The Trophic Action of IL-7 on Pro-T Cells: Inhibition of Apoptosis of Pro-T1, -T2, and -T3 Cells Correlates with Bcl-2 and Bax Levels and Is Independent of Fas and p53 Pathways1

Abstract

Signals from the IL-7R are essential for normal thymocyte development. We isolated thymocytes from early developmental stages and observed that suspensions of pro-T1, -T2, and -T3 cells rapidly died in culture. Addition of IL-7 promoted their survival, but did not induce cell division. Pro-T4 cells did not undergo rapid cell death, and their survival was therefore independent of IL-7. Death in the absence of IL-7 showed the hallmarks of apoptosis, including DNA fragmentation and annexin V binding; however, caspase inhibitors blocked DNA fragmentation, but did not block cell death. The trophic effect of IL-7 was partially inhibited by blocking protein synthesis. The p53 pathway was not involved in this death pathway, since pro-T cells from p53-/- mice also underwent cell death in the absence of IL-7. The Fas/Fas ligand pathway was not involved in cell death, since Fas-deficient pro-T cells died normally in the absence of IL-7, anti-Fas Abs did not protect cells from death in the absence of IL-7, and Fas expression was undetectable on cells at these stages. The IL-7 trophic affect correlated with increased intracellular levels of Bcl-2 and decreased levels of Bax, whereas no Bcl-X(L), Bcl-w, or Bad was detectable. Thus, maintaining a favorable Bcl-2/Bax ratio may account for the trophic action of IL-7.

Full text

The Trophic Action of IL-7 on Pro-T Cells: Inhibition of
Apoptosis of Pro-T1, -T2, and -T3 Cells Correlates with Bcl-2
and Bax Levels and Is Independent of Fas and p53 Pathways
Kyungjae Kim,* Chong-kil Lee,* Thomas J. Sayers,
†
Kathrin Muegge,
†
and Scott K. Durum*
Signals from the IL-7R are essential for normal thymocyte development. We isolated thymocytes from early developmental stages
and observed that suspensions of pro-T1, -T2, and -T3 cells rapidly died in culture. Addition of IL-7 promoted their survival, but
did not induce cell division. Pro-T4 cells did not undergo rapid cell death, and their survival was therefore independent of IL-7.
Death in the absence of IL-7 showed the hallmarks of apoptosis, including DNA fragmentation and annexin V binding; however,
caspase inhibitors blocked DNA fragmentation, but did not block cell death. The trophic effect of IL-7 was partially inhibited by
blocking protein synthesis. The p53 pathway was not involved in this death pathway, since pro-T cells from p53
2/2
mice also
underwent cell death in the absence of IL-7. The Fas/Fas ligand pathway was not involved in cell death, since Fas-deficient pro-T
cells died normally in the absence of IL-7, anti-Fas Abs did not protect cells from death in the absence of IL-7, and Fas expression
was undetectable on cells at these stages. The IL-7 trophic affect correlated with increased intracellular levels of Bcl-2 and
decreased levels of Bax, whereas no Bcl-X
L
, Bcl-w, or Bad was detectable. Thus, maintaining a favorable Bcl-2/Bax ratio may
account for the trophic action of IL-7. The Journal of Immunology, 1998, 160: 5735–5741.
N
ormal T cell development in the mouse and in man has
been shown to depend on signals from the IL-7R (re-
viewed in Ref. 1). This conclusion is based on the phe-
notypes of mice deficient for IL-7R
a
(2, 3), IL-7 itself (4, 5), mice
treated with Abs against IL-7 (6, 7), and deficiencies of the
g
c
component of the IL-7R in humans (8) and mice (9, 10).
