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Cell Death Promotes Priming of Diabetogenic CD8 T Lymphocytes1
,
Departments of
*
Medicine and
Pathology and Laboratory Medicine, British Columbia Research Institute of Children and Women’s Health, University of British Columbia, Vancouver, British Columbia, Canada;
Department of Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada; and
Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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cell destruction in
the nonobese diabetic mouse model of type 1 diabetes.
Cross-presentation of Ag is one means of priming CTLs. The death of
Ag-bearing cells has been implicated in facilitating this mode of
priming. The role of cell death in facilitating the onset of
spontaneous autoimmune diabetes is unknown. Here, we used an adoptive
transfer system to determine the time course of islet-derived Ag
presentation to naive cell-specific CD8 T cells in nonobese
diabetic mice and to test the hypothesis that cell death enhances
the presentation of cell autoantigen. We have determined that
cell death enhances autoantigen presentation. Priming of diabetogenic
CD8 T cells in the pancreatic lymph nodes was negligible before 4 wk,
progressively increased until 8 wk of age, and was not influenced by
gender. Administration of multiple low doses of the cell toxin
streptozotocin augmented in situ cell apoptosis and accelerated the
onset and magnitude of autoantigen presentation to naive CD8 T cells.
Increasing doses of streptozotocin resulted in both increased
pancreatic cell death and significantly enhanced T cell priming.
These results indicate that in situ cell death facilitates
autoantigen-specific CD8 T cell priming and can contribute to both the
initiation and the ongoing amplification of an autoimmune
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells are destroyed by the
cellular immune system (1, 2). The nonobese diabetic (NOD)
mouse is a useful and well-studied animal model for human type 1 DM.
Adoptive T cell transfer studies using T cells from prediabetic NOD
mice have demonstrated that transfer of diabetes into immunodeficient
mice requires both CD4 and CD8 T cells (3, 4, 5, 6). CD8 T cells
are known to participate in pancreatic cell destruction
(7, 8, 9). The importance of CD8 T cells in diabetogenesis is
underscored by the fact that neither DM nor insulitis develop in NOD
mice deficient in MHC class I expression and CD8 T cells (10, 11). Recent data suggest that CD8 T cells play a pivotal role in
the earliest initiative phases of autoimmune pancreatic cell
destruction (12, 13).
The activation of naive (resting and previously unactivated) T cells
requires activation of the TCR and the simultaneous delivery of a
costimulatory signal by a specialized APC . Using adoptive transfer of
naive CD4 T cells specific for a pancreatic cell Ag, it has been
demonstrated that these cells can be primed in the pancreatic lymph
nodes as early as, but not before, 14 days of age (14).
Similarly, CD8 T cells responding to transgenically overexpressed Ags
in the pancreas cannot be primed in mice of 5–10 days of age
(14, 15). The timing and mechanism by which CD8 T cells
are primed to pancreatic cell Ag in spontaneous autoimmunity
remains to be determined. Cross-presentation of Ag, where
tissue-specific Ag is processed by APCs and presented to T cells via
class I MHC, is one means of priming CD8 T cells (16).
Cross-presentation of cellular Ags expressed by tumors
(17), viruses (18), and self
(19), as well as of soluble Ags, has been described
(20). Cell death, specifically by apoptosis, can provide
an excellent source of cellular Ag for cross-presentation (21, 22). Cell death, by either apoptosis (23) or
necrosis (24), can also provide cell-derived signals that
activate APCs and promote immune responses. Therefore, we hypothesized
that cell death could promote the initiation and/or progression of
spontaneous autoimmune diabetes.
The aims of the present study were first to delineate the timing of CD8
T cell priming to pancreatic cell-associated Ag in NOD mice and,
second, to determine whether cell death can promote the priming of
CD8 T cells to a diabetogenic self-Ag. We used an adoptive transfer
system using CD8 T cells derived from a mouse line that transgenically
expresses a TCR of a diabetogenic T cell clone (8). This
clone uses a TCR rearrangement expressed by a preponderance of CD8 T
cells found in early insulitic lesions of NOD mice (13).
