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Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
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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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Anergy is defined as a cellular state in which a lymphocyte is alive
but fails to display certain functional responses when optimally
stimulated through both its Ag-specific receptor and any other
receptors that are normally required for full activation
(12). It is typically characterized by a defect in IL-2
production and T cell proliferation (12). The persistence
of anergic T cells specific for self-Ags in vivo is somewhat puzzling.
Because these T cells are self-specific, a breakdown of mechanisms to
maintain their inactive state could lead to autoimmune reactions.
However, it is conceivable that anergic T cells may subserve a
regulatory role to prevent the induction of immune responses to
self-Ags. We have begun to examine these possibilities using a model of
anergy induction centered on a functionally mature
CD4-CD8- T cell
population that expresses a transgenic TCR-
ß (herein referred to
as DN cells). Development of these DN cells in TCR transgenic mice is
thymus dependent (13) but independent of positively
selecting MHC molecules (13, 14). They are also resistant
to clonal deletion in Ag-expressing mice (14, 15). We have
previously found that, based on their inability to produce IL-2 or to
proliferate (16), DN T cells in this system are rendered
anergic when chronically exposed to their antigenic ligand in vivo. In
the present study, we show that these anergic T cells have a lower
activation threshold for the induction of CD25. Furthermore, they
proliferate vigorously when stimulated with a low-affinity ligand plus
an exogenous source of IL-2. The implications of the existence of such
a population of anergic T cells in inducing autoimmune reactions or in
the suppression of anti-self-reactivity are discussed.
Previous studies have shown that depending on the cell type and anergy induction protocol, specific biochemical defects have been associated with the anergic state(for review, see Ref. 17). In some models, the block in IL-2 production is attributed to a decrease in IL-2 gene transcription (18) and this block results from a failure to activate p21ras (Ras) after TCR ligation. This failure leads to a decrease in the kinase activities of both extracellular signal-regulated protein kinase (ERK) and c-Jun NH2-terminal kinase, which in turn leads to a failure to activate the IL-2 transcription factor AP-1 (18). In other models based on human T cell clones, an impaired intracellular calcium response has been credited with the reduced IL-2 production by means of impaired binding of the NF-AT transcription factor to distal response elements within the IL-2 enhancer (19). However, in the latter human T cell model, AP-1 is hardly observed to be affected (20). Although the above-mentioned alterations in AP-1 or NF-AT function provide a distal biochemical basis for the anergic T cell’s inability to produce IL-2, signaling defects more proximal to the TCR also exist in anergic cells and may be causal to the reduced activation of these transcription factors.
Anergic cells exhibit increased expression of p59fyn(Fyn) along with increased Fyn protein tyrosine kinase (PTK)3 activity (21). Fyn has been reported to be constitutively associated with Cbl in anergic cells (22) and this association has been shown to lead to increased activity of Rap1, a negative regulator of IL-2 transcription (22). More recently, Cbl has been shown to be a negative regulator of ZAP-70 and Cbl-/- cells show increased ZAP-70 PTK activity (23, 24). Cbl binds to ZAP-70 and this binding is dependent on phosphorylation of tyrosine 292 on ZAP-70 (25). Anergic T cells have been reported to be unable to activate ZAP-70 upon TCR ligation (26), but it is unclear whether this is due to negative regulation of ZAP-70 by Cbl in anergic T cells.
Previous models of in vivo induced T cell anergy have been based
largely on either repeated injections of superantigen (9, 27) or adoptive transfer models of T cells from TCR-transgenic
mice (28, 29). However, biochemical analysis of in vivo
anergized T cells is very limited, due in part to the difficulty in
isolating sufficient numbers of purified anergic cells for these
studies. The few studies done to date were performed using
heterogeneous CD4 T cells that have been anergized by repeated
injections of staphylococcal enterotoxin A. These studies showed that
CD4 T cells anergized in this manner exhibit impaired NF-AT, NF-
B,
and AP-1 binding to IL-2 enhancer response elements (30, 31).
