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*
Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095; and
Roche Milano Ricerche, Milano, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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cell-autoantigenic peptides to I-Ag7, as
well as the precursor frequency, functional avidity, and phenotype of
the T cells that recognize these peptides in type 1 diabetes-prone
nonobese diabetic mice. We observed that autoimmunity gradually spreads
from a cell determinant, which had the largest precursor pool of
high avidity T cells, to cell determinants with progressively
smaller and lower avidity T cell precursor pools. This correlation
between the sequential development of spontaneous T cell autoimmunity
and the frequency and avidity of autoantigen-reactive T cells suggests
that the extent to which T cells were negatively selected by the
self-determinants is the key factor determining the spreading
hierarchy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Theoretically, the spreading hierarchy of spontaneous autoimmunity should reflect the different immunogenicities of self-determinants in the target tissue. Studies of immune responses to foreign determinants have shown that foreign determinants often have large pools of high avidity precursor T cells (as they did not contribute to T cell selection) and that the primary factor defining their immunogenicity is the efficiency with which they are displayed on MHC (7). Consequently, the most immunogenic foreign determinants are often those with the highest affinity for MHC. However, these rules may not apply to self-Ags, as the same factors that cause a foreign determinant to be immunogenic promote the induction of T cell tolerance to self-determinants. Indeed, the repertoire of potentially autoreactive T cells should bear the imprint, in terms of both their precursor frequency and avidity, of selection by self-determinants. Accordingly, the self-reactive T cell pool should be trimmed to leave only those T cells that interact with self-Ags below the T cells’ activation thresholds. Given this trimming of the self-reactive T cell repertoire, it is presently unclear what properties of the remaining self-reactive T cells, and of their cognate self-determinants, underlie the predefined patterns in which T cell autoreactivity spreads during the pathogenesis of organ-specific autoimmune diseases.
The nonobese diabetic (NOD) mouse spontaneously develops autoreactive T
cell responses to cell Ags at 
4 wk of age, concurrent with the
onset of insulitis (4, 8, 9, 10, 11, 12, 13). Initially, T cell
autoreactivity appears to be limited in its specificity, but it
gradually spreads among Ag determinants in a defined chronological
pattern, often leading, several months later, to insulin-dependent
diabetes mellitus (IDDM) (4, 8, 9). This sequential
spreading of T cell autoreactivity among cell Ags provides a model
system to dissect the factors that determine the spreading hierarchy.
Toward this, we identified a panel of different cell Ag
determinants that become involved in the autoimmune cascade at
different stages of the disease process in NOD mice. We then
systematically characterized the factors underlying their
immunogenicity, namely, their binding affinity for MHC, as well as the
precursor frequency and functional avidity of reactive T cells in
preautoimmune NOD mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD mice (Taconic Farms, Germantown, NY) were bred under
specific pathogen-free conditions. In our colony, 
85% female NOD
mice spontaneously develop IDDM by 30 wk of age. Only female
mice were used in these studies.
Antigens
The 65-kDa form of mouse glutamic acid decarboxylase (GAD) and
control Escherichia coli -galactosidase were
purified as previously described (8). The autoantigenic
and immunodominant GAD peptides that were tested included
GAD524–543 (also termed GADp35;
SRLSKVAPVIKARMMEYGTT), GAD78–97 (also termed
GADp6; KPCSCSKVDVNYAFLHATDL), and GAD217–236
(also termed GADp15; EYVTLKKMREIIGWPGGSGD) (4, 8, 14).
Peptides from other key cell autoantigens included the
immunodominant peptide of the 65-kDa heat shock protein (HSP), termed
HSPp277 (VLGGGCALLRCIPALDSLTPANED; Ref. 15) and the
immunodominant determinant of insulin,
insulin9–23 (SHLVEALYLVCGERG; Ref.
16). An immunogenic hen egg lysozyme (HEL) peptide
HEL11–25 (AMKRHGLDNYRGYSL; Ref.
17) was used as a control foreign peptide and a mouse
serum albumin (MSA) peptide MSA560–574
(KPKATAEQLKTVMDD; Ref. 18), which contains a nontargeted
determinant, was used as a control self-Ag. All peptides were
synthesized by Multiple Peptide Systems (San Diego, CA) at 
95%
purity.
