephalomyelitis (EAE) in
SJL/J mice. PLP1 was expressed on an Ig, and the resulting Ig-PLP1 chimera when injected in
saline into newborn mice confers resistance to EAE induction later in life. Mice injected with Ig-PLP1 at birth and challenged as adults with PLP1 developed T cell proliferation in the
lymph node but not in the spleen, whereas control mice injected with Ig-W, the parental Ig
not including PLP1, developed T cell responses in both lymphoid organs. The lymph node T
cells from Ig-PLP1 recipient mice were deviated and produced interleukin (IL)-4 instead of
IL-2, whereas the spleen cells, although nonproliferative, produced IL-2 but not interferon (IFN)-. Exogenous IFN-
, as well as IL-12, restored splenic proliferation in an antigen specific manner. IL-12-rescued T cells continued to secrete IL-2 and regained the ability to produce IFN-
. In vivo, administration of anti-IL-4 antibody or IL-12 restored disease severity.
Therefore, adjuvant-free induced neonatal tolerance prevents autoimmunity by an organ-specific regulation of T cells that involves both immune deviation and a new form of cytokine- dependent T cell anergy.
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Introduction |
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Nearly half a century ago, it was shown that mice injected at birth with splenic cells from an allogeneic donor were later able to accept grafts from the same donor (1). Ever since, the neonatal period has been considered a critical window during which an initial contact with antigen will instruct the immune system to ignore such an antigen and not respond to it during a subsequent encounter.
Soon after the discovery that T cells recognize rather short antigenic fragments, tolerization regimens using peptides in IFA were defined that induced clonal unresponsiveness and mediated neonatal tolerance (2, 3). However, recent developments indicated that antigen injection during the neonatal stage can immunize rather than suppress (4, 5). In allogeneic systems it was discovered that graft acceptance by neonatally tolerized animals was due to the development of functionally deviated T cells producing Th2-type cytokines instead of the usual Th1 cytokines produced by T cells of nontolerized mice (6, 7). These Th2 cells are unable to support development of the cytolytic T lymphocytes required for graft rejection (8). Similarly, neonatal in-jection of a self-peptide in IFA, which protected mice from autoimmune disease induction, was found to operate through clonal unresponsiveness in the lymph nodes accompanied by induction of deviated Th2 cells in the spleen (9). The notion that neonatal injection of antigen can immunize is now well accepted, and evidence has accumulated indicating that factors such as the type of APCs (5, 10), the adjuvant into which the antigen is emulsified (9), the dose of antigen (11), and the availability of antigen in vivo (12) could engender various outcomes ranging from inactivation of T cells to the priming of an immune response. Therefore, in the face of this susceptibility to regulation, it is important to further investigate the outcome of neonatal antigen injection, particularly to self-antigens, since neonatal tolerance could provide a potential strategy for prevention of autoimmunity.
IFA, which has been required for tolerance induction by soluble proteins and peptides, may exert adjuvanticity by contributing a slow release of antigen and inducing cytokine production by APCs (13). However, the use of IFA may generate side effects and trigger oil-induced arthritis (14).
Igs are autologous molecules permissive for peptide expression and can function as a delivery system for T cell
epitopes (15). Internalization of Igs into APCs via FcRs
grants the incorporated peptide access to newly synthesized
MHC class II molecules, leading to efficient peptide presentation and T cell activation (19). For instance, when
the proteolipid protein (PLP)1 139-151 peptide sequence
(hereafter referred to PLP1) was expressed on an Ig molecule, the peptide's presentation was increased by 100-fold
(20). Furthermore, the Ig-PLP1 chimera was a potent inducer of PLP1-specific T cell responses both in the spleen
and lymph nodes (20, 21). Since Igs are long-lived molecules in vivo, presentation could persist for a long period of
time. In addition by binding FcR on APCs, Igs can trigger
production of cytokines (22, 23). These functions may provide the Ig delivery system with adjuvant-like properties
that could circumvent the use of IFA for tolerance induction and prevention of autoimmunity. Here, we report
that Ig-PLP1 injected into newborn mice in saline confers
resistance to experimental autoimmune encephalomyelitis (EAE) induction by a novel mechanism defined by IL-4
driven lymph node deviation and an unusual IFN--mediated splenic proliferative unresponsiveness.
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Materials and Methods |
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Mice.
6-8-wk-old SJL/J mice (H-2s) 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, and 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 carried out according to the guidelines of the institutional animal care committee.Peptides.
