Antigen Activation Rescues Recent Thymic
Emigrants from Programmed Cell Death in the
BB Rat
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J Immunol 1998; 160:5757-5764; ;
http://www.jimmunol.org/content/160/12/5757
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References
Sheela Ramanathan, Ken Norwich and Philippe Poussier
Antigen Activation Rescues Recent Thymic Emigrants from
Programmed Cell Death in the BB Rat1
Sheela Ramanathan,* Ken Norwich,† and Philippe Poussier2*‡
T
he BB rat spontaneously develops an autoimmune, insulin-dependent diabetic syndrome that is very similar to
that observed in humans and in the nonobese diabetic
mouse (1). The disease is polygenic in the three species (2), and
one of the diabetes susceptibility genes of the BB rat, lyp, has been
mapped to chromosome 4 (3). Homozygosity for the BB lyp mutation leads to a 10-fold reduction in the number of peripheral
CD41 T cells and a virtual absence of CD81 T cells (4). This T
lymphopenia that results from an intrinsic defect in T cell precursors (5) is necessary, although not sufficient, for the development
of the BB rat diabetic syndrome (2).
Although excessive intrathymic death of T cell precursors has
not been demonstrated in BB rats, there is indirect evidence that
the BB lyp mutation manifests itself at the latest stages of intrathymic T cell development. Two studies reported a significant reduction in the number of single-positive mature CD4281 thymocytes in BB rats, which is consistent with the interpretation that a
large proportion of the precursors of peripheral CD4281 T cells
dies intrathymically (6, 7). In rats, and in the absence of Ag activation, T cells undergo a series of well-characterized changes in
membrane phenotype during the first week following their thymic
Departments of *Medicine and ‡Immunology, and †Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
Received for publication December 3, 1997. Accepted for publication February
10, 1998.
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 grants from the Medical Research Council of Canada
and the Juvenile Diabetes Foundation International to P.P., and the Natural Sciences
and Engineering Research Council of Canada to K.N. S.R. was the recipient of a Hugh
Sellers Postdoctoral Fellowship from Banting and Best Diabetes Centre in Toronto, at
the initiation of this study. She is currently the recipient of a postdoctoral fellowship
from the Juvenile Diabetes Foundation International.
2
Address correspondence and reprint requests to Dr. Philippe Poussier, TorontoWellesley Arthritis and Immune Disorder Research Centre, 620 University Avenue,
Suite 700, c/o Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario
M5G 2 M9, Canada. E-mail address: [email protected]
Copyright © 1998 by The American Association of Immunologists
emigration (8). Specifically, membrane expression of Thy-1 decreases, while that of CD45RC and RT6 increases (8). The number
of circulating T cells expressing Thy-1 is reduced in BB rats, while
RT61CD45RC1 T cells are virtually absent in secondary lymphoid organs (9). These observations suggested that the thymic
output of T cells is reduced and premature death of recent thymic
emigrants (RTE)3 may preclude the up-regulation of CD45RC and
RT6 expression in BB rats (10, 11). A recent study by Zadeh et al.
provides evidence that this interpretation is correct (12). These
authors evaluated the thymic output of T cells and the life span of
RTE in BB rats after in situ labeling of T cell precursors through
intrathymic injection of FITC (12). Accumulation of FITC1 T
cells in secondary lymphoid organs was reduced in BB rats when
compared with age-matched control animals, and was evident as
early as 2 h after the injection of FITC (12). This early detection
strongly suggests that the thymic output of BB rat T cells is reduced, although one cannot formally rule out a normal production
of T cells followed by a massive and precipitous death of most
RTE after exit from the thymus. Moreover, this study demonstrated that the life span of most BB rat RTE is very short and does
not exceed 1 wk (12). This latter observation explains why
thymectomy of adult BB rats is followed by a rapid depletion of 75
to 80% of the peripheral T cells from secondary lymphoid organs.
It is, however, important to note that the origin of the seemingly
long-lived T cells that persist after surgery (13) remains unknown.
In normal animals, the pool of recirculating T cells reaches its
full expansion soon after puberty. Subsequently, the thymus atrophies, the thymic output of T cells is reduced drastically, and the
stability of the PRL is maintained mostly through a slow turnover
of mature T cells (14, 15). The mitotic activity of peripheral T cells
is Ag driven in the case of naive T cells, while that of memory T
3
Abbreviations used in this paper: RTE, recent thymic emigrants; BrdU, bromodeoxyuridine; DP, double-positive; MNC, mononuclear cell; PE, phycoerythrin; PRL,
pool of recirculating T cells; TdT, terminal deoxynucleotidyl transferase; TUNEL,
terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.
0022-1767/98/$02.00
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One of the diabetes susceptibility genes of the BB rat is a mutation at the lyp locus that decreases the thymic output of T cells and
the life span of most recent thymic emigrants (RTE). Consequently, there is a 10-fold reduction in the number of CD41 and CD81
T cells in secondary lymphoid organs. Results presented in this work demonstrate that the BB rat lyp mutation is associated with
an accelerated apoptotic death in vitro of mature CD4182 and CD4281 thymocytes and peripheral T cells. The stability of the
pool of recirculating T cells (PRL) of BB rats over time results from a >10-fold increase in the mitotic activity of T cells as assessed
in vivo by bromodeoxyuridine incorporation. This increased mitotic activity is not observed when BB T cells develop in the context
of a normal sized PRL. MHC haploidentical WF and BB rats differ at minor histocompatibility loci. Intravenous injection of
(WF 3 BB)F1 T cells into euthymic BB rats led to the rejection of donor T cells within 3 wk by unprimed recipients and within
1 wk by primed recipients. This secondary immune response was unaffected by postpriming thymectomy. F1 T cells were not
rejected, but rather expanded after their injection into thymectomized BB rats that had been primed as early as 48 h after
thymectomy. These results strongly suggest that the BB rat PRL is devoid of long-lived naive T cells and that rescue of recent
thymic emigrants from programmed cell death is initiated by Ags, exclusively. The Journal of Immunology, 1998, 160: 5757–
5764.
