? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
Priming and effector dependence on insulin
B:9–23 peptide in NOD islet autoimmunity
Maki Nakayama,1 Joshua N. Beilke,1 Jean M. Jasinski,1 Masakazu Kobayashi,1
Dongmei Miao,1 Marcella Li,1 Marilyne G. Coulombe,1 Edwin Liu,1
John F. Elliott,2 Ronald G. Gill,1 and George S. Eisenbarth1
1Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center (UCHSC), Aurora, Colorado, USA.
2Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
In patients who develop organ-specific autoimmunity, an impor-
tant question is why only certain organs are targeted (1–3). We
believe that there may be single or multiple primary autoantigens
that are specific to a target organ and trigger the autoimmune
response, although there are examples of autoimmunity directed
against autoantigens expressed in multiple tissues for some organ-
specific autoimmune diseases. In type 1A diabetes, which is a pan-
creatic β cell–specific autoimmune disease, insulin and the islet-
specific glucose-6-phosphatase catalytic subunit–related protein
(IGRP) have been identified as β cell–specific autoantigens (4–6).
For type 1A (immune-mediated) diabetes, insulin has been pro-
posed as a key autoantigen (7, 8). Insulin has been shown to be a
target of both T and B lymphocytes with the demonstration of both
insulin autoantibodies (IAAs) and insulin-reactive T cells in patients
with type 1A diabetes, in prediabetic subjects, and in animal models
such as the NOD mouse (9–13). IAAs are frequently detected in sera
of patients and NOD mice before and after diabetes onset (14, 15).
Kent et al. recently reported that CD4+ T cell clones isolated from
pancreatic lymph nodes of patients with type 1 diabetes react with
an insulin A chain peptide (amino acids 1–15) restricted by DR4 (16).
In NOD mice, both CD4+ and CD8+ T cells derived from pancreatic
lymph nodes and pancreatic islets show insulin reactivity (17, 18),
and insulin-reactive T cell clones established from pancreatic islets
show cytotoxicity to pancreatic β cells (19, 20). The observations
that IAA levels are highest in the youngest children developing dia-
betes and usually precede the development of other autoantibodies
(21, 22) and that insulin-reactive T cells are preferentially detected
in younger NOD mice (23) have led to the hypothesis that insulin
may be a crucial autoantigen in initiating islet autoimmunity. Sup-
porting this hypothesis are the separate findings of Jaeckel et al. and
French et al. that targeting T cells reacting with insulin results in
dramatic prevention of type 1 diabetes in NOD mice (24, 25).
Among the insulin epitopes recognized by NOD islet–infiltrat-
ing T cells, insulin B chain amino acids 9–23 (insulin B:9–23) is
reported to be a key peptide (26). Anti–insulin B:9–23 CD4+ TCR
transgenes can induce (BDC12-4.1) (27) or prevent diabetes (2H6)
(28). We recently reported that dual Ins1 and Ins2 knockout NOD
mice that express a mutated insulin transgene — the wild-type
tyrosine at amino acid 16 of the insulin B chain replaced with ala-
nine — are protected from anti-islet autoimmunity and prevent
diabetes (29). These mice lack both endogenous insulin genes and
were rescued from an absolute insulin deficiency by a transgene
expressing the alanine-to-tyrosine mutation in Ins2 under control
of the Rat insulin promoter. Mice carrying the transgene express
an altered form of Ins2 (alanine at position 16 of insulin B chain;
B16:A). These results suggest that insulin, and specifically the
insulin B:9–23 sequence, may be essential for the initiation of the
spontaneous diabetogenic autoimmune process of NOD mice.
Given the prevention of diabetes in NOD mice lacking native
insulin B:9–23 sequences, one obvious question arises: In which
tissues would expression of the native insulin sequence restore
anti-insulin autoimmunity? Besides the target pancreatic β
cells, preproinsulin is reported to be expressed in thymic epithe-
lial, thymic dendritic, and a subset of peripheral dendritic cells,
potential sites at which preproinsulin expression may modulate
insulin autoimmunity (30–32). With the creation of multiple
NOD strains lacking native insulin genes, including strains with
the NOD/SCID mutation, it is now possible to transplant islets
Nonstandard?abbreviations?used: B16:A, alanine at position 16 of insulin B chain;
B16:A-dKO, double Ins1 and Ins2 knockout NOD mice with a mutated B16:A prepro-
insulin transgene; B16:Y, tyrosine at position 16 of insulin B chain; B16:Y-dKO, dou-
ble Ins1 and Ins2 knockout NOD mice with a mutated B16:Y preproinsulin transgene;
IAA, insulin autoantibody; insulin B:9–23, insulin B chain amino acids 9–23.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 117:1835–1843 (2007). doi:10.1172/JCI31368.
1836? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
or bone marrow with native (tyrosine at position 16 of insulin B
chain; B16:Y) or altered B16:A insulin sequences to assess their
influence on IAA production, insulitis, and diabetes. In this study
we found that transplanted islet cells, but not bone marrow cells,
expressing the native B16:Y insulin sequence restored anti-insulin
autoimmunity in mice that lacked native insulin genes. In addi-
tion, immunization with the native B16:Y insulin B:9–23 peptide,
but not the mutated B16:A insulin B:9–23 peptide, rendered CD4+
T cells able to rapidly transfer anti-insulin autoimmunity and dia-
betes to NOD/SCID mice when the recipient mouse also had the
native B16:Y insulin B:9–23 sequence in its islets.
Transplantation of bone marrow with native insulin genes does not restore insu-
lin autoimmunity. Preproinsulin is reported to be expressed in thymic
epithelial, thymic dendritic, and a subset of peripheral dendritic cells
(30–32). In order to determine whether native insulin gene expression
in hematopoietic cells is sufficient to induce anti-islet autoimmunity,
1 × 107 bone marrow cells derived from 4-week-old wild-type B16:Y
NOD mice were transplanted into irradiated 4-week-old double Ins1
and Ins2 knockout mice with a mutated B16:A preproinsulin trans-
gene (B16:A-dKO mice). None of the 5 B16:A-dKO recipients devel-
oped IAAs (Figure 1A). In contrast, 6 of 7 B16:Y NOD recipients of
wild-type B16:Y bone marrow expressed anti-IAAs (Figure 1B). Thus,
bone marrow cells carrying the native B16:Y insulin sequence do not
restore anti-insulin autoimmunity in B16:A-dKO mice.
