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IL-7-Regulated Homeostatic Maintenance of Recent Thymic Emigrants in Association with Caspase-Mediated Cell Proliferation and Apoptotic Cell Death

Rose M. O’Neill^1,*, Jaythoon Hassan^* and Denis J. Reen^*,

^* Children’s Research Centre, Our Lady’s Hospital for Sick Children, Crumlin, Dublin, Ireland; and Dublin Molecular Medicine Centre, and Conway Institute of Biomolecular and Biomedical Research, University College, Dublin, Ireland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Homeostasis of T cells is essential to the maintenance of the T cell pool and TCR diversity. In this study, mechanisms involved in the regulation of cytokine-mediated expansion of naive T cells in the absence of Ag, in particular the role of caspase activation and susceptibility to apoptosis of recent thymic emigrants (RTEs), were examined. Low level caspase-8 and caspase-3 activation was detected in proliferating IL-7-treated cells in the absence of cell death during the first days of culture. Caspase inhibitors suppressed IL-7-induced expansion of RTEs. Low level expression of CD95 and blocking Ab experiments indicated that this early caspase activation was CD95 independent. However, CD95 levels subsequently became dramatically up-regulated on proliferating naive T cells, and these cells became susceptible to CD95 ligation, resulting in high level caspase activation and apoptotic cell death. These results show a dual role for caspases in proliferation and in CD95-induced cell death during Ag-independent expansion of RTEs. This method of cell death in IL-7-expanded RTEs is a previously unrecognized mechanism for the homeostatic control of expanded T cells.

	Introduction

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The developmental and maturation status of T cells as they exit the thymus in early life is not known. CD4⁺, CD45RA⁺ naive T cells, described as recent thymic emigrants (RTEs),² express a number of characteristics that distinguish them from naive T cells that are present later in life. They express high levels of the thymus-associated Ag CD38 and are characterized by decreased proliferative responses to Ag stimulation, decreased cytokine production, increased susceptibility to apoptosis and tolerance induction, as well as increased susceptibility to the immunosuppressive effects of cyclosporin A and glucocorticoids (1, 2, 3, 4, 5, 6, 7). RTEs exiting the thymus are preferentially incorporated into the periphery, causing displacement of resident mature T cells, thereby maintaining the size of the peripheral naive T cell pool (8) and ensuring that the TCR repertoire is kept diverse.

Uniquely, these RTEs proliferate in response to certain cytokines in the absence of Ag stimulation, unlike their adult-derived counterparts (6, 9, 10). These cytokines, which include IL-2, IL-4, and IL-7, play a vital role in the development, expansion, and survival of the lymphoid system (11). Previous work by our group and others has shown that IL-7 can induce expansion and survival of these cells while maintaining a naive phenotype (6, 10, 12). The antiapoptotic effect of IL-7 has been shown to be in part due to its up-regulation of the antiapoptotic protein Bcl-2 (13, 14). The mechanisms by which cytokines induce proliferation of these cells or the mechanisms controlling this expansion have not been identified.

Classically, caspases are a family of cysteine-activated proteases and are the effectors of apoptosis. Recently, the emergence of a role for caspases in cell cycling has also been proposed (15, 16). It has been shown that in mature T cells, upon early TCR stimulation, caspase activity can be detected in nonapoptotic cells, and this activation is required for T cell proliferation (17, 18). Indirect evidence of caspase involvement in cell cycling has come from Fas-associated death domain (FADD) knockout and Bcl-2 transgenic mice, which exhibit impaired mature T cell proliferation (19, 20). This alternative role for caspases may not only be cell cycle specific, but may also be involved in terminal differentiation of cells (21) and can contribute to IL-2 release during T cell activation (22). Control of Ag-driven T cell expansion is exerted through activation-induced cell death (AICD) via CD95 and TNFR-I receptor activation of caspases.

In this study, we considered that similar mechanisms might regulate the expansion of cytokine-driven, Ag-independent expansion of RTEs. We show that CD95-independent caspase activation is associated with the proliferative response of RTEs to IL-7. In addition, ligation of CD95 induced caspase activation, leading to apoptotic cell death. This appears to be an effective mechanism capable of circumventing the antiapoptotic activity of IL-7, resulting in the control of expansion of IL-7-treated RTEs.

