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,
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* Department of Neurology,
Center for Immunology, and
Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas, TX 75390
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production were significantly
increased in mev+/- mice after
immunization with MBP Ac1-11. The frequency of MBP Ac1-11-specific CD4
T cells, analyzed by staining with fluorescently labeled tetramers
(MBP1-11[4Y]: I-Au complexes), was increased in the
draining lymph node cells of mev+/-
mice compared with wild-type mice. In addition,
mev+/- mice developed a more severe
course of EAE with epitope spreading to proteolipid protein peptide
43-64. Finally, expansion of MBP Ac1-11-specific T cells in response to
Ag was enhanced in mev+/- T cells,
particularly at lower Ag concentrations. These data demonstrate that
the level of SHP-1 plays an important role in regulating the activation
threshold of autoreactive T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1) is a cytosolic protein tyrosine phosphatase that is expressed primarily in hemopoietic cells. SHP-1 is involved in negatively regulating T cell development and activation (2, 3, 4, 5). SHP-1 deficiency results in reducing the activation threshold of peripheral T cells and increasing T cell proliferative responses (6, 7). SHP-1-deficient mice, motheaten or viable motheaten (me or mev) mice, have severe defects in immunity and hemopoiesis (8, 9, 10, 11, 12, 13, 14, 15, 16). The me or mev mice have an overgrowth of macrophages and granulocytes (17, 18, 19, 20), abnormal B cell development and polyclonal B cell activation (12, 21, 22), decreased NK cell activity (10, 23), and increased proliferative activity of thymocytes in response to TCR stimulation (24, 25). SHP-1 is also involved in the regulation of cytokine/chemokine signaling and function (26, 27). The mev or me mice develop severe pneumonitis and autoimmunity in early life, leading to premature death (9, 14). Initially thought to be phenotypically normal, me+/- and mev+/- mice have about one-half the functional SHP-1 activity of wild-type (WT) mice (28).
Although SHP-1 plays a crucial role in the immune response, few studies have examined the role of SHP-1 in autoimmune diseases. To study the role of SHP-1 in a prototypical T cell-mediated autoimmune disease, EAE, we immunized H-2u, mev+/- mice with myelin basic protein (MBP Ac1-11) and examined the T cell response to this autoantigen and the clinical signs of EAE. Our data show that a reduction in the level of SHP-1 enhances the T cell response to MBP Ac1-11, exacerbates clinical signs of EAE, and results in spreading to other myelin protein epitopes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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V
8.2 TCR, or V
2.3V8.2 transgenic (Tg), mice were kindly
provided by Dr. J. Goverman (University of Washington, Seattle, WA)
(29). The mev+/- B10.PL
(mev+/-) mice were generated by crossing
mev+/- C57BL/6
(mev+/-.B6) mice with B10.PL WT mice for
more than nine generations. The mev+/-.B6
and B10.PL mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). The mev TCR V8.2 Tg
(Tg.mev+/-) mice were generated
by crossing mev+/- B10.PL with V8.2
TCR mice. These mice were bred and maintained in our animal colony at
the University of Texas Southwestern Medical Center (Dallas, TX) in
compliance with the Animal Studies Committee. All mice were 7–10 wk of
age when experiments were initiated. No spontaneous EAE was observed in
the TCR V8.2 mice bred onto the mev
background (Tg.mev+/-).
Reagents
Whole MBP was prepared from guinea pig spinal cords as previously described, and purity was assessed by SDS-PAGE (30). MBP peptide Ac1-11, myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, and proteolipid protein (PLP) peptide 43-64 were purchased from BioSource International (Camarillo, CA).
Immunization and evaluation of EAE
For induction of EAE, mice were immunized s.c. with MBP Ac1-11 (200 µg/mouse) in an emulsion with CFA. Pertussis toxin (200 ng/mouse) in PBS was injected i.p. at the time of immunization and 48 h later. EAE scoring was performed as previously described (31): 0, no abnormality; 1, a limp tail; 2, moderate hind limb weakness; 3, severe hind limb weakness; 4, complete hind limb paralysis; 5, quadriplegia or premoribund state; and 6, death. A relapse was defined as a sustained increase of at least one full grade in clinical score after the animal had previously improved at least a full clinical score and had stabilized. For short-term studies, the mice were immunized s.c. with MBP Ac1-11 (200 µg/mouse) in an emulsion with CFA. Ten days later, the draining lymph node cells (LNCs) were harvested for various assays.