Transplantation of native B16:Y insulin–bearing islets induces develop-
ment of IAAs. Using islet transplantation, we sought to determine
whether native insulin expression in transplanted islets is suffi-
cient to induce anti-islet autoimmunity in B16:A-dKO mice. Two
hundred islets from native B16:Y insulin–bearing NOD/SCID mice
were transplanted under the kidney capsule of 4-week-old B16:A-
dKO mice. High IAA levels developed within 3 weeks of transplan-
tation in 11 of 13 B16:Y islet recipients (Figure 2A). In contrast, 1
of 8 mice transplanted with B16:A-dKO islets developed IAAs after
transplant, and only at a low titer (P < 0.0001; Figure 2B).
Therefore, islet expression of insulin with the native insulin
B:9–23 sequence, even with ectopic expression under the
kidney capsule, is sufficient to rapidly induce IAAs in NOD
mice lacking native insulin B:9–23 sequences.
Induction of insulitis by B16:Y islet transplant. The B16:Y islet
grafts (Figure 3, A and C), but not the B16:A-dKO islet grafts
(Figure 3, B and D), were completely destroyed by lympho-
cytic infiltration. This suggests that wild-type insulin, with
the native B16:Y insulin B:9–23 sequence, in transplanted
islet grafts is recognized by lymphocytes of B16:A-dKO mice
and that these lymphocytes are able to kill the native insu-
lin–positive islet cells. In addition to analyzing the histology
of the transplanted islet grafts, we also analyzed insulitis of
the endogenous pancreatic islets of the B16:A-dKO recipi-
ent mice. All 5 recipients of B16:Y islets that were sacri-
ficed fewer than 20 weeks after transplant showed insulitis
(Figure 3E) but not recipients of B16:A islets (Figure 3F).
Pancreatic insulitis, however, was transient: 5 of 10 mice
receiving the same B16:Y islet transplants that were sacri-
ficed more than 35 weeks after islet transplant had insu-
litis. Insulitis at less than 20 weeks after transplant was
significantly more severe than at more than 30 weeks after
transplant (P = 0.02, c2 test; Figure 3G). Pancreatic insulitis
of B16:A-dKO mice receiving B16:Y islets was more severe
than that of mice receiving B:16A-dKO islet grafts (P < 0.03, c2 test;
Figure 3G). Importantly, insulitis of mice receiving B16:A-dKO
islets did not differ from unmanipulated B16:A-dKO mice, which
did not receive islet transplants. These results indicate that tran-
sient insulitis of the B16:A pancreas induced by islet transplants is
specific to the native B16:Y insulin sequence of donor islets.
While transplantation of B16:Y islets into B16:A-dKO mice
induced not only graft destruction but also lymphocytic infil-
tration of the recipients’ pancreatic islets, diabetes was not
induced. None of the B16:A-dKO mice that received B16:Y islet
transplants became diabetic (0 of 11, followed up to 35 weeks).
Even though insulitis was induced in the host pancreas, it was
Bone marrow transplant with native B16:Y insulin genes is not sufficient to
restore insulin autoimmunity. Four-week-old irradiated B16:A-dKO (A) or wild-
type B16:Y mice (B) were transplanted (Tx) with bone marrow derived from
4-week-old wild-type NOD mice. Native insulin-positive B16:Y bone marrow
cells did not induce IAAs in B16:A-dKO mice but did induce IAAs in B16:Y
mice. Each line represents an individual recipient. The y axis represents the
micro-IAA assay (mIAA) index in log scale.
Development of IAAs after B16:Y NOD/SCID islet transplant. (A) B16:
A-dKO mice, when transplanted with native B16:Y insulin NOD/SCID
islets, developed IAAs. (B) Mice receiving B16:A-dKO islets did not
express insulin antibodies after transplant, with the exception of 1
mouse. Each line represents an individual mouse. The y axis repre-
sents the micro-IAA assay index in log scale.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
not sufficient to cause diabetes in mice whose pancreatic islets
lacked the native insulin B:9–23 sequence.
Splenocytes from B16:A-dKO recipients of native B16:Y insulin–bearing
islet transplants are diabetogenic. In order to evaluate whether lympho-
cytes from B16:A-dKO mice with B16:Y NOD/SCID islet transplant
can cause diabetes if the host pancreas has the native B16:Y insulin
sequence, we transferred splenocytes from B16:A-dKO mice that
had received islet transplants into wild-type B16:Y NOD/SCID or
B16:A-dKO NOD/SCID mice (Figure 4A). Splenocytes from B16:A-dKO
mice transplanted with B16:Y NOD/SCID islets rapidly induced dia-
betes in B16:Y NOD/SCID mice (5 of 6; Figure 5A), which indicates
that immunization with B16:Y islets generates splenocytes able to
recognize and destroy B16:Y pancreatic islets (i.e., induces autoim-
munity) and that B16:A-dKO recipients of B16:Y islets contain func-
tional native insulin–autoreactive lymphocytes in the absence of dis-
ease. In contrast, when splenocytes were transferred from B16:A-dKO
mice that received B16:A-dKO islets, diabetes occurred in 2 of 8 B16:
Y NOD/SCID recipients, at a later time after splenocyte transfer
(P < 0.01; Figure 5A). This is potentially consistent with the natural
late (i.e., independent of islet transplant) priming that occurred after
transfer of splenocytes into a B16:Y NOD/SCID host from unma-
nipulated B16:A-dKO mice (Figure 5A). In the opposing model, in
which the splenocyte recipients were B16:A-dKO NOD/SCID mice,
diabetes occurred in 2 of 5 mice receiving splenocytes from B16:Y
islet recipients (Figure 5B). In contrast, splenocytes from B16:A-dKO
mice transplanted with B16:A-dKO islets did not induce diabetes or
insulitis upon transfer to B16:A-dKO NOD/SCID mice (Figure 5B).
These results indicate that
the diabetogenic splenocytes
are induced by B16:Y islet trans-
plants, which can destroy β cells
in NOD/SCID mice with either
B16:Y or B16:A pancreatic islets.
Yet, B16:A islet transplants are
unable to generate diabetogenic
lymphocytes, for diabetes did
not develop whether the recipi-
ent NOD/SCID mouse had insu-
lin with B16:A or B16:Y. This is
consistent with the importance
of the native insulin B:9–23
sequence in activating diabeto-
genic anti-islet autoimmunity.
Immunization with insulin B:9–23
peptides induces IAAs but not insulitis.
