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Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5535/Institut Fédératif de Recherche 122, Montpellier, France
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several cytokines that signal through the common 
chain (c),3 IL-2, IL-4, IL-7, and IL-15, have recently been shown to be important for the homeostatic proliferation of CD4+ and CD8+ murine lymphocytes (1, 2, 3, 4). Nevertheless, it is clear that the survival and proliferation of CD4+ and CD8+ T cell populations, as well as naive and memory subsets, are not equivalently regulated by these cytokines. For example, memory CD8+ cells, but not naive or memory CD4+ cells, proliferate in response to IL-15 in mice (5). Moreover, the combination of IL-4 and IL-7 contributes to naive, but not memory, murine CD4+ T cell survival (1).
However, the role of c cytokines must be considered in the context of the entire T cell pool. As the size of this T cell pool remains constant, it is essential that thymic output and peripheral T cell expansion be balanced by lymphocyte death. Thus, under conditions where cytokines promote the proliferation of specific T cell subsets, other cells must die. Additionally, this scenario strongly suggests that cytokines will have distinct effects in lymphopenic and nonlymphopenic animals. Indeed, in lymphopenic mice IL-7 induces the proliferation of both naive CD4+ and CD8+ T cells (6, 7, 8, 9), whereas it appears that in a nonlymphopenic environment, CD8+ T cells undergo more division than CD4+ T cells (10, 11). Moreover, IL-7 has distinct effects on T cells of different maturational stages. We and others have recently shown that in vitro, IL-7 promotes the proliferation of immature naive human T cells that have recently exited from the thymus (recent thymic emigrants (RTE)), whereas mature peripheral blood naive T cells do not divide under the same conditions (12, 13). These results are supported by in vivo mice data demonstrating that RTE have a selective survival advantage over resident peripheral naive T cells for up to 4 wk following thymic export (14). Thus, IL-7 differentially affects CD4+ and CD8+ T cells, the outcome of which is dependent on the maturational state of the cell as well as the overall status of the T cell pool.
It is generally accepted that following TCR engagement, activation-induced cell death (AICD) plays an important role in the termination of an immune response. However, outside the context of TCR engagement, the effects of cytokines on T cell apoptosis have not yet been assessed. The predominant death pathway activated in TCR-stimulated T cells undergoing AICD is mediated through Fas/CD95. Stimulation of CD95 by its ligand results in the formation of a death-inducing signaling complex. Recruitment and activation of caspase 8 in the death-inducing signaling complex induces a proteolytic cascade that leads to apoptosis. Although both CD95 and its ligand (CD95L) are induced in T cells upon engagement of the TCR (15), expression of CD95 is not in itself a measure of the sensitivity of a cell to Fas-mediated apoptosis. Memory peripheral blood T cells are resistant to cell death induced by agonistic 
-Fas mAbs despite detectable levels of surface CD95 (16, 17, 18). Moreover, while T cells acquire sensitivity to AICD following TCR engagement, the responses of naive and memory T cell pools are not equivalent: memory T cells are more resistant to AICD after Ag restimulation than primary Ag-stimulated naive T cells (19, 20, 21).
In this study, we assessed whether the susceptibility of CD4+ T cell subsets to Fas-mediated apoptosis is modified by the presence of cytokines and whether this susceptibility correlates with cytokine-mediated survival and/or proliferation. Although the IL-7 cytokine promotes the survival of naive CD4+ T cells (22, 23, 24), we now show that together with IL-2, it primes naive peripheral blood T cells as well as RTE for Fas-mediated cell death. In contrast, IL-2/IL-7-stimulated memory T cells are significantly more resistant to Fas-induced cell death. The level of Fas-induced cell death does not correlate with proliferation as only RTE divided significantly in response to the combination of these homeostatic c cytokines. However, in contrast to the previously reported inhibitory effect of IL-2 on IL-15-induced proliferation of CD8+ T cells (25, 26), IL-2 potentiated the IL-7-induced proliferation of CD4+ T cells. Thus, the IL-2 and IL-7 cytokines distinctly modulate the proliferation of naive and memory T cell subsets and contribute significantly to the differential susceptibility of these lymphocytes to Fas-induced cell death.
