| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Microbiology, University of Tennessee, Knoxville, TN 37996
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The peptide used in these studies encompasses the amino acid (aa) sequence 139–151 of proteolipid protein (PLP).3 This peptide (thereafter referred to as PLP1) is encephalitogenic and induces experimental autoimmune encephalomyelitis (EAE) in SJL/J mice (23, 24, 25). The altered peptide designated PLP-LR was derived from PLP1 by replacing the TCR contact residues Trp144 and His147 with Leu144 and Arg147, respectively (26). PLP-LR binds to I-As class II molecules equally as well as PLP1, but when presented to PLP1-specific T cell clones and hybridomas it induces inactivation (26). PLP1 and PLP-LR were genetically engineered into the heavy chain variable region of an Ig, and the resulting Ig-PLP1 and Ig-PLP-LR chimeras were efficiently presented to T cells (27). Ig-PLP-LR, like free PLP-LR, antagonizes T cell lines and hybridomas developed against free PLP1 (Ref. 27, and our unpublished results). In vivo, Ig-PLP1 and Ig-PLP-LR induce T cells that are cross-reactive with both PLP1 and PLP-LR (28). However, when injected together into animals, there is a down-regulation of both PLP1 and PLP-LR responses. Interestingly, in vivo down-regulation of PLP1-reactive T cells required higher amounts of Ig-PLP-LR, while much less Ig-PLP1 was needed for full down-regulation of PLP-LR-specific responses (27, 28). These results indicated that Ig-PLP1 and Ig-PLP-LR target common naive T cells with differing avidity of interaction. Based on these observations and the findings that altered peptides endowed with antagonist functions manisfest a fast off rate relative to wild-type ligands (29), it was presumed that PLP1 and PLP-LR might drive differential avidities of interaction with T cells.
Because Ig-PLP1 and Ig-PLP-LR express a 
2b constant region isotype,
they should be able to cross the maternal placenta and drag the
peptides from mother to fetus. Fetal presentation of PLP1 and PLP-LR
provides a system to investigate the role that peptide-driven avidity
plays on T cell development. The results indicate that both Ig-PLP1 and
Ig-PLP-LR when injected into pregnant mothers cross the maternal
placenta and transfer to the fetus. Thymic APCs of neonates born to
mothers that were injected with Ig-PLP1 at day 19 of gestation
stimulated a PLP1-specific T cell hybridoma. Moreover, adult offspring
born to mothers given Ig-PLP1 on days 16, 17, and 18 of gestation could
not mount proliferative and cytokine responses or develop EAE when
challenged with either Ig-PLP1 or Ig-PLP-LR. In contrast, thymic APCs
from neonates born to mothers that were injected with Ig-PLP-LR could
not stimulate a PLP-LR-specific T cell clone. In addition, these
offspring developed proliferative and cytokine responses, as well as
EAE, when challenged with either chimera as adults. The interpretation
we would like to put forth for these results is that PLP1 most likely
supports negative selection and ablates responses to both PLP1 and
PLP-LR, while the altered peptide, PLP-LR, does not. These in vivo
observations favor an avidity model for T cell selection and provide
evidence that altered peptides could support positive selection. In
addition, the splenic APCs of neonates born to mothers that were
injected with Ig-PLP-LR during gestation, like the splenic APCs of
neonate offspring of Ig-PLP1 recipient mothers, stimulate specific T
cell clones. These results indicate that the T cells, which were
selected in a thymus most likely presenting PLP-LR at levels
undetectable by T cell clones, survive in a periphery that supports a
much stronger PLP-LR presentation.
Therefore, the altered peptide, PLP-LR, supports positive selection and, although presented in the periphery in a manner that provides higher avidity, allows the development and generation of specific T cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
SJL/J (H-2s) mice were bred, housed, and maintained in our animal care facility for the duration of experiments according to the guidelines of the University of Tennessee Animal Care Committee. Females were assessed daily for seminal plugs. When found, the female was removed from the breeding cage (labeled as day 2 of gestation) and allowed to develop possible pregnancy until day 16. On day 16 of gestation, the pregnant females were administered as described below.