To determine whether the provi-
sion of just the insulin B:9–23
peptide rather than islets with
B16:A versus B16:Y insulin is
sufficient to restore anti-islet
autoimmunity and diabetoge-
nicity, we immunized 4-week-old
B16:A-dKO mice with 100 μg of
B16:Y insulin B:9–23 peptide or
B16:A insulin B:9–23 peptide in
CFA (Figure 4B). More than 95%
(29 of 30) of B16:A-dKO mice
immunized with B16:Y insulin
B:9–23 peptide developed anti-
IAAs (data not shown). These
antibodies were not absorbed by control tetanus toxin peptide or
B:9–23 peptides, but were absorbed by insulin (Figure 6A). The
absorbing insulin contained the B16:Y insulin B:9–23 sequence;
therefore, insulin is an autoantigen corresponding to this peptide.
All B16:A-dKO mice (15 of 15) immunized with the B16:A insulin
B:9–23 peptide similarly produced IAAs that were not absorbed
by the peptides, but were absorbed by insulin (Figure 6B). As
expected, B16:Y NOD mice immunized with B16:Y insulin B:9–23
peptide also produced IAAs (Figure 6C).
Despite the induction of IAAs, immunization with both insulin
B:9–23 peptides did not induce lymphocytic infiltration of pancre-
atic islets analyzed 10–15 weeks after immunization (Figure 6E).
This suggests that provision of the insulin B:9–23 peptide itself
is insufficient to induce inflammation of endogenous pancreatic
islets lacking the B16:Y insulin B:9–23 sequence. Correlating with
the lack of insulitis, none of the mice progressed to diabetes.
Immunization with B16:Y insulin B:9–23 peptide–induced diabetogenic
splenocytes induces diabetes in NOD/SCID recipients. As outlined in Fig-
ure 4B, immunization with insulin B:9–23 peptides did not induce
insulitis in B16:A-dKO mice. Nevertheless, when we transferred
splenocytes from B16:A-dKO mice immunized with B16:Y peptide,
recipient B16:Y NOD/SCID mice, but not B16:A-dKO NOD/SCID
mice, developed IAAs and diabetes. As shown in Figure 6D and 7A,
splenocytes from B16:A-dKO mice immunized with B16:Y insulin
B:9–23 peptide induced high levels of IAAs within 10–20 days of
transfer to B16:Y NOD/SCID mice. Splenocytes from B16:A-dKO
mice immunized with B16:A insulin B:9–23 peptide did not rap-
Native B16:Y islet transplants induce graft insulitis and severe transient insulitis in endogenous pancre-
atic islets. (A–D) B16:Y NOD/SCID (A and C) and B16:A-dKO (B and D) islets were transplanted under
the kidney capsule of B16:A-dKO mice. (A and B) H&E stain. (C and D) Insulin stain. Two weeks after
transplant, the B16:Y NOD/SCID islet graft showed very little insulin staining and severe lymphocytic
infiltration, whereas the B16:A-dKO islet graft was intact. (E and F) Endogenous pancreatic islets of B16:
Y islet recipients (E), but not B16:A-dKO islet recipients (F), showed marked lymphocytic infiltration 18
weeks after islet transplant. Original magnification, ×100 (A, B, E, and F); ×200 (C and D). (G) More than
10 pancreatic islets from each B16:A-dKO mouse receiving B16:Y NOD/SCID or B16:A-dKO islet trans-
plant were evaluated for lymphocytic infiltration less than 20 weeks or more than 30 weeks after transplant
(n = 5–10). Pancreatic islets from an age-matched unmanipulated B16:A-dKO mouse were also evaluated.
The y axis represents the mean ± SD of the insulitis score.
1838? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
idly induce IAAs in B16:Y NOD/SCID (Figure 7C) or B16:A-dKO
NOD/SCID recipients (Figure 7D). Even splenocytes from mice
immunized with B16:Y insulin B:9–23 peptide did not rapidly
induce IAAs in B16:A-dKO NOD/SCID mice (Figure 7B).
As shown in Figure 8, only the combination of immunization with
B16:Y insulin B:9–23 peptide in B16:A-dKO mice followed by sple-
nocyte transfer to B16:Y NOD/SCID mice caused diabetes in the
recipient. In contrast, these same splenocytes did not induce diabe-
tes in the B16:A-dKO NOD/SCID recipients, which suggests that
pancreatic islet expression of native B16:Y insulin is necessary for
diabetes following immunization with just the peptide in contrast to
immunizing with (i.e., transplanting) whole islets. Splenocytes from
mice immunized with B16:A insulin B:9–23 peptide did not rapidly
transfer diabetes in B16:Y NOD/SCID mice (2 of 8 mice developed
diabetes at 65 and 110 days after transfer; Figure 8; as seen in B16:Y
recipients of splenocytes from nonimmunized mice; see Figure 5A).
Taken together, our results suggest the 2 requirements for efficient
insulin B:9–23 peptide induction of diabetes are priming by the B16:
Y insulin B:9–23 sequence and splenocytes encountering native B16:
Y insulin in the pancreatic islets of the recipient.
B16:Y insulin B:9–23–primed CD4+ T cells are sufficient to transfer
IAA production. Immunization of B16:A-dKO splenocyte donors
with native B16:Y peptide is essential for the rapid induction of
IAAs after splenocyte transfer to B16:Y NOD/SCID mice. Because
the spleen contains CD4+ T cells and non-CD4+ splenic cells (e.g.,
macrophages, B lymphocytes, and dendritic cells) that may take
up B16:Y peptide, we set out to determine which of these subsets
of cells were responsible for inducing IAA production. We trans-
ferred CD4+ T cells isolated from the spleens of B16:A-dKO mice
immunized with B16:Y insulin B:9–23 peptide to NOD/SCID mice.
Because NOD/SCID mice lack B lymphocytes, we also simultane-
ously transferred CD4-depleted splenocytes derived from nonim-
munized B16:A-dKO mice. As shown in Figure 9A, splenic CD4+ T
cells from the insulin B:9–23 peptide–immunized mice with non-
CD4+ cells from nonimmunized mice upon transfer were sufficient
to induce IAAs. In contrast, transfer of non-CD4+ splenocytes from
immunized mice together with CD4+ splenocytes from nonim-
munized mice failed to induce IAAs (Figure 9B), even though the
immunized donor mice had IAAs. These results indicate that the
induction of IAAs after splenocyte transfer is dependent on CD4+ T
cells from the B16:Y insulin B:9–23 peptide–immunized mouse and
non-CD4+ splenocytes do not induce anti-IAAs upon transfer.