All peptides used in these studies were purchased from Research Genetics, Inc. (Huntsville, AL) and purified by HPLC to >90% purity. PLP1 peptide (HSLGKWLGHPDKF) encompasses an encephalitogenic sequence corresponding to amino acid residues 139-151 of PLP (24, 25). PLP1 peptide is presented to T cells in association with I-As MHC class II molecules. PLP2 peptide (NTWTTCQSIAFPSK) encompasses an encephalitogenic sequence corresponding to amino acid residues 178-191 of PLP (26). This peptide is also presented by I-As MHC class II molecules and induces EAE in SJL/J mice.Ig-PLP Chimeras.
Expression of PLP1 peptide on Ig-PLP1 has been described previously (20). Ig-W, the parental Ig not encompassing PLP1 peptide, has been described elsewhere (27). The genes used to construct these chimeras are those coding for the light and heavy chains of the anti-arsonate antibody, 91A3, and the procedures for deletion of the heavy chain CDR3 region and replacement with nucleotide sequences coding for PLP1 are similar to those described for the generation of Ig-NP (27). Both Ig-W and Ig-PLP1 are from IgG2bNeonatal Injections and Adult Immunizations.
Neonatal injections of Ig chimeras were done intraperitoneally in 50-100 µl saline. When the mice reached 7 wk of age they were subjected to immunization with peptide in CFA or to EAE induction. Immunization of adult mice with peptide in CFA was carried out subcutaneously in the foot pads and at the base of the limbs and tail. The peptide was emulsified in a 200 µl PBS/CFA (vol/vol) mixture. 10 d later the mice were killed by cervical dislocation, and the spleens and lymph nodes (axillary, inguinal, popliteal, and sacral) were removed for analysis of proliferative and cytokine responses.Induction of EAE.
EAE was induced by subcutaneous injection in the foot pads and at the base of the limbs with a 200 µl IFA/PBS (vol/vol) solution containing 100 µg free PLP1 peptide and 200 µg Mycobacterium tuberculosis H37Ra. 6 h later 5 × 109 inactivated Bordetella pertussis were given intravenously. After 48 h, another 5 × 109 inactivated B. pertussis were given to the mice. Mice were scored daily for clinical signs as follows: 0, no clinical signs; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis; 4, forelimb paralysis; and 5, moribund or death.Assay of Neonatal Splenic and Thymic Cells for Ig-PLP1 Presentation.
Newborn mice were injected intraperitoneally with 100 µg Ig-PLP1 or Ig-W in 100 µl saline. 2 d later the spleen and thymus were removed, and single cell suspensions were prepared, irradiated (3000 rads), and assayed for stimulation of the PLP1-specific T cell hybridoma 4E3 (28) without addition of exogenous peptide. In brief, graded numbers of splenic or thymic cells were incubated with 5 × 104 4E3 cells, and after 24 h the supernatant was removed and IL-2 production, used as a measure of T cell activation, was determined. [3H]Thymidine incorporation upon incubation of the supernatant with the IL-2-dependent HT-2 cell line was used for detection of IL-2 as described previously (20).Lymph Node and Spleen T Cell Proliferation.
Lymph node and spleen cells were incubated in 96-well flat-bottomed plates at 4 × 105 and 10 × 105 cells/100 µl per well, respectively, with 100 µl of stimulator for 3 d. Subsequently, 1 µCi [3H]thymidine was added per well, and the culture was continued for an additional 14.5 h. The cells were then harvested on glass fiber filters and incorporated [3H]thymidine was counted using the trace 96 program and an InotechAssays for Restoration of Splenic T Cell Proliferation with Exogenous Cytokines.
Spleen cells from mice that were injected at birth with Ig-PLP1 and immunized as adults with PLP1 in CFA were incubated in 96-well flat-bottomed plates at 10 × 105 cells/100 µl per well with 100 µl media containing the stimulator peptide and the exogenous cytokine (IFN-Measurement of Cytokines by ELISA.
Spleen cells were incubated in 96-well round-bottomed plates at 10 × 105 cells/100 µl per well with 100 µl of stimulator for 24 h. Cytokine production was measured by ELISA according to PharMingen's instructions (San Diego, CA) using 100 µl of culture supernatant. Capture antibodies were rat anti-mouse IL-2, JES6-1A12; rat anti-mouse IL-4, 11B11; rat anti-mouse IFN-Measurement of Cytokines by ELISPOT Assay.