Antigens
Peptides. The peptides used in this study were purchased from Res. Genetics (Huntsville, AL) and were purified by HPLC to >90% purity. PLP1 peptide (HSLGKWLGHPDKF) encompasses an encephalitogenic sequence corresponding to aa 139–151 of PLP (23, 24, 25). PLP-LR (HSLGKLLGRPDKF) is an altered peptide form of PLP1 in which the TCR contact residues Trp144 and His147 were replaced with Leu and Arg, respectively (26). PLP1 and PLP-LR are known to bind equally well to I-As class II molecules (26). PLP2 peptide (NTWTTCQSIAFPSK) encompasses the encephalitogenic aa sequence corresponding to residues 178–191 of PLP. PLP2 also binds to I-As MHC class II molecules and induces EAE in SJL/J mice (30).
Ig-PLP chimeras. Ig-PLP1, Ig-PLP-LR, and Ig-PLP2 are chimeras expressing PLP1, PLP-LR, and PLP2, respectively (27). Construction of these chimeras used the genes coding for the light and heavy chains of the anti-arsonate Ab, 91A3, and the procedures for deletion of the heavy chain CDR3 region and replacement with the nucleotide sequence coding for the selected peptide were previously described (27, 28). Ig-W is the parental Ig not encompassing any PLP peptide and was described elsewhere (27).
Large scale cultures of transfectants were conducted in DMEM containing
10% iron-enriched calf serum (Intergen, Purchase, NY). The Ig-PLP
chimeras were subsequently purified on separate rat anti-mouse 
chain Sepharose columns to avoid cross-contamination.
Fetal tolerization of mice with Ig-PLP chimeras
Pregnant mothers were injected i.v. with 100 µg of Ig-PLP1, Ig-PLP-LR, or Ig-W on days 16, 17, and 18 of gestation, and offspring were used for analysis of immune response or induction of EAE at the age of 6–8 wk.
Immunization of mice with Ig-chimera and peptides
Immunizations with Ig-PLP chimeras. Fetal tolerized offspring 6–8 wk old were immunized s.c. in the footpads and at the base of the limbs and tail with 50 µg of Ig-PLP1, Ig-PLP-LR, or Ig-W emulsified in a 200-µl mixture of PBS/CFA (v/v). After 10 days, the mice were sacrificed by cervical dislocation, and the spleens and lymph nodes (axillary, lateral axillary, and popliteal) were removed, single-cell suspensions were prepared, and the T cell-proliferative and cytokine responses were analyzed as described below.
Immunization with PLP peptides. Fetal tolerized offspring 6–8 wk old were immunized as above with 100 µg of PLP1 or PLP2 in a 200-µl mixture of PBS/CFA (v/v), and splenic and lymph node proliferation and cytokine production were analyzed 10 days after immunization.
Assessment of transfer of Ig-PLP1 from mother to fetus
Pregnant mice were given 300 µg of Ig-PLP1 i.v. in the tail vein at day 19 of gestation, and serum from offspring born 2 days later was used for detection of Ig-PLP1 by immunospot assay. Serum (10 µl) was deposited on a nitrocellulose membrane and allowed to adsorb for 30 min at room temperature. The membrane was then blocked with PBS-5% BSA for 1 h and then incubated in a PBS-2% BSA solution containing 1 x 106 cpm/ml 125I-labeled rabbit Abs to PLP1 (27). Subsequently, the membrane was washed with 0.05% Tween 20 in PBS, dried, and exposed to a Cronex film.
Fetal presentation of Ig-PLP chimeras
To assess fetal presentation of the Ig chimeras, splenic and thymic cells from neonates born to mothers that were injected with 300 µg of Ig-PLP1, Ig-PLP-LR, or Ig-W at day 19 of gestation were tested for stimulation of specific T cell hybridomas or clones. Graded numbers of thymic or splenic cells were irradiated (3000 rads) and incubated with 5 x 104 4E3 PLP1-specific T hybridoma or 2.1H8 cross-reactive T cell clone without the addition of exogenous Ag in a total volume of 200 µl. After 24 h, the supernatant was used for cytokine detection.