Purification of the I-Ag7 molecule and peptide binding assay
The I-Ag7 molecule was affinity purified
from detergent lysates of 4G4.7 B cell hybridoma by sequential negative
selection with 34.1.4 and 14.4.4S mAbs followed by elution from OX-6
mAb coupled to protein A-Sepharose, as previously described
(19). Briefly, the lysates of 1011
4G4.7 cells were passed on the 34.1.4 protein A-Sepharose column
followed by a 14.4.4S protein A-Sepharose column, and finally loaded on
to the OX-6 protein A-Sepharose column. Following extensive washing,
the I-Ag7 molecule was eluted and immediately
neutralized. The purity of the eluted proteins was >95% as
demonstrated by two bands with m.w. 33,000 and 28,000 resolved in
SDS-PAGE (corresponding to the 
- and -chains, respectively, of
MHC class II molecules).
Peptides for the analysis of affinity were dissolved at 10 mM in DMSO and diluted with 25% DMSO in PBS for the assay. The peptide HEL10–23 was synthesized with two spacer residues and a biotin molecule at the NH2 terminus and used as the indicator. The indicator (500 nM), together with different concentrations (50 µM-50 pM at 10-fold dilution) of the tested peptides, was coincubated with 200 nM of I-Ag7 molecules in U-bottom polypropylene 96-well plates in binding buffer at room temperature for 48 h. The concentration of the complex of biotinylated indicator peptide HEL10–23/I-Ag7 was determined by an ELISA using prebound OX-6 Abs (10 µg/ml) and streptavidin-alkaline phosphatase and p-nitrophenylphosphate. Competition curves were plotted, and the peptide affinity for MHC molecules was expressed as the peptide concentration required to inhibit the binding of biotinylated peptide by 50% (IC50).
Immunizations
To determine the frequency of autoantigen determinant-reactive precursor T cells and the functional T cell avidity, we immunized 2-wk-old female NOD mice with a control or autoantigen peptide (50 nanomoles per mouse) in 50% CFA (Life Technologies, Grand Island, NY) in the footpad. Nine days after immunization, single cell suspensions were prepared from the draining lymph nodes, and Ag-specific T cell responses were characterized by proliferation or enzyme-linked immunospot (ELISPOT) assays.
Proliferation assay
Nine days following immunization, single cell suspensions were prepared from the draining lymph nodes. Mononuclear cells (4 x 105 cell/well) were stimulated with different concentrations (0.2–70 µM) of Ag peptides in triplicate in FCS-free HL-1 medium (BioWhittaker, Walkersville, MD) and incubated in 96-well microtiter plates at 37°C with 5% CO2 for 96 h. Medium alone (without any Ag) was used as the negative control, and purified protein derivative (PPD; 10 µg/ml) or anti-CD3 (1 µg/ml) was used as the positive control for each mouse. During the last 12–16 h of the 96-h culture period, 1 µCi [3H]thymidine was added to each well. Incorporation of label was measured by liquid scintillation counting. Lymph node T cell proliferation to a specific Ag was plotted as dose-dependent curves, and the functional T cell avidity was presented as the percentage of proliferation.
ELISPOT analysis
The frequency of Ag-specific splenic T cells secreting IFN-
,
IL-4, and IL-5 was determined using a modified ELISPOT technique as
previously described (20, 21). Briefly,
106 splenic mononuclear cells per well were added
(in duplicate) to an ELISPOT plate that had been coated with cytokine
capture Abs and incubated with peptide (20 µM) or whole protein (100
µg) at 24 h for IFN-, or 40 h for IL-4 and IL-5
detection. After washing, biotinylated detection Abs were added, and
the plates were incubated at 4°C overnight. Bound secondary Abs were
visualized using HRP-streptavidin (Dako, Carpinteria, CA) and
3-amino-9-ethylcarbazole. Abs R4-6A2/XMG 1.2-biotin,
11B11/BVD6-24G2-biotin, and TRFK5/TRFK4-biotin (PharMingen, San Diego,
CA) were used for capture and detection of IFN-, IL-4, and IL-5,
respectively.