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Materials and Methods |
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Adult peripheral blood (APB), obtained from healthy adult donors after informed consent, as well as umbilical cord blood (UCB), obtained immediately after delivery of full-term infants (Montpellier, France), was collected in heparinized tubes. CD4+ T cells were purified by negative selection using tetrameric complexes in which one Ab recognizes a surface Ag on B cells, monocytes, NK cells, or CD8+ cells and the other recognizes glycophorin A on the surface of RBCs (RosetteSep; StemSep Technologies, Vancouver, British Columbia, Canada). Non-CD4+ T cells were then pelleted upon Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) separation and nonbound CD4+ T cells were recuperated. To separate naive and memory T cell subsets, CD4+ lymphocytes were incubated with CD45RO (DAKO, Trappes, France) or CD45RA (Immunotech, Marseille, France) mAbs, respectively, in combination with HLA-DR and CD69 mAbs, to eliminate preactivated T cells, followed by addition of mouse IgG-conjugated magnetic beads (Dynal Biotech, Oslo, Norway). Cells not bound to beads, defined as CD45RO- (naive) and CD45RA- (memory) populations, were separated using a magnet (Dynal Biotech) and the purity of each cell isolation was monitored on a FACSCalibur (BD Biosciences, San Jose, CA) after staining with appropriate FITC-conjugated and PE-conjugated mAbs. The purity of the selected populations was >90%. Lymphocytes were cultured in RPMI 1640 medium (Invitrogen, Auckland, New Zealand) supplemented with 10% (v/v) FCS, penicillin, and streptomycin, in the absence or presence of rIL-2 (100 U/ml) (Chiron, Emeryville, CA) and/or IL-7 (10 ng/ml) (PeproTech, London, U.K.). Alternatively, cells were stimulated with immobilized CD3 Ab (UCHT-1, 1 µg/ml; generous gift of D. Cantrell, Imperial Cancer Research Fund, London, U.K.) together with a CD28 Ab (9.3, 1 µg/ml; kindly provided by C. June, University of Pennsylvania, Philadelphia, PA).
Flow cytometry for surface markers
To detect expression of CD69 and CD95, cells were incubated for 20 min on ice with the appropriate PE-conjugated mAb (clones TP1.55.3 and 7C11, respectively; Immunotech), at the indicated time points. Background fluorescence was measured using an Ig isotype control Ab. Cells were washed with PBS (Bio Media, Boussens, France), analyzed on a FACSCalibur (BD Biosciences) and data analysis was performed using CellQuest software (BD Biosciences).
CFSE labeling
Freshly isolated T cell subsets were washed and resuspended in PBS at a concentration of 2.5 x 106 cells/ml for labeling with the fluorochrome CFSE (Molecular Probes, Eugene, OR). Cells were incubated with CFSE at a final concentration of 2.5 µM for 3 min at room temperature. Labeling was terminated by the addition of FCS (30% of total volume), cells were washed twice, and then cultured as indicated. Division was analyzed on a FACSCalibur on the FL-1 wavelength, at the indicated time points.
Anti-Fas Ab-induced apoptosis
To monitor CD95-induced apoptosis, T cell subsets were left untreated (control), or incubated with 100 ng/ml of an agonistic CD95 mAb (clone 7C11; Immunotech) Ab for 16 h at 37°C. To investigate whether Fas-induced apoptosis requires caspase activation, cells were either left untreated or pretreated with the broad spectrum caspase inhibitor Z-VAD-fmk (50 µM; Sigma-Aldrich) for 30 min and then incubated with the agonistic CD95 Ab for 16 h. Cells (5 x 105) were harvested and incubated with annexin FITC solution (200 ng/ml) and propidium iodide (10 µg/ml; Annexin VFITC kit; Immunotech) for 10 min on ice. After washes, apoptotic cells were quantified by FACS analysis, counting annexin V-positive cells.
