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Department of Microbiology and Infectious Diseases, and Julia McFarlane Diabetes Research Centre, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The diabetes-prone nonobese diabetic (NOD) mouse, which spontaneously develops a form of diabetes closely resembling human IDDM, is homozygous for a unique MHC haplotye (H-2g7) that ecodes a single MHC class II molecule (I-Ag7) with unique structure and biochemical behavior (5, 6). Genetic studies have shown that, while necessary, I-Ag7 homozygosity is insufficient for development of diabetes, and that diabetogenesis requires interactions between pathogenic MHC molecules and putative products encoded on as many as 17 different non-MHC-linked loci (2, 3, 4). The mechanisms through which protective non-MHC-linked loci afford diabetes resistance, however, remain poorly understood.
It has been shown that in T cell-dependent autoimmune disorders, such as IDDM, there is a breakdown of T cell tolerance to self autoantigens (7, 8, 9, 10). Studies of T cell tolerance in nonautoimmune disease-prone mice expressing transgenic neo-Ags and neo-Ag-specific TCR transgenes have revealed that autoreactive T cells may undergo intrathymic or peripheral clonal deletion, functional inactivation (anergy), receptor desensitization, down-regulation of TCRs or associated coreceptors, immune deviation, or ignorance (11). A logical hypothesis arising from these studies is that antidiabetogenic genes encode elements that promote the induction of tolerance or ignorance of diabetogenic T cells. Unfortunately, however, the relevance of these protective mechanisms to spontaneous autoimmunity is difficult to test with currently available animal models; most of these models employ TCRs and/or antigenic systems that are either not involved in spontaneous autoimmune diseases (12, 13, 14, 15, 16, 17, 18, 19) or are not highly pathogenic in genetically susceptible backgrounds (20, 21, 22). To overcome some of these limitations, we have generated transgenic mice expressing the MHC class I (Kd)- or MHC class II-restricted (I-Ag7) ß cell-reactive TCR genes of diabetogenic CD4+ and CD8+ T cell clones isolated from pancreatic islets of diabetic NOD mice. These TCRs are involved in spontaneous diabetes, are restricted by diabetogenic MHC molecules, target native, nontransgenic ß cell autoantigens, are highly pathogenic when expressed in the diabetes-prone NOD mouse, and fail to trigger disease in diabetes-resistant mice (23, 24). These are, therefore, useful models with which to probe the mechanisms of action of protective genetic elements.
Our previous studies with I-Ag7-restricted ß
cell-reactive TCR-transgenic mice expressing antidiabetogenic MHC class
II haplotypes have shown that protective MHC class II molecules may
function by inducing the deletion of certain highly pathogenic TCRs
(23). These studies, however, also revealed that positive selection of
pathogenic TCRs in diabetes-resistant backgrounds expressing
diabetogenic MHC haplotypes does not imply autoreactivity, and
suggested that the diabetes resistance of these mice is controlled by
non-MHC-linked genetic elements. The studies presented in this work
were initiated to elucidate the mechanisms of action of non-MHC-linked
antidiabetogenic genes. This was done by following the fate of our two
ß cell-specific TCRs (4.1, I-Ag7 restricted; and 8.3,
Kd restricted) in NOR and (NOD x NOR)F1
mice, both of which are resistant to islet inflammation (insulitis) and
diabetes, despite deriving 
88% of their genome from the NOD mouse,
including two copies of the H-2g7 haplotype (25, 26). These
studies have resulted in the discovery of two mechanisms of
non-MHC-linked control of diabetogenic T cells. One of these mechanisms
is recessive and induces a reduction in the peripheral frequency of
diabetogenic CD8+, but not CD4+, T cells. The
other is dominant and is mediated by immune suppressive functions
provided by endogenous lymphocytes that do not involve the deletion,
anergy, or immune deviation of diabetogenic T cells, nor their
ignorance of ß cells, and that target pathogenic TCRs regardless of
their MHC restriction or fine antigenic specificity. These results
provide an explanation as to how non-MHC-linked gene polymorphisms can
override the susceptibility to an autoimmune disease provided by
pathogenic MHC haplotypes and demonstrate that protective
non-MHC-linked genes may selectively target specific cellular elements
in cellularly complex pathogenic autoimmune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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8.3-NOD and 4.1-NOD mice, expressing Kd- or
I-Ag7-restricted ß cell-reactive TCR transgenes, derived
from the CD8+ and CD4+ T cell clones NY8.3 and
NY4.1, respectively, have been described (23, 24). NOR/Lt mice were
purchased from The Jackson Laboratory (Bar Harbor, ME). Diabetic
8.3-NOD or 4.1-NOD mice were outcrossed with NOR/Lt mice
(H-2g7 and 
88% genetically identical to NOD
mice, including the diabetes susceptibility loci Idd2,
idd3, Idd6, Idd7, Idd8, and
Idd10, but homozygous for diabetes-resistance alleles at
Idd5, Idd9/11 and Idd13) (26) to
generate 8.3-F1 and 4.1-F1 mice, respectively.
NOR/Lt mice are also homozygous for Idd4r, but this
locus does not afford diabetes resistance in NOR mice (26). Diabetic
8.3-F1 and 4.1-F1 mice were then backcrossed
with NOR/Lt mice for up to seven generations, to generate 8.3-NOR and
4.1-NOR mice. Mice of the N5 backcross were homozygous for NOR alleles
at Idd5, Idd9/11, and Idd13 loci.
RAG-2- NOR mice were generated by backcrossing
the RAG-2 mutation of RAG-2-
C57BL/6/129 mice (a gift from F. Alt, Boston Children’s Hospital,
Boston, MA) onto the NOR background for 10 generations, followed by
intercrossing N10 heterozygotes (typed as homozygous for NOR alleles at
Idd5, Idd9/11, and Idd13) (the
RAG-2 locus lies 13 cM away from the
Idd13-containing region on chromosome 2).
RAG-2- 8.3-F1 and
RAG-2- 4.1-F1 mice were generated
by intercrossing RAG-2- 8.3-NOD or
RAG-2- 4.1-NOD mice (24) with
RAG-2- NOR or RAG-2+/-
NOR mice. 8.3-TCRß-transgenic NOR mice were produced by backcrossing
8.3-TCRß-transgenic (NOD x NOR)F1 mice (27) with
NOR mice for up to nine generations. Fas+/-
NOD.lpr mice were generated by backcrossing the
Faslpr gene of
B6.MRL-Faslpr mice (from The
Jackson Laboratory) onto the NOD background for up to seven
generations. Fas- NOD.lpr mice were
generated by intercrossing heterozygous mice of the N3 to N7
generations. Mice were screened for inheritance of the transgenes and
mutated and wild-type RAG-2 or Fas alleles by PCR
of tail DNA. NOR/NOD polymorphisms at Idd5,
Idd9/11, and Idd13 loci were determined by PCR
using primers for D1Mit46 (Idd5), D4
Mit11 (Idd9/11), D2Mit144, and
D2Mit490 (Idd13) (26), in the absence (for
D2Mit490) or presence of [32P]dCTP (for all
other markers). D2 Mit490 lies between the Idd13
(3cM) and RAG-2 loci (10 cM). PCR products were resolved in
4% NuSieve agarose gels (D2Mit490) or in 7 M urea/6%
acrylamide gels (other markers). All mice were housed in a specific
pathogen-free facility.
