By
From the Department of Microbiology and Infectious Diseases and Julia McFarlane Diabetes Research Centre, Faculty of Medicine, Health Sciences Centre, The University of Calgary, Calgary, Alberta T2N 4N1, Canada
Certain major histocompatibility complex (MHC) class II haplotypes encode elements providing either susceptibility or dominant resistance to the development of spontaneous autoimmune diseases via mechanisms that remain undefined. Here we show that a pancreatic beta cell-reactive, I-Ag7-restricted, transgenic TCR that is highly diabetogenic in nonobese diabetic mice (H-2g7) undergoes thymocyte negative selection in diabetes-resistant H-2g7/b, H-2g7/k, H-2g7/q, and H-2g7/nb1 NOD mice by engaging antidiabetogenic MHC class II molecules on thymic bone marrow-derived cells, independently of endogenous superantigens. Thymocyte deletion is complete in the presence of I-Ab, I-Ak + I-Ek or I-Anb1 + I-Enb1 molecules, partial in the presence of I-Aq or I-Ak molecules alone, and absent in the presence of I-As molecules. Mice that delete the transgenic TCR develop variable degrees of insulitis that correlate with the extent of thymocyte deletion, but are invariably resistant to diabetes development. These results provide an explanation as to how protective MHC class II genes carried on one haplotype can override the genetic susceptibility to an autoimmune disease provided by allelic MHC class II genes carried on a second haplotype.
Insulin-dependent diabetes mellitus (IDDM),1 a prototype of organ-specific autoimmune diseases, results from
selective destruction of pancreatic beta cells by a T lymphocyte-dependent autoimmune process in genetically
predisposed individuals (1). Genetic susceptibility and/or
resistance to most autoimmune disorders, including IDDM,
is associated with highly polymorphic genes of the MHC
complex and, to a lesser extent, with polygenic modifiers on other chromosomes (2).
Population and animal studies have suggested that the
MHC class II-linked susceptibility and resistance to IDDM
are inherited as dominant traits with incomplete penetrance
(2, 3). In humans, the MHC-associated IDDM susceptibility and resistance are predominantly, but not exclusively,
determined by polymorphisms at the human leukocyte antigen (HLA) DQB1 locus. Alleles encoding DQ The precise mechanisms through which MHC genes
provide autoimmune disease susceptibility and resistance,
however, remain mysterious. MHC molecules are cell-surface receptors that present short fragments of self and foreign proteins to T lymphocytes and play a pivotal role in
instructing T lymphocytes maturing in the thymus how to discriminate between self- and nonself-antigens (19, 20). Thymocytes bearing TCRs capable of recognizing self-peptide-MHC complexes with high affinity/avidity die or are
rendered unresponsive to antigenic stimulation (21). In
contrast, thymocytes bearing TCRs capable of engaging
self-peptide-MHC complexes with intermediate affinity/
avidity survive and are exported to the peripheral lymphoid organs as cells capable of recognizing foreign antigens bound to self-MHC molecules (25). On the basis of some of
this knowledge, a number of authors hypothesized that
MHC molecules providing resistance to autoimmune diseases, including IDDM, might do so by mediating the clonal
deletion or anergy of autoreactive T cells (8, 11, 31). Studies
in MHC-congenic NOD mice and in I-A-, I-E-, and
TCR-transgenic NOD mice, however, did not find evidence of T cell tolerance induction, and suggested that the
diabetes resistance provided by protective MHC genes
likely involved the induction of immunoregulatory functions
(10, 13, 16, 18, 32, 33).
The studies presented here were initiated to test the hypothesis that diabetes-resistant genetic backgrounds express
non-MHC-linked genetic elements other than endogenous superantigens that are tolerogenic for diabetogenic T
cells. To that end, we followed the fate of an NOD islet-derived, beta cell-specific, I-Ag7-restricted transgenic TCR
in diabetes-prone NOD mice, and in diabetes-resistant F1
hybrid mice lacking endogenous superantigens binding to
the transgenic TCR. In NOD mice, T cells expressing the
transgenic TCR underwent positive thymic selection and
triggered a dramatic acceleration of the onset of IDDM. In
certain F1 hybrid strains, however, the same cells underwent negative selection and the mice did not develop
IDDM. Contrary to what we expected, the thymocyte deletion and IDDM resistance observed in these mice cosegregated as a single locus trait with MHC haplotypes known to provide dominant resistance to IDDM in MHC-transgenic and/or -congenic NOD mice (H-2b and H-2k). Thymocyte deletion in these mice was not mediated by endogenous superantigens, since it was absent in single-chain
TCR- Generation of TCR Transgenes.
The TCR- Production of Transgenic Mice.
After removing prokaryotic sequences by digestion with PvuI and SalI (TCR- Antibodies and Flow Cytometry.
Hybridomas secreting mAbs
H57-597 (anti-TCR- Cloning and Sequencing of TCR- Preparation of CD8+ T Cell-depleted Splenic and Islet-derived T
Cells.
Spleens were disrupted into single cell suspensions and
red blood cells lysed in 0.87% ammonium chloride. Remaining
cells were washed with complete medium (CM: RPMI 1640 media containing 10% heat-inactivated fetal bovine serum [GIBCO
BRL, Gaithersburg, MD], 50 U/ml penicillin, 50 µg/ml streptomycin [Flow Labs., McLean, VA], and 50 µM 2-ME [Sigma
Chemical Co., St. Louis, MO]) and then depleted of CD8+ T
cells using anti-CD8 mAb (53-6.7)-coated magnetic beads as described (35). Islet-infiltrating CD4+ T cells were isolated from
acutely diabetic 4.1-NOD mice essentially as described previously
(35), analyzed by flow cytometry, depleted of CD8+ T cells by
negative selection with anti-CD8 mAb-coated immunobeads, expanded in rIL-2-containing CM for 1-2 wk, and used for in
vitro and in vivo studies.
Proliferation Assays.
Pancreatic islets from 5-8-wk-old nontransgenic male NOD mice were dispersed into single cells by incubation in Ca2+- and Mg2+-free PBS containing 0.125% trypsin
and 3 mM EGTA at 37°C for 3 min. 2 × 104 splenic or islet-derived CD4+ cells were incubated, in triplicate, with chains
with serine, alanine, or valine at position 57 provide susceptibility, whereas those encoding DQ
chains with aspartic acid at position 57 provide resistance with differing
degrees of dominance (1, 2). In mice, susceptibility and resistance to spontaneous IDDM are also linked to the MHC
(H-2). The diabetes-prone nonobese diabetic (NOD) mouse,
which spontaneously develops a form of diabetes closely
resembling human IDDM, is homozygous for a unique
H-2 haplotype (H-2g7). This haplotype carries a nonproductive I-E
gene and encodes an I-A
d/I-A
g7 heterodimer in which the histidine and aspartic acid found at positions 56 and 57 in most murine I-A
chains (the counterpart of human DQ
chains) are replaced by proline and
serine, respectively (4, 5). Studies of congenic NOD mice
expressing non-NOD MHC haplotypes, and of NOD
mice expressing I-E
d, modified I-A
g7, I-A
k/I-A
k, or
I-A
d transgenes, have demonstrated that MHC class II
molecules encoded by H-2 haplotypes derived from NOD
or IDDM-resistant mice play a direct role in providing either susceptibility or resistance to spontaneous IDDM, respectively (6).
