ORIGINAL ARTICLE
On the Pathogenicity of Autoantigen-Specific T-Cell
Receptors
Amanda R. Burton,1 Erica Vincent,1 Paula Y. Arnold,1 Greig P. Lennon,1 Matthew Smeltzer,2
Chin-Shang Li,3 Kathryn Haskins,4 John Hutton,5 Roland M. Tisch,6 Eli E. Sercarz,7
Pere Santamaria,8 Creg J. Workman,1 and Dario A.A. Vignali1
OBJECTIVE—Type 1 diabetes is mediated by T-cell entry into
pancreatic islets and destruction of insulin-producing ␤-cells. The
relative contribution of T-cells specific for different autoantigens is
largely unknown because relatively few have been assessed in vivo.
RESEARCH DESIGN AND METHODS—We generated mice
possessing a monoclonal population of T-cells expressing 1 of 17
T-cell receptors (TCR) specific for either known autoantigens
(GAD65, insulinoma-associated protein 2 (IA2), IA2␤/phogrin,
and insulin), unknown islet antigens, or control antigens on a
NOD.scid background using retroviral-mediated stem cell gene
transfer and 2A-linked multicistronic retroviral vectors (referred
to herein as retrogenic [Rg] mice). The TCR Rg approach
provides a mechanism by which T-cells with broad phenotypic
differences can be directly compared.
RESULTS—Neither GAD- nor IA2-specific TCRs mediated T-cell
islet infiltration or diabetes even though T-cells developed in
these Rg mice and responded to their cognate epitope. IA2␤/
phogrin and insulin-specific Rg T-cells produced variable levels
of insulitis, with one TCR producing delayed diabetes. Three
TCRs specific for unknown islet antigens produced a hierarchy of
insulitogenic and diabetogenic potential (BDC-2.5 ⬎ NY4.1 ⬎
BDC-6.9), while a fourth (BDC-10.1) mediated dramatically accelerated disease, with all mice diabetic by day 33, well before
full T-cell reconstitution (days 42–56). Remarkably, as few as
1,000 BDC-10.1 Rg T-cells caused rapid diabetes following adoptive transfer into NOD.scid mice.
CONCLUSIONS—Our data show that relatively few autoantigen-specific TCRs can mediate islet infiltration and ␤-cell destruction on their own and that autoreactivity does not
From the 1Department of Immunology, St. Jude Children’s Research Hospital,
Memphis, Tennessee; the 2Department of Biostatistics, St. Jude Children’s
Research Hospital, Memphis, Tennessee; the 3Division of Biostatistics, Department of Public Health Sciences, University of California, Davis, Californnia; the 4Department of Immunology, University of Colorado Health Sciences
Center, Denver, Colorado; the 5Barbara Davis Center for Childhood Diabetes,
University of Colorado, Aurora, Colorado; the 6Department of Microbiology
and Immunology, University of North Carolina, Chapel Hill, North Carolina;
the 7Division of Immune Regulation, Torrey Pines Institute for Molecular
Studies, San Diego, California; and the 8Julia McFarlane Diabetes Research
Centre and the Department of Microbiology and Infectious Diseases, Institute
of Infection, Immunity and Inflammation, University of Calgary, Calgary,
Alberta, Canada.
Corresponding author: Dr. Dario Vignali, Department of Immunology, St.
Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 381052794. E-mail: [email protected].
Received for publication 12 August 2007 and accepted in revised form 20
February 2008.
Published ahead of print at http://diabetes.diabetesjournals.org on 21 February 2008. DOI: 10.2337/db07-1129.
C.J.W. and D.A.A.V. share senior authorship of this article.
FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein;
HBSS, Hank’s Balanced Salt Solution; FBS, fetal bovine serum; IA2, insulinoma-associated protein 2; ILN, inguinal lymph node; HEL, Hen egg white
lysozyme; PE, phycoerythrin; PLN, pancreatic lymph node; TCR, T-cell
receptor.
© 2008 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
DIABETES, VOL. 57, MAY 2008
necessarily imply pathogenicity. Diabetes 57:1321–1330, 2008
A
utoimmune type 1 diabetes is a chronic, complex autoimmune disease mediated by the infiltration of autoreactive lymphocytes into the
pancreas, resulting in the destruction of the
insulin-producing ␤-cells in the islets of Langerhans (1).
The nonobese diabetic (NOD) mouse is a spontaneous
murine model of type 1 diabetes and involves many of the
same autoantigens targeted by human T-cells (1–5). CD4⫹
T-cells appear to be essential in both the early and late
stages of the disease, as evidenced by the ability of
multiple CD4⫹ T-cell clones to transfer disease (6 – 8).
Additionally, anti-CD4 therapy can prevent the onset of
disease in NOD mice (9).
Over two dozen islet specific autoantigens have been
implicated in the initiation and/or pathogenesis of diabetes
(6). While T-cell responses to many of these antigens have
been studied (e.g., GAD [10 –13], insulin [14,15], the tyrosine phosphatase-like protein insulinoma-associated
protein 2 (IA2) [islet cell antigen 512] [16 –18], and IA2␤
[phogrin] [19,20]), their relative contribution to disease
initiation or pathogenesis remains unclear. Furthermore,
the specificities of the majority of diabetogenic T-cell
clones isolated from NOD mice are still unknown (8). A
key question that remains unresolved is the extent to
which autoantigenicity and pathogenicity are linked.
