IL-21 Sustains CD28 Expression on IL-15-Activated Human Naive CD8+ T Cells
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免疫学杂志 2005年第14期
Abstract
Human naive CD8+ T cells are able to respond in an Ag-independent manner to IL-7 and IL-15. Whereas IL-7 largely maintains CD8+ T cells in a naive phenotype, IL-15 drives these cells to an effector phenotype characterized, among other features, by down-regulation of the costimulatory molecule CD28. We evaluated the influence of the CD4+ Th cell-derived common -chain cytokine IL-21 on cytokine-induced naive CD8+ T cell activation. Stimulation with IL-21 did not induce division and only slightly increased IL-15-induced proliferation of naive CD8+ T cells. Strikingly, however, IL-15-induced down-modulation of CD28 was completely prevented by IL-21 at the protein and transcriptional level. Subsequent stimulation via combined TCR/CD3 and CD28 triggering led to a markedly higher production of IL-2 and IFN- in IL-15/IL-21-stimulated cells compared with IL-15-stimulated T cells. Our data show that IL-21 modulates the phenotype of naive CD8+ T cells that have undergone IL-15 induced homeostatic proliferation and preserves their responsiveness to CD28 ligands.
Introduction
Immunological protection to pathogens relies on the formation and maintenance of a diverse pool of T cells, each bearing a unique TCR. Following maturation in the thymus, naive T cells enter the periphery where they must survive for future encounters with Ag. Apart from the contribution of thymic emigrants, the number of mature T cells is tightly regulated through cell survival, proliferation, and apoptosis. This process, known as T cell homeostasis, is largely controlled by two cytokines, IL-7 and IL-15, belonging to the common (c)2 -chain-dependent cytokine family that also includes IL-2, IL-4, IL-9, and IL-21 (1, 2, 3).
For CD8+ T cells, it has been shown that the maintenance of the naive T cell population in the periphery is mainly dependent on IL-7 concurrently with signals from low affinity interactions between TCR and self-peptide-MHC (4, 5, 6). In addition, recent studies performed both in humans and mice suggest that IL-15 also contributes to the homeostasis of naive CD8+ T cells (7, 8, 9, 10, 11, 12). Although both cytokines induce proliferation in an Ag-independent manner, IL-15 drives naive CD8+ T cells to differentiate into effector-type cells, whereas IL-7 preserves their naive characteristics (11, 12, 13, 14).
IL-21 is a newly identified member of the c-chain family produced by activated CD4+ T cells. Its effects are mediated through a class I cytokine family receptor, IL-21R, which associates with the common cytokine receptor chain for intracellular signaling transduction. The IL-21R is widely expressed in lymphoid tissues and shows homology with the -chain of IL-2/15R (15, 16, 17). It has been demonstrated that IL-21 has pleiotropic effects, influencing both humoral and cell-mediated immune responses by regulating dendritic cell (DC), NK cell, B and T cell function (15, 18, 19, 20, 21).
IL-21 is a four helix-bundle cytokine structural related with IL-15 (15). It has been shown in mice that IL-21 inhibits IL-15-mediated expansion of NK cells and memory CD8+ T cells (22). IL-21 is produced specifically by CD4+ T cells following immune activation in an environmental context where IL-15 is likely being expressed (23, 24). Thus, is likely that T and NK cells are exposed to both cytokines simultaneously in a physiological setting. In this study, we analyze whether IL-21 modulates IL-15-induced homeostatic proliferation in human naive CD8+ T cells. We show that IL-21 moderately augments IL-15-driven expansion of naive CD8+ T cells. Noticeably, IL-21 prevents down-modulation of CD28 and enhances IFN- production in naive CD8+ T cells that undergo IL-15-mediated proliferation.