Some
ab
T cell development occurs in about 40% of IL-7R
2/2
mice, suggesting that alternative pathways can also support T cell
development; however, the peripheral T cells that eventually ac-
cumulate in these leaky mice do not proliferate in response to
stimuli (11). The
gd
T cell lineage is completely undetectable in all
IL-7R
2/2
mice (3, 12) based on a failure to rearrange the TCR
g
locus (13, 14) or a failure to express these genes (15). The devel-
opment of B lymphocytes is also severely impaired in IL-7R
a
2/2
mice and
g
c
2/2
mice, but not in
g
c
-deficient humans reflecting the
IL-7 independence of human B cells (16). NK development is
normal in IL-7R
2/2
mice (12).
The earliest stages in murine T cell development (before ex-
pression of CD4, CD8, and CD3) have been distinguished based
on CD44 and CD25 expression (reviewed in Ref. 17): pro-T1
(CD44
1
CD25
2
), pro-T2 (CD44
1
CD25
1
), pro-T3 (CD44
2
CD25
1
),
and pro-T4 (CD44
2
CD25
2
). Expression of c-Kit, the receptor for
stem cell factor, corresponds with expression of CD44 at these
pro-T cell stages. The CD25
1
stages are deficient in IL-7R
2/2
mice and in mice treated with anti-IL-7. This suggests that one
requirement for IL-7R signals occurs before or during the pro-T2
stage. IL-7 has been reported to induce proliferation of early T
cells (18) and could therefore play a role in the expansion as well
as differentiation or survival of these cells. To clarify the nature of
the IL-7R signal requirement, pro-T cells at different stages were
isolated from the thymus and examined for the effects of IL-7 on
survival and cell cycle, and the cell death process that occurred in
the absence of IL-7 was characterized.
Materials and Methods
Mice
C57BL/6 and MRL-lpr/lpr mice were housed in a specific pathogen-free
environment. Mice were mated overnight and checked for plugs the fol-
lowing day, which was designated day 1 of gestation. On the indicated day
of gestation, mothers were killed by CO
2
asphyxiation, and embryos were
killed by chilling on ice. Thymi were removed from embryos using a dis-
secting microscope. Rag-2
2/2
(19) and p53
2/2
(20) mice were bred in our
facility from breeders purchased from The Jackson Laboratory (Bar
Harbor, ME).
Preparation and culture of fetal thymocytes
Fetal thymus lobes were obtained from embryos on day 14, 15, 16, 17, or
18 of gestation. Cell suspensions were prepared by gentle disruption with
a micropipette after treatment with 0.2% collagenase (Sigma, St. Louis,
MO) dissolved in PBS containing 20% heat-inactivated FCS (HyClone,
Logan, UT) for1hat37°C. The cells were cultured in RPMI 1640 sup-
plemented with 2 mM glutamine, 100 U/ml penicillin, 100
m
g/ml strepto-
mycin, 50
m
M 2-ME, and 10% heat-inactivated FCS. Fetal thymocytes
(4 3 10
5
/200
m
l/well) were cultured in 96-well U-bottom plates in the
presence or the absence of IL-7 (50 ng/ml; PeproTech, Rocky Hill, NJ) for
the indicated length of time. Hamster anti-Fas (Jo2; 10
m
g/ml; PharMin-
gen, San Diego, CA) was added to some cultures. Caspase inhibitors, z-
VAD-FMK and z-DEVD-FMK (20
m
M; Enzyme Systems Products, Dub-
lin, CA), were preloaded into thymocytes at 0°C for 30 min before placing
cells in culture together with inhibitors at 37°C.
Anti-Fas control treatment
Renca cells were stimulated with IFN-
g
(100 U/ml for 18 h) to increase
their susceptibility to fas-mediated killing. Renca cells were then labeled
with
51
Cr and incubated with d11S cells that express FasL
2
and hamster
anti-Fas (10
m
g/ml) for 16 h, and the release of
51
Cr was determined.
*Laboratory of Molecular Immunoregulation and
†
Science Applications International
Corporation, Division of Basic Sciences, National Cancer Institute, Frederick, MD
21702
Received for publication August 28, 1997. Accepted for publication February 9, 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
Address correspondence and reprint requests to Dr. Scott K. Durum, Laboratory of
Molecular Immunoregulation, National Cancer Institute, Building 560, Room 31-45,
Frederick Cancer Research Facility, Frederick, MD 21702-1201.