ELISPOT assay was used to measure cytokines produced by lymph node T cells during antigen stimulation as previously described (9). HA-multiscreen plates (Millipore, Bedford, MA) were coated with 100 µl/well 1 M NaHCO3 buffer containing 2 µg/ml capture antibody. After an overnight incubation at 4°C the plates were washed three times with sterile PBS and free sites were saturated with DMEM culture media containing 10% fetal calf serum for 2 h at 37°C. Subsequently, the blocking media was removed and 5 × 105 lymph node cells/100 µl per well were added along with 100 µl of antigen and incubated for 24 h at 37°C in a 7% CO2 humidified chamber. The plates were then washed and 100 µl of biotinylated anticytokine antibody (1 µg/ml) was added. The anticytokine antibody pairs used here were those described for the ELISA technique. After overnight incubation at 4°C the plates were washed and 100 µl of avidin-peroxidase (2.5 µg/ml) was added. The plates were then incubated for 1 h at 37°C. Subsequently, spots were visualized by adding 100 µl of substrate (3-amino-9-ethylcarbazole, from Sigma Chemical Co., St. Louis, MO) in 50 mM acetate buffer, pH 5.0, and counted under a dissecting microscope. ![]() |
Results |
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The nucleotide
sequence coding for PLP1 peptide was genetically inserted
into the variable region of an Ig heavy chain gene and the
resulting gene chimera was transfected together with the gene encoding the parental light chain into the non-Ig-
secreting myeloma B cell line, SP2/0 (20). Selection with
the appropriate drugs allowed for the generation of transfectants producing complete Ig molecules with PLP1 peptide embodied within the heavy chain CDR3 region.
Affinity chromatography-purified Ig-PLP1 was efficiently presented to PLP1-specific T cell hybridomas (20) and induced in vivo T cell responses in both the lymph nodes and
spleen that were of Th1 type and produced both IL-2 and
IFN- (20, 21).
To assay for induction of neonatal tolerance, 1-d-old mice were injected with Ig-PLP1 in saline, and 7 wk later were challenged with a disease-inducing regimen of free PLP1 peptide and scored daily for paralysis. As can be seen in Fig. 1 A, the mice were resistant to induction of paralysis and developed mild monophasic clinical signs of EAE. The mean maximal disease severity was 2.7 ± 0.5 with 0% mortality rate. However, mice that were injected with Ig-W, the parental wild-type Ig not encompassing any PLP peptide (27), developed severe clinical signs of EAE with a 4.2 ± 0.9 mean maximal disease severity accompanied by a 40% mortality. The surviving mice from the group that received Ig-W at birth exhibited a relapsing and remitting disease that persisted to day 100, whereas animals that were injected with Ig-PLP1 did not display any relapses after recovery from the initial mild disease (Fig. 1 B).
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To investigate the mechanism underlying tolerance induction in newborn mice, we first determined if Ig-PLP1 is presented by neonatal APCs in vivo. Mice were injected with Ig- PLP1 at birth, and 2 d later thymic and splenic cells were irradiated and assayed for stimulation of the PLP1-specific T cell hybridoma 4E3 (28) without addition of exogenous antigen. Fig. 2 shows that both thymic and splenic cells from mice that were injected with Ig-PLP1 stimulated the 4E3 hybridoma, whereas cells from mice that received Ig-W instead of Ig-PLP1 did not. These results indicate that Ig-PLP1 was taken up and processed by neonatal APCs and that PLP1-I-As complexes were displayed on the surface of these APCs. Next, we investigated the consequences of such neonatal Ig-PLP1 presentation on the outcome of a later challenge with PLP1 peptide. Newborn mice were injected at birth with either Ig-PLP1 or the control Ig-W, challenged with PLP1 peptide in CFA when they reached 7 wk of age, and then examined for proliferative responses in both lymph node and spleen at day 10 after challenge. The results in Fig. 3 indicate that those animals, that received Ig-PLP1 at birth developed proliferative responses to PLP1 peptide in the lymph node but not the spleen, whereas mice that were injected with Ig-W developed proliferative responses in both lymphoid organs. Neither group responded to the negative control PLP2 peptide, corresponding to amino acid residues 178- 191 of PLP (26). Therefore, exposure to Ig-PLP1 during the neonatal stage affects the response to a challenge with PLP1 peptide and leads to lymph node proliferation and splenic unresponsiveness.
