Unmasking Genes in a Type 1 Diabetes–Resistant Mouse
Strain That Enhances Pathogenic CD8 T-Cell Responses
John P. Driver, Yi-Guang Chen, Weidong Zhang, Seblewongel Asrat, and David V. Serreze
OBJECTIVE—Nominally resistant mouse strains such as
C57BL/6 (B6) harbor latent type 1 diabetes susceptibility genes
uncovered in outcross to disease-susceptible NOD mice. How-
ever, identification of possible recessively acting B6-derived
susceptibility genes is limited because very few F2 progeny
derived from outcrossing this strain with NOD develop sponta-
neous autoimmune diabetes. Thus, we assessed whether a trans-
genic T-cell receptor (TCR) disease transfer model allowed the
mapping of recessively acting B6 genetic loci that in the proper
context contribute to diabetes.
RESEARCH DESIGN AND METHODS—CD8 T-cells trans-
genically expressing the diabetogenic AI4 TCR were transferred
into 91 (NODxB6.H2g7)F1xB6.H2g7first-backcross (BC1) females.
A genome-wide scan was performed for loci affecting clinical di-
abetes and insulitis severity.
RESULTS—A major locus on chromosome 11 in tight linkage
with the marker D11Mit48 (logarithm of odds score = 13.2)
strongly determined whether BC1 progeny were susceptible to
AI4 T-cell–mediated diabetes. Mice homozygous versus hetero-
zygous for B6 markers of this chromosome 11 genetic locus were,
respectively, highly susceptible or resistant to AI4-induced insu-
litis and diabetes. The genetic effect is manifest by host CD4
T-cells. Microarray analyses of mRNA transcript expression iden-
tified a limited number of candidate genes.
CONCLUSIONS—The distal region of chromosome 11 in B6
mice harbors a previously unrecognized recessively acting gene
(s) that can promote autoreactive diabetogenic CD8 T-cell
responses. Future identification of this gene(s) may further aid
the screening of heterogeneous humans at future risk for
diabetes, and might also provide a target for possible disease
interventions. Diabetes 60:1354–1359, 2011
and NOD mice, disease pathogenesis requires interactions
with multiple other susceptibility (Idd) genes (1). This is
illustrated by the fact that C57BL/6 background mice
congenic for the NOD-derived H2g7MHC haplotype (B6.
H2g7) are normally diabetes-resistant (2). However, cer-
tain T-cell receptor (TCR) molecules contributing to di-
abetes development in NOD mice paradoxically exert even
greater pathogenic activity when transgenically expressed
in the B6.H2g7strain (3,4). This finding, along with previous
complex (MHC) haplotypes are the strongest
contributor to T-cell–mediated autoimmune
type 1 diabetes development in both humans
linkage analyses and congenic approach studies (5,6), in-
dicate normally diabetes-resistant strains harbor some
genes that in the proper combination can actually contrib-
ute to aggressive disease development.
Analyses of F2 rather than first backcross (BC1) prog-
eny is the preferable approach for mapping diabetic genes,
because this allows for the identification of both suscepti-
bility or resistance variants from NOD mice or the outcross
partner strain. However, identifying possible recessively
acting B6-derived susceptibility genes after the outcross to
NOD has been hampered because very few F2 progeny
develop spontaneous diabetes even when the H2g7MHC is
fixed in all segregants (7). Nevertheless, analyses of prog-
eny from a first backcross to B6.H2g7mice revealed re-
cessive alleles from this strain on chromosomes 1, 2, 7, and
15 promoting the diabetogenic activity of CD4 T-cells
transgenically expressing the BDC2.5 TCR (3). We now
report a similar BC1 strategy using another transgenic TCR
that reveals at least one additional B6 origin recessive Idd
susceptibility gene on chromosome 11 contributing to the
peripheral activation of pathogenic CD8 T-cells.
RESEARCH DESIGN AND METHODS
Mice. NOD/ShiLtDvs mice are maintained in a specific pathogen-free research
colony. B6.H2g7mice are maintained at the N8 backcross generation (4). NOD
mice transgenically expressing the TCR from the diabetogenic CD8+T-cell
clone AI4 plus a functionally inactivated Rag1 gene (NOD.Rag1null.AI4) have
been described (8).