To characterize lymph node T cell responses in Ag-primed mice, draining
lymph node mononuclear cells (4 x 105 cells
per well) were challenged with different concentrations of control or
autoantigen peptides, 20 µg/ml PPD, or 1 µg/ml anti-CD3. The
frequency of Ag-induced spot-forming colonies (SFC) is expressed
as dose-dependent curves. The proportion of high, intermediate, and low
avidity T cells in an Ag-specific repertoire was determined based on
the average number of SFC in response to 0.2, 0.7–2.0, and 2.0 µM
Ag (respectively) divided by the average maximal number of Ag-specific
SFC responses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies of the determinant spreading of autoimmunity during the development of IDDM in NOD mice used T cell proliferation assays (8, 9), which are limited in sensitivity and may not have detected low frequency autoreactive T cells. To obtain a higher resolution analysis of the spreading hierarchy, we used an ELISPOT assay capable of detecting a single autoreactive T cell within a million, and which unlike proliferation assays, is not affected by the presence of regulatory T cells (22).
At different stages of NOD mouse development, we characterized splenic
T cell responses to a panel of cell-autoantigenic determinants. The
tested cell autoantigens included GAD78–97,
GAD217–236, GAD524–543,
insulin9–23, and HSPp277, which have been
implicated as being key target determinants (4, 8, 14, 16, 23, 24, 25). Control Ags included an I-Ag7
binding nontargeted self-peptide from mouse serum albumin,
MSA560–574, as well as a foreign peptide from
hen egg lysozyme, HEL11–25.
We did not detect a splenic T cell response to any of these Ags in
3-wk-old NOD mice (Fig. 1
). Beginning at
4 wk of age, IFN--secreting T cells reactive to whole GAD and
GAD524–543 were detected. At 6–8 wk of age,
spontaneous autoimmunity spread to GAD78–97 and
HSPp277. By 12 wk of age, T cell responses to
GAD217–236 and
insulin9–23 appeared (Fig. 1). All of these
autoreactivities were Th1 type, with only IFN- SFC, and no
detectable IL-4 and IL-5 SFC (data not shown). No responses to self-Ag
MSA560–574 or foreign determinant
HEL11–25 were detected at any age. Thus, ELISPOT
analysis of T cell frequency and phenotype confirm previous
observations (4, 8) that the hierarchy of determinant
spreading among this panel of autoantigenic peptides is
GAD524–543 (an early target), followed by
GAD78–97 and HSPp277 (intermediate targets), and
eventually by GAD217–236 and
insulin9–23 (late targets).
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To dissect the factors that determine the spreading hierarchy, we
first tested whether the strength of MHC binding could determine the
immunogenicity of these cell Ag determinants. Using a competitive
peptide binding assay, we observed that autoantigen peptides displayed
a range of binding affinities for I-Ag7 (Fig. 2). The hierarchy of control and cell
autoantigen peptide binding affinities for I-Ag7
(expressed as IC50) is
MSA560–574 (0.4 µM) <
insulin9–23 (0.6 µM) <
HEL11–23 and GAD217–236
(0.7 µM) < GAD524–543 (0.8 µM) <
GAD78–97 (1.3 µM) < HSPp277 (10.7 µM).
This ranking of binding affinity of the autoantigen peptides for
I-Ag7 displays no relationship to the
chronological pattern in which spontaneous autoreactive T cell
responses spread, either intramolecularly (within GAD) or
intermolecularly (Fig. 1
). For example, both early
(GAD524–543) and late
(GAD217–236) target determinants of GAD have
fairly high affinities for I-Ag7, and a late
target determinant (insulin9–23), has a higher
affinity for I-Ag7 than an intermediate target
determinant (HSPp277). Thus, the sequential spreading of
autoimmunity among cell Ag determinants is independent of the
affinity for I-Ag7 of the determinant.
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We next tested whether the clonal size of autoantigen-specific T
cells in the preimmune repertoire could explain the observed spreading
hierarchy. We immunized NOD mice with a high dose of a peptide (50 nM
in CFA) to maximally load the I-Ag7 molecules so
that Ag presentation should not be a limiting factor. NOD mice were
immunized at 14 days of age, before the onset of the endogenous
autoimmune response. Nine days after the immunization, the frequency of
peptide-specific T cells was determined in the draining lymph node by
ELISPOT. Under these conditions, the induced T cell response provides a
measurement of the size of an Ag-specific T cell pool in preautoimmune
NOD mice.