Results
Distinct responsiveness of naive CD8+ T cells to IL-7, IL-15, and IL-21
IL-15 and IL-7 are two key cytokines involved in the Ag-independent proliferation of human naive CD8+ T cells (10, 11, 12, 13). A previous study showed that the CD4+ T cell-derived cytokine IL-21 inhibits the IL-15-mediated proliferation of mouse memory CD8+ T cells (22). We considered examining IL-21 effects on cytokine-driven expansion of human naive CD8+ T cells. We started by analyzing the responsiveness of adult total CD8+ T cells to the c-dependent cytokines, IL-2, IL-7, IL-15, and IL-21. The proliferative response was measured by monitoring cell division of CFSE-labeled PBMCs cultured for 7 days in the presence of graded concentrations of cytokines. As depicted in Fig. 1A, IL-21 did not induce Ag-independent division of CD8+ T cells at any of the concentrations tested. In contrast, IL-7 and IL-15 were able to induce a clear response in CD8+ T cells, IL-7 being a more potent mitogenic stimulus. CD8+ T cells were unresponsive to IL-2 although a clear response was observed at the higher concentration in CD4+ T cells (divided cells underwent six to seven divisions, precursor frequency 4–10%) (data not shown). We next examined whether IL-21 influenced IL-15-mediated expansion of CD8+ T cells, as reported previously (22). PBMCs were labeled with CFSE and cultured with an optimal concentration of IL-15 (10 ng/ml) alone or in presence of a range concentration of IL-21. In contrast with the report by Kasaian et al. (22), IL-21 enhanced the IL-15-dependent expansion of total CD8+ T cells in a dose-dependent manner (Fig. 1, B and C). Having established that IL-21 on its own did not induce expansion but increased IL-15-mediated expansion of total adult CD8+ T cells, we determined the effect of IL-21 on cytokine-driven expansion of human naive CD8+ T cells. The proliferative response was measured by monitoring cell division of CFSE-labeled cord blood-derived naive CD8+ T cells cultured in the presence of IL-7, IL-15, and IL-21 alone or combining two cytokines. IL-21 did not induce Ag-independent proliferation of naive CD8+ T cells. Moreover, IL-21 had no effect on T cell survival as a similar reduction in T cell cellularity was observed in cells cultured in medium (Fig. 2). In contrast, IL-7 stimulated the majority of naive CD8+ T cells to enter cell cycle, with 86% of responding cells. IL-15 revealed a less potent stimulus compared with IL-7, with 40% of the cells initiating cell cycling. However, IL-15-responding cells underwent more rounds of cell divisions than those cultured in IL-7. IL-7-mediated proliferation was not influenced by IL-21. In contrast, IL-21 moderately increased IL-15-mediated expansion with a slight augmentation in the precursor frequency and in the cell yield (Fig. 2). Overall, our data show that CD8+ T cells display distinct responsiveness to IL-7 and IL-15. Moreover, IL-21 slightly affects the Ag-independent expansion of human naive CD8+ T cells induced by IL-15.
We have previously shown that IL-15-activated naive CD8+ T cells acquire the ability to produce IFN-, express key cytotoxic molecules such as perforin and granzyme B, and acquire cytotoxic potential (12). It has been reported that in murine T cells IL-21 increases IL-15-induced IFN- production (22, 31). To further address the IL-21 effects on the functionality of IL-15-activated naive CD8+ T cells, we measured the production of IFN- following combined TCR/CD3 and CD28 stimulation (Fig. 7A, lower panel). We found that IL-21 significantly augmented IFN- production of IL-15-activated T cells, independently of CD28 signaling. Moreover, in concordance with the results obtained with culture supernatants following combined TCR/CD28 stimulation, an increased number of IL-2 and IFN--producing cells was observed in IL-15/IL-21-activated cells following PMA/ionophore/CD28 stimulation (Fig. 7B). Further, activation with IL-15 plus IL-21 augmented IL-2 and IFN- production on a per-cell basis (Fig. 7B). Analyses on the expression of key cytotoxic effector molecules revealed that IL-21 had no effect on IL-15-induced up-regulation of perforin and granzyme B (Fig. 7C). In agreement with this finding we found no differences when IL-15 and IL-15/IL-21-activated cells were compared in redirected cytotoxicity assays (data not shown).