Provision of the B16:Y insulin B:9–23 sequence by transgenesis induces IAAs
and insulitis. In order to explore whether induction of IAAs following
provision of B16:Y insulin B:9–23 sequences to a B16:A-dKO mouse
is dependent upon postnatal B:9–23 exposure, we evaluated an addi-
tional model for induction of IAAs. We created double Ins1 and Ins2
knockout NOD mice with a mutated B16:Y preproinsulin transgene
Summary of experiments with transfer of splenocytes into NOD/SCID recipients for mice immunized with islets (A) or insulin B:9–23 peptides
(B). DM, diabetes mellitus.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
(B16:Y-dKO mice) driven off the same promoter as our mutated B16:
A preproinsulin transgene. Introducing this transgene with the B16:Y
insulin B:9–23 sequence into B16:Y-dKO mice resulted in expression
of IAAs (Figure 10A). More than half of B16:Y-dKO mice (5 of 10)
developed high levels of IAAs, whereas 2 of 31 B16:A-dKO mice devel-
oped IAAs at low levels (P < 0.01). In addition, B16:Y-dKO mice devel-
oped insulitis as severe as that of age-matched wild-
type NOD mice, whereas age-matched B16:A-dKO
mice did not (P < 0.01; Figure 10, B–D). We evalu-
ated mice for islet infiltration between 10 and 22
weeks of age. Most B16:A-dKO mice did not have
any lymphocytic infiltration, and 2 of 9 mice had
peri-islet infiltration, which was observed in less
than 20% of the islets in individual mice. In con-
trast, all of the 4 B16:Y-dKO mice developed severe
insulitis, and 50%–70% of the islets demonstrated
intraislet infiltration. These results indicate that
even genetically induced expression of B16:Y pro-
insulin restores anti-islet autoimmunity as well as
postnatal provision via islet transplant.
We have evaluated 3 phenotypes (anti-IAA, insu-
litis, and development of diabetes) in multiple
NOD-derived animal models by using varying
combinations of islet transplants with different insulin B:9–23
sequences and corresponding insulin B:9–23 sequences in the recipi-
ent pancreatic islets (Tables 1 and 2). Our results highlight a dramat-
ic dependence on the presence or absence of the native B16:Y insu-
lin sequence, consistent with the hypothesis that the insulin B:9–23
peptide is a key determinant of NOD autoimmune diabetes and that
Rapid induction of diabetes with splenocytes from mice transplanted with B16:Y islets
transferred into NOD/SCID mice with B16:Y. Splenocytes from B16:A-dKO mice that
received B16:Y NOD/SCID islets, B16:A-dKO islets, or no transplant (unmanipulated)
were transferred into wild-type B16:Y NOD/SCID mice (A) or B16:A-dKO NOD/SCID mice
(B). P < 0.01, B16:Y islets versus B16:A-dKO islets in B16:Y NOD/SCID recipients.
Development of IAAs but not insulitis by immunization with insulin B:9–23 peptide. (A–D) Serum from B16:A-dKO mice immunized with native
B16:Y insulin B:9–23 peptide (A), B16:A-dKO mice immunized with altered B16:A insulin B:9–23 peptide (B), wild-type B16:Y NOD mice
immunized with native B16:Y insulin B:9–23 peptide (C), and wild-type B16:Y NOD/SCID mice that received splenocytes from insulin B:9–23
peptide–immunized B16:A-dKO mice (D) was incubated with I125-insulin in the presence of tetanus toxin peptide (TT), native B16:Y insulin B:9–23
peptide, B16:A insulin B:9–23 peptide, or human insulin. Each line represents an individual mouse. (E) More than 10 pancreatic islets from each
B16:A-dKO mouse immunized with B16:Y insulin B:9–23 peptide (n = 10), B16:A insulin B:9–23 peptide (n = 4), or PBS (n = 10) in CFA were
evaluated for lymphocytic infiltration. The y axis represents the mean ± SD of the insulitis score.
1840? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
islet expression of the native insulin sequence, but not expression in
bone marrow–derived cells, induces anti-islet autoimmunity. Even
transgenic provision of the B16:Y insulin B:9–23 sequence restored
IAAs and insulitis in B16:A-dKO mice. Thus, postnatal exposure to
the native B16:Y proinsulin sequence in B16:A-dKO mice was not
essential for inducing anti-islet autoimmunity, although it is pos-
sible that postnatal exposure may enhance the immune response.
It is noteworthy that islets differing at a single amino acid of the
insulin molecule (i.e., B16:Y versus B16:A) differed in their ability to
induce diabetogenic T lymphocytes (Tables 1 and 2).
Transplantation of native insulin–positive bone marrow cells did
not induce the development of IAAs in B16:A-dKO mice. This sug-
gests that native insulin expression in hematopoietic cells, espe-
cially antigen-presenting cells such as dendritic cells, may not be
sufficient to initiate anti-insulin autoimmunity. However, there is
a caveat that the level of insulin expression in such hematopoietic
cells may not have been sufficient to break tolerance compared
with insulin expression in islets.
It was easier to induce IAAs than insulitis in B16:A-dKO mice,
and diabetes only occurred upon transfer of splenocytes from
immunized B16:A-dKO hosts into NOD/SCID mice. Thus, simply
immunizing B16:A-dKO mice with islets or peptide with the B16:
Y insulin B:9–23 sequence was not sufficient to induce diabetes
(Table 1), which was not surprising given the lack of the B16:Y
insulin B:9–23 sequence in the pancreatic islets of the recipient
mouse. Of note, only transplantation of islets bearing the B16:Y
insulin B:9–23 sequence, but not immunization with the B16:Y
insulin B:9–23 peptide, induced insulitis. Islets may be more effi-
cient inducers of pathogenic T cells than peptides for many rea-
sons. Islet transplant provides the whole preproinsulin molecule
(33), other antigens such as IGRP (5, 34, 35), and per-
haps specific danger signals related to the surgical pro-
cedure not provided by CFA (36). As shown in Table 2,
when transferring splenocytes from islet-transplanted
mice to NOD/SCID recipients, induction of insulitis
and rapid diabetes required donor islets to have the
B16:Y insulin B:9–23 sequence, and induction of dia-
betes with peptide immunization required the B16:Y
insulin B:9–23 sequence in both the peptide and the
NOD/SCID recipient. Thus, priming by B16:Y insulin
B:9–23 was sufficient to initiate anti-islet autoimmu-
nity for disease transfer to NOD/SCID, and diabetes
induction was specific to the B16:Y insulin B:9–23
sequence in pancreatic islets. We believe it is likely that
in addition to CD4+ T cells, CD8+ T cells participate in
the generation of diabetes as shown through the previ-
ously described insulin B:15–23 CD8 epitope (37).