Adoptive transfer of diabetes. Indicated female mice were sublethally ir-
radiated (600 R) and injected intravenously with 1 3 107NOD.Rag1null.AI4
splenocytes to induce diabetes. In one experiment, recipients were injected
intraperitoneally with 250 mg/mouse of the CD25-depleting PC61.5 antibody
1 day before AI4 T-cell transfer. In another experiment, donor cells were
prelabeled with 2.5 mmol/L carboxy fluorescein succinimidyl ester (CFSE).
After 4 days, viable AI4 T-cells from collagenase D-digested spleen and pan-
creatic lymph nodes were identified by flow cytometry using CFSE and a CD8-
specific antibody (53–6.7). Expression of various T-cell surface markers was
assessed using antibodies specific for CD44 (IM7.8.1), CD25 (PC61.5), and
CD62 L (MEL-14). Another experiment used female mice injected with AI4
T-cells preactivated in culture for 3 days with 100 nmol/L antigenic mimotope
peptide YFIENYLEL and 50 units/mL interleukin 2.
In a separate experiment, female B6.H2g7mice were lethally irradiated
(1,200 R) and injected intravenously with 5 3 106bone marrow cells from
BC1 progeny heterozygous or homozygous for the microsatellite marker
D11Mit48. Fourteen weeks later, recipient mice were sublethally irradiated
and injected intravenously with 1 3 107NOD.Rag1null.AI4 splenocytes to in-
duce diabetes. In another study, B6.H2g7, NOD and F1 mice all homozygous
for the Rag1nullmutation were injected with 1 3 107NOD.Rag1null.AI4 sple-
nocytes. Finally, magnetic bead purified CD4 T-cells from D11Mit48B6/B6or
D11Mit48NOD/B6BC1 progeny were cotransferred with 1 3 107NOD.Rag1null.
AI4 splenocytes into B6.H2g7.Rag1nullrecipients. Recipients were either killed
at the indicated time or monitored for diabetes development.
Assessment of diabetes and insulitis. Diabetes was assessed by daily
monitoring of glycosuria with Ames Diastix (Bayer, Diagnostics Division,
Elkhart, IN), with disease onset defined by two consecutive values of $3.
Previously described criteria (9) were used to establish insulitis scores ranging
from 0 (individual islet with no leukocytic infiltration, normal b-cell mass) to 4
(complete destruction) for the indicated mice.
Genotyping and linkage analyses. BC1 progeny were genotyped as pre-
viously described (10) for 131 single nucleotide polymorphisms (SNP) at
From The Jackson Laboratory, Bar Harbor, Maine.
Corresponding author: David V. Serreze, email@example.com.
Received 25 June 2010 and accepted 13 January 2011.
This article contains Supplementary Data online at http://diabetes.
? 2011 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
1354DIABETES, VOL. 60, APRIL 2011 diabetes.diabetesjournals.org
approximately 20-Mb intervals across the genome (National Center for Bio-
technology Information [NCBI] build 37 marker positions). Linkage markers
for genes controlling insulitis severity and diabetes development in response
to AI4 T-cell transfer were identified as previously described (11). PCR typing
of the polymorphic D11Mit48 microsatellite marker was also done.
Microarray analyses. CD4 T-cells were purified from BC1 progeny either
heterozygous or homozygous for D11Mit48 and cotransferred into female B6.
H2g7.Rag1nullrecipients with 1 3 107NOD.Rag1null.AI4 splenocytes. RNA was
isolated from CD4 T-cells sorted from spleens of these recipients by flow
cytometry ;3 weeks after adoptive transfer. Three biologic replicates were
generated for both genotypes, with each sample emanating from purified CD4
T-cells pooled from two donor mice. Microarray analysis of comparative CD4
T-cell gene expression was conducted using the Affymetrix 430v2 GeneChip
array (Santa Clara, CA). R/Bioconductor software summarized the probe
intensities for each gene using the robust multiaverage method (12,13).
R/MAANOVA software (Churchill Group, Bar Harbor, ME) was used to gen-
erate lists of differentially expressed genes between the tested samples (14).