The results of our investigations show that priming of diabetogenic CD8
T cells occurs in the pancreatic lymph nodes and is undetectable before
3–4 wk of age and then progressively increases with age to reach
maximal levels at 8 wk of age. An increased incidence of apoptosis in
the pancreas after the administration of low doses of the cell
toxin streptozotocin (STZ) augmented CD8 T cell priming and allowed it
to occur at ages when it does not occur spontaneously. These results
suggest that cell death precedes the priming of T cells in
autoimmune diabetes and implicate in situ cell death in the
facilitation of autoantigen-specific CD8 T cell priming in
autoimmunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD mice were obtained from Taconic Farms (Germantown, NY) and bred in a specific pathogen-free environment. The 8.3-NOD mice, expressing the rearranged TCR genes of the diabetogenic CTL clone NY8.3, have been previously described (8) and were bred at the University of Calgary (Calgary, Alberta, Canada). C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All animal experiments were approved by the Animal Care Committee of the University of British Columbia. EL4 thymoma and E.G7-OVA cells (EL4 cells transfected to express OVA) (20) were obtained from the American Type Culture Collection (Manassas, VA) and cultured in complete medium (CM) consisting of RMPI 1640 medium (Invitrogen, Burlington, Ontario, Canada) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 5 x 10-5 M 2-ME, and 10% FCS.
Adoptive transfers and cytofluorometric analysis
Naive 8.3 CD8 T cells were isolated from 4- to 5-wk-old 8.3-NOD
mice. Single-cell suspensions were prepared from peripheral lymph nodes
and spleen, and CD8 T cells were purified using immunomagnetic cell
separation kits (Miltenyi Biotec, Auburn, CA) according to the
manufacturer’s specifications. Purified 8.3 CD8 T cells were labeled
with 5 µmol/L CFSE (Molecular Probes, Eugene, OR) in PBS for 10 min.
The labeled cells (10 x 106) were
transferred to NOD recipients by tail vein injection (for 8-wk-old
animals) or i.p. (for younger animals). Three-channel FACS analysis was
performed on a FACScalibur flow cytometer (BD Biosciences, San Jose,
CA) using Cell Quest software (BD Biosciences). Briefly, 200,000 cells
were analyzed after staining with appropriate Abs or tetramer.
Kb-OVA tetramers were synthesized as described
(25). Abs to CD44, CD25, and TCR V8.1/8.2 were purchased
from Cedarlane (Hornby, Canada). Abs to CD8
(clone 53-6.7) and CD69
were purchased from BD PharMingen (San Diego, CA). The Ab to TCR V
8.1/8.2 recognizes the V-chain used by the 8.3 TCR (8).
In vitro stimulation of purified 8.3 CD8 T cells was conducted in the
presence of 1 µg/ml of the indicated peptides and CM using irradiated
(3000 rad) syngeneic splenocytes as APCs.
Immunizations and cytotoxicity assay
C57BL/6 mice (5–6 wk old) were immunized in the flank by s.c. injection with 100 µg OVA V (Sigma-Aldrich, St. Louis, MO) in PBS with or without 5 x 106 EL4 thymoma cells that had been either irradiated or freeze thawed for four cycles. The gamma irradiation consisted of 20,000 rads followed by incubation for 4 h in serum-free medium and induced apoptosis in 82% of cells at the time of injection, as confirmed by Annexin V positivity (BD PharMingen). Mice were boosted at 1 wk and sacrificed at day 14. EL4 cells were Mycoplasma negative, as tested by PCR (American Type Culture Collection). For tetramer analysis, spleen cells were depleted of RBCs and analyzed by fluorescence cytometry. For CTL generation, 5 x 106 splenocytes were stimulated with 2 x 106 irradiated E.G7-OVA cells and cultured in 10 ml CM for 5 days. Human rIL-2 (20 IU/ml) (BD PharMingen) was added on day 3. Chromium release assays were conducted using 51Cr-labeled E.G7-OVA cells as targets. Plates were incubated for 4 h at 37°C. Supernatants were collected and gamma irradiation was measured. Specific lysis of target cells was calculated using the following formula: [(release by CTL - medium release)/(detergent release - medium release)] x 100. Spontaneous release from target cells was always <20%.
Peptide synthesis
The peptides TUM (KYQAVTTTL), NRP-A7 (KYNKANAFL), and OVA (SIINFEKL) were prepared using N-(9-fluorenyl)methoxycarbonyl chemistry and purified by reversed phase HPLC to >80% purity at the Nucleic Acid and Peptide Synthesis Facility of the University of British Columbia.