Our data show that IL-21 preserves CD28 responsiveness of naive CD8+ T cells that have undergone IL-15-induced proliferation
Discussion
Previous studies have shown that IL-21, a recently identified cytokine produced by activated CD4+ Th cells, affects both humoral and cell-mediated immune responses (23). IL-21 promotes the full maturation of NK cells and enhances effector function of NK and memory CD8+ T cells (19, 21, 22, 31, 32, 33). Interestingly, clear interspecies differences exist with respect to the biological effect of IL-21. In particular, in contrast to what has been observed in humans, IL-15-mediated expansion of murine NK cells is inhibited by IL-21 (15, 22). A similar inhibitory effect was seen on the proliferation of IL-15-activated murine memory CD8+ T cells (22). Herein, we examined the effects of IL-21 on IL-15-induced expansion and differentiation of human naive CD8+ T cells. We demonstrated that IL-21 slightly increased IL-15-mediated expansion. However, IL-21 prevented IL-15-induced CD28 down-regulation with a concomitant maintenance of CD28 functionality. Moreover, IL-21 augmented the IL-15-induced IFN- production in naive CD8+ T cells.
IL-21 has been shown to enhance the proliferation of Ag-driven naive and memory T cells (22, 34, 35). However, in the absence of TCR activation, IL-21 is not able to induce proliferation, in contrast to other c chain signaling cytokines such as IL-2, IL-7, and IL-15 (10, 12, 13, 15). Consistently, we observed that IL-21 had no effect on the division of human naive CD8+ T cells. We found no inhibitory effect of IL-21 on IL-15-driven expansion of naive CD8+ T cells (Fig. 2) whereas others have reported that IL-15-induced proliferation of murine memory cells is lowered in the presence of IL-21 (22). A possible explanation to reconcile both observations is that effects of IL-21 might depend on the activation or differentiation state of the T cell (23). Alternatively, discrepancies could result from differences between human and mouse. However, in concordance with our data, a recent article by Zeng et al. (33) has shown that IL-21 synergies with IL-15 promoting the expansion of both naive and memory murine CD8+ T cells. As suggested in the latter report, differences in the concentration of IL-21 used in the course of the experiments by Kasaian et al. (22) may explain the discrepancies observed.
Previous studies have shown that IL-21 regulates phenotypic changes in the later stage of NK cell maturation (15, 19). Yet, despite the numerous reports on the role of IL-21 on the function of T cells (21, 22, 31, 32, 33, 35, 36), a modulatory effect of IL-21 on the cell surface phenotype of human T cells has not been ascertained hitherto. Our results demonstrated that on naive CD8+ T cells, IL-21 prevented both the IL-15-induced down-regulation of CD28 and the up-regulation of its ligand, CD80 (Figs. 4, 5, and 6B). It has been reported that IL-21 inhibits DC maturation, blocking the LPS-induced up-regulation of the CD28 ligands, CD80 and CD86 (18), suggestive of a similar mechanism between T cells and DCs. Strikingly, IL-15-induced CD28 down-regulation was still observed, both at the transcriptional and protein level, when the interaction of CD28 with its ligands was blocked (Fig. 6, C and D). Thus, we would favor the hypothesis that IL-15 suppresses the transcription of the CD28 gene independent of receptor-ligand interaction. Several observations have shown that CD28 expression can be down-modulated by cytokines such as IL-15 and TNF- on CD8+ and CD4+ T cells, respectively (12, 14, 37). TNF- represses the transcriptional activity of the CD28 promoter by inhibiting the binding of nuclear complexes that recognize two regulatory sequence motifs, site and , within the CD28 promoter (37). It would be interesting to investigate whether IL-15-induced CD28 down-regulation occurs through a similar mechanism. It has been shown that IL-4 prevents the loss of CD28 expression in long-term TCR-activated CD8+ T cells (38). IL-4 and IL-21 belong to the c-chain-dependent cytokine family and bind to overlapping epitopes on the c chain (39). Indeed, we found that, in contrast to IL-2 and IL-7 (Fig. 5), IL-15-induced down-regulation of CD28 was not only blocked by IL-21, but also by IL-4 (our unpublished data).