2
Abbreviations used in this paper: FasL, Fas ligand.
Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

Flow cytometric analysis and cell sorting with Abs
For Ab staining, cells were harvested, washed in a staining solution of PBS
containing 5% FCS and 0.1% NaN
3
, and resuspended in 50
m
l of staining
solution containing 0.5
m
g of rat mAb 2.4G2 (anti-mouse Fc
g
RII, PharM-
ingen) and 10% normal mouse serum to reduce nonspecific binding of Abs
to Fc receptors. Cell suspensions were stained with mAbs (1/200 dilution)
for 20 min at 4°C. Abs were purchased from PharMingen, including R-
phycoerythrin-conjugated anti-CD44 (clone IM7), FITC-conjugated anti-
CD25 (clone 7D4), FITC-anti-CD4 (RM4-5), and R-phycoerythrin-conju-
gated anti-mouse CD8
a
(clone 53-6.7). Cells were washed and fixed in 1%
paraformaldehyde in PBS, and analyzed on a FACStar Plus (Becton Dick-
inson Immunocytometry System, Mountain View, CA), gating out dead
cells by forward low angle scatter and low right angle scatter. For sorting
cells, the same staining method was used without fixation, and cells were
sorted on a FACStar Plus or a modified FACSII (Becton Dickinson).
Pro-T1 (CD44
1
CD25
2
) cells were sorted from day 14 thymocytes. Pro-T2
(CD44
1
CD25
1
) cells were sorted from day 14 or 15 thymocytes. Pro-T3
(CD44
2
CD25
2
) cells were sorted from day 16 thymocytes or from
Rag2
2/2
adult thymocytes. Pro-T4 (CD4
2
CD8
2
CD44
2
CD25
2
) cells
were sorted from day 17 thymocytes. From p53
2/2
mice a mixture of
pro-T2 and pro-T3 cells (CD4
2
CD8
2
CD25
1
) was sorted from thymocytes
from adult mice. The purity of sorted populations was generally .98%.
Sorted cells were then cultured overnight with or without IL-7.
Intracellular staining of Bcl-2, Bcl-X
L
, Bax, and Bad
Single-cell suspensions were permeabilized with saponin buffer (PBS con-
taining 1% BSA (Sigma) and 0.04% saponin) for 20 min, then incubated
with monoclonal anti-mouse Bcl-2 (clone 3F11, PharMingen), anti-mouse
Bcl-X
L
(clone 4, Transduction Laboratory, Lexington, KY), anti-mouse
Bad (clone 2G11, PharMingen), or a polyclonal anti-mouse Bax (Santa
Cruz Biotechnology, Santa Cruz, CA) for 30 min on ice. Isotype-matched
control Abs were purified mouse IgG2b (clone 49.2 PharMingen), purified
polyclonal hamster IgG (PharMingen), or purified rabbit IgG. Cells were
washed twice with saponin buffer, incubated with FITC anti-hamster IgG
(clone G94-56, PharMingen), FITC anti-mouse IgG2b (clone R12-3
PharMingen), or FITC-anti-rabbit IgG (Santa Cruz Biotechnology) for 30
min at 4°C, and washed once with saponin buffer and once with PBS
containing 1% BSA.
Annexin V staining and viability
Annexin V binding was performed with a commercial kit (Clontech, Palo
Alto, CA). Cells were collected, then resuspended in annexin V binding
buffer containing annexin V-FITC (1
m
g/ml) and propidium iodide (2
m
g/
ml) for 10 min at room temperature in the dark, washed, and analyzed by
microfluorometry. To analyze for viability, cells were incubated with pro-
pidium iodide, then the percentage of cells excluding the dye was deter-
mined by flow microfluorometry.