5758
Materials and Methods
Animals
Age- and sex-matched inbred Wistar Furth (WF) and diabetes-prone BB
rats were purchased from Charles River (Frederick, MD) and from University of Massachusetts (Worcester, MA), respectively. BB and WF rats
share the same MHC haplotype (RT1u), but differ at minor histocompatibility loci and are congenic for the two allelic forms of CD45, RT7.1 for
BB rats and RT7.2 for WF animals. (WF 3 BB)F1 animals were bred in
our animal facility. All animals were housed in specific pathogen-free conditions. BB rats were tested three times per week for the presence of glycosuria and ketonuria. Once the animals became glycosuric, the diagnosis
of diabetes was made on the basis of hyperglycemia (blood glucose .16.7
mM) for 2 consecutive days. Diabetic rats were treated with s.c. implants
of insulin (Linplant; University of Toronto, Ontario, Canada). Thymectomy and sham thymectomy were performed as previously described (19).
mAbs, three-color immunofluorescence, and FACS analysis
The mAbs used in this study were affinity purified from hybridoma culture
supernatants on rat anti-mouse Ig- or mouse anti-rat Ig-Sepharose and then
conjugated with FITC, biotin, or PE using standard procedures. These
mAbs included anti-CD8 (MRC-OX8; (20)), anti-CD4 (W3/25; (20)), antiCD25 (MRC-OX39; (21)), anti-Thy-1.1 (MRC-OX7; (20)), and anti-CD5
(MRC-OX19; (22)), which were kindly provided by Dr. A. A. Like
(Worcester, MA) with the permission of Drs. A. F. Williams and D. Mason
(Oxford, U.K.). R73, a hybridoma secreting a mAb specific for a nonpolymorphic determinant of rat TCR-ab (23), was a gift of Dr. T. Hünig (Martinsried, Germany). The rat hybridomas NDS58 (anti-RT7.1) and 8G6.1
(anti-RT7.2) (24) were provided by Dr. D. Greiner (Worchester, MA) and
Dr. M. Newton (Oxford, U.K.), respectively. G4.18, a mAb specific for
CD3e; OX-18, a mAb specific for a nonpolymorphic determinant of the rat
MHC class I Ag RT1A; and streptavidin PE/Texas Red Tandem were
purchased from PharMingen (San Diego, CA) and Southern Biotechnology
Associates (Brimingham, AL), respectively.
Suspensions of mononuclear cells (MNC) were incubated with biotinylated mAb, followed by streptavidin PE/Texas Red Tandem. PE-labeled
and FITC-conjugated mAbs were then added simultaneously. Viable cells
were gated using forward and side angle scatter and analyzed flow cytometrically with a FACScan (Becton Dickinson, San Jose, CA).
Assessment of apoptotic death among T cell subsets
Purification of T cell subsets was achieved by negative selection, as described previously (7, 25). Briefly, unfractionated T cells and CD41 T cells
were purified from lymph node MNC using Cellect T cell columns (Biotex,
Calgary, Alberta), per manufacturer’s instructions. These columns were
also used to prepare mature CD4182 and immature CD4282 thymocyte
subsets, or immature CD4282 and CD4281 together with mature CD4281
thymocyte subsets from total thymocytes. Double-positive (DP) CD4181
thymocytes were obtained by depletion of cells expressing high levels of
MHC class I molecules on their surface through a two-step rosetting procedure, as described previously (7). The purity of the samples was assessed
by multicolor cell surface immunofluorescence and FACS analysis (26),
and was always .98.5% for thymocyte subsets and WF T cells, and .86%
for BB T cells.
The various T cell subsets (106 cells/well) were cultured in 1 ml of
Iscove’s modified Dulbecco’s medium containing 5% FCS in 24-well
plates at 37°C for various periods of time. At the end of the culture, the
proportion of T cells undergoing apoptosis was determined by detecting
DNA breaks through terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay (27, 28) and flow
cytometry. Briefly, cells were first surface labeled with a FITC-conjugated
mAb specific for CD4, CD8, or TCR-ab. Cells were then washed with
PBS, fixed in 100 ml of ice-cold 70% ethanol for 15 min, washed in PBS,
and incubated in PBS containing 1% paraformaldehyde for 15 min on ice.
Cells were washed once in PBS, and once in buffer (100 mM cacodylic
acid, 0.2 mM cobalt chloride, 0.1 mM DTT, and 100 mg/ml BSA, pH 6.8),
and then incubated in 25 ml TdT buffer containing 0.1 U/ml TdT and 5 mM
biotinylated 21dUTP, for 30 min at 37°C. After washing in PBS and FCS,
cells were incubated with streptavidin PE for 15 min, washed, and analyzed
flow cytometrically.
Assessment of Bcl-2 and Bcl-x expression in T cell subsets
Bcl-2 and Bcl-x expression was assessed in freshly isolated and activated
T cell subsets. Freshly isolated cells consisted of unfractionated thymocytes, as well as purified CD4181 DP thymocytes and lymph node T cells.
Purified lymph node CD41 T cells were activated with 1 mg/ml of Con A
(Sigma, St. Louis, MO) in the presence of irradiated WF splenocytes (in a
T cell:APC ratio of 100:1) for 3 days in vitro. Activated T cells (.99%
pure) were recovered by density-gradient centrifugation. Activated and
freshly isolated T cells were lysed at 5 3 107 cells/ml in buffer containing
150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, and
50 mM Tris at pH 7.5, supplemented with 8 mg/ml aprotinin, 2 mg/ml
leupeptin, and 170 mg/ml PMSF. After a 45-min incubation on ice, postnuclear fractions were obtained by centrifugation of lysates at 13,000 3 g
for 10 min. A total of 1.5 3 106 cell equivalent proteins was resolved on
12.5% SDS-PAGE and transferred to nitrocellulose by electroblotting. The
blots were blocked with 5% nonfat milk and 0.5% Tween-20 in Tris-buffered saline for 1 h at room temperature. The blots were then probed with
a 1/1000 dilution of rabbit polyclonal anti-Bcl-2 serum (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Bcl-x serum (a generous gift from Dr.