STZ treatment
Animals were injected i.p. with STZ (Sigma-Aldrich), freshly prepared in citrate buffer (0.01 mmol/L; pH 4.5), and used at the indicated doses.
Identification and quantification of apoptotic cells
The TUNEL method for labeling DNA strand breaks was used to
quantify the number of apoptotic cells in sections of pancreas from
mice aged 2 wk, as has been described (26).
Pancreata were removed immediately after killing and were fixed
in Bock’s fixative for 48 h at room temperature. The tissue was
washed in PBS and then in 70% alcohol and was embedded in paraffin
using standard histological techniques. Microwave irradiation was used
for Ag retrieval. The ApopTag Kit (Intergen, Purchase, NY) was then
used to identify the apoptotic cells. Visualization of positive cells
was achieved with 3,3'-diaminobenzidine tetrahydrochloride as the
substrate. To provide a negative control for the TUNEL, TdT was omitted
from the labeling mix. The cell origin of the apoptosis was
verified by double staining for insulin using guinea pig
anti-porcine insulin Ab (DAKO, Mississauga, Ontario, Canada), a
biotinylated anti-guinea pig IgG as secondary Ab (Vector
Laboratories, Burlington, Ontario, Canada), and the chromogen New
Fuschin (DAKO). Cells with brown nuclei (TUNEL positive) and pink
cytoplasm (insulin positive) were identified as apoptotic cells.
Apoptosis was quantified in all cells present in stained sections
from each animal, each section being separated from the next by at
least 40 µm. Approximately 1000 cells were counted for each
animal by a single observer, blinded to the treatment status of the
animal. The number of apoptotic cells present was expressed as a
percentage of the total number of cells observed.
Assessment of diabetes development
Blood glucose was measured in tail vein blood using Elite glucostrips and glucometer (Bayer, Etobicoke, Canada). Animals were considered to be overtly diabetic when two consecutive blood glucose measurements exceeded 14 mmol/L.
Statistical analysis
Groups were compared using one- or two-tailed Student’s t tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytotoxic T cells are known to infiltrate pancreatic islets, where
they participate in pancreatic cell damage (7, 8, 9). A
significant fraction of CD8 T cells in NOD mice express highly
homologous TCR -chains (V17 and J42 elements, joined by the
N-region sequence M-R-D/E) (7, 13, 27) and are cytotoxic
to cells in the context of the H-2Kd MHC
class I molecule. The 8.3-NOD mice were generated using the TCR and
genes of one such H-2Kd-restricted cell
cytotoxic CD8 T cell clone isolated from the islets of an acutely
diabetic NOD mouse (8). We first purified 8.3 CD8 T cells
from 8.3-NOD mice using either negative or positive selection.
Phenotype analysis using the activation markers CD69, CD25, and CD44
revealed that our positive selection protocol yielded both resting and
naive CD8 T cells. By combinatorial peptide library screening, an
agonist peptide (NRP-A7, KYNKANAFL) for the 8.3 TCR has been
identified (28). The purified cells up-regulated early
activation markers in response to agonist peptide but not to a control
peptide (TUM, KYQAVTTTL) (Fig. 1
; Ref.
29). Adoptive transfer of fluorochrome-labeled Ag-specific
T cells has been used as a method to track the activation of T cell in
vivo and can be used to determine the location and timing of cognate Ag
presentation (30). Using this method, it has been
determined that diabetogenic CD4 T cells encounter cognate Ag in the
pancreatic lymph nodes in adult but not in neonatal (10-day-old) mice
(14). Similarly, transgenic expression of OVA in
pancreatic islets leads to OVA-specific CTLs in adult but not in
10-day-old mice. To determine whether diabetogenic CD8 T cells
recognizing an endogenous pancreatic cell Ag are primed in the
pancreatic lymph nodes, we labeled purified naive 8.3 CD8 T cells with
the fluorescent dye CFSE. We then adoptively transferred 10 x
106 of these cells into 8-wk-old NOD mice by tail
vein injection. Within 72 h after transfer, cells that had
proliferated at least once (as indicated by serial CFSE dye dilution)
were detected in the pancreatic lymph nodes but not in the mesenteric
lymph nodes or spleens of these mice (Fig. 2). Thus, naive 8.3 CD8 T cells
proliferate and are first activated (primed) in the pancreatic draining
lymph nodes of prediabetic NOD mice.