Cell cycle analysis
Cells were placed in a detergent buffer (21) and an equal volume of staining
buffer (propidium iodide, 50
m
g/ml). Cells were mixed by inversion, in-
cubated at room temperature in the dark for 1 h, and analyzed by flow
microfluorometry.
Results
Thymocytes from mouse embryos on day 14 of gestation were
cultured with or without IL-7 for various times. The recovered
cells were examined for viability by exclusion of propidium io-
dide, which stains the nuclei of either necrotic or apoptotic cells.
Apoptotic cells were detected by binding annexin V to their
plasma membranes. As shown in Figure 1, these freshly explanted
thymocytes began to die by apoptosis within 2 h, and by 20 h, 90%
had undergone apoptotic death. IL-7 protected about half the cells
from apoptotic death at 20 h, and its survival effects could be seen
as early as 6 h. In .20 experiments, IL-7 protected 50 to 80% of
the cells from death at the 20 h point. Further culture in IL-7
beyond 20 h (not shown) showed that with each additional day,
about half of the cells died. We noted that culture density had an
effect on viability, with IL-7 being less effective as culture density
was lowered.
The thymocyte population on day 14 of gestation is comprised
of the first two stages of pro-T cell development, pro-T1 and pro-
T2, in approximately equal proportions. We sorted the cells into
pro-T1 and pro-T2 based on the CD25 and CD44 markers and
tested each population for the effect of IL-7 using cell cycle anal-
ysis. In this analysis, apoptotic cells were visualized based on
DNA fragmentation to subG1 quantities of DNA. As shown in
Figure 2, both pro-T1 and pro-T2 populations were protected from
apoptotic death by IL-7. We also visualized DNA fragmentation
by gel electrophoresis and observed a comparable IL-7 effect (not
shown). It is clear from this analysis that IL-7 did not promote
cycling of pro-T cells, as shown by the decline in the proportion of
cells in S and G
2
-M during culture in IL-7 compared with that at
the start of the culture (see also Fig. 6). The percentages of pro-T2
cells in G
1
, S, and G
2
-M phases when initially isolated were 56.5,
37.5, and 6.1%, respectively. After IL-7 culture, they were 81.5,
16.5, and 1.9%, respectively. Thus, cell division declined during
culture in IL-7, and its activity is best described as “trophic” rather
than “growth” for pro-T cells.
The later stages of pro-T cell development were then compared
with earlier stages for responsiveness to IL-7. Pro-T3 cells were
isolated by sorting from day 16 embryos using CD25 and CD44
markers. As shown in Figure 3, pro-T3 cells were protected by
IL-7 from cell death during culture. Of the first three stages (pro-
T1, -T2, and -T3), the most dramatic IL-7 trophic effects were
consistently observed at pro-T2.
Pro-T4 cells were sorted from day 17 embryos by selecting for
CD4
2
, CD8
2
, CD25
2
, and CD44
2
cells. As shown in Figure 3,
these cells survived independently of IL-7 in culture, and addition
of IL-7 produced only a slight increase in survival that was not
observed consistently. Pro-T4 cells also rapidly differentiated in
culture (not shown), expressing CD4 and CD8 after overnight cul-
ture, and this, too, was unaffected by IL-7. Pro-T4 cells were also
cycling much more rapidly than pro-T1, -T2, and -T3 cells (not
shown), and this cycling was unaffected by IL-7.
FIGURE 1. IL-7 protects day 14 embry-
onic thymocytes from apoptotic death. Thy-
mocytes were cultured with or without IL-7
for varying times. Recovered cells were ana-
lyzed by flow microfluorometry for viability
using exclusion of propidium iodide (left) and
apoptosis using binding of annexin-FITC
(right).
5736 TROPHIC ACTION OF IL-7 ON PRO-T CELLS

After the pro-T4 stage, thymocytes express CD4 and CD8. We
examined CD4
1
CD8
1
cells for IL-7 trophic effects (not shown)
and observed that, like pro-T4 cells, these cells survived indepen-
dently of IL-7 for several days in vitro. We conclude that the
trophic effects of IL-7 end with the pro-T3 stage.