Western blot analysis
Cells were collected at the indicated time points and immediately lysed in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM glycerolphosphate, 20 mM sodium pyrophosphate, 7.5 µg/ml aprotinin, and 200 µM PMSF). Cells that were stimulated immediately after isolation were incubated in the presence of either IL-2 (103 U/106 cells), IL-7 (100 ng/106 cells) or both cytokines (103 U/106 cells and 100 ng/106 cells, respectively) for 30–60 min at 37°C. Protein lysates were resolved on SDS-PAGE gels and transferred electrophoretically to Protran Nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were blocked for 1 h at room temperature in TBS (150 mM NaCl, 20 mM Tris, pH 7.5, 0.05% Tween 20) containing 5% milk and incubated with either a polyclonal Ab recognizing Tyr694 phosphorylated STAT 5 (1/1000 dilution; Cell Signaling Technology, Beverley, MA) or an extracellular signal-regulated kinase (Erk-2) mAb (1/2000 dilution; BD Transduction Laboratories, Lexington, KY) for 1 h at room temperature. Blots were then incubated with goat anti-rabbit or anti-mouse secondary Ab linked to peroxidase (Amersham, Arlington Heights, IL), respectively, and immunoreactive proteins were visualized using the ECL detection system (Amersham).
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c cytokines
The naive T cells present in UCB have previously been defined as RTEs on the basis of TCR excision circle levels (12) whereas naive CD4+ T cells present in the peripheral circulation of adults often emigrated from the thymus years earlier. We and others have previously shown that RTE from UCB, but not adult CD4+ lymphocytes, proliferate in response to the IL-7 cytokine, despite high receptor expression (IL-7R and c chains) on all subsets (12, 13). The range of biological activities induced by a second c cytokine, IL-2, on RTE is not known, but its effects on mature CD4+ T lymphocytes are pleiotropic with responses including proliferation, protection from apoptosis, increased susceptibility to AICD, and limited T cell expansion (26, 27, 28, 29). Using the CFSE dilution method to precisely monitor cell division, we first compared the proliferative responses of RTE as well as mature naive (TN) and memory (TM) CD4+ T cells from adult peripheral blood (APB) to IL-2, IL-7, and the combination of these two c cytokines. IL-2 alone did not stimulate cell division of either TN or TM whereas 30% of RTE divided at least once under these conditions (1). As previously reported (12, 13) IL-7 stimulated division of RTE but only minimal proliferation was observed in APB CD4+ lymphocytes. Nevertheless, significantly more RTE underwent three or more divisions in the presence of both c cytokines as compared to IL-7 alone (55 vs 20% in a representative experiment; Fig. 1). Proliferation of APB CD4+ lymphocytes was also increased but to a much lower level, with only 15–25% of cells having undergone one or more divisions. Notably, in all cases where proliferation was observed, the kinetics of c cytokine-induced division were much slower than that observed following TCR stimulation with a first division noted after 72–96 h as compared to 30–48 h in Ag-stimulated cells (data not shown). Moreover, the effects of IL-2 and IL-7 are additive as supplementing higher concentrations of either of these cytokines alone did not modify proliferation profiles in any of the T cell subsets (data not shown).
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c cytokine-stimulated CD4+ lymphocyte subsets
Immunological responsiveness is controlled by a balance between proliferation and cytokine secretion on the one hand, and apoptotic cell death, on the other. However, the vast majority of studies performed to date have monitored apoptotic cell death in the context of TCR stimulation. As the contribution of the Fas-mediated cell death pathway plays an important role in controlling peripheral T cell immune responses, it was important to analyze the effects of c cytokines on the susceptibility of CD4+ T lymphocyte subsets to Fas-induced apoptosis in the absence of MHC/peptide signals.
We first monitored the levels of Fas on freshly isolated CD4+ lymphocyte subsets by flow cytometry. As previously reported (30, 31, 32), Fas was not detected on RTE (Fig. 2A). Moreover, Fas was not detected on TN although it was present at low levels on a significant percentage of TM. Expression of Fas was not in itself an intrinsic marker of T cell activation, as expression of an unrelated activation marker, CD69, was similar in all three CD4+ T lymphocyte subsets (Fig. 2A).