Our studies indicate that it is CD4+ T cells from the
splenocytes of immunized donor mice that transfer
production of IAAs to NOD/SCID recipients. CD4+
T cells from a nonimmunized splenocyte donor were
unable to induce IAAs in NOD/SCID recipients. Anti-
IAAs appeared within 20 days, but only when the
recipient mouse islets had the B16:Y insulin B:9–23
sequence. The same was true for the rapid induction
of diabetes with insulin B:9–23 peptide immuniza-
tion and splenocyte transfer. The speed with which
IAAs appeared suggests that B lymphocytes of even
the nonimmunized B16:A donor mice exist, awaiting
nontolerant CD4+ T lymphocytes (i.e., B16:Y primed)
to help rapidly produce IAAs. Alternatively, the transferred B lym-
phocytes may rapidly acquire insulin with native insulin B:9–23
sequences from the B16:Y NOD/SCID host (38). Of note, the IAAs
were not simply antibodies to the insulin B:9–23 peptide and were
not absorbed with the peptide, but were absorbed with intact insu-
lin, similar to the IAAs of regular NOD mice or NOD and Balb/c
mice directly immunized with B:9–23 peptide (39). The ability of
CD4+ T cells from B16:Y insulin B:9–23–immunized splenocyte
donors alone to induce IAAs and diabetes should provide an assay
to further define these pathogenic helper T lymphocytes.
Only wild-type B16:Y NOD/SCID mice receiving splenocytes of B16:A-dKO mice
immunized with native B16:Y insulin B:9–23 peptide rapidly produce IAAs. Spleno-
cytes from B16:A-dKO mice immunized with native insulin B:9–23 peptide (A and B)
or mutated B16:A insulin B:9–23 peptide (C and D) were transferred to wild-type B16:
Y NOD/SCID (A and C) or B16:A-dKO NOD/SCID mice (B and D). IAAs were mea-
sured weekly after splenocyte transfer. Each line represents an individual mouse.
Development of diabetes after splenocyte transfer from insulin B:9–23
peptide–immunized B16:A-dKO mice. Splenocytes from mice immunized
with native B16:Y insulin B:9–23 peptide, but not with mutated B16:A
insulin B:9–23 peptide, rapidly transferred diabetes to wild-type B16:Y
NOD/SCID mice. B16:A-dKO NOD/SCID mice did not develop diabetes
following transfer of splenocytes from mice immunized with either native
B16:Y insulin B:9–23 or mutated B16:A insulin B:9–23 peptides.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
A potentially unique feature of the insulin B:9–23 peptide is
that TCR recognition of this epitope appears to be primarily
determined by specific conserved Vα and Jα sequences without
defined conservation of the N region of the α chain or TCR β
chain (40). Recognition with such a simplified TCR motif raises
the possibility that there may be a high precursor frequency of
thymic T cells reacting with insulin B:9–23. Insulin is a mol-
ecule that circulates at low concentrations but is expressed at
extremely high concentrations within pancreatic β cells (41).
The general hypothesis we are pursuing is that insulin B:9–23
peptide is recognized by a low-stringency common TCR, which
increases the ease of breaking CD4+ T cell tolerance to insulin,
and that such loss of tolerance leads to production of IAAs and
specific β cell destruction.
Our present results indicate that loss of tolerance to
the B16:Y insulin B:9–23 sequence at sites distinct from
the pancreas and pancreatic lymph node (i.e., renal
capsule or subcutaneous sites) can rapidly engender
diabetogenic T lymphocytes and anti-IAAs. This raises
the possibility that environmental mimotopes of such a
peptide may contribute to diabetes by activating CD4+ T
cell reactivity to a primary autoantigen. Once such toler-
ance to insulin or other islet molecules is lost, additional
autoantigens are likely to be targeted. Whether there
are additional epitopes of insulin or other molecules
as central to the development of diabetes in the NOD
mouse warrants further study. At present, it is unknown
whether there exists a similar dominant epitope of insu-
lin or another islet molecule for human type 1 diabetes.
Our findings suggest that it may be fruitful to search for
such a determinant, especially in individuals with a fixed
HLA, such as DR3/DR4 heterozygous diabetics, which
comprise 30%–50% of patients with type 1 diabetes.
Mice. B16:A-dKO mice were established as previously described
(42). Briefly, Ins1 and Ins2 knockout NOD mice were separately
established by breeding the original insulin knockouts (kindly
provided by J. Jami, INSERM, Paris, France; ref. 43) onto NOD/Bdc
mice using speed congenic techniques (44). Mutated B16:A
NOD mice were produced by microinjection of mutated B16:A
Ins2 cDNA constructs ligated to rat insulin 7 promoter (pRIP7)
directly into NOD fertilized eggs (42). Ins1 knockouts, Ins2
knockouts, and B16:A NOD mice were combined and geno-
typed for the native insulin genes and the mutated insulin
transgene (42). Native B16:Y Ins2 cDNA constructs ligated to
pRIP7 were created by directly replacing nucleotides (GCC for
alanine) in the vector carrying B16:A transgene with TAC for
tyrosine using site-directed mutagenesis (Stratagene) and were microinject-
ed into NOD fertilized eggs. B16:Y-dKO mice were established in the same
manner as B16:A-dKO mice (42). NOD/SCID mice (NOD.CB17-Prkdcscid/J)
were purchased from The Jackson Laboratory (stock no. 001303). B16:A-dKO
mice were crossed with NOD/SCID mice, and (B16:A-dKO × NOD/SCID)
F1 mice were intercrossed to obtain B16:A-dKO NOD/SCID mice. The
NOD/SCID mutation was confirmed by PCR amplification of extracted
genomic DNA (forward primer, GACTAGAAAGCTAGAGAGCT; reverse
primer, AGTTATAACAGCTGGGTTGGC) followed by incubation with Alu I
(Invitrogen) at 37°C for 2 hours. A final product of 239 bp was considered
wild type; the NOD/SCID mutation was 209 bp.