The role of p53 in the apoptotic response to IL-7 deprivation
was tested for two reasons. First, p53 mediates one type of thy-
mocyte apoptosis, that induced by dsDNA breaks (20, 22, 23).
Second, p53
2/2
mice develop thymic lymphomas at a very high
frequency (24, 25), suggesting that they evade the normal death
mechanisms. Although the phenotype of such p53
2/2
thymomas is
primarily CD4
1
CD8
1
, an IL-7-independent stage, we neverthe-
less considered it possible that the actually transformed cell was a
pro-T cell because such thymic lymphomas also arise in rag
2/2
p53
2/2
mice (24) (S. Cande`ias and S. Durum, unpublished obser-
vation) that do not develop beyond the IL-7-dependent pro-T3
stage. However, a role for p53 was ruled out by using pro-T cells
from p53
2/2
mice, as shown in Figure 4. Thus, a similar depen-
dency on IL-7 was observed comparing p53
2/2
thymocytes to
their heterozygous littermates. Note that cell death (in the absence
of IL-7) was not as extensive in this experiment as that shown in
Figure 3; this is because adult pro-T cells die more slowly in cul-
ture than their fetal counterparts. We also tested fetal pro-T cells
from p53
2/2
mice (not shown) and similarly showed that they are
IL-7 dependent. It can be noted (Fig. 4) that p53
2/2
thymocytes
survive better in culture than do their heterozygous counterparts.
This improved survival of cultured p53
2/2
cells was not observed
in studies using unfractionated thymocytes or pre-B cells (20, 22,
23); however, we consistently observed it in pro-T cells and acti-
vated mature T cells (S. Cande`ias and S. Durum, unpublished ob-
servations), and it has been noted in embryonic fibroblasts (26).
A cascade of caspases is a feature of the apoptotic response, and
inhibiting this cascade has been shown to block cell death in many
studies. Caspase inhibitors were added to thymocyte cultures, and
the effect on IL-7 deprivation was studied. Two different inhibitors
were used: z-VAD, which inhibits caspases 1 and 4, and z-DEVD,
which inhibits caspases 3, 6, 7, 8, and 10. As shown in Figure 5
(left panel), neither caspase inhibitor prolonged life, as assessed by
propidium iodide exclusion. However, both caspase inhibitors no-
tably reduced DNA fragmentation (right panel). Thus, IL-7 de-
privation activates caspases (recognized by these inhibitors), lead-
ing to DNA fragmentation; however, an additional mechanism
must also be involved in cell killing when IL-7 is withdrawn; this
mechanism could involve caspases not blocked by these inhibitors
or may be caspase independent.
FIGURE 2. Effect of IL-7 on apoptosis and cell
cycle in stage 1 and stage 2 pro-T cells. Thymocytes
from day 14 embryos were sorted into pro-T1 and
pro-T2 subsets using CD25 and CD44 markers. Cells
were cultured for 24 h with or without IL-7. Recov-
ered cells were analyzed for DNA content.
FIGURE 3. Effect of IL-7 on survival of subsets of pro-T cells. Thy-
mocytes from different days of gestation were sorted into the indicated
populations using CD25 and CD44 markers. Pro-T1 and -T2 cells were
sorted from suspensions of day 14 embryonic thymus, pro-T3 cells were
sorted from day 16 embryonic thymus, and pro-T4 cells were sorted from
day 17 embryonic thymus. Cells were cultured for 24 h with or without
IL-7 and analyzed for viability using exclusion of propidium iodide.
FIGURE 4. Thymocyte death from IL-7 deprivation is not dependent on
p53. Adult thymocytes (from 8-wk-old p53
2/2
or p53
1/2
mice) were
sorted to yield the CD25
1
CD4
2
CD8
2
fraction (pro-T2 and -T3). Cells
were cultured for 24 h and analyzed for viability using exclusion of pro-
pidium iodide.