Lawrence Boise, Miami, FL) for 1 h at room temperature. After washing,
the blots were incubated with horseradish peroxidase-conjugated protein A
(Bio-Rad, Richmond, CA) for 1 h. Western blots were developed with the
enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
Assessment of thymic output of T cells
To evaluate the output of T cells from the thymus in vivo, thymocytes were
labeled intrathymically with FITC (Sigma), as described by Scollay et al.
(29). Any stress-related effect on T cell turnover was prevented by studying
animals 2 wk after a bilateral adrenalectomy. Following surgery, the animals were maintained on 0.15 M NaCl. Fifteen days later, a short upper
thoracotomy was performed under general anesthesia to expose the thymus, and 10 ml of a FITC solution (1 mg/ml in PBS) was injected into two
sites of each thymic lobe using a 0.5-ml insulin syringe and a 28 –1/2-gauge
needle. Animals were sacrificed 24 h after the FITC injection. Lymphocytes were isolated from the thymus, spleen, blood, and pooled (cervical,
axillary, inguinal, paraaortic, and mesenteric) lymph nodes, and counted.
The proportion of FITC1 T lymphocytes, i.e., RTE, among the MNC of
each of these lymphoid organs was then determined by three-color immunofluorescence and flow cytometry. The absolute number of RTE was then
calculated according to the formula: absolute number of RTE 5 (total
number of FITC1 peripheral T cells)/(fraction of FITC1 thymocytes).
T cell turnover
5-Bromo-29-deoxyuridine (Sigma) was dissolved in PBS (10 mg/ml), and
100 mg/kg of body weight/day was injected i.p. as two injections given at
8:00 a.m. and 8:00 p.m. for various periods of time. BrdU incorporation
into the DNA of peripheral T cells was assessed as follows (15, 30). MNC
pooled from secondary lymphoid organs were incubated with PE-labeled
anti-CD3e mAb. T cells were then sorted using a FACStar (Becton Dickinson). Sorted T cells were washed and fixed by dropwise addition of 70%
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cells can be triggered by cross-reactive Ags as well as non-TCRmediated signals (16). It is not known whether the size of the BB
rat PRL remains stable, albeit at a reduced level when compared
with normal rats, throughout life. In the event that this is the case,
the question follows as to whether this stability is obtained through
a sustained thymic output of T cells, an up-regulation of the peripheral expansion of T cells, or a combination of both mechanisms. It has been shown that an elevated proportion of BB rat
peripheral T cells expresses surface markers of activation (17, 18),
while cell cycle analysis has revealed a twofold increase in the
proportion of Thy-11 T cells that are in S/G2/M phase (12). These
two observations suggest that a high mitotic activity of peripheral
T cells may contribute to the stability of the BB rat PRL after
puberty.
This study was undertaken to characterize the factors that contribute to the homeostasis of the BB rat PRL. Although the proportion of apoptotic cells among freshly isolated T cells is normal
in BB rats, we demonstrate that the lyp mutation carried by this
strain is associated with an accelerated rate of apoptosis in vitro
among single-positive mature thymocytes and peripheral T cells.
The size of the BB rat PRL remains stable throughout life, despite
a physiologic thymic involution and a parallel reduction in the
thymic output of T cells. We provide evidence that long (.48 h)-lived
naive T cells are undetectable in the BB rat and that the PRL
stability of this animal is obtained through a 15- to 20-fold increase
in the mitotic activity of peripheral T cells.
T CELL HOMEOSTASIS IN THE DIABETIC BB RAT
The Journal of Immunology
5759
FIGURE 1. Apoptosis in subsets of thymocytes and peripheral T cells. T cell subsets
were purified and cultured, as described in
Materials and Methods. The proportion of
TUNEL-positive cells before (solid line) and
after 18 h (broken line) of culture was determined flow cytometrically. Results are from
a single experiment and are representative of
three different experiments.
the question: After n days, what fraction of T cells will have divided zero
time, once, twice, ... n times? The total number of cells after n days is equal
to N0(1 1 e)n. However, if we write this expression in the form N0[(1 1
e) 1 2e]n, and expand the expression using the binomial theorem, then the
respective terms in the expansion will provide the numbers we require.
@~1 2e! 1 2e# n 5~1 2 e! n 1 n ~1 2e!n21 ~2e! 1
~1 2 e! n 2 2~2e! 2 1 . . . .
Then the fraction of cells that have divided zero times in n days (from first
term on right-hand side) 5 (1 2 e)n/(1 1 e)n; the fraction of cells that have
divided once in n days (from second term on right-hand side) 5
n ~1 2 e!n 2 1~2 e!
, etc.
~11 e!n
Preparation of radiation chimeras
Forty-day-old WF rats were exposed to 950 rad of g-irradiation from a
137
Cs source (Gamma cell 40; Atomic Energy of Canada, Ottawa, Ontario,
Canada), and within 24 h, these animals were reconstituted with an i.v.
injection of 55 3 106 T-depleted BM cells of BB and WF origin, in a 4:1
ratio. Hemopoietic reconstitution was assessed 5 to 7 wk later by surface
immunofluorescence and FACS analysis of PBL using mAbs specific for
RT7.1, RT7.2, TCR-ab, and rat Ig k. Once the number of peripheral blood
T cells had been restored, radiation chimeras were thymectomized, and one
of their cervical lymph nodes was excised at the same time. The proportion
of BB and WF T cells among thymocytes and lymph node MNC at the time
of thymectomy was determined flow cytometrically. Ten days after
thymectomy, the animals were pulsed with BrdU for 24 h and then killed.