Differentially expressed genes were identified by using ts, a modified t statistic
incorporating shrinkage estimates of variance components from within the
FIG. 1. NOD and B6.H2g7but not (NODxB6.H2g7)F1 mice succumb to AI4 T-cell–induced diabetes. A: Incidence of diabetes in 6- to 8-week-old
female NOD, B6.H2g7, F1, and (F1xB6.H2g7)BC1 recipient mice that were sublethally irradiated (600 R) and injected intravenously with 1 3 107
NOD.Rag1null.AI4 splenocytes. Incidence of diabetes in BC1 recipients was significantly different (P < 0.0001) from NOD, B6.H2g7, and F1 mice. B:
Insulitis scores (0 = no insulitis to 4 = no remaining islet cell mass) for surviving nondiabetic NOD.Rag1null.AI4 splenocyte recipients. Insulitis
severity was significantly greater in surviving NOD mice compared with F1 and BC1 recipients according to the Mann-Whitney test. C: In vivo
proliferation and activation of CFSE-labeled NOD.Rag1null.AI4 T-cells at 4 days after transfer in pancreatic lymph nodes of NOD, B6.H2g7, and F1
mice. D: Incidence of diabetes in 6- to 8-week-old sublethally irradiated female NOD and F1 recipients of in vitro activated AI4 T-cells.
J.P. DRIVER AND ASSOCIATES
diabetes.diabetesjournals.orgDIABETES, VOL. 60, APRIL 2011 1355
expressing the TCR from the diabetogenic AI4 CD8 T-cell
clone and also homozygous for the Rag1nullmutation
(NOD.Rag1null.AI4). Adoptively transferred AI4 T-cells
from such donors rapidly induce diabetes in both sub-
lethally irradiated NOD and B6.H2g7mice (16). Thus, we
were surprised AI4 T-cells failed to transfer diabetes or
significant levels of insulitis to sublethally irradiated
(NODxB6.H2g7)F1 hybrids (Fig. 1A and B). This difference
was not due to varying post-transfer stimulation of AI4
T-cells within pancreatic lymph nodes, because the pro-
liferation of such effectors at this site in F1 hybrids was
similar to that of NOD mice and even greater than in B6.
H2g7recipients (Fig. 1C). Furthermore, no differences in
expression of CD44, CD25, and CD62 L activation markers
were detected between AI4 T-cells isolated from the parental
or F1 mice (Fig. 1C and data not shown). However, AI4
T-cells preactivated in culture rapidly transferred diabetes to
F1 recipients (Fig. 1D). Hence, the F1 genetic environment
allows for less efficient initial pathogenic activation of di-
abetogenic CD8 T-cells than in either parental strain.
F1 hybrid resistance to AI4-induced diabetes indicates
the NOD and B6.H2g7genomes harbor separate recessively
acting alleles supporting pathogenic CD8 T-cell acti-
vation. To map such unrecognized recessive B6 alleles,
NOD.Rag1null.AI4 splenocytes were transferred into 91
sublethally irradiated female (NODxB6.H2g7)F1xB6.H2g7
BC1 progeny that were then monitored for diabetes de-
velopment and also genotyped. Diabetes developed in 40%
of BC1 segregants at a rate similar to both NOD and B6.
H2g7mice (Fig. 1A). Nondiabetic BC1 mice were exam-
ined for insulitis levels. Similar to F1 hybrids, insulitis
levels were low in nondiabetic BC1 mice (Fig. 1B). This
sharp dichotomy among BC1 progeny suggested that
rather than contributions from multiple loci, which would
result in a broad spectrum of insulitis scores, a limited
number of genes rendered B6.H2g7mice susceptible to
AI4 T-cell–induced diabetes. Indeed, a SNP-based genome-
wide one-dimensional scan of BC1 progeny revealed only
one B6 genomic region on chromosome 11 that was highly
linked to diabetes susceptibility and insulitis (logarithm of
odds [LOD] score = 13.2; Fig. 2A).
On the basis of original typing of SNP markers only, the
95% CI for the region of interest was originally narrowed to
a 3.6-Mb segment at the distal end of chromosome 11
(112.6–116.2 Mb), ;40 Mb below the previously identified
Idd4 locus (Fig. 2B and C). Susceptibility to AI4 T-cell–
induced diabetes/insulitis was associated with homozy-
gosity for B6 markers within this region of chromosome
11, with LOD scores increasing up to and including the
most distal NOD/B6 distinguishing SNP (rs3675087 at
116.2 Mb) that was typed. However, it remained possible
that a gene(s) controlling susceptibility to AI4 transfer
resided distally to the 116.2-Mb position. Thus, BC1 prog-
eny were regenotyped for the polymorphic microsatellite
marker D11Mit48 located at 117.76 Mb. Linkage between
B6 homozygosity at D11Mit48 and diabetes/insulitis was
very similar to that for rs3675087 (Supplementary Table 1).
