Download Activation, Differentiation, and Cell Death on Murine Chromosome 7

Genetic Dissection of Sle Pathogenesis: Sle3
on Murine Chromosome 7 Impacts T Cell
Activation, Differentiation, and Cell Death
This information is current as
of June 18, 2017.
Chandra Mohan, Ying Yu, Laurence Morel, Ping Yang and
Edward K. Wakeland
J Immunol 1999; 162:6492-6502; ;
http://www.jimmunol.org/content/162/11/6492
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References
Genetic Dissection of Sle Pathogenesis: Sle3 on Murine
Chromosome 7 Impacts T Cell Activation, Differentiation, and
Cell Death1
Chandra Mohan,2* Ying Yu,† Laurence Morel,† Ping Yang,† and Edward K. Wakeland2*
A
rich series of immunological events underlies the pathogenesis of lupus (1–3). Both generalized B cell hyperactivity and T cell-driven B cell autoantibody production have been well documented in murine and human lupus.
Likewise, generalized T cell abnormalities, as well as help rendered by clonally expanded Ag-specific Th cells, are both likely to
contribute to pathology, as discussed below. These different immunological aberrations ultimately lead to the formation of pathogenic antinuclear Abs (ANAs),3 the hallmark of this disease. Although the genetic basis of these mechanisms has long remained a
mystery, recent murine studies are just beginning to reveal their
intricacies.
Susceptibility to lupus in murine models has been mapped to
several loci, as recently reviewed (4 – 6). Our studies have focused
on the NZM2410 strain, derived from a cross between New Zealand Black (NZB) and New Zealand White (NZW) mice (7). This
lupus-prone strain demonstrates splenomegaly, a significant expansion of activated CD41 T cells and B cells, high-titered ANAs,
and early-onset immune-complex glomerulonephritis (GN) (7, 8).
To map these immunological traits, a (NZM2410 3 C57BL/6
*Simmons Arthritis Research Center and Center for Immunology, University of
Texas Southwestern Medical Center, Dallas, TX 75235; and †Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610
Received for publication October 22, 1998. Accepted for publication March 9, 1999.
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.
1
This work was supported by National Institutes of Health Grants PO1 AI39824 and
RO1 AR44894 and by a grant from the National Arthritis Foundation.
2
Address correspondence and reprint requests to Drs. Chandra Mohan and Edward K.
Wakeland, Simmons Arthritis Research Center and the Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235-8884. E-mail addresses: [email protected] and [email protected]
3
Abbreviations used in this paper: ANA, anti-nuclear Ab; NZB, New Zealand Black;
NZW, New Zealand White; GN, glomerulonephritis; B6, C57BL/6; PI, propidium
iodide; AICD, activation-induced cell death; LN, lymph node; TTP, tristetraprolin.
Copyright © 1999 by The American Association of Immunologists
(B6)) 3 NZM2410 backcross analysis was undertaken several
years ago (9). That mapping study revealed that genomic intervals
on chromosomes 1 (Sle1), 4 (Sle2), 7 (Sle3), and 17 (Sle4) were
strongly linked to lupus nephritis. Of relevance to this study, the
locus on chromosome 7 (Sle3) showed significant linkage to GN,
with peak linkage at the pink eye-dilution ( p) locus (X 2 5 16.7;
p , 1024; logarithm of the odds score 5 4). Other independent
mapping studies have also mapped lupus susceptibility to murine
chromosome 7, close to Sle3. Using an (NZB 3 NZW)F2 mapping
panel, Kono et al. (10) mapped a lupus susceptibility locus, Lbw5,
in the vicinity of Sle3. Subsequently, the same workers mapped
susceptibility to lymphadenopathy and anti-dsDNA production in
another disease model, using an (MRL.Faslpr 3 B6.Faslpr)F2 analysis, to Lmb3, again in the vicinity of Sle3 (11). Using an (MRL/
lpr 3 CAST/Ei) backcross panel, Watson et al. (12) mapped susceptibility to lupus nephritis to a more centromeric locus on
murine chromosome 7, close to ckmm. Thus, this genomic interval
on murine chromosome 7 appears to harbor a gene(s) that potentially dictates some critical pathogenic mechanisms leading to lupus in several different disease-prone strains.
Deciphering the relative immunopathological contributions of
these loci to disease has been greatly facilitated by the generation
and characterization of congenic mice bearing these individual lupus susceptibility intervals (4, 13–17). Adopting this powerful approach, we have recently shown that Sle1 triggers loss of tolerance
to chromatin, apparently without any generalized abnormalities in
lymphocyte activation, differentiation, or apoptosis (15, 17). In
contrast, Sle2 leads to generalized B cell hyperactivity, elevated
levels of splenic and peritoneal B1a cells, and increased serum
polyclonal/polyspecific IgM, but with no evidence of IgG ANAs,
T cell defects, or GN (15, 16). This study focuses on B6.NZMc7
mice, which are B6 mice rendered congenic for the NZM2410derived Sle3 interval, as diagrammed in Fig. 1. Our initial studies
with this congenic strain had revealed increased serum levels of
polyclonal/polyspecific IgM and IgG Abs, accompanied by an
0022-1767/99/$02.00
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Polyclonal, generalized T cell defects, as well as Ag-specific Th clones, are likely to contribute to pathology in murine lupus, but
the genetic bases for these mechanisms remain unknown. Mapping studies indicate that loci on chromosomes 1 (Sle1), 4 (Sle2), 7
(Sle3), and 17 (Sle4) confer disease susceptibility in the NZM2410 lupus strain. B6.NZMc7 mice are C57BL/6 (B6) mice congenic
for the NZM2410-derived chromosome 7 susceptibility interval, bearing Sle3. Compared with B6 controls, B6.NZMc7 mice exhibit
elevated CD4:CD8 ratios (2.0 vs 1.34 in 1- to 3-mo-old spleens); an age-dependent accumulation of activated CD41 T cells (33.4%
vs 21.9% in 9- to 12-mo-old spleens); a more diffuse splenic architecture; and a stronger immune response to T-dependent, but
not T-independent, Ags. In vitro, Sle3-bearing T cells show stronger proliferation, increased expansion of CD41 T cells, and
reduced apoptosis (with or without anti-Fas) following stimulation with anti-CD3. With age, the B cells in this strain acquire an
activated phenotype. Thus, the NZM2410 allele of Sle3 appears to impact generalized T cell activation, and this may be causally
related to the low grade, polyclonal serum autoantibodies seen in this strain. Epistatic interactions with other loci may be required
to transform this relatively benign phenotype into overt autoimmunity, as seen in the NZM2410 strain. The Journal of Immunology, 1999, 162: 6492– 6502.
