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Departments of
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Pediatrics and
Pathology, Center for Immunology, Washington University School of Medicine, St. Louis, MO 63110
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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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and lack of Zap-70 activation. These phenomena have led us and others
to conclude that the T cell signaling triggered by these APLs is both
qualitatively and quantitatively different from signaling with the
antigenic ligand (8, 9, 10, 11). Many other investigations strongly imply that both positive and negative selection of developing thymocytes involves similar recognition events (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). For example, in a class I-restricted system, the data show that antagonists promoted positive selection of OVA-transgenic T cells in fetal thymic organ culture (FTOC) (12). In a class II-specific system, FTOC results implicate antagonists in negative selection (21). Others have used an in vitro cell culture system to examine the role of suboptimal ligands in thymic selection. Their data implicate antagonists in negative selection, and suggest that negative selection of thymocytes as a measure of peptide activity is a more sensitive assay than antagonism of peripheral T cells (22).
Using a TCR ß-chain only transgenic mouse, we previously reported the effect of an endogenous TCR antagonist on T cell development and peripheral tolerance (23). The data showed that antagonist peptides could promote the negative selection of high avidity thymocytes. Peripheral antagonism was not observed unless the number of antagonist peptide/MHC complexes was increased by adding exogenous peptide (24). In summary, APL/MHC complexes clearly play a role in thymic selection. The rules governing their influence in vivo remain to be established.
In the present study, we describe the functional consequences for T cells when an endogenous antagonist is expressed in the thymus and the periphery. We have utilized the 3.L2 TCR-transgenic mouse (3.L2tg), which is specific for hemoglobin (Hb)(64–76)/I-Ek, and is antagonized by A72/I-Ek. A clonotypic Ab (CAB) was used to follow 3.L2tg-specific responses. High levels of the endogenous antagonist were achieved by expressing the antagonist as a membrane protein in all MHC class II-positive cells. The data demonstrate that the predominate effect in vivo is peripheral TCR antagonism. We do not see enhanced positive selection under the conditions of these experiments.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have generated a transgenic mouse line (A72tg) that expresses a membrane form of hen egg-white lysozyme (HEL) containing Hb(64–76) N72A as a 13-amino acid epitope tag (mHEL/A72). The peptide Hb(64–76) N72A (henceforth called A72) is an APL of the self-Ag Hb(64–76), and has been shown to be a strong antagonist of 3.L2 T cell responses (25). The N72A substitution occurs in the P5 position as determined by crystallography (26), which is the TCR contact residue located in the center of the peptide/MHC ligand (27). The construction of a mHEL/Hb(64–76) chimeric gene has been described elsewhere (24). The asparagine to alanine substitution at position 72 of Hb(64–76) was created by PCR mutagenesis with the following nonoverlapping oligonucleotides: 5'-GAAGGCC-TGAAAAACCGTAACACC-3' (coding) and 5'-CGCAAAGGCAGTTATCACCTTTTT-GC-3' (noncoding). The mutation is contained in the 5' end of the noncoding oligonucleotide, and the two oligonucleotides are directly adjacent to one another. Sequencing in both directions confirmed the substitution.
We sought to maximize presentation of the antagonist ligand by
expressing mHEL/A72 in all APCs. The mHEL/A72 construct was subcloned
into the EcoRI site of pDOI-5 (Fig. 1
, panel A),
which contains the MHC E
promoter upstream of a fragment of the
rabbit ß globin gene. The E promoter has been effective in
expressing transgenes only in class II-positive cells (28, 29). From
this plasmid, a 5.2-kb BglI fragment was isolated and used
to inject the male pronuclei of fertilized B6.AKR oocytes. Founders
were obtained and bred to the 3.L2tg mouse, which is specific for
Hb(64–76)/I-Ek, (G. J. Kersh et al., manuscript in
preparation), or to the 3A9 TCR-transgenic mouse, which is specific for
HEL(46–61)/I-Ak (30). Progeny were screened by PCR
analysis of purified tail digest DNA, and all progeny analyzed were
heterozygous for the relevant transgenes, including those for the
TCR.
