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Sections of Infectious Diseases and Immunobiology, Yale University School of Medicine, New Haven, CT 06520; and
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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cells in InsHA mice. Previous studies have demonstrated that BALB/c
and B10.D2 mice that express this transgene exhibit tolerance of
HA and retain only low-avidity CD8+ T cells specific for
the dominant peptide epitope of HA. In this study, we present data that
demonstrate a deficiency in peripheral tolerance within the
CD8+ T cell repertoire of NOD-InsHA mice. CD8+
T cells can be obtained from NOD-InsHA mice that exhibit high avidity
for HA, as measured by tetramer (KdHA) binding and dose
titration analysis. Significantly, these autoreactive CD8+
T cells can cause diabetes very rapidly upon adoptive transfer into
NOD-InsHA recipient mice. The data presented demonstrate a retention in
the repertoire of CD8+ T cells with high avidity for islet
Ags that could contribute to autoimmune diabetes in NOD
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells and represents a failure on the
part of the immune system to maintain self-tolerance. Susceptibility to
diabetes is determined by both environmental and genetic factors.
Genetic predisposition is polygenic, with as many as 15–20 genes
involved in progression to type I diabetes in mice and humans
(1). These observations suggest that autoimmune disease
represents a situation in which numerous events conspire to override
multiple checkpoints that normally maintain self-tolerance
(2).
One of the best rodent models of autoimmune diabetes is the nonobese
diabetic mouse (NOD). These mice develop spontaneous diabetes that is
mediated by both CD4+ and
CD8+ T cells (3, 4, 5, 6, 7, 8, 9). The strongest
genetic determinants of autoimmune diabetes in both mice and man is the
MHC genotype. The presence of certain MHC class II molecules, HLA-DQ8
in humans and I-Ag7 in NOD mice, is strongly
associated with disease susceptibility (10, 11, 12). These
molecules share the same nonaspartic acid substitution at residue 57 in
the -chain. As a consequence, the role of CD4+
T cells and the MHC class II molecules they recognize has been a major
focus of research in diabetes. Several laboratories have demonstrated
an enhanced proliferative response to self-Ags within the
CD4+ T cell compartment (13, 14, 15).
However, there is strong evidence that spontaneous autoimmune diabetes
that occurs in NOD mice is mediated not only by
CD4+ T cells, but also by
CD8+ T cells. NOD mice bred to MHC class
I-deficient mice
(2m-/-) or treated
with anti-CD8 Abs develop neither insulitis nor diabetes
(16, 17, 18, 19, 20). Also, it was found that proinsulin-specific
CD8+ T cell populations from NOD mice can cause
diabetes in the absence of CD4+ T cell help
(21) and accumulate in the islets of young NOD mice
(22).
The current study was undertaken to determine the extent to which there
may be a defect in tolerance to islet Ags within the
CD8+ T cell repertoire of the NOD mouse. To
address this question we have used a model pancreatic Ag, the
hemagglutinin (HA) molecule of influenza virus, that is expressed on
cells in the pancreatic islets under control of the rat insulin
promoter (InsHA). When this molecule is expressed in BALB-InsHA or
B10.D2-InsHA mice, tolerance is induced and maintained even after
immunization with influenza virus (23, 24, 25, 26, 27). One hallmark
of successful tolerance in this model is a fundamental alteration in
the HA-specific repertoire such that all CD8+ T
cells specific for HA exhibit low avidity for HA compared with the
HA-specific response of conventional mice (23, 24, 27). We
have previously reported that peripherally induced tolerance plays a
major role in eliminating high-avidity CD8+ T
cells specific for HA. HA-specific naive T cells encounter Ag that is
cross-presented by professional APCs in the lymph nodes draining the
pancreas. This results in their activation and is followed by their
functional elimination (25, 26).
We have investigated the degree of tolerance to HA within the CD8+ T cell compartment of NOD-InsHA mice. We show that under identical stimulatory conditions, high-avidity CD8+ T cells specific for this Ag can be retrieved from NOD-InsHA mice, but not from BALB-InsHA mice. Importantly, these HA-specific CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into irradiated NOD-InsHA recipient mice. These data demonstrate a deficiency in self-tolerance within the CD8+ T cell repertoire of NOD mice such that CD8+ T cells with high affinity for an islet Ag are retained within the repertoire. This may help explain why NOD mice develop diabetes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and NOD/Shi were purchased from the breeding colony of The Scripps Research Institute (La Jolla, CA). InsHA-transgenic mice were generated and characterized as previously described (23) and were bred onto the BALB/c background for at least 10 generations (BALB-InsHA) or onto the NOD background for 13 generations (NOD-InsHA).