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c cytokine-exposed T cell subsets is not known. Exposure to either IL-2 or IL-7 alone resulted in a small increase in Fas expression on both RTE and TN (Fig. 2B). Notably though, ex vivo culture in the absence of cytokines also resulted in a small augmentation of Fas levels on these T cells, likely due to the presence of FCS in the media (compare Fig. 2, A and B, untreated cells). Nevertheless, concomitant exposure to both c cytokines resulted in the induction of significant levels of Fas on these cells, with 
80% of RTE and TN expressing this molecule in a representative experiment (Fig. 2B). This increase in Fas expression was not associated with activation per se as CD69 levels were only marginally augmented following cytokine stimulation (Fig. 2B). Although basal Fas levels were higher on TM than on the other CD4+ lymphocyte subsets, Fas expression on these former cells was not augmented by either IL-2, IL-7, or the combination of IL-2 and IL-7 (Fig. 2B). Moreover, CD69 levels remained unchanged on TM in the presence of c cytokines. It should be noted though that this phenomenon was specific to c cytokine stimulation as both Fas and CD69 levels were significantly augmented under conditions of Ag stimulation (data not shown). Thus, the responsiveness of CD4+ lymphocytes to treatment with the IL-2 and IL-7 cytokines, as measured by Fas induction, differs significantly between RTE and naive T cell subsets, on the one hand, and memory T cell subsets, on the other. Susceptibility of CD4+ lymphocyte subsets to Fas-mediated apoptosis
Expression of Fas on T lymphocytes is not in itself a measure of the susceptibility of these cells to Fas-mediated apoptosis (30, 35). The differential susceptibility of activated CD4+ lymphocytes to Fas-mediated apoptosis is not solely dependent on cell surface CD95 levels, (36) but rather may correlate with actin polarization (35) as well as other changes at the cell surface and in the internal cell environment. Therefore, it was important to directly determine whether these c cytokines sensitize CD4+ lymphocytes to Fas-engagement and whether cell death correlates with the induction of cell surface CD95 expression. CD4+ T cell populations were cultured ex vivo during a short period, 5–7 days, in the absence or presence of these c cytokines. Sensitivity to Fas-mediated induction of apoptosis was then monitored following addition of the agonistic anti-CD95 Ab, 7C11.
As previously reported, a significantly higher proportion of RTE, as compared to TN or TM, died when c cytokines were not added to the media (22, 24). However, under these conditions, no further apoptosis was induced in any of the subsets upon Fas engagement. The addition of IL-7, but not IL-2, significantly inhibited the level of spontaneous cell death observed in the ex vivo-maintained RTE. In the presence of either cytokine alone, there was only a minimal level of Fas-induced cell death within TN and TM populations (Fig. 3A). A higher level of cell death was observed in Fas-engaged RTE pretreated with IL-7 (10–40%), but these levels were consistently lower than those detected following Ag activation (55–90%) (Fig. 3A and data not shown). However, upon pretreatment with a combination of the IL-2 and IL-7 cytokines, Fas engagement resulted in significant levels of cell death in both TN and RTE (45–90%), equivalent to that observed in Ag-activated cells (Fig. 3, A and B and data not shown). In contrast, IL-2/IL-7-treated memory T cells were much less susceptible to Fas-mediated cell death, with apoptosis ranging from 20% to a maximum of 45% (Fig. 3, A and B and data not shown). It should be noted that a rather broad range of apoptosis was observed in independent experiments, likely due to variability between T lymphocytes isolated from different donors. Nevertheless, the differences in Fas-mediated apoptosis detected in each culture condition remained constant. Thus, the combination of IL-2 and IL-7 cytokines primed naive T cells as well as RTE for Fas-mediated apoptosis whereas memory cells were relatively refractory to this process.
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c cytokine-stimulated CD4+ lymphocyte subsets is dependent upon caspase activation
Caspases, a group of cysteine proteases, play a critical role in the Ag-driven regulation of T cell death (reviewed in Ref.37), and as such, it was important to determine their involvement in the Fas-mediated cell death observed in IL-2/IL-7-stimulated CD4+ T cell subsets. Addition of a pan-caspase inhibitor, Z-VAD-fmk, did not block the low spontaneous level of cell death observed in any of the IL-2/IL-7-stimulated CD4+ subsets (data not shown). However, it almost completely inhibited -Fas-induced cell death (Fig. 4). Thus, induction of caspases appears to regulate the Fas-mediated cell death observed in the context of IL-2/IL-7 cytokine stimulation.
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c cytokine-stimulated CD4+ T cell subsets
Signaling pathways activated via c cytokines are transduced via Janus kinases (JAKs) and STATs. Both IL-2 and IL-7 are known to activate JAK1 and JAK3, as well as STAT 5 which translocates to the nucleus where it functions as a transcription factor (reviewed in Ref.38). Indeed, STAT 5 has been shown to be involved in long-term survival of an IL-2-dependent cell line and promotes the sensitization of IL-2-treated T cells to AICD (27, 28). Nevertheless, the vast majority of studies assessing the activation of STAT 5 and its role in IL-2-mediated apoptosis have been performed in cell lines or in Ag-stimulated T cells. Therefore we studied the IL-2/IL-7-induced activation of STAT 5 in the context of non-antigen stimulated CD4+ T lymphocyte subsets.