IL-21 has been reported to be produced by activated CD4+ T cells, predominantly in Th2 cells. Moreover, it has been shown that IL-21 inhibits the production of IFN- in naive Th cell precursors (40). In contrast to what has been reported for CD4+ T cells, IL-21 augments both IFN- production and cytolytic function of CD8+ T cells (21, 22, 31, 36). We have previously shown that IL-15 induces IFN- production in naive CD8+ T cells upon secondary stimulation (12). In concordance with previous studies (22, 31, 33), our results showed that addition of IL-21 enhanced IL-15-induced IFN- production in T cells. A recent paper showed that granzyme B is preferentially induced in murine naive CD8+ T cells following short-time activation with IL-15 and IL-21 (33). Differently, in our experiments IL-21 had little effect on other functional properties induced by IL-15, such as the expression of perforin and granzyme B (Fig. 7). Rather, our results suggest that IL-21 contributes to enhance particular IL-15-induced effector cytokines in naive CD8+ T cells. Importantly, IL-21 preserves the function of CD28, a costimulatory molecule of paramount importance for the establishment of an effective T cell-mediated immune response. IL-21 has been primarily described as a T cell costimulatory cytokine enhancing TCR-mediated proliferation and function of CD8+ T cells (15, 22, 22, 33). We would consider an analysis of the influence of IL-21 on the regulation of CD28 during Ag-dependent immune responses of great interest.
It is relevant to note that the biological effects induced by IL-15 and IL-21 may depend on their concurrent availability within the lymphoid microenvironment. Although they are produced by distinct cell types, parallel mechanisms might regulate the level of both cytokines. IL-15 is produced by activated monocytes/macrophages, DCs, and epithelium mainly under stress conditions (e.g., infection) whereas IL-21 is produced specifically by CD4+ T cells following immune activation (23, 24). Thus, we would envision that both cytokines can be present simultaneously in sites of infection.
In summary, we demonstrated that IL-21, a CD4+ Th-derived cytokine, exerts specific immunoregulatory properties in naive CD8+ T cells that have undergone IL-15-driven homeostatic proliferation. Importantly, IL-21 preserves the ability of naive CD8+ T cells to respond to costimulatory ligands on future encounters with activated APCs expressing the cognate Ag. In addition, IL-21 can potentiate specific effector T cell functions, such as IFN- production, induced by IL-15. Hence, IL-21 may serve as an immunomodulatory cytokine in IL-15-mediated activation of human naive CD8+ T cells.
Acknowledgments
We thank Drs. Eric Eldering, Ester M. M. van Leeuwen, and Robert H. Hoek for critical reading of the manuscript. We thank Si-La Yong for all technical support.
Disclosures
The authors have no financial conflict of interest.
Footnotes
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. Nuno L. Alves, Laboratory for Experimental Immunology, Academic Medical Center, G1-133, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail address: N.M.LagesAlves@amc.uva.nl
2 Abbreviations used in this paper: c, common ; DC, dendritic cell.
Received for publication February 8, 2005. Accepted for publication May 6, 2005.
References
Fry, T. J., C. L. Mackall. 2001. Interleukin-7: master regulator of peripheral T-cell homeostasis?. Trends Immunol. 22: 564-571.
Schluns, K. S., L. Lefrancois. 2003. Cytokine control of memory T-cell development and survival. Nat. Rev. Immunol. 3: 269-279.
Marrack, P., J. Kappler. 2004. Control of T cell viability. Annu. Rev. Immunol. 22: 765-787.
Schluns, K. S., W. C. Kieper, S. C. Jameson, L. Lefrancois. 2000. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat. Immunol. 1: 426-432.