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NOD mice develop an inflammatory infiltrate of the islets
(insulitis) consisting of T and B lymphocytes, macrophages, and
dendritic cells (DCs). Onset of diabetes in female NOD mice occurs from
12 wk of age onwards (2). The spontaneous onset of
insulitis seems to be a fixed checkpoint at approximately days 18–21
(31). It has been shown that diabetogenic CD4 T cells are
primed in the pancreatic lymph nodes before islet infiltration
(14). To establish the time course of priming of naive
diabetogenic CD8 T cells in NOD mice, we adoptively transferred
CFSE-labeled naive 8.3 CD8 T cells into male and female NOD mice aged
1–8 wk. The percentage of CFSE-labeled cells that had undergone at
least one cell division was then quantified (Fig. 3). The priming of diabetogenic CTLs in
the pancreatic lymph nodes was negligible (3% ± 0.9% of adoptively
transferred cells at 1 wk, n = 8) until 4 wk of age (at
which time it was 11% ± 6%, n = 7). Priming then
progressively increased until 8 wk of age (when it reaches a maximum of
42% ± 11%, n = 6). Autoantigen presentation to
diabetogenic CD8 T cells between 4 and 8 wk of age was not influenced
by gender, as shown by the fact that priming of adoptively
transferred cells into male and female NOD recipients was similar.
Thus, as is the case for diabetogenic CD4 T cells, there is a
developmental regulation of the presentation of endogenous pancreatic
cell Ags to diabetogenic CD8 T cells with minimal presentation of
Ag before 3–4 wk of age. Interestingly, this period of activation
follows a period of remodeling and physiologic apoptosis of the
pancreatic islets that peaks at 2 wk of age (32, 33).
Thus, in situ cell death precedes the priming of diabetogenic CD8 T
cells. Whether this local wave of cell death plays a role in the
initiation of autoimmunity (33) or whether Ag presentation
is developmentally blocked to minimize autoimmunity is not clear
(16).
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Cell death by either apoptosis or necrosis can make cellular
contents available for uptake by professional APCs and may thus
facilitate the priming of cells to self-Ag. Cell death may also act as
a stimulus to create mature immunogenic DCs (24, 34). To
determine whether cell death has adjuvant properties in addition to
providing a means of accessing cell-associated Ag, C57BL/6 mice were
immunized with the nominal soluble Ag OVA in the presence or absence of
either apoptotic or necrotic cells. We chose to use OVA as the nominal
Ag because its immunodominant CTL epitope, SIINFEKL, in the context of
the H-2Kb class I MHC molecule, is well defined
(35). CTL responses were monitored by the enumeration of
OVA (SIINFEKL)-specific CTLs using fluorescence cytometry and
Kb-OVA multimeric complexes. CTL responses
were confirmed using a standard chromium release assay (Fig. 4). Whereas C57BL/6 mice immunized with
soluble protein did not mount significant CTL responses, mice immunized
with soluble OVA co-injected with either irradiated (apoptotic) or
freeze thawed (necrotic) EL4 thymoma cells mounted good CTL responses.
These responses were not due to tumor-specific factors because similar
results were obtained with the co-injection of autologous irradiated or
freeze thawed fibroblasts (data not shown). These results suggest that
cellular material released by either apoptotic or necrotic cells can
potentiate the priming of CTLs to soluble co-administered Ag.