Cell culture
Draining lymph nodes and/or spleens, as noted, from different mice were harvested, and single-cell suspensions were obtained by pressing the tissue through a wire mesh screen. The cells were cultured (4 x 106 cells/ml) in complete medium for the times specified in the text or figure legends. MBP, MBP peptide Ac1-11, or PLP peptide 43-64 (concentrations are indicated in the text or figure legends) were used as stimulating Ags.
Cytokine detection
IFN-, IL-4, and IL-10 were measured by ELISA. ELISA plates
(Immunol 2; Dynatech Laboratories, Chantilly, VA) were coated with 2
µg/ml (50 µl/well) IFN-, IL-4, or IL-10 mAb (BD PharMingen, San
Diego, CA) in 0.1 M carbonate buffer (pH 8.2) overnight at 4°C. The
plates were blocked with 200 µl of 10% FBS in PBS for 2 h. A
total of 100 µl of supernatant was added at various dilutions titered
to the linear portion of the absorbance/concentration curve in
duplicate and incubated overnight at 4°C. After the plates were
washed four times with PBS and Tween 20 (0.05%), 100 µl of
biotinylated anti-cytokine detecting mAbs (directed to a different
determinant than the first Ab used to coat ELISA plates) at 1 µg/ml
in PBS and 10% FBS were added for 45 min at room temperature. Then,
100 µl of avidin-peroxidase (2.5 µg/ml) was added and incubated for
30 min. Subsequently, the peroxidase substrate ABTS (6) in
0.1 M citric buffer (pH 4.35) in the presence of
H2O2 was added, and the
absorbance was measured at 405 nm.
Staining of splenocytes and LNCs with MBP1-11[4Y]:I-Au tetramers
The generation of a soluble construct of I-Au with covalently tethered -11[4Y] peptide has been described elsewhere (32). An analogous tetramer containing OVA 323-339 bound to I-Au was also constructed (OVA 323-339 binds tightly to I-Au) (33). Tetrameric complexes of MBP1-11[4Y]:I-Au or OVA 323-339:I-Au were prepared by incubation with PE-conjugated streptavidin (Sigma-Aldrich, St. Louis, MO) and used to stain splenocytes and LNCs as previously described (34), with slight modifications. Briefly, 3 x 106 cells were incubated with the tetrameric complexes in the presence of Ab mix for 30 min at 37°C. In every assay, CD80-FITC (16-10A1, Armenian hamster IgG), CD4-PerCP (RM4-5, rat IgG2a), CD62 ligand (CD62L)-APC (MEL-14, rat-IgG2A), and the corresponding Ig-isotype control were used (BD Biosciences, Mountain View, CA). Analysis of staining was performed using a FACScan (BD Biosciences) and the CellQuest (BD Biosciences) and WinMDI 2.8 analysis software (The Scripps Research Institute, La Jolla, CA).
Purification of CD4 T cells by negative selection
TCR V8.2, V2.3 Tg splenocytes were purified by negative
selection using R&D Systems (Minneapolis, MN) mouse T cell CD4 subset
column kit (MCD4C-1000). Briefly, 2 x 108
splenocytes in 2 ml of column buffer were incubated with 1 ml of
mixture Ab for 15 min at room temperature. The cells were washed twice
with column buffer and resuspended into 2 ml and loaded into the
prewashed column. After the cells were suspended in the column and were
incubated for 10 min at room temperature, a total of 10 ml of column
buffer was used to elute the cells from the column. The cells were
centrifuged at 250 x g for 5 min and suspended into
culture medium. The purity of CD4+ T cells was
determined by FACS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the effect of SHP-1 on the development of EAE, both male
and female mev+/- and WT B10.PL mice were
immunized s.c. with MBP Ac1-11 in CFA. Mice were examined daily for
clinical signs of EAE. The mice initially developed EAE on day 10 after
immunization. Overall, mev+/- mice had a
higher incidence of disease and developed a more severe course of EAE
(males, p = 0.032; females, p = 0.045;
Fig. 1
). The mean maximal EAE clinical
score in mev+/- males was 5, whereas it
was only 3.5 in WT mice. The mev+/- mice
demonstrated more relapses, whereas WT mice generally exhibited a
monophasic course of disease (Fig. 1). Male
mev+/- mice experienced 17 relapses
compared with 10 relapses for the WT mice. Female
mev+/- mice experienced nine relapses
compared with five for the WT mice. Thus, reduced levels of SHP-1 in
mev+/- mice exacerbated the clinical
course of EAE and increased the number of relapses.