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The peptides used in this study were synthesized, purified, and analyzed as previously described (25). The peptide sequences (in single letter amino acid code) are: GKKVITAFNEGLK (Hbßd(64–76)); NTDGSTDYGILQINSR (HEL(46–61)); and ADLIAYLKQATK (MCC(92–103)).
T cell hybridomas and stimulation assay
The generation and characterization of the 3.L2–12 (Hb(64–76)/I-Ek-specific), 3A9 (HEL(46–61)/I-Ak-specific), and 2B4 (MCC(92–103)/I-Ek-specific) hybridomas have been described (27, 31, 32). Hybridomas were cultured in RPMI containing 10% heat-inactivated FCS, 2 mM Glutamax (Gibco-BRL, Gaithersburg, MD), 2 x 10-5 M 2-ME, 50 µg/ml gentamicin. The 3.L2-12 and 3A9 hybridomas were used to assay for the presence of stimulatory ligands on splenocytes from A72-transgenic mice. Increasing numbers of splenocytes (10–106 cells/well) in 100 µl were added in triplicate to flat-bottom 96-well microtiter plates containing 1 x 105 3A9 or 3.L2-12 hybridoma cells in 100 µl of media. The cells were incubated for 24 h, and stimulation of the hybridoma was ascertained by determining the level of IL-2 released, using the IL-2-dependent cell line CTLL-2, as described (27). To measure the ability of I-Ek on splenocytes from the various mouse strains to present exogenous peptide, a hybridoma assay was performed with 1 x 105 2B4 hybridoma cells/well mixed with 5 x 105 splenocytes/well plus increasing amounts of MCC peptide. Stimulation of the hybridoma was ascertained as described above.
Purification of T cells and stimulation of purified T cells
T cells were purified by passing a single cell suspension of splenocytes over a nylon wool column as follows. Three grams of fine, thread-like nylon wool were used to pack a 60-cc syringe to make a column. The column was rinsed with RPMI and then incubated with enough R10 FCS to cover the nylon wool for 1 h. A suspension of splenocytes from two spleens was prepared (300 x 106 cells) and added to the column in a volume of 8 ml. Enough media was drained to lower the level to the top of the nylon wool. The loaded columns were incubated for 1 h at 37°C, and 50 ml of R10 FCS was added. The media was drained over 1 h at a rate of approximately one drop per second. The T cells were collected by centrifugation and analyzed by FACs analysis for class II expression with the 14-4-4S Ab. No class II expression was detected, implying no contamination with APCs (not shown). These purified T cells were then used in T cell proliferation assays at a concentration of 1 x 105 cells/well with 5 x 105 splenocytes/well as APCs, plus Hb(64–76) peptide at concentrations ranging from 0.001 to 100 µM. These assays were incubated for 48 h, pulsed with 0.4 µCi/well of [3H]TdR for 18 h, and then harvested. Proliferation was measured as cpm incorporated, and each point represents the mean of triplicate wells.
Primary T cell proliferation
Proliferation assays of primary T cells were performed in complete media at 37°C with 5% CO2 as described (24). Briefly, 5 x 105 splenocytes/well were incubated with peptide for 48 h. The cells were then pulsed with 0.4 µCi/well of [3H]TdR for 18 h and then harvested. Proliferation was measured as cpm incorporated (mean of triplicate wells).
Antibodies
Cells were stained for flow cytometry with the following Abs: phycoerythrin (PE) anti-mouse CD4 (PharMingen, San Diego, CA); FITC anti-mouse CD8a (PharMingen); biotin 3.L2 clonotypic Ab (G.J. Kersh et al., manuscript in preparation); biotin 3A9 clonotypic Ab (D. A. Peterson, unpublished observations); biotin F10.6.6 (33); biotin 14-4-4S (34); TRI- COLOR (TC) streptavidin (Caltag, San Francisco, CA); and PE streptavidin (Caltag).