Only female mice were used for experiments. All mice were bred and
housed in a pathogen-free animal colony at the UCHSC Center for Com-
parative Medicine. All experiments were approved by the University of
Provision of the native B16:Y insulin B:9–23 sequence by transgenesis induces IAAs
and insulitis. (A) B16:A-dKO and B16:Y-dKO mice were measured for the development
of IAAs every 2–3 weeks between 4 and 30 weeks of age. Each symbol represents
the peak level of mIAA index for individual mice. B16:Y-dKO mice developed IAAs
(P < 0.01 versus B16:A-dKO). (B) Insulitis scoring of B16:A-dKO mice, B16:Y-dKO
mice, and wild-type NOD mice between 10 and 22 weeks of age. B16:Y-dKO
mice developed insulitis significantly more severe than did B16:A-dKO mice
(P < 0.01) and as severely as did wild-type NOD mice. (C and D) Pancreatic histology
(H&E; original magnification, ×100) of B16:A-dKO (C) and B16:Y-dKO (D) mice.
Development of IAAs in wild-type B16:Y NOD/SCID mice
after splenocyte transfer. (A) Splenic CD4+ T cells from B16:
A-dKO mice immunized with native B16:Y insulin B:9–23
peptide were transferred to wild-type B16:Y NOD/SCID
mice along with non-CD4+ splenocytes from unmanipulated
double insulin-knockout mice. (B) Splenic CD4+ T cells from
unmanipulated mice were transferred to wild-type B16:Y
NOD/SCID mice along with a non-CD4+ splenocyte popula-
tion from mice immunized with B16:Y insulin B:9–23 peptide.
Each line represents an individual mouse.
1842? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
Colorado at Denver Health Sciences Center (UCDHSC), (Denver, Colo-
rado, USA) Animal Care and Use Committee.
Bone marrow transplantation. Bone marrow was harvested from femurs
and tibias of 4-week-old NOD mice, and red blood cells were removed
using RBC lysis buffer (Sigma-Aldrich). To deplete mature T and B
cells, harvested bone marrow was incubated with CD4+, CD8+, and
B220+ MicroBeads (Miltenyi Biotec), and the labeled cells were mag-
netically depleted by AutoMACS (Miltenyi Biotec). After cell sorting, we
confirmed that the contamination of CD4+, CD8+, or B220+ cells was
below 5% by flow cytometric analysis using anti-CD4 (clone H129.19;
BD Biosciences — Pharmingen), anti-CD8 (clone 53-6.7; BD Biosci-
ences — Pharmingen), and anti-B220 (clone RA3-6B2; BD Biosciences
— Pharmingen) antibodies.
Four-week-old B16:A-dKO and wild-type B16:Y NOD mice received 450
rads of radiation from the cesium irradiator twice at a 4-hour intervals
prior to bone marrow injection. T cell– and B cell–depleted bone marrow
(1 × 107 cells) was intravenously injected.
Islet transplantation. Pancreatic islets were isolated from adult NOD/SCID
or B16:A-dKO mice by collagenase digestion (type V; Sigma-Aldrich) of
the pancreas and purification by Histopaque (Sigma-Aldrich). We grafted
4-week-old B16:A-dKO mice with 200 islets beneath the left kidney sub-
capsular space (45).
Peptide immunization. HPLC-purified B16:Y insulin B:9–23 peptide
(SHLVEALYLVCGERG) and the mutated B16:A insulin B:9–23 peptide
(SHLVEALALVCGERG) were purchased from Invitrogen. The peptides
were dissolved in sterile saline and adjusted to a neutral pH. Four-week-
old B16:A-dKO mice were subcutaneously immunized with 100 μg of B16:
Y insulin B:9–23 or B16:A insulin B:9–23 peptide
emulsified with CFA.
Adoptive splenocyte transfer experiments. Spleno-
cytes were isolated from B16:A-dKO mice trans-
planted with either B16:Y or B16:A-dKO islets
or immunized with either B16:Y insulin B:9–23
peptide or B16:A insulin B:9–23 peptide, and red
blood cells were removed using RBC lysis buffer
(Sigma-Aldrich). Splenocytes (3 × 107) were trans-
ferred intraperitoneally to 6- to 8-week-old B16:Y
NOD/SCID or B16:A-dKO NOD/SCID mice.
For the CD4+ T cell transfer experiment, CD4+ T
cells or non-CD4+ T cells were isolated from spleno-
cytes by depletion of magnetically labeled non-CD4+
T cells using CD4 T cell isolation kit or CD4+ T
cells using CD4 MicroBeads, respectively (Miltenyi
Biotec). We used AutoMACS (Miltenyi Biotec) for
cell sorting. An aliquot of isolated CD4+ T cells or
non-CD4+ T cells was stained with anti-CD4 antibody (clone H129.19, BD
Biosciences — Pharmingen), and the purity was assessed as greater than 95% by
flow cytometric analysis. CD4+ T cells from peptide-immunized B16:A-dKO
mice and non-CD4+ T cells from nonimmunized B16:A-dKO mice (1.5 × 107
and 4 × 107 cells, respectively) were intraperitoneally transferred to 6- to
8-week-old NOD/SCID mice. Similarly, CD4+ T cells from nonimmunized
mice and non-CD4+ T cells from immunized mice were intraperitoneally
transferred to NOD/SCID mice.
Measurement of micro-IAA. B16:A-dKO mice were bled before and every 2
weeks after transplantation or immunization to measure anti-IAAs. Recipi-
ent B16:Y NOD/SCID or B16:A-dKO NOD/SCID mice were bled before
and weekly after splenocyte transfer. To measure spontaneous develop-
ment of IAAs, B16:A-dKO and B16:Y-dKO mice were bled every 2–3 weeks
between 4 and 30 weeks of age. IAA levels were measured with the 96-well
filtration plate micro-IAA assay previously described (10) and expressed as
an index. A value of 0.01 or greater was considered positive.
To investigate the absorption by insulin B:9–23 peptide, 20,000 cpm
of human 125I-insulin (GE Healthcare) was incubated with 5 μl of serum
overnight along with 100 μg/ml of B16:Y insulin B:9–23 or B16:A insulin
B:9–23 peptide, precipitated with protein A/G sepharose (GE Health-
care), and counted in a TopCount beta counter (Packard).
