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Department of Microbiology, University of Tennessee, Knoxville, TN 37996
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We purchased 5- to 8-wk-old SJL/J mice (H-2s) from Harlan-Sprague-Dawley (Frederick, MD). The mice were housed in our animal facility for the duration of these experiments.
Antigens
Peptides. All of the peptides used in this study were purchased from Research Genetics (Huntsville, AL) and purified by HPLC to >90% purity. PLP1 peptide (HSLGKWLGHPDKF) corresponds to aa residues 139–151 of PLP. PLP1 is a T cell peptide that is presented by I-As class II molecules and is encephalitogenic in H-2s mice (16, 17). PLP-LR peptide (HSLGKLLGRPDKF) is a mutant form of PLP1, in which the major TCR-contacting residues, Trp144 and His147, were replaced with Leu and Arg, respectively (7). PLP-LR binds to I-As equally as well as PLP1 and has been defined as a TCR antagonist peptide (7). PLP2 peptide (NTWTTCQSIAFPSK) corresponds to aa residues 178–191 of PLP and is also presented by I-As class II molecules (22).
Ig-PLP chimeras.
Nucleotide sequences encoding PLP1, PLP-LR, and PLP2 peptides were
inserted in place of the D segment within the complementarity
determining region 3 of the VH gene of the 91A3
anti-arsonate Ab using a previously described PCR mutagenesis
procedure (23). The 91A3 VH-PLP chimeric genes were then
ligated to the exons encoding a BALB/c 
2b constant region (C2b)
to form complete heavy chains (21). The 91A3 VH-PLP-C2b
genes were cotransfected with the parental 91A3 light chain gene into
the non-Ig-secreting myeloma B cell line SP2/0 to express complete Ig
molecules. The resulting Ig chimeras were designated Ig-PLP1,
Ig-PLP-LR, and Ig-PLP2, respectively. Transfectoma cells producing 2 to
4 µg/ml of Ig-PLP chimera were grown to large scale in 2-L roller
bottles, and the chimeras were purified from culture supernatant by
affinity chromatography on columns made of rat anti-mouse 
chain
coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) as
described previously (23). Each Ig chimera was purified using separate
columns to avoid cross contamination.
Immunization of mice with Ig chimeras
Mice (five per group) were immunized s.c. in the foot pads and at the base of the limbs and tail with Ig-PLP chimera emulsified in a 200 µl mixture of PBS/CFA (1:1 v/v). The mice were sacrificed after 10 days by cervical dislocation, the spleens and lymph nodes (axillary, lateral axillary, and popliteal) were removed, single-cell suspensions were prepared, and the T cell proliferative response and cytokine production were assessed as described below.
Assays for spleen and lymph node proliferative responses
Lymph node and spleen 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 of [3H]thymidine was added per well, and the culture was continued for an additional 14.5 h. The cells were subsequently harvested on glass fiber filters, and incorporated [3H]thymidine was counted using the trace 96 program and an Inotech beta counter (Wohlen, Switzerland). Unless indicated otherwise, the stimulators were used at the following defined optimal concentrations: PLP1, PLP-LR, and PLP2 peptides at 15 µg/ml and purified protein derivative (PPD) of Mycobacterium tuberculosis at 5 µg/ml.
ELISA screening for spleen cytokine production
Spleen cells were incubated in 96-well round-bottom plates at
10 x 105 cells/100 µl/well with 100 µl of
stimulator, as described above, for 24 h. Cytokine production was
measured by ELISA according to the manufacturer’s instructions
(PharMingen, San Diego, CA) using 100 µl of culture supernatant. The
capture Abs used 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). The biotinylated anti-cytokine
Abs used were rat anti-mouse IL-2 (JES6-5H4), rat anti-mouse
IL-4 (BVD6-24G2), rat anti-mouse IFN- (XMG1.2), and rat
anti-mouse IL-10 (JES5-16E3). 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 mouse rIL-2,
rIL-4, rIFN-, and rIL-10 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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indicate that Ig-PLP1, like PLP1
peptide, induced a specific T cell response to PLP1 peptide. Similarly,
Ig-PLP-LR, like PLP-LR peptide, induced a specific T cell response to
PLP-LR peptide. Neither the Ig chimera nor the free peptides induced T
cells that significantly reacted with the negative control PLP2, a
peptide that is also presented by I-As class II
molecules. Surprisingly, however, the response induced by Ig-PLP1
cross-reacted with PLP-LR peptide, while the response induced by
Ig-PLP-LR cross-reacted with PLP1. The responses induced with free PLP1
or free PLP-LR were not cross-reactive under these experimental
conditions (Fig. 1). Stimulation of cells from PLP-LR-immunized mice
with a higher concentration of PLP1 peptide resulted in low but
significant proliferation (24). These observations prompted us to
investigate whether Ig-PLP-LR acts on Ig-PLP1 in a dose
dependent-fashion, and whether it is subjected to counterregulation by
Ig-PLP1. As can be seen in Figure 2,
lymph node T cells from a new group of mice that were immunized with
Ig-PLP1 proliferated equally well to free PLP1 and PLP-LR peptides.