Flow cytometry
Single cell suspensions of thymocytes or splenocytes were stained in PBS supplemented with 0.5% BSA and 0.1% sodium azide. Cells (1 x 106/sample in 100 µl) were incubated on ice for 1 h with the biotinylated or directly labeled Abs, washed, and incubated for 30 min on ice with the streptavidin-fluorochrome conjugate when appropriate. Cells were then washed again, fixed in 1% paraformaldehyde, and analyzed on a FACScan (Becton Dickinson, Mountain View, CA) flow cytofluorometer using CELLQuest (Becton Dickinson) software. Samples were gated on live cells, and 100,000 events collected per sample.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To achieve high levels of antagonist A72/I-Ek
complexes on the surface of all APCs, we utilized the established
expression vector pDOI-5, which contains the MHC E promoter, to
drive transcription of our chimeric mHEL/A72 gene in transgenic mice
(Fig. 1
A). This E promoter has been quite
effective in expressing transgenes at physiologic levels in all class
II-positive cells in both the thymus and the spleen (28, 29). Two
founders were obtained and characterized. The A72.45 mouse line (A72tg)
expressed the mHEL/A72 protein in both the medulla and cortex of the
thymus, as detected by immunohistochemistry with the anti-HEL Ab
F10.6.6 (data not shown). A single cell suspension of splenocytes from
the A72.45 line was also stained with F10.6.6 and with 14-4-4S
(anti-I-Ek). FACS analysis showed expression of
mHEL/A72 on all class II cells (Fig. 1B). The A72.48
mouse line expressed mHEL/A72 on only a subset of APCs (not shown) and
was not examined further.
Membrane proteins are efficiently processed and presented in the class
II pathway (35, 36). We have previously shown that mHEL/Hb(64–76) can
be stably expressed in CH27 cells and that mHEL/Hb(64–76)-transfected
CH27 cells strongly stimulated both Hb(64–76) and HEL(46–61)-specific
hybridomas, demonstrating efficient processing and presentation of the
relevant determinants (24). In the present experiments, by design, we
could not directly examine the presentation of the A72 ligand because
it is an antagonist with no agonist activity (Transgenic mice
expressing mHEL/N72 (wild-type Hb) and mHEL/T72 (weak agonist)
conclusively demonstrate processing and presentation of Hb epitopes
from this chimeric construct in vivo (data not shown.)) We could,
however, utilize the HEL(46–61) determinant. Based on the above, we
expected that splenocytes from the A72tg mouse would stimulate the 3A9
T cell hybridoma, which is specific for HEL(46–61)/I-Ak.
Conversely, the same splenocytes should fail to stimulate the 3.L2-12 T
cell hybridoma, which is specific for Hb(64–76)/I-Ek but
antagonized by the A72 substitution (25). The results of these
experiments are shown in Figure 1C and conform to our
expectations. As few as 1 x 104 splenocytes will
stimulate 3A9, while 1 x 106 splenocytes fail to
stimulate 3.L2-12. These results demonstrate that mHEL/A72 is highly
expressed in peripheral APCs and is processed and presented.
Expression of mHEL/A72 in the thymus results in a small decrease in cells bearing the clonotypic receptor
To test the effect of the endogenous antagonist on the development
of 3.L2tg thymocytes, we bred the A72tg mouse to the 3.L2tg mouse and
performed three-color FACS analysis on thymocytes from mice that were 3
to 12 wk of age. Cells were stained with mAbs against CD4, CD8, and the
3.L2 clonotypic receptor (Fig. 2). The
data show that 3.L2tg and 3.L2tg x A72tg mice are the same in
terms of the percentage of total cells and the overall number of cells
in each compartment (Fig. 2A). However, when we gated
on the CD4-single positive (SP) cells or the CD4/CD8 double positive
(DP) cells, a reduction in thymocytes bearing the highest levels of the
clonotypic receptor was seen (Fig. 2B). This loss
represents approximately 15 to 20% of the CD4-SP cells with the 3.L2
TCR (right panel). Loss of cells
with the clonotypic specificity clearly occurs as early in thymocyte
development as the DP stage, suggesting that overall, the A72 ligand
provides either weak negative selection or antagonism of positive
selection. Although we cannot formally distinguish between the two
possibilities, the consequence is the same, namely loss of cells with
the highest levels of the clonotypic receptor. This experiment was
repeated four times (Table I).