STAT 5 phosphorylation was not detected in freshly isolated CD4+ T lymphocytes, irrespective of the subset. Upon short-term activation with IL-2 (30 to 60 min), significantly higher levels of STAT 5 phosphorylation were induced in APB memory T cells than in naive T cells and only minimal levels were observed in RTE from UCB (Fig. 5A). It is interesting to note that in all these populations, expression of the -chain of the IL-2R (CD25) was barely detectable (<10%), albeit by conventional FACS analysis (not shown). In contrast to IL-2-mediated phosphorylation, IL-7 induced STAT 5 phosphorylation in RTE as well as in naive and memory APB subsets (Fig. 5A). Following ex vivo culture (6 days) in the presence of c cytokine(s), the same general trends were observed. There was a significantly higher level of STAT 5 phosphorylation in IL-2-stimulated memory T cells than in either naive cells or RTE (Fig. 5B). Furthermore, IL-7 consistently induced significantly higher levels of STAT 5 phosphorylation in naive and RTE subsets as compared with memory cells, despite expression of IL-7R and c chains on all subsets (13). Nevertheless, as compared with IL-7 alone, the combination of IL-2 and IL-7 did not appear to increase STAT5 phosphorylation in RTE or naive T cells, at either an early or "steady state" time point (Fig. 5, A and B). Altogether, these data show that although both IL-2 and IL-7 can transduce a signal via STAT 5 in CD4+ T cells, the STAT 5 signaling cascade is differentially regulated in naive and memory CD4+ T cell subsets.
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Discussion |
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To date, the effects of rIL-7 on mature human T cells have been analyzed exclusively on peripheral blood-derived lymphocytes cultured ex vivo. Under these conditions, the cytokine milieu is more strictly controlled but the effects are clearly biased by the culture conditions as well as the methods used to isolate the selected populations (47, 48). In our study, naive and memory CD4+ T cell populations were isolated by negative selection on the basis of CD45RA and CD45RO expression, respectively. Nevertheless, it should be noted that in vivo some CD45RO cells convert back to CD45RA expression upon entering a quiescent state and thus, CD45RA T lymphocytes from UCB are the only population which is believed to be "truly" naive (49, 50, 51, 52, 53). That being said, >95% of the isolated naive CD4+ T cell populations used in these studies, albeit from UCB or from APB, coexpressed CD62L and CD27, two other markers associated with a naive phenotype (data not shown). It remains to be determined whether other factors and subpopulations, such as T regulatory cells, which may differ between UCB and APB affect the cytokine responsiveness of these naive T cells.
With regard to distinctions between in vitro and in vivo responsiveness to IL-7, it is notable that IL-7 can prime T cells to respond to suboptimal TCR activation (23, 54, 55). In this manner, it can promote homeostatic proliferation in vivo following interaction of the TCR with low affinity ligands that generally do not induce cell cycle entry (56, 57). As the interaction of T cells with a low affinity Ag is significantly less likely to occur in vitro than in vivo, this may explain the more constrained effects of rIL-7 on human T cells cultured ex vivo. However, under the ex vivo conditions used by ourselves and others, rIL-7 does promote a significant division of human RTE although it does not induce division of naive or memory peripheral CD4+ human T cells (12, 13, 58). In fact, proliferation of mature peripheral blood CD4+ T cells cultured in the presence of IL-7 ex vivo has only been observed following the addition of IL-15, and even then, only memory cells proliferate (58). Thus, responsiveness to a combination of the IL-7 and IL-15 c cytokines appears to be progressively acquired during T cell differentiation in the periphery.