Histology. The pancreata and islet grafts transplanted under kidney
capsules were fixed in 10% formalin and embedded in paraffin. Paraf-
fin-embedded tissue sections were stained with H&E, and sections from
islet grafts were also stained with polyclonal guinea pig anti-insulin
antibodies (Millipore) followed by incubation with a peroxidase-labeled
anti–guinea pig IgG antibody (Kierkegaard & Perry Laboratories Inc.).
To evaluate insulitis, more than 10 pancreatic islets from an individual
mouse were randomly selected, and each islet was scored as 0 (no insu-
litis), 0.25 (peri-islet insulitis), and 1 (intra-islet insulitis) by the same
reader blinded to the group of mice. The insulitis score was calculated as
follows: ([0.25 × no. islets with peri-islet insulitis] + no. islets with intra-
islet insulitis)/total no. estimated islets.
Diabetes. The blood glucose levels of B16:A-dKO mice were measured
every 2 weeks, and recipient NOD/SCID mice that received splenocytes
were measured twice per week with the FreeStyle blood glucose monitor-
ing system (TheraSense). Mice were considered diabetic after 2 consecutive
blood glucose values greater than 250 mg/dl.
Statistics. The incidence of IAAs and insulitis scores were analyzed with
the c2 test. Survival curves were analyzed with the log-rank test. Statistical
tests used PRISM software (version 3.02; GraphPad Software). A P value
less than 0.05 was considered significant.
Transplantation of islets and immunization with insulin B:9–23
peptide in B16:A-dKO mice
B16:A 85% (11/13) 0.38 ± 0.15 0%
B16:A 13% (1/8) 0.09 ± 0.21 0% (0/7)
100% (15/15) 0.14 ± 0.13
0.11 ± 0.12 0% (0/6)
Transfer of splenocytes from immunized B16:A-dKO mice to NOD/SCID mice
0.69 ± 0.28
0.07 ± 0.06
0.26 ± 0.24
0.55 ± 0.28
0.11 ± 0.09
ND, not determined. AWithin the group, 25% (2 of 8) were followed up for 35 weeks. BWithin the
group, 37% (3 of 8) were followed up for 35 weeks.
research article Download full-text
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
This work was supported by grants from the NIH to G.S. Eisenbarth
(DK32083, DK55969, and DK62718) and to E. Liu (DK064605);
by a Diabetes Endocrine Research Center grant from the National
Institute of Diabetes and Digestive and Kidney Diseases, NIH to
G.S. Eisenbarth (P30 DK57516); by a mentor-based fellowship
from the American Diabetes Association to M. Kobayshi; by a grant
from the Juvenile Diabetes Foundation to G.S. Eisenbarth (JDRF1-
2006-16) and an advanced postdoctoral fellowship to M. Nakayama
(JDRF10-2006-51); and by program funds to G.S. Eisenbarth from
the Children’s Diabetes Foundation.
Received for publication December 29, 2006, and accepted in
revised form March 20, 2007.
Address correspondence to: George S. Eisenbarth, Barbara Davis
Center for Childhood Diabetes, University of Colorado Health Sci-
ences Center, 1775 North Ursula Street, Mail Stop B140, PO Box
6511, Aurora, Colorado 80045-6511, USA. Phone: (303) 724-6847;
Fax: (303) 724-6839; E-mail: email@example.com.
J.N. Beilke’s present address is: Department of Microbiology and
Immunology, UCSF, San Francisco, California, USA.
1. Todd, J.A., Bell, J.I., and McDevitt, H.O. 1987.
HLA-DQB gene contributes to susceptibility and
resistance to insulin-dependent diabetes mellitus.
2. Morel, P.A., et al. 1988. Aspartic acid at position 57
of the HLA-DQ beta chain protects against type I
diabetes: a family study. Proc. Natl. Acad. Sci. U. S. A.
3. Corper, A.L., et al. 2000. A structural framework
for deciphering the link between I-Ag7 and auto-
immune diabetes. Science. 288:505–511.
4. Narendran, P., Mannering, S.I., and Harrison, L.C.
2003. Proinsulin — a pathogenic autoantigen in
type 1 diabetes. Autoimmun. Rev. 2:204–210.
5. Lieberman, S.M., et al. 2003. Identification of the
beta cell antigen targeted by a prevalent population
of pathogenic CD8+ T cells in autoimmune diabetes.
Proc. Natl. Acad. Sci. U. S. A. 100:8384–8388.
6. Yang, J., et al. 2006. Islet-specific glucose-6-
phosphatase catalytic subunit-related protein-reac-
tive CD4+ T cells in human subjects. J. Immunol.
7. Jasinski, J.M., and Eisenbarth, G.S. 2005. Insulin as
a primary autoantigen for type 1A diabetes. Clin.
Dev. Immunol. 12:181–186.
8. Pugliese, A. 2005. The insulin gene in type 1 diabetes.
IUBMB Life. 57:463–468.
9. Palmer, J.P., et al. 1983. Insulin antibodies in insu-
lin-dependent diabetics before insulin treatment.
10. Yu, L., et al. 2000. Early expression of antiinsulin
autoantibodies of humans and the NOD mouse: evi-
dence for early determination of subsequent diabetes.
Proc. Natl. Acad. Sci. U. S. A. 97:1701–1706.
11. Alleva, D.G., et al. 2001. A disease-associated cel-
lular immune response in type 1 diabetics to an
immunodominant epitope of insulin. J. Clin. Invest.
12. Pinkse, G.G., et al. 2005. Autoreactive CD8 T cells
associated with beta cell destruction in type 1 diabetes.
Proc. Natl. Acad. Sci. U. S. A. 102:18425–18430.
13. Wegmann, D.R., Norbury-Glaser, M., and Daniel, D.
1994. Insulin-specific T cells are a predominant com-
ponent of islet infiltrates in pre-diabetic NOD mice.
Eur. J. Immunol. 24:1853–1857.
14. Barker, J.M., et al. 2004. Prediction of autoantibody
positivity and progression to type 1 diabetes: Dia-
betes Autoimmunity Study in the Young (DAISY).
J. Clin. Endocrinol. Metab. 89:3896–3902.
15. Bonifacio, E., et al. 2001. International workshop on
lessons from animal models for human type 1 dia-
betes: identification of insulin but not glutamic acid
decarboxylase or IA-2 as specific autoantigens of
humoral autoimmunity in nonobese diabetic mice.