In this study, we have examined early intracellular signaling events in in vivo anergized DN cells. We demonstrate that these anergic cells exhibit differential requirements in mobilizing intracellular calcium and in the activation of the ERK mitogen-activated protein (MAP) kinase pathway. These biochemical characteristics were associated with an elevated level of Fyn, but not Lck, expression in these anergic T cells. The inefficient mobilization of intracellular calcium was associated with defects in ZAP-70 and linker for the activation of T cells (LAT) phosphorylation in response to TCR signaling. Efficient ERK1/2 activation was associated with efficient induction of the CD69 activation marker and phosphorylation of SLP-76. A model for the regulation of TCR-signaling pathways in these anergic T cells is proposed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeders for the H-2b 2C TCR-transgenic mice (4, 32) were kindly provided by Dr. Denis Loh (then at the University of Washington, St. Louis, MO). The H-2b 2C TCR-transgenic mice have been backcrossed to the C57BL/6 background. DBA/2 (H-2d) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). H-2b/d 2C mice were F1 mice obtained by mating DBA/2 mice with H-2b 2C TCR mice. All animals were maintained in the animal facility at the University of British Columbia in the Microbiology and Immunology building.
Cells
Lymph node cells were harvested from transgenic mice. Purification of DN cells was as described previously (16). The purified DN cells were typically >95% CD4-CD8-Ig- and expressed exclusively the 2C TCR, which was detected by the 1B2 mAb (33). The peptide transporter-deficient cell lines T2-Ld and T2-Kb (34) were derived by transfecting the human (T x B) hybridoma T2 with Ld or Kb. The T2-Ld or T2-Kb cells were used as APC for the p2Ca peptide. The p2Ca peptide (LSPFPFDL) was synthesized by the Nucleic Acid Service Laboratory at the University of British Columbia. Cells were cultured in IMDM (Life Technologies, Burlington, Ontario, Canada) supplemented with 5 x 10-5 M 2-ME, 10% FCS (Life Technologies), and antibiotics.
Proliferation assays
Proliferation assays were performed by culturing 1 x 104 purified DN cells with 5 x 104 mitomycin-treated T2-Ld or T2-Kb cells with indicated concentrations of the p2Ca peptide. Cells were cultured in triplicates in a volume of 0.20 ml in 96-well round-bottom plates. For assessment of proliferation, 1 µCi of [3H]thymidine was added to each culture well in the last 6 h of a 72-h culture period.
Whole-cell lysate and immunoprecipitation studies
DN cells were prepared and resuspended in IMDM supplemented with 0.25% FCS at 107 cells/ml. Cells were prewarmed to 37°C, stimulated with 10 µg/ml 2C11 for the indicated time period, washed with ice-cold PBS, and then lysed in lysis buffer. Lysis buffer consisted of the following: TNE (10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA) pH 7.60, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM sodium orthovanadate, 1 mM sodium molybdate, 2 mM PMSF, and 1% Triton X-100. For studies on LAT, Brij 97 was substituted for Triton X-100. Cells were lysed on ice for 10 min. Lysates were clarified by centrifugation at 14,000 rpm for 10 min before immunoprecipitation. Lysates were incubated for 2 h with the appropriate Ab and 20 µl of packed protein A-Sepharose CL-4B (Amersham Pahrmacia Biotech, Baie d’Urfe, Quebec, Canada). Immune complexes were washed 3x with lysis buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Western blotting was conducted with the indicated Abs and developed with the appropriate HRP-conjugated secondary Abs and the enhanced chemiluminescence (Amersham Pharmacia Biotech) detection system. Where indicated, blots were stripped for 40 min at 55°C in stripping solution: 62.5 mM Tris-HCl (pH 6.8), 2.0% SDS, and 100 mM 2-ME.
Spot densitometry quantification was done using the AlphaImager 1200 v4.03 software on an Alpha Imager 1200 (Alpha Innotech, San Leandro, CA).