The Journal of Immunology
6493
FIGURE 1. The NZM2410 strain spontaneously develops lupus. In a backcross study
involving this strain, three non-MHC genomic
intervals showed significant linkage to autoantibody production and/or GN: Sle1 on chromosome 1, Sle2 on chromosome 4, and Sle3 on
chromosome 7 (9). In the latter study, peak
linkage for Sle3 was noted at the p locus (28
cM). By repeated backcrossing and microsatellite marker-based selection, the 95% confidence
interval flanking Sle3 on NZM2410 chromosome 7 has been bred onto the B6 background
(14). The introgressed chromosome 7 interval
encompassing Sle3 is shown in white, with termini at D7 MIT56 and D7 MIT62. Lbw5 (10)
and Lmb3 (11) are lupus susceptibility loci
mapped by other groups to the same genomic
interval.
Materials and Methods
Mice
B6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME)
and subsequently bred in our animal colony. The derivation of B6 congenic
mice bearing NZM2410-derived lupus susceptibility intervals has been detailed previously (14). As depicted in Fig. 1, B6.NZMc7 mice are B6 mice
congenic (homozygotes) for a 40-cM interval on murine chromosome 7,
encompassing the 95% confidence interval flanking Sle3, with termini at
D7 MIT56 and D7 MIT62. The entire congenic interval is derived from the
NZW parent of the NZM2410 strain. All mice used for this study were bred
and housed in a conventional colony, under identical conditions, at the
University of Florida Department of Animal Resources. As no sex differences were noted in the expression of any of the phenotypes described,
both male and female mice were used in this study. Nevertheless, for the
in vivo antigenic challenge experiments, female mice were used.
Cell preparation
Splenocytes were depleted of RBCs using 0.83% NH4Cl, and single-cell
suspensions were prepared. Total splenic B cells were prepared as described before (17, 18), using pretitrated amounts of anti-Thy-1 (Accurate
Chemicals, New York, NY) and rabbit complement (Accurate Chemicals)
to lyse T cells, and were typically 85–95% pure. Splenic T cells were
prepared as described (17, 18). Briefly, red cell-depleted splenocytes were
loaded onto nylon wool columns (Robbins Scientific, Sunnyvale, CA) and
incubated at 37°C for 45 min. Nonadherent cells were washed through and
then incubated with pretitrated amounts of anti-I-Ab (clone K25– 8.7, Accurate Chemicals) and anti-CD24 (PharMingen, San Diego, CA) on ice for
45 min. Ab-bound cells were lysed with rabbit complement (Accurate
Chemicals), yielding T cells with .90% purity. Bone marrow cells were
obtained by flushing femurs and tibia with medium. Peritoneal cavity cells
were obtained by injecting medium into the peritoneal cavity and extracting the cells with transfer pipettes. Prepared single-cell suspensions were
counted and used for culture or flow cytometric analysis, as described
below.
Flow cytometric analysis, sorting, and Abs
Flow cytometric analysis (FACS) was performed as described previously
(16, 19). Briefly, cells were first blocked with staining medium (PBS, 5%
horse serum, and 0.05% azide) containing 10% normal rabbit serum. Cells
were then stained on ice with optimal amounts of FITC, PE, or biotinconjugated primary mAbs diluted in staining medium for 30 min. Follow-
ing two washes, biotin-conjugated Abs were revealed using streptavidin-PE (Life Technologies, Grand Island, NY) or streptavidin-Quantum
Red (Sigma, St. Louis, MO). Cell staining was analyzed using a FACScan
(Becton Dickinson, San Jose, CA). Dead cells were excluded on the basis
of scatter characteristics and propidium iodide (PI) uptake, and 10,000
events were acquired per sample. The following dye- or biotin-coupled Abs
were obtained from PharMingen and used at pretitrated dilutions: CD4
(RM4-5), CD5 (53-7.3), CD8 (Ly-2), CD23 (B3B4), CD24 (M1/69), CD25
(7D4), CD43 (S7), CD44 (IM7), CD45R/B220 (RA3-6B2), CD62L
(MEL14), CD69 (H1.2F3), CD80/B7-1 (16-10A1), and CD86/B7-2 (GL1).
Cell stimulation assays
F(ab9)2 goat anti-mouse IgM (Cappel, Durham, NC) and LPS (Sigma) were
used for B cell stimulation at concentrations from 0.1 to 100 mg/ml. Likewise, graded doses of anti-CD3 (PharMingen), rIL-2 (Chiron Therapeutics,
Emeryville, CA) or keyhole limpet hemocyanin (KLH; Calbiochem, La
Jolla, CA) were added to assess T cell responses to stimuli. For these
assays total splenocytes or splenic T cells (5 3 105/well) were cultured for
72 h in 200-ml cultures, in serum-free HL-1 medium (HyCor Biomedicals,
Irvine, CA), with or without added stimuli. Lymphocyte response was assessed either by assaying the extent of proliferation, using [3H]TdR incorporation (ICN Biomedicals, Costa Mesa, CA) over the last 18 h of culture
or by measuring by ELISA the amount of IL-2, IL-4, or IFN-g secreted at
the indicated time points postculture.
For the experiments depicted in Fig. 4, 5 3 105 splenocytes per well
were cultured with anti-CD3 and anti-CD28 (1 mg/ml each, PharMingen).
At 24, 48, and 72 h postculture, the absolute numbers of CD41 and CD81
T cells in culture were determined by counting live (i.e., PI-excluding) cells
and by FACS analysis. For assaying activation-induced cell death (AICD),
total splenocytes were stimulated with anti-CD3 and anti-CD28 for 48 h.
Then, blasts (consisting of .90% T cells) were purified over Ficoll,
washed, and recultured with plate-bound anti-CD3, with or without antiFas (2 mg/ml, clone Jo2, PharMingen) or control hamster IgG (PharMingen). After the indicated culture periods, the percentage of apoptosis was
determined, as described below.