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The number of clonotype-positive splenocytes in 3.L2tg x A72tg mice is also slightly reduced
Given the 15 to 20% reduction in CD4-SP thymocytes bearing the
clonotypic receptor described above, we expected to observe a similar
reduction in the number of CD4+CAB+ splenocytes
and in the overall level of the clonotypic receptor in the periphery.
On average, the number of CD4+CAB+ splenocytes
was reduced by 15 to 20% (Table I). However, in two experiments, both
the 3.L2tg and 3.L2tg x A72tg mice were virtually identical in
terms of both the number of CD4+CAB+ cells and
the level of clonotypic receptor on those cells (Fig. 3, A and B, taken
from Expt. 3). Furthermore, in those experiments where the 3.L2tg
x A72tg mice had fewer CAB+ splenocytes than their 3.L2tg
littermates, the reduction in CAB+ cells was not selective
for cells with the highest levels of TCR, as it was in the thymus (not
shown).
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. Also, 3.L2tg
splenocytes do not proliferate when stimulated with as much as 100 µM
of A72 peptide (G. J. Kersh et al., manuscript in preparation).
Instead, it seems plausible that inefficient negative selection in the
thymus of 3.L2tg x A72tg mice allows escape of a few cells with
high levels of the clonotypic receptor, which gradually accumulate in
the periphery and may reach the levels seen in the 3.L2tg mouse by 10
to 12 wk of age.
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We wanted to examine the proliferative response of 3.L2tg T cells
to Hb(64–76) in 3.L2tg x A72tg mice. It follows from the
preceding data that the proliferative response of splenocytes from
3.L2tg x A72tg mice should be the same as 3.L2tg controls,
particularly in those 3.L2tg x A72tg mice with equal numbers of
CD4+CAB+ splenocytes and equal levels of
the clonotypic receptor. In these mice, a direct comparison is not
complicated by reduced numbers of CAB+ T cells (Table I,
Expts. 3 and 4). When 3.L2tg x A72tg splenocytes from these mice
are stimulated with Hb(64–76) peptide, their proliferative response is
significantly reduced relative to 3.L2tg splenocytes. A representative
experiment showing a sevenfold reduction in the proliferative response
at 0.1 µM Hb(64–76) is shown in Figure 5A. This decrease in
proliferation in the presence of the antagonist A72 ligand was a
consistent feature of all experiments (Table I) and on average ranged
from three- to eightfold, depending on the concentration of agonist
ligand. We conclude that the proliferative response of 3.L2tg T cells
in the presence of the endogenous antagonist A72 is significantly
suppressed.
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Three possible mechanisms could account for this marked decrease in proliferative response to Hb(64–76) seen in 3.L2tg x A72tg mice: anergy (38, 39), peripheral antagonism (5, 40), or MHC competition. Regarding the third possibility, studies have demonstrated that continuous delivery of an endogenously expressed membrane protein results in only a fraction of class II molecules (6–15%) loaded with a specific epitope (41, 42). Thus, it is highly unlikely that the endogenous antagonist A72 could occupy enough I-Ek binding sites to inhibit the binding of the exogenously loaded agonist Hb(64–76) and give the appearance of a shift in the dose-response curve. Furthermore, we demonstrated that there was no competition for MHC binding by loading 3.L2tg and 3.L2tg x A72tg splenocytes with MCC(92–103), another well-characterized I-Ek epitope (43), and then using these splenocytes to stimulate the 2B4 hybridoma (I-Ek/MCC(92–103) specific). If the endogenous antagonist A72 was inhibiting the binding of exogenous Hb(64–76) peptide, then it should also inhibit the binding of MCC(92–103). The stimulation of 2B4 by MCC(92–103), from 0.01 µM to 10 µM, was identical on both populations of APCs, thus ruling out competition for MHC binding as an explanation for the observed shift (data not shown).