-transgenic H-2g7/b and H-2g7/k F1 mice. Studies
of bone marrow chimeras, I-A
b-deficient H-2g7/b mice
and I-Ak-transgenic NOD mice demonstrated that the thymocyte deletion and IDDM resistance of these mice resulted from the ability of the transgenic TCR to engage I-Ab,
I-Ak, and possibly, I-Ek molecules on thymic bone marrow-derived cells. Additional experiments with H-2g7/q-
and H-2g7/nb1-congenic NOD mice revealed that this highly
pathogenic TCR was also deleted in the presence of I-Aq
and I-A/I-Enb1 molecules, which also provide dominant resistance to autoimmune diabetes in non-TCR-transgenic
NOD mice. Our unexpected results provide an explanation
as to how protective MHC class II molecules may provide
resistance to spontaneous T cell-mediated autoimmune disorders, i.e., by removing certain highly pathogenic autoreactive T cells.
and -
cDNAs
of NY4.1 were cloned by anchored PCR and multiple recombinants sequenced as described (34). The cDNAs were then amplified by PCR using primers containing L-V and J-C intron sequences, the corresponding splice donor and acceptor sites and
convenient restriction sites, cloned into pBS-SK+ (Stratagene, La
Jolla, CA), and sequenced. Inserts with the expected sequences
were released from the vector by digestion with ClaI and NotI
(TCR-
) or XhoI and NotI (TCR-
). The 4.1-VDJ
sequence was subcloned into a TCR-
shuttle vector carrying the endogenous TCR-
enhancer and 5
regulatory sequences (3A9
; gift
from M. Davis, Stanford University, Stanford, CA). The 4.1-VJ
cDNA was subcloned into PRE53
(from M. Davis). A 2.4-kb
ClaI-KpnI fragment from 4.1-VJ
/PRE53
, containing 5
regulatory sequences and the 4.1-VJ
sequence, was then coligated
into pBS-SK+ with a genomic 11.9-kb KpnI-NotI fragment
from AN6.2
(provided by S. Hedrick, University of California
San Diego, San Diego, CA), containing the four C
exons and
the downstream TCR-
enhancer.
) or ClaI and
SacII (TCR-
), the constructs (21.5- and 14.5-kb, respectively)
were injected into fertilized (SJL × C57BL/6) F2 eggs (DNX,
Princeton, NJ). Offspring were screened for integration of the
transgenes by Southern blotting using VDJ
and VJ
cDNA
probes. Transgenic founder mice (4.1-AN6A3-TCR-
/
, 4.1-AN6B3-TCR-
and 4.1-AN6B7-TCR-
) were backcrossed
with NOD/Lt mice (I-Ag7, I-E
; Jackson Laboratory, Bar Harbor, ME) for 3-5 generations to generate TCR-
/
-, TCR-
-,
and TCR-
-transgenic NOD mice. TCR-
-, TCR-
-, and
TCR-
/
-transgenic NOD mice (of the N5 backcross) were
crossed with SJL/J (S; I-As, I-E
), C57BL/6 (B; I-Ab, I-E
),
C58/J (C; I-Ak, I-Ek), NOD.H-2g7/q (I-Ag7/q, I-E
), or NOD.H-2g7/nb1 (I-Ag7/nb1, I-Enb1) mice (Jackson Laboratory), to generate
TCR-transgenic F1 mice or H-2 heterozygous TCR-transgenic
NOD mice. TCR-
/
-transgenic F1 (4.1-F1) mice were also
backcrossed with NOD, C57BL/6, or C58/J mice, to generate
H-2g7, H-2g7/b, H-2g7/k 4.1 mice with 75% NOD genotype, and
H-2b or H-2k 4.1 mice with 75% C57BL/6 or C58/J genotypes,
respectively. I-A
b-deficient 4.1-(N × B) F1 mice were generated by crossing I-A
b
C57BL-6 mice (Taconic, Germantown,
NY) with 4.1-NOD mice.
2 microglobulin (
2m)
4.1-(N × B) F1 mice were obtained by breeding the
2m mutation of
2m
NOD/Lt mice (Jackson Laboratory) into 4.1-NOD mice,
to obtain
2m
4.1-NOD mice, and then by crossing these mice
with
2m
C57BL/6 mice (Taconic). CD8-
H-2g7/b mice
were obtained by crossing CD8-
mice (I-Ab, I-E
) (gift from
T. Mak, University of Toronto, Toronto, Canada) with 4.1-NOD mice, followed by crossing CD8-
/+ 4.1-H-2g7/b mice
with CD8-
H-2b mice. I-Ak-transgenic 4.1-NOD mice were
obtained by crossing I-A
k/I-A
k-transgenic NOD mice (from
G. Morahan and J.F.A.P. Miller, The Walter and Eliza Hall Institute, Victoria, Australia) with 4.1-NOD mice. Mice were screened
for inheritance of the transgenes, mutated alleles, and MHC haplotypes by PCR of tail DNA (4.1
, 4.1
,
2m, I-Ag7, I-Ak) and/
or by flow cytometry (4.1
, CD8-
, I-Ab, I-As, Kd, Kk). All mice
were housed in a specific pathogen-free facility.
), 53-6.7 (anti-CD8-
), B220 (anti-B
cells), and M1/70 (anti-Mac-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Anti-
Lyt-2 (CD8-
)-PE (53-6.7), anti-L3T4-FITC (IM7), or anti- L3T4-biotin (CD4; H129.19), anti-V
11-FITC (RR3-15),
anti-H-2Kd-FITC (SF1-1.1), and anti-I-A
b-biotin (25-9-3)
were purchased from PharMingen (San Diego, CA). Purified
mouse anti-H-2Kk (11-4.1) was from Becton Dickinson (San
Jose, CA). Mouse IgG-absorbed FITC-conjugated goat anti-rat
IgG and FITC-conjugated goat anti-mouse IgG were from
CALTAG Labs. (South San Francisco, CA) and Becton Dickinson, respectively. Streptavidin-PerCP was from Becton Dickinson. Thymi, spleens, and islet-derived T cells were analyzed by
three-color flow cytometry using a FACScan® as described (35).
cDNAs.