Cell death assays
Purified splenic B cells or T cells, or T cell blasts, prepared as detailed
above, were cultured, with or without added stimuli. Aliquots of this culture were assayed at 0, 24, 48, and 72 h for the percentage of apoptotic cells
in culture, using hypotonic lysis and PI incorporation, as described before
(20). Briefly, the cell pellets were resuspended in hypotonic PI solution (3.4
mM sodium citrate, 50 mg/ml PI, 0.1% Triton X-100, 1 mM Tris, and 0.1
mM EDTA) and analyzed by FACS, using logarithmic scales. Apoptotic
nuclei were distinguished by their hypodiploid DNA content and scatter
characteristics, after excluding the debri. Cell death was also assayed by
monitoring annexin binding, as described (21). Annexin is known to bind
to phosphatidylserine exposed on the outer leaflets of cells undergoing
apoptosis. For assaying this, lymphocytes or T cell blasts were stained with
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18% incidence of severe (i.e., .50% of glomeruli affected) immune complex-mediated GN (15). By comparing the cellular phenotypes seen in the B6.NZMc7 strain with those of age-matched
B6 controls, the present study aims to understand the pathogenic
mechanisms through which Sle3 might contribute to disease.
6494
Sle3 IMPACTS T CELL ACTIVATION AND APOPTOSIS
PE- or biotin-labeled anti-CD4, anti-CD8, or anti-B220, as described
above. Following a washing step, the cells were incubated with annexinFITC (PharMingen) in HEPES-containing buffer and then analyzed on a
flow cytometer.
IL ELISAs
IL produced in culture was assayed by ELISA, as described previously
(17). The following reagents were purchased from PharMingen: anti-IL-2;
anti-IL-4; anti-IFN-g; biotinylated anti-IL-2; biotinylated anti-IL-4; biotinylated anti-IFN-g; and rIL-2, rIL-4, and IFN-g standards. Briefly, Immulon
I plates (Dynatech Laboratories, Chantilly, VA) precoated with the “capture” Ab (anti-IL-2, anti-IL-4, or anti-IFN-g) were blocked and then incubated for 2 h with culture supernatants (diluted 1:2) or serial dilutions of
the rIL standard. Captured IL was detected using biotin-coupled anti-IL,
avidin-alkaline phosphatase (1:10,000, Sigma), and p-nitrophenylphosphate substrate (Sigma). ODs were converted to pg/ml using the derived
standard curve.
In vivo immunization
FIGURE 2. Splenic sections from B6 (top) and B6.NZMc7 (bottom)
were immunostained with anti-B220 (purple) and anti-CD3 (orange).
Shown sections were obtained from 9- to 12-mo-old mice and are representative of five to eight mice of each strain. Magnification, 310.
Immunochemistry
Immunochemistry on splenic sections was performed, as described previously (8). Briefly, fragments of spleens were snap frozen in liquid nitrogen
and stored at 270°C, until time of sectioning. Cryosections 0.4 – 0.6 mm
were fixed in acetone and air dried. Slides were rehydrated with TBS-0.1%
BSA and then blocked with 5% normal rabbit serum and 0.3% H202/100%
MeOH. T cells were tagged by sequential incubations with biotin-coupled
anti-CD3 (1 mg/ml, PharMingen) and avidin-alkaline phosphatase. B cells
were identified using FITC-coupled anti-B220 (5 mg/ml, PharMingen) and
peroxidase-coupled anti-FITC (Boehringer Mannheim). In addition, the
size and numbers of germinal centers in the spleens of the immunized mice
were assessed using biotin-coupled peanut agglutinin. All incubations were
performed for 30 min at room temperature in a moist chamber. Sections
were developed using the peroxidase substrate diaminobenzidine (Sigma)
and the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium (Sigma) and then washed and mounted using
Permount (Fisher, Pittsburgh, PA).
Statistics
Data obtained for the B6.NZMc7 mice were compared with those from the
control B6 mice using the Student’s t test. For all experiments, the mean
(6SEM) values observed are shown.
Results
Lymphoid organs and lymphocyte subsets in B6.NZMc7 mice
Sle3 did not seem to impact the development or cellular composition of the primary lymphoid organs. Thus, the marrow and thymus from B6.NZMc7 mice exhibited the appropriate distributions
of B cells and T cells, respectively, at the different developmental
stages (data not shown). All differences observed were confined to
the secondary lymphoid organs. Although B6.NZMc7 mice did not
differ from B6 controls in the size or cellularity of their spleens, the
T cell and B cell zones in these mice were poorly organized, with
frequent intermingling of T cell and B cell foci, compared with B6
spleens, as illustrated in Fig. 2. However, this immunohistological
phenotype was a relatively late event, being apparent only in the
older age group (9- to 12-mo-old mice). B6.NZMc7 spleens did
not differ from B6 spleens in the numbers or size of germinal
centers after immune challenge (data not shown).
Table I illustrates the distribution of T cell and B cell subsets in
the peripheral lymphoid organs of B6.NZMc7 mice. Because the
total splenocyte numbers did not differ between the 2 strains, any
observed differences in lymphocyte percentages also reflect differences in absolute numbers. The B6.NZMc7 spleens did not differ
significantly from B6 controls in the numbers of B1, B2, or total T
cells, at any age. However, B6.NZMc7 spleen and lymph node
(LN) had significantly greater percentages and numbers of CD41
T cells relative to CD81 T cells, leading to elevated CD4:CD8
ratios compared with age-matched B6 controls (Fig. 3A; Table I).