To distinguish between anergy and antagonism, we examined the inherent
reactivity of the T cells. T cells were purified from 3.L2tg and
3.L2tg x A72tg mice and cultured with B6.AKR splenocytes plus
peptide, or with A72tg splenocytes plus peptide. We reasoned that
anergized cells would be less reactive, regardless of the source of
APCs. Alternatively, peripheral antagonism should be seen only when
A72tg splenocytes were used as APCs. The two populations of T cells
gave identical responses with B6.AKR splenocytes plus Hb(64–76)
peptide as APCs (Fig. 5B). Antagonism was observed
for both populations of T cells when A72tg splenocytes plus Hb(64–76)
peptide were used as APCs. Furthermore, the difference between 3.L2tg T
cells + B6.AKR APCs and 3.L2tg T cells + A72tg APCs at 0.1
µM is about sevenfold, which fits well with the cumulative data
presented in Table I.
Clearly then, the two purified populations of T cells have quite similar responses under these two conditions. In the absence of the A72 ligand, both populations respond identically to stimulation with Ag, ruling out anergy. In the presence of the A72 ligand, both populations of T cells have an identical decrease in their proliferative response. These data demonstrate that the decreased proliferative response in 3.L2tg x A72tg mice is an example of peripheral antagonism with an endogenous APL.
Expression of mHEL/A72 in the thymus is sufficient to negatively select transgenic T cells specific for HEL(46–61)/I-Ak
One possible explanation for our unexpected observation that the A72 ligand had a minimal effect in the thymus but was still able to antagonize 3.L2tg T cell responses in the periphery was that expression of the mHEL/A72 protein in the periphery was far greater than in the thymus. Two lines of evidence argue against this explanation. First, immunohistochemical staining of the thymus and spleen with an anti-HEL Ab indicated that expression of mHEL/A72 was equivalent on APCs in the thymus and the spleen (not shown).
Another proof that this was not the explanation was obtained by
demonstrating processing and presentation of the HEL(46–61) epitope by
thymic APCs. The A72tg mouse was bred to the 3A9 TCR-transgenic mouse,
which expresses a TCR specific for HEL(46–61)/I-Ak
(30). In progeny expressing both transgenes, thymocytes with the 3A9
TCR should be negatively selected by APCs expressing the strong agonist
ligand HEL(46–61)/I-Ak. The data from this experiment are
shown in Figure 6. Virtually all DP
thymocytes are deleted (Fig. 6A, right
panel). There was a 75% reduction in CD4-SP cells
when comparing this mouse to a 3A9 mouse (center
panel). Furthermore, gating on the CD4-SP populations
and then analyzing for the presence of the 3A9 TCR reveals that none of
these CD4-SP cells in 3A9 x A72tg mice carry the clonotypic
receptor (Fig. 6B). Similar results were obtained by
Akkaraju et al. (44) when they bred the 3A9 TCR-transgenic mouse to a
mouse expressing mHEL under the control of a MHC class I promoter. In
double transgenic mice, there were virtually no DP thymocytes and only
9% CD4-SP thymocytes. They concluded that this is the result of
negative selection on the strong agonist ligand HEL(46–61).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These findings demonstrate that not all self-peptide/MHC ligands capable of interacting with a given TCR result in positive selection or tolerance by activation-induced cell death. T cells interacting with the endogenous self-antagonist A72 clearly mature and encounter the A72 ligand in the periphery, with the consequence being inhibition of specific immune responses. This suggests that the avidity threshold for negative selection of the 3.L2tg TCR is approximately that achieved in the A72tg mouse. On balance, it is better for reactive T cells with this avidity to mature and generate a more diverse T cell repertoire than to be deleted in the thymus. The potential tradeoff for this increase in diversity is the inhibition of primary immune responses, an effect that can be overcome by increasing the concentration of stimulating Ag. This phenotype has not been described in other systems, and may be more relevant to class II-restricted responses. In at least one class I system, T cells that react with a TCR antagonist early in development become tolerant by down-regulating CD8 (37). Down-regulation of CD4 was not observed in our experiments. Lastly, our results suggest that peripheral antagonism could play a role in the maintenance of self-tolerance. For example, the autoimmune potential of self-reactive cells could be held in check by the presence in the periphery of tissue-specific endogenous antagonists (17).
