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Departments of
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Medicine and
Immunology, and
Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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 CD4-8+ thymocytes in BB rats, which is consistent with the interpretation that a large proportion of the precursors of peripheral CD4-8+ 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 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 RT6+CD45RC+ 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 FITC+ 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 cells can be triggered by cross-reactive Ags as well as non-TCR-mediated 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-1+ 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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 x 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)), anti-CD25 (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-
ß (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 CD3
; 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 CD4+ 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 CD4+8- and immature CD4-8- thymocyte subsets, or immature CD4-8- and CD4-8+ together with mature CD4-8+ thymocyte subsets from total thymocytes. Double-positive (DP) CD4+8+ 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-ß. Cells
were then washed with PBS, fixed in 100 µl 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 µg/ml BSA, pH 6.8), and then incubated in 25 µl TdT
buffer containing 0.1 U/µl TdT and 5 µM 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 CD4+8+ DP thymocytes and lymph node T cells. Purified lymph node CD4+ T cells were activated with 1 µg/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 x 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 µg/ml aprotinin, 2 µg/ml leupeptin, and 170 µg/ml PMSF. After a 45-min incubation on ice, postnuclear fractions were obtained by centrifugation of lysates at 13,000 x g for 10 min. A total of 1.5 x 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 µl 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 FITC+ 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 = (total number of FITC+ peripheral T cells)/(fraction of FITC+ thymocytes).
T cell turnover
5-Bromo-2'-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-CD3 mAb. T cells were then sorted
using a FACStar (Becton Dickinson). Sorted T cells were washed and
fixed by dropwise addition of 70% 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-CD3 mAb. FITC+ and
FITC- T cells were then sorted, and the frequency of
BrdU+ cells among these two T cell subsets was determined
using a biotinylated anti-BrdU mAb and streptavidin PE/Texas Red
Tandem.
Preparation of radiation chimeras
Forty-day-old WF rats were exposed to 950 rad of 
-irradiation
from a 137Cs 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 x 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-ß, and rat Ig 
. 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 x
107 irradiated (2000 rad) or nonirradiated T cells of
(WF x 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-ß.
Recipients were then thymectomized or sham thymectomized. Two weeks
after surgery, the animals received an i.v. injection of 3 x
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 BrdU+ 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 per day that divide. is obtained from a 24-h
BrdU pulse. We asked 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 + )n. However, if we
write this expression in the form N0[(1 + ) +
2]n, and expand the expression using the binomial
theorem, then the respective terms in the expansion will provide the
numbers we require.
 |
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)n/(1 + )n; the fraction of cells
that have divided once in n days (from second term on
right-hand side) =
 |
) +
2]n, except the first (representing day 0), since each
cell that divides will pick up BrdU.
A theoritical expression for the number of BrdU+ cells
found on day n of a pulse experiment is given by the sum of all terms
in the binomial expansion of [(1-) + 2]n except
the first (representing day 0), since each cell that divides will pick
up BrdU.
A theoritical expression for the number of BrdU+ 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 BrdU+ by the flow cytometer.
Statistics
Statistical analysis of significance was performed by unpaired Student’s t test.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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It has been demonstrated recently, using the technique of in situ
labeling of thymocytes with FITC, that the 24-h accumulation of
FITC+ 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 (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).
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). During the same period, however, the proportion of
CD4+8- and CD4-8+
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 CD4+ and CD8+ 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 CD4+8+ thymocytes, but virtually in
all mature single-positive thymocytes and peripheral T cells (32). In
contrast, bcl-x is expressed in
CD4+8+ thymocytes, but is not expressed in
single-positive thymocytes 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).
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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 age-matched 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.
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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 cells that express
activation markers, specifically CD25, in BB rats (Table I). We,
therefore, assessed the turnover of BB rat peripheral T cells.
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-1+ 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-1+ 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-1+ 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-1+ 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-1+ 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. FITC+ T cells were sorted
and labeled with anti-BrdU Ab, and the proportion of
FITC+ 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 ± 1.2%
of unfractionated peripheral T cells had also incorporated BrdU at the
end of a 24-h pulse in euthymic BB rats (n = 4).
In contrast, <1% (n = 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
non-RTE in BB rats.
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, at the end of a 24-h BrdU
pulse, 13.9 ± 2.9% of BB rat Thy-1- 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
BrdU+ T cells was slow in secondary lymphoid organs of WF
rats, as <1% of peripheral T cells had cycled in 1 day, and
BrdU+ T cells accounted for roughly 25% of unfractionated
T cells in these animals at the end of a 13-day pulse. The accumulation
of BrdU+ 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.
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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 BrdU+ T cells that had
accumulated in secondary lymphoid organs during a 15-day pulse. More
than 90% of BrdU+ 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 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.
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 donor-derived 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 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.
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). 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 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 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 x 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 CD3, 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 x 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 x 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
cross-reactive Ags and cytokines (16, 41, 42, 43).
|
five percent of these T cells fail to incorporate BrdU during the
course of a 15-day pulse (Fig. 4A). To determine
whether these BrdU- 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 x 107
F1 T cells into BB rats 2 wk after thymectomy was not
followed by the disappearance of donor-derived 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 x
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 x 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Philippe Poussier, Toronto-Wellesley 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:
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.
Received for publication December 3, 1997. Accepted for publication February 10, 1998.
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