Tan, J. T., E. Dudl, E. LeRoy, R. Murray, J. Sprent, K. I. Weinberg, C. D. Surh. 2001. IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc. Natl. Acad. Sci. USA 98: 8732-8737
Goldrath, A. W., P. V. Sivakumar, M. Glaccum, M. K. Kennedy, M. J. Bevan, C. Benoist, D. Mathis, E. A. Butz. 2002. Cytokine requirements for acute and basal homeostatic proliferation of naive and memory CD8+ T cells. J. Exp. Med. 195: 1515-1522.
Lodolce, J. P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9: 669-676
Kennedy, M. K., M. Glaccum, S. N. Brown, E. A. Butz, J. L. Viney, M. Embers, N. Matsuki, K. Charrier, L. Sedger, C. R. Willis, et al 2000. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J. Exp. Med. 191: 771-780.(Nuno L. Alves1,*,, Fernan)
Human naive CD8+ T cells are able to respond in an Ag-independent manner to IL-7 and IL-15. Whereas IL-7 largely maintains CD8+ T cells in a naive phenotype, IL-15 drives these cells to an effector phenotype characterized, among other features, by down-regulation of the costimulatory molecule CD28. We evaluated the influence of the CD4+ Th cell-derived common -chain cytokine IL-21 on cytokine-induced naive CD8+ T cell activation. Stimulation with IL-21 did not induce division and only slightly increased IL-15-induced proliferation of naive CD8+ T cells. Strikingly, however, IL-15-induced down-modulation of CD28 was completely prevented by IL-21 at the protein and transcriptional level. Subsequent stimulation via combined TCR/CD3 and CD28 triggering led to a markedly higher production of IL-2 and IFN- in IL-15/IL-21-stimulated cells compared with IL-15-stimulated T cells. Our data show that IL-21 modulates the phenotype of naive CD8+ T cells that have undergone IL-15 induced homeostatic proliferation and preserves their responsiveness to CD28 ligands.
Introduction
Immunological protection to pathogens relies on the formation and maintenance of a diverse pool of T cells, each bearing a unique TCR. Following maturation in the thymus, naive T cells enter the periphery where they must survive for future encounters with Ag. Apart from the contribution of thymic emigrants, the number of mature T cells is tightly regulated through cell survival, proliferation, and apoptosis. This process, known as T cell homeostasis, is largely controlled by two cytokines, IL-7 and IL-15, belonging to the common (c)2 -chain-dependent cytokine family that also includes IL-2, IL-4, IL-9, and IL-21 (1, 2, 3).
For CD8+ T cells, it has been shown that the maintenance of the naive T cell population in the periphery is mainly dependent on IL-7 concurrently with signals from low affinity interactions between TCR and self-peptide-MHC (4, 5, 6). In addition, recent studies performed both in humans and mice suggest that IL-15 also contributes to the homeostasis of naive CD8+ T cells (7, 8, 9, 10, 11, 12). Although both cytokines induce proliferation in an Ag-independent manner, IL-15 drives naive CD8+ T cells to differentiate into effector-type cells, whereas IL-7 preserves their naive characteristics (11, 12, 13, 14).
IL-21 is a newly identified member of the c-chain family produced by activated CD4+ T cells. Its effects are mediated through a class I cytokine family receptor, IL-21R, which associates with the common cytokine receptor chain for intracellular signaling transduction. The IL-21R is widely expressed in lymphoid tissues and shows homology with the -chain of IL-2/15R (15, 16, 17). It has been demonstrated that IL-21 has pleiotropic effects, influencing both humoral and cell-mediated immune responses by regulating dendritic cell (DC), NK cell, B and T cell function (15, 18, 19, 20, 21).
IL-21 is a four helix-bundle cytokine structural related with IL-15 (15). It has been shown in mice that IL-21 inhibits IL-15-mediated expansion of NK cells and memory CD8+ T cells (22). IL-21 is produced specifically by CD4+ T cells following immune activation in an environmental context where IL-15 is likely being expressed (23, 24). Thus, is likely that T and NK cells are exposed to both cytokines simultaneously in a physiological setting. In this study, we analyze whether IL-21 modulates IL-15-induced homeostatic proliferation in human naive CD8+ T cells. We show that IL-21 moderately augments IL-15-driven expansion of naive CD8+ T cells. Noticeably, IL-21 prevents down-modulation of CD28 and enhances IFN- production in naive CD8+ T cells that undergo IL-15-mediated proliferation.