This pattern was noted at all ages tested. Thus, 1- to 3-mo-old and
4- to 6-mo-old B6.NZMc7 spleens exhibited mean splenic CD4:
CD8 ratios of 2.0, and 1.77, respectively, compared with the corresponding B6 ratios of 1.34 and 1.22 ( p , 0.05 and p , 0.001,
respectively). Likewise, B6.NZMc7 LN also exhibited significant
increases in the absolute numbers of CD41 T cells, but not CD81
T cells, compared with B6 controls. Thus, for example, 4- to 6-moold B6.NZMc7 nodes exhibited 31.7% CD41 T cells and mean
CD4:CD8 ratios of 1.07, compared with the corresponding B6 values of 23.9% and 0.82, respectively ( p , 0.01). Interestingly, the
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B6 and B6.NZMc7 mice (four or five mice per group, age 2.5 mo) were
injected i.p. with DNP-KLH (100 mg/mouse; Calbiochem) in CFA and
boosted 2 weeks later with 100 mg/mouse DNP-KLH in IFA. Immunized
mice were seromonitored for anti-hapten Abs for several weeks thereafter,
as illustrated in Results. At sacrifice, the spleens were stained with peanut
agglutinin to assess the size and numbers of germinal centers, as described
below. In addition, the T cell response to the immunogen was gauged by
measuring IL production (as detailed above) upon rechallenge with KLH in
vitro. To assess response to thymus-independent Ags, independent groups
of mice (four per group) were challenged i.p. with trinitrophenol (TNP)LPS (20 mg/mouse, Sigma) and seromonitored for anti-hapten response for
2 mo postchallenge. Briefly, serum anti-DNP and anti-TNP Abs were determined by assaying serial dilutions of the test sera on DNP-BSA-coated
(Calbiochem) or TNP-BSA-coated (Accurate Chemicals) Immulon-II
plates (Dynatech), respectively. Bound Abs were revealed with alkaline
phosphatase-conjugated anti-mouse IgM or anti-mouse IgG (Boehringer
Mannheim, Indianapolis, IN), utilizing p-nitrophenylphosphate as a substrate. The isotypes of anti-DNP Abs were determined by using isotypespecific enzyme conjugates. The avidity of these serum Abs was determined by gauging the amount of competitor (e-DNP-lysine, Sigma) that
was required for reducing the ELISA reactivity to DNP-BSA by 50%
(ID50).
The Journal of Immunology
6495
Table I. Lymphocyte subsets in secondary lymphoid organsa
Mice
1–3 Months Old
B6
B6.NZMc7
4–6 Months Old
9–12 Months Old
B6
B6.NZMc7
B6
B6.NZMc7
b
Spleens
Total cells (31026)
% B cells
% B1a cells
% CD41
% CD81
CD4:CD8 ratio
% CD691CD4 T cellsb
Lymph nodes
%B-cells
% CD41
% CD81
CD4:CD8 ratio
% CD691CD4 T cellsa
101 6 4.4
53.4 6 1.8
6.1 6 0.8
18.7 6 1.0
13.9 6 1.2
1.34 6 0.05
15.8 6 1.3
93.1 6 7.1
55.7 6 2.2
5.3 6 1.7
20.8 6 0.7*
10.3 6 0.7*
2.0 6 0.6*
20.6 6 1.6*
96.3 6 5.9
49.5 6 1.4
3.9 6 0.8
17.7 6 0.7
14.4 6 0.8
1.22 6 0.06
21.8 6 1.3
114 6 11.3
46.4 6 1.9
4.0 6 1.0
22.1 6 0.8***
12.5 6 0.9*
1.77 6 0.13***
23.9 6 2.5*
93.5 6 3.5
51.1 6 1.7
5.1 6 0.5
18.3 6 0.6
15.8 6 0.7
1.16 6 0.04
21.9 6 2.1
105 6 5.9
46.9 6 2.6
4.4 6 0.4
21.7 6 1.4*
13.9 6 1.2
1.56 6 0.12**
33.4 6 2.1***
34.7 6 2.8
26.2 6 1.0
22.6 6 1.3
1.16 6 0.1
21.6 6 3.0
26.8 6 3.3
36.2 6 1.5***
22.9 6 1.3
1.58 6 0.1*
21.3 6 2.3
35.0 6 3.3
23.9 6 0.6
29.0 6 1.1
0.82 6 0.03
27.8 6 1.4
29.6 6 4.7
31.7 6 2.1**
29.6 6 1.5
1.07 6 0.06**
26.1 6 2.6
41.2 6 3.3
18.9 6 1.4
23.3 6 1.8
0.81 6 0.07
35.9 6 3.5
23.6 6 2.6***
23.5 6 2.2*
29.4 6 3.1
0.80 6 0.05
39.6 6 4.9
T cell:B cell ratios and the absolute numbers of T cells were significantly elevated in B6.NZMc7 LN compared with B6 controls,
at all ages, as illustrated in Table I.
Sle3 impacts lymphocyte activation
B6.NZMc7 splenic T cells also appeared to be spontaneously activated, having increased expression of CD69, compared with B6
T cells (Table I and Fig. 3A). This phenotype became progressively stronger with age. Thus, in 9- to 12-mo-old B6.NZMc7
spleens, on the average, 33.4% of CD41 T cells expressed CD69,
compared with 21.9% of B6 CD41 T cells ( p , 0.001). A similar
trend was seen with the expression of two other activation markers,
CD25 and CD44 (data not shown). Although splenic CD81 T cells
from B6.NZMc7 mice also appeared to be more activated than
their B6 counterparts, these differences were not as pronounced as
those seen with CD41 T cells. In contrast, no significant differences were seen in the activation status of B6 and B6.NZMc7 LN
T cells (Table I).
In addition to the T cells, B6.NZMc7 B cells also appeared to be
spontaneously activated. As illustrated in Fig. 3B, B6.NZMc7
splenic B cells from 9- to 12-mo-old mice exhibited significantly
higher levels of surface I-Ab, CD44, and B7-2. This phenotype was
late in onset, as B cells from younger B6 and B6.NZMc7 spleens
did not differ from each other, by surface phenotype. At all ages
tested, B6.NZMc7 spleens did not differ from B6 spleens with
respect to the numbers or percentages of B1a or B1b cells. Likewise, there were no significant differences in the numbers or percentages of B1a or B1b cells in their peritoneal cavities (data not
shown).
We next assessed the functional status of B6.NZMc7 lymphocytes. B6.NZMc7 and B6 splenic B cells showed a similar proliferative response to anti-IgM and LPS (data not shown). However,
when stimulated with anti-CD3 and anti-CD28, B6.NZMc7 T cells
behaved differently. As illustrated in Fig. 4, B6.NZMc7 CD41 T
cells demonstrated significantly increased expansion in culture,
compared with B6 CD41 T cells. Thus, by 72 h postculture,
B6.NZMc7 CD41 T cells exhibited more than a 3-fold increase in
absolute numbers ( p , 0.004). A similar trend was not noted for
CD81 T cells. Indeed, in all B6 cultures, there were always more
CD81 T cells than CD41 T cells 72 h poststimulation, but the
reverse was true with B6.NZMc7 T cells, as can be seen in Fig. 4.