All mice were bred and maintained under specific pathogen-free conditions in The Scripps Research Institute vivarium. All experimental procedures were conducted according to the guidelines laid out in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Cell lines
The SV40-transformed H-2d cell line B10.D2 was maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% (v/v) heat-inactivated FCS (Gemini Biological Products, Calabasas, CA), 2 mM glutamine (Life Technologies), 5 x 10-5 M 2-ME (Sigma, St. Louis, MO), and 50 mg/ml gentamicin (Gemini Bio-Products). Cells were cultured in a humidified incubator at 37°C with 5% (v/v) CO2 and were used as targets in CTL assays. The Bio.oz cell line was originally obtained from B. Knowles (University of Pennsylvania, Philadelphia, PA).
Peptide
HA influenza virus A/PR/8/34 (H1N1) peptide (518IYSTVASSL526, restricted by H-2Kd) was synthesized by the core facility of The Scripps Research Institute using an Applied Biosystems model 430A synthesizer (Foster City, CA). Purity was >85%, as determined by mass spectrometry and reversed-phase HPLC analysis on a Vydac C18 column (Vydac, Hesperia, CA).
Virus
Influenza virus A/PR/8/34 H1N1 (PR8) was grown in the allantoic cavity of 10- to 11-day-old hen’s eggs. Upon isolation, the allantoic fluid was titrated for HA using chicken RBC and stored at -70°C.
Production of effector CTL and adoptive transfers
Eight-week-old mice were injected i.p. with 1200 HA units of influenza virus A/PR/8 in the form of allantoic fluid. Three weeks later, mice were sacrificed, and responder splenocytes were seeded into 24-well tissue culture plates at 7 x 106 cells/well in 1 ml complete RPMI 1640. APC splenocytes were irradiated (3000 rad) and then pulsed for 1 h with 5 nM KdHA peptide in a humidified incubator at 37°C with 5% (v/v) CO2. After washing three times in complete RPMI 1640 to remove unbound peptide, 1 ml containing 6 x 106 cells was added to the responder splenocytes and cultured in a humidified incubator at 37°C with 5% (v/v) CO2. For adoptive transfers, effector CTLs were injected i.v. into sublethally irradiated (600 rad) 4-wk-old BALB-InsHA or NOD-InsHA.
CD4+ T cell depletion
CD4+ T cells were depleted in vitro by incubating splenocytes with anti-CD4 Ab (RL172) supernatant on ice for 1 h. Cells were then centrifuged, the supernatant was discarded, and cells were resuspended in Low-Tox rabbit complement (Accurate Chemical and Scientific, Westbury, NY), and incubated for an additional hour at 37°C. Cells were then washed three times in complete RPMI 1640 and used for primary cultures. Depletion was complete as checked by staining cells with FITC-conjugated anti-CD4 Abs and analyzing with a FACScan and CellQuest software (BD Biosciences, Mountain View, CA).
Cytotoxicity assay
Target cells were prepared by incubating B10.D2 cells at 37°C with 200 µCi sodium 51Cr (NEN, Boston, MA) for 1 h in the presence or the absence of various concentrations of KdHA peptide as indicated. Target cells were washed four times, resuspended in complete RPMI 1640, and seeded into 96-well plates at 1 x 104 cells/well in 100 µl. Effector CTL were harvested 6 days after stimulation and seeded into duplicate wells containing the target cells at various E:T cell ratios, making a final volume of 200 µl. Plates were incubated at 37°C in a humidified incubator with 5% (v/v) CO2 for 5 h. Plates were centrifuged, and 100 µl supernatant was removed from each well to assess isotope release using an ICN Isomedic gamma radiation counter (VWR Scientific, San Diego, CA). Relative cytotoxic activity (percentage) was calculated as: 100 x (sample release - spontaneous release)/(maximum release - spontaneous release).