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cell toxin STZ enhances the priming of 8.3 CD8 T cells in
2-wk-old NOD mice
Next we wanted to know whether increased cell death at the tissue
level could promote the priming of autoreactive CD8 T cells. STZ, a
D-glucopyranose derivative of
N-methyl-N-nitrosourea, is cytotoxic to
pancreatic cells. When delivered at high doses (200 mg/kg), STZ
causes complete cell destruction and insulin depletion. At low
concentrations and when administered in multiple low doses, STZ can
initiate a delayed diabetic state in susceptible strains that is
largely dependent on immune mechanisms (36, 37, 38). High
doses of STZ in vitro can cause necrosis of cells
(39). Low doses of STZ, however, initiate an apoptotic
pathway of cell death. Multiple low doses of STZ have been shown to
increase in situ apoptotic cell death in the pancreas
(26). Because cell death may increase cellular Ag
availability and have adjuvant properties, we treated 2-wk-old mice
with STZ and assessed cell-specific T cell priming in the
pancreatic lymph nodes. Mice were treated with either STZ (40 mg/kg)
daily for 3 days or with citrate buffer alone before adoptive transfer
of naive 8.3 CD8 T cells. Adoptively transferred cells were analyzed 4
days later (Fig. 5). Priming was detected
by CFSE dilution and up-regulation of the early activation marker,
CD69. Although there was minimal priming in 2- to 3-wk-old animals
treated with citrate buffer alone (n = 5), both
proliferation and CD69 up-regulation of 8.3 CD8 T cells were observed
in the pancreatic lymph nodes of mice treated with STZ
(n = 11). Comparison of the means of CFSE-labeled cells
having undergone at least one division revealed that STZ significantly
enhanced priming (p = 0.007). Similar results
were found using 4-wk-old mice (n = 5 for both test and
control groups; p = 0.016). These results suggest that
STZ enhances the priming of diabetogenic CD8 T cells to endogenous
pancreatic cell Ag and allows this priming to occur at a time when
there is normally minimal Ag presentation and priming.
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cell autoantigen correlates with
cell death
STZ-induced pancreatic cell toxicity is dependent on initial
glucose transporter-2-mediated STZ transport into the cell
(40, 41) and subsequent poly(A)DP-ribose polymerase
(PARP)-dependent depletion of NAD+ within the
cells (42, 43, 44). Because susceptibility to low-dose STZ is
strain specific, we first determined whether 4-wk-old NOD mice given
five daily doses of STZ develop accelerated diabetes. Seven of 10
STZ-treated mice developed diabetes (blood glucose 
14 mmol/L
over two consecutive readings) at age 6 wk. Nine of 10 developed
diabetes by age 7 wk and 10 of 10 did so at 8 wk, compared with zero of
10 citrate buffer-treated mice (Fig. 6.).
A delay of 1–2 wk between STZ administration and diabetes onset
suggests an immune contribution to diabetes development and is
consistent with the delayed onset observed in standard susceptible
strains. Our finding that multiple low-dose STZ can accelerate the
onset of diabetes in NOD mice is in agreement with previous
observations (45). To confirm that the administered STZ
was inducing apoptotic cell death of pancreatic cells, 2-wk-old
mice were treated with three repeated injections of STZ using varying
doses. Pancreatic tissue was then removed, processed for histology, and
double stained for insulin to label cells and by the TUNEL
technique to identify apoptotic cells. The number of apoptotic
pancreatic cells correlated positively with the dose of STZ (Fig. 7). Identically treated littermates
received naive 8.3 CD8 T cells after the three injections of STZ to
assess levels of priming in the pancreatic lymph nodes. Again, priming
increased with increasing doses of STZ, and the percentage of
proliferating cells correlated with the incidence of apoptotic
pancreatic cells in situ. We conclude that low doses of STZ induce
apoptotic death of pancreatic cells. Furthermore, the magnitude of
in situ cell death correlates with CD8 T cell priming to a natural
pancreatic cell self-Ag in the pancreatic lymph nodes. To determine
whether increased cell death at an early time point could result in
the priming of diabetogenic T cells, 9-day-old NOD mice were next
treated with a single injection of STZ at a dose of 80 mg/kg. Adoptive
transfer of CFSE-labeled 8.3 CD8 T cells was performed the next day,
with analysis 4 days later. Although this treatment was toxic to the
mice, in the two of five mice that survived this treatment, 14 and 34%
of adoptively transferred cells detectable in the pancreatic lymph
nodes had proliferated. We conclude that chemically induced pancreatic
cell death can facilitate priming of diabetogenic CD8 T cells at a
time point when it does not occur naturally and before the peak of
physiologic apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells (19, 46).