Antibodies
Abs used include the following: anti-Fyn (sc-16),
anti-ZAP-70 (sc-574), anti-ERK1/2 (sc-94), and anti-pERK1/2
(sc-7383) from Santa Cruz Biotechnology (Santa Cruz, CA). 4G10
anti-phosphotyrosine (catalogue no. 05-321) and anti-LAT
(catalogue no. 06-807) were purchased from Upstate Biotechnology (Lake
Placid, NY). Anti-CD4 (GK1.5), anti-CD8 (53.67), anti-CD3
(145-2C11), and anti-CD25 (PC61) were obtained from American Type
Culture Collection (Manassas, VA). Anti-CD69 (catalogue no. 01502D) was
obtained from PharMingen (San Diego, CA). Sheep anti-SLP-76
polyclonal Abs were a kind gift from Dr. Koretsky (University of Iowa,
Iowa City, IA) (35). The 2C TCR Id-specific mAb 1B2
hybridoma was a kind gift from Dr. Eisen (Cambridge, MA)
(33). The anti-TCR-
mAb (G3), specific for the
cytoplasmic domain of , was produced in our laboratory
(36). The anti-Lck Ab 54.3B is a peptide-specific
(N-terminal residues 3–147) rabbit antisera generated in our
laboratory (37).
Intracellular calcium
Flow cytometry was used to measure intracellular calcium levels in cells loaded with the calcium-binding dye fluo-3-acetoxymethyl ester (Molecular Probes, Eugene, OR) using the Chronys software package (Becton Dickinson, Mountain View, CA) as described previously (38).
CD69 and CD25 flow cytometry
Single-cell suspensions of lymph node cells were prepared. Purified DN cells (1 x 106) were stimulated with 5 x 105 mitomycin C-treated T2-Ld or T2-Kb cells plus the indicated concentration of the p2Ca peptide in a 24-well plate in a volume of 2.0 ml. No exogenous IL-2 was added. After a culture period of 40 h, the cells were collected and stained with biotinylated anti-CD69 or anti-CD25 mAb followed by streptavidin-Tricolor and analyzed with the FACScan flow cytometer using Lysis II software (Becton Dickinson). A total of 15,000 events was analyzed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anergic H-2b/d 2C DN cells proliferate in response to a low-affinity ligand and exogenous IL-2
The hypoproliferative response of Ag-stimulated anergic T cells
has been reported to be reversible in some systems by the addition of
IL-2 (17, 43). In agreement with our previous results
(16), nonanergic DN cells from H-2b
2C mice exhibit a significant proliferative response to stimulation
with the high-affinity p2Ca/Ld ligand even
without the addition of exogenous sources of IL-2 (Fig. 1
). The addition of exogenous IL-2 to
these cultures led to a relatively modest increase in the proliferative
response (Fig. 1). As previously reported, the anergic DN cells from
H-2b/d 2C mice showed a minimal proliferative
response when stimulated only with high concentrations of the
p2Ca/Ld ligand. Strikingly, when exogenous IL-2
was added to p2Ca/Ld-stimulated cultures, they
proliferated better than the nonanergic DN cells, particularly at low
concentrations of the p2Ca/Ld ligand (Fig. 1).
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1000-fold lowering in the affinity for the
latter interaction (42). Since very low concentrations of
the high-affinity p2Ca/Ld ligand were able to
induce a vigorous proliferative response in anergic DN cells in the
presence of exogenous IL-2, we tested the hypothesis that the anergic
DN cells may in fact have a lower activation threshold than the
nonanergic DN cells. The results in Fig. 2 support such a hypothesis. It was found
that nonanergic DN cells were unable to proliferate when stimulated
with various concentrations of the low-affinity
p2Ca/Kb ligand, even in the presence of
exogenously added IL-2. In contrast, the anergic DN cells already
exhibit a small but significant proliferative response to the
p2Ca/Kb ligand even in the absence of exogenous
IL-2; this small proliferative response was also elicited by the
high-affinity ligand (Fig. 1
). In the presence of exogenously added
IL-2, these cultures mount a vigorous proliferative response (Fig. 2).