The number, surface phenotype, and BrdU incorporation of WF and BB T
cells present in the secondary lymphoid organs of these athymic radiation
chimeras were assessed flow cytometrically.
Immunization of BB rats
BB rats were immunized with one i.v. injection of 3 3 107 irradiated (2000
rad) or nonirradiated T cells of (WF 3 BB)F1 (F1) origin. Control animals
received the same number of syngeneic T cells. Recipients were bled
weekly for 1 mo to assess the fate of donor-derived T cells among PBL by
flow cytometry using mAbs specific for RT7.1, RT7.2, and TCR-ab. Recipients were then thymectomized or sham thymectomized. Two weeks
after surgery, the animals received an i.v. injection of 3 3 107 F1 T cells.
The fate of F1 cells among PBL was then followed weekly by flow cytometry. During the course of the prospective follow-up, some animals
from the various groups were killed, and their lymphoid organs (lymph
nodes, spleen, intestinal epithelium, lamina propria, and Peyer’s patches)
were examined for the presence of donor-derived T cells by flow
cytometry.
Modeling of BrdU kinetics
For BrdU kinetics, we made use of a simple model of cell disappearance
using the binomial theorem to derive theoretical expressions for the fraction of BrdU1 T cells in both the pulse and chase experiments.
We considered a number of T cells, N0, at zero time, and a fraction of
cells e per day that divide. e is obtained from a 24-h BrdU pulse. We asked
n~n 2 1!
2
A theoretical expression for the number of BrdU1 cells found on day n of
a pulse experiment is given by the sum of all terms in the binomial expansion of [(1 2 e) 1 2e]n, except the first (representing day 0), since each
cell that divides will pick up BrdU.
A theoritical expression for the number of BrdU1 cells found on day n
of a pulse experiment is given by the sum of all terms in the binomial
expansion of [(12e) 1 2e]n except the first (representing day 0), since each
cell that divides will pick up BrdU.
A theoritical expression for the number of BrdU1 cells found on day n
of a chase experiment is given by the first 2 terms of the binomial expansion. No terms beyond 2 need be taken for any day, n, since after one cell
division, the cell contains too little BrdU to be registered as BrdU1 by the
flow cytometer.
Statistics
Statistical analysis of significance was performed by unpaired Student’s
t test.
Results and Discussion
Increased susceptibility of BB rat T cell subsets to apoptosis
It has been demonstrated recently, using the technique of in situ
labeling of thymocytes with FITC, that the 24-h accumulation of
FITC1 RTE in secondary lymphoid organs of BB rats is reduced
profoundly when compared with age-matched controls (12). This
observation strongly suggests that a high proportion of mature thymocytes of BB rats dies intrathymically. To gain further insights
into the pathophysiology of the BB rat T lymphopenia, we determined whether freshly isolated thymocyte and peripheral T cell
subsets of BB rats die prematurely by apoptosis in vitro using the
TUNEL technique (Fig. 1). This technique is based on the fact that
nuclei of apoptotic cells contain DNA strand breaks, and thus can
incorporate nucleotides, such as dUTP-bio, in the presence of TdT.
The proportion of apoptotic cells among freshly isolated thymocytes and peripheral T cells was #1% in both BB and WF animals
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ice-cold ethanol. After a 30-min incubation on ice, cells were washed with
PBS and incubated in PBS containing 1% paraformaldehyde and 0.01%
Tween-20 for 30 min at room temperature. Cells were pelleted and exposed
to 50 Kunitz units of DNase I (Sigma) in 1 ml of 0.15 M NaCl, 4.2 mM
MgCl2, pH 5, for 15 min at room temperature. After washing, cells were
incubated with FITC-conjugated anti-BrdU mAb (Becton Dickinson) and
analyzed by fluoroflow cytometry.
To assess the frequency of RTE entering into cycle daily, some adrenalectomized animals were injected intrathymically with FITC, thymectomized 24 h later, and given BrdU immediately and 12 h after thymectomy.
These animals were killed 24 h postthymectomy, and MNC pooled from
secondary lymphoid organs were incubated with PE-labeled anti-CD3e
mAb. FITC1 and FITC2 T cells were then sorted, and the frequency of
BrdU1 cells among these two T cell subsets was determined using a biotinylated anti-BrdU mAb and streptavidin PE/Texas Red Tandem.
5760
T CELL HOMEOSTASIS IN THE DIABETIC BB RAT
mocytes nor in resting peripheral T cells (33, 34). These observations are consistent with the hypothesis that up-regulation of bcl-x
serves to maintain DP thymocytes before positive selection (33). In
the absence of positive selection, bcl-x expression is down-regulated, and DP thymocytes die by apoptosis (33). In contrast, DP
thymocytes that undergo positive selection up-regulate bcl-2 expression and survive. It is striking in this context that the T lymphopenic process of the BB rat starts manifesting itself at precisely
this stage of intrathymic T cell development. However, our analysis of Bcl-2 and Bcl-x expression in thymocyte and peripheral T
cell subsets failed to detect any abnormality in BB rats (Fig. 2).
Normal involution of BB rat thymus
(Fig. 1). This is in agreement with the previous observation of
Doukas et al. that the proportion of apoptotic cells among cortical
and medullary thymocytes of BB rats is similar to that of normal
rats, as assessed by histochemistry (31).
After 18 h of culture, 40% of CD4181 DP thymocytes isolated
from both WF and BB rats were undergoing apoptosis (Fig. 1).
During the same period, however, the proportion of CD4182 and
CD4281 mature BB thymocytes undergoing apoptosis was twofold higher than that observed among WF thymocytes (Fig. 1). The
difference between the two strains was even more striking among
peripheral T cells. More than 50% of BB T cells were apoptotic
after 18 h of culture vs ,5% among WF T cells (Fig. 1). Thus, in
the absence of stimulation, BB rat single-positive mature thymocytes and peripheral T cells die prematurely by apoptosis in vitro.