	Materials and Methods

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Reagents

IL-7 was purchased from R&D Systems (Oxford, U.K.). Blocking CD95 (clone ZB4) Ab was purchased from Medical and Biological Laboratories (Nagoya, Japan). Agonistic CD95 (clone CH-11) was purchased from Immunotech (Cedex, France).

T cell purification

Cord blood (CB) as a source of RTEs was collected from the umbilical vein, immediately after delivery, in uncomplicated pregnancies at term. Venous peripheral blood was obtained from healthy adult (AD) volunteers. Mononuclear cells were isolated by Ficoll-Hypaque (Lymphoprep; Nycomed, Oslo, Norway) density-gradient centrifugation and resuspended in PBS/4% FCS. CD4⁺ T cells were purified by negative selection using an immunomagnetic CD4⁺ enrichment kit (Stemsep; StemCell Technologies, Vancouver, British Columbia, Canada), as per manufacturer’s instructions. Briefly, 1–1.5 x 10⁷ cells were resuspended in PBS (without calcium or magnesium)/4% FCS. The mononuclear cells were incubated with CD4⁺ enrichment Ab cocktail mix at room temperature for 15 min. Magnetic colloid was then added to the cells, and they were further incubated for 15 min at room temperature. Following this incubation, the cells were passed through a magnetic column, and nonbound CD4⁺ T cells were collected. The resulting populations were >99% viable, 90–95% CD3⁺ and CD4⁺; they contained <5% CD8⁺, CD16⁺, CD19⁺, CD14⁺, and HLA-DR⁺ cells.

Cell culture

Cells were cultured in RPMI 1640 (Biowhittaker, Wokingham, U.K.) supplemented with HEPES (10 µM), L-glutamine (4 mM), gentamicin (50 µg/ml), and 10% FCS. IL-7 (10 ng/ml) was added to cultures at day 0. Lymphocyte proliferation was assessed by [³H]thymidine incorporation assay. Briefly, cells were cultured at 2 x 10⁵ cells/well in 96-well flat-bottom plates; 0.3 µCi [³H]thymidine (Amersham Biosciences, Amersham, Buckinghamshire, U.K.) was added for the last 18 h of culture. The cells were harvested and counted using a 1450 Microbeta PLUS liquid scintillation counter (Wallac, Gaithersburg, MD).

Analysis of cell survival/apoptosis

Following culture, cells were washed with cold PBS and resuspended in 200 µl of 1x annexin V-binding buffer (Biosource International, Nevilles, Belgium) and 2 µl of annexin V (IQ Products, Groningen, The Netherlands) for 10 min at 4°C. Cells were centrifuged and resuspended in 400 µl of PBS and 1 µl of propidium iodide (PI) (Intergen, Oxford, U.K.). Cell survival/apoptosis was assessed using flow cytometry.

Caspase activity

The level of caspase-3 and caspase-8 activation was examined using the CaspaTag flow cytometric based assay (Caspatag-Intergen, Oxford, U.K.) for detecting activated caspases in living cells. With this assay system, based on irreversible inhibitor binding to active caspases (23), caspase-positive cells appear as a separate peak or as a shoulder of the baseline peak showing increased fluorescence intensity. At specified times, 3 x 10⁵ CD4⁺ T cells were removed from culture and incubated with 30 µl of 30x FAM-DEVD-FMK substrate (caspase-3) or FAM-LETD-FMK substrate (caspase-8) for 1 h at 37°C in the dark. Subsequently, cells were centrifuged and washed twice in 1x wash buffer. Finally, cells were resuspended in 400 µl of 1x wash buffer; 1 µl of PI was added; and the cells were analyzed by flow cytometry. Caspase-3 activity was also detected using PhiPhilux flow cytometric assay (Oncoimmunin, College Park, MD). This assay is based on specific substrate cleavage by active caspase-3 (23). Briefly, resting or treated cells were washed, then incubated with the substrate (10 µM) for 1 h at 37°C, followed by another wash with the dilution buffer, according to manufacturer’s instructions. The cells were then analyzed by flow cytometry.