Consistent with previous reports, we find that there is a developmental
regulation of priming to Ags present in the pancreata of NOD mice
(14, 15). Developmental regulation of priming exists in
both NOD mice and in nonautoimmune mouse strains and is specific for
the pancreas. Although priming of OVA-specific CD8 T cells to
transgenically expressed Ag in the pancreas does not occur in the
pancreatic lymph nodes in 10-day-old mice, it does occur in the lymph
nodes draining the kidneys, which also express the Ag
(14). This suggests organ-specific mechanisms for the
developmental regulation of Ag presentation. The priming of naive
diabetogenic CD8 T cells that we noted in NOD mice precedes the
commonly observed onset of insulitis in genetically unmanipulated mice
(47). This is also subsequent to a natural increase in the
incidence of apoptotic pancreatic cells in the pancreas that peaks
at 2 wk of age (32). Acceleration of diabetes and
insulitis in NOD mice over-expressing TNF- on cells is preceded
by increased apoptotic death of these cells (47). It is
possible that this endogenous cell death subsequently allows the
priming of naive diabetogenic CD8 T cells. We chose to explore this
possibility in subsequent experiments.
The observation that priming does not differ between male (which have
an incidence of diabetes of 
30%) and female (80% of which will
develop diabetes) NOD mice suggests that the timing of presentation of
self-Ag does not identify mice that will later develop diabetes. This
finding is consistent with the observation that the onset of insulitis
does not differ between male and female NOD mice. Therefore, the
difference between male and female NOD mice (with respect to their
equal priming ability and differing susceptibilities to diabetes
development) must reside either within the avidity/affinity of
self-reactive T cells generated or in the existence of differential
suppressor or homeostatic mechanisms to control these self-reactive T
cell populations. It recently has been shown that the progression of
benign to destructive insulitis is characterized by an increase in the
avidity/affinity of diabetogenic CD8 T cells for a mimotope
target (48). Furthermore, CD8 T cells that exhibit high
avidity for a model pancreatic Ag can be isolated from NOD mice bearing
the model Ag on the pancreas. In contrast, BALB/c mice, bearing the
same model Ag on the pancreas, retain only low-avidity CD8 T cells for
the immunodominant epitope (49). Although priming to
self-Ag must occur to initiate autoimmunity, these observations
underscore the importance of events subsequent to the priming of
autoreactive T cells in the development of a full autoimmune
phenotype.
Apoptotic cell death can enable Ag uptake by professional APCs (21, 22). Cell death can thus enhance the availability and quantity of self-Ag to be sampled and detected by the immune system. For instance, apoptotic cells derived from the gut can be transported to the T cell areas of the mesenteric lymph nodes by a subpopulation of DCs (50). Such sampling of apoptotic cells has been associated with tolerance induction. An apoptotic cell load can also promote the maturation of DCs (23). Indeed, injection of large numbers of apoptotic cells has been associated with the induction of autoimmune responses (51). Therefore, we sought to determine whether cell death can also enhance immune responses to co-administered Ag in an adjuvant-like fashion. Our results show that co-injection of dead cells with soluble Ag does indeed result in the enhanced generation of effector cytotoxic CD8 T cells specific for the co-injected Ag. The mechanism of this adjuvancy is still unclear, but the release of endogenous heat shock proteins from necrotic cells (52) and the release of cytokines and other "danger" signals from cells undergoing apoptosis may be contributory.
Whereas priming of diabetogenic CD8 T cells is negligible in 2-wk-old
animals, it is clearly detected in 4-wk-old animals. Enhanced priming
at 4 wk of age compared with 2 wk of age may be attributable to
multiple factors. Activation of APCs by interaction with CD4 T cells
and subsequent DC licensing after CD40-CD40 ligand interactions is one
such possibility (53, 54, 55). However, neither co-injection
of naive diabetogenic CD4 T cells nor co-injection of an activating
CD40 Ab resulted in enhanced or earlier CD8 T cell priming in 2-wk-old
mice (data not shown). This suggests that other mechanisms, such as Ag
availability or the presence of currently undefined signals that
promote DC maturation, are operant. Autoreactive CD8 T cell priming was
found to occur subsequent to the natural increase in cell death in
the pancreas (32). Whether this sequencing is causally
related is unclear. Because cell death can both increase sampling of
self-Ag and have adjuvant properties, we next determined whether
increasing cell death by administration of a pancreatic cell toxin
could enhance priming. STZ has been shown to increase the incidence of
pancreatic cell apoptosis both in situ (26) and in a
cell line in vitro (39). The resistance of
PARP-deficient mice to the cell cytotoxic effects of STZ
(42, 43, 44) suggests that a primary mode of action of this
toxin is via PARP-mediated NAD+ depletion, which
subsequently induces cell death. Our results demonstrate that
multiple low doses of STZ facilitate priming of diabetogenic CD8 T
cells in 2-wk-old mice, at an age when negligible priming otherwise
occurs.