Immunization with the control self-peptide
MSA560–574 primed the lowest number of T cells,
as expected for a well presented self-Ag (Fig. 3). In contrast,
GAD524–543 primed an even greater number of
peptide-reactive T cells than the control foreign Ag determinant
HEL11–25. GAD524–543 also
induced a significantly greater number of T cell responses than did
GAD78–97, which in turn induced more T cells
than GAD217–236. Therefore, when Ag
presentation is not limiting, the immunogenicity of determinants within
a single autoantigen (GAD) follows the order in which autoimmunity
spreads intramolecularly among these determinants during disease
development. Similarly, HSPp277, an intermediate target along with
GAD78–97, primed an intermediate level of T cell
responses. However, the frequency of T cell responses to the late
target insulin9–23 was significantly higher than
that of both HSPp277 and GAD78–97. These data
suggest that the size of the precursor T cell pool may be an important
factor in establishing the spreading hierarchy. However, the lack of
complete correlation between the size of precursor T cell pools and the
spreading hierarchy suggests that some other factor(s) contribute to
the establishment of the spreading hierarchy.
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During the development of self-tolerance, self-determinants shape the size of precursor T cell pools and limit the avidity of self-reactive T cells remaining in the repertoire. Notably, the immunogenicity of a determinant depends on both the clonal size and the avidity of reactive T cells. Therefore, we next examined the functional avidity of autoantigen-reactive T cells in the preimmune repertoire. High avidity T cells become activated at low peptide concentrations, and vice versa. Accordingly, functional T cell avidity for a determinant can be measured by the dose response to the peptide, and defined as the peptide dose at which a 50% maximal response is observed (26).
As described above, 14-day-old NOD mice were immunized with a
control or autoantigen peptide in CFA, and their draining lymph node T
cells were tested for proliferative responses to different
concentrations of the Ag (Fig. 4A). The relative strength of
T cell responses primed by different concentrations of each peptide is
presented in Fig. 4B. The dose-response curve for
GAD524–543 was similar to that of the foreign
determinant HEL11–25, and both of these
responses were severalfold higher than that primed by other
self-determinants (Fig. 4A). Both
GAD524–543 and HEL11–25
induced 50% maximal T cell proliferation at a concentration of 1
µM. The intermediate target determinants
GAD78–97 and HSPp277 required 8 and 13 µM
(respectively), whereas the late target determinants
GAD217–236 and
insulin9–23 required 17 and 19 µM
(respectively) to induce 50% of maximal T cell proliferation.
Accordingly, the order of functional T cell avidity, as determined by
proliferative responses, matched the developmental sequence of
spontaneous T cell immunity to the tested autoantigen determinants.
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The minimal amount of Ag required to induce significant T cell
IFN--secreting SFC varied widely among the different tested Ags
(Fig. 5A). Only 0.2-µM
peptide was sufficient to elicit frequent IFN--secreting
colonies to GAD524–543 and foreign Ag
HEL11–25.In contrast, 0.7- to 2.0-µM
concentrations of peptide were required to elicit significant IFN- T
cell responses to intermediate target determinants
(GAD78–97 and HSPp277), and 7 µM or higher
concentrations of peptide were needed to elicit significant responses
to the late target determinants (GAD217–236 and
insulin9–23), as well as to the control self-Ag
MSA560–574 (Fig. 5A). Thus, both the
ELISPOT analysis and proliferation assays provide evidence of
significant differences in the overall functional avidity of T cells
reactive to the early, intermediate, and late cell target
determinants.
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B). Notably,
the GAD524–543-reactive T cell repertoire, like
that of HEL11–25, contained a large proportion
of high avidity T cells. Other autoantigen-reactive precursor T cell
pools were comprised of very few, or no, high avidity T cells. Instead,
the intermediate target determinant-reactive T cell pools consisted of
medium-low avidity T cells, and the late target determinant-reactive T
cell pools were comprised predominantly of low avidity T cells.