Cell phenotyping

Cells were removed from culture, washed in 1x PBS, resuspended in 100 µl of PBS/0.1% BSA, and incubated with 10 µl of TNFR-I PE (R&D Systems), 5 µl of CD95 PE (BD PharMingen, Oxford, U.K.), 5 µl of CD4⁺ FITC, or CD45RA⁺ FITC mAbs (BD Biosciences, Oxford, U.K.) for 30 min at 4°C. Cells were washed and resuspended in PBS/0.1% BSA and analyzed by flow cytometry.

Cell death induction

To determine CD95-mediated apoptosis, IL-7-treated and untreated CD4⁺ T cells were incubated with 500 ng/ml of agonistic CD95 (clone CH-11; Immunotech, Marseille, France) Ab for 24 h during the indicated incubation times. Flow cytometric analysis for apoptosis was then performed, as described above.

Western blot analysis

Following stimulation, cells were lysed in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue) and heated to 95–100°C for 5 min. Protein extracts corresponding to 0.5–1 x 10⁶ cells of protein were loaded per lane and separated on a 10% SDS-PAGE gel, transferred onto nitrocellulose membrane, and immunodetected with mAb directed against cellular FLIP (c-FLIP) (1/1000; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were stripped and reprobed with anti-actin Ab (1/200; Santa Cruz Biotechnology). The bands were visualized using an ECL system (Amersham). Densitometric readings of captured images of the bands were obtained using Gelworks ID Advanced software (Medical Supply Company, Dublin, Ireland). Results of c-FLIP levels were expressed relative to actin levels (loading control), in which the lowest actin level was arbitrarily assigned a value of 1.

	Results

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Caspase activation in IL-7-stimulated RTEs

IL-7 has been shown to be essential in the homeostasis of CD4⁺ RTEs (6, 9, 10). IL-7 induced a dose-dependent proliferative response in CD4⁺ RTEs from newborns, but had no effect on CD4⁺, CD45RA⁺ T cells from adults (data not shown). One of the most potent signaling cascades involved in cell death is that of the caspases. These families of cysteine proteases have been well established as effectors of apoptosis. Recently, caspase activation has also been associated with cell proliferation (17, 18). We examined the level of caspase activity in the IL-7 model of T cell proliferation, and possible roles for caspases in the expansion of RTEs. Using an irreversible inhibitor-based flow cytometric assay (23), caspase-8 and caspase-3 activities were detected in AD PBMCs after 4 days in culture with CD3/IL-2, similar to the results of others (17) (Fig. 1A). Caspase-8 activity was detected in living RTEs in the presence of IL-7 after 24-h incubation, while both caspase-8 and caspase-3 activation were present after 3 days of culture (Fig. 1B). No such activation was detected in IL-7-treated AD CD4⁺ T cells, suggesting that the caspase activity was associated with proliferating cells (data not shown).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Caspase activity in adult mononuclear cells and RTEs. A, Adult mononuclear cells were cultured at 1 x 106 cells/ml in the absence or in the presence of CD3/IL-2 for 4 days. Caspase-8 and caspase-3 activity was detected in the living gate, as determined by forward light scatter (FSC)/side light scatter (SSC), using Caspatag substrates and flow cytometric analysis. Data are representative of one other experiment. B, RTEs were cultured at 1 x 106 cells/ml in the absence or in the presence of IL-7 (10 ng/ml) for 3 days. At days 1 and 3, cells were removed and caspase-8 and caspase-3 activity was measured in the living gate, as determined by FSC/SSC, by flow cytometry. The average mean fluorescence intensity values for four experiments are shown ± 1 SEM.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. IL-7 induced caspase-3 activity in CD4+ T cells using the Phiphilux assay system. RTEs were cultured at 1 x 106 cells/ml in the absence or in the presence of IL-7 (10 ng/ml) for 3 days. Cells were removed, and caspase-3 activity was measured in the living gate, as determined by FSC/SSC, using flow cytometry. The average MFI values for three experiments are shown ± 1 SEM.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Inhibition of IL-7 induced proliferation of RTEs by caspase inhibitors. A, RTEs (1 x 106/ml) were pretreated for 1 h with the caspase inhibitors ZVAD-fmk, IETD-fmk, and DEVD-CHO at three concentrations (100, 10, and 1 µM) for 1 h and then treated with IL-7 (10 ng/ml) for 3 days. [3H]Thymidine incorporation was measured over the last 18 h of culture. Values represent the mean percentage inhibition ± SEM (n = 3). B, Survival of RTEs treated with caspase inhibitors, and IL-7 was assessed at day 3 using annexin V/PI staining and flow cytometric analysis. Percentage survival was measured as the percentage of cells detected within the annexin V/PI-negative gate (n = 2).