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).
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Next, we examined T cell proliferation and cytokine production in
mev+/- and WT mice in response to MBP
Ac1-11 challenge. Both mev+/- and WT mice
were immunized s.c. with MBP Ac1-11 in CFA. Ten days later, draining
LNCs were collected and single-cell suspensions were made. LNCs were
cultured in the presence of MBP Ac1-11. The
mev+/- mice exhibited increased T cell
proliferation to MBP Ac1-11 compared with WT mice (Fig. 3A). The difference was more
apparent in males than in females. In addition, the production of
IFN- was markedly increased in mev+/-
mice compared with WT mice (Fig. 3B). Therefore, reduced
SHP-1 levels in the heterozygous, mev+/-
mice resulted in an increased T cell response to the priming
Ag.
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The mev+/- mice demonstrated more
severe EAE and enhanced T cell responses to immunizing Ags. We examined
whether increased T cell responses lasted over the course of EAE. To
confirm our hypothesis, we tested T cell proliferation and cytokine
production 8 wk after immunization with MBP Ac1-11. As expected, T cell
proliferative responses to both MBP and MBP Ac1-11 were markedly
increased in mev+/- mice compared with WT
mice. Interestingly, we observed that T cells from
mev+/- mice responded to PLP 43-64
peptide, whereas T cells from WT mice did not respond detectably to PLP
43-64 (Fig. 4A). In addition,
mev+/- splenocytes produced more IFN-
in response to MBP, MBP Ac1-11, and PLP 43-64 stimulation (Fig. 4B). This suggested that endogenous priming to PLP 43-64 had
resulted in epitope spreading. We also examined whether a response to
PLP 43-64 existed in mev+/- mice, which
were immunized with MBP Ac1-11 for 10 days, and in naive V8.2 TCR
mev+/- (Tg
mev+/-) mice. T cells did not respond to
PLP 43-64 peptide in these two situations (data not shown). Therefore,
epitope spreading to PLP 43-64 occurred during chronic EAE in
mev+/- mice and may have contributed to
the increased severity of disease and relapse rate.
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The mev+/- mice had shown an
increased response to MBP Ac1-11 and enhanced signs of disease. Using
MBP1-11[4Y]:I-Au tetramers, which have been
shown in previous studies (32, 34) to detect MBP
Ac1-11-specific T cells, we next examined the frequency of MBP
Ac1-11-specific T cells after immunization with MBP Ac1-11. Ten days
later, the draining LNCs were harvested and stained with anti-CD4,
CD62L, CD80 mAbs and MBP1-11[4Y]:I-Au
tetramers. The number of tetramer-positive cells was reproducibly
higher in mev+/- mice compared with WT
mice (Fig. 5). The percentage of
CD62Llow cells and B7-1-positive cells in
tetramer-positive cells was comparable between
mev+/- and WT mice (data not shown).
These data suggest that the increased T cell proliferation and IFN-
production could be attributed to the higher frequency of MBP
Ac1-11-specific T cells after immunization in
mev+/- mice compared with WT mice. The
precursor frequency of MBP Ac1-11-specific T cells in nontransgenic
mice is undetectable. To analyze precursor frequencies in naive mice,
it is therefore necessary to use mice Tg for the -chain of a TCR
specific for MBP Ac1-11. These V8.2 Tg mice have a much higher
frequency of MBP Ac1-11-specific cells (
0.5% of
CD4+ T cells; Fig. 6) than do nontransgenic mice. We
analyzed the same cell surface markers in naive Tg
mev+/- and Tg WT mice. There was no
difference in the number of tetramer-positive MBP Ac1-11-specific T
cells, the expression of B7-1, or CD62L expression between naive Tg
mev+/- and Tg WT mice (Fig. 6 and data
not shown). These data demonstrate that the autoantigen-specific T
cells are not activated in naive Tg
mev+/- mice, and the baseline precursor
frequency of MBP Ac1-11-specific T cells is the same between
mev+/- and WT in the V8.2 TCR Tg mice.
Thus, the increased number of Ag-specific cells after immunization in
mev+/- mice relative to WT mice (Fig. 5)
is most likely because of increased expansion in vivo, rather than
because of differences in precursor frequencies.