5737The Journal of Immunology

FasL signals through Fas, inducing apoptotic death in peripheral
T cells, and this pathway also appears to operate in late pro-T4 and
early double-positive cells (27). Two methods were used to test
whether Fas is involved in death from IL-7 deprivation. First, anti-
Fas Ab was added to cultures of thymocytes to determine whether
it blocked cell death in the absence of IL-7. Second, pro-T cells
from MRL-lpr/lpr mice, which lack functional Fas, were also
tested. As shown in Figure 6 (left), anti-Fas did not block the death
of pro-T cells, nor did the MRL-lpr/lpr thymocytes fail to die in
the absence of IL-7. As a positive control for anti-Fas blocking, the
same Ab was shown to inhibit the FasL-Fas killing pathway of
FasL, expressing d11S killers on the Fas-expressing Renca target
cells (Fig. 6, right). Although this Ab can have agonist activity and
kill Fas targets, we do not observe this unless it is secondarily
cross-linked (T. Sayers unpublished observation). Further evi-
dence against a Fas mechanism is our failure to detect Fas expres-
sion by flow microfluorometry before or after culture of pro-T cells
(not shown). Thus, Fas is not involved in the death of pro-thymo-
cytes deprived of IL-7.
The relative concentrations of antiapoptotic (e.g., Bcl-2) and
proapoptotic members (e.g., Bax) of the Bcl-2 family determine
whether a cell will live or die. Intracellular staining was performed
for the antiapoptotic factors Bcl-2 and Bcl-X
L
and for the pro-
apoptotic factors Bax and Bad. As shown in Figure 7 (left), freshly
isolated cells expressed high levels of Bcl-2 and Bax, whereas no
Bcl-X
L
or Bad was detected. During culture (Fig. 7, right panels),
Bcl-2 levels declined sharply, and IL-7 reduced this decline. Bax
levels increased during culture, and IL-7 prevented this increase.
Thus, IL-7 preserved the ratio of Bcl-2 to Bax at a level interme-
diate between that at initiation and that of cultures deprived of
IL-7. Therefore, the trophic action of IL-7 on day 14 thymocytes
correlated with its maintaining a favorable Bcl-2/Bax ratio.
Discussion
We analyzed the effect of IL-7 on survival of pro-T cells. IL-7
protected pro-T1, -T2, and -T3 cells from rapid apoptotic death,
whereas pro-T4 cells survived independently of IL-7. IL-7 did not
induce cell cycling of pro-T cells. There was no evidence for the
involvement of p53 or Fas pathways in cell death. Inhibiting
caspases inhibited DNA fragmentation, but did not protect cells
from death. IL-7 protected pro-T cells from a decline in Bcl-2 and
a rise in Bax, suggesting that the Bcl-2/Bax ratio could be the basis
of the trophic action of IL-7.
These findings complement the recent reports that a bcl-2 trans-
gene restores
ab
T cell development in IL-7R
2/2
mice (28, 29) and
in
g
c
2/2
mice (30), demonstrating that Bcl-2 can substitute for
some IL-7 activities. It has also recently been reported that IL-7
regulates Bcl-2 levels in pro-T cells, and that this regulation is
FIGURE 5. Effects of caspase inhibitors on cell death and DNA fragmentation in the absence of IL-7. Thymocytes from day 14 embryos were cultured
with or without IL-7 and caspase inhibitors. After 24 h, recovered cells were analyzed for viability using exclusion of propidium iodide (left) and DNA
content (right).
FIGURE 6. Thymocyte death from IL-7
deprivation is not dependent on Fas. Thymo-
cytes from day 14 embryos were cultured
with or without IL-7 and anti-Fas Ab, and
cell viability was measured 24 h later (left).