Because of the chemical reactivity of STZ, it has been suggested that
one possible mechanism by which STZ may accelerate diabetes is through
the generation of neoantigens. As the 8.3 CD8 T cell recognizes a
self-Ag, our finding suggests that STZ enables and enhances the
presentation of a chemically unmodified self-Ag. This is consistent
with recent studies using mice that overexpress CD80 and a model Ag
derived from lymphocytic choriomeningitis virus on pancreatic cells
(56). The ability to enhance priming of 8.3 CD8 T cells
with STZ at and before 2 wk of age suggests that developmental
expression of the autoantigen may not be a limiting factor. Rather, the
processing and display of the Ag may be modulated. Using increasing
concentrations of STZ, we confirmed that a consequence of
administration of this agent was a corresponding increase in apoptotic
cell death in situ. The positive correlation between in situ cell
death of pancreatic cells with priming of a diabetogenic CD8 T cell
in the pancreatic lymph nodes is consistent with the hypothesis that
cell death promotes self-Ag processing. Such cell death may also
release factors that promote either T cell priming or APC maturation.
As multiple low doses of STZ accelerate immune-mediated diabetes in NOD
mice (Ref. 45 and Fig. 6
), such priming likely results in
activation and not tolerance induction. Although we detected an
increase in apoptotic cell death after low-dose STZ administration, we
cannot rule out a concomitant increase in necrotic cell death.
Tissue damage induced by activated cytotoxic T cells can promote
priming to tissue Ags (57). Autoantibodies to epitopes
generated by granzyme B released during cytotoxic cell assault are a
unifying theme in many autoimmune diseases (58). Although
these observations argue for the importance of cellular cytotoxic
mechanisms in the development of autoimmunity, these observations do
not explain how the initial wave of CTLs may become activated. It has
been proposed that increased cell death of keratinocytes after UV
exposure may be one trigger for systemic autoimmunity in individuals
predisposed to photosensitive lupus erythematosus (59, 60). Recently, the in vivo prevention of apoptosis using the
irreversible caspase inhibitor
carbobenzoxyvalylalanylaspartyl-(-O-methyl)fluoromethylketone
has been shown to decrease the severity of glomerular disease in a
mouse model of systemic lupus erythematosus (61). Our
present study is the first to link increased parenchymal cell death (in
the absence of CTL assault or viral damage) to the increased priming of
autoreactive T cells in the draining lymph nodes of affected tissues
and the subsequent acceleration of spontaneous autoimmmune diabetes.
Our findings suggest that strategies aimed at decreasing pancreatic
cell death may delay the onset of autoimmunity. Clinical trials of one
such strategy, using nicotinamide, are already underway
(62). Nicotinamide can prevent cyclophosphamide-induced
pancreatic cell apoptosis (63) and can delay or
prevent spontaneous diabetes in NOD mice (63, 64). Our
observations suggest that one mechanism contributing to the protective
actions of nicotinamide may be to diminish T cell priming by decreasing
in situ cell death. This hypothesis is currently being tested in
our laboratory. The early timing of the cell-specific T cell
priming that we have observed would suggest that such strategies, to be
optimally effective, may need to be initiated in very young predisposed
individuals several years before the clinical onset of diabetes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jan P. Dutz at the current address: The Skin Care Center, 835 West Tenth Avenue, Vancouver, British Columbia V5Z 4E2, Canada. E-mail address: dutz{at}interchange.ubc.ca
3 Abbreviations used in this paper: DM, diabetes mellitus; NOD, nonobese diabetic; STZ, streptozotocin; CM, complete medium; DC, dendritic cell; PARP, poly(A)DP ribose polymerase.
Received for publication September 19, 2001. Accepted for publication November 13, 2001.
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References |
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,
n = 11) or citrate buffer (
,
n = 5) daily for 3 days, and ability to prime 8.3
CD8 T cells was then assessed. Values represent the means ± SEM.
STZ significantly enhanced priming in 2-wk-old mice
(p = 0.007) and 4-wk-old mice
(n = 5 each; p = 0.016). FL1,
Fluorescence.