Results
Distinct responsiveness of naive CD8+ T cells to IL-7, IL-15, and IL-21
IL-15 and IL-7 are two key cytokines involved in the Ag-independent proliferation of human naive CD8+ T cells (10, 11, 12, 13). A previous study showed that the CD4+ T cell-derived cytokine IL-21 inhibits the IL-15-mediated proliferation of mouse memory CD8+ T cells (22). We considered examining IL-21 effects on cytokine-driven expansion of human naive CD8+ T cells. We started by analyzing the responsiveness of adult total CD8+ T cells to the c-dependent cytokines, IL-2, IL-7, IL-15, and IL-21. The proliferative response was measured by monitoring cell division of CFSE-labeled PBMCs cultured for 7 days in the presence of graded concentrations of cytokines. As depicted in Fig. 1A, IL-21 did not induce Ag-independent division of CD8+ T cells at any of the concentrations tested. In contrast, IL-7 and IL-15 were able to induce a clear response in CD8+ T cells, IL-7 being a more potent mitogenic stimulus. CD8+ T cells were unresponsive to IL-2 although a clear response was observed at the higher concentration in CD4+ T cells (divided cells underwent six to seven divisions, precursor frequency 4–10%) (data not shown). We next examined whether IL-21 influenced IL-15-mediated expansion of CD8+ T cells, as reported previously (22). PBMCs were labeled with CFSE and cultured with an optimal concentration of IL-15 (10 ng/ml) alone or in presence of a range concentration of IL-21. In contrast with the report by Kasaian et al. (22), IL-21 enhanced the IL-15-dependent expansion of total CD8+ T cells in a dose-dependent manner (Fig. 1, B and C). Having established that IL-21 on its own did not induce expansion but increased IL-15-mediated expansion of total adult CD8+ T cells, we determined the effect of IL-21 on cytokine-driven expansion of human naive CD8+ T cells. The proliferative response was measured by monitoring cell division of CFSE-labeled cord blood-derived naive CD8+ T cells cultured in the presence of IL-7, IL-15, and IL-21 alone or combining two cytokines. IL-21 did not induce Ag-independent proliferation of naive CD8+ T cells. Moreover, IL-21 had no effect on T cell survival as a similar reduction in T cell cellularity was observed in cells cultured in medium (Fig. 2). In contrast, IL-7 stimulated the majority of naive CD8+ T cells to enter cell cycle, with 86% of responding cells. IL-15 revealed a less potent stimulus compared with IL-7, with 40% of the cells initiating cell cycling. However, IL-15-responding cells underwent more rounds of cell divisions than those cultured in IL-7. IL-7-mediated proliferation was not influenced by IL-21. In contrast, IL-21 moderately increased IL-15-mediated expansion with a slight augmentation in the precursor frequency and in the cell yield (Fig. 2). Overall, our data show that CD8+ T cells display distinct responsiveness to IL-7 and IL-15. Moreover, IL-21 slightly affects the Ag-independent expansion of human naive CD8+ T cells induced by IL-15.
We have previously shown that IL-15-activated naive CD8+ T cells acquire the ability to produce IFN-, express key cytotoxic molecules such as perforin and granzyme B, and acquire cytotoxic potential (12). It has been reported that in murine T cells IL-21 increases IL-15-induced IFN- production (22, 31). To further address the IL-21 effects on the functionality of IL-15-activated naive CD8+ T cells, we measured the production of IFN- following combined TCR/CD3 and CD28 stimulation (Fig. 7A, lower panel). We found that IL-21 significantly augmented IFN- production of IL-15-activated T cells, independently of CD28 signaling. Moreover, in concordance with the results obtained with culture supernatants following combined TCR/CD28 stimulation, an increased number of IL-2 and IFN--producing cells was observed in IL-15/IL-21-activated cells following PMA/ionophore/CD28 stimulation (Fig. 7B). Further, activation with IL-15 plus IL-21 augmented IL-2 and IFN- production on a per-cell basis (Fig. 7B). Analyses on the expression of key cytotoxic effector molecules revealed that IL-21 had no effect on IL-15-induced up-regulation of perforin and granzyme B (Fig. 7C). In agreement with this finding we found no differences when IL-15 and IL-15/IL-21-activated cells were compared in redirected cytotoxicity assays (data not shown).