The hallmark of preclinical type 1 diabetes is the infiltration of CD4⫹ T-cells into the pancreatic islets. While
many cell types participate in the disease process, CD4⫹
T-cells command a central role in the initiation, regulation,
and progression of the disease. Clearly autoantigen specificity plays a critical role in governing CD4⫹ T-cell entry
into islets. However, the ability to study these processes
using unmanipulated T-cell populations that have a broad
range of insulitic and diabetogenic potential has been
greatly limited. T-cell receptor (TCR) transgenic (Tg) mice
are powerful tools for the analysis of autoimmune diseases
such as type 1 diabetes. However, only five major histocompatibility complex class II–restricted, autoantigenspecific Tg NOD lines have been described (BDC-2.5,
BDC-6.9, NY4.112-4.1, and a GAD286 –300 Tg), providing a
limited phenotypic range (21–25). We developed a new
approach for the rapid generation of TCR Tg mice, refered
to as retrogenic (Rg) mice, using retroviral-mediated stem
cell gene transfer and novel self-cleaving 2A peptide–
linked multicistronic retroviral vectors that express both
TCR chains plus a fluorescent protein marker in a single
vector (26 –30). This approach allows us to generate and
analyze multiple TCR Rg mice on a variety of defined
genetic backgrounds in less than 2 months. This is partic1321
AUTOREACTIVITY VS. PATHOGENICITY IN TCR Rg MICE
ularly important in studying diabetes, for which background genes have a significant effect on disease outcome
(31). Furthermore, the phenotype of Tg mice can be
affected by additional variables such as the level and
timing of TCR expression and the possibility of insertional
mutagenesis by the integrated transgene. These issues are
eliminated in Rg mice because the same expression system is used for all TCRs; multiple, independently generated mice are analyzed; and retroviral integration is
widespread. This facilitates the direct comparison between multiple TCRs.
The goal of this study was to assess the link between
autoreactivity versus pathogenicity of multiple autoantigen-specific TCRs using the Rg system. We chose to clone
and generate a panel of Rg mice expressing twelve different TCRs specific for four known autoantigens (various
epitopes of GAD65, IA2, phogrin, and insulin) and four
different TCRs specific for unknown islet antigens. All four
known autoantigens have been implicated in the disease
onset, but their relative contribution is still controversial.
With this panel of Rg mice, we addressed two important
questions. First, are all autoreactive TCRs pathogenic and
capable of initiating insulitis and diabetes in the absence
of other T-cells and B-cells? Second, is there a defined
relationship between the extent of insulitis, the time of
diabetes onset, and the incidence of diabetes?
RESEARCH DESIGN AND METHODS
NOD.scid mice were obtained from The Jackson Laboratory and bred
in-house. B6.H2g7 mice were provided by Christophe Benoist and Diane
Mathis (Joslin Diabetes Center, Boston, MA) (31) and crossed onto an
RAG-1⫺/⫺ background. Diabetes incidence was monitored on a biweekly,
weekly, or more frequent basis by testing for the presence of glucose in the
urine by Clinistix (Bayer, Elkhart, IN). Mice testing positive by Clinistix were
then tested with a One Touch Ultra glucometer (Lifescan, Milpitas, CA) for
blood glucose levels and were considered diabetic if their blood glucose was
⬎200 mg/dl for retroviral-mediated stem cell gene transfers or 400 mg/dl for
adoptive transfers. All mice were bred and housed at the St. Jude Animal
Resources Center (Memphis, TN) in a Helicobacter-free specific pathogen free
facility following state, national, and institutional mandates. The St. Jude
Animal Resources Center is accredited by the American Association for the
Accreditation of Laboratory Animal Care. All animal experiments followed
animal protocols approved by the St. Jude institutional animal care and use
committee.
TCR retroviral constructs. All TCRs were generated as 2A-linked single
open reading frame inserts using RT-PCR and cloned into a murine stem cell
virus– based retroviral vector with a green fluorescent protein (GFP) marker
as previously described (26,28 –30). Details of cloning strategies and primer
sequences are available on request ([email protected]). TCRs were
cloned by PCR using plasmid or cDNA obtained from Phogrin13 and Phogrin18 T-cell clones; 10.23 hybridoma from John Hutton (Barbara Davis Center
for Diabetes, Aurora, CO); BDC-6.9 and BDC-10.1 T-cell clones from Kathryn
Haskins (University of Colorado Health Sciences Center, Denver, CO); BDC2.5 TCR plasmids from Luc Teyton (Scripps Research Institute, La Jolla, CA);
NY4.1 TCR plasmids from Pere Santamaria (University of Calgary, Alberta,
Canada); 1A4 TCR plasmids from Roland Tisch (University of North Carolina,
Chapel Hill, NC); 530.45.19 hybrid from Eli Sercarz (Torrey Pines Institute for
Molecular Studies, San Diego, CA); and PA15.14B12, PA19.5E11, PA18.10F10,
PA17.9G7, PA18.9H7 and PA21.14H4 hybridomas generated in our lab by Paula
Arnold. The 12-4.4v1 and 12-4.1 TCR constructs were generated de novo from
the published sequence data (12-4.1 accession numbers: DQ172905 for ␣-chain
and DQ180320 for ␤-chain) and information generously provided by George
Eisenbarth (Barbara Davis Center for Childhood Diabetes). For TCR with
unknown V␣ and V␤ usage, V␤ was determined by flow cytometry using
V␤-biotinylated antibodies and streptavidin phycoerythrin (PE). V␣ usage was
determined by RT-PCR with V␣ primers, cloning into the PCR Blunt II-TOPO
vector (Invitrogen, Carlsbad, CA), and sequencing as previously described
(32). All TCR 2A-linked vectors utilize the PTV1.2A sequence except for
PA21.14H4, NY4.1, and PA18.10F10, which utilize the TaV.2A sequence
(27,28). The BDC-10.1 and BDC-6.9 vectors contain a gly-ser-gly linker
between the C␣ region and the PTV1.2A sequences. The BDC-2.5 vector lacks
1322
this linker, while the remaining vectors contain a gly between the C␣ region
and the 2A sequences.
Retroviral-mediated stem cell gene transfer, flow cytometric analysis,
and cell sorting. Retroviral-mediated stem cell gene transfer was performed
as previously described (26 –30). At 35, 70, and 140 days posttransplant,
spleens, inguinal lymph nodes (ILNs), and pancreatic lymph nodes (PLNs)
were harvested, processed, counted, and stained with TCR␤(H57)-PE/and
CD4-APC (BD Biosciences Pharmingen, San Diego, CA). For functional
assays, splenocytes were stained with CD4-PE (BD Biosciences Pharmingen,
San Diego, CA) and sorted with a fluorescence-activated cell sorter (FACS) for
GFP⫹/CD4⫹ on a MoFlow (Dako, Fort Collins, CO).