Diabetes
Diabetes was assessed by measuring urine glucose levels with
Diastix strips (Miles, Ontario, Canada) twice weekly. Animals were
considered diabetic after two consecutive readings 
3+.
Cell lines, Abs, and flow cytometry
L1210-Fas+ and L1210-Fas- cells were
provided by Dr. P. Goldstein (Centre National de la Recherche
Scientifique, Marseille, France). NIT-1 NOD insulinoma cells were a
gift from Dr. E. Leiter (The Jackson Laboratory).
L929-Kd transfectants were provided by Dr. J.
Yewdell (National Institutes of Health, Bethesda, MD). Hybridomas
secreting mAbs GK1.5 (anti-CD4) and 53-6.7 (anti-CD8) were
obtained from the American Tissue Culture Collection (Manassas, VA). A
hybridoma secreting the Vß8.1/8.2-specific mAb KJ16 was a gift from
P. Marrack (National Jewish Center, Denver, CO). Anti-Lyt-2 (CD8
)
phycoerythrin (53-6.7), anti-L3T4 FITC (IM7), anti-L3T4 biotin
(CD4) (H129.19), anti-CD2 biotin (RM2-5), anti-CD5 (53-7.3)
biotin, anti-CD11a biotin (M17/4), anti-CD24 biotin (M1/69),
anti-CD28 biotin (37.51), anti-CD44 FITC (IM7), anti-CD45RB
FITC (23G2), anti-L-selectin biotin (CD62L) (Mel-14), anti-CD69
biotin (H1.2F3), anti-Vß8.1/8.2 FITC (MR5-2), and
anti-H-2Kd FITC (SF1-1.1) were purchased from
PharMingen (San Diego, CA). Anti-IL-2R FITC (CD25) (AMT13) was
purchased from Cedarlane Laboratories (Hornby, Ontario, Canada). Mouse
IgG-absorbed FITC- or biotin-conjugated goat anti-rat IgG, and
FITC-conjugated goat anti-mouse IgG were obtained from Caltag (San
Francisco, CA) and Becton Dickinson (San Jose, CA), respectively.
Streptavidin-PerCP (peridinin chlorophyll protein) was obtained
from Becton Dickinson. Goat polyclonal anti-granzyme B IgG was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Guinea pig
polyclonal anti-swine insulin antisera and biotinylated
anti-guinea pig IgG were obtained from Dako (Carpinteria, CA).
Biotinylated swine anti-goat IgG was obtained from Cedarlane
Laboratories. Thymi, spleens, and islet-derived T cell lines were
analyzed by three-color flow cytometry using a FACScan (Becton
Dickinson), as described by Verdaguer et al. (27).
Proliferation and limiting dilution assays
Splenocytes from 8.3-NOD mice or 4.1-NOD were depleted of
CD4+ or CD8+ T cells, respectively, using
anti-CD4 mAb (GK-1.5)- or anti-CD8 mAb (53-6.7)-coated magnetic
beads (27), adjusted to 104 CD8+ or
CD4+ T cells/100 µl of complete medium (CM: RPMI 1640
media containing 10% heat-inactivated FBS (Life Technologies, Grand
Island, NY), 50 U/ml penicillin, 50 µg/ml streptomycin (Flow
Laboratories, McLean, VA), and 50 µM 2-ME (Sigma, St. Louis, MO)) and
incubated, in triplicate, with 
-irradiated (3000 rad) islet cells
(3–100 x 103/well) in 96-well tissue culture plates
for 3 days at 37°C in 5% CO2. Cultures of
4.1-CD4+ T cells received 105 irradiated
splenocytes from nontransgenic NOD mice, as feeders. Cultures were
pulsed with 1 µCi of [3H]thymidine during the last
18 h of culture and harvested. Thymidine incorporation was
measured by scintillation counting, and specific proliferation was
calculated as described previously (27). Proliferation assays using
plate-bound anti-Vß8.1/8.2 mAb (KJ16) were done as described
earlier (24), except that IL-2 was omitted. To determine the frequency
of ß cell-reactive CD8+ T cells, 12 replicate cultures of
four 10-fold serial dilutions of splenocytes
(101–105 cells/well) were stimulated with
irradiated NOD islets (8/well) for 4 days, expanded in rIL-2 for 10
days, and restimulated with islets and rIL-2. The resulting cultures
were split and challenged with NIT-1 or L929-Kd
cells for 24 h, and the supernatants were collected to measure the
contents of TNF-. Cultures that secreted TNF- in response to
NIT-1, but not L929-Kd, cells were considered to contain
ß cell-reactive CD8+ T cells (24). To determine the
frequency of ß cell-reactive CD4+ T cells, four 12
replicate cultures of 10-fold serial dilutions of CD8+ T
cell-depleted splenocytes (101–105 cells/well)
were stimulated with 2.5 x 103 irradiated islet cells
and 2 x 105 irradiated NOD splenocytes for 4 days,
followed by rIL-2 for 10 days and a second restimulation with islet
cells, splenocytes, and rIL-2 (27). Control plates received rIL-2 and
splenocytes, but not islet cells. Growth was scored microscopically.
Frequencies were calculated with Poisson statistics.
Generation of spleen- and islet-derived CD8+ T cell lines and clones
CD4+ T cell-depleted spleen cells were stimulated with irradiated NOD islets for 3 to 4 days, and the activated cells expanded with 0.5 U/ml rIL-2 (Takeda, Osaka, Japan) for 10 to 14 days. Growing cultures were assayed for serine esterase content (27) and used as effectors in cytotoxicity assays, or restimulated twice with irradiated NOD islets and rIL-2, to generate ß cell-specific CD8+ T cell lines. Islet-derived CD8+ T cell lines and clones were generated as described by Verdaguer et al. (24). Growing clones were assayed within 15 days of cloning for serine esterase content, and serine esterase+ clones were expanded by stimulation with irradiated NOD islets and rIL-2. Some experiments employed islet cell suspensions (containing endocrine cells and infiltrating T cells) as effectors in cytotoxicity assays.