The roles of c cytokines in T cell homeostasis are nonredundant (reviewed in Refs.59 and 60). We now show that in human T cells, IL-2 enhances IL-7-induced proliferation of RTE as well as mature naive and memory CD4+ T cells. Nevertheless, this effect is significantly more pronounced in RTE than in naive or memory populations. In mature TN and TM subsets, the contribution of IL-2 to proliferation is dependent on the presence of IL-7, as IL-2 alone does not induce cell division. This effect may be of important physiological significance as IL-7-stimulated mature T cells appear to be primed to secrete IL-2 (61). With regard to the pleiotropic nature of IL-2, it is interesting to note that this cytokine has recently been shown to limit the expansion of murine CD8+ T cells stimulated with another c cytokine, IL-15 (25, 26). In contrast, we find that IL-2 enhances IL-7-induced proliferation of human CD4+ T cells. These distinct effects may allow IL-2 to contribute to a differential regulation of CD4+ and CD8+ T cell expansion.
As discussed above, our data and that of many other groups support an important role for IL-7 in homeostatic T cell proliferation. Indeed, treatment of nonlymphopenic monkeys with a short course of rIL-7 resulted in a significant increase in peripheral T cell numbers (9). Nevertheless, this effect was transient and T cell counts returned to baseline within 10 days after termination of treatment (9). As homeostasis is defined as the maintenance of a fixed number of T cells, this outcome is not unexpected and suggests an important amount of cell death following IL-7 therapy. However, prior to the experiments reported here, studies assessing whether IL-7 modulated T cell susceptibility to apoptotic signals had not been performed. We find that IL-7 alone increased Fas-mediated cell death of RTE, albeit to relatively low levels. In marked contrast, RTE and naive T cells exposed to a combination of IL-2 and IL-7 were extremely susceptible to Fas-induced cell death, to levels equivalent to that observed following TCR activation. Despite equivalent levels of surface Fas, IL-2/IL-7-stimulated memory T cells were significantly more resistant to Fas-induced cell death. Intriguingly, similar differences have been reported for TCR-induced cell death in naive and memory T cell subsets: specifically, Ag receptor-restimulated memory cells are more resistant to AICD than restimulated naive T cells (19, 20). In the previously studied TCR-stimulated cells as well as in the c cytokine-stimulated cells studied here, the apoptotic cell death induced in all T cell subsets required activation of caspases. Altogether, these data strongly suggest that the response of CD4+ T cells to Fas engagement is determined by a previous antigenic history, with a selective advantage observed for lymphocytes having acquired memory/effector functions.
Susceptibility of c cytokine-stimulated cells to Fas-mediated cell death did not correlate with proliferation per se, but rather was inversely correlated with the maturation state of the cell. Thus, under in vivo conditions of lymphopenia, c cytokines might be expected to promote a more robust division of RTE. However, if global compensatory mechanisms result in excessive T cell numbers, cytokine-stimulated cells, at least those stimulated by IL-2 and IL-7, would be susceptible to Fas-mediated cell death. As memory T cells were more resistant to this latter process, they would be selectively maintained as compared to naive T cells. With regard to RTE, they would also die at high levels but their loss would be balanced by a high initial rate of c cytokine-induced proliferation.
It is well-established that cytokines transduce signals via multiple protein kinase pathways. As the CD4+ T cell populations studied here demonstrated distinct IL-2/IL-7-mediated biological responses, it was reasonable to assess whether these downstream effects were regulated at the level of proximal signaling intermediates. The Erk mitogen-activated protein kinase pathway has been shown to be stimulated by IL-2 but not by IL-7 (62) and indeed, IL-7 did not induce mitogen-activated protein kinase activation in either RTE or naive/memory T cell subsets (data not shown). In contrast, the JAK/STAT and phosphatidylinositol-3 kinase/Akt pathways play essential roles in the cytokine signaling cascades activated via both IL-2 and IL-7 (38). Akt activation and subsequent Bcl-2 expression appears to be necessary for IL-2-mediated T cell survival whereas STAT 5 is apparently required for both proliferation and sensitivity to AICD (27, 28). We found that although STAT 5 phosphorylation was induced by both IL-2 and IL-7 in all CD4+ T cell subsets, the magnitude of the response differed significantly in these populations. IL-2 induced significantly higher levels of STAT 5 phosphorylation in memory T cells than in naive T cells or RTE, at both early (30–60 min) and late (6 days) time points. In contrast, IL-7-mediated STAT 5 phosphorylation was observed in all CD4+ populations at early time points but was more pronounced in naive T cells and RTE following 6 days of culture. There was no synergy observed when the two cytokines were used in combination. As cytokine-induced proliferation and Fas-mediated apoptosis were most pronounced in distinct populations, IL-7-induced RTE and IL-2/IL-7-induced memory T cells, respectively; our data strongly suggest that STAT 5 phosphorylation per se is not responsible for the diverse downstream biological responses observed in these T cell subsets. Thus, the activation of STAT 5 via different c cytokines does not result in homogeneous cell environments. Rather, we hypothesize that because of the initial "signatures" of these T cell subsets, they attain distinct "physiological states" following cytokine stimulation, even if the same signaling intermediates are activated. The precise associations between the recruitment of various c cytokine-induced signaling pathways and the susceptibility of the various T lymphocyte subsets to apoptosis/proliferation remain to be determined.