Caspase-8 lies upstream of caspase-3 and is activated by cell surface receptors containing death domains. These receptors include TNFR-I and CD95. However, previous work has shown that resting T cells, in particular naive T cells, express little or no TNFR-I or CD95 (24, 25, 26). Expression of CD95 and TNFR-I in the presence of IL-7 was measured over time. Initially, RTEs expressed low levels of both TNFR-I and CD95 (Fig. 4, A and B, left panel). However, upon stimulation with IL-7, there was a significant increase in CD95 levels in RTEs at day 3 (Fig. 4B, right panel; Table I), while there was no change in TNFR-I levels (Fig. 4A, right panel; Table I). Two populations of CD95-expressing cells, one low and one high, which did not alter over time in the presence of IL-7, were observed in AD CD4⁺ T cells (Fig. 4C). The high levels of CD95 expression in AD CD4⁺ T cells were associated with CD45RO⁺ memory T cells (Fig. 5B). However, in IL-7-treated RTEs, high levels of CD95 were associated with the CD45RA⁺ population, which continued to express CD45RA⁺ in the presence of IL-7 (Fig. 5A). Suppressing CD95 signaling with blocking Ab was used to assess the importance of CD95 activation in the caspase activity of proliferating cells. Inhibition of CD95 signaling had minimal or no effect on cell proliferation (Fig. 6A) or caspase-8 activation of IL-7-treated RTEs (Fig. 6B), suggesting CD95-independent caspase activation. This Ab was completely effective in blocking caspase activation in a Jurkat cell line control system (Fig. 6C).

View larger version (33K): [in this window] [in a new window]   FIGURE 4. TNFR-I and CD95 expression in IL-7-treated RTEs or AD CD4+ T cells. Cells (1 x 106 cells/ml) were cultured in the absence (untreated) or presence of IL-7 for 3 days, and surface marker expression was measured by flow cytometry. A, RTEs at days 1 and 3 of culture were stained with CD4 FITC/ TNFR-I PE. B, RTEs at days 1 and 3 of culture, stained with anti-CD4 FITC/CD95 PE. C, AD CD4+ T cells at days 1 and 3 of culture stained with anti-CD4 FITC/CD95 PE.

View larger version (45K): [in this window] [in a new window]   FIGURE 5. CD95 expression associated with the CD45RA+ population in RTEs. RTEs (A) and AD CD4+ T cells (B) (1 x 106 cells/ml) were cultured for 3 days in the absence (untreated) or presence of IL-7 (10 ng/ml). Cells were stained with CD4 FITC/CD95 PE or CD45RA FITC/CD95 PE Abs. Data are representative of four separate experiments.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Blocking CD95 activation had no effect on proliferation or caspase-8 activity in RTEs. A, Proliferation of IL-7-treated RTEs in the presence of blocking CD95 Ab or control Ab. RTEs (2 x 105 cells/well) were cultured with IL-7 (10 ng/ml) and blocking CD95 (ZB-4) (1 µg/ml) or control Ab (1 µg/ml) for 3 days. [3H]Thymidine incorporation was measured over the last 18 h of culture (n = 2). B, RTEs cultured as in A were assessed for caspase-8 activity in the living gate, as determined by FSC/SSC. Data are representative of one other experiment. C, Jurkat T cells were either untreated (filled histogram) or treated with agonistic CD95 (CH-11) (50 ng/ml) (open histogram) or pretreated with blocking CD95 Ab (ZB4) (1 µg/ml) for 1 h before being treated with agonistic CD95 Ab (CH-11) (50 ng/ml) (heavy open histogram) for 16 h and then assessed for caspase-8 activity. This is representative of one other experiment.