Adoptive transfers. At 28 days posttransplant, GFP⫹CD4⫹TCR⫹ splenocytes
from NOD.scid TCR Rg mice were purified by FACS and injected intravenously into NOD.scid recipient mice.
Islet isolation. Pancreata were perfused by injecting 3 ml collagenase P (1.5
␮g/ml in Hanks’ Balanced Salt Solution [HBSS]) (Roche, Indianapolis, IN),
harvested, and placed in 3–5 ml collagenase P on ice. The pancreata were then
incubated at 37°C for 16 –20 min, after which 7 ml Hanks’ balanced salt
solution (HBSS) with 5% fetal bovine serum (FBS) was added and the tissues
pelleted at 1,000 rpm for 3 min. The pellet was washed twice with 7 ml 5%
FBS/HBSS and resuspended in 7 ml 5% FBS/HBSS. Islets were handpicked and
dissociated with 1 ml cell dissociation buffer (Invitrogen, Carlsbad, CA) and
incubated at 37°C for 5 min. After vortexing, the dissociation process was
repeated twice. Cells were placed in 10 ml 5% FBS/HBSS, centrifuged for 10
min at 1,300 rpm, resuspended in 5% FBS/HBSS, and irradiated at 3,000 rads.
Functional assays. To determine the activation index of TCRs with known
specificities, 5 ⫻ 105 splenocytes of mice at 70 or 140 days posttransplant were
cultured with or without peptide in either supplemented Eagle’s minimum
essential medium or RPMI with 10% FBS for 48 h in 96-well flat-bottom plates.
In some instances, splenocytes were FACS sorted as described above, and
2.5 ⫻ 104 purified T-cells were cultured with irradiated (3,000 rads) NOD/LtJ
splenocytes with or without peptide for 48 h. The wells were then pulsed with
1␮Ci/well of [3H]-thymidine for the final 8 or 24 h and then harvested. The
peptides used were: IA2678 – 688, GAD217–236, GAD284 –300, GAD206 –220, GAD510 –524,
GAD524 –538, GAD530 –543, Hen egg white lysozyme (HEL)11–25, insulin B9 –23,
IA2␤640 – 659, and IA2␤755–777. For TCRs of unknown specificity, 2.5 ⫻ 104 purified
GFP⫹/CD4⫹ splenic T-cells were cultured with 5.0 ⫻ 104 irradiated purified islets
from 11- to 19-week-old NOD/LtJ mice in supplemented Eagle’s minimum
essential medium with 10% FBS in 96-well round-bottom plates for 72 h, pulsed
with 1␮Ci/well of [3H]-thymidine for 18 h, and then harvested.
Insulitis scores. Pancreata of NOD.scid TCR retrogenic mice were harvested
at 28, 70, and 140 days posttransplant; placed into 10% buffered formalin; and
embedded in paraffin; 4 ␮m–thick sections were cut at 150-␮m step-sections
and stained with hematoxylin and eosin at the St. Jude Histology Core Facility.
An average of 90 –100 islets per mouse were scored in a blinded manner using
the following metric: no insulitis (normal islet and no infiltration), periinsulitis (infiltration on edges of islet or 0 –20% of islet infiltrated) or insulitis
(infiltration of 30 –100% of islet).
Statistics. The time to diabetes (survivor function) for each group of mice
was estimated using the Kaplan-Meier log-rank test both overall and pairwise
within each experiment. The overall Type I error rate for each experiment was
controlled at the 0.05 level using the Bonferroni adjustment for multiple
comparisons.
RESULTS
Cloning and expression of ␤-cell–specific TCRs.
GAD65 is one of the principal autoantigens in human type
1 diabetes and was one of the first autoantigens implicated
in diabetes in NOD mice (10,11). Nevertheless, its role as
a target autoantigen remains controversial (24,26,33,34).
We cloned and expressed seven different TCRs specific for
the major immunogenic GAD epitopes and others that
have been proposed to modulate disease progression
(Table 1) (35,36). All of the GAD-specific TCRs, with the
exception of 1A4, were derived by immunizing NOD mice
with peptide. 1A4 was isolated from the islets of a nonimmunized, nondiabetic NOD mouse. Four different TCRs
based on relevant epitope immunogenicity were chosen:
PA15.14B12 and PA19.5E11 (specific for GAD206 –220, the
primary immunogenic epitope of GAD65) and 1A4 and
PA17.9G7 (which covered the second and third most
immunogenic epitopes GAD221–235 and GAD284 –300, respectively) (35,36). We also cloned and expressed three differDIABETES, VOL. 57, MAY 2008
A.R. BURTON AND ASSOCIATES
4.7
4.7
4.4
4.5
1.4
6.2
13.3
13.3
V␣
12
8.2
8.2
4
10
10
12
4
2
V␤
34.2 ⫾ 12.7
1.0 ⫾ 0.04
22.8 ⫾ 10.2
NA**
NA**
NA**
NA**
7.8 ⫾ 5.0
2.5 ⫾ 0.5
ND
29.2 ⫾ 18.3
21.4 ⫾ 7.9
ND
18.9 ⫾ 3.0
5.9 ⫾ 3.1
2.0 ⫾ 0.5
15.0 ⫾ 2.0
SI ⫾ SD*
9
5
10
—
—
—
—
2
4
—
2
4
—
2
5
6
3
n†
ND
ND
ND
6.8/93.0
0.3/0
9.5/87.0
29.7/56.7
1.3/0
ND
ND
ND
ND
ND
ND
ND
ND
ND
28 days‡
0.9/0
2.5/0.6
2.0/1.0
27.1/58.8
20.9/22.7
NA††
37.1/50.7
2.1/0
14.5/13.8
0/0
0.6/0
0.4/0
0/0
1.0/0
1.6/1.6
0.8/1.2
41.2/46.0
70 days‡
0/0
0.3/0
11.4/5.6
NA††
24.4/24.8
NA††
24.9/12.3
6.1/1.2
6.0/3.2
0/0
0.3/0
1.1/0
ND
0/1.5
0/0
2.8/1.0
33.3/20.0
140 days‡
12
11
11
4
8
7
10
17
10
2
8
10
2
10
8
12
7
n
0
0
0
100
56
100
71
0
0
0
0
0
0
0
0
0
33
Incidence§
(%)
—
—