Thymocytes from C57BL/6 mice are com-
pared with the Fas-deficient strain MRL-lpr/
lpr. As a positive control for anti-Fas block-
ade of cell death, D11S cells were mixed
with IFN-treated Renca cells labeled with
51
Cr, with or without anti-Fas, and cell death
was measured 18 h later (right).
5738 TROPHIC ACTION OF IL-7 ON PRO-T CELLS

deficient in IL-7
2/2
mice (31). Taken together with our observa-
tion that IL-7 influences the Bcl-2/Bax ratio in vitro, it suggests
that these regulators of apoptosis could mediate the IL-7 response.
It has previously been shown that IL-7 enhanced the Bcl-2 levels
in cell lines (32, 33), and our study extends this to pro-T cells,
which have a physiologic requirement for IL-7R
a
signals. How-
ever, Bcl-2 induction may not completely explain the trophic ef-
fects of IL-7 on pro-T cells in embryonic life. Mice lacking Bcl-2
have relatively normal numbers of thymocytes at birth; however,
these numbers rapidly decline by 1 mo (34). The decline in thy-
mocytes after birth in these mice has been attributed to differences
in stem cells that seed the thymus before and after birth (35). Thus,
it was proposed that Bcl-2 was not required for embryonic thy-
mopoiesis generated by stem cells derived from fetal liver. At
birth, thymopoiesis is subsumed by a stem cell arriving from bone
marrow, and it or its progeny are dependent on Bcl-2. Perhaps
another Bcl-2 family member is also induced by IL-7 in fetal cells.
We sought two other Bcl-2 family members; Bcl-X
L
protein was
not detected in pro-T cells (Fig. 7), nor was bcl-w (36) mRNA
detectable by Northern blotting (not shown). Remaining family
members that have not been evaluated are Mcl-1 (37) and A1 (38).
The trophic activity of IL-3 on a cell line was attributable to phos-
phorylation of Bad (39); this mechanism would be resistant to
cycloheximide, as the IL-7 trophic effect is in part; however, we
did not detect Bad in pro-T cells (Fig. 7).
Blocking caspase activity is reported to inhibit apoptosis in
many cell types (reviewed in Ref. 40). It is therefore surprising that
caspase inhibitors, although inhibiting DNA fragmentation, did not
preserve the viability of these pro-T cells. Several lines of evidence
suggest that these cells die by apoptosis, including the phenotype
of the cells (Figs. 1 and 2) and the observation that Bcl-2 protects
them (28–30). One possibility is that death is mediated by caspases
that are insensitive to the effects of these inhibitors. Another pos-
sibility is that the death pathway does not involve caspases at all.
This is reminiscent of cytotoxic T cell killing, which is primarily
mediated by perforin; caspase inhibitors block DNA fragmenta-
tion, but not cell death, which in that case is caused by pores in the
plasma membrane (41).
It was observed that wortmannin inhibited the trophic action of
IL-7 on a pre-B cell line, suggesting that phosphatidylinositol 3-ki-
nase is required (42). The same study noted that a tyrosine site on
IL-7R
a
that mediated activation of phosphatidylinositol 3-kinase
was required for the trophic activity in pre-B cells. However, we
did not observe that wortmannin inhibited the trophic activity of
IL-7 on pro-T cells (data not shown). Hence, the seemingly similar
activities of IL-7 on survival of early T and B cells may involve
different signaling pathways and, by extension, different survival
and death pathways.
The Fas pathway did not appear to mediate death from IL-7
deprivation. It has been reported that Fas expression does not begin
until the CD4
1
CD8
1
stage. We have confirmed this and also ob-
served that its expression is not induced during IL-7 deprivation
(not shown). A recent report implicates the Fas system in the death
of cells that fail to productively rearrange their TCR
b
genes (27).