51Cr release assays
Target cells (L1210-Fas+, L1210-Fas-,
and single NOD or NOR islet cells) (5 x 105) were
labeled with 51Cr sodium chromate (DuPont/NEN, Boston, MA)
and seeded at 104 cells per 100 µl/well. Some experiments
(those in Fig. 5
F) employed islet cells derived from islets
that had been preincubated overnight with supernatants from 3-day-old
islet-derived CD8+ T cell lines, or with rIL-1
(103 U/L), to increase their susceptibility to Fas-mediated
cytotoxicity (28). Effector cells (islet-infiltrating T cells,
islet-derived serine esterase- CD8+ T cell
lines, or islet-derived serine esterase+ CD8+ T
cell clones; 100 µl) were added to each well, in duplicate, at
several E:T ratios. Plain medium or 1% Triton X-100 was added to sets
of target cells for examination of spontaneous and total cell lysis,
respectively. The plates were incubated at 37°C for 8 h, and the
supernatants were collected at this point for determination of
specific 51Cr release (24).
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mRNAs
The TCR-chain cDNA molecules of islet-stimulated splenic
CD8+ T cells from 8.3-TCRß-transgenic NOD and
8.3-TCRß-transgenic NOR mice were amplified by anchor PCR, cloned,
and sequenced, as previously described (27).
Cytokine RT-PCR
Total cellular RNA from islet-derived, CD4+ T cell-depleted T cell lines was reverse transcribed using oligo(dT)12–18 (Life Technologies) as a primer, and the resulting cDNAs were amplified by PCR (27).
In vitro and in vivo cytokine secretion
Splenic CD4+ T cells (2 x
104/well) were incubated with -irradiated NOD islet
cells (105/well) and splenocytes (105/well) in
96-well plates for 48 h at 37°C in 5% CO2. The
supernatants (100 µl/well) were assayed for IL-2, IL-4, IFN-,
and/or TGF-ß1 content by ELISA using commercially available kits
(Genzyme Diagnostics, Cambridge, MA). Determination of intraislet
cytokine content was done as described in Cameron et al. (29), with
modifications. Briefly, purified islets from nondiabetic mice were
homogenized in PBS containing protease inhibitors (PMSF (1 mM),
leupeptin (20 µM), pepstatin A (10 µM), sodium azide (0.2%), and
EDTA (100 µM)), and centrifuged at 10,000 rpm to remove debris. The
supernatants were adjusted at 200 µg of total protein/ml, and 100
µl of each sample was used in duplicate to measure the cytokine
content by ELISA.
Histopathology and immunohistochemistry
Pancreata (one-half) were fixed in formalin, embedded in paraffin, sectioned at 4.5 µm, stained with hematoxylin and eosin, and examined for inflammation. The degree of insulitis was evaluated by scoring 15–30 islets/mouse using previously described criteria (24). The second half of each pancreas was snap frozen in liquid nitrogen, immersed in OCT, sectioned at 6–7 µm, fixed in cold acetone for 10 min, incubated with hydrogen peroxide to block endogenous peroxidase activity, and stained with anti-CD4 (GK1.5) and anti-CD8 (53-6.7) mAbs, followed by biotinylated anti-rat Ig Abs and horseradish peroxidase (HRP)-streptavidin conjugate (Dimension Laboratories, Mississauga, ON, Canada). The slides were developed with diaminobenzidine (DAB; Sigma).
To determine the percentages of granzyme B+ cells in islets, mice were euthanized and perfused with PBS and 4% paraformaldehyde in PBS. The pancreas of each mouse was then fixed overnight in 4% paraformaldehyde at 4°C, immersed in 20% sucrose overnight at 4°C, snap frozen in liquid nitrogen, and processed for immunopathology, as described above. Tissue sections were incubated with goat polyclonal anti-granzyme B IgG, biotinylated swine anti-goat IgG, and HRP-streptavidin; developed with DAB; and counterstained with hematoxylin.
In vivo bromodeoxyuridine (BrdU) labeling
Mice were given two i.v. injections of 200 µl of a 4 mg/ml solution of 5-bromo-2'-deoxyuridine (BrdU; Calbiochem, La Jolla, CA) 4 h apart. The pancreas and spleen of each mouse were collected 12 h later. Quantitation of the percentage of islet-infiltrating cells and splenocytes incorporating BrdU was done on frozen tissue using a BrdU staining kit (Calbiochem).
Statistical analyses
Statistical analyses were performed using Mann-Whitney Uand 
2 tests and by simple regression.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown that 8.3-CD8+ T cells are highly
diabetogenic in 8.3-NOD mice (24). Since these cells require the
assistance of endogenous CD4+ T cells to accelerate
diabetes (24), we reasoned that studies with the 8.3-TCR would be the
most informative. To test the hypothesis that non-MHC-linked genes
control the diabetogenic activity of 8.3-CD8+ T cells, we
first crossed 8.3-NOD mice with NOR/Lt mice to generate 8.3-(NOD
x NOR)F1 (8.3-F1) mice. NOR/Lt mice are
homozygous for the prodiabetogenic H-2g7 haplotype
and are 88% genetically identical to NOD/Lt mice, but unlike NOD/Lt
mice and the 8.3-NOD mice used for these studies (homozygous for NOD
alleles at the three Idd regions that provide diabetes
resistance in NOR mice), are homozygous for antidiabetogenic alleles at
Idd5, Idd9/11, and Idd13 loci. The
incidence of diabetes in 8.3-F1 mice was significantly
lower than in 8.3-NOD mice, both in females and in males
(p < 0.0002) (Fig. 1). These differences in diabetes
incidence between 8.3-NOD and 8.3-F1 mice were not due to
genetic heterogeneity in the 8.3-NOD mouse population used as donor of
the transgenes for this study, since the incidence of diabetes in the
8.3-TCR-transgenic littermates of the transgene donors was similar to
the current incidence of diabetes in our 8.3-NOD colony (78% vs 71%
in females, and 60% vs 52% in males, respectively). As expected, none
of the 20 female nontransgenic F1 littermates that were
followed developed diabetes (data not shown). We then backcrossed the
8.3-TCR of a diabetic 8.3-F1 male mouse onto the NOR/Lt
background for up to seven generations, to generate 8.3-NOR mice.
Cohorts of mice from each backcross were followed for diabetes
development. As expected, female 8.3-NOR mice of the N5-N7 backcrosses
(homozygous for NOR alleles at Idd5, Idd9/11, and
Idd13) also developed diabetes less frequently than female
8.3-NOD mice (p < 0.0002) (Fig. 1).