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8.2 TCR Tg
mev+/- T cells compared with V8.2 TCR Tg WT
T cells
Naive LNCs and splenocytes from V8.2 TCR Tg WT and V8.2 TCR
Tg mev+/- mice were stained with
anti-CD4, B7-1, CD62L, and MBP Ac1-11[4Y] tetramer before and 4
days after culture with different concentrations of MBP Ac1-11. Before
the cells were cultured with MBP Ac1-11, the number of
tetramer-positive cells was comparable between V8.2 TCR Tg
mev+/- and V8.2 TCR Tg WT mice (Fig. 6
). After 4 days of culture with MBP Ac1-11, the tetramer-positive
cells had expanded significantly more in
mev+/- mice than those from WT mice. The
tetramer-positive cells increased 9- and 6-fold in
mev+/- mice when the MBP Ac1-11
concentrations were 0.4 and 0.8 µg/ml, respectively (Fig. 6), whereas
there was only a 2-fold increase of tetramer-positive T cells in
mev+/- mice compared with WT T cells when
5 µg/ml of MBP Ac1-11 was used for stimulation (Fig. 6). The number
of CD62Llow tetramer-positive T cells was also
increased in mev+/- mice compared with WT
mice after stimulation with MBP Ac1-11 (data not shown). Similar data
were obtained with LNCs (data not shown). These data indicate that a
reduced level of SHP-1 leads to a reduction in T cell activation
threshold, and they clearly demonstrate that at lower Ag
concentrations, mev+/- T cells expand
more readily than do WT T cells.
T cell proliferation and cytokine production are increased in
heterozygous V8.2 TCR Tg mev+/- mice
MBP Ac1-11-specific T cells were demonstrated to be at similar
levels in naive mev+/- and WT V8.2 TCR
Tg mice (Fig. 6). We examined T cell proliferation and IFN-
production by V8.2 TCR Tg mev+/- and
V8.2 TCR Tg WT T cells after stimulation with different
concentrations of MBP Ac1-11. LNCs and splenocytes from V8.2 TCR Tg
mev+/- or V8.2 TCR Tg WT mice were
cultured with MBP Ac1-11. T cell proliferation was measured by
3H incorporation, and IFN- production was
examined by ELISA. The T cell proliferation to MBP Ac1-11 was
significantly increased in V8.2 TCR Tg
mev+/- compared with V8.2 TCR Tg WT
mice (Fig. 7A). IFN-
secretion was markedly higher for V8.2 TCR Tg
mev+/- cells than for V8.2 TCR Tg WT
cells in response to MBP Ac1-11 (Fig. 7B). Our data indicate
that V8.2 TCR Tg mev+/- mice have a
higher T cell proliferative response to MBP Ac1-11 compared with
V8.2 TCR Tg WT mice. Naive V8.2 TCR Tg
mev+/- mice have comparable MBP
Ac1-11-specific cells compared with WT mice, and the T cells are not
activated in naive mice. Therefore, the higher T cell responses
observed in V8.2 TCR Tg mev+/- mice
are most likely because of the lower threshold for activation.
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To address whether APCs play a role in the difference of T cell
activation between mev+/- and WT mice, we
purified MBP Ac1-11-specific TCR Tg CD4 T cells by negative
selection and stimulated CD4 T cells with MBP Ac1-11 in the presence of
irradiated splenocytes from either WT or
mev+/- mice. The T cell proliferation in
response to MBP Ac1-11 was comparable between cultures with WT and
mev+/- APCs (Fig. 8A). After 3 days of culture,
the percentage of MBP Ac1-11-specific tetramer-positive
CD4+ cells was similar between the cultures with
WT and mev+/- APCs (Fig. 8B).
Interestingly, the production of IFN- was lower in the
mev+/- APC culture compared with WT APC
culture (Fig. 8C). Similar results were observed using
V8.2 TCR Tg T cells (Fig. 8D). These data clearly
demonstrate that the increased T cell response in
mev+/- mice is not because of alterations
in APC function, with T cell proliferation and expansion being
essentially identical. These data also would imply that the increased
IFN- production noted in the mev+/-
cultures must be because of effects related to the T cell, because
mev+/- APC-stimulating WT T cells
produced less IFN- than did WT APC-stimulating WT T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50% of SHP-1 activity
(28). Therefore, mev+/- mice
provide a useful tool for studying the effect of reduced SHP-1 levels
on the pathogenesis of EAE.