The TCR-
cDNA
molecules of splenic CD4+ T cells were cloned and sequenced by
anchored PCR as described previously (35).
-irradiated (3,000 rad) islet cells (3-100 × 103/well) and unfractionated
NOD splenocytes (105/well), as a source of antigen and APCs,
respectively, in 96-well round-bottomed tissue culture plates for
3 d at 37°C in 5% CO2 in rIL-2-free CM. Cultures were pulsed
with 1 µCi of [3H]thymidine during the last 18 h of culture and
harvested. The incorporated thymidine was measured by scintillation counting. Specific proliferation was calculated by substracting background proliferation (cpm of cultures containing islet
cells plus APCs alone and cpm of cultures of T cells alone) from
islet cell-induced proliferation (cpm of cultures containing T
cells, APCs, and islet cells).
stimulation, 96-well flat-bottomed plates
were precoated overnight at 4°C with serial dilutions of purified anti-TCR-
mAb (H57-597; 0.3-10 µg/ml) in 50 mM Tris-HCl, 150 mM NaCl (pH 9.5), and washed three times in CM.
CD4+ T cells (2 × 104) were added to each well in triplicate, incubated for 48 h, pulsed with [3H]thymidine for 18 h, harvested,
and analyzed by scintillation counting.
Histology and Immunopathology.
The body-tail of each pancreas was divided into two pieces. One piece was fixed in formalin, embedded in paraffin, sectioned at 4.5 µM, and stained with
hematoxylin and eosin. The degree of insulitis was evaluated by
scoring 15-30 islets/mouse in a blinded fashion using the following criteria: 0, normal islet; 1, peri-insulitis; 2, mononuclear cell
infiltration in <25% of the islet; 3, mononuclear cell infiltration
in 25-50% of the islet; 4, >50% of the islet infiltrated. A second
piece of tissue was snap frozen, immersed in OCT, sectioned at
6-7 µM, and stored at 80°C for immunopathology. Sections
were fixed in cold acetone for 10 min and stained with anti-CD4
(GK1.5), anti-CD8 (53-6.7), anti-Mac-1, and anti-B220 mAbs,
followed by anti-rat IgG-FITC, or with anti-rat IgG-FITC
alone, as described (35).
Adoptive T cell Transfer. CD8+ T cell-depleted islet-derived CD4+ T cells from diabetic 4.1-NOD mice (5 × 106 cells/ mouse) were transfused into the tail veins of scid-NOD/Lt (Jackson Laboratory) in 200 µl of PBS, pH 7.2. Transfused mice were followed for development of IDDM by monitoring blood glucose levels with Glucostix and a glucometer (Miles Canada, Etobicoke, Ontario). Mice were killed at IDDM onset for flow cytometry and immunopathological studies.
Bone Marrow Chimeras. Bone marrow chimeras were generated following standard protocols (36). In brief, bone marrow suspensions (5-10 × 106 cells) from donor mice (transgenic NOD or [N × B] F1 mice) were injected into the tail vein of recipient mice (nontransgenic NOD, [N × B] F1 or [N × S] F1 mice) treated with two doses of 500 rads 3 h apart from a 137Cs source (Gammacell; Atomic Energy of Canada, Ottawa, Ontario). Chimeric mice were killed 5-6 wk after bone marrow transplantation.
Statistical Analyses.
Statistical analyses were performed using
Mann-Whitney U and 2 tests.
A CD4+ T cell clone (NY4.1) that
was derived from pancreatic islets of a diabetic NOD
mouse and that recognized a putative beta cell autoantigen
in the context of I-Ag7 (37) was chosen as donor of the
TCR transgenes. This T cell clone transcribed one functional TCR- rearrangement, carrying V
11 and J
2.4 sequences and one functional TCR-
rearrangement, carrying a novel V
gene (V
x4.1) and the J
33 element (These sequence data are available from EMBL/GenBank/DDBJ
under accession numbers U80816 and U80817). These TCR
rearrangements were subcloned into genomic TCR-
and
-
shuttle vectors carrying endogenous TCR enhancers and
5
-regulatory sequences. The resulting genomic constructs
were then used to produce transgenic mice. Founders expressing the transgenes (4.1-AN63A-TCR-
/
, 4.1-AN6B3-
TCR-
, and 4.1-AN6B7-TCR
) were crossed with NOD
mice for several generations to generate 4.1-TCR-
/
-,
4.1-TCR-
-, and 4.1-TCR-
-transgenic NOD mice, respectively.
Three-color cytofluorometric studies showed that >90% CD4+CD8 thymocytes (Fig. 1 A)
and splenic CD4+ T cells (Fig. 1 B) from 4.1-NOD mice
expressed V
11+ TCRs, compared to ~6% of the
CD4+CD8
thymocytes and splenic CD4+ T cells from
nontransgenic littermates, thus indicating TCR-
transgene expression. Although we do not have a transgenic
TCR-
chain-specific antibody and thus cannot directly
quantitate TCR-
transgene expression, five different lines
of evidence suggest that the transgenic TCR-
chain is expressed on a sizeable fraction of thymocytes and peripheral
T cells of 4.1-NOD mice. First, CD4+CD8+ thymocytes
from 4.1-NOD mice expressed higher levels of total TCR-
/
(as determined by staining with an anti-C
mAb) than
CD4+CD8+ thymocytes from TCR-
-transgenic NOD
mice (mean fluorescence intensities: 59 ± 12 versus 31 ± 3, respectively; P <0.001), suggesting early TCR-
chain
expression (23, 38, 39). Second, thymocyte development
in 4.1-NOD mice, but not TCR-
-transgenic NOD
mice, was skewed towards the CD4+CD8
subset (Fig. 1
A), compatible with TCR-
transgene-dependent positive
selection of 4.1-CD4+ thymocytes. Third, skewing of thymocytes into the CD4+CD8
subset occurred in transgenic mice expressing the selecting I-Ag7 molecule, but not
in transgenic mice expressing only nonselecting I-A molecules (i.e., I-As, see below), as seen with other MHC class
II-restricted TCR-
/
-transgenic models (38, 40). Fourth,
37 out of 37 4.1-NOD TCR-
cDNA sequences, generated from splenic CD4+ T cell-derived RNA by anchored
PCR, were TCR-
transgene-derived. Finally, splenic
CD4+ T cells from 4.1-NOD, but not TCR-
-transgenic
or nontransgenic, NOD mice proliferated in a dose-dependent manner in response to irradiated NOD islet cells (Fig.