Much recent evidence indicates that the composition and size of the naive and memory T cell pools are regulated by competition for limiting resources, including cytokines (reviewed in Refs.59 and 60). In this study, we demonstrate that the IL-2 and IL-7 c cytokines are important players in the regulation of differential CD4+ T cell subset proliferation in the context of nonlymphoid tissues. Moreover, these c cytokines differentially modulate the susceptibility of CD4+ T cells to Fas-induced cell death. In this manner, IL-2 and IL-7 can promote homeostatic expansion while limiting the size of the overall T cell pool. The finding that recent thymic emigrants are the most susceptible to the proliferative effects of these c cytokines (12, 13) may allow for maintenance of optimal diversity, especially under conditions of lymphopenia. Nevertheless, it remains to be determined whether the RTE in neonates respond in the same manner as RTE that develop in adults with T cell depletion. The potential use of recombinant IL-2 and IL-7 molecules as immunorestorative and immunoenhancing agents for these lymphopenic patients, especially patients with HIV infection and those undergoing chemotherapy (reviewed in Refs.63 and 64), makes it crucial that the wide range of effects of these cytokines be explored. Cytokines that mediate the balance between proliferation and apoptosis will regulate the size and diversity of RTE, as well as mature naive and memory T cell pools in the peripheral circulation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Naomi Taylor, Institut de Génétique Moléculaire de Montpellier, 1919 Route de Mende, 34293 Montpellier, Cedex 5, France. E-mail address: taylor{at}igm.cnrs-mop.fr
3 Abbreviations used in this paper: c, common chain; RTE, recent thymic emigrant; AICD, activation-induced cell death; APB, adult peripheral blood; UCB, umbilical cord blood; Erk, extracellular signal-regulated kinase; TN, naive CD4+ T cell; TM, memory CD4+ T cell; JAK, Janus kinase.
Received for publication January 28, 2003. Accepted for publication April 18, 2003.
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F. Baratelli, Y. Lin, L. Zhu, S.-C. Yang, N. Heuze-Vourc'h, G. Zeng, K. Reckamp, M. Dohadwala, S. Sharma, and S. M. Dubinett Prostaglandin E2 Induces FOXP3 Gene Expression and T Regulatory Cell Function in Human CD4+ T Cells J. Immunol., August 1, 2005; 175(3): 1483 - 1490. [Abstract] [Full Text] [PDF]
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I. Rochman, W. E. Paul, and S. Z. Ben-Sasson IL-6 Increases Primed Cell Expansion and Survival J. Immunol., April 15, 2005; 174(8): 4761 - 4767. [Abstract] [Full Text] [PDF]
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 J.-D. Lelievre, F. Petit, D. Arnoult, J.-C. Ameisen, and J. Estaquier Interleukin 7 Increases Human Immunodeficiency Virus Type 1 LAI-Mediated Fas-Induced T-Cell Death J. Virol., March 1, 2005; 79(5): 3195 - 3199. [Abstract] [Full Text] [PDF]
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E. Dondi, G. Roue, V. J. Yuste, S. A. Susin, and S. Pellegrini A Dual Role of IFN-{alpha} in the Balance between Proliferation and Death of Human CD4+ T Lymphocytes during Primary Response J. Immunol., September 15, 2004; 173(6): 3740 - 3747. [Abstract] [Full Text] [PDF]
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) or treated with an agonistic anti-Fas Ab (7C11, 100 ng/ml; 
) for 16 h at 37°C. Cell death was analyzed by annexin V/PI staining. Data from one of four experiments with similar results are shown. B, Representative staining profiles of IL-2/IL-7-stimulated cells following Fas-induced cell death are shown.