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Injection of free PLP1 peptide in saline at birth had no effect on the outcome of a challenge with PLP1 at 7 wk of age. Indeed, when newborn mice were injected with PLP1 peptide in saline and challenged at 7 wk of age with PLP1 in CFA, both the lymph node and spleen developed T cell proliferative responses (Fig. 4). These responses were comparable to those obtained in mice that were not subject to any injection at birth but were immunized with PLP1 peptide in CFA at 7 wk of age. In contrast, mice injected at birth with free PLP1 peptide emulsified in IFA and challenged with PLP1 peptide in CFA at the age of 7 wk developed T cell proliferation in the spleen but were unresponsive in the lymph node (Fig. 4). The slight reduction in the splenic response in mice recipient of PLP1/IFA at birth versus those injected with PLP1/saline is not statistically significant (unpaired Student's t test analysis, P > 0.1). The results obtained with PLP1/IFA injection are in good agreement with data reported for other peptides (9). Consequently, injection of Ig-PLP1 in saline at birth displays an organ-specific regulation of the T cell that is different from the regulation induced by injection of free peptide in IFA or saline (Fig. 4).
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Examination of the cytokine production in the
lymph node and spleen of mice injected at birth with Ig-
PLP1 and challenged at 7 wk of age with PLP1 peptide in
CFA revealed yet another unexpected result. As can be
seen in Fig. 5, the lymph node T cells from the Ig-W recipient group produced IL-2 but not IL-4 or IFN-, whereas T cells from the Ig-PLP1 recipient group produced IL-4 instead of IL-2. This cytokine production
was specific for PLP1 peptide, since PLP2 was unable to
stimulate the cells for cytokine production. In the spleen,
the Ig-W group produced both IL-2 and IFN-
upon
stimulation with PLP1, whereas cells from the Ig-PLP1 group, although nonproliferative, produced IL-2 and
dropped IFN-
to undetectable levels (Fig. 6). In vitro
stimulation with PLP2 peptide had no effect on cytokine
production. IL-4 was undetectable with either stimulation
in both groups of mice (Fig. 6). IL-10 was undetectable in
all groups of mice (data not shown).
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Because the splenic cells from
mice that received Ig-PLP1 at birth produced IL-2 but
could not proliferate or secrete IFN- upon stimulation
with PLP1 peptide, we reasoned that the defect in proliferation might be related to the deficiency in IFN-
. To address this issue, mice that received Ig-PLP1 at birth were challenged with PLP1 peptide in CFA, and the splenic cells
were in vitro stimulated with PLP1 peptide in the presence
of exogenous IFN-
or IL-12 (an inducer of IFN-
) and
assayed for proliferation. As can be seen in Fig. 7 A, exogenous IFN-
restored proliferation of the spleen cells upon
stimulation with PLP1 peptide. The restoration of proliferation is antigen specific, since addition of exogenous IFN-
did not restore proliferation when in vitro stimulation was
carried out with PLP2 peptide. Furthermore, IL-12 was
also able to restore splenic proliferation (Fig. 7 B). IL-12
restoration of proliferation also proved to be antigen specific and did not develop when in vitro stimulation was
carried out with PLP2 peptide. The restoration of T cell
proliferation by IL-12 was completely inhibited when the in
vitro stimulation was carried out in the presence of 5 µg/ml
of the anti-CD4 mAb, GK 1.5, indicating that the proliferating cells are CD4 T cells (data not shown). In addition, the anti-I-As mAb, 10-2.16, inhibited IL-12 restoration of
proliferation, indicating the requirement for peptide presentation (data not shown).
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Although IFN--restored splenic T cell proliferation did
not induce IL-4 production, the production of IL-2 was
slightly reduced in comparison to stimulation without cytokine addition (Table 1, IFN-
). In addition, exogenous
IL-12, which restored proliferation but slightly reduced the
amount of IL-2, restored production of IFN-
by the splenic
T cells (Table 1, IL-12). Restoration of IFN-
production
was antigen specific because it required stimulation with
PLP1 and did not occur when PLP2 was used for stimulation.
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To evaluate the contribution of lymph node IL-4 and splenic anergy to the resistance against disease induction, mice, neonatally tolerized with Ig-PLP1, were subjected to EAE induction while exposed to anti-IL-4 mAb or rIL-12. As can be seen in Fig. 8, administration of 11B11 rat anti-mouse IL-4 mAb 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 11B11 mAb, like the Ig-PLP1 neonatally tolerized mice that were not given anti-IL-4 during disease induction, did not restore paralysis. The mice treated with anti- IL-4 mAb had a 4.0 ± 0.9 mean maximal disease severity, a score comparable to the 4.2 ± 0.9 obtained with the Ig-W tolerized mice, whereas those treated with the rat IgG instead of 11B11 mAb had a mean severity of 2.7 ± 0.2, which is comparable to the 2.7 ± 0.5 of the mice tolerized with Ig-PLP1 but not treated with anti-IL-4. The rate of mortality was 40% in the anti-IL-4 treated mice and 0% in those treated with the rat IgG. Overall, in vivo neutralization of IL-4 restores severe EAE.