1 C). The islet cell-induced proliferation of 4.1- and control CD4+ T cells was quite variable between experiments,
perhaps due to variability in the quality of the islet cell
preparations; however, the differences within individual
experiments were reproducible. Taken together, these results provide strong evidence that in 4.1-NOD mice, the
4.1-TCR specificity is expressed appropriately, and that, in
the presence of the selecting I-Ag7 molecule, 4.1-TCR-
/
transgene expression fosters the positive selection of beta
cell-reactive CD4+ T cells.
Early Onset of IDDM in 4.1-NOD Mice.
To determine
whether positive selection of the beta cell-specific TCR in
4.1-NOD mice had any pathogenic significance, we followed 4.1-NOD mice of the N3-N5 backcrosses of the
founder mouse onto the NOD background, and nontransgenic NOD mice for development of IDDM. As shown in
Fig. 2 A, 4.1-NOD mice developed IDDM much earlier
than nontransgenic NOD mice (IDDM onset at 43.6 ± 13 versus 119 ± 26 d in females, and at 56 ± 27 versus 157 ± 28 d in males; P <0.0001) (Fig. 2 A). In males, transgene
expression also increased the incidence of IDDM (73.3 versus 45.7%; P <0.05). The kinetics of disease penetrance
in the transgenic and nontransgenic populations were,
however, remarkably similar (Fig. 2 A). Disease acceleration in 4.1-NOD mice required coexpression of the TCR- and -
transgenes, since the few 4.1-TCR-
-NOD mice
that became diabetic (3/7, 43%) did so significantly later
than 4.1-NOD mice (103 ± 20 versus 46 ± 19 d; P
<0.01). These results contrast with those obtained with the
only other existing beta cell-specific TCR-transgenic
NOD mouse model (32), which, when housed under specific pathogen-free conditions, develops IDDM less frequently and significantly later (10-15% at 6 mo) than 4.1-NOD mice (41). Taken together, our results indicate that
the 4.1-TCR-
/
is highly diabetogenic in the NOD
background, and suggest that the events that trigger accelerated diabetogenesis in 4.1-NOD mice are similar to those
that trigger IDDM in nontransgenic NOD mice.
To investigate the mechanisms underlying disease acceleration in 4.1-NOD mice, we then followed the progression of insulitis in prediabetic and diabetic mice. Histopathological studies of pancreata from 3- and 6-wk-old
prediabetic 4.1-NOD mice showed that acceleration of diabetes in these mice was a result of faster progression, but
not earlier onset, of islet inflammation (Fig. 2 B). As expected, the insulitis lesions of diabetic 4.1-NOD mice contained more CD4+ and fewer CD8+ T cells (but similar
numbers of B cells and macrophages [not shown]), than those
of diabetic nontransgenic NOD mice (Fig. 2 C). Islet-derived CD4+ T cells from 4.1-NOD mice expressed high
levels of the transgene-encoded V11+ chain (Fig. 2 D), proliferated in response to NOD islet cells in vitro (Fig. 2 E),
and transcribed messenger RNA for IL-2 and IFN-
, but
not IL-4 (data not shown). These data indicate that these
cells were transgenic, beta cell reactive, and of the Th1 type, as expected. Moreover, purified islet-derived CD4+
T cells from three different diabetic 4.1-NOD mice were
able to transfer IDDM into three different scid-NOD mice
shortly after transfusion (36 ± 12 d) in the absence of
CD8+ T cells in the inflamed islets (Fig. 2 F). We thus
conclude that expression of the 4.1-TCR in the NOD
background promotes the selection of highly diabetogenic
CD4+ Th1 cells and their accelerated recruitment into
pancreatic islets, leading to massive beta cell destruction
and IDDM within the first few weeks of life.
The exquisite pathogenicity of the
4.1-TCR provided us with a powerful tool with which to
test our initial hypothesis that diabetes-resistant backgrounds encode non-MHC-linked elements other than
endogenous mouse mammary tumor virus superantigens (vSAgs) that are tolerogenic for diabetogenic T cells. To
investigate this, we crossed 4.1-NOD mice (H-2g7) with
SJL/J (H-2s), C57BL/6 (H-2b), and C58/J mice (H-2k); F1
mice resulting from crosses of nontransgenic NOD mice
with these strains express the diabetogenic I-Ag7 molecule,
but are diabetes-resistant, and do not delete V11+ T cells
(42). The lack of vSAg-mediated deletion of V
11+ or
V
x4.1+ cells in these backgrounds was confirmed by the
fact that the thymocyte profiles of single-chain TCR-
- or
TCR-
-transgenic F1 mice and those of TCR-
- or
TCR-
-transgenic NOD mice, respectively, were indistinguishable; as shown in Fig. 3, the percentages of thymic
and splenic CD4+CD8
V
11+ cells in TCR-
-transgenic
(Fig. 3 A) or nontransgenic F1 mice (Fig. 3 B) were virtually identical to (if not greater than) those seen in TCR-
-transgenic or nontransgenic NOD mice, respectively.
The flow cytometric profiles of thymocytes (Fig. 4 A)
and splenocytes (Fig. 4 B) from 4.1-(N × S) F1 mice (n = 8) were comparable to those seen in 4.1-NOD mice (Fig.
1), indicating that the 4.1-TCR specificity also undergoes
positive selection in H-2g7/s mice. In contrast, all 4.1-(N × B) F1 mice (n = 22) and most 4.1-(N × C) F1 mice (n = 16/19) had only one-third to one-half the number of thymocytes seen in 4.1-NOD mice (and 4.1-[N × S] F1 mice),
and displayed flow cytometric profiles of thymocytes and
splenocytes compatible with negative selection of the 4.1-TCR, as compared with other TCR-/
-transgenic models (23, 24, 39, 43) (Fig. 4). When compared to 4.1-NOD
mice, 4.1-(N × B) F1 and 4.1-(N × C) F1 mice (also referred to as "deleting") displayed a significant reduction in
the percentage of CD4+CD8
thymocytes, a reduction in
the percentage of CD4+CD8
thymocytes expressing the
transgene-encoded V
11 element, and an increase in the percentage of CD4
CD8
thymocytes (Fig. 4 A). In the spleen,
4.1-NOD mice had significantly more CD4+ T cells and
more CD4+ T cells expressing V
11+ TCRs than deleting
mice (Fig. 4 B). The few V
11+CD4+ T cells that matured
in deleting mice expressed half as many transgenic TCR-
chains on the cell surface (but comparable numbers of total
TCR-
/
complexes) as V
11+CD4+ T cells of 4.1-NOD mice, both in the thymus and in the spleen (P
<0.003; data not shown), suggesting that in deleting mice
these cells were selected on endogenous TCR chains that
had bypassed allelic exclusion, as seen in other models (23,
24, 39, 43).