For control purposes, thymic and splenic cells from mice born to untreated mothers were used to assess presentation of the chimeras in vitro. In this case, 5 x 105 thymic or splenic cells were irradiated (3000 rads) and incubated with graded amounts of Ig chimera or free peptide and 5 x 104 T cells for 24 h. Subsequently, cytokine detection in the supernatant was conducted by ELISA.
Assays for spleen and lymph node proliferative responses
Lymph node and splenic cells were incubated in 96-well flat-bottom plates at 4 and 10 x 105 cells/100 µl/well, respectively, with 100 µl of stimulator for 3 days. Subsequently, 1 µCi [3H]thymidine was added per well, and the incubation was continued for an additional 14.5 h. The cells were then harvested onto glass fiber filters, and the incorporated [3H]thymidine was counted using the trace 96 program on an Inotech (Wohlen, Switzerland) beta counter. The stimulators were used at the defined optimal concentrations of 15 µg/ml for PLP1, PLP-LR, and PLP2. A control of media without stimulator was included for each mouse and used as background.
Enzyme-linked immunospot (ELISPOT) assay
The cytokines produced by lymph node T cells were measured by
ELISPOT assay as described (31). Briefly, HA multiscreen plates
(Millipore, Bedford, MA) were coated with 100 µl/well 1 M
NaHCO3 buffer containing 2 µg/ml capture Ab (PharMingen,
San Diego, CA). The capture Abs were rat anti-mouse IL-2,
JES6-1A12; rat anti-mouse IL-4, 11B11; and rat anti-mouse
IFN, R4–6A2. After an overnight incubation at 4°C, the plates
were washed three times with sterile PBS, and then free sites were
blocked for 2 h at 37°C with 100 µl/well DMEM containing 10%
calf serum. Subsequently, 50 µl of medium were removed and 5 x
105 lymph node cells/50 µl/well and stimulator (100 µl)
were added. After a 24-h incubation, the plates were washed and 100
µl of biotinylated anti-cytokine Ab (1 µg/ml) were added. The
biotinylated anti-cytokine Abs were rat anti-mouse IL-2,
JES6-5H4; rat anti-mouse IL-4, BVD6-24G2; and rat anti-mouse
IFN, XMG1.2 (PharMingen). After overnight incubation at 4°C, the
plates were washed and 100 µl of 2.5 µg/ml avidin-peroxidase
(Sigma) were added. After a 1-h incubation at 37°C, the plates were
washed, visualized by addition of 100 µl of 3-amino-9-ethylcarbazole
(Sigma) in 50 µM acetate buffer (pH 5.0), allowed to dry, and counted
under a dissecting microscope. The stimulators were used at the defined
optimal concentrations of 15 µg/ml for PLP1, PLP-LR, and PLP2. A
control of media without stimulator was included for each mouse and
used as background.
ELISA for detection of cytokines
Detection of cytokine in culture supernatant was conducted by
ELISA according to the PharMingen standard protocol. The
anti-cytokine Ab pairs used here are those described for ELISPOT.
The OD405 was measured on a SpectraMAX 340 counter
(Molecular Devices, Menlo Park, CA) using SoftMAX Pro version 1.2.0
software. Graded amounts of recombinant mouse IL-2, IL-4, and IFN
were included in all experiments to construct standard curves. The
concentration of cytokines in culture supernatants was estimated by
extrapolation from the linear portion of the standard curve. The
stimulators were used at the defined optimal concentrations of 15
µg/ml for PLP1, PLP-LR, and PLP2. A control of media with no
stimulator was included for each mouse and used as background.
Measurement of IL-2 by activation of IL-2-dependent HT-2 cells
Measurement of IL-2 was done by [3H]thymidine incorporation of the IL-2-dependent HT-2 cell line. Briefly, 100 µl of culture supernatant were incubated with 1 x 104 HT-2 cells for 17 h. One microcurie of [3H]thymidine was then added per well, and the culture was continued for 12 h. The cells were then harvested onto glass fiber filters, and the incorporated [3H]thymidine was counted using the trace 96 program on an Inotech beta counter.