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Administration of IL-12 also restores disease severity in Ig-PLP1 neonatally tolerized mice (Fig. 9). Indeed, mice injected with Ig-PLP1 during the neonatal stage and exposed to rIL-12 during disease induction developed a pattern of paralysis much more severe than mice that did not receive rIL-12. In fact, the severity followed a profile similar to that of the susceptible mice recipient of Ig-W during the neonatal stage. The mean maximal disease severity in these mice was 3.8 ± 0.8 and the rate of mortality was 29%. These values are significantly higher than the 2.7 ± 0.5 mean maximal disease severity and 0% death obtained in mice that were not administered with rIL-12.
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Interestingly, the spleen cells from mice tolerized with Ig-PLP1 at birth and immunized with PLP1 peptide at adult life re-acquired proliferative capabilities when the animals were given rIL-12 during peptide immunization (Fig. 10). Indeed, the splenic T cell proliferative responses in these mice were significantly higher than those obtained in the mice that were not treated with rIL-12. In addition, the proliferation was optimal as it was only slightly higher than the proliferation of cells provided rIL-12 in vitro only and moderately lower than the proliferation of cells exposed to rIL-12 both in vivo and in vitro (Fig. 10).
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Discussion |
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Ig-PLP1, an Ig molecule expressing the encephalitogenic sequence 139-151 of PLP, injected in saline into newborn mice was efficiently presented by neonatal thymic and
splenic APCs in vivo (Fig. 2). Consequently, the mice
showed resistance to EAE induction later in life (Fig. 1).
Examination of the T cell responses to PLP1 in mice that
received Ig-PLP1 at birth and challenged with PLP1 at 7 wk
of age indicated a strong peptide-specific proliferative response in the lymph node but a markedly reduced splenic
proliferation (Fig. 3). Control animals injected at birth with
Ig-W, the parental Ig not encompassing PLP1 peptide, developed proliferative responses in both lymphoid organs
(Fig. 3). In the lymph node there was a cytokine deviation
from IL-2, in Ig-W recipient mice, to IL-4 in Ig-PLP1 recipient mice (Fig. 5). In the spleen, the control mice injected with Ig-W at birth produced both IL-2 and IFN-, whereas Ig-PLP1 recipient mice, despite the absence of T
cell proliferation produced IL-2 but IFN-
was undetectable (Fig. 6). The nonproliferative splenic T cells were able
to recover from this status and proliferated when stimulated
with PLP1 peptide in the presence of IFN-
or IL-12 (Fig.
7). The restoration of proliferation by exogenous cytokines
was inhibited by anti-CD4 and anti-MHC class II antibodies indicating that the target cells are CD4-positive T cells
requiring peptide presentation for reacquisition of the proliferative status. In addition, these splenic T cells, once recovered, became able to produce IFN-
(Table 1). In vivo, when the Ig-PLP1 tolerized mice were given anti-IL-4
mAb during disease induction, the severity of EAE was restored (Fig. 8). Similarly administration of rIL-12 during
peptide immunization or disease induction restored both
splenic proliferation (Fig. 10) and disease severity (Fig. 9).
A summary of these results is illustrated in Table 2. The
overall conclusion that could be drawn from these data is
that the placing of a peptide in the context of an Ig for delivery to the neonatal immune system circumvents the use
of IFA to confer resistance to disease induction later in life.
Moreover, the results reveal a new mechanism operating neonatal tolerance. As indicated in Table 2, the lymph
node T cells were deviated and produced IL-4 instead of
IL-2, and the splenic T cells, although nonproliferative,
produced IL-2 and regained the ability to proliferate when
provided IFN-
or IL-12. We wish to define this phenomenon as IFN-
-mediated anergy.