These results support the hypothesis that the anergic DN cells have a
lower activation threshold when compared with nonanergic DN cells.
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We sought independent support for the hypothesis that anergic DN
cells have a lower activation threshold than nonanergic DN cells. The
expression of activation markers associated with T cell activation
following stimulation of these cells with either the high- or
low-affinity ligand was assessed. DN cells from
H-2b or H-2b/d 2C mice were
activated with either the high- or low-affinity ligand in the absence
of exogenously added IL-2 and the expression of CD25 and CD69 on these
cells was determined after 40 h of stimulation. The results in
Fig. 3 show that the anergic DN cells
were able to undergo blastogenesis (increase in forward scatter) and
up-regulate CD25 and CD69 in response to either the high- or the
low-affinity ligand. In contrast, only the high-, but not low-,
affinity ligand was able to induce blastogenesis, CD25 and CD69
expression in the nonanergic DN cells. These results are consistent
with the proliferation data in Figs. 1 and 2 and further support the
hypothesis that the anergic DN cells have a lower activation threshold
in comparison to the nonanergic DN cells. Furthermore, the vigorous
proliferation that ensued when exogenous IL-2 was added to these
stimulated cultures indicated that these anergic DN cells retain the
biochemical machinery that is required for normal proliferation.
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The above findings suggest that the anergic DN cells were able to
mediate signaling from the TCR which led to effective induction of CD25
and CD69 expression. We have shown in a recent study that Ag-activated
anergic DN cells were defective in IL-2 production (16).
Stimulation of anergic and nonanergic DN cells with an anti-CD3
mAb led to similar findings. Thus, anti-CD3-stimulated anergic
DN cells (8 x 105/well in 0.2 ml with 10
µg/ml of immobilized 2C11 mAb for 18 h) produced less IL-2 on a
per cell basis (3.5 U/ml) when compared with similarly stimulated
nonanergic DN cells (10 U/ml). The proliferative response of
anti-CD3-stimulated anergic DN was also lower when compared with
similarly stimulated nonanergic DN cells (Fig. 4). To determine the extent of the
signaling defects in the anergic cells, we first compared whole-cell
lysate phosphorylation profiles from anti-CD3-stimulated
H-2b 2C and H-2b/d 2C DN
cells. We noted that a major difference in the phosphorylation of a
36-kDa protein was observed (Fig. 5A). The apparent molecular
mass and rapid phosphorylation of this protein in the
H-2b DN cells suggested this protein to be the
LAT adaptor molecule (44).
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B). In this experiment, LAT was
also hypophosphorylated in anti-CD3-stimulated anergic DN cells.
This hypophosphorylation of LAT could be accounted for in part by the
lower amount of LAT that was precipitated from the anergic DN cells
(Fig. 5B). Densitometric quantitation of the data indicated
that when the lesser amount of immunoprecipitated LAT was taken into
consideration, LAT phosphorylation was reduced by about 60% relative
to that observed in the nonanergic DN cells. The level of expression of
LAT in anergic and nonanergic DN cells was also determined by Western
blotting of whole-cell lysates, and the data were quantified by
densitometric analysis (Fig. 5C). This analysis showed a
34% reduction in LAT expression in the anergic population. These data
indicate that the hypophosphorylation of LAT in the anergic DN cells
upon TCR stimulation is due to two factors: 1) the lower expression of
LAT and 2) the less efficient phosphorylation of LAT upon TCR
stimulation. ZAP-70 phosphorylation is reduced in H-2b/d 2C DN cells
To explain the LAT phosphorylation defect observed in the
H-2b/d 2C DN cells, we examined signaling events
known to be upstream of LAT phosphorylation. ZAP-70 has LAT as a major
substrate and phosphorylation of LAT by ZAP-70 is a major event in T
cell activation (44). We first determine the expression
level of ZAP-70 in anergic and nonanergic DN cells and found it to be
fairly equivalent (Fig. 6A).