Surprisingly, however, 8.3-NOR males of the N5-N7 backcrosses displayed
an increased incidence of diabetes when compared with
8.3-F1 (but not 8.3-NOD) mice (p <
0.02) (Fig. 1). In both female and male 8.3-NOR mice (N5-N7
generations), there was a decrease in the average age at onset of the
disease when compared with 8.3-NOD and/or 8.3-F1 mice
(37 ± 22 days vs 43 ± 26 days and 68 ± 31 days for
females, p < 0.04; 30 ± 11 days vs 83 ± 35
and 41 ± 15 days for males, p < 0.0001). No
obvious differences in disease incidence or age at diabetes onset were
noted between N2, N3, or N4 mice and N5-N7 mice. In males, for example,
the incidence and age at onset of diabetes for each generation were:
N2, 2/4 mice at 39 ± 6 days; N3, 3/11 mice at 34 ± 6 days;
N4, 2/5 mice at 35 ± 4 days. It therefore appears that NOR/Lt
mice bear one or more chromosomal regions that, either alone or in
combination, afford dominant protection from 8.3-CD8+ T
cell-induced diabetes in 8.3-F1 mice. The fact that this
dominant protective effect is lost in male 8.3-NOR mice suggests that
NOR/Lt mice also bear recessive prodiabetogenic genes that in males,
but not females, can override the antidiabetogenic function of these
dominant protective elements.
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Three-color cytofluorometric studies of thymocytes revealed that
the resistance of 8.3-F1 and 8.3-NOR mice to
8.3-CD8+ T cell-induced diabetes was not a result of
deletion or abnormal maturation of 8.3-CD8+ thymocytes. The
thymi of these mice contained similar numbers of thymocytes and similar
percentages of the four major thymocyte subsets as the thymi of 8.3-NOD
mice (Fig. 2A). Likewise, no
differences were found between the individual thymocyte subsets of all
of these mice with respect to cell surface levels of the transgenic TCR
(Fig. 2A) and several differentiation markers, including
CD5, MHC class I (Kd), CD24, CD44, and CD69 (data not
shown).
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B); however, the spleens (and, to a lesser extent, the
lymph nodes) of 8.3-NOR mice contained significantly fewer
CD8+ T cells than the spleens (or lymph nodes) of 8.3-NOD
mice (Fig. 2B, and data not shown)
(p < 0.0005). This reduction in the percentage
of splenic CD8+ T cells in 8.3-NOR mice (defined as a
splenic CD4+:CD8+ T cell ratio greater than the
average plus 2 SDs of the 8.3-NOD values) segregated as a recessive
trait: it occurred in none of 6 F1 mice, in 4 of 10 N2
mice, and in 11 of 13 N4-N5 mice (8.3-F1 vs 8.3-N2,
p < 0.009; 8.3-N2 vs 8.3-N4/N5, p <
0.0001). This phenotype was not due to CD8 coreceptor down-regulation
by 8.3-CD8+ T cells, since virtually all of the
CD4-CD8- splenic cells of these mice were
Vß8.1- (data not shown), and could not be accounted for
by disproportionate expansion of 8.3-CD8+ T cells in the
spleens of 8.3-NOD vs 8.3-NOR mice, since the splenic CD8+
T cells of 8.3-NOD (and 8.3-NOR) mice did not express activation
markers ((24) and data not shown). Furthermore, the splenic
periarteriolar lymphoid sheaths of 8.3-NOD mice contained very few
actively proliferating cells, as determined by immunopathologic
analyses of BrdU-injected mice, and the number of BrdU-incorporating
cells in the spleens of 8.3-NOD and 8.3-NOR mice was similar (data not
shown). Interestingly, this reduction in the percentage of splenic
CD8+ T cells was accompanied by a reduction in the overall
ability of the splenic CD8+ T cells of 8.3-NOR vs 8.3-NOD
mice to proliferate in response to islet stimulation (Fig. 3A). Subsequent limiting
dilution (Fig. 3B) and proliferation assays (Fig. 3C) using islet Ag or a plate-bound anti-Vß8.1 mAb as
stimuli, respectively, demonstrated that the lower proliferative
responsiveness of 8.3-NOR-derived CD8+ T cells to islet
stimulation was not due to anergy, but rather to a reduction in the
peripheral frequency of ß cell-reactive CD8+ T cells
(1/70–90 vs 1/17–30 CD8+ T cells in 8.3-NOR and
8.3-NOD mice, respectively). Paradoxically, however, 8.3-NOR mice
developed an accelerated onset (but not an increased incidence) of
diabetes. This suggested that this reduction in the peripheral
frequency of ß cell-reactive CD8+ T cells in 8.3-NOR mice
might increase with age, and that it would have to reach a certain
threshold to be able to afford diabetes protection. Time course studies
confirmed that deletion of 8.3-CD8+ T cells in 8.3-NOR mice
was indeed an age-dependent phenomenon: contrary to what happened in
8.3-NOD mice, in which the percentage of splenic CD8+ T
cells increased with age (r = 0.682, p
< 0.0001), the percentage of splenic CD8+ T cells in
8.3-NOR mice did not increase as the mice grew older (r
= 0.266). Studies with nontransgenic and TCRß-transgenic NOR mice
confirmed that the deleting phenotype of 8.3-NOR mice was not mediated
by an endogenous superantigen binding to the 8.3-TCRß-chain; the
percentages of Vß8.1/8.2+CD8+ T cells in the
spleens of NOR and 8.3-TCRß-transgenic NOR mice were similar to those
seen in the spleens of NOD and 8.3-TCRß-transgenic NOD mice,
respectively (Fig. 3D). Taken together, these results
suggested that: 1) 8.3-CD8+ T cells undergo partial
deletion in 8.3-NOR mice; and 2) this phenotype segregates as a
recessive trait. Our next set of experiments thus focused on
investigating this phenotype further. The dominant diabetes resistance
observed in 8.3-F1 mice, which does not involve T cell
deletion, will be addressed further below.