We examined the effects of decreased SHP-1 on the pathogenesis of EAE
by immunizing mev+/-
H-2u mice with MBP Ac1-11 in CFA. The
mev+/- mice had an exacerbated course of
EAE that was associated with an increased T cell proliferative response
to the priming Ag and increased IFN- production. Increased severity
of EAE in mev+/- mice may be because of
1) an increased number of encephalitogenic MBP Ac1-11-specific T cells,
2) epitope spreading, or 3) a lower threshold for T cell activation.
These are not mutually exclusive possibilities.
Cumulative data showed that SHP-1 negatively modulates the signaling
function of the TCR. The proliferation and IL-2 production induced by
TCR engagement are markedly increased in
mev T cells relative to WT T cells
(3, 25). In mature T cells, downstream delivery of TCR
stimulatory signals requires the initial activation of the Src family
protein tyrosine kinases Lck and Fyn, with consequent tyrosine
phosphorylation of the TCR CD3 and 
subunits, recruitment of the
ZAP-70 tyrosine phosphokinase, and the sequential activation of
signaling effectors that transduce the signal to the nucleus. SHP-1
negatively regulates the activities of Lck, Fyn (25), and
phosphatidylinositol 3-kinase (36), therefore regulating T
cell activation. Our data clearly show that T cells from
mev+/- mice have increased proliferative
responses and IFN- production after MBP Ac1-11 stimulation compared
with WT mice. Encephalitogenic T cells are typically of a Th1
phenotype, and IFN- is crucial for developing inflammatory lesions
and demyelination in the CNS (37). Thus,
mev+/- mice may have more severe EAE
because of increased production of IFN- by encephalitogenic T
cells.
The increased T cell proliferation and IFN- production in
mev+/- mice may be because of the
increased the number of MBP Ac1-11-specific T cells, decreased
threshold of T cell activation because
mev+/- mice have reduced SHP-1 activity,
or both. Our data show that the number of MBP Ac1-11-specific T cells
is higher in mev+/- mice after
immunization with MBP Ac1-11/CFA. The increased number of MBP
Ac1-11-specific T cells could partially explain the enhanced
proliferation and cytokine production in
mev+/- mice after immunization with MBP
Ac1-11. However, the number of MBP Ac1-11-specific T cells is
comparable between naive Tg mev+/- mice
and Tg WT mice, whereas T cell proliferation and IFN- production are
higher in Tg mev+/- mice after MBP Ac1-11
stimulation. This suggests that even when starting with equal numbers
of MBP Ac1-11-specific T cells, the
mev+/- T cells have an inherent advantage
in expanding in response to Ag. Johnson et al. (6)
reported that SHP-1 contributes to establishing thresholds for TCR
signaling in thymocytes and naive T cells. In their Tg MHC class
I-restricted TCR system, both the number and the percentage of single
positive CD8+ thymocytes were significantly
increased in SHP-1-deficient mice vs WT mice. In addition, expression
of the activation marker CD44 was significantly higher in mutant mice
than in WT mice, indicating that in vivo loss of SHP-1 leads to an
increased basal level of activation of mature CD8 T cells. The CD8
cells showed hyperproliferation but an equivalent cytolytic activity in
SHP-1-deficient mice in response to stimulation with cognate peptide.
Similar data were reported by Carter et al. (7). Lack of
SHP-1 revealed alterations in the percentages of thymocyte
subpopulations; me/me thymocytes undergo negative selection
to stimulation at lower concentrations of Ag compared with WT
thymocytes and were hypersensitive to stimulation by specific Ag. In
our system, the percentage of CD62Llow T cells
was comparable between mev+/- and WT mice
in the V8.2 TCR Tg mice (data not shown). Our data indicate that T
cells were activated at the same level before immunization in Tg
mev+/- and Tg WT mice. Thus, prior
activation of mev+/- TCR Tg T cells did
not contribute to their proliferative advantage. Although we could not
analyze the activation state of MBP Ac1-11-specific T cells in B10.PL
WT or mev+/- mice for technical reasons,
it is reasonable to assume that the observations in V8.2 Tg mice
concerning CD62L expression and cell number are relevant to
nontransgenic mice. It is also clear from our data that
mev+/- T cells expand better in response
to antigenic stimulation both in vitro and in vivo (
Figs. 3–7).