Splenic T cells from these mice failed to respond to free PLP-LR
peptide stimulation (Fig. 3). However,
when an additional group of mice was immunized with Ig-PLP-LR, both
lymph node and splenic cells proliferated to PLP1 as well as to PLP-LR
peptide (Figs. 2 and 3). Ig-W, a wild-type molecule not carrying any
PLP peptide, failed to induce a T cell response in either lymphoid
tissue (Figs. 2 and 3). When mice were immunized with a mixture
containing equal amounts of Ig-PLP1 and Ig-PLP-LR, the T cell responses
were greatly decreased in comparison with immunization with either
chimera alone (Figs. 2 and 3). Furthermore, the PLP1-specific lymph
node responses were lower than responses obtained in mice immunized
with Ig-PLP1 alone and represented only 50% of the PLP1-specific lymph
node response of mice immunized with Ig-PLP-LR. Surprisingly, the
PLP-LR response was at BG levels (Fig. 2). Accordingly, although the
responses to the Ig chimeras share cross-reactivity between PLP1 and
PLP-LR peptides, immunization with mixtures of Ig chimeras yielded
down-regulation rather than additive responses. In fact, the data argue
for an opposite down-regulation among Ig-PLP1 and Ig-PLP-LR. This
down-regulation appeared to be dose-dependent, because mice that were
injected with a mixture of 50 µg of Ig-PLP1 and 150 µg of Ig-PLP-LR
failed to respond to PLP1 and mounted responses to PLP-LR that were
reduced to the levels observed with mice injected with Ig-PLP1 alone
(Fig. 2). Previously, we had concluded that this PLP-LR response was
normal (21), but in comparison with the responses of animals injected
with Ig-PLP-LR alone, there is a 50% reduction indicating the
down-regulatory effect of Ig-PLP1 on a high dose of Ig-PLP-LR
(Fig. 2).
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).
Similarly, when Ig-PLP1 and Ig-PLP-LR were separately coinjected into
animals with Ig-PLP2, a chimera presented by I-As class II
molecules, the response to either peptide was unaffected (Fig. 2).
In the spleen, like in the lymph nodes, the proliferative responses
were not additive (Fig. 3). Rather, an opposite down-regulatory effect
between Ig-PLP1 and Ig-PLP-LR was seen. Although a coinjection of Ig-W
with either Ig-PLP1 or Ig-PLP-LR did not affect either response, a
coinjection of Ig-PLP2 with Ig-PLP1 increased reactivity to PLP-LR
among the T cells induced by Ig-PLP1. Whether or not a bystander effect
(24, 25) emanating from PLP2-induced T cells helped Ig-PLP1-induced
PLP-LR-reactive T cells to migrate to the spleen remains to be
investigated.
To further investigate the opposing down-regulation among Ig-PLP1 and
Ig-PLP-LR, splenic Ag-induced cytokine responses were measured in
animals immunized with either a single Ig chimera or a combination of
both. Upon in vitro stimulation with PLP1 peptide, T cells from
Ig-PLP1-immunized mice produced IL-2, IFN-, and small amounts of
IL-4 (Fig. 4). However,
stimulating the same cells with PLP-LR yielded minimal IL-2
and undetectable IFN- or IL-4 (Fig. 4). Spleen cells from
Ig-PLP-LR-immunized mice generated IL-2 but no IFN- or IL-4 upon
stimulation with PLP1 peptide. Moreover, PLP-LR peptide stimulation
produced only a minimal IL-2 response. In mice immunized with equal
amounts of Ig-PLP1 and Ig-PLP-LR, all cytokine production was reduced
to minimal or BG levels upon stimulation with either peptide.