This death presumably occurs in cells at stage pro-T3, which, ac-
cording to our findings, also die when deprived of IL-7. The signal
for a successful
b
gene rearrangement is thought to emanate from
the pre-TCR, which incorporates the successfully produced
b
-chain. Thus, the trophic signals from pre-TCR may be different
from those of IL-7R, and this is substantiated by the failure of a
bcl-2 transgene to rescue T cells in rag
2/2
mice (which cannot
generate
b
gene rearrangements), whereas it protects IL-7R
2/2
thymocytes (28). Consistent with this interpretation, it has been
reported that Bcl-2 cannot protect cells from Fas-mediated killing
(43, 44). We also found no evidence for IL-7 serving as a cofactor
together with the pre-TCR signal, since pro-T4 cells survived in-
dependently of IL-7; pro-T4 cells also rapidly proliferated and dif-
ferentiated into CD4
1
CD8
1
cells within 18 h in the absence of
IL-7 (not shown).
We recently observed that the IL-7R activates the
a
4
b
1
integrin,
increasing its affinity for the extracellular matrix protein fibronec-
tin (45). Integrins, in turn, are known to provide viability signals to
some types of cells. However, we tested whether fibronectin could
augment the trophic effect of IL-7 on pro-T cells and could not
detect such an effect (data not shown). IL-7 produced by thymic
epithelial cells appears to be bound to extracellular matrix in the
thymus (45), which raises questions about the molecular form,
solubility, and concentration of the IL-7 actually encountered by
pro-T cells. Thus, we do not know whether the concentrations of
IL-7 used in our experiments fall into the physiologic or the phar-
macologic range.
We did not observe an induction of cell cycle progression by
IL-7, but, rather, found a completion of the cycle and accumulation
of cells in G
1
. There are reports that IL-7 induced the growth of
pro-T cells (for example, Ref. 46), but these effects may be attrib-
utable to the presence of other stimuli that in some studies were
FIGURE 7. Effect of IL-7 on levels of
Bcl-2 and Bax. Intracellular staining for
Bcl-2, Bcl-X
L
, Bax, and Bad was performed
on thymocytes before and after culture in
IL-7 for the indicated times. Right, Data are
expressed as specific staining, obtained by
subtracting the mean specific fluorescence
(on a linear scale) of the control staining at
each time point.
5739The Journal of Immunology

intentionally added or in other cases may have been produced
endogenously; it is also possible that in long term cultures, a sub-
population of cells that grows in response to IL-7 eventually dom-
inates; however, it can be seen from our results (and inferred from
the bcl-2 transgenic mice noted above (28–30)) that this is not the
prevailing response. Stem cell factor has been identified as a potent
cofactor that, together with IL-7, induces rapid growth (47). Other
cytokines may be produced by the thymocytes during culture in
our studies. The evidence for such autocrine factors is that cultur-
ing the cells at high density in U-bottom wells promoted the tro-
phic effects of IL-7, and conditioned medium from high cell den-
sity cultures promoted the survival of low cell density cultures (not
shown).
Differentiation of thymocytes has also been reported to be in-
duced by IL-7. VDJ recombination is promoted by the IL-7R in
thymocytes (3, 14, 48, 49) and pro-B cells (reviewed in Ref. 1; 42).
This effect is partly attributable to inducing rag1 and rag2 expres-
sion (48, 50, 51) and perhaps also to enhancing locus accessibility,
especially of the TCR
g
locus. We examined whether IL-7 induced
expression of CD25 on pro-T1 cells, since CD25
1
cells are absent
in IL-7R
2/2
mice, but this was not detectable (not shown). How-
ever, we did observe significant induction of CD8
a
and -
b
surface
expression on pro-T cells after overnight culture with IL-7 (K.
Kim, manuscript in preparation).
In conclusion, IL-7 has a trophic effect on several early stages of
pro-T cells, promoting survival without growth. Bcl-2 and Bax
levels are associated with these effects, but there may be other
mechanisms that remain to be identified for this type of biologic
activity.
Acknowledgments
We thank Drs. R. Fenton and J. Oppenheim for comments on the manu-
script, Dr. R. Kirken for wortmannin, L. Finch for flow cytometry, and
R. Wiles for technical assistance.
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