—
52
81
27
46
—
—
—
—
—
—
—
—
—
60
ID-5¶
9
10
10
19
9
15
21
16
8
5
10
13
4
5
8
9
6
n
Diabetes
13.3
10
16
11
4
4
15
11
6
Peri-insulitis/Insulitis
Antigen
specificity
10.7
10.1
15.1
1.5
13.1
9.2
13.1
13.1
Function
GAD206–220
GAD206–220
GAD217–236
GAD284–300
GAD510–524
GAD524–538
GAD530–543
Insulin
B9–23
Insulin
B9–23
IA2676–688
IA2␤640–659
IA2␤755–777
Islet Ag
Islet Ag
Islet Ag
Islet Ag
HEL11–25
TCR usage
TABLE 1
The expression and insulitogenic and diabetogenic potential of the T-cell receptors used
TCR
PA15.14B12
PA19.5E11
1A4
PA17.9G7
PA18.10F10
PA18.9H7
530.45.19
12–4.1
12–4.4v1储
10.23
Phogrin13
Phogrin18
BDC-2.5
BDC-6.9
BDC-10.1
NY4.1
PA21.14H4
*Stimulation index (SI) of splenocytes activated with peptide. †Mice analyzed. ‡Percentage of islets showing either peri-insulitis (defined as infiltration around islet or 0 –20% of islet
infiltrated) or insulitis. The data are represented as peri-insulitis/insulitis at 28, 70, or 140 days posttransplant. §Incidence of diabetes. ¶Number of days posttransplant that 50% of the
mice that became diabetic had a blood glucose level ⬎400mg/dl. 储The 12– 4.4 sequence used in this study was reconstructed from information obtained from the study by Wegmann et
al. (15). However, as a result of changes in J␣ nomenclature since publication of their article, an alternate J␣ was used in its construction (a modified J␣42 关SGGSNYKLTFGKGTKLSVKS兴
rather than J␣53 关SGGSNYKLTFGKGTLLTVTP兴). It is important to note that 12– 4.4v1 retains insulin reactivity and mediates significant islet infiltration and thus represents a very useful
research reagent. As this is now a variant of the original 12– 4.4, we refer to this TCR as 12– 4.4v1. **Not applicable (NA) because the peptide specificity of the TCR is not known. The
inapplicable TCRs were activated with whole islets to determine functionality. ††Not applicable because the mice were all diabetic by 70 and 140 days posttransplant. ND, not determined.
1323
DIABETES, VOL. 57, MAY 2008
AUTOREACTIVITY VS. PATHOGENICITY IN TCR Rg MICE
104
104
IA4
NOD Control
20.3%
103
103
102
102
102
101
101
101
100
100
4
10
101
102
103
104
PA18.10F10
GAD510-524
64.5%
103
TCRβ
GAD217-236
6.3%
104
100
100
4
10
101
103
102
104
12-4.1
InsulinB9-23
14.0%
103
103
100
100
4
10
102
102
101
101
101
101
102
103
104
Phogrin13
IA2β640-659
17.0%
103
100
100
4
10
101
102
103
104
Phogrin18
IA2β755-777
57.8%
103
100
100
4
10
102
102
101
101
101
101
102
103
104
BDC-10.1
Islet Ag
8.5%
103
100
100
4
10
101
102
103
104
NY 4.1
Islet Ag
22.4%
103
100
100
4
10
102
102
101
101
101
101
102
103
104
100
100
101
102
103
104
100
100
103
104
10.23
IA2718-728
37.6%
101
102
103
104
BDC-2.5
Islet Ag
0.87%
101
102
103
104
PA21.14H4
HEL11-25
40.9%
103
102
100
100
102
103
102
100
100
4
10
101
103
102
100
100
4
10
PA17.9G7
GAD284-300
18.6%
101
102
103
104
CD4
FIG. 1. Expression of Rg T-cells in NOD.scid mice. Flow cytometric analysis of splenocytes from female NOD.scid TCR Rg mice 32–39 days
posttransplant was performed. Splenocytes were gated on live (based on forward/side scatter) GFPⴙ cells and dot plots of CD4 vs. TCR
(anti–TCR␤-H57) expression depicted. The numbers in the top right quadrant refer to the percentage of TCRⴙCD4ⴙ T-cells. Note that the level
of TCR expression and the number of T-cells was more variable among BDC-2.5 Rg mice, with the example depicted representative of those on
the low end (See Fig. 2 for a mean of all mice examined).
1324
DIABETES, VOL. 57, MAY 2008
A.R. BURTON AND ASSOCIATES
10000
Spleen
#GFP+/TCR+/CD4+
(x103) Cells
1000
100
10
1
10000
Inguinal Lymph Node
#GFP+/TCR+/CD4+
(x102) Cells
1000
100
10
11
10000
ND*
Pancreatic Lymph Node
#GFP+/TCR+/CD4+
(x102) Cells
1000
100
10
11
ND*
FIG. 2. Reconstitution levels of TCR retrogenic mice. The spleens,
ILNs, and PLNs from the NOD.scid TCR Rg mice were removed and
single cell suspensions prepared, counted, and stained for TCR and
CD4 expression. The number of Rg T-cells was determined from the
percentage of GFPⴙ/TCRⴙ/CD4ⴙ cells and the total number of cells in
the spleen and lymph nodes. The data represents the means ⴞ SE of
3– 6 mice per group. *Not determined.
ent TCRs specific for the COOH-terminus portion of
GAD65 thought to be important in the initiation of islet
infiltration and ␤-cell destruction, two of which—
PA18.9H7 and 530.45.19 — cover the GAD524 –538 and
GAD530 –543 epitope, respectively, and PA18.10F10, specific
for GAD510 –524, just upstream of epitope GAD524 –543 (Table
1). 530.45.19 and PA18.9H7 are particularly intriguing
because it has been reported that spontaneously arising,
GAD-reactive T-cells in NOD mice use V␤4⫹ (530.45.19)
and are specific for the 530 –543 epitope, while TCRs that
recognize GAD524 –538 are primarily generated by immunization, use V␤12 (PA18.9H7) and may have regulatory
activity (10,13,34).