Flow cytometry
CTLs were stained 4 days after stimulation, purified through a Ficoll-Paque gradient (Pharmacia Biotech, Uppsala, Sweden), and then washed in FACS buffer (HBSS containing 1% (w/v) BSA; Sigma) and 0.02% (w/v) sodium azide (Sigma). Cells were incubated on ice with PE-conjugated KdHA tetramer and FITC-conjugated anti-CD8 (BD PharMingen, La Jolla, CA) for 1 h. Tetramers were produced as previously described, except that KdHA peptide was used (24). Cells were washed three times with FACS buffer. Propidium iodide (Sigma) was added after the final wash at 1 mg/ml to exclude dead cells in all experiments. Cells were analyzed with a FACScan and CellQuest software (BD Biosciences)
Analysis of blood glucose level
The glucose concentration in blood obtained from the retro-orbital plexus of mice was measured using the Accu-ChekIII (Roche, La Jolla, CA). Animals were considered diabetic if blood glucose levels were >300 mg/dl.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have revealed that BALB-InsHA and B10.D2-InsHA
mice are profoundly tolerant to HA and do not develop diabetes even
after infection with influenza virus (23). To determine
whether CD8+ T cells in NOD mice undergo
tolerance to a model pancreatic Ag, we produced NOD mice expressing the
same InsHA transgene (NOD-InsHA). NOD-InsHA females develop spontaneous
diabetes at a similar rate and incidence as normal NOD females (70%
incidence by 28 wk of age) (28). Also, the level of HA
expression on pancreatic cells is similar for NOD-InsHA and
BALB-InsHA mice, as measured by flow cytometry using anti-HA Abs
(data not shown).
To assess the HA-specific CD8+ T cell repertoire
in NOD-InsHA mice, recipient animals were immunized with influenza
virus A/PR/8/34 H1N1 (PR8) that contained the same HA molecule as that
used by the transgene. Three weeks after priming, spleen cells from
BALB/c or NOD mice were restimulated in vitro with a concentration of
KdHA peptide that has previously been shown to be
optimal for stimulating high-avidity HA-specific
CD8+ T cells (27). As previously
shown, CD8+ T cells from BALB/c, but not
BALB-InsHA, mice respond vigorously to HA (Fig. 1
A). In contrast to BALB-InsHA, a good response to HA was
detectable in NOD-InsHA mice (Fig. 1B). However, the
response by NOD-InsHA mice was clearly weaker than that of
nontransgenic NOD responders, suggesting that at least partial
tolerance to HA had occurred as a result of expression of the InsHA
transgene. It is also important to note that not every adult NOD-InsHA
animal responded to HA, suggesting variability in the degree of
tolerance induction.
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Previous studies of the mechanism of tolerance to HA in BALB-InsHA mice
indicated that tolerance does not occur until several weeks after
birth. This correlated with the ability of naive HA-specific
CD8+ T cells to first detect HA Ag within the
draining lymph nodes of the pancreas (25). Consistent with
these previous results, both BALB-InsHA and NOD-InsHA neonates respond
vigorously to HA (Fig. 1
, C and D). These results
strongly suggest that the peripheral deletion of HA-specific
CD8+ T cells that normally occurs in BALB-InsHA
and B10.D2-InsHA over time (25), does not occur in
NOD-InsHA mice.
HA-specific CD8+ T cells in NOD-InsHA mice have high avidity toward self-Ags
Previous studies from this laboratory have demonstrated that the
most significant difference between HA-specific
CD8+ T cells from BALB-InsHA or B10.D2-InsHA mice
and nontransgenic mice is a reduction in T cell avidity for HA
(24, 27). This reduction is attributable to elimination of
T cells that express TCRs with high affinity for HA. The residual
low-avidity T cells are unable to cause diabetes (27). The
observed high level of cytotoxicity by HA-specific CTLs from NOD-InsHA
mice could be attributable to the presence of either a large number of
CD8+ T cells with low avidity for HA or a small
number of HA-specific cells with high avidity for HA. To distinguish
between these alternative possibilities, the avidity of the HA-specific
CD8+ T cells from NOD-InsHA was compared with
that of HA-specific CD8 T cells from NOD mice on the basis of their
lytic activity on targets pulsed with varying concentrations of
KdHA peptide (Fig. 2A). Both NOD and NOD-InsHA responded with comparable
avidities, although it took more NOD-InsHA CTLs to obtain
comparable lysis (3- to 10-fold). This suggested that the
HA-specific CD8+ T cells from NOD and NOD-InsHA
mice differed quantitatively, but were comparable in their avidity
for HA.