Immunoprecipitation studies of ZAP-70 found it to be tyrosine
phosphorylated in nonanergic DN cells upon TCR ligation (Fig. 6B). Previous studies have shown that phosphorylation of
ZAP-70 is associated with its activation (45, 46). In
contrast, tyrosine phosphorylation of ZAP-70 upon TCR ligation was
found to be impaired in the H-2b/d 2C DN cells
(Fig. 6B). In this experiment, we observed more tyrosine
phosphorylation of ZAP-70 despite the fact that slightly less ZAP-70
was precipitated from TCR-stimulated nonanergic cells. This observation
strengthens the conclusion that ZAP-70 is hypotyrosine phosphorylated
in anergic DN cells upon TCR stimulation. However, precipitation of
less ZAP-70 from TCR-stimulated nonanergic cells was not a consistent
finding since in repeat experiments similar amounts of ZAP-70 were
precipitated from nonstimulated and stimulated cells (data not
shown).
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chain to be doubly phosphorylated to
allow binding of the tandem Src homology 2 domains present in ZAP-70
(45). These phosphorylation events occur via activation of
the Src kinases, Lck and Fyn, and their subsequent recruitment to the
TCR- chains, their autophosphorylation, and then subsequently their
phosphorylation of the immunoreceptor tyrosine-based activation motifs
within the TCR chains (45, 47). We examined whether the
hypophosphorylation of ZAP-70 in anergic DN cells is related to the
less efficient phosphorylation of TCR- chains upon TCR stimulation.
Fig. 7 indicates the fairly equivalent
level of expression of the TCR- chain in anergic and nonanergic DN
cells. Quantitation of p21 and p23 by densitometric analysis yielded
the following results: percent decrease in p21 tyrosine phsophorylation
in H-2b/d 2C DN cells relative to
H-2b 2C DN cells at 3, 10, and 15 min after TCR
stimulation was 31%, 5%, and 37%, respectively; the corresponding
values for p23 were decreases of 55%, 30%, and 50%, respectively.
These data suggest that there is less p23 in anergic DN cells after TCR
stimulation. The reduced induction of p23, which represents fully
phosphorylated TCR- chains (48), may explain in part
the less efficient recruitment and activation of ZAP-70 to the TCR-CD3
signaling complex in TCR-stimulated anergic DN cells.
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). Such
an increase in expression was not observed for Lck (Fig. 8).
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One major consequence of LAT phosphorylation is the TCR-mediated
activation of phospholipase C (PLC)-
1 and the Ras pathway (49, 50). Having observed a defect in the phosphorylation of LAT in
TCR-stimulated anergic DN cells, and since PLC-1 activation is
required for the induction of a sustained increase in intracellular
calcium, we examined the ability of these cells to mobilize
intracellular calcium in response to TCR ligation. In this respect, the
anergic DN cells were found to be inefficient in mobilizing
intracellular calcium when compared with nonanergic DN cells (Fig. 9). This observation supports the notion
that one consequence of defective LAT phosphorylation in anergic DN
cells is a failure to mobilize intracellular calcium efficiently in
response to TCR ligation.
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The adaptor molecule SLP-76, like LAT, is a linker molecule
closely associated with proximal TCR signaling (reviewed in Ref.
51). SLP-76 also undergoes tyrosine phosphorylation upon
TCR engagement and is also a substrate of ZAP-70 (52).
SLP-76 has been shown to associate with the Src homology 3 domain of
Grb2 via proline-rich motifs and is essential for the coupling of
TCR-regulated PTKs to downstream signaling pathways (53).