DIABETES, VOL. 57, MAY 2008
Insulin has also been implicated in the diabetogenic
response. A considerable number of T-cells infiltrating the
islets react to insulin, with ⬎90% recognizing the insulin
B9 –23 epitope (15,37). Recently it has been shown that
insulin I/II null NOD mice expressing an insulin transgene
with a mutation in the critical insulin B9 –23 epitope did not
develop disease, suggesting that it is a primary autoantigenic epitope (38). T-cell clones generated against insulin
B9 –23 both accelerate diabetes onset in NOD mice and
confer disease following adoptive transfer into NOD.scid
mice (15). The 12-4.1 and 12-4.4v1 TCRs were originally
isolated from the islets of prediabetic NOD mice and were
reconstructed by PCR from published sequences (see
RESEARCH DESIGN AND METHODS and Table 1 for details) and
expressed in NOD.scid mice.
Insulinoma-associated tyrosine phosphatase-like protein (IA2) or islet cell antigen 512 is found in the secretory
granules of ␤-cells (17,39) and is a potential target of CD4⫹
T-cells (16,18,40). We cloned the TCR 10.23, which is
specific for IA2 and was derived through immunizations
with whole protein. We later identified the epitope as
IA2676 – 688 (data not shown). In addition, given the implication of IA2␤ (phogrin)-reactive CD4⫹ T-cells in disease
pathogenesis (19,20,41), two phogrin-specific TCRs, phogrin13 and phogrin18, were cloned from T-cell lines established by immunization with whole protein (41).
Included in this study were clonotypic BDC-2.5, BDC6.9, BDC-10.1, and NY4.1 TCRs specific for undefined
␤-cell epitopes. BDC-2.5, BDC-6.9, and BDC-10.1 were the
first diabetogenic CD4⫹ T-cell clones described and were
originally isolated from the spleen and lymph nodes of
diabetic NOD mice by Haskins and colleagues (8,42,43),
while NY4.1 was one of six CD4⫹ T-cell clones that were
isolated from the islets of acutely diabetic NOD mice and
were reactive to islet cells (44). These clones are highly
diabetogenic upon transfer into NOD.scid mice or when
expressed as a transgene in NOD mice (BDC-2.5, BDC-6.9,
and NY4.1) (21–23,45,46). We have previously generated
BDC-2.5 and NY4.1 Rg mice (26), which were included in
this study as positive controls for the other TCR Rg mice.
Likewise, Rg mice expressing PA21.14H4, an H-2Ag7–
restricted, HEL11–25-specific TCR, were included as negative controls.
TCR Rg mice were established using NOD.scid mice as
both bone marrow donors and recipients. This enabled us
to examine the ability of Rg T-cells to infiltrate the islets
and mediate diabetes in the absence of other T-cells or
B-cells. For each TCR, we examined expression and
reconstitution levels, T-cell proliferative capacity, and the
incidence of insulitis and diabetes in the Rg mice.
Expression and function of ␤-cell–specific TCRs. All
of the TCRs were expressed in mice and had comparable
levels of GFP (Fig. 1 and data not shown). However, there
was variability in TCR expression levels compared with
the NOD control and in the number of GFP⫹/TCR⫹/CD4⫹
Rg T-cells in the spleen, ILN, and PLN (Figs. 1 and 2). This
is likely due to the influence of thymic selection and the
availability of selecting ligands, as well as homeostatic and
antigen-driven proliferation in the periphery. Importantly,
TCR expression level and T-cell numbers were consistent
within each group, suggesting that this variability was due
to intrinsic characteristics of each TCR rather then experimental variance. Also, there was a correlation between
the number of T-cells in the spleen compared with the ILN
and PLN within each TCR Rg group (Fig. 2).
The GAD65-specific TCRs were reactive to and specific
1325
AUTOREACTIVITY VS. PATHOGENICITY IN TCR Rg MICE
A
CPM (x102)
CPM (x102)
80
60
40
200
PA19.5E11
20
20
0
10-2
10-1
100
µM Peptide
101
B
0 -2
10
100
50
600
5µM GAD284-300
5µM HEL11-25
50
CPM (x102)
No Antigen
75
10-3
10-2 10-1 100
µM Peptide
101
D
C
100
CPM (x102)
150
0
10-4
101
10-1
100
µM Peptide
Phogrin18
IA2β755-777
GAD284-300
InsulinB9-23
IA2676-688
HEL11-25
Phogrin18
400
200
600
400
CPM (x102)
40
PA18.10F10
100
CPM (x102)
120
200
20
15
10
25
5
0
PA17.9G7
PA21.14H4
0
0.0001 0.001 0.01
0. 1
µM Peptide
1.0
10.0
0
Islets
+
No
T cells
+
BDC-2.5
+
BDC-10.1
+
PA21.
14H4
FIG. 3. T-cells from TCR retrogenic NOD.scid mice are functional and specific for their cognate antigen. A: 5 ⴛ 105 splenocytes from TCR NOD.scid
Rg mice were incubated with varying concentrations of peptide for 48 h, pulsed with 1 ␮Ci, and harvested 24 h later. Background counts from
T-cells alone were subtracted at each antigen concentration. Data are representative of two experiments. B: GFPⴙ/CD4ⴙ T-cells from PA17.9G7
and PA21.14H4 Rg mice were purified by FACS and incubated with or without 5 ␮mol/l GAD284 –300 or HEL11–25 peptide presented by irradiated
NOD splenocytes for 48 h, pulsed with 1 ␮Ci, and harvested 18 h later. Data represent means ⴞ SE of three independent experiments. C:
Phogrin18 Rg T-cells were purified by FACS based on GFP and CD4 expression and incubated with a titration of various peptides presented by
irradiated NOD splenocytes for 48 h, pulsed with 1 ␮Ci, and harvested 18 h later. Data represent means ⴞ SE of three independent experiments.
D: BDC2.5, BDC10.1, and PA21.14H4 Rg T-cells were sorted and incubated with or without irradiated 11- to 19-week-old NOD/LtJ islets for 72 h,
pulsed with 1␮Ci, and harvested 18 h later. The data represent the means ⴞ SE of 2– 4 independent experiments.
for their respective peptides, albeit with differing efficiencies (Table 1 and Fig. 3A and B). While 12-4.1 and 12-4.4v1
recognize the same epitope (insulin B9 –23), the significant
difference in stimulation index observed is likely due to
differences in cognate ligand affinity caused by differences
in complimentarity-determining region/V␤ usage (Table 1).