We next examined a possible role for IL-7-induced CD95 expression in the eventual death of RTEs (Fig. 7, A and B). In the presence of agonistic CD95 (CH-11) Ab, cell death occurred in RTEs cultured in IL-7 in a time-dependent manner, at days 1 (15 ± 9%), 3 (33 ± 13%), and 7 (55 ± 25%), respectively (Fig. 7, A and B). There was a concomitant increase (1- to 2-fold log order higher) in the levels of caspase-8 and caspase-3 activity present in IL-7-treated cells undergoing CD95-induced apoptosis (Fig. 7C).

View larger version (20K): [in this window] [in a new window]   FIGURE 7. CD95 induced cell death in RTEs following long-term culture in IL-7. A, RTEs (1 x 106 cells/ml) were cultured in the absence (untreated) or presence of IL-7 (10 ng/ml) for 7 days. At the time points specified, 1 x 105 cells were removed and cultured with agonistic CD95 (CH-11) (500 ng/ml) Ab for 16 h. Percentage survival was assessed as the number of cells that were in the annexin V/PI-negative gate. Data are the mean ± 1 SEM of five separate experiments. B, This is a representative dot plot (five experiments) of FSC/SSC parameters (left panels) and annexin V/PI staining (right panels) of IL-7 (lower panels) and IL-7 plus agonistic CD95 Ab (lower panels)-treated cells after 7 days of culture. C, RTEs treated with IL-7 (filled histogram) or IL-7 + CD95 (open histogram) were assessed at day 7 for caspase-8 and caspase-3 activity. Data are representative of four experiments. D, Western blot analysis of c-FLIP expression in RTEs treated with IL-7 for 7 days. Relative values of c-FLIP/actin were calculated using densitometry. Values are the mean of three separate experiments ± 1 SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
One of the most potent manifestations of the importance of controlling expansion of T cells is seen in the thymus in which apoptosis is fundamental to the selection of mature thymocytes and maintenance of viable cell numbers. Similarly in the periphery, cell numbers following Ag-driven expansion are controlled through AICD. It is now clear that in addition to Ag-driven expansion in the periphery, various populations of T cells at different stages of differentiation or development can proliferate in response to selected cytokines in the absence of Ag. This responsiveness is considered as homeostatic regulation for the maintenance of T cell populations, or in the case of RTEs, for their differentiation into more mature T cells. There has been no discussion of how Ag-independent expansion of T cells might be controlled, a regulatory process that may be just as fundamental a mechanism for maintaining T cell homeostasis as in the case of Ag-driven T cell expansion. The proliferative response of human newborn naive T cells to IL-7 provided us with a useful model to explore the regulation of expansion of these cells and mechanisms for controlling Ag-independent expansion of naive T cells. IL-7 was originally identified as a stromally derived growth factor for B cells (27), and subsequently has been shown to have several different roles. These include lymphopoiesis (28), survival (14, 29), quiescence (30, 31), enhancing the cytolytic function of mature T cells (32), and cell cycling both in RTEs (6, 12) and mature T cells following suboptimal stimulation with anti-CD3 Abs in vitro (33, 34). In initial experiments, we confirmed that proliferation of RTEs in response to IL-7 is dose dependent (data not shown), in which a single dose can induce long-term survival of RTEs (data not shown).

Analogous to AICD, we considered that caspase-mediated cell death would be central to the biological control of Ag-independent expansion of RTEs.

Caspases are cysteine-activated proteases and are produced as proenzymes containing an amino-terminal prodomain and the p20 and p10 domains that are cleaved to form the active enzyme as an active tetramer of two p20-p10 heterodimers; each heterodimer contains two active sites (35). There is growing evidence for the participation of caspases and other apoptosis regulators not only in cell death, but also in the control of other cell processes. Earliest reports of these alternative roles for caspases came from studies identifying caspase-3 processing in nonapoptotic PBMCs after polyclonal activation (15, 16). Further support came from studies of FADD, a protein associated with upstream events in caspase activation. FADD^-/- mice displayed impaired T cell proliferation upon activation, despite normal IL-2 production (36). More recently, Kennedy et al. (18) found that caspase-8, but not caspase-3, processing could be detected within 4 h of stimulation. In addition, inhibitors of caspase activation blocked T cell proliferation and IL-2 production. Mukerjee et al. (22) have subsequently demonstrated that caspases may be involved in calcineurin A cleavage, which contributes to IL-2 production. Alam et al. (17) demonstrated that 4 days after T cell activation, selective processing and activation of downstream caspases (caspase-3, -6, and -7) could be detected. They also found that caspase inhibitors could partially inhibit CD3-induced T cell proliferation. However, the recently published findings by Chun et al. (37) of a novel mutation in caspase-8 that resulted in lymphocyte activation, leading to human immunodeficiency, much more definitively links caspase activation directly with lymphocyte proliferation. Homozygous individuals for the specific caspase-8 mutation were defective for lymphocyte apoptosis, homeostasis, and in their activation of T lymphocytes, B lymphocytes, and NK cells, which lead to immunodeficiency.