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-chains are undetectable in 8.3-TCRß-transgenic NOR mice
To confirm that the reduction in the peripheral frequency of
8.3-CD8+ T cells in 8.3-NOR mice was not an artifact of the
high peripheral frequency of clonotypic 8.3-CD8+ T cells,
we investigated whether this phenotype also occurred in single-chain
8.3-TCRß-transgenic NOR mice. We chose to use these mice to address
this issue for several reasons: 1) unlike 8.3-TCRß-transgenic NOD
mice, 8.3-TCRß-transgenic NOR mice do not develop diabetes (0%
incidence, unpublished observations); 2) when compared with 8.3-NOD
mice, 8.3-TCRß-transgenic NOD mice only have a minor increase in the
peripheral frequency of ß cell-reactive CD8+ CTL (27);
and 3) the splenic CD8+ T cells of 8.3-TCRß-transgenic
NOD mice display a highly heterogeneous TCR repertoire, yet most of
the islet-associated CD8+ T cells in acutely diabetic
animals bear endogenously derived TCR-chains identical to the
8.3-TCR-chain, indicating that expression of the 8.3-TCRß
transgene fosters the maturation of some 8.3-CD8+ T cells
(27). To investigate whether 8.3-TCRß-transgenic NOR mice also have a
reduction in the peripheral frequency of 8.3-CD8+ T cells
when compared with 8.3-TCRß-transgenic NOD mice, we compared the
endogenous TCR repertoire of ß cell-reactive CD8+ T
cells in 8.3-TCRß-transgenic NOR and 8.3-TCRß-transgenic NOD mice.
To do this, we stimulated splenic CD8+ T cells from several
8.3-TCRß-transgenic NOD and 8.3-TCRß-transgenic NOR mice with NOD
islets in the presence of rIL-2 and sequenced multiple anchor
PCR-generated TCR cDNAs from each line. As shown in Fig. 3E, the 8.3-TCR sequence was the predominant TCR
sequence within each of the three different 8.3-TCRß-transgenic NOD
lines studied (46/60 cDNAs). In contrast, while some of the 49 TCR
cDNAs derived from four 8.3-TCRß-transgenic NOR lines encoded
homologous junctional (CDR3) amino acid sequences, none of them encoded
the 8.3-TCR-chain or the 8.3-TCR-CDR3 region sequence. These
results therefore demonstrated that the reduction in the peripheral
frequency of 8.3-TCR-bearing CD8+ T cells in 8.3-NOR mice
was not an artifact of transgenesis. Furthermore, these data suggested
that the putative CD8+ T cell-deleting element(s) of NOR
mice preferentially target(s) ß cell-reactive
Vß8.1+CD8+ T cells bearing the highly
diabetogenic 8.3-TCR-chain, as opposed to all ß cell-reactive
CD8+ T cells regardless of TCR usage.
Diabetes resistance in 8.3-NOR mice is not associated with insulitis resistance or immune deviation
To investigate whether the diabetes resistance of 8.3-NOR mice was
associated with resistance to insulitis and/or local immune deviation,
we determined whether nondiabetic 8.3-NOR mice (at least 1 SD older
than the average age at onset of diabetes in these mice; <2.5%
chances of ever becoming diabetic) developed insulitis, and whether the
insulitis lesions of these mice were quantitatively and/or
qualitatively similar to those of nondiabetic 8.3-NOD mice.
Histopathologic and RT-PCR studies revealed that 8.3-NOR mice had
severe insulitis (Fig. 4A),
that the insulitis lesions of these mice had
CD4+:CD8+ T cell ratios similar to those seen
in 8.3-NOD mice (Fig. 4B), and that the CD8+ T
cells derived from these lesions displayed cytokine profiles similar to
those derived from the insulitis lesions of 8.3-NOD mice (Fig. 4C). These cytokine profiles were confirmed by measuring the
intraislet content of IL-2, IL-4, and IFN- in 8.3-NOD and 8.3-NOR
mice: the insulitis lesions of both types of mice contained IFN-
(120–1200 pg/mg), but undetectable levels of IL-4 and IL-2 (data not
shown) (note that IL-2 was barely detectable by RT-PCR). These data
demonstrated that 8.3-CD8+ T cells do not undergo immune
deviation in 8.3-NOR mice, and that they accumulate in the islets of
these mice quite efficiently.
|
Cytofluorometric studies of 8.3-F1 mice indicated that
their diabetes resistance could not be accounted for by deletion of
8.3-CD8+ T cells (Figs. 1 and 2). Our next set of
experiments focused on attempting to elucidate the mechanism(s)
underlying the genetically dominant diabetes resistance of these mice.
Since the pancreatic islets of these mice were severely infiltrated and
contained abundant CD8+ T cells (data not shown), we
considered three possible mechanisms of diabetes resistance in these
mice: 1) 8.3-CD8+ T cells undergo anergy in situ; 2)
8.3-CD8+ T cells fail to differentiate into ß
cell-cytotoxic T cells (CTLs); and/or 3) the local 8.3-CD8+
CTLs are unable to kill NOR ß cells because these cells do not
express the target autoantigen, or are resistant to CTL-induced
apoptosis. These questions were investigated by studying nondiabetic
mice at ages beyond which their chances of developing diabetes were
<5% (>10 wk).
To determine whether 8.3-CD8+ T cells of 8.3-F1
mice undergo anergy in situ, we compared the percentage of
proliferating cells (incorporating exogenous BrdU) in islets of
nondiabetic 8.3-F1 and 8.3-NOD mice. As shown in Fig. 5A, no significant differences
were noted between these mice at any of three different age points. To
determine whether these cells differentiated into CTLs in situ, we
compared the percentages of insulitic cells from nondiabetic
8.3-F1 and 8.3-NOD mice that expressed granzyme B (a marker
of cytotoxic granule content), as detected by immunohistochemistry. As
shown in Fig. 5B, no differences were observed in these
studies. The ability of 8.3-NOR CD8+ T cells to
differentiate into CTL in situ was confirmed by comparing the
percentages of CD8+ T cell clones containing serine
esterase activity that could be isolated from islets of these two types
of mice. As shown in Fig. 5C, islet-derived CD8+
T cell lines from 8.3-F1 mice contained as many serine
esterase+ clones as lines derived from 8.3-NOD mice.
Differentiation of these islet-derived clones into CTL was not an
artifact of in vitro stimulation, because none of 41 clones derived
from splenic CD8+ T cells of 8.3-NOD mice using the same
stimulation protocol expressed serine esterase activity (Fig. 5C). Subsequent cytotoxicity assays using serine
esterase+CD8+ CTL clones from
8.3-F1 and 8.3-NOD mice demonstrated that the diabetes
resistance of 8.3-F1 mice could not be accounted for by an
inability of 8.3-F1-derived 8.3-CD8+ T cells to
kill ß cells, or by an inherent resistance of NOR ß cells to
perforin-based cytotoxicity (Fig. 5D); NOR ß cells were at
least as, if not more, susceptible to CTL-induced lysis as NOD ß
cells.