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B). HA-specific CTLs from NOD-InsHA bound tetramer as
well as those from NOD CTLs (mean fluorescence intensity, 134 vs 142),
although there were fewer HA-specific CTLs present in the NOD-InsHA
cultures (11 vs 40%). Taken together, these data indicate that
NOD-InsHA mice exhibit the presence of reduced numbers of HA-specific
cells with high avidity, rather than large numbers of
CD8+ T cells with low avidity, for HA. HA-specific CD8+ T cell reactivity in unprimed NOD-InsHA mice, but not NOD
These results demonstrate a significant HA response in NOD-InsHA
mice that had been primed in vivo with influenza virus. To determine
whether HA-specific CD8+ T cells could also be
detected in unprimed NOD-InsHA mice, KdHA
tetramers were used in an attempt to detect HA-specific
CD8+ T cells in the spleen, lymph nodes, and
pancreas of NOD-InsHA mice. This failed, presumably due to the low
frequency of these cells in vivo. To increase the numbers of such
cells, splenocytes from unprimed NOD-InsHA were stimulated in vitro
with KdHA peptide-pulsed APCs. After three
stimulations, HA reactivity was measured in a CTL assay using target
cells pulsed with KdHA peptide (Fig. 3). By this method we could detect HA-specific CD8+
T cells from two of the seven NOD-InsHA tested. In contrast, none of
the cultures from NOD mice developed HA-specific CTL.
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Another measure of the degree to which InsHA mice are tolerant to
HA is the fact that the HA-specific CD8+ T cells
from B10.D2 or BALB/c, but not B10.D2-InsHA or BALB/c-InsHA mice, can
cause diabetes upon adoptive transfer into InsHA mice (27)
(Table I). To determine whether the HA-specific CD8+ T
cells retrieved from NOD-InsHA mice could lead to cell destruction
and diabetes, HA-reactive CTLs obtained from NOD-InsHA were
adoptively transferred into irradiated NOD and NOD-InsHA mice (Table I). As shown in Table I, HA-reactive CTL lines from NOD-InsHA were able
to cause diabetes very rapidly in irradiated NOD-InsHA recipient mice.
No diabetes was observed if the recipients did not express HA in the
pancreas, indicating that cell destruction was Ag specific.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purpose of this study was to determine whether the CD8+ T cell compartment in NOD mice is responsive to self-Ags. Previous data have demonstrated reactivity to certain self-Ags within the CD4 compartment, such as glutamic acid decarboxylase, carboxypeptidase, insulin, 60-kDa heat shock protein, and peripherin (13, 14, 15). It was further shown that expression of an unusual MHC class II I-Ag7 is necessary for autoproliferation to occur, but is not sufficient, since CD4+ T cells from I-Ag7-positive B10 mice do not exhibit autoproliferation nor do CD4+ T cells from NOD mice that lack I-Ag7 molecules (32, 33). This suggests that multiple NOD genes are required for CD4+ T cell autoproliferation to occur. However, the progression toward autoimmune diabetes requires not only CD4+ T cells, but also CD8+ T cells (16, 17, 18, 19, 20). It was therefore of interest to investigate whether CD8+ T cells exhibit autoreactivity to islet Ags.
In this study, we present data demonstrating a defect in the peripheral deletion of CD8+ T cells specific for a model pancreatic self-Ag expressed in NOD mice. This defect results in the presence of autoreactive HA-specific CD8+ T cells that have high avidity toward this self-Ag and are very aggressive in causing autoimmune diabetes upon adoptive transfer into irradiated recipient mice. Such CD8+ T cells are normally eliminated from adult InsHA mice, as they are not found in either BALB-InsHA or B10.D2-InsHA mice (23, 24, 27). Although a defect in the induction of peripheral tolerance was found in NOD-InsHA mice, clearly some tolerance did occur, since the CTL response toward HA was significantly lower in some NOD-InsHA mice compared with that in NOD mice. It is tempting to speculate that such variability in CTL responses may parallel the significant variability that occurs in the age of onset of diabetes in NOD animals.
The frequency of HA-reactive CD8+ T cells in
unprimed NOD-InsHA is low, and several rounds of in vitro stimulation
were required for their detection (Fig. 3). Although HA-specific
CD8+ T cells are also present in unprimed NOD
mice, no HA-specific CD8+ T cells were detected
in cultures from NOD mice. One explanation for this could be that some
HA-specific CD8+ T cells in NOD-InsHA mice are
endogenously primed by pancreatic HA and that this facilitates their
ability to respond in vitro to stimulation with HA peptide.