Selective caspase processing has also been demonstrated in terminal erythroid differentiation (21). One of the means by which caspases may be involved in cell cycling or erythroid differentiation may be due to selective activation of target molecules. Under normal apoptotic conditions, activation of caspases such as caspase-3 results in the activation of various molecules, including poly(ADP-ribose) polymerase, cell division control 2 kinase Wee1, and the DNA fragmentation factor 45. However, studies on cell-cycling lymphocytes showed that only certain substrates such as poly(ADP-ribose) polymerase and Wee1 were cleaved (17). In the case of erythroid differentiation, proteins involved in nuclear integrity were selectively activated (21). Strength of activation of the caspase cascade may be one of the mechanisms by which selectivity is achieved with any further amplification of the proteolytic process leading to complete substrate activation and cell demise.

Based on these findings, it was decided to examine the role of caspases in both the proliferative response and the control of T cell expansion in response to IL-7. To do this, caspase activity was measured using caspase-specific fluorogenic substrates. Initially, we confirmed the observations of Alam et al. (17) by showing that CD3/IL-2 treatment of AD T cells induces activation of caspase-8 and caspase-3 within 2 days of stimulation and is strongly detectable after 4 days (Fig. 1A). In IL-7-induced proliferating RTEs, caspase-8 activation is seen in viable cells after 24-h stimulation, while caspase-3 activation is delayed, appearing at 2–3 days (Fig. 1B). This would suggest that caspase-8 activation occurs upstream of caspase 3, indicating a similar cascade as seen in apoptotic pathways. A role for caspases in the proliferation of these cells was further advanced through the use of caspase inhibitors. Each of the three inhibitors studied caused similar levels of inhibition of cell proliferation, suggesting a role for caspase activation in IL-7-induced expansion of RTEs. However, care must be taken about drawing conclusions with regard to the role of specific caspase inhibitors, as these inhibitors have the ability to titrate all accessible caspases (18). Given that at the high doses of caspase inhibitors used (100 µM) only partial inhibition occurred, it appears that caspase activation is not an absolute requirement for cell proliferation to occur. However, the fact that our system is a TCR-independent signaling system involving _c-chain cytokines adds further to the complexity of the mechanism through which caspases may be mediating their effects.

The mechanism through which caspases are activated in proliferating cells is not known. In cells undergoing apoptosis, caspase-8 is recruited to the membrane and activated via the protein FADD. FADD is itself activated by one of two receptors, TNFR-I or CD95 (Fas). Activation of TNFR-I via TNF- or CD95 via Fas ligand results in the recruitment and activation of procaspase-8 to caspase-8, which leads downstream to caspase-3 activation and apoptosis. Early studies demonstrated that FADD dominant-negative mice had unaltered negative selection, but were defective in activation through the TCR and CD28 (38). This was the first indication that FADD and Fas signaling could be involved in cellular responses other than apoptosis. The likely mechanism by which Fas signaling could be directed toward a role in proliferation would be via caspase-8 and c-FLIP, the natural inhibitor of Fas-induced cell death. Two cellular homologues have been characterized (39). FLIP (L), the full-length 55-kDa form of FLIP, shows overall structural homology to caspase-8, containing two death effector domains that interact with FADD, and an inactive caspase-like domain. FLIP (S), an alternatively spliced short form of FLIP, contains only the two death effector domains and has lower antiapoptotic capacity (40). It has been shown that FLIP can mediate the activation of NF-B and extracellular signal-regulated kinase, thus providing a switch to a proliferative signal. The direct mechanism by which the interaction between FLIP and extracellular signal-regulated kinase/NF-B occurs is currently unknown. TCR activation in conjunction with CD95 costimulation could result in a signaling cascade toward proliferation via FLIP. Then, depending on the ratio of caspase-8 to FLIP levels, Fas-FADD complexes might recruit either caspase-8 homocomplexes or caspase-8 FLIP heterocomplexes. In the latter case, FLIP could stop apoptotic signaling events downstream of caspase-8 activation. In the context of long-term stimulation of the TCR, FLIP is involved in the production of IL-2, which is important in the expansion of the T cells and eventually results in the down-regulation of FLIP, thus allowing activation-induced cell death to occur (41).