Sanger sequence analysis (www.sanger.ac.uk) indicated
there are NOD/B6 polymorphisms in protein-encoding
regions of two genes distal to D11Mit48, but these were
not covered by the SNP typing panel available to us. For
this reason, we redefined the support interval controlling
differential sensitivity to type 1 diabetes induced by
transferred AI4 T-cells to between 112.6 Mb and the end of
chromosome 11 (121.8 Mb; Fig. 2C).
We tested whether genotyping BC1 mice for D11Mit48
alone could predict susceptibility to AI4 T-cell–induced
diabetes. An additional cohort of 31 BC1 mice was geno-
typed for the D11Mit48 polymorphism before receiving
FIG. 2. A single genetic locus primarily contributes to B6.H2g7suscep-
tibility to AI4 T-cell–induced diabetes. Whole genome (A) and chromo-
some 11–specific (B) LOD score analysis of SNP markers linked to
diabetes susceptibility and insulitis severity in sublethally irradiated
BC1 recipients of 1 3 107NOD.Rag1null.AI4 splenocytes. Horizontal
lines depict LOD scores indicative of 1, 5, 10, and 63% linkage support
thresholds. The 1 and 63% thresholds, respectively, indicate significant
and suggestive linkage. The lower bold line indicates the 95% CI for
linkage. C: Schematic representation of the distal region of chromo-
some 11. The positions of the markers (Mb) are based on NCBI build
37. The distance between markers is not drawn to scale.
NOVEL C57BL/6-DERIVED SUSCEPTIBILITY LOCUS
1356DIABETES, VOL. 60, APRIL 2011diabetes.diabetesjournals.org
FIG. 3. A polymorphic gene(s) in close linkage with the D11Mit48 microsatellite marker controls susceptibility to AI4 T-cell–induced diabetes
through effects on a CD4 T-cell population other than CD25+Tregs. A: Mice homozygous for the B6 allele (B6/B6) vs. heterozygous (NOD/B6) for
D11Mit48 were, respectively, highly susceptible and resistant to AI4 T-cell–induced diabetes. Results represent three independent experiments.
B: Insulitis scores are shown for nondiabetic heterozygous and homozygous BC1 NOD.Rag1null.AI4 splenocyte recipients. C and D: B6.H2g7mice
previously reconstituted with bone marrow from D11Mit48B6/B6or D11Mit48NOD/B6BC1 segregants are, respectively, susceptible or resistant to
diabetes and insulitis induced by subsequently infused AI4 T-cells. E: Incidence of diabetes in 6- to 8-week-old female B6.H2g7.Rag1null, NOD.
Rag1null, and F1.Rag1nullrecipients of 1 3 107NOD.Rag1null.AI4 splenocytes. F: Incidence of diabetes in 6- to 8-week-old sublethally irradiated
NOD and F1 recipients of 1 3 107NOD.Rag1null.AI4 splenocytes. Recipients were also injected intraperitoneally with a CD25-depleting antibody
(PC61 250 mg/mouse) 1 day before AI4 T-cell transfer. G and H: B6.H2g7.Rag1nullmice infused with purified CD4+T-cells from D11Mit48B6/B6
or D11Mit48NOD/B6BC1 segregants are, respectively, susceptible or resistant to diabetes and insulitis induced by subsequently infused AI4 T-cells.
J.P. DRIVER AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 60, APRIL 20111357
NOD.Rag1null.AI4 splenocytes. None of the 15 heterozy-
gous (D11Mit48NOD/B6) animals developed diabetes and
usually no more than mild peri-insulitis (Fig. 3A and B).
Conversely, 14 of 16 homozygous (D11Mit48B6/B6) mice
developed diabetes, and the remaining two mice were se-
verely insulitic (Fig. 3A and B). This simple segregation
pattern strongly indicates that a recessively acting B6 or-
igin gene(s) tightly linked to D11Mit48 is a primary con-
tributor to the pathogenic activation of diabetogenic CD8
T-cells, whereas the NOD allelic variant actually domi-
nantly suppresses this process. However, the protective
effect of this NOD chromosome 11 allelic variant must
normally be masked by the large number of other diabetes
susceptibility genes characterizing this strain.