Although the present study showed a defect in peripheral tolerance in the NOD mouse, we cannot exclude the possibility that there are additional defects that may impair thymic tolerance toward self-Ags in NOD mice. However, if this is the case, it is unlikely that the basis for any such defect in tolerance within the CD8 compartment is the same as the linear avidity model, first proposed by Fathman and coworkers (34). According to this model, the instability of the I-Ag7 molecule is believed to contribute to defective negative selection, which, in turn, is attributed to the autoproliferation in the CD4 compartment. However, the restriction element in the HA model (Kd) is the same in BALB/c and B10.D2 mice and therefore is unlikely to contribute to defective negative selection in the CD8 compartment.
The increased reactivity to HA in NOD-InsHA animals within the CD8+ T cell compartment may be due to a number of different factors. It is possible that the presence of CD4+ T cell help prevents the peripheral deletion of autoreactive CD8+ T cells (35, 36, 37, 38). In a different transgenic model of CD8+ T cell tolerance it was shown that autoreactive CD4+ T cells could prevent peripheral deletion of CD8+ T cells induced by cross-presentation of self-Ags. This resulted in autoimmunity, rather than tolerance (38). Since NOD animals contain many autoreactive CD4+ T cells, this could prevent the normal induction of CD8+ T cell tolerance in the periphery. We are currently testing this hypothesis.
Another possibility that could explain the defective peripheral tolerance of autoreactive CD8+ T cells is a general defect in T cell apoptosis and function. It has been shown that both mature NOD T lymphocytes as well as immature thymocytes can resist a variety of apoptosis-inducing stimuli, such as cyclophosphamide, dexamethasone, IL-2 deprivation, and activation-induced cell death (39, 40, 41, 42, 43, 44, 45, 46, 47, 48). This defect in programmed cell death could contribute to defective thymic and peripheral deletion of autoreactive T cells in NOD mice.
A third possibility could be a defect in APC function. Recent data suggest a crucial role for professional APCs in the induction and maintenance of T cell tolerance by cross-presentation of self-Ags (30, 31). In both NOD mice and diabetic patients, a defect in maturation and function was found in monocyte-derived dendritic cells (49, 50, 51, 52). It is possible that this defect in maturation leads to inefficient cross-presentation of self-Ag and therefore inefficient presentation of self-Ags to CD8+ T cells. We are currently investigating this possibility.
In conclusion, we demonstrate a defect in the induction of
CD8+ T cell peripheral tolerance in NOD mice,
leading to the presence of high-avidity, islet-specific autoreactive
CD8+ T cells that may contribute to autoimmune
diabetes. In future experiments we will use CD8+
T cells from a TCR transgenic line specific for HA to elucidate
the mechanisms responsible for such a tolerance defect. As there is no
a priori reason to expect that tolerance to conventional islet Ags
differs from tolerance toward HA, it is likely that NOD mice are
defective in peripheral tolerance within their CD8 compartment toward
other islet Ags as well. Indeed, the recent demonstration of
high-avidity, cell-specific CD8+ T cells in
NOD mice may reflect such a defect (22, 53).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Linda A. Sherman, Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, IMM-15, La Jolla, CA 92037. E-mail address: lsherman{at}scripps.edu
3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; HA, hemagglutinin; InsHA, transgenic mouse expressing the HA from influenza virus on pancreatic islet cells under the control of rat insulin promoter.
Received for publication May 1, 2001. Accepted for publication May 7, 2001.
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Y. Zhang, B. O'Brien, J. Trudeau, R. Tan, P. Santamaria, and J. P. Dutz In Situ {beta} Cell Death Promotes Priming of Diabetogenic CD8 T Lymphocytes J. Immunol., February 1, 2002; 168(3): 1466 - 1472. [Abstract] [Full Text] [PDF]
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), BALB-InsHA
(A, 
), NOD (B, •), NOD-InsHA
(B, 
), BALB/c-InsHA neonates (C, 
),
and NOD-InsHA neonates (D, 
) were cultured with
homologous, irradiated (3000 rad) APCs pulsed with 5 nM
KdHA peptide. After 6 days, specific lysis was tested in a
5-h chromium release assay on B10.D2 targets (H-2Kd
positive) pulsed with 25 µM KdHA peptide. Background
lysis on target cells without Ag was <16% at the highest E:T cell
ratio. Data are representative of three independent experiments.