Since only a small percentage of the insulitic T cells of
8.3-F1 and 8.3-NOD mice contained cytotoxic granules, we
reasoned that 8.3-CD8+ T cells might primarily kill ß
cells via the Fas pathway, as proposed for other CD8+ T
cells (30). In that case, the diabetes resistance of 8.3-F1
mice might result from the relative inability of the insulitic
CD8+ T cells of 8.3-F1 vs 8.3-NOD mice to
effect Fas-dependent cytotoxicity and/or from resistance of NOR ß
cells to Fas-induced apoptosis. To investigate this, we first
determined whether 8.3-CD8+ T cells within islet cell
suspensions from both types of mice could kill Fas+
targets. As shown in Fig. 5E, cells from both types of mice
killed Fas+ (but not Fas-) fibroblasts with
similar efficiency. We then tested the ability of 4–5-day-old serine
esterase- islet-derived T cell lines from 8.3-NOD mice to
kill L1210-Fas+ and L1210-Fas- fibroblasts,
and NOD, NOR, and NOD.lpr (Fas-) ß cells,
both in the absence and presence of cytokines that potentiate
Fas-mediated cytotoxicity (28). As shown in Fig. 5F, these
lines efficiently killed L1210-Fas+ cells,
Fas+NOD islet cells, and Fas+NOR islet cells,
but not L1210-Fas- fibroblasts or Fas- NOD
ß cells; thus, 8.3-F1 CD8+ CTLs can kill
Fas-expressing targets, and NOR and NOD ß cells are equally
susceptible to Fas-mediated cytotoxicity.
Diabetes resistance in 4.1-(NOD x NOR)F1 and 4.1-NOR mice
The results of these experiments suggested that the genetically
dominant resistance of (NOD x NOR)F1 mice to diabetes
was mediated by a novel mechanism. This prompted us to ask one
fundamental question: does this mechanism selectively target
8.3-CD8+ T cells, or does it also target other highly
diabetogenic T cells regardless of phenotype or antigenic specificity?
To address this question, we followed the fate of another highly
pathogenic, but I-Ag7-restricted, ß cell-specific TCR
(4.1-TCR) (23) in (NOD x NOR)F1 (4.1-F1)
and NOR (4.1-NOR) mice derived from a diabetic 4.1-NOD mouse.
Interestingly, both 4.1-F1 and 4.1-NOR mice (N2-N7
generations) displayed much lower incidences of diabetes than 4.1-NOD
mice (p < 0.0001 for both females and males)
(Fig. 6A). No obvious
differences in disease incidence were noted between 4.1-TCR-transgenic
mice of the N2-N4 and N5-N7 backcrosses (the latter typed as homozygous
for NOR alleles at Idd5, Idd9/11, and
Idd13 loci) (females, 4/13 and 2/11, respectively; males,
1/10 and 0/7, respectively). The 4.1-F1 and 4.1-NOR mice
that developed diabetes did so slightly later than 4.1-NOD mice (i.e.,
60 ± 26 vs 76 ± 49 vs 47 ± 21 days, respectively). As
expected, the incidence of diabetes in the nontransgenic F1
littermates that were followed was 0% at 32 wk (data not shown). Thus,
like 8.3-F1 mice, 4.1-F1 mice also display
dominant resistance to diabetes.
|
We then investigated whether the diabetes resistance of
4.1-F1 mice was also dissociated from known forms of T cell
tolerance. Cytofluorometric, functional, and pathologic studies of
nondiabetic mice (studied at ages beyond which the chances of
developing diabetes were <9%; see Fig. 6) demonstrated that their
diabetes resistance was not due to deletion, anergy, or ignorance; the
4.1-CD4+ T cells of 4.1-F1 (not shown),
4.1-NOR, and 4.1-NOD mice matured similarly (Fig. 6B),
proliferated equally well in response to NOD (and NOR, data not shown)
islet cell stimulation in vitro (Fig. 6C), contained similar
numbers of ß cell-reactive CD4+ T cells (Fig. 6D), and had similar insulitogenic activity (Fig. 6, E and F). As in 8.3-F1 and 8.3-NOR
mice, there was no evidence of immune deviation in the periphery of
nondiabetic 4.1-F1 or 4.1-NOR mice (i.e., differentiation
of 4.1-CD4+ T cells into nonpathogenic Th2 cells): although
there was considerable variation in the levels of IFN- secreted by
CD4+ T cells from different 4.1-NOD mice, the splenic
4.1-CD4+ T cells of >11-wk-old 4.1-F1 and
4.1-NOR mice (<4% chances of ever becoming diabetic) consistently
secreted IL-2 and/or IFN-, but not IL-4, in response to islet
stimulation (Table I).
|
These results did not rule out the possibility that
4.1-CD4+ T cells undergo immune deviation in situ.
Alternatively, since induction of Th2 responses against ß cell
autoantigens can inhibit diabetes progression in prediabetic NOD mice
and disease recurrence in islet-grafted NOD mice (31), and diabetes
does not develop in IL-4-treated mice (32), it was also possible that
the genetic resistance of 4.1-F1 or 4.1-NOR mice was due to
recruitment of IL-4-secreting cells to islets. To address these
possibilities, we quantitated the IL-2, IL-4, and IFN- content of
islet extracts from nondiabetic mice of all three mouse strains by
ELISA. These experiments revealed that while the islets of nondiabetic
4.1-NOD mice contained significant levels of both IL-4 and IFN-, the
islets of nondiabetic 4.1-F1 and 4.1-NOR mice (>11 wk of
age; <4% chances of developing diabetes) contained IL-2 and/or
IFN-, but undetectable levels of IL-4 (Table II). Islets from 4.1-F1 and
4.1-NOR mice contained lower levels of IFN- than islets from 4.1-NOD
mice, but the significance of these differences is unclear (Table II).
Next, since TGF-ß1 has been shown to have suppressive effects on the
action of Th1 and Tc1 cells in vitro and in vivo, and can
suppress the development of experimental autoimmunity in mice (33, 34, 35),
it was also important that we determined whether the diabetes
resistance of 4.1-F1 mice was associated with recruitment
of TGF-ß1-producing cells to islets. The levels of TGF-ß1 in islets
of seven different 4.1-F1 and 4.1-NOR mice, however, were
barely detectable (1.8 ± 2 pg/mg) (data not shown). The mechanism
that prevents diabetogenesis in F1 (and NOR) mice does not
therefore promote local immune deviation and does not involve the
recruitment of IL-4- or TGF-ß1-producing cells to islets.