No differential survival was observed among the various T cell
subsets of BB and WF rats at the end of an 18-h culture. Specifically, the proportions of CD41 and CD81 T cells expressing RT6,
Thy-1, and CD45RC on their surface remained similar during the
period of observation (data not shown).
It has been shown that the expression of bcl-2 and bcl-x is developmentally regulated in the thymus. During intrathymic T cell
development, bcl-2 is expressed in only 5 to 10% of CD4181
thymocytes, but virtually in all mature single-positive thymocytes
and peripheral T cells (32). In contrast, bcl-x is expressed in
CD4181 thymocytes, but is not expressed in single-positive thyTable I. Age-related changes in T cell subsets of WF and BB rats a
RTE
(3 1026)
Thymocytes
(3 1026)
Age
(months)
PRL
(3 1026)
% CD251 T Cells
WF
BB
WF
BB
WF
BB
WF
BB
1
(n 5 3)b
2
970 6 256
1120 6 179
5.9 6 0.5
4.1 6 0.2*
162.3 6 5.8
22.2 6 2.5*
ND
ND
700 6 134
710 6 36
4
6
368 6 49
98 6 28
487 6 24
191 6 22*
6.3 6 1.2
(n 5 11)
1.0 6 0.3
1.1 6 0.2
3.9 6 0.1*
(n 5 8)
1.4 6 0.5
1.3 6 0.2
190.1 6 7.3
(n 5 11)
172.2 6 11.1
168.1 6 14.0
34.5 6 2.0*
(n 5 8)
20.6 6 5.2†
22.3 6 4.7†
3.6 6 0.8
(n 5 11)
5.6 6 1.3
4.1 6 0.5
8.5 6 2.2*
(n 5 11)
14.3 6 2.0†
25.9 6 5.6†
a
Numbers of T cells were determined by flow cytometry.
n 5 3 unless specified.
* p , 0.01 compared with age-matched WF rats; and
†
p , 0.05 compared with 2-mo-old BB rats.
b
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FIGURE 2. Bcl-2 and Bcl-x expression in various T cell subsets of WF
and BB rats. Lysates from 1.5 3 106 T cells of the indicated subsets were
subjected to SDS-PAGE and Western blot analysis with antisera to Bcl-2
and Bcl-x, as described in Materials and Methods. Bcl-2 expression was
analyzed in unfractionated thymocytes and peripheral T cells (upper panel), while Bcl-x expression was assessed in unfractionated and DP thymocytes, as well as Con A-activated peripheral CD41 T cells (lower panel).
The size of the PRL of adult BB rats, although considerably
smaller than that of age-matched controls, remains relatively stable
for several months (Table I). A possible mechanism through which
this stability could be maintained during adulthood is a lack of
thymic involution after puberty that occurs at 2 mo in rats. We
therefore examined the age-related changes in the size of the thymus and the thymic output of T cells (Table I). The number of
thymocytes decreased by a factor of 10 between the ages of 1 and
6 mo in WF rats. Concomitantly, the daily accumulation of RTE in
secondary lymphoid organs declined to a comparable extent (Table
I). Although the number of thymocytes found in 1- to 2-mo-old
WF and BB rats was similar, the thymus atrophied more slowly in
BB than in WF rats (Table I). However, this differential progression of thymic atrophy had little influence on the daily thymic
output of T cells. Thus, while the number of thymocytes present in
6-mo-old BB rats was slightly larger than that found in agematched WF rats, the daily thymic output of T cells was similar in
both strains (Table I). It has been shown in mice that Ab-mediated
depletion of peripheral T cells does not result in a compensatory
increase in the thymic output of T cells (35). Our results strongly
suggest that, in rats, the size of the PRL also has little influence on
the regulation of the thymic output of T cells.
Having established that thymic involution proceeds in BB rats in
a physiologic manner, we tested the possibility that an extrathymic
source of T cells could contribute to the stability of the BB rat PRL
over time. This possibility was ruled out since T cells accounted
for less than 1% of MNC in 15- to 20-wk-old BB rats that had been
either adult thymectomized, lethally irradiated, and reconstituted
with syngeneic fetal liver, or thymectomized at birth (data not
shown).
An alternative mechanism for compensating the age-related decline in thymic output of T cells would be an increase in the mitotic activity of BB rat peripheral T cells. In support of this hypothesis, it is striking to observe an inverse correlation between the
daily thymic output of T cells and the proportion of peripheral T
The Journal of Immunology
5761
Table II. Daily turnover of T cell subsets in euthymic and athymic
(ATX) rats
% BrdU1 T Cells
Euthymic rats (n 5 4)
CD31
CD31 Thy 12
ATX rats (n 5 4)
RTEb
CD31c
WF
BB
,1
,1
14.9 6 1.2
13.9 6 2.9
1.9 6 0.2
2.2 6 1
8.7 6 0
18.2 6 2.3
a
a
Euthymic or adult thymectomized rats received two i.p. injections of BrdU 12 h
apart and were killed 12 h after the second injection. CD31 and CD31, Thy 12
subsets were sorted and the proportion of BrdU1 cells was determined by flow cytometry.
b
To determine the turnover of RTE, rats were injected intrathymically with FITC,
thymectomized 24 h later, and then pulsed with BrdU for 24 h. The proportion of
BrdU1 cells among sorted FITC1 T cells (RTE) was determined by flow cytometry.
c
A 24-h BrdU pulse was performed 10 days after thymectomy.
Elevated mitotic activity of peripheral T cells in the BB rat
Cell cycle analysis of BB rat peripheral T cells through propidium
iodide staining has shown previously that the proportion of Thy-11 T
cells in S/G2/M phase is increased by a factor of 2 in BB rats when
compared with controls (12). This observation, combined with the
reduction in both the number of peripheral Thy-11 T cells and the size
of the PRL, has led to the interpretation that a high proportion of BB
rat RTE undergoes nonproductive proliferation (12). One must, however, be cautious before drawing this conclusion. First, Thy-11 T cells
represent a heterogenous T cell population in terms of age, since it
takes ;7 days after thymic emigration for membrane expression of
Thy-1 to become undetectable by flow cytometry (8). Furthermore,
when the PRL turnover is in a steady state, the proportion of Thy-11
T cells in S/G2/M phase is influenced not only by the proportion of
cells that are activated, but also by the death rate of Thy-11 T cells
and their life span.