EAE induction
Fetal tolerized mice 6–8 wk old were induced for EAE by s.c. injection in the foot pads and at the base of the limbs and tail with a 200-µl IFA/PBS (v/v) solution containing 200 µg of Mycobacterium tuberculosis H37Ra and 200 µg of Ig-PLP1, 200 µg of Ig-PLP-LR, or 100 µg of free PLP1. Six hours later, 5 x 109 inactivated Bordetella pertussis were given i.v. After 48 h, a second dose of 5 x 109 inactivated B. pertussis was administered. 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.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To ascertain that the Ig-PLP chimeras could cross the maternal
placenta and transfer from mother to fetus, pregnant mice were injected
i.v. with Ig-PLP1 at day 19 of gestation, and the serum of offspring
born 2 days later was tested for the presence of Ig-PLP1. Fig. 1
shows that the serum of mice born to
mothers that were injected with Ig-PLP1 during pregnancy like the
control purified Ig-PLP1 bound rabbit Abs to PLP1 peptide. This spot
blot system was specific because the serum from offspring born to
mothers injected with Ig-W instead of Ig-PLP1 during gestation did not
bind the rabbit Abs. These data indicate that Ig-PLP1 crossed the
maternal placenta and reached the fetal circulation. Subsequently, we
asked whether the transferred Ig-PLP chimeras could reach the fetal
lymphoid organs for presentation. To address this issue, thymic and
splenic cells from neonates born to mothers injected with either
Ig-PLP1 or Ig-W during gestation were assayed, without the addition of
Ag, for the ability to activate the PLP1-specific T cell hybridoma,
4E3. Fig. 2 shows that both thymic and
splenic cells from mice born to Ig-PLP1 recipient mothers stimulated
the production of IL-2 by the 4E3 hybridoma whereas cells from mice
born to Ig-W recipient mothers did not. These data indicate that the
transferred Ig-PLP1 was taken up by APCs and that I-As-PLP1
complexes were generated in both the central and peripheral fetal
lymphoid organs.
|
|
Expression of MHC molecules on fetal APCs begins at day 14 of
gestation (32, 33), and TCR rearrangement and expression on T cells
follows 3 days later (34, 35, 36, 37). Therefore, T cell selection commences at
day 17 of fetal life. To investigate the consequences of Ig-PLP1 fetal
presentation on the development of specific T cells, pregnant mothers
were injected with Ig-PLP1 at days 16, 17, and 18 of gestation, and the
offspring were immunized as adults with either Ig-PLP1 or PLP1 peptide
in CFA. Ten days later, the lymph node and spleen cells were assessed
for proliferative and cytokine responses. Fig. 3 shows that fetal presentation of
Ig-PLP1 dramatically reduces the lymph node and splenic proliferative
responses of adult offspring to immunization with either Ig-PLP1 or
free PLP1. In contrast, mice born to mothers that received the parental
Ig-W instead of Ig-PLP1 developed specific responses in both lymphoid
organs to either immunization. These results indicate that fetal
presentation of Ig-PLP1 neither primes specific T cells nor allows the
development of memory cells. Rather, the precursor cells are not
present and/or incapable of responding to challenge with Ag.
Stimulation of cells in the presence of exogenous IL-2 did not restore
the proliferative response of cells from offspring born to
Ig-PLP1-injected mothers (not shown).
|
). IL-4 was undetectable
in all groups of mice (not shown). Additionally, although these mice
were completely resistant to EAE induction by Ig-PLP1, those born to
mothers who received Ig-W during pregnancy developed, like the
offspring born to unmanipulated mothers, a standard monophasic EAE
(Fig. 5a). When free PLP1
peptide was used for disease induction, the mice born to mothers that
received Ig-W developed a severe clinical relapsing remitting form of
EAE with relapses occurring up to day 100. Those offspring born to
mothers that received Ig-PLP1 during gestation developed a milder
monophasic form of EAE that did not relapse through day 120 (Fig. 5b). Overall, Ig-PLP1 transfers from mother to fetus, and
the consequent fetal presentation of PLP1 peptide influences T cell
development.
|
|
We then chose this fetal delivery and presentation strategy to
investigate the role of altered peptides on T cell development in vivo.