Our data show that IL-21 preserves CD28 responsiveness of naive CD8+ T cells that have undergone IL-15-induced proliferation
Discussion
Previous studies have shown that IL-21, a recently identified cytokine produced by activated CD4+ Th cells, affects both humoral and cell-mediated immune responses (23). IL-21 promotes the full maturation of NK cells and enhances effector function of NK and memory CD8+ T cells (19, 21, 22, 31, 32, 33). Interestingly, clear interspecies differences exist with respect to the biological effect of IL-21. In particular, in contrast to what has been observed in humans, IL-15-mediated expansion of murine NK cells is inhibited by IL-21 (15, 22). A similar inhibitory effect was seen on the proliferation of IL-15-activated murine memory CD8+ T cells (22). Herein, we examined the effects of IL-21 on IL-15-induced expansion and differentiation of human naive CD8+ T cells. We demonstrated that IL-21 slightly increased IL-15-mediated expansion. However, IL-21 prevented IL-15-induced CD28 down-regulation with a concomitant maintenance of CD28 functionality. Moreover, IL-21 augmented the IL-15-induced IFN- production in naive CD8+ T cells.
IL-21 has been shown to enhance the proliferation of Ag-driven naive and memory T cells (22, 34, 35). However, in the absence of TCR activation, IL-21 is not able to induce proliferation, in contrast to other c chain signaling cytokines such as IL-2, IL-7, and IL-15 (10, 12, 13, 15). Consistently, we observed that IL-21 had no effect on the division of human naive CD8+ T cells. We found no inhibitory effect of IL-21 on IL-15-driven expansion of naive CD8+ T cells (Fig. 2) whereas others have reported that IL-15-induced proliferation of murine memory cells is lowered in the presence of IL-21 (22). A possible explanation to reconcile both observations is that effects of IL-21 might depend on the activation or differentiation state of the T cell (23). Alternatively, discrepancies could result from differences between human and mouse. However, in concordance with our data, a recent article by Zeng et al. (33) has shown that IL-21 synergies with IL-15 promoting the expansion of both naive and memory murine CD8+ T cells. As suggested in the latter report, differences in the concentration of IL-21 used in the course of the experiments by Kasaian et al. (22) may explain the discrepancies observed.
Previous studies have shown that IL-21 regulates phenotypic changes in the later stage of NK cell maturation (15, 19). Yet, despite the numerous reports on the role of IL-21 on the function of T cells (21, 22, 31, 32, 33, 35, 36), a modulatory effect of IL-21 on the cell surface phenotype of human T cells has not been ascertained hitherto. Our results demonstrated that on naive CD8+ T cells, IL-21 prevented both the IL-15-induced down-regulation of CD28 and the up-regulation of its ligand, CD80 (Figs. 4, 5, and 6B). It has been reported that IL-21 inhibits DC maturation, blocking the LPS-induced up-regulation of the CD28 ligands, CD80 and CD86 (18), suggestive of a similar mechanism between T cells and DCs. Strikingly, IL-15-induced CD28 down-regulation was still observed, both at the transcriptional and protein level, when the interaction of CD28 with its ligands was blocked (Fig. 6, C and D). Thus, we would favor the hypothesis that IL-15 suppresses the transcription of the CD28 gene independent of receptor-ligand interaction. Several observations have shown that CD28 expression can be down-modulated by cytokines such as IL-15 and TNF- on CD8+ and CD4+ T cells, respectively (12, 14, 37). TNF- represses the transcriptional activity of the CD28 promoter by inhibiting the binding of nuclear complexes that recognize two regulatory sequence motifs, site and , within the CD28 promoter (37). It would be interesting to investigate whether IL-15-induced CD28 down-regulation occurs through a similar mechanism. It has been shown that IL-4 prevents the loss of CD28 expression in long-term TCR-activated CD8+ T cells (38). IL-4 and IL-21 belong to the c-chain-dependent cytokine family and bind to overlapping epitopes on the c chain (39). Indeed, we found that, in contrast to IL-2 and IL-7 (Fig. 5), IL-15-induced down-regulation of CD28 was not only blocked by IL-21, but also by IL-4 (our unpublished data).