Together, these data show that the tested cell Ags have had very
different impacts on the frequency and the avidity of
autoantigen-reactive precursor T cells in preautoimmune NOD mice.
Importantly, both the overall functional avidity and the extent to
which each autoantigen-reactive T cell pool is comprised of high
avidity T cells correlate precisely with the developmental sequence in
which spontaneous T cell immunity will arise in these mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells specific for foreign Ags often respond to the foreign
determinants that have the best MHC binding properties. In contrast,
self-reactive T cells are likely to be negatively selected for well
presented self-peptides, and those that are allowed to persist may be
only weakly responsive to such determinants. This was elegantly
demonstrated in studies of T cell repertoires in myelin basic protein
(MBP)-expressing and MBP-deficient mice. The peptides
MBP78–87 and MBP121–140
are the immune-dominant determinants when MBP is a foreign Ag, in
MBP-deficient H-2k and H-2u
mice, respectively (26, 27). However, the same
determinants cull most of their reactive T cells, and are only weakly
immunogenic determinants in H-2k and
H-2u MBP-expressing strains. Instead, other
determinants of MBP, which have larger pools of reactive T cells
available, can more efficiently induce EAE. Little is known about the
impact of cell Ags on the reactive T cell repertoire in animals
that are susceptible to spontaneous cell autoimmunity.
Using a highly sensitive ELISPOT assay and a panel of peptides that
included the major target determinants of different cell
autoantigens, we confirmed that autoreactive T cell responses arise in
a defined chronological order during NOD mouse development. Next, we
showed that the binding affinity of these peptides for
I-Ag7 had no relationship with the pattern in
which spontaneous autoimmunity spreads. Furthermore, we did not observe
a correlation between the affinity of autoantigen peptides for
I-Ag7 and the size of their reactive precursor T
cell pools in preautoimmune NOD mice. These findings are consistent
with previous reports that the ability of autoantigen determinants to
prime immune responses in NOD mice is unrelated to their binding
affinity for I-Ag7 (28, 29). Thus,
unlike immune responses to foreign Ags, the pattern in which
spontaneous T cell autoreactivity emerges during the course of the
disease is independent of the affinity of the autoantigen determinant
for the I-Ag7 molecule.
This lack of correlation between MHC binding affinity and the
immunogenicity of cell Ags (as reflected by the spreading
hierarchy) indicates that the preimmune T cell repertoire was shaped by
the endogenous self-Ag during tolerance development. To evaluate the
impact of each target determinant on the T cell repertoire, we primed
preautoimmune mice with an excess of each determinant, thereby
activating the full avidity spectrum of reactive T cells, and providing
a measure of the clonal size of autoantigen-reactive T cells that
escaped negative selection. We found that the clonal size of
autoantigen-reactive precursor T cells correlated with the sequence of
spontaneous T cell autoimmunity, with the exception of
insulin9–23. Although T cell autoreactivity to
insulin9–23 appears late, the clonal size of
insulin9–23-reactive T cells is significantly
larger than that of earlier target determinants.
The differences in the clonal size of precursor T cells responding to autoantigens can partially explain the spreading hierarchy. If the autoreactive T cells were simultaneously primed by endogenous autoantigen, they may become detectable sequentially as the largest, followed by progressively smaller, autoantigen-reactive T cell pools, expanded to detectable levels. However, the precursor frequencies differ by only 2.5-fold between the early and late target determinants, and it is difficult to imagine how this factor alone could account for the 8 wk that separate the appearance of these autoreactivities. Moreover, this scenario does not fully explain the determinant spreading hierarchy, as a complete correlation was only seen after accounting for T cell avidity.