Furthermore, differences in IA2- and phogrin-specific TCR
expression in 10.23, phogrin13, and phogrin18 Rg mice
may explain the differing reactivity observed (Table 1 and
Figs. 1 and 3A). However, Rg T-cells were antigen specific
and T-cells isolated from the spleen and PLN reacted
equivalently, as exemplified with phogrin18 T-cells (Fig.
3C and data not shown). Finally, all of the unknown islet
antigen–specific TCRs—BDC-2.5, BDC-6.9, BDC-10.1, and
NY4.1—were expressed at detectable levels (Fig. 1). The
level of TCR expression was more variable among some of
the Rg mice. This was particularly evident with BDC-2.5
T-cells and may, in part, be due to the relatively low level
of TCR expression seen in these mice compared with
other Rg T-cells (Figs. 1 and 2). The functionality of these
Rg T-cells was determined by measuring proliferation in
response to purified, irradiated islets from 11- to 19-weekold NOD mice. BDC-10.1 Rg T-cells were strongly reactive
to irradiated islets, while BDC-2.5 T-cells responded
weakly compared with the control HEL-specific
PA21.14H4 Rg T-cells, Rg T-cells alone, and islets alone
(Fig. 3D and data not shown). In contrast, minimal reactivity was seen with BDC-6.9 and NY4.1 Rg T-cells, suggesting that either these islets express insufficient levels of
the antigenic epitope to elicit proliferation in vitro or,
more likely, that T-cells derived from these Rg mice are
less sensitive than the parent T-cell clones.
1326
Insulitis and incidence of diabetes of Rg mice. Next,
we evaluated the extent of insulitis 28, 70, and/or 140 days
post– bone marrow transplant and the incidence of diabetes for 140 days in the 17 TCR Rg mouse lines. Essentially,
no peri-insulitis or insulitis was observed in any of the
GAD-specific Rg mice (Table 1 and Fig. 4). Likewise,
minimal infiltration of IA2676 – 688–specific 10.23 T-cells or
IA2␤/phogrin640 – 659–specific phogrin13 T-cells was seen in
the respective Rg mice. This was surprising because the
phogrin-specific T-cell clones phogrin15 and phogrin12
infiltrate rat islets transplanted under the kidney capsule
and destroy ␤-cells (41). However, demonstrable periinsulitis (⬃11%) and insulitis (⬃6%) were observed in
IA2␤/phogrin755–777–specific phogrin18 Rg mice, although
this was not manifested until 140 days posttransfer (Table
1 and Fig. 4). The differential ability of these phogrinspecific T-cells to infiltrate islets may be due to differences
in epitope recognition or TCR affinity/avidity (Table 1).
Not surprisingly, none of these Rg mice developed diabetes (Table 1).
Analysis of the two insulin B9 –23 Rg mice was particularly intriguing. While significant peri-insulitis and insulitis
were observed, insulitis was greater in the 12-4.1 versus
the 12-4.4v1 Rg mice (Table 1). Furthermore, diabetes
developed in 33% of the 12-4.1 Rg mice (ID50 – 60 days),
while none of the 12-4.4v1 Rg mice became diabetic. The
difference in diabetes incidence may be due to differences
in the relative avidity of the corresponding T-cells to
antigen (Table 1). The peri-insulitis/insulitis score appeared to decline by 140 days posttransfer. Given that a
proportion of the 12-4.1 Rg mice becomes diabetic, it is
possible that this reduced insulitis score is due to the
DIABETES, VOL. 57, MAY 2008
A.R. BURTON AND ASSOCIATES
No Insulitis
28 Days
Peri-Insulitis
70 Days
Insulitis
140 Days
Islet Score (%)
100
75
50
25
0
FIG. 4. Insulitis incidence in TCR Rg NOD.scid mice. Hematoxylin and eosin–stained pancreata of NOD.scid retrogenic mice were scored for
lymphocytic infiltration of the islets at 28, 70, and 140 days posttransplant. Insulitis was scored (⬃100 islets per mouse) using the following
metric: no insulitis (normal islet; no infiltration), peri-insulitis (infiltration on edges of islet or 0 –20% of islet infiltrated), or insulitis
(infiltration of 30 –100% of islet). The average score of 4 –17 mice (Table 1) is presented with SEs (⬃800 slides in total). An alternative scoring
system was also used for comparison, which gave comparable results (BDC10.1– 0.85 ⴞ 0.04, BDC2.5– 0.89 ⴞ 0.02, NY4.1– 0.59 ⴞ 0.08, BDC6.9, and
14H4 – 0) (48).
reduced disease incidence in the remaining mice. However, this cannot account for the decline in the 12-4.4v1 Rg
mice and suggests that the insulitis resolves with time in
these mice.
As previously reported, BDC-2.5 and NY4.1 Rg mice
developed significant insulitis ⬃28 days posttransfer,
which is remarkable given that it takes ⬃50 days for
complete bone marrow reconstitution and maximal T-cell
population of the periphery. There was also substantial
insulitis in the BDC-10.1 Rg mice (87% for BDC-10.1 vs. 93%
for BDC-2.5 at 28 days posttransfer [Table 1 and Fig. 4]).
Indeed, there was a correlation between the percentage of
infiltrating CD4⫹ Rg T-cells and the severity of insulitis and
incidence of diabetes (BDC-10.1, 5.9% CD4⫹ T-cells in the
islets; BDC-2.5, 5.1%; 12-4.1, 3.6%; phogrin18, 0.6%; and
PA21.14H4, 0.2%).
Diabetes incidence in the BDC-2.5 Rg mice was 100%
with an ID50 of 52 days. Curiously, the rate of diabetes
development in the NY4.1 Rg mice was comparable (ID50 –
46 days) but the incidence was only 71% (Table 1 and Fig.