Altered peptides mutated at the TCR contact residues have a faster off
rate than wild-type peptides and presumably interact with the TCR with
lower affinity (29, 38). PLP-LR, a peptide derived from PLP1 by
mutation of TCR contact residues 144W and 147H to 144L and 147R,
respectively, binds to I-As MHC class II molecules equally
as well as PLP1 peptide but generates an altered peptide ligand that
antagonizes PLP1-specific T cell lines and hybridomas (26, 27). T cell
antagonism most likely is the consequence of low affinity TCR-ligand
interaction and TCR spoiling (29, 38, 39). Altered peptides, used as
tools to modulate affinity, provide useful ligands to investigate the
avidity model of T cell selection. Recently, PLP-LR peptide was
expressed on an Ig molecule, and the resulting Ig-PLP-LR chimera, like
Ig-PLP1, induced T cells that were cross-reactive with both PLP1 and
PLP-LR peptides (28). Since Igs can transfer from mother to fetus, PLP1
and PLP-LR, expressed on Ig, could access the developing fetal thymus
and provide an in vivo system to study the effect of differential
avidity on T cell development. To address this issue, pregnant mice
were injected with either Ig-PLP1 or Ig-PLP-LR on days 16, 17, and 18
of gestation, and the offspring born to these mothers were immunized
with either Ig-PLP1 or Ig-PLP-LR. Ten days later their proliferative
and cytokine responses to stimulation with PLP1 and PLP-LR were
assessed. Fig. 6 shows that fetal
injection of Ig-PLP-LR has little or no down regulatory effect on
either lymph node or splenic proliferative responses to a challenge
with Ig-PLP1 or Ig-PLP-LR, in fact there is a slight proliferative
enhancement when the stimulator is PLP-LR. In contrast, when Ig-PLP1
was injected into the pregnant mothers the offspring’s proliferative
responses to PLP1 and PLP-LR were markedly reduced, whether the
immunogen was Ig-PLP1 or Ig-PLP-LR (Fig. 6). Overall, mother
sensitization with Ig-PLP1 precludes the responses to both Ig-PLP1 and
Ig-PLP-LR, while sensitization with Ig-PLP-LR, the chimera carrying the
altered peptide, does not significantly reduce the proliferative
responses to either chimera. Similar results were obtained at the
cytokine production level (Fig. 7). While
fetal administered Ig-PLP1 abrogated the offsprings’ cytokine
responses to both Ig-PLP1 and Ig-PLP-LR, fetal injection of Ig-PLP-LR
did not modify the IL-2 response, and the amount of cytokine produced
in response to either Ig-PLP1 or Ig-PLP-LR was comparable to that
obtained in mice born to mothers that were injected with Ig-W during
gestation (Fig. 7). IL-4 was undetectable in all groups of mice (not
shown). Fetal presentation of Ig-PLP1 and its consequent effect on T
cell development is specific and does not affect the development of
other T cell precursors using I-As class II molecules for
selection and maturation. This conclusion is drawn from the observation
that injection of Ig-PLP1 during gestation does not interfere with the
proliferative and cytokine responses to PLP2 peptide (Fig. 8). Indeed, offspring from mothers that
were injected with Ig-PLP1 during gestation developed lymph node and
splenic proliferative and cytokine responses to a challenge with PLP2
peptide that were similar to the responses of offspring born to mothers
that received Ig-W instead of Ig-PLP1 during pregnancy. Offspring born
to mothers injected with Ig-PLP1 during pregnancy resisted induction of
EAE by both Ig-PLP1 and Ig-PLP-LR (Fig. 9). In contrast, offspring born to
mothers that received Ig-PLP-LR during pregnancy developed clinical
signs of EAE when induced with either Ig-PLP1 or Ig-PLP-LR.