To determine whether the chromosome 11 gene(s) con-
trols diabetes susceptibility through effects on hematopoietic
cells, NOD.Rag1null.AI4 splenocytes were transferred into
B6.H2g7mice previously reconstituted with bone marrow
from D11Mit48B6/B6or D11Mit48NOD/B6BC1 progeny. Only
recipients of D11Mit48B6/B6but not D11Mit48NOD/B6bone
marrow developed diabetes (Fig. 3C) or significant levels of
insulitis (Fig. 3D). Therefore, the D11Mit48-linked gene(s)
controlling susceptibility to AI4 T-cell–mediated diabetic
functions through a hematopoietic cell population(s).
Next, we established this gene(s) controls type 1 di-
abetes susceptibility through a lymphocyte population(s).
This was determined by demonstrating NOD.Rag1null.
AI4 splenocytes transferred diabetes with equal efficiency
to B6.H2g7, NOD, and F1 mice all homozygous for the
Rag1nullmutation, eliminating all endogenous lympho-
cytes (Fig. 3E). We tested whether regulatory T-cells
(Tregs) were the lymphocyte population rendering F1
mice resistant to AI4-mediated diabetes. NOD and F1 mice
were treated with a CD25-depleting antibody 1 day before
receiving NOD.Rag1null.AI4 splenocytes. This eliminated
most CD4+CD25+Tregs for the duration of the 15-day
postadoptive transfer period during which AI4 T-cells
normally induce diabetes (Supplementary Fig. 1). Anti-
CD25–treated F1 mice remained resistant to AI4-induced
diabetes (Fig. 3F).
We next tested whether the chromosome 11 gene(s)
controls type 1 diabetes susceptibility through effects on
a CD4 T-cell population other than Tregs. Total CD4
T-cells purified from D11Mit48B6/B6or D11Mit48NOD/B6
BC1 progeny were cotransferred with NOD.Rag1null.AI4
splenocytes into B6.H2g7.Rag1nullrecipients. Only recipi-
ents of D11Mit48B6/B6but not D11Mit48NOD/B6CD4 T-cells
developed AI4 T-cell–induced diabetes or high levels of
insulitis (Fig. 3G and H). Therefore, the D11Mit48-linked
gene(s) controlling susceptibility to AI4 T-cell–mediated di-
abetic functions through a non-Treg CD4 T-cell population(s).
We used microarray-based comparisons of mRNA
transcript levels to identify candidates for a CD4 T-cell–
expressed gene(s) within the chromosome 11 support
interval regulating pathogenic activation of diabetogenic
AI4 CD8 T-cells. This was accomplished by recovering CD4
List of polymorphic genes within the chromosome 11 interval 112.6 to 121.8 Mb that are differentially expressed in CD4 T-cells purified
from D11Mit48B6/B6vs. D11Mit48NOD/B6genotyped BC1 progeny
over B6/B6 Fs P
117.8Suppressor of cytokine
21.4 0.0529 Inhibits activation and/or differentiation
pathways in macrophages, dendritic cells,
116.7Major facilitator superfamily
domain containing 11
Solute carrier family 9
0.0387 Multifunctional adaptor protein, recruiting
cytoplasmic signaling proteins and
membrane receptors/transporters into
functional complexes. Defective regulation
is linked with susceptibility to psoriasis
0.0088 Calcium ion binding, nucleotide metabolism
Nuclear protein localization
RAB37, member of RAS
cDNA sequence BC018473
CD300 antigen like family
115.02.1 0.0023 GTPase expressed in mast cells
0.0089 Member of an immunoglobulin superfamily
gene cluster that may serve as an inhibitory
receptor to regulate the maturation and
differentiation of immune cells, helping
to contain inflammation
0.0055 Component of the transmembrane laminin
*Marker positions were taken from NCBI build 37.1 (www.ncbi.nlm.nih.gov). †See http://harvester.fzk.de/harvester.
NOVEL C57BL/6-DERIVED SUSCEPTIBILITY LOCUS
1358DIABETES, VOL. 60, APRIL 2011diabetes.diabetesjournals.org