5A). Insulitis development in the BDC-6.9 Rg mice was
delayed, with no insulitis at day 28 but substantial insulitis
at days 70 and 140 posttransfer. Consistent with this,
diabetes onset in the BDC-6.9 Rg mice was delayed (ID50 –
81 days; incidence –56%), in accordance with observations
made with their Tg counterparts (22). Diabetes onset in
the BDC-10.1 Rg mice was rapid and highly penetrant with
all the mice hyperglycemic by day 33 (ID50 – 27 days) (Fig.
5A). Given the diabetogenic potential of the BDC-10.1 TCR
and the flexibility of the Rg system, BDC-10.1 Rg T-cells
were generated on a C57BL/6 background using the
B6g7.RAG-1⫺/⫺ mice. Interestingly, BDC-10.1 Rg T-cells on
a nonautoimmune background mediated diabetes, albeit at
a slower rate (ID50 ⫺ 76 days) and reduced frequency
(80%) (Fig. 5A).
To further evaluate the diabetogenic potency of the
BDC-10.1 Rg T-cells, GFP⫹CD4⫹ T-cells were sorted from
BDC-10.1, NY4.1, and the control TCR PA21.14H4 Rg mice
and adoptively transferred into NOD.scid mice (Fig. 5B).
As expected, the HEL-specific T-cell recipients failed to
develop diabetes, while mice receiving NY4.1 Rg T-cells
initially developed diabetes by day 55 posttransfer, and by
DIABETES, VOL. 57, MAY 2008
day 138 all recipients were diabetic. Strikingly, all of the
BDC-10.1 T-cell recipients developed diabetes by day 13
posttransfer. Finally, we adoptively transferred titrated
numbers of purified BDC-10.1 Rg T-cells into NOD.scid
mice to define the minimal number required to mediate
disease (Fig. 5C). Remarkably, only 50,000 BDC-10.1 Rg
T-cells were required to cause rapid diabetes onset in 100%
of recipients by day 13, while as few as 1,000 BDC-10.1 Rg
T-cells caused diabetes in 80% of recipients. Taken together, these data highlight the increased diabetogenic
potential of BDC-10.1 T-cells.
DISCUSSION
Type 1 diabetes is a complex disease mediated by T-cells
of multiple specificities, many of which have yet to be
determined. Important questions remain concerning what
governs T-cell islet entry, what makes a T-cell diabetogenic, and what the relationship is between autoreactivity
and pathogenicity. While the generation of TCR Tg mice
has provided important insight into the disease process,
these studies represent a limited repertoire of potential
autoantigen epitope-specific TCRs that might mediate type
1 diabetes. Our study demonstrates the utility of the Rg
approach for the generation and analysis of mice expressing a large panel of TCRs with varied specificity. These
data show that for the panel of TCRs studied, relatively
few promote islet entry and ␤-cell destruction in the
absence of other T-cells and B-cells. Indeed, it is noteworthy that four of the five TCRs that promoted diabetes when
expressed in Rg mice recognize unknown antigens.
It has recently been suggested that GAD-reactive T-cells
may not be as critical to the initiation and progression of
type 1 diabetes as originally thought (10,11,33). Our data
support this view. Despite the high immunogenicity of
GAD epitopes (206 –220, 221–235 and 284 –300) and the
large number of Rg T-cells in the spleens and lymph nodes,
no ␤-cell destruction was seen in the seven GAD-reactive
TCR Rg mouse lines analyzed. While some peri-insulitis
was observed in some mice, particularly those expressing
TCR specific for distal GAD epitopes (e.g., 530.45.19
specific for GAD530 –543), this was not significantly greater
1327
AUTOREACTIVITY VS. PATHOGENICITY IN TCR Rg MICE
A
Recipients (n)
TCR
(n)
12-4.1
75
BDC-6.9
50
NY4.1
PA21.14H4
12-4.1
BDC-6.9
NY4.1
BDC-2.5
BDC-10.1
25
0.00115 0.00005 0.00001 0.00376 0.02287 0.10960
0.04665 0.00013 0.00088 0.06317 0.43964
0.16156 0.00001 0.00049 0.12318
0.91864 0.00000 0.04511
0.01916 0.00004
0.00005
p ≤ 0.002381*
*Bonferroni correction for multiple comparisons
100
0
B
25
100
50
75
Days Post-Transplant
TCR # Purified T cells
PA21.14H4 5x104 cells
NY4.1
5x104 cells
BDC-10.1 5x104 cells
125
150
(n)
(3)
(4)
(5)
0
25
NY4.1
BDC-10.1
Incidence of Diabetes (%)
Recipients
BDC-2.5 NOD.SCID
(19)
(15)
BDC-10.1 NOD.SCID
BDC-10.1 B6g7.RAG-1-/- (12)
BDC-2.5
(16)
(6)
(9)
(21)
BDC-10.1
NOD.SCID
NOD.SCID
NOD.SCID
NOD.SCID
BDC-10.1 (B6g7)
Incidence of Diabetes (%)
TCR
PA21.14H4
12-4.1
BDC-6.9
NY4.1
0
PA21.14H4 0.00815 0.01767
NY4.1 0.00346
p ≤ 0.0167*
*Bonferroni correction for multiple comparisons
50
75
100
0
C
25
50
100
75
Days Post-Transfer
Incidence of Diabetes (%)
1x106 Cells
1x105 Cells
5x104 Cells
1x104 Cells
5x103 Cells
25
50
75
100
0
10 20
30 40 50 60 70 80
Days Post-Transfer
1x105 Cells
0
5x104 Cells
(n)
(5)
(5)
(5)
1x104 Cells
# BDC-10.1
1x104 cells
5x103 cells
1x103 cells
5x103 Cells
(n)
(5)
(5)
(5)
150
1x103 Cells
# BDC-10.1
1x106 cells
1X105 cells
5x104 cells
125
0.00270 0.00270 0.00270 0.00270 0.04953
0.02147 0.00863 0.27789 0.36849
0.01431 0.00270 0.13361
0.93701 0.38696
0.83114
p ≤ 0.00333*
*Bonferroni correction for multiple comparisons
90 100
FIG. 5. Diabetes incidence in TCR Rg NOD.scid mice. A: NOD.scid and B6.g7.RAG-1ⴚ/ⴚ TCR retrogenic mice were monitored for diabetes onset for
140 days. B: GFPⴙCD4ⴙTCR␤ⴙ cells were FACS purified from BDC-10.1, NY4.1, and PA21.14H4 NOD.scid Rg mice 28 days posttransplant,
adoptively transferred into NOD.scid recipient mice, and monitored for diabetes. C: A titration of purified BDC-10.1 NOD.scid Rg T-cells were
transferred into NOD.scid mice and monitored for diabetes. The tables of P values on the right were generated using the log-rank test both overall
and pairwise within each experiment. The P value was based at the 0.05 significance level using the Bonferroni adjustment for multiple
comparisons.