In both the B6 and B6.NZMc7 cultures, 72 h poststimulation, the
T cells were uniformly large in size and exhibited similarly high
levels of CD69 and Fas and reduced levels of L-selectin, typical of
activated T cell blasts (data not shown).
The increase in absolute numbers of B6.NZMc7 CD41 T cells
in culture could reflect an increased production of nascent T cells
and/or reduced apoptosis of activated T cells. Indeed, both appear
to be contributing, as illustrated in Figs. 5 and 6. Compared with
B6 T cells, B6.NZMc7 T cells showed significantly increased proliferation, as assessed by [3H]TdR incorporation, in response to
stimulation with anti-CD3 (Fig. 5A), and IL-2 (Fig. 5B). These
experiments were performed with 2- to 4-mo-old mice, and their
splenic T cells did not differ in the surface levels of CD3 or IL-2R
(data not shown). However, B6.NZMc7 T cells produced similar
quantities of IL-2, IL-4, and IFN-g upon stimulation (data not
shown).
Sle3 impacts T cell apoptosis
Apoptosis was studied by two complementary methods, hypotonic
lysis with PI incorporation (20) and annexin binding (21), as detailed in Materials and Methods. In the former method, apoptotic
cells were identified by their hypodiploid DNA content and light
scatter characteristics. The latter method is based on the principle
that dying cells expose phosphatidylserine on the outer membrane
leaflets, which can be tagged by annexin binding. This method has
the added advantage of allowing double or triple staining with
other surface markers. This permits one to gauge cell death in
selected lymphocyte populations.
By both methods, B6 and B6.NZMc7 splenic B cells showed
similar rates of apoptosis, with or without anti-IgM stimulation, as
illustrated in Fig. 6A. Likewise, splenic T cells from both strains
showed similar rates of spontaneous apoptosis in culture, with no
stimuli added. In contrast, B6.NZMc7 T cells exhibited reduced
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a
The percentages of different lymphocyte subsets in the spleens and LN of 1- to 3-mo-old, 4- to 6-mo-old, and 9- to 12-mo-old B6 and B6.NZMc7 mice are tabulated. Data
represent the mean (6SEM) values obtained from the indicated numbers of mice.
b
Numbers for spleens: for 1- to 3-mo-old mice, n 5 16 –36 for B6 and n 5 10 –28 for B6.NZMc7; for 4- to 6-mo-old mice, n 5 21–29 for B6 and n 5 8 –15 for B6.NZMc7;
and for 9- to 12-mo-old mice, n 5 22–30 for B6 and n 5 14 –20 for B6.NZMc7.
c
Displayed percentages represent the % of CD4 T cells that expressed CD69.
d
Data were obtained from inguinal LN, with a similar trend being noted in other LN. B6 and B6.NZMc7 nodes did not differ in total cell numbers.
e
Numbers for LN: for 1- to 3-mo-old mice, n 5 24 for B6 and n 5 17 for B6.NZMc7; for 4- to 6-mo-old mice, n 5 21 for B6 and n 5 9 for B6.NZMc7; and for 9- to
12-mo-old mice, n 5 15 for B6 and n 5 8 –17 for B6.NZMc7.
*p , 0.05, significantly different from B6 values; **p , 0.01, significantly different from B6 values; and ***p , 0.001, significantly different form B6 values.
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Sle3 IMPACTS T CELL ACTIVATION AND APOPTOSIS
FIGURE 3. A, Two-dimensional plots depict the expression of CD4,
CD8, and CD69 on B6 or B6.NZMc7 splenic lymphocytes, as determined
by flow cytometry. Top, Individual dots in the upper right quadrants represent CD691CD41 T cells. Bottom, Percentages of cells in the lower right
(i.e., CD41 T cells) and upper left (i.e., CD81 T cells) were used to derive
the CD4:CD8 ratios. Shown data were obtained from 9-mo-old mice and
are representative of data obtained from 14 –30 mice of each strain at this
age, as detailed in Table I. B, Mean fluorescence intensity of I-Ab, CD44,
and B7-2 expression on B2201 splenic B cells from 9- to 12-mo-old B6
(black bars) and B6.NZMc7 (gray bars) mice. Each bar represents the mean
(6SEM) value of the mean fluorescence intensity recorded in three individual mice of each strain, stained and analyzed within the same experiment. For the shown experiment, B6.NZMc7 B cells had significantly
higher expression levels of I-Ab (p , 0.02), CD44 (p , 0.05), and B7-2
(p , 0.05). These data are representative of four independent FACS staining experiments.
AICD. For these experiments, splenocytes were first stimulated for
48 h with anti-CD3 and anti-CD28. Then, the lymphoblasts were
Ficoll purified from the cultures, washed, and recultured on antiCD3-coated wells for the indicated durations. Importantly, the 6 h
data points should accurately reflect the apoptotic rates of the
plated T cell blasts, as this time lapse is too short for any cell
divisions to have occurred. Importantly, the B6.NZMc7 T cell
blasts consistently showed reduced apoptotic rates compared with
the B6 T cell blasts, at the 6 h and 40 h time points.
This was confirmed to occur with both CD41 and CD81 T cells
by the annexin binding experiments (Fig. 6, B and C). Thus, both
CD41 and CD81 T cell blasts showed impaired AICD, even in the
presence of anti-Fas Abs. B6 and B6.NZMc7 T cells showed no
consistent difference in the expression levels of Fas or FasL (data
not shown). Although T cell blasts from the lupus-prone
NZM2410 strain exhibited a similar phenotype (Fig. 6, B and C),
they were very sensitive to Fas-mediated apoptosis. The latter observation correlates well with the heightened expression of Fas on
NZM2410 blasts, compared with B6 or B6.NZMc7 T cell blasts
(data not shown).