Coinjecting Ig-W with either chimera had no measurable effect on the
cytokine production pattern (Fig. 4). Significant amounts of IL-4 and
IFN- were evident upon stimulation with PLP-LR peptide when the
animals were given a 3:1 ratio of Ig-PLP-LR to Ig-PLP1, although the
splenic proliferative responses and IL-2 production were at BG levels
(Fig. 4). Consequently, the excess of Ig-PLP-LR may lead to a mixed but
PLP-LR-dominant TCR triggering that induces cells which are able to
produce cytokine but which exhibit no proliferative response.
Incomplete or mixed signaling, which is a form of interference with
signal one, was shown to have a more pronounced down-regulatory effect
on proliferation than on cytokine production (1). None of the
immunization regimens illustrated in Figure 4 induced detectable levels
of IL-10 (data not shown).
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, both lymph node and spleen cells from
mice immunized with Ig-PLP1 or Ig-PLP-LR proliferated equally as well
to stimulation with a single peptide as to a mixture of PLP1 and
PLP-LR. The proliferative response to the mixture, in most cases, was
even higher than the response to a single peptide stimulation (Fig. 5).
Similarly, IL-2 production was not decreased when spleen cells were
stimulated with varying mixtures of PLP1 and PLP-LR (Fig. 6). On the contrary, IL-2 production was
higher in most cases of stimulation with peptide mixture than
stimulation with a single peptide. Although both proliferation and IL-2
production were often higher when the stimulation was conducted with
the peptide mixture, additive responses were not observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Indeed, T cells from mice immunized with either Ig-PLP1 or
Ig-PLP-LR reacted with both PLP1 and PLP-LR (Fig. 1). This
cross-reactivity was specific to PLP1 and PLP-LR peptide and was not
observed when the cells were stimulated with PLP2, a peptide also
presented by I-As class II molecules like PLP1 and
PLP-LR. The splenic T cell responses of mice immunized with Ig-PLP-LR
were also cross reactive with both peptides, while the responses
induced with Ig-PLP1 showed no cross-reactivity with PLP-LR. The
mechanism underlying the loss of cross-reactivity among Ig-PLP1-induced
T cells subsequent to migration to the spleen is unknown. Efficient peptide loading onto MHC class II molecules by the Ig chimeras could generate significant amounts of MHC/peptide complexes (19). In addition, the binding and internalization of Igs into APCs via FcRs may up-regulate the expression of costimulatory molecules. Under these circumstances, an immunization with Ig-peptide chimeras would be able to prime a larger T cell repertoire, including low affinity T cells.
When Ig-PLP1 and Ig-PLP-LR were administered together into mice, the
lymph node as well as the splenic proliferative T cell responses were
markedly reduced (Figs. 2 and 3). In addition, IL-2 production was
reduced to BG levels (Fig. 4). These data indicated that Ig-PLP1 and
Ig-PLP-LR exerted adverse reactions on one another, leading to the
down-regulation of both T cell responses. Competition for
internalization into APCs via FcRs cannot account for the opposing
effects between Ig-PLP1 and Ig-PLP-LR, because coinjecting either
chimera with Ig-W, the parental Ig encompassing an identical Fc region
(the site that mediates binding to FcRs) as Ig-PLP1 and Ig-PLP-LR had
no down-regulatory effect on either T cell response (Figs. 2 and 3).
Similarly, since PLP2 peptide, like PLP1 and PLP-LR, is presented by
I-As class II molecules (22), and because Ig-PLP2 had no
effect on the responses induced by Ig-PLP1 or Ig-PLP-LR, the opposite
down-regulation between Ig-PLP1 and Ig-PLP-LR would appear to be
Ag-specific and would most likely not involve competition for binding
to I-As class II molecules (Figs. 2 and 3).
The explanation we wish to put forth for this opposite down-regulation between Ig-PLP1 and Ig-PLP-LR is that clonal expansion requires an optimal serial triggering with an homogeneous peptide (i.e., all or most of the receptors on a single naive T cell must engage one type of peptide to expand). The simultaneous stimulation of naive T cells with peptides encompassing subtle differences at the TCR contact residues, which may be occurring during immunizations involving mixtures of Ig-PLP1 and Ig-PLP-LR, fails to cause T cell expansion and in vitro proliferation.