Resting CB CD4⁺ T cells expressed little or no TNFR-I, as has been shown by others (24), and these levels do not change in the presence of IL-7. However, when CD95 expression was examined, we found that while there were low/absent levels on resting (day 0) CD4⁺ T cells, these cells up-regulated CD95 levels equivalent to those of AD memory T cells after 3 days in culture with IL-7. Previous work had established that activated CB T cells were less susceptible to Fas-induced cell death due in part to the lower expression of CD95 on naive T cells (25). Thus, it was very surprising to see this increase in CD95 expression at day 3 in response to IL-7, which remained associated with a naive T cell phenotype. Human thymocytes can up-regulate CD95 upon IL-7 plus IFN- stimulation (42), and our observations in respect of IL-7-induced CD95 expression in naive CD4 T cells at birth are further evidence for a thymic phenotype carryover into the periphery in early life, and the fact that at birth these cells as RTEs are at a unique developmental state.

Although one of the major roles for CD95 in peripheral T cells is the process of AICD, its role in homeostatic regulation of T cells in an Ag-independent regulated system had not previously been considered. It is known that within 2 days of TCR stimulation, cycling T cells become sensitive to CD95-induced apoptosis, which has been attributed to a decrease in FLIP levels (43). Ligation of CD95 with Ab on CB CD4⁺ T cells cultured in IL-7 resulted in a time-dependent development of sensitivity to CD95-induced cell death in a manner similar to AICD. However, unlike that reported for AICD, this effect appears not to be mediated through a down-regulation of c-FLIP (Fig. 7D). It has recently been shown that the ratio of c-FLIP to CD95 can determine the level of CD95-induced cell death (44). Therefore, in the RTE model, the dramatic up-regulation of CD95 expression in the presence of unaltered c-FLIP levels appears to be sufficient to render these cells susceptible to CD95-induced cell death. Overall, a dual role for caspases in cell proliferation and cell death is evident in the RTE model of responsiveness to IL-7.

These data indicate that T cells that proliferate in response to cytokines in the absence of Ag may be under expansionary control in a manner similar to Ag-expanded T cells. There is marked lymphocytosis at birth and during the first week of life in humans. The mechanism by which this lymphocytosis is quickly down-regulated to circulating adult cell numbers during the first month of life is presently unknown. RTEs are not in cell cycle phase or proliferating while circulating at birth. It is known from animal studies that RTEs exiting the thymus initially home to lymph nodes, and that expansion of these cells is likely to occur at sites of IL-7 production such as skin, gut, and the liver. Paradoxically, these organs through the presence of macrophages and immature dendritic cells expressing CD95L (45, 46) could also provide a mechanism for limiting the expansion of immature RTEs in the periphery. Although the need for restriction of Ag-independent cell expansion under normal physiological conditions may be limited, it is, however, likely to be an important mechanism to prevent uncontrolled bystander T cell expansion in lymphoid tissues as part of the adaptive immune response.

	Acknowledgments

 
We gratefully acknowledge the mothers from the Coombe Women’s Hospital, who consented to CB being used for these studies, and the expert CB collection skills of Julie Grantham.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Rose O’Neill, Our Lady’s Hospital for Sick Children, Crumlin, Dublin 12. E-mail address: Rmoneill{at}hotmail.com

² Abbreviations used in this paper: RTE, recent thymic emigrant; AD, healthy adult; AICD, activation-induced cell death; CB, cord blood; c-FLIP, cellular FLIP; FADD, Fas-associated death domain; FSC, forward light scatter; PI, propidium iodide; SSC, side light scatter.

Received for publication June 17, 2002. Accepted for publication February 28, 2003.

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