We took an alternative approach to evaluate in vivo the proportion of RTE that cycle daily. Animals were thymectomized 24 h
after intrathymic injection of FITC. Immediately after thymectomy, they were pulsed for 24 h with BrdU. FITC1 T cells were
sorted and labeled with anti-BrdU Ab, and the proportion of
FITC1 RTE that had incorporated BrdU in 24 h was determined by
flow cytometry. As illustrated in Table II, the proportion of cycling
RTE that had accumulated in 24 h was four- to fivefold higher in
BB rats than in WF animals (Table II). However, this increase in
the proportion of cycling T cells was not restricted to RTE, since
14.9 6 1.2% of unfractionated peripheral T cells had also incorporated BrdU at the end of a 24-h pulse in euthymic BB rats (n 5
4). In contrast, ,1% (n 5 4) of WF peripheral T cells incorporated
BrdU during the same period. RTE account for up to 20% of peripheral T cells in the BB rat (data not shown). Our results, therefore, demonstrate that most cycling T cells are found among nonRTE in BB rats.
Evaluation of BrdU incorporation in peripheral T cells of euthymic animals is complicated by the continuous input of RTE that
could have divided and, hence, incorporated BrdU at some point
during their intrathymic development. Two approaches were taken
to avoid this potential problem. First, we analyzed the daily rate of
division among peripheral Thy-12 T cells, i.e., T cells that had
emigrated from the thymus at least 5 to 7 days earlier, in euthymic
animals (8). As illustrated in Table II, at the end of a 24-h BrdU
pulse, 13.9 6 2.9% of BB rat Thy-12 T cells incorporated BrdU
compared with ,1% in WF animals.
The second approach was to evaluate the rate of division of
peripheral T cells in thymectomized rats. As illustrated in Figure
3A, the results were similar to those obtained with the first approach. Thus, the accumulation of BrdU1 T cells was slow in
secondary lymphoid organs of WF rats, as ,1% of peripheral T
cells had cycled in 1 day, and BrdU1 T cells accounted for roughly
25% of unfractionated T cells in these animals at the end of a
13-day pulse. The accumulation of BrdU1 T cells was considerably faster in BB rats. These cells accounted for roughly 20% of
peripheral T cells after 24 h of BrdU administration, and .90% of
T cells at the end of a 13-day pulse (Fig. 4A). These results demonstrate that a high mitotic activity of peripheral T cells is an
important component of the homeostasis of the BB rat PRL.
Cycling T cells are short-lived in BB rats
Despite the high mitotic activity of T cells in athymic BB rats,
there is no discernible increase in the size of the PRL of these
animals over time (data not shown). This implies that the progeny
of these cells are also short-lived. This was confirmed by our analysis of the rate of disappearance of BrdU1 T cells that had accumulated in secondary lymphoid organs during a 15-day pulse.
More than 90% of BrdU1 T cells disappeared from the secondary
lymphoid organs of BB rats within the first 2 wk of the chase
period (Fig. 4B). In contrast, the proportion of BrdU-labeled T
cells decreased very slowly in WF animals during the same interval (Fig. 3B). The t1/2 of activated T cells was found to be approximately 4.9 days in BB rats and 69 days in WF animals. It has
been shown that the life span of BB rat RTE does not exceed 1 wk
(12). Our results strongly suggest that the life span of BB rat T
cells is very short after cell division, which explains why the size
of the BB rat PRL remains small despite the high mitotic activity
of these cells. It has been shown in normal animals that Ag-induced activation and expansion of peripheral T cells are followed
by the apoptotic death of a large proportion of the responding T
cells (36). Consequently, the size of the PRL remains constant over
time. We cannot rule out that this physiologic activation-induced T
cell death contributes to the high rate of death observed among
cycling T cells in BB rats. However, activation of normal T cells
passively transferred to immunodeficient animals results in the expansion of the pool of donor-derived T cells over time (37). This
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
cells that express activation markers, specifically CD25, in BB rats
(Table I). We, therefore, assessed the turnover of BB rat peripheral
T cells.
FIGURE 3. Kinetics of accumulation (A) and disappearance (B) of
BrdU1 T cells in secondary lymphoid organs of adult thymectomized WF
rats. Daily i.p. injections of BrdU were administered for 13 to 15 days.
Pooled splenic and lymph node MNC were stained with anti-CD3e, and T
cells were purified with a FACStar. The disappearance of BrdU-labeled T
cells was followed after a pulse for 15 days. Three to four animals were
analyzed at each time point. Detection of BrdU incorporation and flowcytometric analysis of BrdU1 T cells were performed as described in Materials and Methods.
5762
T CELL HOMEOSTASIS IN THE DIABETIC BB RAT
FIGURE 4. Kinetics of accumulation (A) and disappearance (B) of
BrdU1 T cells in secondary lymphoid organs of adult thymectomized BB
rats. The curves are theoretical curves obtained with our mathematical
model using e 5 0.14 per day and assuming that cell death will occur with
the same probability in BrdU1 or BrdU2 cells. The theoretical curves may
be seen to conform quite closely with the measured data.
The high mitotic activity of BB rat peripheral T cells is
secondary to the T lymphopenia
The fate of normal T cells after passive transfer to histocompatible
recipients depends on the size of the recipient PRL (37). When the
size of the PRL is normal, a large proportion of the injected T cells
disappears soon after transfer, and the mitotic activity of the donorderived T cells that persist is similar to that of recipient T cells
(14). In contrast, transfer of T cells to “T-less” recipients is followed by the persistence of a large proportion of donor-derived T
cells and a considerable, Ag-driven expansion of these cells, which
results in the partial restoration of the size of the PRL (37, 38).