IL-21 has been reported to be produced by activated CD4+ T cells, predominantly in Th2 cells. Moreover, it has been shown that IL-21 inhibits the production of IFN- in naive Th cell precursors (40). In contrast to what has been reported for CD4+ T cells, IL-21 augments both IFN- production and cytolytic function of CD8+ T cells (21, 22, 31, 36). We have previously shown that IL-15 induces IFN- production in naive CD8+ T cells upon secondary stimulation (12). In concordance with previous studies (22, 31, 33), our results showed that addition of IL-21 enhanced IL-15-induced IFN- production in T cells. A recent paper showed that granzyme B is preferentially induced in murine naive CD8+ T cells following short-time activation with IL-15 and IL-21 (33). Differently, in our experiments IL-21 had little effect on other functional properties induced by IL-15, such as the expression of perforin and granzyme B (Fig. 7). Rather, our results suggest that IL-21 contributes to enhance particular IL-15-induced effector cytokines in naive CD8+ T cells. Importantly, IL-21 preserves the function of CD28, a costimulatory molecule of paramount importance for the establishment of an effective T cell-mediated immune response. IL-21 has been primarily described as a T cell costimulatory cytokine enhancing TCR-mediated proliferation and function of CD8+ T cells (15, 22, 22, 33). We would consider an analysis of the influence of IL-21 on the regulation of CD28 during Ag-dependent immune responses of great interest.
It is relevant to note that the biological effects induced by IL-15 and IL-21 may depend on their concurrent availability within the lymphoid microenvironment. Although they are produced by distinct cell types, parallel mechanisms might regulate the level of both cytokines. IL-15 is produced by activated monocytes/macrophages, DCs, and epithelium mainly under stress conditions (e.g., infection) whereas IL-21 is produced specifically by CD4+ T cells following immune activation (23, 24). Thus, we would envision that both cytokines can be present simultaneously in sites of infection.
In summary, we demonstrated that IL-21, a CD4+ Th-derived cytokine, exerts specific immunoregulatory properties in naive CD8+ T cells that have undergone IL-15-driven homeostatic proliferation. Importantly, IL-21 preserves the ability of naive CD8+ T cells to respond to costimulatory ligands on future encounters with activated APCs expressing the cognate Ag. In addition, IL-21 can potentiate specific effector T cell functions, such as IFN- production, induced by IL-15. Hence, IL-21 may serve as an immunomodulatory cytokine in IL-15-mediated activation of human naive CD8+ T cells.
Acknowledgments
We thank Drs. Eric Eldering, Ester M. M. van Leeuwen, and Robert H. Hoek for critical reading of the manuscript. We thank Si-La Yong for all technical support.
Disclosures
The authors have no financial conflict of interest.
Footnotes
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. Nuno L. Alves, Laboratory for Experimental Immunology, Academic Medical Center, G1-133, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail address: N.M.LagesAlves@amc.uva.nl
2 Abbreviations used in this paper: c, common ; DC, dendritic cell.
Received for publication February 8, 2005. Accepted for publication May 6, 2005.
References
Fry, T. J., C. L. Mackall. 2001. Interleukin-7: master regulator of peripheral T-cell homeostasis?. Trends Immunol. 22: 564-571.
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