|
|
|
|
Because Ig-PLP1 was presented by fetal thymic APCs in vivo and
neither proliferative/cytokine response nor EAE could develop
subsequent to challenge with either Ig-PLP1 or Ig-PLP-LR, it is likely
that PLP1/PLP-LR-specific T cell precursors were incapacitated or
negatively selected during development. Anergy is most likely not
responsible for the Ig-PLP1-mediated suppression of proliferative
response because offspring born to Ig-PLP1-treated mothers do not
restore their proliferative response when peptide stimulation is
conducted in the presence of IL-2 (not shown). The absence of a
down-regulatory effect on T cell development by Ig-PLP-LR could suggest
that Ig-PLP-LR was not presented by fetal APCs or that presentation
took place, but the generated ligand could not drive negative
selection. To investigate this issue, a readout system for Ig-PLP-LR
fetal presentation was needed. Because PLP-LR and Ig-PLP-LR are
antagonist for PLP1-specific hybridomas, we needed a PLP-LR-specific T
cell clone to assess the in vivo presentation of Ig-PLP-LR. To this
aim, mice were immunized with Ig-PLP-LR, and the lymph node cells were
cycled through stimulation/resting until reactivity with PLP-LR peptide
was apparent and a line was established. Subsequently, the line was
cloned by limiting dilution, and wells positive for cell growth were
tested for proliferation to PLP-LR and PLP1. One clone, designated
2.1H8, proliferated to Ig-PLP1, Ig-PLP-LR, PLP1, and PLP-LR (not shown)
and produced IL-4 in response to these stimulators (Fig. 10). This production of IL-4 is Ag
specific and does not occur when 2.1H8 is stimulated with Ig-PLP2 or
PLP2 peptide, Ags that are also presented by I-As class II
molecules like PLP1 and PLP-LR peptides. 2.1H8 was then used to assess
fetal presentation of Ig-PLP-LR. Mice were injected with Ig-PLP-LR or
Ig-PLP1 on day 19 of pregnancy, and the thymic and splenic cells from
offspring born on day 21 were assayed for stimulation of the 2.1H8 T
cell clone. Fig. 11a indicates that
thymic APCs from offspring born to Ig-PLP1 recipient mothers induced
IL-4 production by the 2.1H8 clone, while thymic APCs from offspring
born to Ig-PLP-LR recipient mothers did not stimulate IL-4 production.
In contrast, splenic cells from both types of offspring stimulated IL-4
production by the 2.1H8 clone (Fig. 11b). Finally, while
neonatal thymic APCs incubated in vitro with Ig-PLP1 stimulated the
2.1H8 clone, those incubated with Ig-PLP-LR did not induce IL-4
production by the 2.1H8 cells (Fig. 11c). In contrast,
neonatal splenic APCs, whether incubated with Ig-PLP1 or with
Ig-PLP-LR, stimulated the 2.1H8 T cell clone (Fig. 11d).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and 11).
Ig-PLP1 was presented in both the fetal thymus and the spleen to an
extent that was detectable by T cell clones and hybridomas (Figs. 2 and 11). However, although Ig-PLP-LR was transferred from mother to fetus
as suggested by its presentation by splenic APCs to a specific T cell
clone, it was most likely presented in the thymus in a manner that
could not be detected by the T cell clone (Fig. 11). In vitro, whereas
Ig-PLP1 stimulated the PLP1/PLP-LR-specific T cell clone when presented
by neonatal splenic or thymic APCs, Ig-PLP-LR stimulated the T cell
clone only when the APCs were of splenic origin (Fig. 11). Similarly,
free PLP-LR peptide stimulated the T cell clones only when the APCs
were of thymic origin. These observations indicate that presentation of
altered peptides by fetal thymic APCs is less potent than that of
peripheral presentation. It is worth noting that the 2.1H8 T cells are
more sensitive to stimulation with PLP-LR or Ig-PLP-LR than PLP1 or
Ig-PLP1 (Fig. 10). The inability of the thymic APCs to stimulate 2.1H8
when Ig-PLP-LR is used as Ag could be related to a diminished
internalization and/or processing of Ig-PLP-LR or to an inadequate
expression of costimulatory molecules on fetal thymic APCs. However,