These observations demonstrate that the size of the PRL has a
profound influence on the mitotic activity of T cells. To determine
whether the high mitotic activity of BB rat T cells results from an
intrinsic abnormality of T cell precursors in this strain or is simply
a reflection of the regulatory mechanisms that normally control the
homeostasis of the PRL, we analyzed the fate of BB rat T cells in
a nonlymphopenic environment.
Several weeks after hemopoietic reconstitution of lethally irradiated rats with mixed bone marrow originating from BB and normal donors, the size of the recipient PRL is similar to that of
The elevated mitotic activity of BB rat peripheral T cells is
initiated by Ag
To examine the role of Ag in the mitotic activity of BB rat T cells
and the possibility that some naive BB rat T cells are long-lived,
we took advantage of a fortuitous observation. It has been demonstrated that BB and WF rats share the same MHC (2). Furthermore, it has been shown that passive transfer of WF T cells to
diabetes-prone BB rats results in the partial correction of the recipient T lymphopenia by expansion of donor-derived T cells, and
prevents the development of diabetes in recipients (40). Accordingly, the proliferative response of T cells from both strains to
reciprocal APC is comparable with their response to syngeneic
APC in a one-way primary MLR (data not shown). Surprisingly,
when we tried to protect BB rats from diabetes through passive
transfer of WF T cells, the recipients became diabetic and showed
no evidence of T cell reconstitution. Our in vitro and in vivo observations, therefore, demonstrate that differences at minor histocompatibility loci exist between BB and WF strains. The immune
response of BB rats to the molecules encoded by these loci was
Table III. Development of BB T cells in euthymic and athymic (BB 1 WF)3 WF radiation chimeras a
% T Cells Among
LN MNC
Pre-TX
53.7 6 4.9
b
% BB-Derived T Cells
Among LN MNC
% BB-Derived B Cells
Among LN MNC
% BrdU1 LN T Cells
Post-TX
Pre-TX
Post-TX
Pre-TX
Post-TX
Pre-TX
Post-TX
56.3 6 10.6
8.1 6 2.3
0.8 6 0.4
73.6 6 9.1
69.9 6 11.0
1.1 6 0.3
0.8 6 0.4d
c
1.3 6 0.5c
a
Lethally irradiated (950 rad) WF animals were reconstituted with 1.1 3 107 and 4.4 3 107 T-depleted bone marrow of WF and BB origin, respectively. Six to eight weeks
after reconstitution, recipients were thymectomized and one of their cervical lymph nodes was excised to assess the proportion of donor-derived T cells flow cytometrically. Ten
days after thymectomy (TX), radiation chimeras was given two i.p. injections of BrdU, 12 h apart, and were killed 12 h after the second injection. The proportion of WF2,
BB-derived and BrdU1 T cells among peripheral MNC was determined flow cytometrically.
b
n 5 6.
c
BrdU1 cells among WF T cells.
d
BrdU1 cells among BB T cells.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
observation demonstrates that the balance between expansion and
apotosis of T cells following activation is influenced by the size of
the PRL. The small size of the BB rat PRL remains constant despite the high mitotic activity of T cells. It strongly suggests that
the BB rat lyp mutation by itself contributes to the high rate of
death observed among cycling T cells in this animal.
unmanipulated normal rats (39). Importantly, while the ratio of BB
to non-BB cells among the thymocytes of these radiation chimeras
is similar to that in the bone marrow inoculum, the number of cells
of BB origin among peripheral T cells is disproportionately low
(39). The question follows as to whether a high proportion of these
BB rat T cells is cycling. WF rats were lethally irradiated and
reconstituted with T-depleted bone marrow of WF and BB origin
in a ratio of 1:4. Five weeks after reconstitution, the PRL of these
chimeras had been restored. While the proportion of cells of BB
and WF origin among peripheral B cells was similar to that of the
bone marrow inoculum (Table III), only 8% of peripheral T cells
were BB derived. Importantly, only 0.8 to 1.1% of both WF and
BB rat T cells had incorporated BrdU at the end of a 24-h pulse.
This observation demonstrates that when BB rat T cells develop in
the context of a normal sized PRL, a very low proportion of them
divide. Therefore, the increased mitotic activity of peripheral T
cells observed in euthymic and athymic BB rats is not an intrinsic
feature of the BB rat T cells.
The radiation chimeras were then thymectomized, and the fate
of WF and BB peripheral T cells was assessed 10 days after
thymectomy. As expected, the proportion of T cells among peripheral MNC remained stable after thymectomy. However, virtually all peripheral T cells were now of WF origin in these athymic
radiation chimeras (Table III). This observation illustrates the important contribution of cycling T cells to the PRL of euthymic and
athymic BB rats. Moreover, it raises the questions as to whether
there are any long-lived naive T cells in BB rats and what is the
stimulus responsible for the high mitotic activity of peripheral T
cells in this animal.
The Journal of Immunology
5763
FIGURE 5. Role of Ags in the mitotic activity of
BB rat T cells. The experimental protocol is as depicted
in the figure. Groups of five to seven BB rats were
primed i.v. with 3 3 107 nonirradiated (groups A and
B), or irradiated (2000 rad; groups D and E) (WF 3
BB)F1 or BB (group C) T cells. Groups D and E were
thymectomized and sham thymectomized, respectively,
2 wk before priming. Groups B and C were thymectomized 5 wk after priming. The animals were bled
weekly, and the proportion of F1 T cells among peripheral blood T cells was determined by flow cytometry.
All groups were challenged with an i.v. injection of 3 3
107 F1 T cells 7 wk after priming. The numbers represent the mean percentage 6 1 SD of F1 T cells among
peripheral blood T cells.
rived T cells, but rather by the rapid expansion of these cells.