Sle3 augments the immune response to antigenic challenge
The immune responsiveness of these mice was assessed by challenging them with a T-dependent (DNP-KLH), or a T-independent
(TNP-LPS) Ag. As illustrated in Fig. 7, upon secondary challenge
with DNP-KLH, B6.NZMc7 mice showed significantly elevated
levels of IgM (about 3–5 fold) and IgG (nearly two-fold) anti-DNP
Abs, for several months postchallenge. The anti-DNP Abs in B6
and B6.NZMc7 mice did not differ in their avidity for the hapten
(Fig. 7E) or the IgG subclass distribution (data not shown). Importantly, B6.NZMc7 T cells responded more vigorously to rechallenge with KLH by IL production (IL-2 production is depicted
in Fig. 7F). However, these two strains did not differ in the relative
quantities of IL-4 vs IFN-g produced upon rechallenge, suggesting
that Sle3 does not impact the Th1/Th2 balance (data not shown).
B6 and B6.NZMc7 immune-challenged mice did not differ in the
numbers or sizes of germinal centers, as revealed by staining with
peanut agglutinin. Finally, B6 and B6.NZMc7 mice showed a similar humoral response when challenged with the T-independent
Ag, TNP-LPS (data not shown).
Discussion
End-organ damage incited by selected subsets of ANAs is likely to
represent a final common pathogenic event in all models of murine
lupus, as well as in human lupus. Studies performed over the past
3 decades have collectively suggested that the generation of pathogenic ANAs is not due to a single immunological aberration, but
indeed represents the consequence of a multitude of underlying
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FIGURE 4. For these experiments, 5 3 105 splenocytes from 2- to
4-mo-old mice were cultured with anti-CD3 and anti-CD28 (1 mg/ml each).
At 0, 24, 48, and 72 h postculture, the absolute numbers of CD41 and
CD81 T cells in culture were determined by enumerating viable (i.e., PIexcluding) cells in culture and by FACS analysis using anti-CD4 and antiCD8 Abs. Each data point represents the mean (6SEM) of triplicate cultures. Shown are the absolute numbers of CD41 T cells from B6 (F) and
B6.NZMc7 (E), and CD81 T cells from B6 () and B6.NZMc7 mice (ƒ).
In the depicted experiment, B6.NZMc7 cultures showed significantly
higher numbers of CD41 T cells, compared with B6 cultures, at 48 h (p ,
0.001) and 72 h (p , 0.004) postculture. This experiment is representative
of five independent experiments.
The Journal of Immunology
pathogenic mechanisms, as reviewed in References 1–3. In particular, generalized B cell hyperactivity and T cell-driven autoantibody production by autoreactive B cells are both likely to contribute to ANA formation.
Several investigators have examined the features of T cells in
the various murine models of lupus, as well as in human SLE. Of
relevance to this work, an expansion of activated CD41 T cells has
been well documented in several lupus-prone (or genetically related) strains, including (NZB 3 NZW) F1, (NZB 3 SWR) F1,
NZB.H2bm12, NZM2410, NZW, and Faslpr/lpr mice, as well as in
human lupus (Refs. 22–25 and C. Mohan, unpublished observations). Although several of the mouse models are also characterized by elevated CD4:CD8 ratios, this has not been a characteristic
feature of human SLE (reviewed in Ref. 25). Experiments involving knockouts, Ab-mediated blocking, and in vitro cultures have
clearly demonstrated the critical role played by Th cells in driving
B cell autoantibody production in murine and human lupus (26 –
32). Importantly, parallel studies have documented generalized abnormalities in T cell signal transduction (33–37) and apoptosis
(38 – 43) in murine and human lupus.
FIGURE 6. A, Purified B6 (black bars) or B6.NZMc7 (white bars)
splenic B cells, T cells, or Ficoll-purified T cell blasts (see Materials and
Methods) were cultured with or without anti-IgM (anti-u) or anti-CD3 for
the indicated durations. Following this culture, the percentage of apoptotic
cells in culture was assayed flow cytometrically after hypotonic lysis and
PI incorporation, as detailed in Materials and Methods. Each bar represents
the mean (6SEM) percentage apoptosis detected in B cells or T cells
obtained from three individual mice, for each strain, or the mean of triplicate cultures of T cell blasts purified from individual B6 or B6.NZMc7
mice. The experiment shown was performed with cells obtained from 2- to
4-mo-old mice and is representative of at least three independent experiments. In the shown experiment, B6.NZMc7 T cell blasts show significantly reduced levels of apoptosis at 6 h (p , 0.004) and 40 h (p , 0.02)
poststimulation. B, Alternatively, annexin binding was used to quantitate
cell death in CD41 or CD81 T cells. T cell blasts were purified from B6,
B6.NZMc7, or NZM2410 spleens (as detailed in Materials and Methods)
and recultured for 6 h on anti-CD3-coated wells. The percentages of annexin-binding CD41 T cell blasts (which are annotated in the histograms
shown) and CD81 T cell blasts were determined by flow cytometry. The
shown plots are representative of data obtained from 4 –13 individual mice
of each strain. C, The extent of cell death among CD41 or CD81 T cell
blasts in restimulated cultures was determined by annexin binding, as demonstrated in Fig. 6B. In addition, some cultures also received anti-Fas (2
mg/ml) or control hamster Ig. Each bar represents the mean (6SEM) percentage of annexin binding seen in triplicate cultures of T cell blasts obtained from B6 (white bars), B6.NZMc7 (striped bars), or NZM2410
(cross-hatched bars) mice. In this experiment, compared with the B6 controls, significantly less annexin binding was seen among B6.NZMc7 CD41
T cell blasts (p , 0.03) and CD81 T cell blasts (p , 0.003) 6 h postrestimulation. Likewise, reduced Fas-mediated cell death was also seen in
B6.NZMc7 CD41 (p , 0.0005) and CD81 (p , 0.02) T cell blasts, compared with B6 T cell blasts. The presented data are representative of three
or four independent experiments.
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FIGURE 5. A, Splenocytes (5 3 105) from 2- to 4-mo-old B6 (black
bars) or B6.NZMc7 (white bars) mice were cultured with graded doses of
anti-CD3 for 72 h. The extent of lymphocyte proliferation was ascertained
by [3H]TdR incorporation during the last 18 h of culture. Each bar represents the mean (6SEM) cpm incorporated by triplicate cultures. In this
experiment, B6.NZMc7 T cells showed a significantly increased proliferative response to anti-CD3, compared with B6 controls, at doses of 0.1
(p , 0.04), 1 (p , 0.02), and 10 mg/ml (p , 0.01). This experiment is
representative of six independent experiments. B, Purified splenic T cells
(2 3 105) from 2- to 4-mo-old mice were cultured with graded doses of
rIL-2 for 72 h. The extent of lymphocyte proliferation was ascertained by
[3H]TdR incorporation during the last 18 h of culture. Each bar represents
the mean (6SEM) cpm incorporated by triplicate cultures. In the shown
experiment, B6.NZMc7 T cells showed a significantly heightened proliferative response to rIL-2, compared with B6 T cells (p , 0.02). This
experiment is representative of three independent experiments.