Ig-PLP1 which is subject to the same events, still generated sufficient
avidity, leading to activation of the 2.1H8 clone. The failure of
thymic APCs of Ig-PLP-LR-tolerized mice to activate the 2.1H8 T cell
clone may be the result of weak avidity supported by PLP-LR peptide in
the thymic environment. Differential avidity may have been responsible
for the discrepancy of the responses in mice receiving Ig-PLP1 fetal
tolerization procedure vs those receiving Ig-PLP-LR. Indeed, offspring
born to mothers that were injected with Ig-PLP1 during gestation
presented PLP1 in the thymus as well as the spleen during fetal
development but were unable to mount responses or develop EAE when
challenged as adults with either Ig-PLP1 or Ig-PLP-LR (
Figs. 3–5). The
residual proliferative and cytokine responses observed in these mice
when they were challenged with free PLP1 peptide instead of Ig-PLP1
could be related to a difference in the T cell repertoire generated by
free peptide vs Ig-PLP1 (Figs. 3 and 4). This statement is supported by
the data illustrated in Fig. 5, which demonstrates that free PLP1
induces EAE with a relapsing remitting pattern of paralysis different
from the monophasic pattern induced by Ig-PLP1. Consequently, fetal
tolerization with Ig-PLP1 had a partial effect on the T cell response
that led to a decrease in disease severity and absence of relapses when
the mice were induced for EAE with free peptide (Fig. 5b).
In contrast, offspring born to mothers that received Ig-PLP-LR during
pregnancy presented PLP-LR peptide in the spleen, but their thymic APCs
were unable the stimulate the 2.1H8 clone (Fig. 11). Moreover, these
mice responded to both peptides when challenged with Ig-PLP1 or
Ig-PLP-LR (Figs. 7 and 8) and developed EAE when induced with either Ig
chimera (Fig. 9). The question of how PLP-LR-specific T cell precursors, positively selected in the thymus, are not activated by the strong peripheral presentation of Ig-PLP-LR remains unanswered. One possibility is that T cells, once exiting the thymus, are subject to a second round of selection (i.e., peripheral tolerance) (40, 41, 42, 43) and that only those cells that readjust their activation avidity to a level higher than the avidity provided by peripheral presentation survive (44, 45). Knowing that the TCR is not subject to mutation, that the T cell may not re-rearrange the TCR in the periphery, and that most T cell responses to peptides are not restricted to a particular V-gene usage, the postulate of readjustment in the avidity required for activation becomes more attractive (22, 45, 46). In addition, T cells still develop in transgenic mice expressing a single TCR and low levels of the corresponding Ag (46). However, when the presentation of Ag was enhanced to generate an avidity that surpasses the readjusted threshold, the T cells became activated (22, 46).
Fig. 12 shows a schematic diagram that
illustrates arbitrary levels of avidity for the different developmental
stages of the T cell. The diagram reflects a coordination of events
with the level of avidity. In the thymus, cells expressing a TCR that
is unable to engage a MHC-peptide complex will die by neglect (5, 47).
However, if the TCR can mediate an interaction with thymic APCs with an
avidity below the threshold for negative selection, then the cell will
be positively selected. For instance, thymic APCs from fetuses
receiving Ig-PLP-LR could not stimulate 2.1H8 clones. Consequently,
positive selection had taken place, and T cell responses to PLP-LR were
obtained when the mice were challenged with Ig-PLP-LR. In vitro,
although splenic APCs pulsed with Ig-PLP-LR did stimulate the T cell
clone, thymic APCs did not. Therefore, although we have no direct
evidence for thymic presentation of Ig-PLP-LR, we think that it was
processed and that the peptide was loaded onto MHC molecules, but the
resulting complexes, because of a possible fast off rate, supported an
avidity that could not drive activation of the 2.1H8 T cell clone (48).