Previous studies have also shown that tyrosine phosphorylation of
PLC-1 and the Ras-signaling pathway are defective in
SLP-76-/- T cells (52). The
anergic DN cells exhibit efficient induction of CD69 (Fig. 3), which
has been shown to be dependent on the Ras-signaling pathway
(52). However, induction of calcium mobilization in
TCR-stimulated anergic DN cells was shown to be defective (Fig. 9). To
reconcile these findings, we hypothesize that LAT and SLP-76 may depend
differentially on ZAP-70 for their phosphorylation. We also propose
that phosphorylated SLP-76 may be sufficient to link TCR-signaling
pathways to the Ras-signaling pathway in anergic DN cells. To test this
hypothesis, we examined SLP-76 phosphorylation in anergic and
nonanergic DN cells upon TCR ligation (Fig. 10). SLP-76 was immunoprecipitated from
these two cell types before and after TCR stimulation. It was found
that similar amounts of SLP-76 were precipitated from
H-2b and H-2b/d 2C DN (Fig. 10). Quantitation of the data in Fig. 10 indicates that SLP-76 tyrosine
phosphorylation in H-2b/d 2C DN cells after TCR
stimulation was decreased by 12% relative to
H-2b 2C DN cells. This observation is consistent
with the notion that phosphorylation of SLP-76 is less dependent on
activated ZAP-70. Alternatively, it is conceivable that other
uncharacterized pathways are responsible for SLP-76 phosphorylation in
anergic DN cells.
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) suggest that the Ras-signaling pathway is
intact in these cells. Since ERK1/2 phosphorylation and activation
occurs downstream of the Ras-signaling pathway, we sought independent
confirmation of activation of the Ras-signaling pathway by examining
the phosphorylation of ERK1/2 in nonanergic and anergic DN cells upon
TCR ligation. The results in Fig. 11
indicate that ERK1/2 phosphorylation occurs normally in anergic DN
cells upon TCR stimulation. Quantitation of the data in Fig. 11 showed
that the level of phosphorylation of ERK1/2 in
H-2b/d DN cells was within 10% of that observed
in H-2b DN cells. These data are consistent with
the hypothesis that the Ras-signaling pathway and ERK1/2 are activated
normally in TCR-stimulated anergic DN cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biochemical changes observed in the anergic DN cells following TCR
stimulation are remarkably similar to the biochemical phenotype of T
cells that have been stimulated by partial agonists. TCR partial
agonist ligands were shown to activate the Ras/MAP kinase pathway by an
alternative pathway that is independent of ZAP-70 kinase activity
(54) and LAT phosphorylation (55). In this
model, Grb2-SOS complexes were found to associate with incompletely
phosphorylated p21 phospho-TCR-; this observation provides a
possible explanation for the activation of the Ras/MAP kinase pathway
in the absence of ZAP-70 activation and phosphorylation of LAT
(55). Interesting, we found that despite the incomplete
activation of all of the TCR-signaling pathways, these signals were
sufficient to promote the induction of CD25 in anergic cells and
support their proliferation in the presence of exogenous IL-2.
A critical defining characteristic of T cell anergy is an inability to produce IL-2 upon TCR ligation. We have shown previously that Ag-stimulated anergic DN cells are defective in IL-2 production (16). Distinct regulatory regions exist in the 5' promoter region of the IL-2 gene, and these distinct regions can be bound by varied nuclear factors to initiate IL-2 transcription. These factors include the AP-1 and NF-AT proteins. The AP-1 family of nuclear factors include members of the Fos and Jun families. Fos and Jun can bind DNA as the AP-1 complex and have been implicated in the control of IL-2 transcription. AP-1 dependent DNA binding and IL-2 gene transcription are deficient in some forms of anergy (18). In conjunction with AP-1, NF-AT is also involved in IL-2 transcription and can bind the IL-2 promoter at an NF-AT site when complexed with AP-1. In contrast to Fos and Jun, which are regulated by ERK and c-Jun NH2-terminal kinase activation (56, 57), NF-AT exists in the cytoplasm in an inactive phosphorylated form. TCR-mediated activation of calcium signaling leads to activation of calcineurin, a phosphatase able to dephosphorylate NF-ATp, allowing NF-AT to enter the nucleus and bind DNA in the presence of AP-1 (58). Thus, the nature of IL-2 transcriptional regulation is such that different mechanisms may exist to control the production of IL-2. As mentioned above, a failure to activate the AP-1 complex has been reported in many forms of anergy. As well, a failure to activate NF-AT has also been reported (20) and this form of anergy has been referred to as calcium-blocked anergy to contrast it from Ras-blocked anergy (reviewed in Ref. 17). Our findings are consistent with the form of anergy we have studied to be calcium blocked and not Ras blocked.