Several studies have implicated a role for APLs in thymic selection. The first reports demonstrated that peptide antagonists of mature T cells could induce positive selection of transgenic T cells with the same receptor in FTOC (12). Subsequent studies demonstrated that T cells positively selected on an APL down-regulated their CD8 coreceptor, again implicating APLs in positive selection and coreceptor down-regulation as a mechanism of self-tolerance in the periphery (37). Page et al. used the deletion of DP thymocytes in culture after stimulation with Ag as a measure of negative selection, and found that negative selection could be induced by ligands of lower affinity than those required for full T cell activation (22). Spain et al. have also shown in FTOC that an antagonist peptide can decrease positive selection, either through negative selection or by antagonizing positive selection (21). Sebzda et al. have shown that positive and negative selection could be induced by different concentrations of the stimulatory peptide (14), and that an APL with moderate agonist properties could mediate positive selection in FTOC (45). However, in another study, T cells selected on low concentrations of the agonist peptide were shown to be nonfunctional (46). Taken together, these data demonstrate that APLs can exert some influence on developing thymocytes in vitro. Importantly, this effect seems to correlate with the affinity of TCR/ligand interaction (47), implying that for any given peptide, the balance between positive and negative selection will depend on the avidity of the interaction of the specific T cell with thymic APCs (13, 18, 48). Factors influencing this overall avidity include the binding affinity of the TCR and MHC/peptide pair, the concentration of the coreceptor, and the concentration of the relevant ligand on that APC. These studies clearly predict that APLs will have an effect on thymic selection in vivo.
In our in vivo studies with the endogenous antagonist A72, we do not see enhanced positive selection. One explanation for this observation is that positive selection of the 3.L2tg TCR may already be maximized on the B6.AKR background. Should this be the case, adding an additional positively selecting ligand would have no effect. It is important to point out that our experiments only test the effect of adding an antagonist to the endogenous pool of ligands, and not the independent function of the antagonist. Given this caveat, our results are therefore most consistent with the in vitro results of Spain et al. (21) and Page et al. (22), with a number of important differences that are highlighted above. We see a small but consistent reduction in the number of thymocytes with high levels of the clonotypic receptor in 3.L2tg x A72tg mice. We favor weak negative selection, rather than antagonism of positive selection, as the explanation for a number of reasons. Loss of CAB+ cells in the presence of the endogenous antagonist occurs at the CD4+CD8+ stage of development, and negative selection occurs at this stage in other TCR-transgenic systems (21, 49). Furthermore, the A72 peptide induces deletion of CD4+CD8+ thymocytes in a suspension culture deletion assay, albeit at very high concentrations of peptide (G. J. Kersh et al., manuscript in preparation). The A72 peptide does not induce either the 3.L2–12 hybridoma, the 3.L2 clone, or 3.L2tg T cells to produce IL-2 at any concentration. Given the above, it seems plausible that negative selection is responsible for the loss CAB+ CD4+CD8+ cells, although as mentioned we cannot rule out antagonism of positive selection.
