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. 2009 Oct 1;87(1):43–49. doi: 10.1189/jlb.0209086

IL-21 preferentially enhances IL-15-mediated homeostatic proliferation of human CD28+ CD8 memory T cells throughout the adult age span

Huy Nguyen 1, Nan-ping Weng 1,1
PMCID: PMC2801617  PMID: 19797296

Abstract

An age-related decline in human immune response is marked by the accumulation of CD28– CD8 T cells, which is attributed to repeated antigenic stimulation and to homeostatic proliferation mediated by cytokines such as IL-15. However, the identity of the cytokines that are responsible for the maintenance of CD28 expression is less known. Here, we report the role of IL-21 in the regulation of IL-15-mediated growth and CD28 expression of CD8 memory T cells of young and old donors. We showed that IL-21 drives more IL-15-stimulated cells to enter cell division and to undergo apoptosis. Furthermore, IL-21 preferentially enhanced IL-15-induced proliferation of CD28+ CD8 memory T cells over their CD28– counterparts, as CD28+ cells expressed higher levels of IL-15R and IL-21R and greater pSTAT5 upon IL-15 and IL-21 stimulation. In addition, IL-21 reduced IL-15-induced CD28 down-regulation in CD8 memory T cells. Finally, the ability of proliferation and survival in response to homeostatic cytokines IL-15 and IL-21 of CD28+ CD8 memory T cells was well-maintained with age. Together, these findings suggest that IL-21 enhances IL-15-mediated proliferation of CD8 memory T cells, particularly CD28+ memory T cells, and also serves as an antagonist to the IL-15-induced increase of CD28– CD8 T cells.

Keywords: cytokines, aging

Introduction

CD8 T cells play an essential role in the adaptive immune response against intracellular pathogens and cancerous growths. The ability of CD8 T cells to protect the host declines with age as a result of a decrease in the number of naïve cells and in the diversity of the TCR repertoire and an increase in the number of dysfunctional memory cells, such as CD28– CD8 T cells [1]. Accumulation of CD28– CD8 T cells presents one of the most consistent changes in human immune cells with aging [2, 3]. This age-associated increase of CD28– CD8 T cells has been attributed to repeated or chronic antigenic stimulation [4]. More recent studies also suggest that homeostatic cytokines such as IL-15 can also induce CD28– CD8 T cells in vitro [5, 6]. How cytokines regulate growth and generation of CD28– CD8 T cells is not fully understood.

As a key costimulatory receptor, CD28 provides signals that are required along with the TCR/MHC interaction for complete activation of T cells and thus, for generation of an effective T cell response to antigen [7]. All newly generated, naïve T cells express CD28, as seen in newborns, and loss of CD28 expression in T cells, particularly often in CD8 T cells, occurs after antigenic activation [3]. As a consequence of cumulative stimulations through life, CD28– CD8 T cells increase progressively with age and become the majority of CD8 T cells in 70- to 80-year-old adults [3]. Although these CD28– CD8 T cells display proliferation defects in response to antigenic stimulation and a reduced diversity of the TCR repertoire [8], they appear to have increased cytotoxic activity and may even have some regulatory effects [2, 9].

Homeostatic cytokines, especially the cytokine-receptor γc family including IL-7 and IL-15, play an essential role in the maintenance of memory T cells [10,11,12]. It has been shown that IL-7 is essential for survival of thymocytes and mature T cells, and IL-15 is essential for the maintenance of CD8 memory T cells. IL-21 is a newly identified member of this cytokine family and is produced mainly by activated CD4 T cells and has pleiotropic effects on humoral and cell-mediated immune responses [13]. IL-21 enhances effector function of NK and CD8 T cells [14], induces apoptosis [15], and is capable of sustaining CD28 expression in IL-15-cultured, CD8 naïve T cells [16]. Recently, we showed that IL-15-induced proliferation of CD8 memory T cells results in loss of CD28 expression, mainly in actively dividing CD8 memory T cells in vitro [6]. However, it is not known what role IL-21 may play in IL-15-mediated homeostatic proliferation and in the regulation of CD28 expression by CD8 memory T cells nor whether the effect of IL-21 on CD8 memory T cells changes with age.

To address these questions, we evaluated the effects of IL-21 on the growth and CD28 expression of IL-15-stimulated CD8 memory T cells from young and old donors. Our results showed that IL-21 preferentially enhances IL-15-stimulated growth of CD28+ CD8 memory T cells over its CD28– counterparts and that IL-21 also reduces the IL-15-mediated down-regulation of CD28 in CD8 memory T cells. Furthermore, the preferential enhancement of IL-15-mediated proliferation results, at least in part, from the higher expression of IL-15R and IL-21R and higher levels of induced pSTAT5 in CD28+ CD8 T cells relative to CD28– CD8 T cells. Finally, the ability of proliferation of CD28+ CD8 memory T cells in response to IL-15 + IL-21 was well-maintained with age.