The immunogenicity of self-determinants should also depend on the
avidity of reactive precursor T cells. Indeed, the rate of TCR
phosphorylation and the subsequent proliferative response of T cells
are in part modulated by the avidity of the TCR for the MHC/peptide
complex (30, 31). Furthermore, T cell avidity may be an
especially critical factor in determining the stage at which T cells
reach their activation threshold as the autoimmune process gradually
develops in the islets of NOD mice. We found that the precursor T cell
pool reactive to the early target GAD524–543,
like that of HEL11–25, had high overall avidity,
and proportionally was comprised of a large percentage of high avidity
T cell clones. Subsequently, T cell autoreactivity spreads to cell
Ag determinants that had progressively lower overall T cell avidity,
and whose respective precursor T cell pools were increasingly comprised
of low avidity T cell clones. Thus, the frequency of high avidity
autoantigen-reactive T cells (in terms of both their actual number and
the proportion to which they comprised the Ag-specific repertoire)
correlated precisely with the spreading hierarchy of spontaneous T cell
autoimmunity. These findings suggest that the extent of selection by
self-determinants, which impacts both the size of T cell precursor
pools and their avidity, is the major factor responsible for
establishing the spreading hierarchy.
Conceivably, the preautoimmune T cell repertoire of NOD mice contains
cell-reactive T cells with a range of different avidities, but
which fail to interact with self-Ags at sufficient levels for
activation. As an inherent perturbation develops in their islets, the
high avidity cell Ag-reactive T cells, which require less Ag and
costimulation, should be the first to exceed their activation
thresholds and expand (32, 33). These high avidity T cells
should also have faster expansion kinetics (30, 31). This
early wave of proinflammatory activity creates a microenvironment (via
the local release of cytokines, the recruitment of activated APC pools,
and the up-regulation of T cell activation-associated
molecules), which supports the activation of lower avidity T cells
(1). The activation of the large high avidity T cell
response against GAD appears to be crucial for establishing and driving
a self-perpetuating autoimmune response, as the early inactivation or
circumvention of the GAD-specific T cell response prevents the
development of autoimmunity to cell Ags and insulitis (8, 34). The first wave of autoreactive T cells generates further
proinflammatory positive feedback, so that T cells with progressively
lower avidities are recruited and participate as effector cells. Such
sequential activation of T cells based on their avidity may underlie
the observed spreading of cell autoreactivity in NOD mice during
disease progression.
Recently, GAD524–543 was found to contain two
determinants, a stronger determinant that is recognized by the
spontaneous autoimmune response, and a weaker determinant that is
recognized by regulatory T cells (see figures 2 and 4 within Ref.
35). Thus, the large precursor pool reactive to
GAD524–543 may reflect reactivity to both of
these determinants. However, given that the precursor pool reactive to
GAD524–543 is 2-fold larger than that to the
other tested Ags, the precursor pool reactive to the stronger,
spontaneously recognized determinant of
GAD524–543 should still be larger than
any other tested determinant.
Previous studies of EAE have suggested that the frequency of precursor T cells contributes to the spreading hierarchy (T cell avidity was not evaluated) (6, 36). However, in our spontaneous model of IDDM, the size of the precursor T cell pools did not completely correlate with the spreading hierarchy. Notably, myelin Ags have been shown to extensively shape the T cell repertoire by negative selection, and it appears that low avidity T cells mediate the disease (26, 37, 38). Accordingly, the frequency of myelin-reactive T cells may be the major factor in determining their immunogenicity and encephalogenicity following immunization with myelin Ags. In contrast, NOD mice have deficiencies in tolerance development (12, 39), and many high avidity self-reactive T cells may be allowed to persist. Theoretically, when a perturbation gradually arises in their islets, it is the high avidity T cells that should first become activated and drive subsequent spreading. Thus, in spontaneous autoimmune disease, the avidity of the autoantigen-reactive T cell repertoire may be the most important factor in determining the spreading hierarchy.
In summary, T cell autoreactivity was first detected to a cell
determinant that had both the largest precursor T cell pool and the
highest proportion of high avidity T cells. With disease progression, T
cell autoreactivity spread to cell Ag determinants with
progressively smaller and lower avidity T cell pools. The hierarchy of
determinant spreading partially correlated with the size of precursor T
cell pools, and perfectly matched the order of the frequency of high
avidity T cells. These features of Ag-reactive T cell repertoires are
all shaped by the immunogenicity of cell Ags during tolerance
development. Thus, our findings suggest that the extent to which T
cells were negatively selected by the self-determinants is the key
factor determining the spreading hierarchy of spontaneous T cell
autoimmunity. These results may provide insights into the pathogenesis
of organ-specific autoimmune diseases and aid in the design of
stage-specific immunotherapies.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daniel Kaufman, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Medical School, Box 173517, University of California, Los Angeles, CA 90095-1735. E-mail address: dkaufman{at}mednet.ucla.edu
3 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; GAD, glutamic acid decarboxylase; HEL, hen egg lysozyme; HSP, heat shock protein; IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; SFC, spot-forming colonies; MSA, mouse serum albumin; PPD, purified protein derivative; MBP, myelin basic protein; ELISPOT, enzyme-linked immunospot.