Proliferation assays using splenic CD4+ T cells as responders and irradiated NOD islet cells and splenocytes as
antigen and APCs, respectively, revealed the absence of
beta cell-reactive CD4+ T cells in the periphery of deleting, but not nondeleting, 4.1-F1 mice (Fig. 5 A). Lack of
proliferation in these assays was the result of deletion,
rather than anergy, since peripheral CD4+ T cells from deleting and nondeleting 4.1-F1 mice proliferated similarly in
response to plate bound anti-TCR-/
mAb (Fig. 5 B).
The absence of diabetogenic 4.1 T cells in the peripheral lymphoid organs of deleting mice was confirmed by the
observation that, like their nontransgenic littermates, 4.1-F1
hybrid mice developed neither diabetes nor insulitis (Table 1).
|
Taken together, these data indicate that: (a) C57BL/6 and C58/J mice carry genes encoding elements capable of mediating the deletion of the diabetogenic 4.1-TCR; (b) the deleting element(s) expressed by these strains has complete or incomplete penetrance, respectively; and (c) these elements are not encoded by vSAgs and target T cells coexpressing both chains of the diabetogenic TCR.
Thymocyte Deletion and Resistance to Insulitis and IDDM Cosegregate with H-2b and H-2k.Previous studies have suggested that the IDDM resistance provided by certain non-NOD MHC class II genes, including I-Ab and I-Ak, involves the induction of immunoregulatory functions rather than the deletion or anergy of autoreactive T cells (10, 16, 18, 32, 33, 44). Accordingly, we hypothesized that thymocyte deletion in 4.1-F1 hybrid mice would be mediated by elements encoded by non-MHC-linked genes. To test this hypothesis, we backcrossed 4.1-F1 mice with NOD mice and investigated whether thymocyte deletion and IDDM resistance in the transgenic offspring (4.1-F2 mice) segregated away from the H-2 haplotypes of the deleting backgrounds (H-2b and H-2k). All 4.1-F2 mice were killed at IDDM onset, or at 10 wk if nondiabetic, to determine: (a) their H-2 phenotypes, (b) the occurrence of thymocyte deletion; and (c) the degree of insulitis. Unexpectedly, we found that deletion of 4.1 thymocytes segregated as a single-locus trait linked to the MHC; it occurred in H-2g7/b (16/16 mice, 100%) or H-2g7/k mice (9/15 mice, 60%; incidence of deletion comparable to that seen in [N × C] F1 mice), but never in H-2g7/g7 mice (0/24 mice, 0%) (Table 2). Like deleting 4.1-F1 mice, deleting 4.1-F2 mice did not harbor detectable beta cell-reactive CD4+ T cells in the spleen (data not shown). Furthermore, deleting H-2g7/b or H-2g7/k 4.1-F2 mice developed neither IDDM nor insulitis, whereas H-2g7/g7 4.1-F2 mice developed moderate to severe insulitis (100%) and diabetes (~50%) within 10 wk (Table 2). These data thus indicate that the deleting elements of C57BL/6 and C58/J mice are encoded by genes tightly linked to their H-2b and H-2k complexes, and demonstrate that insulitis and diabetes resistance in 4.1-F2 mice carrying these haplotypes results from deletion of diabetogenic thymocytes.
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The data presented above suggested that the deletion
of transgenic thymocytes and the IDDM resistance observed in 4.1-F1 and -F2 mice might be mediated by
MHC class I and/or class II molecules encoded by the protective H-2 haplotypes. To determine whether 4.1 thymocyte deletion required the engagement of MHC class I molecules, we followed the maturation of 4.1 thymocytes
in CD8-- or
2m-deficient 4.1-(N × B) F1 mice (H-2g7/b),
which either do not express the MHC class I-binding CD8
coreceptor on thymocytes, or lack MHC class I molecules,
respectively. These mice deleted transgenic thymocytes as
efficiently as wild-type 4.1-F1 mice (data not shown), thus
indicating that deletion of transgenic thymocytes was not
mediated by MHC class I molecules.
Since H-2g7/b mice do not express I-E molecules, we
reasoned that deletion in these mice might be mediated by
I-Ab. To test this notion, we followed the development of
4.1 thymocytes in I-Ab-deficient 4.1-(N × B) F1 mice;
except for the I-A
b mutation, I-A
b-deficient 4.1-(N × B) F1 and 4.1-(N × B) F1 mice are genetically identical.
Selective abrogation of I-A
b expression in 4.1-(N × B)
F1 mice restored, at least in part, the positive selection of
the transgenic TCR, as evidenced by (a) significant increases in the percentage of V
11+ thymocytes (Table 3),
(b) significant increases in the ratio of CD4+CD8
to
CD4
CD8
T cells in the thymus and in the ratio of
CD4+ to CD8+ T cells in the spleen (Table 3), (c) the reappearance of beta cell-reactive CD4+ T cells in the spleen
(Fig. 6, left), and (d) the reemergence of insulitis (Table 3).
The overall positive selection of the 4.1-TCR in I-A
b-
deficient 4.1-(N × B) F1 mice, however, was less efficient
than in 4.1-NOD mice; I-A
b-deficient 4.1-(N × B) F1
mice had more CD4
CD8
thymocytes than, and half the
splenic CD4+ T cells of, age-matched (3-5-wk-old) 4.1-NOD mice (Table 3). Furthermore, the peripheral CD4+
T cells of I-A
b-deficient 4.1-(N × B) F1 mice proliferated less efficiently in response to NOD islet cells than the
peripheral CD4+ T cells of 4.1-NOD mice (Fig. 6, left). Finally, the insulitis lesions of I-A
b-deficient 4.1-(N × B)
F1 mice were milder than those seen in 4.1-NOD mice
and did not lead to IDDM in any of the 15 mice that were
followed (Table 3). Therefore, the I-A
b gene is not the
only protective element present in (N × B) F1 mice, but
its expression is sufficient in and of itself to induce deletion
of 4.1 thymocytes.
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To investigate whether the H-2k-dependent deletion of
thymocytes in 4.1-(N × C) F1 mice was also I-A mediated, we crossed 4.1-NOD mice with I-Ak/I-A
k (I-Ak)-
transgenic NOD mice, to generate 4.1/I-Ak-NOD mice.