The failure of anergic DN cells to mobilize intracellular calcium upon
TCR ligation is likely due to their inability to phosphorylate LAT, and
this is most likely due to their failure to optimally activate ZAP-70
(Fig. 5). Similar reductions in phosphorylation of a 38-kDa molecule,
possibly LAT, have previously been reported in anergic Th1 cells
(59). LAT is phosphorylated by ZAP-70 upon TCR ligation,
leading to recruitment of multiple signaling molecules that culminates
in the activation of calcium- and Ras-dependent pathways
(50). In this study, we have shown that whereas defective
LAT phosphorylation affected calcium-dependent pathways in anergic DN
cells, it does not seem to affect the Ras-signaling pathway in these
cells.
We also observed efficient tyrosine phosphorylation of SLP-76 upon TCR
ligation in the anergic cells (Fig. 9). Because SLP-76 is a substrate
for ZAP-70 (52), this observation suggests that either
suboptimally activated ZAP-70 is sufficient to phosphorylate SLP-76 but
not LAT, or alternatively SLP-76 may be phosphorylated by other
mechanisms that remain to be defined. Previous studies have shown that
SLP-76 tyrosine phosphorylation is required for optimal SLP-76 function
including Vav recruitment to SLP-76 (reviewed in Ref. 51).
SLP-76-deficient Jurkat T cells exhibit a marked reduction in PLC-1
tyrosine phosphorylation, intracellular calcium mobilization, and ERK
activation (52). Since ERK activation, but not calcium
mobilization, is normal in TCR-stimulated anergic DN cells, these
findings support the notion that phosphorylated SLP-76 alone, in the
absence of phosphorylated LAT, may be sufficient to activate the Ras
pathway. However, it is insufficient to activate intracellular calcium
mobilization.
We have shown that the p23 form of the TCR- chain is phosphorylated
to a lesser extent in TCR-stimulated anergic DN cells. It is unclear
whether this is a consequence of the increased expression of Fyn
relative to Lck. The less efficient phosphorylation of p23 may lead to
less efficient recruitment and activation of ZAP-70. Alternatively,
and/or in addition to this mechanism, the failure to fully activate
ZAP-70 and hence phosphorylate LAT may be due to the presence or
activation of a negative regulator of ZAP-70. Previously, constitutive
association of Cbl with Fyn has been observed in anergic T cells
(22). We found that anergic DN cells have an increased
basal level of Fyn expression and this may lead to more efficient
phosphorylation of Cbl. Recent studies have shown that Cbl acts as a
negative regulator of ZAP-70 (23, 24). Furthermore,
tyrosine phosphorylation of LAT and SLP-76 was also found to be
sustained in TCR-stimulated Cbl-/- thymocytes
(60, 61). It is therefore conceivable that the suboptimal
phosphorylation of ZAP-70 in TCR-stimulated anergic DN cells is due in
part to increased Fyn expression and recruitment of Cbl to the
TCR-signaling complex. In this scenario, the hypophosphorylation of
ZAP-70 is a consequence of negative regulation by Cbl. This possibility
is currently under investigation.