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T cell deviation has previously been associated with neonatal tolerance in allogeneic (6, 7), viral (11), and peptide/ IFA (9) antigen systems. In the Ig-PLP1 model, there is a
unique organ-specific T cell regulation characterized by a
deviation in the lymph node and an unusual IFN--mediated anergy in the spleen. In fact, when free PLP1 peptide
was injected in saline at birth, the response to challenge
with PLP1 in CFA was normal (Fig. 4). Moreover, when
free PLP1 peptide was injected into newborn mice in IFA,
a challenge with PLP1 in CFA induced significant proliferation in the spleen but the lymph node was unresponsive (Fig. 4). Similar results were reported in an MBP/IFA
model, where the proliferative splenic T cells were deviated and produced Th2-type cytokines (9). The lymph
node IL-4 may play an important role in the resistance to
disease induction. This statement is supported by the observation that neutralization of IL-4 by the administration of
anti-IL-4 antibody restores the severity of disease. In addition, an IL-4-mediated bystander effect (29) may have
been responsible for the suppression of epitope spreading
and related relapses (30, 31) in the Ig-PLP1-tolerized mice.
The second striking observation is that the splenic T cells
of mice injected with Ig-PLP1 at birth and challenged with
PLP1 peptide as adults could not proliferate (Fig. 3 B) or
produce IFN- (Fig. 6 C). However, there was production
of IL-2 (Fig. 6 A). Moreover, these cells regained the ability to proliferate when supplied with IFN-
or IL-12, a cytokine defined as an inducer of IFN-
(Fig. 7; references
32, 33). This phenomenon may qualify for the term anergy, with the distinction that the T cells produce IL-2 but
are unable to produce IFN-
. Therefore, we propose the
term IFN-
-mediated anergy to differentiate it from standard T cell anergy (34). The fact that the T cells produce
IFN-
when they regain proliferative capacity upon supply
of IL-12 further justifies the involvement of IFN-
in this
form of unresponsiveness. Moreover, since administration
of rIL-12 was able to restore both in vivo splenic T cell
proliferation and disease severity, it maybe that bystander
reactivation of these cells is responsible for the residual EAE
observed in Ig-PLP1 tolerized mice. Also, the question as
to the factors involved in the initiation and perpetuation of
these altered cellular responses mediating resistance to disease induction remains unanswered. On a speculative basis, it could be reasoned that neonatal presentation of Ig-PLP1
involves specific APCs that strongly attach Ig-PLP1 and
coordinately regulate expression of costimulatory molecules and/or cytokine production. Under these circumstances, the T cell-APC interactions and the local cytokine
environment could be affected, leading to the generation of
T cells for which differentiation is still susceptible to regulation. During challenge with antigen these cells would be
subject to organ-specific regulation. Alternatively, neonatal
presentation of Ig-PLP1 could be generating T cells deficient in IFN-
production. During antigen challenge those
restimulated in regional lymph nodes would be subject to
the effects of CFA and possibly default to the Th2 pathway,
whereas those restimulated systemically remain dependent
on IFN-
for proliferation and differentiation.
The deficiency in IFN- and the dependency of splenic
T cell proliferation on such a cytokine constitute another
puzzle in these observations. IL-12, a key cytokine for the
differentiation of T cells into effector Th1 cells (35, 36) restored proliferation and IFN-
production by splenic T
cells (Fig. 7 B and Table 1). It could be possible that the
splenic T cells, although producing the growth factor IL-2,
are defective in the biochemical signals required for differentiation and consequent IFN-
production.
Overall, neonatal injection of antigen, generally thought of as a strategy for induction of unresponsiveness appears to function for immunization in this Ig-peptide model as well as in other systems (4, 5, 9). In addition, since immune deviation provides the possibility for bystander T cell downregulation (29, 37), the Ig-peptide strategy may evolve as a means of vaccination without the need for adjuvant against autoimmune diseases involving multiple antigens.
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Footnotes |
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Address correspondence to Habib Zaghouani, The University of Tennessee, Department of Microbiology, M409 Walters Life Sciences Bldg., Knoxville, Tennessee 37996. Phone: 423-974-4025; Fax: 423-974-4007; E-mail: hzagh{at}utk.edu
Received for publication 17 April 1998 and in revised form 7 August 1998.
We would like to thank Jacque Caprio for technical support.
This work was supported by grant RG2778A1/1 (to H. Zaghouani) from the National Multiple Sclerosis Society, and by a grant (to H. Zaghouani) from Astral, Inc., a subsidiary of Alliance Pharmaceutical Corp. (San Diego, CA).
Abbreviations used in this paper EAE, experimental autoimmune encephalomyelitis; PLP, proteolipid protein.
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