It is instructive to consider the findings presented herein together with our previous studies describing the effects of endogenous antagonists. Our earlier studies utilized a TCR ß-chain-transgenic mouse in which Hb(64–76) is an antagonist, and Hb(64–76) T69S (Ser69) is the agonist. Expression of the antagonist Hb(64–76) in vivo resulted in elimination of high avidity Ser69-reactive cells, demonstrating negative selection by an endogenous antagonist (23). In the periphery, low avidity Ser69-reactive cells appeared, but the level of endogenous Hb(64–76)/I-Ek complexes was too low to antagonize the response of these Ser69-reactive cells without the addition of exogenous peptide (24). In the present study, expression of the endogenous antagonist resulted in a profound lack of central tolerance in which high avidity Ag-specific T cells mature, and are antagonized by endogenous A72/I-Ek complexes in the periphery. Taken together, these results illustrate the broad range of biologic activity attributable to endogenous antagonists. The differential-avidity model of thymic selection predicts that those antagonists that negatively select will have a higher affinity for their cognate TCR than will those antagonists that are neutral or that positively select (16). While we cannot examine this directly in the Ser69 system due to the oligoclonal T cell response to Ser69, we can determine the kinetics of TCR/ligand interactions in the 3.L2 system. Based on the above, the prediction is that thymic expression of 3.L2 TCR antagonist ligands with greater biologic activity (and presumed greater affinity) will result in increased negative selection. We conclude that these in vivo data are consistent with the view that there is a correlation between the affinity of antagonist/TCR interactions and the magnitude of their central effect (47).
In summary, we have presented data examining the in vivo effects of expressing one particular 3.L2 TCR antagonist at one fixed level in MHC class II-positive cells in the thymus and spleen. Surprisingly, the predominant effect is tolerance by peripheral antagonism, rather than central tolerance by negative selection. We see no evidence for positive selection of Ag-specific T cells in addition to that already achieved on the B6.AKR background. Since only a single transgenic founder was examined, our results may be affected to some degree by quantitative effects, which are determined by the level of transgene expression, the site of integration, and the details of tissue-specific expression. Such quantitative effects will need to be addressed before the role of APLs in vivo can be firmly established. Caveats not withstanding, the unexpected findings described herein highlight the importance of this in vivo approach, which we are extending to a spectrum APLs of Hb(64–76). The avidity model predicts that those ligands that bind more weakly than I-Ek/A72 to the 3.L2tg TCR may result in enhanced positive selection, and those with stronger interactions should increase negative selection (16). We postulate that the antagonist I-Ek/A72 has an avidity positioned directly between these two possibilities.
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Acknowledgments |
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promoter; Kathy Frederick, Darren
Kreamalmeyer, and Donna Thompson for help breeding the mice; Dr. Gil
Kersh, Dr. Claude Daniel, Ellen Neumeister, and Devraj Basu for helpful
discussion and sharing unpublished results; Drs. Talal Chatila, Osami
Kanagawa, Jonathan Katz, and Skip Virgin for review of the manuscript;
and Jerri Smith for her assistance in the preparation of the
manuscript. |
Footnotes |
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2 Scholar of the Child Health Research Center of Excellence in Developmental Biology at Washington University School of Medicine (HD33688).
3 Address correspondence and reprint requests to Dr. Paul M. Allen, Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8118, St. Louis, MO 63110.
4 Abbreviations used in this paper: APL, altered peptide ligand; Hb, hemoglobin; CAB, clonotypic Ab; A72, APL of (64–76) with an alanine for asparagine substitution at position 72; DP, double positive (CD4+CD8+); SP, single positive; HEL, hen egg-white lysozyme; mHEL/A72, chimeric membrane protein containing HEL and the APL A72; A72tg, transgenic mouse expressing mHEL/A72 in all class II-positive cells; 3.L2tg, 3.L2 TCR-transgenic mouse; FTOC, fetal thymic organ culture; PE, phycoerythrin; TC, tri-color.