Except for the presence of the I-Ak transgenes in 4.1/I-Ak-
NOD mice, 4.1-NOD and 4.1/I-Ak-NOD mice have virtually identical genetic backgrounds. We found that 4.1-NOD mice had significantly more thymocytes (data not shown) and greater ratios of CD4+CD8
to CD4
CD8
thymocytes and of CD4+ to CD8+ splenocytes than 4.1/
I-Ak-NOD mice (Table 3), results compatible with deletion of 4.1 thymocytes in 4.1/I-Ak-NOD mice. This deletion, however, was incomplete; unlike the splenic CD4+ T
cells of deleting H-2g7/k 4.1-F2 mice, the few CD4+ T cells
that matured in 4.1/I-Ak-NOD mice expressed high levels
of the transgenic TCR-
chain, and these cells proliferated
in response to beta cells in vitro (Tables 2 and 3; and Fig. 6,
middle). Furthermore, unlike deleting H-2g7/k 4.1-F2 mice,
4.1/I-Ak-NOD mice developed moderate insulitis, suggesting that these cells were also responsive to antigen stimulation in vivo (Table 3). The insulitis lesions of these
mice, however, were less severe than those of 4.1-NOD
mice, and did not lead to diabetes in any of the nine mice
that were followed (Table 3). It appears, then, that (a) deletion of thymocytes in H-2g7/k 4.1 mice is triggered, at least
in part, by I-Ak molecules, and (b) in addition to causing
partial deletion of 4.1 thymocytes, I-Ak molecules may
somehow abrogate the diabetogenic potential of the 4.1 T
cells that escape deletion.
We next asked whether the 4.1-TCR could engage additional antidiabetogenic MHC class
II molecules during thymocyte development. We therefore
crossed 4.1-NOD mice with NOD mice congenic for H-2q
or H-2nb1 haplotypes, which provide dominant resistance
to diabetes in nontransgenic NOD.H-2g7/q or nb1 mice (7,
15), and followed the fate of the 4.1-TCR in the TCR-transgenic offspring. As shown in Table 3 and Fig. 6 (right) 4.1-NOD.H-2g7/q and 4.1-NOD.H-2g7/nb1 mice had phenotypes compatible with partial or complete deletion of the
4.1-TCR, respectively. When compared to 4.1-NOD
mice, 4.1-NOD.H-2g7/q mice had twofold fewer thymocytes (data not shown), increased percentages of CD4
CD8
thymocytes, and reduced percentages of thymic and
splenic CD4+CD8
T cells (Table 3). The splenic CD4+
T cells of these mice proliferated in response to NOD islet
cells in vitro, but did so less efficiently than those of 4.1-NOD mice (Fig. 6, right). Furthermore, these mice only
developed very mild periinsulitis and never became diabetic (Table 3). In contrast, thymocyte deletion in 4.1-NOD.H-2g7/nb1 mice was complete; these mice had a
three- to fourfold reduction in the absolute number of thymocytes (data not shown), dramatically increased percentages of CD4
CD8
thymocytes, and reduced percentages
of thymic and splenic V
11+CD4+CD8
T cells (Table
3). These mice did not contain detectable beta cell-reactive
CD4+ T cells in the spleen (Fig. 6, right) and developed
neither diabetes nor insulitis (Table 3). Thymocyte deletion
in these mice was not mediated by putative NOD vSAgs (i.e.,
mouse mammary tumor virus superantigen 17) binding to
V
11 and H-2q or H-2nb1 MHC class II molecules, since
the spleens of nontransgenic NOD. H-2g7/q and NOD.H-2g7/nb1 littermates harbored more V
11+CD4+ T cells than
nontransgenic NOD mice (20 ± 7 and 12 ± 2% versus 6 ± 1%, P <0.008 and P <0.04, respectively).
To elucidate whether in vivo deletion
of 4.1 thymocytes was "restricted" by I-Ag7 molecules, we
next studied thymocyte maturation in 4.1 mice homozygous for either nonselecting/nondeleting (H-2s) or deleting
(H-2b or H-2k) MHC haplotypes. As expected, 4.1 thymocytes underwent neither positive nor negative selection
in H-2s 4.1 mice; these mice had significantly fewer
CD4+CD8 thymocytes (but not more CD4
CD8
thymocytes) than H-2g7/s 4.1 mice (compare Fig. 4, left and 7, left; P <0.005). In contrast, the thymocyte profiles of H-2b
and H-2k 4.1 mice were compatible with deletion; these
mice had fewer thymocytes and greater percentages of
CD4
CD8
thymocytes than H-2s 4.1 mice (Fig. 7, middle
and right). Therefore, unlike the positive selection of 4.1 T
cells in selecting (I-Ag7+) mice, the I-Ab- and I-Ak-mediated deletion of 4.1 thymocytes in 4.1-F1 mice is not I-Ag7-
restricted.
Thymocyte Deletion in 4.1-F1 Mice Is Preceded by Positive Selection and Is Mediated by Hematopoietic Cells.
Previous studies have shown that the factors underlying the MHC-linked
resistance to spontaneous IDDM predominantly reside in
the bone marrow (17, 33, 44). To determine whether
deletion of diabetogenic thymocytes in 4.1-F1 mice was
mediated by hematopoietic cells or by thymic epithelial
cells, we transfused bone marrow from deleting H-2g7/b- or
selecting H-2g7-4.1 mice into lethally-irradiated nontransgenic NOD (H-2g7) or (N × B) F1 mice (H-2g7/b), respectively, and followed the fate of 4.1 thymocytes in the chimeras. As shown in Fig. 8, the mice that received marrow
from H-2g7/b-4.1 mice (expressing I-Ab only on hematopoietic cells), but not those that received marrow from
H-2g7-4.1 mice (expressing I-Ab only on thymic epithelial
cells), had a phenotype compatible with deletion (low thymocyte CD4+CD8/CD4
CD8
ratios and small percentages of V
11+CD4+CD8
thymocytes). It thus appears that deletion of thymocytes in 4.1-F1 hybrid mice is
preceded by their positive selection on radioresistant thymic epithelial cells, and is mediated by hematopoietic cells.
This finding raised one final question: if deletion is preceded by positive selection, why does it also occur in mice homozygous for the deleting H-2 haplotypes, which lack the selecting I-Ag7 molecule? We reasoned that the 4.1-TCR might actually perceive the deleting I-A molecules (i.e., I-Ab) expressed on thymic epithelial cells as selecting. To test this hypothesis, we followed the fate of 4.1 thymocytes arising from marrow of H-2g7-4.1 mice in lethally irradiated H-2s/b mice, which express deleting (I-Ab) and nonselecting/nondeleting (I-As), but not selecting (I-Ag7), MHC class II molecules on thymic epithelial cells. As shown in Fig. 8 (right), the thymocyte profiles of recipient mice were indistinguishable from those of H-2g7/b mice transplanted with marrow from H-2g7-4.1 mice, which were nondeleting (Fig. 8, middle). It thus appears that in heterozygotes, the 4.1-TCR senses the I-Ab molecules expressed by thymic epithelial cells as selecting, rather than as deleting.