Our data showed that there was a difference in T cell expansion between mev+/- and WT mice, which we have interpreted to be because of decreased signaling threshold through the TCR in mev+/- T cells. Another explanation could be that there is a difference in apoptosis by activation-induced cell death between mev+/- and WT mice. Recently, Zhang et al. (38) reported that mev T cells are more sensitive than WT T cells to induction of programmed cell death after TCR stimulation. The increased apoptosis in mev T cells was mediated through up-regulated Fas-Fas ligand interaction and induction of the Fas signaling cascade. In their studies (38), the expression of Fas ligand on mev T cells is markedly increased with anti-CD3 stimulation, whereas the Fas ligand expression is only mildly increased on WT T cells when stimulated through anti-CD3. In our study, if programmed cell death in mev+/- was increased compared with WT T cells, one would anticipate greater expansion of WT compared with mev+/- T cells. This was not the case. The other possibility is that programmed cell death is greater in mev+/- T cells than in WT cells, but that proliferation or expansion is dramatically greater in mev+/- T cells because of decreased signaling threshold, resulting in our present observations.
Costimulatory signals play a crucial role in T cell activation. Manipulation of B7 pathways could alter T cell activation, eventually influencing the outcome in animal models of autoimmunity. It has been shown that SHP-1 does not influence the functions of CTLA-4 and CD28 (3). Therefore, exacerbated EAE and increased T cell responses in mev+/- mice are unlikely because of influences of SHP-1 on costimulatory signals.
EAE has a relapsing-remitting course of paralysis that is very similar
to the clinical profile observed in MS (39). MHC class
II-restricted, Ag-specific T cells are crucial for the pathogenesis of
EAE. Several studies have demonstrated that changes occur in the Ag
specificity of neuroantigen-specific proliferative responses during the
course of EAE. Proliferative responses to additional encephalitogenic
myelin epitopes have been reported to arise after the initial acute
phase of EAE (40). Relapses could result from activation
of T cells specific for endogenous myelin epitopes released during the
acute phase of disease, which was initiated by the priming
encephalitogen (40, 41, 42). Thus, reactivity to neuroepitopes
other than that used to induce the initial clinical episode, or epitope
spreading, may contribute to the relapsing course of clinical relapsing
EAE (43, 44). Epitope spreading has also been proposed to
contribute to the pathogenesis of spontaneous autoimmune diabetes in
nonobese diabetic mouse (45, 46). Recently, Karandikar et
al. (47) reported that down-regulation of epitope
spreading is mediated by CTLA-4 in relapsing EAE. Neville et al.
(48) showed that treating Theiler’s virus-induced
demyelinating disease in SJL mice with CTLA-4 Ig or anti-B7-1 and
B7-2 Abs significantly enhanced clinical disease severity. Epitope
spreading to myelin epitopes was accelerated as a result of the
increased availability of myelin epitopes, leading to a more severe
chronic disease course (48). In our system,
mev+/- mice have increased severity of
EAE with more relapses. Eight weeks after immunization with MBP Ac1-11,
the splenocytes from mev+/- mice
responded not only to MBP Ac1-11 itself, but also to the PLP 43-64
peptide. However, there was no response to PLP 43-64 observed 10 days
after mev+/- mice were immunized with MBP
Ac1-11. This suggests that the response to PLP 43-64 was because of
endogenous presentation of this epitope after the acute onset of CNS
inflammation. Thus, reduced levels of SHP-1 result in a reduction in
the threshold of activation for PLP 43-64-reactive T cells and
enhancement of epitope spreading in
mev+/- mice. Increased production of
IFN- by the infiltrating MBP-Ac1-11-specific T cells may also play a
role in enhanced epitope spreading.
In summary, our results show that SHP-1 activity plays an important role in EAE pathogenesis through regulation of autoreactive T cell activation. Reduction of the expression of SHP-1 leads to a lower T cell activation threshold, increases expansion of autoreactive T cells, and enhances processes such as epitope spreading. These factors result in mev+/- mice developing more severe clinical EAE with increased relapses. These results also suggest that factors that lower the T cell activation threshold may also have implications for human autoimmune diseases such as MS.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael K. Racke, Department of Neurology and Center for Immunology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9036. E-mail address: Michael.Racke{at}utsouthwestern.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; SHP-1, Src homology 2 domain-containing protein tyrosine phosphatase 1; me, motheaten; mev, viable motheaten; WT, wild type; MBP, myelin basic protein; Tg, transgenic; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein; LNC, lymph node cell; CD62L, CD62 ligand.
Received for publication November 13, 2001. Accepted for publication March 5, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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