Because Ig-PLP-LR was presented in the fetal spleen and the mice
developed normal responses when challenged as adults with either
chimera, the T cells must have readjusted their avidity of activation
to a level higher than that provided by the peripheral presentation of
Ig-PLP-LR (which activates the mature 2.1H8 T cell clone). Finally, the
avidity required for activation should be higher than the threshold for
negative selection, in that fetuses presenting Ig-PLP-LR in the fetal
spleen were able to develop T cell responses when challenged with
Ig-PLP-LR as adults. Meanwhile, Ig-PLP1 generated a fetal thymic
presentation avidity capable of activating mature T cells, but could
not subsequently immunize the mice because the T cells were negatively
selected. Overall, this strategy provides a system that confirms that
altered peptides can mediate T cell selection in vivo and sheds light
on the necessity for peripheral readjustment of the presentation
avidity. When used with TCR transgenic cells, this in vivo strategy
will permit investigation of T cell selection and peripheral
readjustment avidity at the single cell level.
|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Habib Zaghouani, University of Tennessee, Department of Microbiology, M409 Walters Life Sciences Building, Knoxville, TN 37996. E-mail address:
3 Abbreviations used in this paper: PLP, proteolipid protein; EAE, experimental allergic encephalomyelitis; ELISPOT, enzyme-linked immunospot; SFU, spot-forming unit.
Received for publication September 30, 1998. Accepted for publication February 18, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß thymocytes: is cortical thymic epithelium an obligatory participant in the presentation of major histocompatibility complex protein?. Immunol. Rev. 135:133.[Medline]
genes during thymic development. J. Exp. Med. 164:1.This article has been cited by other articles:
|
|
 |
|
  P. Yu, C. L. Haymaker, R. D. Divekar, J. S. Ellis, J. Hardaway, R. Jain, D. M. Tartar, C. M. Hoeman, J. A. Cascio, A. Ostermeier, et al. Fetal Exposure to High-Avidity TCR Ligand Enhances Expansion of Peripheral T Regulatory Cells J. Immunol., July 1, 2008; 181(1): 73 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Pack, A. E. Cestra, B. Min, K. L. Legge, L. Li, J. C. Caprio-Young, J. J. Bell, R. K. Gregg, and H. Zaghouani Neonatal Exposure to Antigen Primes the Immune System to Develop Responses in Various Lymphoid Organs and Promotes Bystander Regulation of Diverse T Cell Specificities J. Immunol., October 15, 2001; 167(8): 4187 - 4195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Li, K. L. Legge, B. Min, J. J. Bell, R. Gregg, J. Caprio, and H. Zaghouani Neonatal Immunity Develops in a Transgenic TCR Transfer Model and Reveals a Requirement for Elevated Cell Input to Achieve Organ-Specific Responses J. Immunol., September 1, 2001; 167(5): 2585 - 2594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Min, K. L. Legge, J. J. Bell, R. K. Gregg, L. Li, J. C. Caprio, and H. Zaghouani Neonatal Exposure to Antigen Induces a Defective CD40 Ligand Expression that Undermines Both IL-12 Production by APC and IL-2 Receptor Up-Regulation on Splenic T Cells and Perpetuates IFN-{{gamma}}-Dependent T Cell Anergy J. Immunol., May 1, 2001; 166(9): 5594 - 5603. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L. Legge, B. Min, J. J. Bell, J. C. Caprio, L. Li, R. K. Gregg, and H. Zaghouani Coupling of Peripheral Tolerance to Endogenous Interleukin 10 Promotes Effective Modulation of Myelin-activated T Cells and Ameliorates Experimental Allergic Encephalomyelitis J. Exp. Med., June 12, 2000; 191(12): 2039 - 2052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Anderson, L. B. Nicholson, K. L. Legge, V. Turchin, H. Zaghouani, and V. K. Kuchroo High Frequency of Autoreactive Myelin Proteolipid Protein-specific T Cells in the Periphery of Naive Mice: Mechanisms of Selection of the Self-reactive Repertoire J. Exp. Med., February 28, 2000; 191(5): 761 - 770. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||
) and PLP2 (
)
were assessed. Lymph node cells were used at 4 x 105
cells/well, and splenic cells were used at 1 x 106
cells/well. The stimulators PLP1 and PLP2 were used at 5, 15, 30
µg/ml, and the indicated results represent those obtained with the
optimal concentration of 15 µg/ml. Each column represents the
mean ± SD of five individually tested mice.
)
were assessed. Each column represents the mean ± SD of five
individually tested mice.