16. Kent, S.C., et al. 2005. Expanded T cells from pan-
creatic lymph nodes of type 1 diabetic subjects rec-
ognize an insulin epitope. Nature. 435:224–228.
17. Halbout, P., Briand, J.P., Becourt, C., Muller, S., and
Boitard, C. 2002. T cell response to preproinsulin I
and II in the nonobese diabetic mouse. J. Immunol.
18. Chen, W., et al. 2001. Evidence that a peptide span-
ning the B-C junction of proinsulin is an early
autoantigen epitope in the pathogenesis of type 1
diabetes. J. Immunol. 167:4926–4935.
19. Daniel, D., Gill, R.G., Schloot, N., and Wegmann, D.
1995. Epitope specificity, cytokine production pro-
file and diabetogenic activity of insulin-specific T
cell clones isolated from NOD mice. Eur. J. Immunol.
20. Wong, F.S., et al. 1999. Identification of an MHC
class I-restricted autoantigen in type 1 diabetes by
screening an organ-specific cDNA library. Nat. Med.
21. Yu, L., et al. 1996. Anti-islet autoantibodies develop
sequentially rather than simultaneously. J. Clin.
Endocrinol. Metab. 81:4264–4267.
22. Ziegler, A.G., Hummel, M., Schenker, M., and Boni-
facio, E. 1999. Autoantibody appearance and risk for
development of childhood diabetes in offspring of
parents with type 1 diabetes: the 2-year analysis of the
German BABYDIAB study. Diabetes. 48:460–468.
23. Trudeau, J.D., et al. 2003. Prediction of sponta-
neous autoimmune diabetes in NOD mice by
quantification of autoreactive T cells in periph-
eral blood. J. Clin. Invest. 111:217–223. doi:10.1172/
24. Jaeckel, E., Lipes, M.A., and von Boehmer, H. 2004.
Recessive tolerance to preproinsulin 2 reduces but
does not abolish type 1 diabetes. Nat. Immunol.
25. French, M.B., et al. 1997. Transgenic expression of
mouse proinsulin II prevents diabetes in nonobese
diabetic mice. Diabetes. 46:34–39.
26. Daniel, D., and Wegmann, D.R. 1996. Protection of
nonobese diabetic mice from diabetes by intranasal
or subcutaneous administration of insulin peptide
B-(9-23). Proc. Natl. Acad. Sci. U. S. A. 93:956–960.
27. Jasinski, J.M., et al. 2006. Transgenic insulin (B:9-
23) T-cell receptor mice develop autoimmune dia-
betes dependent upon RAG genotype, H-2g7 homo-
zygosity, and insulin 2 gene knockout. Diabetes.
28. Du, W., et al. 2006. TGF-β signaling is required for
the function of insulin-reactive T regulatory cells.
J. Clin. Invest. 116:1360–1370. doi:10.1172/JCI27030.
29. Nakayama, M., et al. 2005. Prime role for an insulin
epitope in the development of type 1 diabetes in
NOD mice. Nature. 435:220–223.
30. Faideau, B., et al. 2006. Tolerance to proinsulin-2
is due to radioresistant thymic cells. J. Immunol.
31. Garcia, C.A., et al. 2005. Dendritic cells in human
thymus and periphery display a proinsulin epitope
in a transcription-dependent, capture-independent
fashion. J. Immunol. 175:2111–2122.
32. Steptoe, R.J., Ritchie, J.M., Jones, L.K., and Harri-
son, L.C. 2005. Autoimmune diabetes is suppressed
by transfer of proinsulin-encoding Gr-1(+) myeloid
progenitor cells that differentiate in vivo into rest-
ing dendritic cells. Diabetes. 54:434–442.
33. Pugliese, A. 2004. Central and peripheral autoanti-
gen presentation in immune tolerance. Immunology.
34. Ouyang, Q., et al. 2006. Recognition of HLA class
I-restricted beta-cell epitopes in type 1 diabetes.
35. Krishnamurthy, B., et al. 2006. Responses against
islet antigens in NOD mice are prevented by tol-
erance to proinsulin but not IGRP. J. Clin. Invest.
36. Gallucci, S., and Matzinger, P. 2001. Danger signals:
SOS to the immune system. Curr. Opin. Immunol.
37. Wong, F.S., Moustakas, A.K., Wen, L., Papadopoulos,
G.K., and Janeway, C.A., Jr. 2002. Analysis of struc-
ture and function relationships of an autoantigenic
peptide of insulin bound to H-2K(d) that stimulates
CD8 T cells in insulin-dependent diabetes mellitus.
Proc. Natl. Acad. Sci. U. S. A. 99:5551–5556.
38. Noorchashm, H., Greeley, S.A., and Naji, A. 2003.
The role of T/B lymphocyte collaboration in
the regulation of autoimmune and alloimmune
responses. Immunol. Res. 27:443–450.
39. Abiru, N., et al. 2001. Peptide and major histocom-
patibility complex-specific breaking of humoral toler-
ance to native insulin with the B9-23 peptide in diabe-
tes-prone and normal mice. Diabetes. 50:1274–1281.
40. Simone, E., et al. 1997. T cell receptor restriction of
diabetogenic autoimmune NOD T cells. Proc. Natl.
Acad. Sci. U. S. A. 94:2518–2521.
41. Harashima, S., Clark, A., Christie, M.R., and Not-
kins, A.L. 2005. The dense core transmembrane
vesicle protein IA-2 is a regulator of vesicle number
and insulin secretion. Proc. Natl. Acad. Sci. U. S. A.
42. Nakayama, M., et al. 2004. The establishment of
native insulin-negative NOD mice and methodol-
ogy to distinguish specific insulin knockout geno-
types and a B:16 alanine preproinsulin transgene.
Ann. N. Y. Acad. Sci. 1037:193–198.
43. Duvillié, B., et al. 1997. Phenotypic alterations in
insulin-deficient mutant mice. Proc. Natl. Acad. Sci.
U. S. A. 94:5137–5140.
44. Moriyama, H., et al. 2003. Evidence for a primary islet
autoantigen (preproinsulin 1) for insulitis and dia-
betes in the NOD mouse. Proc. Natl. Acad. Sci. U. S. A.
45. Nicolls, M.R., Coulombe, M., Beilke, J., Gelhaus,
H.C., and Gill, R.G. 2002. CD4-dependent gen-
eration of dominant transplantation tolerance
induced by simultaneous perturbation of CD154
and LFA-1 pathways. J. Immunol. 169:4831–4839.