Received for publication February 5, 2001. Accepted for publication April 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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phosphorylation. Science 281:572. cells. Science 284:1183. genes. J. Immunol. 166:2982.This article has been cited by other articles:
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A. P. Olcott, J. Tian, V. Walker, H. Dang, B. Middleton, L. Adorini, L. Washburn, and D. L. Kaufman Antigen-Based Therapies Using Ignored Determinants of {beta} Cell Antigens Can More Effectively Inhibit Late-Stage Autoimmune Disease in Diabetes-Prone Mice J. Immunol., August 1, 2005; 175(3): 1991 - 1999. [Abstract] [Full Text] [PDF]
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H. Reijonen, R. Mallone, A.-K. Heninger, E. M. Laughlin, S. A. Kochik, B. Falk, W. W. Kwok, C. Greenbaum, and G. T. Nepom GAD65-Specific CD4+ T-Cells with High Antigen Avidity Are Prevalent in Peripheral Blood of Patients With Type 1 Diabetes Diabetes, August 1, 2004; 53(8): 1987 - 1994. [Abstract] [Full Text] [PDF]
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 J K Kalsi, J Grossman, J Kim, P Sieling, D W Gjertson, E F Reed, F M Ebling, M Linker-Israeli, and B H Hahn Peptides from antibodies to DNA elicit cytokine release from peripheral blood mononuclear cells of patients with systemic lupus erythematosus: relation of cytokine pattern to disease duration Lupus, July 1, 2004; 13(7): 490 - 500. [Abstract] [PDF]
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V. P. Yeung, J. Chang, J. Miller, C. Barnett, M. Stickler, and F. A. Harding Elimination of an Immunodominant CD4+ T Cell Epitope in Human IFN-{beta} Does Not Result in an In Vivo Response Directed at the Subdominant Epitope J. Immunol., June 1, 2004; 172(11): 6658 - 6665. [Abstract] [Full Text] [PDF]
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S. A. Curran, O. M. FitzGerald, P. J. Costello, J. M. Selby, D. J. Kane, B. Bresnihan, and R. Winchester Nucleotide Sequencing of Psoriatic Arthritis Tissue before and during Methotrexate Administration Reveals a Complex Inflammatory T Cell Infiltrate with Very Few Clones Exhibiting Features That Suggest They Drive the Inflammatory Process by Recognizing Autoantigens J. Immunol., February 1, 2004; 172(3): 1935 - 1944. [Abstract] [Full Text] [PDF]
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 L. H. Butterfield, A. Ribas, V. B. Dissette, S. N. Amarnani, H. T. Vu, D. Oseguera, H.-J. Wang, R. M. Elashoff, W. H. McBride, B. Mukherji, et al. Determinant Spreading Associated with Clinical Response in Dendritic Cell-based Immunotherapy for Malignant Melanoma Clin. Cancer Res., March 1, 2003; 9(3): 998 - 1008. [Abstract] [Full Text] [PDF]
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J. Tian, A. P. Olcott, and D. L. Kaufman Antigen-Based Immunotherapy Drives the Precocious Development of Autoimmunity J. Immunol., December 1, 2002; 169(11): 6564 - 6569. [Abstract] [Full Text] [PDF]
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P. Halbout, J.-P. Briand, C. Becourt, S. Muller, and C. Boitard T Cell Response to Preproinsulin I and II in the Nonobese Diabetic Mouse J. Immunol., September 1, 2002; 169(5): 2436 - 2443. [Abstract] [Full Text] |
5. T cell responses to positive controls PPD and anti-CD3 were
similar in all groups. B, Data shown are the percentages
of high, intermediate, and low avidity T cells in each Ag-specific
repertoire (as described in Materials and Methods).