The first striking observation of this study was the finding that positive selection of the 4.1-TCR in NOD mice
caused a dramatic acceleration of the onset of IDDM (by
~3 mo). This was surprising to us considering the observations of Katz et al. in transgenic NOD mice expressing another beta cell-specific and I-Ag7-restricted transgenic TCR
(BDC-2.5) (32, 41). BDC-2.5 mice do not develop an accelerated onset of diabetes (32, 41) and, when housed under specific pathogen-free conditions (41), develop diabetes
less frequently than 4.1-NOD mice (15 versus 74%). We are confident that beta cell destruction in 4.1-NOD mice
was triggered and/or effected by CD4+ T cells expressing
the 4.1-TCR since recombination activating gene 2-deficient 4.1-NOD mice, which cannot rearrange endogenous TCR genes, develop IDDM as early, and as frequently, as
recombination activating gene-2+ 4.1-NOD mice (Verdaguer, J., D. Schmidt, B. Anderson, A. Amrani, and P. Santamaria, manuscript in preparation). Most surprising, however, was the observation that 4.1 thymocytes undergo
deletion in diabetes-resistant H-2g7/b-, H-2g7/k-, H-2g7/q-,
and H-2g7/nb1-4.1-NOD mice by engaging antidiabetogenic I-A (I-Ab, I-Ak, I-Aq) and, possibly, I-E molecules (I-Ek,
I-Enb1) on thymic APCs, particularly since previous studies
did not find evidence for deletion of autoreactive T cells in
congenic or transgenic NOD mice expressing these MHC
class II molecules (7, 15, 16, 32, 33, 44). This deletion was
clearly not mediated by endogenous superantigens binding
to V11, since it was absent in TCR-
-transgenic and
nontransgenic mice expressing the deleting MHC haplotypes, and segregated as a single locus trait (MHC linked) in
backcross studies. Thus, the 4.1-TCR is unique in that it is
both highly diabetogenic and promiscuous.
Abrogation of I-Ab expression in TCR-transgenic I-A
b
(N × B) F1 mice clearly restored the positive selection of
the 4.1-TCR; however, it did not completely eliminate its
negative selection, as suggested by the greater percentage of
CD4
CD8
thymocytes in these mice versus age-matched
4.1-NOD mice. Partial deletion of transgenic thymocytes
in I-A
b- (N × B) F1 mice, which express the C57BL/6-derived I-A
b gene, may be a result of engagement of
I-A
g7/I-A
b complexes by the 4.1-TCR with an affinity/
avidity approaching the threshold for deletion, or perhaps,
to other tolerogenic factors unique to the C57BL/6 background. Similarly, thymocyte deletion in H-2g7/k 4.1-F1
and -F2 mice, which express both I-Ak and I-Ek molecules,
was clearly more efficient than that observed in I-Ak-transgenic
4.1-NOD mice, raising the possibility that thymocyte deletion in the former also involves the engagement of I-Ek
and/or I-E
k/I-E
g7 molecules by the 4.1-TCR. An alternative, but not mutually exclusive, possibility is that the
timing and levels of I-Ak expression in I-Ak-transgenic 4.1-NOD mice are different than the timing and levels of expression of endogenous I-Ak molecules in H-2g7/k 4.1-F1
or -F2 mice. In this respect, it is worth noting that, unlike
wild-type H-2k mice, I-Ak-transgenic NOD mice do not
express I-Ak molecules on bone marrow cells (they do,
however, express them on bone marrow-derived APCs),
and that the thymic cortical epithelial cells of I-Ak-transgenic NOD mice express lower levels of I-Ak than those of
H-2k mice (13). Whatever the explanation for these differences, the MHC-induced T cell tolerance in H-2g7/b and
H-2g7/k 4.1-F1 and -F2 mice was complete. Deletion of the
4.1-TCR was also observed in 4.1-NOD.H-2g7/nb1 mice
(complete deletion), which express I-Anb1 and I-Enb1 molecules, and in 4.1-NOD.H-2g7/q mice (partial deletion),
which express I-Aq, but not I-E, molecules. It thus appears
that, unlike many other MHC class II-restricted TCR
specificities (32, 38, 40), the highly diabetogenic 4.1-TCR
can engage several distinct MHC class II molecules in the
thymus with totally different consequences, i.e., positive
selection or dominant negative selection.
The extensive promiscuity of the 4.1-TCR raises a series
of important questions. How can 4.1 thymocytes recognize
so many different MHC class II molecules? Does the 4.1-TCR recognize each of these different molecules bound to
specific peptides? Or to a common peptide? Or, does it
bind to all of them regardless of the molecular nature of the
bound peptides? Is there anything unique about the molecular structure of the deleting I-A molecules that makes
them function as such? We do not yet have answers to these questions, but we have some clues. For example, we
know that the diabetogenic 4.1-TCR is not a classic alloreactive TCR; the MHC molecules that mediated deletion
of the 4.1-TCR when presented by thymic APCs did not
trigger proliferation of naive and preactivated CD4+ T cells
from 4.1-NOD mice when presented by peripheral APCs
from deleting F1 backgrounds (our unpublished data). Since
thymocyte tolerance is a more sensitive response than peripheral T cell activation (43), these results need not imply
that the putative peptide-MHC class II complexes that mediate thymic deletion in our system are expressed solely by
a specialized thymic APC subpopulation; they may also be
present on peripheral APCs, but may not be able to trigger
mature T cell proliferation. We also know that the I-A
chains of all the deleting I-A molecules that we have tested so far (I-Ab, I-Ak, and I-Aq) share residues at positions 57 (aspartic acid; Asp) and 61 (tryptophan). Interestingly, I-Ek
and I-Enb1 molecules, which are encoded on the deleting
H-2k and H-2nb1 haplotypes, respectively, have the same
residues at these two positions (49). I-Anb1, which is encoded on the deleting H-2nb1 haplotype (but may or may
not be engaged by the 4.1-TCR), is also Asp-57+ and, like
I-Ab, I-Ak, and I-Aq, has a bulky hydrophobic residue at
position 61 (phenylalanine). We are not certain as to whether
the presence of aspartic acid at I-E
and/or I-A
position
57, which is negatively associated with human and murine
IDDM (1, 2), is neccessary to trigger deletion of the 4.1-TCR. However, we know it is not sufficient, since I-As is also
Asp-57+, but it is nondeleting.