It was previously demonstrated that PLP1-specific T cell hybridomas
generated from T cell clones obtained by immunization with free PLP1
peptide were not cross-reactive with PLP-LR (7). Rather, the hybridomas
were antagonized by PLP-LR (7). Similarly, an interaction of these T
cell hybridomas with Ig-PLP-LR led to their inactivation and to an
inhibition of cytokine production (21). The T cells induced by either
Ig-PLP1 or Ig-PLP-LR were, as demonstrated in Figure 1, cross-reactive
with both peptides, and these T cells proliferate and produce cytokines
in response to stimulation with either peptide. Because of this
cross-reactivity, these T cells proliferated and produced cytokines
when they were stimulated with a mixture of PLP1 and PLP-LR in vitro
(Figs. 5 and 6). All of the cells in a single Ig-peptide chimera
immunization regimen are primed by one peptide, and in vitro
stimulation of these cells with a mixture of peptides neither inhibited
nor led to additive responses. These results suggest that the response
induced by immunization with a single Ig-peptide chimera comprises T
cells expressing cross-reactive TCR rather than distinct populations
specific for individual peptides. Consequently, these Ag-experienced,
cross-reactive T cells, unlike naive T cells, are resistant to mixed
TCR triggering by PLP1 and PLP-LR. Monoclonal T cells, whether clones
or hybridomas, that are generated by repeated in vitro peptide
stimulation are likely to be more sensitive to antagonism because of
their higher affinity to the selecting peptide. PLP1-specific T cell
hybridomas and lines were in fact antagonized by Ig-PLP-LR and PLP-LR
peptide (7, 21).
Overall, naive T cells, although potentially cross-reactive with both
agonist and antagonist peptides, resist mixed TCR triggering during the
first Ag exposure and appear to support an opposite down-regulation
among peptides with subtle differences at the TCR contact residues. A
cell-signaling analysis has demonstrated that TCR occupancy without a
sustained calcium signal could form the basis for TCR spoiling and
antagonist interference with TCR triggering by an agonist (14, 15).
Some altered peptides, however, may trigger signals that could support
distinct or partial activation patterns (reviewed in 13 . In our
case of opposite antagonism, one may speculate that both PLP1 and
PLP-LR, when presented by Igs, trigger productive signals that lead to
T cell activation and expansion, as evidenced by the induction of
specific T cell responses. These signals could be closely related and
support cross-reactivity at the level of Ag-experienced T cells.
However, naive T cells undergoing mixed signaling are discriminatory
and may not tolerate these closely related signals emanating from
peptides with such subtle aa differences. Whether this observation
implies that the signaling machinery, exerting a stringent control of
TCR triggering at the level of naive T cells, evolves with some
flexibility to support cross-reactivity by Ag-experienced T cells
remains to be investigated. However, it has been previously suggested
that neuroantigen-specific naive T cells have more stringent activation
requirements than T cells that have encountered the Ag (26). In
addition, it was recently demonstrated that T cells can be activated by
peptides that are unrelated in sequence to their selecting peptide
(27). Finally, we would like to emphasize that the delivery of peptides
on Igs for active immunization could result in responses that are
qualitatively different from those induced by free peptides (Fig. 1).
The binding of Igs to FcRs on APCs recruited to the site of injection
by CFA could trigger specific factors that could influence T cell-APC
interactions, possibly resulting in the preferential expansion of
cross-reactive T cells. In the absence of adjuvant, the enhanced
presentation of antagonist peptides by Igs may be efficient for the
down-regulation rather than for the induction of autoreactive T cells
and the amelioration of autoimmune disease.
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Acknowledgments |
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Footnotes |
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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; PPD, purified protein derivative; aa, amino acid; BG, background.
Received for publication November 14, 1997. Accepted for publication February 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 267:515. and lack of zap70 recruitment in APL-induced T cell anergy. Cell 79:913.[Medline]
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), or PLP2 (
). The resulting proliferation was measured
by [3H]thymidine incorporation. Cells incubated without
stimulator were used as BG. Each mouse was individually tested in
triplicate wells for each stimulator, and the indicated cpms represent
the mean ± SD after the deduction of BG cpms.