|
The remarkable similarity between the results of studies with
4.1-F1 and 8.3-F1 mice prompted us to
investigate whether the diabetes resistance of these mice was due to a
form of immune suppression involving suppressor lymphocytes other than
Th2 (IL-4-producing) or Th3 (TGF-ß1-producing) cells. Since the
diabetogenic potential of 4.1-CD4+ and 8.3-CD8+
T cells in NOD mice is not negatively affected by the presence of
endogenous (nontransgenic) lymphocytes able to compete with the
transgenic T cells for Ag recognition (24), this possibility could be
tested by comparing the natural history of diabetes in
RAG-2- 8.3-F1,
RAG-2- 8.3-NOD, RAG-2-
4.1-F1, and RAG-2- 4.1-NOD mice;
these mice cannot rearrange Ig or endogenous TCR genes and thus express
monoclonal TCR repertoires (24). Interestingly,
RAG-2- 8.3-F1 and
RAG-2- 4.1-F1 mice developed
diabetes almost as frequently, and as early as
RAG-2- 8.3-NOD and
RAG-2- 4.1-NOD mice, respectively (Fig. 7). As expected, the incidence of
diabetes in RAG-2+/- 4.1-F1
littermates was much lower: only 2 of 11
RAG-2+/- 4.1-F1 females (18%) and
none of 8 RAG-2+/- 4.1-F1 males
(0%) developed diabetes (p < 0.015 vs
RAG-2- 4.1-NOD females, and p
< 0.004 vs RAG-2- 4.1-NOD males) (Fig. 7). The
same trend was noted in RAG-2+/-
8.3-F1 mice; only 1 of 3 females, 33%, and none of 4
males, 0%, developed diabetes, incidences comparable with those seen
in RAG-2+/+ 8.3-F1 mice (Fig. 1). It
should be pointed out that direct comparison of the incidences of
diabetes in RAG-2+/- and
RAG-2-/- 8.3-F1 mice is
inappropriate in the context of this study, since the diabetogenic
potential of 8.3-CD8+ T cells is dramatically reduced in
the absence of CD4+ T cells bearing endogenous TCRs (24).
Taken together, these findings demonstrated that the diabetes
resistance of 8.3-F1 and 4.1-F1 mice is, at
least in part, the result of a form of lymphocyte-mediated suppression
not involving IL-4 or TGF-ß1 that targets diabetogenic
CD8+ and CD4+ T cells regardless of their fine
antigenic specificity and MHC restriction.
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reduction in the peripheral frequency of 8.3-CD8+ T
cells that was observed in 8.3-NOR, but not 8.3-F1, mice
increased with age and was not mediated by endogenous superantigens
binding to the transgenic TCRß-chain. Studies of
8.3-TCRß-transgenic NOR mice revealed that this phenotype was not an
artifact of the high peripheral frequency of 8.3-CD8+ T
cells in 8.3-TCR-transgenic mice when compared with nontransgenic
animals, and that it preferentially targeted CD8+ T cells
expressing TCR-chains with CDR3 sequences identical to those of the
CTL clone donating the 8.3-TCRß transgene, rather than all
autoreactive CD8+ T cells regardless of TCR usage. While
the underlying mechanisms remain unclear, we suspect that this
reduction in the peripheral frequency of 8.3-CD8+ T cells
in 8.3-NOR mice is caused by deletion of 8.3-CD8+ T cells
in the periphery. Since initiation of diabetogenesis in nontransgenic
NOD mice requires CD8+ T cells (36, 37, 38), and the 8.3-TCR
uses a TCR-CDR3 sequence that is highly homologous to TCR-CDR3
sequences used by many NOD islet-derived ß cell-cytotoxic
CD8+ T cells (27, 39), it is possible that some of the
diabetes resistance of nontransgenic NOR mice results from their
ability to delete the most pathogenic of all autoreactive
CD8+ T cells, rather than all autoreactive CD8+
T cells regardless of their pathogenicity. The incomplete penetrance of
8.3-CD8+ T cell deletion and diabetes resistance in 8.3-NOR
mice does not argue against this view; the high frequency of
8.3-CD8+ T cells in 8.3-NOR mice may overwhelm the mice’s
deleting machinery, particularly since NOR mice also appear to bear
recessive prodiabetogenic genes. The complete absence of 8.3-TCR
sequences in TCR cDNA libraries generated from islet-reactive T cell
lines of 8.3-TCRß-transgenic NOR mice, which have a much lower
frequency of ß cell-reactive CD8+ T cells and do not
develop diabetes, supports this interpretation of the data. Whatever
the relative role of this phenomenon in the genetic resistance of
NOR/Lt mice to spontaneous IDDM, these results demonstrate the
existence of non-MHC-linked genetic elements other than endogenous
superantigens that can control the fate of pathogenic autoreactive
CD8+ T cells in the periphery. Importantly, these elements
target diabetogenic CD8+ T cells while sparing diabetogenic
CD4+ T cells.
While we do not yet know the nature of these genetic elements,
interpretation of our findings vis-à-vis the results of previous
genetic studies provides some clues. It has been shown that IDDM
resistance in (NOD x NOR)F2 mice segregates with
Idd5, Idd9, and Idd13 (26). Although
Idd5r, linked to ctla4 and
cd28 in chromosome 1, is associated with increased
susceptibility of T cells to cyclophosphamide-induced apoptosis in vivo
(40), two lines of evidence suggest that this reduction in the
peripheral frequency of 8.3-CD8+ T cells in 8.3-NOR mice
and the Idd5r-encoded susceptibility to
cyclophosphamide-induced apoptosis are unrelated phenomena: 1) the
putative gene within the Idd5r region of NOR mice
that controls apoptosis susceptibility/resistance is of NOD origin
(40); and 2) 8.3-CD8+ T cells from 8.3-NOR and 8.3-NOD mice
show equal susceptibility to reactivation-induced apoptosis in response
to several different stimuli, including islet cells and anti-TCR or
anti-Fas mAbs (our unpublished observations). Although a
contribution of Idd5 to the "deleting" phenotype of
8.3-NOR mice cannot be ruled out, the fact that deletion of
diabetogenic T cells in NOR mice selectively targets MHC class
I-restricted CD8+ T cells, but not MHC class II-restricted
CD4+ T cells, makes Idd13 a more attractive
candidate: Idd13 contains the dimorphic
ß2m
(ß2-microglobulin) locus; NOD and NOR mice express
different ß2m isoforms; and these isoforms account for
conformational differences between the otherwise identical
Kd and Db MHC class I molecules of these mice
(41, 42, 43). Since the Idd13r-controlled diabetes
resistance of NOR mice resides in a radio-resistant nonhemopoietic cell
type, perhaps the ß cell itself (44), and since NOR ß cells display
a somewhat greater susceptibility to 8.3-CTL-induced lysis than NOD ß
cells (see Fig. 5), it would be reasonable to speculate that the
reduction in the peripheral frequency of 8.3-CD8+ T cells
in 8.3-NOR mice results from engagement of target
Kd/ß2mb complexes on ß cells or
APCs with an affinity/avidity that surpasses a deleting threshold. This
view is compatible with two observations: 1) "deletion" segregates
as a recessive trait (i.e., two copies of the
ß2mb molecule would increase the avidity of
the TCR-MHC class I interaction and hence the chances of reaching the
deleting threshold); and 2) in 8.3-TCRß-transgenic NOR mice,
"deletion" does not systematically target all ß cell-reactive
CD8+ T cells, but rather only those bearing the pathogenic
8.3-TCR-CDR3 sequence. This interpretation would also provide an
explanation for the paradoxical acceleration of IDDM in the few 8.3-NOR
mice that developed IDDM: in young 8.3-NOR mice, in which the reduction
in the peripheral frequency of 8.3-CD8+ T cells is minimal,
such an increased affinity would actually promote IDDM. The reasons
behind the increased incidence (and accelerated onset) of diabetes in
8.3-NOR vs 8.3-F1 male mice might also be due to this.