Whatever the specific residues involved, the unexpected
promiscuity of the highly pathogenic 4.1-TCR raises the
intriguing possibility that pathogenicity of autoreactive
TCRs and their ability to cross-react with different MHC
class II molecules are related phenomena. At the present
time, it is difficult to envision how promiscuity may lead to
pathogenicity. It is possible, however, that promiscuous autoreactive TCRs, like the 4.1-TCR, are primarily selected
(positively and/or negatively) by reaction with MHC residues rather than with peptide residues, as recently proposed
for alloreactive TCRs (50). Like the latter (50), these promiscuous autoreactive TCRs may then be able to engage a
larger range of peripheral self-peptide-selecting MHC
complexes above the affinity threshold required for mature T cell activation than nonpromiscuous autoreactive TCRs,
resulting in increased chances for pathogenicity. This
would predict that promiscuity would not be unusual
among those autoreactive TCRs with the highest pathogenic potential (i.e., those that trigger diabetes), and that
MHC molecules providing dominant resistance to a given
autoimmune disease would do so predominantly by removing the most pathogenic autoreactive T cells rather
than all autoreactive T cells, regardless of their pathogenicity (i.e., those recruited during amplification of the autoimmune response). This postulate would provide an explanation as to why the beta cell-reactive and I-Ag7-restricted
BDC-2.5-TCR of Katz et al. (32), which does not accelerate IDDM onset in NOD mice (32, 41), did not undergo tolerance in H-2g7/b F1 mice or in I-E-trangenic NOD
mice (32). It would also account for the presence of mildly
insulitogenic, but not diabetogenic, T cells in some congenic NOD.H-2g7/b, NOD.H-2g7/q, or NOD.H-2g7/nb1 mice
(7, 15, 16), I-Ad-transgenic NOD mice (18), NOD mice
reconstituted with bone marrow from I-E-transgenic
NOD mice (33), and I-Ak-transgenic NOD mice (12, 13).
In evaluating the relevance of our findings in 4.1 mice with respect to the MHC-linked susceptibility and resistance to spontaneous IDDM in non-TCR-transgenic mice, one should consider two additional aspects. The first aspect has to do with the pathophysiological consequences of thymocyte selection in 4.1 mice. There was an absolute correlation between deletion of thymocytes in H-2g7/b and H-2g7/k 4.1-F2 mice and resistance to insulitis and diabetes; deleting offspring of the second backcross of 4.1-F1 mice to NOD mice never developed insulitis, whereas all their nondeleting littermates developed moderate to severe insulitis, and ~50% of those which inherited two H-2g7 haplotypes became diabetic (a significant percentage given the polygenic nature of murine IDDM; reference 2). The same was true for 4.1-NOD.H-2g7/q and 4.1-NOD.H-2g7/nb1 mice, which developed mild periinsulitis or no insulitis, respectively, and did not become diabetic. It is noteworthy that these observations are highly reminiscent of the resistance of NOD.H-2g7/b, NOD.H-2g7/q, or NOD.H-2g7/nb1-congenic mice, and of I-E-, I-Ad-, and I-Ak-transgenic NOD mice to insulitis and/or IDDM (6, 16). The second aspect relates to the geography and timing of negative selection of diabetogenic thymocytes in 4.1 mice. As in other models of thymocyte selection (51, 52), negative selection of thymocytes in 4.1-F1 mice was preceded by positive selection and was mediated by hematopoietic cells. This observation also accords with the widely accepted notion that the factors underlying the MHC-linked resistance to spontaneous IDDM predominantly reside in the bone marrow (17, 33, 44). Taken together, then, these data lend strong support to the hypothesis that the MHC-associated resistance to insulitis and IDDM of non-TCR-transgenic mice (and perhaps humans) may also result from negative selection of highly diabetogenic thymocytes.
Nonetheless, deletion of diabetogenic thymocytes does
not account for all the protection afforded by MHC class II
molecules. It appears, also, that in the absence of additional
susceptibility factors and/or in the presence of additional
resistance factors, positive selection of autoreactive T cells
bearing pathogenic TCRs does not automatically imply autoreactivity. These interpretations stem from three observations. First, I-Ak/4.1-NOD mice developed insulitis but
not diabetes, suggesting that engagement of the partially
deleting I-Ak molecules by the few 4.1 T cells that escaped
deletion/anergy might have fostered their differentiation
into nondiabetogenic Th-cells (perhaps of the Th2 type, or
of the Th1 type, but incapable of effecting beta cell damage). Second, unlike their H-2g7 littermates, the H-2g7/k
4.1-F2 mice that did not tolerate the 4.1-TCR (40%) only
developed nondiabetogenic insulitis. The reasons behind
the diabetes resistance of these mice may be similar to those
argued above for I-Ak/4.1-NOD mice. Alternatively, progression from moderate to severe insulitis and overt diabetes may require a double dose of the beta cell antigen-I-Ag7
complex(es) targeted by diabetogenic T cells, as proposed
in nontransgenic systems (16). Finally, I-Ab-deficient 4.1- (N × B) F1 mice developed insulitis but remained diabetes-resistant, and 4.1-(N × S) F1 mice, which also exported functional 4.1 T cells to the periphery, developed
neither diabetes nor insulitis. Whether the diabetes resistance of these mice is due to I-Ag7 hemizygosity, to the
presence of additional C57BL/6- or SJL-derived resistance
elements, or to "dilution" of NOD-derived recessive susceptibility factors, is currently under investigation.
Whatever the nature and function of these additional protective elements turn out to be, our observations in 4.1-TCR-transgenic mice have unearthed what, at the outset of this study, seemed to be an unlikely proposition, namely, the existence of a relationship between thymocyte selection of highly pathogenic T cells and the MHC-linked susceptibility and resistance to one spontaneous autoimmune disease, IDDM. Our results do not imply that deletion is the only mechanism for autoimmune disease protection afforded by MHC class II molecules, but explain how protective MHC genes carried on one haplotype (e.g., I-Ab in mice and DQA1*0102/DQB1*0602 in humans) can override the genetic susceptibility provided by MHC genes carried on a second haplotype (e.g., I-Ag7 in mice and DQA1*0301/DQB1*0302 in humans).
Address correspondence to Dr. Pere Santamaria, Department of Microbiology and Infectious Diseases, Faculty of Medicine. The University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1. Phone: 403-220-8735; FAX: 403-270-8520; E-mail:psantama{at}acs.ucalgary.ca
Received for publication 27 May 1997.
J. Verdaguer was supported by a postdoctoral fellowship from CIRIT (Comissió Interdepartamental de Reçerca i Innovació Tecnològica, Generalitat de Catalunya, Barcelona, Spain). P. Santamaria is a scholar of the Medical Research Council of Canada. This research was supported by a grant from the Medical Research Council of Canada.We thank T. Mak for providing CD8- mice; G. Morahan and J.F.A.P. Miller for providing I-Ak-transgenic NOD mice; M. Davis and S. Hedrick for TCR shuttle vectors; M. Nagata and J.-W. Yoon for providing NY4.1 cells and for exciting discussions; T. Utsugi for advice on histopathology, for providing rIL-2,
and for helpful suggestions; R. Sangha and R. Sparkes for help with histology; R. Dawson and L. Mock for excellent animal care; L. Bryant for excellent assistance with flow cytometry; H. Kominek for editorial assistance.
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