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Sle3 IMPACTS T CELL ACTIVATION AND APOPTOSIS
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FIGURE 7. B6 or B6.NZMc7 mice 2.5 mo old (four or five mice per group) were injected i.p. with DNP-KLH (100 mg/ml) in CFA and boosted 2 weeks
later. The mice were bled on days 0 (preimmune), 14 (primary response), and 21 (secondary response), and at intervals thereafter, as indicated in the figure.
The experiments shown in plots A through E are representative of four independent challenge experiments. In all cases, the shown data represent the mean
(6SEM) ELISA ODs in the B6 and B6.NZMc7 study groups, consisting of four or five mice each, respectively. A, The levels of IgM anti-DNP Abs in
serum (diluted 1:300) were assayed at serial time points as shown. B6.NZMc7 sera exhibit significantly higher levels of IgM anti-DNP Abs than B6 sera
on days 21 (p , 0.03), 98 (p , 0.01), and 128 (p , 0.003) postchallenge. B, Sera obtained on day 21 postchallenge were assayed for IgM anti-DNP Abs,
using serial dilutions of the sera. C, The levels of IgG anti-DNP Abs in serum (diluted 1:2500) were assayed at serial time points, as shown. Compared
with B6 controls, B6.NZMc7 sera show significantly higher IgG anti-DNP Ab levels at days 21 (p , 0.05) and 51 (p , 0.01) postchallenge. D, Sera
obtained on day 21 postchallenge were assayed for IgG anti-DNP Abs, using serial dilutions of the sera. E, The avidity of the postchallenge anti-DNP Abs
was gauged by determining the concentration of e-DNP-lysine (mM) that was required to inhibit serum binding to DNP-BSA on the ELISA plates by 50%
(ID50). Sera from day 21 postchallenge diluted 1:2500 were used for these assays. Each bar represents the mean (6SEM) ID50 value obtained in the B6
or B6.NZMc7 study group, which each consisted of four or five mice. F, To assess the T cell response to the immunogen, splenic T cells (5 3 105/well)
purified 4 mo postimmunization were stimulated with graded doses of KLH, presented by irradiated, T-depleted, syngeneic splenocytes (5 3 105/well).
Shown are the triplicate mean (6SEM) levels of IL-2-produced 48-h poststimulation by T cells purified from an individual B6 or an individual B6.NZMc7
mouse immunized in the same experiment. These data are representative of data obtained from four immunized mice of each strain.
The Journal of Immunology
6499
Superimposed on this generalized (i.e., polyclonal) T cell activation, a select few potentially pathogenic Th clones are likely to
be further expanded in an Ag-driven fashion. This concept is supported by TCR clonotype analyses (44 – 46) and the demonstration
of T cell antigenic specificities to a variety of nuclear, DNA-binding proteins, including histones, nucleosomes, ribosomal P2, and
DNase I, as well as ANA-derived idiopeptides (18, 47–53). It is
likely that the combined events of generalized, polyclonal T cell
activation and the focused expansion of a few Ag-specific T cell
clones are together responsible for driving ANA production by
pathogenic B cell clones and/or end-organ disease.
The present study advances an interval on murine chromosome
7 as harboring one such gene that may be responsible for the intrinsic, generalized T cell abnormality that characterizes lupus. As
discussed earlier, mapping studies indicate that this genomic interval confers susceptibility to ANA and/or GN in several murine
models of lupus, including the NZM2410 strain (9 –12). To elucidate the specific immunological contributions of Sle3 independent
of other lupus susceptibility loci, this genomic interval has been
selectively bred onto the B6 background. Analysis of these
B6.NZMc7 congenic mice reveals that Sle3 leads to an increase in
the absolute numbers of splenic and LN CD41 T cells and a reduction in CD81 T cells, leading to elevated CD4:CD8 ratios (Table I). Although the total numbers of splenic (CD41 and CD81) T
cells are relatively similar between the 2 strains, an age-dependent
increase is observed in the absolute numbers of activated CD41 T
cells in B6.NZMc7 spleens. Indeed, this in vivo phenotype is recapitulated in vitro, as demonstrated by the accumulation of activated CD41 T cells in culture, upon stimulation. Both increased
cell proliferation and reduced AICD appear to contribute to this
accumulation.
This accumulation of activated T cells over time may be causally related to the B cell surface phenotype (i.e., increased expres-
sion of activation markers; Fig. 3B). Indeed, this perpetual, low
grade stimulation of bystander B cells may be responsible for the
low grade, polyclonal humoral autoimmunity seen in this strain
(15). Finally, as one would predict, Sle3 augments the immune
response to T-dependent, but not T-independent, Ags (Fig. 7).
Thus far, no differences have been noted in the functional status or
the subset distributions (B1a, B1b, and B2) of B cells between B6
and B6.NZMc7 mice. Ongoing experiments, utilizing bone marrow transfers and B cell- or T cell-deficient mice, are aimed at
dissecting out the relative contributions of B cells, T cells, and
non-lymphocytes to the observed phenotypes in this strain.
It is important to note that these phenotypes are also present in
the original NZM2410 strain, as well as in other lupus-prone
strains, as discussed above. Indeed, in the NZM2410 strain, these
phenotypes are even more pronounced (C. Mohan et al., manuscript in preparation). Also, in contrast to the lupus-prone strains,
the B6.NZMc7 mice show relatively low levels of ANAs and a
lower penetrance of GN (15). Additional genes that differ between
the B6 and NZM2410 strain are likely to account for these differences. Indeed, it appears that in the presence of genes that actively
breach tolerance to chromatin (e.g., Sle1), loci such as Sle3 can
have a dramatic impact on the amplification and diversification of
the incipient autoimmune response. This notion is supported by the
robust humoral and cellular autoimmune phenotypes seen in
B6.NZMc1/c7 bicongenic mice (Fig. 8) (90).