Received for publication December 4, 1997. Accepted for publication February 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and lack of Zap 70 recruitment in APL-induced T cell anergy. Cell 79:913.[Medline]
Phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 267:515.This article has been cited by other articles:
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  L. M. Relland, M. K. Mishra, D. Haribhai, B. Edwards, J. Ziegelbauer, and C. B. Williams Affinity-Based Selection of Regulatory T Cells Occurs Independent of Agonist-Mediated Induction of Foxp3 Expression J. Immunol., February 1, 2009; 182(3): 1341 - 1350. [Abstract] [Full Text] [PDF]
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C. Fujimoto, C.-R. Yu, G. Shi, B. P. Vistica, E. F. Wawrousek, D. M. Klinman, C.-C. Chan, C. E. Egwuagu, and I. Gery Pertussis Toxin Is Superior to TLR Ligands in Enhancing Pathogenic Autoimmunity, Targeted at a Neo-Self Antigen, by Triggering Robust Expansion of Th1 Cells and Their Cytokine Production J. Immunol., November 15, 2006; 177(10): 6896 - 6903. [Abstract] [Full Text] [PDF]
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 H. Kao and P. M. Allen An antagonist peptide mediates positive selection and CD4 lineage commitment of MHC class II-restricted T cells in the absence of CD4 J. Exp. Med., January 3, 2005; 201(1): 149 - 158. [Abstract] [Full Text] [PDF]
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D. Haribhai, B. Edwards, M. L. Williams, and C. B. Williams Functional Reprogramming of the Primary Immune Response by T Cell Receptor Antagonism J. Exp. Med., December 6, 2004; 200(11): 1371 - 1382. [Abstract] [Full Text] [PDF]
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F. F. Shih, L. Mandik-Nayak, B. T. Wipke, and P. M. Allen Massive Thymic Deletion Results in Systemic Autoimmunity through Elimination of CD4+ CD25+ T Regulatory Cells J. Exp. Med., February 2, 2004; 199(3): 323 - 335. [Abstract] [Full Text] [PDF]
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 B. Murphy, J. Yu, Q. Jiao, M. Lin, T. Chitnis, and M. H. Sayegh A Novel Mechanism for the Immunomodulatory Functions of Class II MHC-Derived Peptides J. Am. Soc. Nephrol., April 1, 2003; 14(4): 1053 - 1065. [Abstract] [Full Text] [PDF]
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D. Haribhai, D. Engle, M. Meyer, D. Donermeyer, J. M. White, and C. B. Williams A Threshold for Central T Cell Tolerance to an Inducible Serum Protein J. Immunol., March 15, 2003; 170(6): 3007 - 3014. [Abstract] [Full Text] [PDF]
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F. R. Santori, S. M. Brown, Y. Lu, T. A. Neubert, and S. Vukmanovic Cutting Edge: Positive Selection Induced by a Self-Peptide with TCR Antagonist Activity J. Immunol., December 1, 2001; 167(11): 6092 - 6095. [Abstract] [Full Text] [PDF]
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V. Raman, F. Blaeser, N. Ho, D. L. Engle, C. B. Williams, and T. A. Chatila Requirement for Ca2+/Calmodulin-Dependent Kinase Type IV/Gr in Setting the Thymocyte Selection Threshold J. Immunol., December 1, 2001; 167(11): 6270 - 6278. [Abstract] [Full Text] [PDF]
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F. R. Santori, I. Arsov, and S. Vukmanovic Modulation of CD8+ T Cell Response to Antigen by the Levels of Self MHC Class I J. Immunol., May 1, 2001; 166(9): 5416 - 5421. [Abstract] [Full Text] [PDF]
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C. B. Williams, D. L. Engle, G. J. Kersh, J. Michael White, and P. M. Allen A Kinetic Threshold between Negative and Positive Selection Based on the Longevity of the T Cell Receptor-Ligand Complex J. Exp. Med., May 17, 1999; 189(10): 1531 - 1544. [Abstract] [Full Text] [PDF]
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D. R. Plas, C. B. Williams, G. J. Kersh, L. S. White, J. M. White, S. Paust, T. Ulyanova, P. M. Allen, and M. L. Thomas Cutting Edge: The Tyrosine Phosphatase SHP-1 Regulates Thymocyte Positive Selection J. Immunol., May 15, 1999; 162(10): 5680 - 5684. [Abstract] [Full Text] [PDF]
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 S. C. Jameson T cell receptor antagonism in vivo, at last PNAS, November 24, 1998; 95(24): 14001 - 14002. [Full Text] [PDF]
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D. Basu, C. B. Williams, and P. M. Allen In vivo antagonism of a T cell response by an endogenously expressed ligand PNAS, November 24, 1998; 95(24): 14332 - 14336. [Abstract] [Full Text] [PDF]
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