Consequently, donor-derived T cells accounted for the majority of
recirculating T cells in thymectomized BB recipients 1 wk after
priming (Fig. 5, group C). A similar result was obtained when BB
rats were immunized as early as 48 h after thymectomy, i.e., at a
time when the size of PRL is still .50% of what it is in euthymic
animals (data not shown). This observation strongly suggests that
thymectomy of BB rats is followed by the rapid disappearance of
naive T cells, disabling thymectomized recipients’ response to, and
elimination of, F1 T cells. An alternative explanation is that the
frequency of Ag-specific naive T cells is too low in thymectomized
BB rats to prevent the expansion of F1 T cells. We therefore tried
to expand Ag-specific T cells in thymectomized BB rats.
BB rats were immunized 2 wk after thymectomy with 3 3 107
irradiated F1 T cells (Fig. 5, group D). Irradiated F1 T cells were
used to circumvent the problem of expansion of donor-derived T
cells observed in group C animals. As expected, irradiated F1 T
cells disappeared from PBL within a few days after injection (data
not shown). Seven weeks after priming, group D animals were
challenged with 3 3 107 nonirradiated F1 T cells. As illustrated in
Figure 5, the expansion of nonirradiated F1 T cells in these primed
thymectomized recipients was similar to that observed in unprimed
thymectomized animals (Fig. 5, groups C and D). This expansion
of F1 T cells after challenge could not be attributed to the inability
of irradiated F1 T cells to prime Ag-specific precursors. As illustrated in Figure 5 (group E), irradiated F1 T cells were able to
prime specific T cells in euthymic BB rats since nonirradiated F1
T cells were cleared from the PBL of these recipients in ,2 wk
after challenge.
These results strongly suggest that the PRL of BB rats is devoid
of long-lived naive T cells, and that the continuous thymic output
of T cells is crucial for maintaining a diverse T cell repertoire in
this strain. Furthermore, our inability to prime BB rat T cells 48 h
after thymectomy shows that the process leading to the death of
these cells in a few days becomes irreversible within the few hours
that follow their thymic emigration. We have shown that, despite
the short life span of activated T cells, long-term immunologic
memory can be maintained in BB rats through an elevated turnover
of memory T cells. If besides Ags, nonspecific stimuli were involved in the initial activation of BB rat RTE, this rescue from
programmed cell death would be expected to affect RTE randomly,
and hence would perpetuate among activated T cells the diversity
of the RTE repertoire. This possibility is inconsistent with our
inability to prime BB rat T cells of defined specificity shortly after
thymectomy. Therefore, our results strongly suggest that rescue of
RTE from programmed cell death is initiated by Ags, exclusively.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
used to assess the longevity of naive T cells and the role of Ags in
the mitotic activity of peripheral T cells in this strain.
T cells of (BB 3 WF)F1 origin were used as a source of Ag for
two reasons. Since the recipients are lymphopenic, the rejection or
expansion of F1 T cells could be easily monitored in secondary
lymphoid organs by flow cytometry using mAbs specific for CD3e,
RT7.1 and RT7.2. Moreover, the use of F1 T cells alleviates the
potential problem of graft vs host disease. After immunization of
euthymic BB rats with one i.v. injection of 3 3 107 F1 T cells, it
took ;3 wk for donor-derived T cells to become undetectable
among PBL (Fig. 5, groups A and B). Group A animals were then
sham thymectomized, while group B animals were thymectomized. Both groups of animals were challenged 7 wk after priming
with a second i.v. injection of 3 3 107 F1 T cells. The depletion of
donor-derived T cells from PBL occurred in both groups in ,1 wk
after challenge, and hence was accelerated compared with what
was observed after priming (Fig. 5, groups A and B). Of note, this
disappearance of F1 T cells was not restricted to the PBL compartment. One month after priming and two weeks after challenge,
F1 T cells could not be detected among MNC isolated from other
lymphoid organs (data not shown). Furthermore, we could not detect any expansion of the recipient-derived PRL after immunization with F1 T cells. These results demonstrate that euthymic BB
rats can mount a primary and a secondary T cell-mediated immune
response. The secondary immune response was comparable in euthymic animals of group A and those of group B that were thymectomized before challenge. This observation therefore rules out any
significant contribution of a continuous output and recruitment of
naive T cells to the response observed after challenge. Furthermore, it demonstrates that despite the short life span of cycling T
cells in BB rats, these animals can generate long lasting immunologic memory, suggesting that this immunologic memory is maintained through the continuous cycling of Ag-primed T cells. Although we could not detect residual F1 T cells in the secondary
lymphoid organs of BB rats 4 wk after priming, we cannot rule out
the persistence of WF-derived Ags and their role in the maintenance of immunologic memory. However, it has been demonstrated recently in normal animals that, even in the absence of their
specific Ag, memory T cells turn over rapidly in response to crossreactive Ags and cytokines (16, 41– 43).
Twenty to twenty-five percent of peripheral T cells persist after
thymectomy of adult BB rats (Ref. 13 and our present results), and
;five percent of these T cells fail to incorporate BrdU during the
course of a 15-day pulse (Fig. 4A). To determine whether these
BrdU2 cells are long-lived naive T cells, we assessed the capacity
of T cells of thymectomized BB rats to respond to Ag (Fig. 5,
group C). Injection of 3 3 107 F1 T cells into BB rats 2 wk after
thymectomy was not followed by the disappearance of donor-de-
5764
Acknowledgments
We thank Dr. Lawrence Boise, University of Miami, for anti-Bcl-x antiserum.
We thank Michael Julius, Jayne Danska, and Casey Fox for critical reading of
this manuscript; Jonathan Guberman for his contribution to the mathematical
modeling of T cell turnover; and Claude Cantin (Cytometrics, Toronto, Ontario, Canada) for expert assistance with flow cytometry.
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T CELL HOMEOSTASIS IN THE DIABETIC BB RAT