The observation that anergic DN cells proliferated extensively in
response to stimulation with a low-affinity ligand and an exogenous
IL-2 source (Fig. 2) has important implications in the role of anergic
T cells in the induction or regulation to anti-self-responses. This
observation indicates that the anergic cells have a significantly lower
activation threshold than their nonanergic counterparts. In this
regard, the anergic DN cells behave like "memory" cells. The high
expression level of CD44 and CD45RB on these cells is also consistent
with the conclusion that they have been activated by Ag in vivo
(16). However, the expansion of Ag-stimulated anergic DN
cells is dependent on an exogenous IL-2 source. One can envision a
scenario where the anergic DN cell and a normal T cell both bind to the
same APC. The anergic DN cell would come in contact with IL-2 produced
by the activated bystander T cell. This scenario could potentially lead
to autoimmune consequences. We have begun to investigate this
possibility and our preliminary experiments indicate that even after
activation with Ag and IL-2, the activated anergic DN cells are unable
to produce their own IL-2. Thus, the expansion of these activated DN
cells is dependent on a constant supply of exogenous IL-2. This may
limit the autoimmune potential of this population of anergic T cells.
This consideration raises the interesting possibility that such a form
of T cell anergy may serve to down-regulate anti-self-immune
responses. One can envision a scenario whereby the anergic DN cell and
a self-reactive conventional T cell bind to the same APC. The lower
activation threshold of the DN cell will lead to more efficient
induction of CD25 on the anergic DN cell and in this activated state
the DN cell serves as a "sponge" to soak up IL-2 in the vicinity.
We propose that competition for the limited amount of IL-2 will inhibit
proliferation of the self-reactive T cells. Importantly, the
anti-self-immune response mediated by the activated DN cells is
self-limiting since its maintenance is dependent on exogenous sources
of IL-2.
The anergic state that we have described for the DN cells differ from
that described for conventional CD4 and CD8 T cells that have been
anergized in vivo in that the latter is irreversible by the addition of
exogenous sources of IL-2 (27, 62, 63). However, in vivo
anergized CD4 T cells can also serve to down-regulate neighboring
immune responses through the release of IL-10 (64). This
observation further emphasized the potential importance of anergic T
cells in the down-regulation of autoimmune responses. The relevance of
this population of anergic DN cells in normal mice remains to be
determined. It has been suggested that the ß DN cells in
TCR-transgenic mice may result from the premature expression of the
and ß TCR transgenes in the 
lineage (65).
Therefore, we need to entertain the possibility that T cells may
potentially provide a source of regulatory T cells for the prevention
of autoimmunity. We hypothesize that these cells may perform
this important function using mechanisms that are similar to the ones
that we have proposed. This possibility is currently under
investigation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hung-Sia Teh, Department of Microbiology and Immunology, University of British Columbia, Room 300, 6174 University Boulevard, Vancouver, British Columbia Canada V6T 1Z3. E-mail address:
3 Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; PTK, protein tyrosine kinase; MAP, mitogen-activated protein; LAT, linker for the activation of T cells; PLC, phospholipase C; ZAP, TCR -associated protein.
Received for publication September 17, 1999. Accepted for publication December 29, 1999.
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ß T cells that contains autoreactive cells. J. Exp. Med. 174:1001.ß T cells and positively selected CD8 T cells expressing the same TCR and the induction of anergy in CD4-CD8- ß T cells in antigen-expressing mice. J. Immunol. 163:1222.B transcription factors. Proc. Natl. Acad. Sci. USA 93:979. chain. J. Immunol. Methods 170:261.[Medline]
subunit: regulation of TCR-associated tyrosine kinase activity by TCR . Mol. Cell. Biol. 13:5771. molecule. J. Immunol. 151:777.[Abstract]
phosphorylation. Science 281:572.1 and the Ras pathway. Immunity 9:617.[Medline]
1 in an SLP-76-deficient T cell. Science 281:413.ß T cell receptor can replace the receptor in the development of lineage cells. Immunity 5:343.[Medline]
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