However, we favor the alternative possibility that NOR/Lt mice also
bear recessive prodiabetogenic genes that in males, but not females,
can override the antidiabetogenic function of the dominant protective
element(s). Ongoing studies of Idd13r-congenic
8.3-NOD mice should answer some of these questions.
Peripheral deletion of diabetogenic CD8+ T cells, however, is clearly not the only mechanism of diabetes resistance operating in NOR mice; 8.3-F1 mice are diabetes resistant, but do not delete transgenic 8.3-CD8+ T cells. Experiments with another highly diabetogenic, but I-Ag7-restricted, ß cell-specific TCR revealed that the diabetes resistance of F1 mice is primarily determined by one (or more) dominant protective element(s) that interfere(s) with the pathogenic activity of diabetogenic CD8+ and CD4+ T cells, regardless of their MHC restriction and antigenic specificity. This genetic element does not function by causing the deletion, anergy, or immune deviation of the transgenic T cells, by interfering with their insulitogenic activity, or by blocking their differentiation into CTLs in situ, but instead by promoting a form of immune suppression that is affected by mature B cells or endogenous T cells. The nature of the lymphocyte type and the specific mechanisms through which it prevents 4.1-CD4+ and 8.3-CD8+-induced ß cell death are not yet known, but there are several possibilities. B cells constitute a substantial fraction of islet- infiltrating lymphocytes in our TCR-transgenic mice (24), and thus may be able to mediate this local immunosuppressive effect. Although B lymphocytes have a greater capacity than other APC types to preferentially activate Th2 cytokine responses (45), the absence of local immune deviation in TCR-transgenic NOR mice suggests that this is not the mechanism by which these cells might function. Alternatively, since anti-idiotypic and anti-TCR-V region Abs can dampen T cell-induced autoimmune responses (46, 47), B cells might afford diabetes protection by mounting powerful local anti-idiotypic responses in NOR (but not NOD) mice (i.e., against the diabetogenic TCRs). The fact that B cell-deficient NOD mice are resistant to diabetes (48), however, argues against a role for B cells in this immunosuppressive response, as this would imply that B cells have qualitatively opposed functions in NOD vs NOR mice. Rather, we favor the alternative, but not exclusive, possibility that this protective function is affected by T cells; T cells capable of suppressing autoimmune diabetes have been described, and some of these cells have actually been isolated from NOD mice (49, 50, 51, 52, 53, 54). Since disease suppression in TCR-transgenic F1 mice is dissociated from local production of IL-4 and TGF-ß1, these suppressor T cells would more likely be related to a recently described type of antidiabetogenic CD4+ Th1 cells (54) or to anti-idiotypic T cells (55, 56, 57), than to Th2, Th3, or IL-4-producing TCR+CD4-CD8- cells (58). It is noteworthy that this protective mechanism is not very efficient at suppressing CD8+ T cell-induced diabetes in male 8.3-NOR mice, suggesting that NOR/Lt mice also bear recessive genes that potentiate the diabetogenic activity of pathogenic CD8+ T cells in males.
Too little is known about the antidiabetogenic loci of NOR mice, to speculate as to whether this form of genetically dominant resistance to diabetes is encoded on one of these loci, on a combination of them, or on other unknown loci. Nonetheless, since the NOR mouse derives some of its genetic material from C57BL/6 and DBA/2 mice (25, 26), it is likely that these elements are also present in other genetic backgrounds. This mechanism may account, in part, for the diabetes resistance of 4.1-(NOD x C57BL/6.I-Aßb-)F1 mice, which were completely resistant to spontaneous IDDM, despite not being able to delete 4.1 thymocytes (23). It would also be reasonable to expect that less powerful forms of these protective elements also exist in mice that are susceptible to autoimmunity. These variants, which would be able to suppress some autoreactive T cells, but not the most pathogenic ones (i.e., 8.3-CD8+ and 4.1-CD4+ T cells), might account for the dramatic acceleration (and increased incidence) of diabetes in BDC-2.5-NOD.scid vs BDC-2.5-NOD mice, which express another, less diabetogenic, ß cell-reactive TCR (22). They may also account for dramatic differences in the incidence of experimental autoimmune encephalomyelitis in RAG-1+ vs RAG-1- mice expressing a myelin basic protein-specific TCR (21).
In summary, this study has uncovered the existence of two mechanisms of non-MHC-linked genetic control of diabetogenic T cells. The fact that one of these mechanisms targets both CD4+ and CD8+ T cells, regardless of their molecular make-up and fine antigenic specificity, suggests that its failure may account for the clustering of multiple autoimmune disorders in affected individuals or their relatives (4, 59). Reductionist approaches such as the one used in this study should help define the mechanisms of action of specific non-MHC-linked chromosomal regions associated with spontaneous autoimmunity.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 J.V. and A.A. made equal contributions to this paper.
3 Address correspondence and reprint requests to Dr. Pere Santamaria, Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1. E-mail address:
4 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; ß2m, ß2-microglobulin; BrdU, 5-bromo-2'-deoxyuridine; CDR, complementarity-determining region; CM, complete medium; DAB, diaminobenzidine; HRP, horseradish peroxidase; NOD, nonobese diabetic; NOR, nonobese resistant; RAG-2, recombination-activating gene-2; PerCP, peridinin chlorophyll protein.
Received for publication September 18, 1998. Accepted for publication January 27, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain CDR3 sequences. J. Immunol. 154:2494.[Abstract]
-1 and -2 superdomain. Immunogenetics 30:361.[Medline]
24JQT cells in type 1 diabetes. Nature 391:177.[Medline]
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