1328
DIABETES, VOL. 57, MAY 2008
A.R. BURTON AND ASSOCIATES
compared with HEL-specific Rg mice (PA21.14H4). While
we cannot rule out a role for GAD-reactive T-cells in the
later stages of pathogenesis or in concert with other
T-cells (34), our data suggest that T-cells expressing the
current panel of clonotypic TCRs specific for GAD are not
capable of initiating insulitis and diabetes on their own.
Furthermore, several studies suggest that certain GADreactive TCRs are expressed by regulatory T-cells, an issue
that was not addressed in this study. While the IA2-specific
Rg T-cells did not infiltrate the islets, suggesting that IA2
may not be an important autoantigenic epitope in the early
stages of type 1 diabetes, both phogrin-specific T-cell
populations infiltrated islets to varying degrees. However,
although phogrin18 mediated significant peri-insulitis/insulitis, likely due to increased TCR expression and T-cell
number, this required 140 days to develop. Taken together,
these data suggest that some of the most commonly
studied type 1 diabetes autoantigens—GAD, IA2 and IA2␤/
phogrin—may have a reduced role in initiating type 1
diabetes.
On the other hand, the importance of insulin as an
autoantigen (38,47) was reaffirmed in this study. Both
insulin-specific TCRs conferred significant insulitis, while
only 12-4.1 caused diabetes. Given that these TCRs recognize the same epitope, the phenotypic distinctions observed are likely due to differences in TCR V␤ (V␤2 vs.
V␤12) and complimentarity-determining region usage,
and/or affinity/avidity as the 12-4.1 Rg T-cells have a
considerably higher stimulation index against the insulin
B9 –23 epitope. This suggests that while autoantigen and
epitope availability are important factors in mediating the
disease process, TCR affinity is likely an important factor.
Differences in the diabetogenicity of TCRs from BDC2.5, BDC-6.9, and BDC-10.1 in Rg mice would not have
been predicted from their pathogenicity as T-cell clones,
which is very similar in adoptive transfers into young NOD
or NOD.scid recipients (8,43). The hierarchy of insulitic
and diabetogenic potential exhibited by the three TCRs
that had previously been expressed as transgenes was
largely as expected (BDC-2.5 ⬎ NY4.1 ⬎ BDC-6.9) (21–23).
The most surprising observation was the diabetogenic
potential of Rg T-cells expressing the BDC-10.1 TCR.
These data suggest that this TCR may recognize a critical
epitope in disease etiology and/or may have an optimal
affinity/avidity for maximal islet infiltration and ␤-cell
destruction. It is important to note that despite the autoreactivity of BDC-2.5, BDC-10.1, and NY4.1, all three TCRs
were expressed, and Rg T-cells escaped thymic selection
(albeit to a lesser extent in the spleen compared with other
Rg T-cells). This suggests that diabetogenicity was attributed to the specificity of the TCR and not to differences in
the levels of TCR expression or the number of Rg T-cell
present in mice.
Taken together, these data suggest that autoantigenspecific TCRs can be segregated into three phenotypic
groups: 1) TCRs that fail to mediate T-cell islet entry but
may play a role in the later stages of the disease (e.g., TCRs
specific for GAD and IA2), 2) TCRs that mediate islet
infiltration but not ␤-cell destruction and thus may only
contribute significantly to type 1 diabetes if potentiated by
diabetogenic T-cells (e.g., certain phogrin- and insulinspecific TCRs), and 3) TCRs that mediate (to varying
degrees) insulitis and ␤-cell destruction and thus may be
key initiators/propagators of type 1 diabetes (e.g., certain
TCRs specific for insulin and unknown islet antigens).
Understanding the molecular distinctions between these
DIABETES, VOL. 57, MAY 2008
three populations will be critically important. In addition,
gaining insight into the levels and timing of autoantigen
expression/availability as well as the location/phenotype
of the APCs presenting autoantigens will be important and
may explain the differences we observed between the autoreactive Rg T-cells. In conclusion, our data suggest that
autoreactivity does not imply pathogenicity, as most autoreactive TCRs, on their own, appear to be nonpathogenic.
ACKNOWLEDGMENTS
J.H was supported by National Institutes of Health grants
R01 DK052068 and P30 DK57516. P.S. was supported by
the Canadian Institutes of Health Research and is a
Scientist of the Alberta Heritage Foundation for Medical
Research. D.A.A.V. was supported by funds from the
Juvenile Diabetes Research Foundation International (12004-141 [The Robert and Janice Compton Research Grant
in honor of Elizabeth S. Compton] and 1-2006-847), a pilot
project from the Cooperative Study Group for Autoimmune Disease Prevention (U19 AI050864-05 [George
Eisenbarth, principal investigator]), the St. Jude Cancer
Center Support CORE grant (CA-21765), and the American
Lebanese Syrian Associated Charities.
We are very grateful to Kate Vignali, Yao Wang, and
Smaroula Dilioglou for technical assistance; to the Vignali
lab for assistance with harvesting bone marrow; to Richard Cross, Jennifer Rogers, and Yuxia He for flow cytometry analysis; to the St. Jude Hartwell Center staff for oligo
synthesis and DNA sequencing, the staff of the St. Jude
ARC Histology Laboratory, and the Animal Husbandry
Unit and Flow Cytometry and Cell Sorting Shared Resource facility staff for AutoMACS. We thank Luc Teyton
and Christophe Benoist for the BDC-2.5 TCR plasmids and
Christophe Benoist and Diane Mathis for the B6.H2g7 mice.
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