Interestingly, induced mutations of several other T cell molecules have been shown to impact some of the same phenotypes
modulated by Sle3. Following T cell activation, several molecules
are known to regulate the delicate balance between resting and
activated T cells. In particular, activated T cells are purged from
the immune system via Fas/FasL-mediated AICD, as reviewed in
Ref. 54. IL-2 plays a pivotal role in facilitating this process, in
addition to its previously documented roles in T cell activation and
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FIGURE 8. The lupus susceptibility interval on murine chromosome 7, harboring Sle3, appears to primarily impact T cell activation and differentiation.
In the context of an otherwise normal B6 genome, Sle3 leads to spontaneous T cell activation, reduced AICD, and skewed CD4:CD8 ratios. This failure
to efficiently purge activated (presumably, anti-self) T cells from the immune system may account for the various phenotypes noted in this strain, including
the age-dependent accumulation of activated CD41 T cells (Table I); heightened humoral and cellular immune response to antigenic challenge (Fig. 7);
spontaneous B cell activation (Fig. 3B); and polyclonal, humoral autoimmunity (15). More importantly, the T cell phenotypes impacted by Sle3 could have
grave disease implications when this interval is bred onto a background that exhibits incipient autoimmunity. The B6.NZMc1 strain, congenic for Sle1,
exhibits breakage in tolerance to chromatin (17). Although the latter strain exhibits anti-H2A/H2B/DNA ANAs, it does not develop the full spectrum of
ANAs seen in lupus or GN. One could posit that introducing Sle3 onto the B6.NZMc1 strain may quantitatively or qualitatively augment the ANA response,
leading to GN. The characterization of B6.NZMc1/c7 bicongenic mice has recently revealed that this is indeed the case (90).
6500
lished observations). With respect to CD22, it is possible that allelic differences between B6 (CD22.2 allele) and NZB/NZW/
NZM2410 strains (CD22.1 allele) may be associated with
functional differences in B cell signaling (89). Further work is
needed to assess the contributions of these allelic differences to the
phenotypes seen in B6.NZMc7 mice and to the pathogenesis of
lupus in the NZM2410 strain.
Although Sle3 has been described as a single locus in this paper,
it is certainly possible that additional genes within this interval are
contributing to the observed phenotypes. Ongoing studies with
newer congenics bearing shorter genomic intervals are in progress
to address the relative contributions of the different regions within
the NZM2410-derived chromosome 7 susceptibility interval. Assigning a robust phenotype to these subinterval congenics would
greatly boost our efforts to narrow down the vicinity of the culprit
gene(s). The eventual identification of the culprit gene(s) on murine chromosome 7 promises to enrich our understanding of how
specific defects in T cell function could impact systemic autoimmunity in general and pathogenic ANA formation in particular.
Acknowledgments
We thank Drs. Eric Sobel and Joel Schiffenbauer for critical review of the
manuscript and Feiyan Liu for technical assistance.
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differentiation. The importance of these molecules has been elegantly demonstrated by targeted deletion of these molecules in
mice. Thus, mutations in Fas/FasL lead to impaired peripheral T
cell tolerance and massive lymphoproliferation, with full-blown
systemic autoimmunity (on the appropriate genetic background)
(55, 56). Knockouts of IL-2, IL-2R, the common cytokine receptor
g-chain (gc), and gc–associated tyrosine kinase (Jak3) all result in
impaired lymphocytic homeostasis (as exemplified by an age-dependent accumulation of activated/memory CD41, but not CD81,
T cells), often accompanied by features of autoimmunity (57– 64).
Through the use of TCR transgenic mice, this expansion of activated CD41 T cells in several of these models has been attributed
to impaired T cell AICD (65– 69). CTLA4 is yet another molecule
that plays a critical role in peripheral T cell tolerance (70), as
CTLA42/2 mice display massive lymphoproliferation, overt autoimmunity, and early mortality (71, 72). Additional molecules
that impact immune responsiveness, T cell activation, and CD4:
CD8 ratios include members of the NF-kB/RelB family, ikB, and
ZAP-70 (73–77).
Although the above molecules do not map to the study interval
on murine chromosome 7, they do suggest that Sle3 could be impinging on the same biochemical pathways underlying T cell expansion and AICD. Signaling studies with B6.NZMc7 mice are
currently in progress, to explore this further. Equally important,
these other molecules have to be viewed as potential candidate
genes for human lupus or for systemic autoimmunity in general. T
cells with low anti-self avidity are likely to escape thymic censorship and exit to the periphery, where they are likely to encounter
self-Ags. Anti-self T cells with aberrant thresholds for expansion/
cell death (such as those with Sle3 or allelic variants of the other
molecules described above), would be more readily triggered into
activation and may not be as readily purged from the immune
system. Such inefficiency in peripheral T cell tolerance may be
expected to manifest differently, depending on the genetic background. Thus, on the B6 background, this might simply lead to a
low grade polyclonal T cell activation, leading to serological and
cellular phenotypes similar to those observed in B6.NZMc7 mice.
On the other hand, the presence of additional susceptibility genes
could transform this phenotype into a more pathological one, as
discussed above (Fig. 8). Importantly, similar epistatic relationships with background genes have also been described for other
molecules that impact T cell homeostasis. For instance, the
Faslpr/lpr allele leads to low grade humoral autoimmunity on the
B6 background, but to high grade, pathological autoimmunity on
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The study interval on chromosome 7 harbors several candidate
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Interestingly, TGF-b2/2 mice exhibit multifocal inflammatory
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diffuse splenic architecture, lymphadenopathy, and serum ANAs
(84 – 87). On the other hand, aberrant Bcl3 expression has been
commonly associated with B cell lymphoproliferation and leukemogenesis (88). As some of the phenotypes impacted by these
candidate molecules overlap with those observed in B6.NZMc7
mice, it is important to ascertain whether any of these represent
Sle3. Thus far, we have failed to detect any expression differences
or coding region polymorphisms in the TTP and TGF-b genes,
between B6 and NZM2410/NZW (L. Morel and Y. Yu, unpub-
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Sle3 IMPACTS T CELL ACTIVATION AND APOPTOSIS