MATERIALS AND METHODS

Isolation of CD8 T cell subsets from human peripheral blood

Total CD8 T cells, CD8 memory T cells, and CD28– and CD28+ CD8 memory T cells were isolated from peripheral blood of normal volunteers by immunomagnetic separation and by cell sorting as described previously [6]. In brief, blood was obtained from healthy adults (age between 24 and 68 years old), young donors (<30 years old), and aged volunteers (>70 years old) of the NIA Clinical Research Branch (Baltimore, MD, USA) under Institutional Review Board-approved protocols, and PBMC were isolated by Ficoll gradient centrifugation (GE Healthcare, UK). CD8 T cells were then enriched by removing other types of cells in PBMC through incubation with a panel of mouse mAb against CD4, CD19, CD11b, CD14, CD16, MHC class II, erythrocytes, and platelets. Antibody-bound cells were removed subsequently by incubation with anti-mouse, IgG-conjugated magnetic beads (Qiagen, Valencia, CA, USA). CD8 memory T cells were isolated further by the anti-CD8 antibody-conjugated beads (Invitrogen, Carlsbad, CA, USA). A cell sorter (MoFlo, Dako-Cytomation, Denmark) was used for isolating CD28+ and CD28– CD8 memory T cells for specific use, described in Results. The purities of isolated CD8 T cells were >96%.

Analysis of cell surface receptors and apoptosis

Fluorescent dye-labeled antibodies were purchased from commercial sources: anti-CD8 (TRI-COLOR®, Invitrogen) and CD28 (APC, PE-Cy5, BD PharMingen, San Diego, CA, USA). Antibodies against CD8 (fluorescein, FITC, and APC), CD28 (FITC, PE), CD45RA (FITC, PE-Cy5), and CD62 ligand (APC) were from eBioscience (San Diego, CA, USA). Apoptosis analysis was performed using antibodies against Annexin V-PE or FITC and 7-AAD, their isotype and fluorescent dye-matched controls, and anti-CD132 (γc; PE) was purchased from BD Biosciences (San Jose, CA, USA). For surface marker analysis, freshly purified and IL-15/21-treated CD8 subsets were incubated with three or four different fluorescent dye-conjugated antibodies and prepared for FACS analysis, according to manufacturers’ instructions. The staining profiles were acquired by FACScan or FACSCalibur (BD Biosciences) and analyzed by FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Cell proliferation assay

Cell division tracking dye, CFSE (Invitrogen), was used to measure the proliferation of cells as described previously [17]. In brief, CD8 memory T cells and subsets were incubated with 5 μM/ml CFSE for 10 min at 37°C, washed with RPMI 1640 once, and cultured at 1 × 106 cells/ml in RPMI 1640 supplemented with 10% FBS and penicillin (10 U/ml)/streptomycin (10 μg/ml; Invitrogen) in the presence of rhIL-15 (50 ng/ml) and rhIL-21 (10 or 25 ng/ml; PeproTech, Rocky Hill, NJ, USA) on 12-well or 24- or 48-well flat-bottom plates (BD Biosciences), depending on the number of cells available. The proliferative responses of the cultured CD8 T cell subsets were analyzed on Days 7, 14, 21, and 28 by FACScan. The division rate and/or proliferation index were analyzed by FlowJo software (Tree Star, Inc.).

RT-PCR

The procedure of real-time qRT-PCR was described previously [18]. In brief, total RNA was extracted from freshly isolated CD8 T cell subsets or after culture with IL-15 alone or IL-15 plus IL-21 by Stat 60 (Tel-Test), based on manufacturers’ protocol and quantified by Nanodrop (Nanodrop Technologies, Wilmington, DE, USA). cDNA synthesis was conducted using 1 μg total RNA with RT (Super-Script II or III, Invitrogen), based on the manufacturer’s instructions. The levels of mRNA of each gene were determined by real-time qPCR in a 20-μl vol with 0.1 μM primers using a SyBr Green kit on ABI Prism 7300 (Applied Biosystems, Foster City, CA, USA). The specificity of amplified RT-PCR products was confirmed by identification of a single and correct size band on an agarose (2.5%) gel after electrophoresis and/or a single peak on a dissociation curve of the PCR product. The primers were made from Integrated DNA Technologies, and their sequences were listed below: CD28 (forward: 5′-AGGCTGCTCTTGGCTCTCAACT-3′ and backward: 5′-ACCGCATTGTCGTACGCTACA-3′), IL15RA (forward: 5′-GAGCCTCTCCCCTTCTGGAA-3′ and backward: 5′-GGCCGCTGTGTTGTTTGAG-3′), IL21R (forward: 5′-TGTGGAGGCTATGGAAGAAGATATG-3′ and backward: 5′-GTGCACCCACCCATTTCTTG-3′), IL2RB (forward: 5′-TCATCATCTTAGTGTACTTGCTGATCA-3′ and backward: 5′-GGTGTTACACTTCAGGACCTTCTTC-3′), IL2RG (forward: 5′-GGAGCAATACTTCAAAAGAGAATCCT-3′ and backward: 5′-CCCATGGAGCCAACAGAGAT-3′), ACTB (forward: 5′-CCTGGCACCCAGCACAA-3′ and backward: 5′-GCCGATCCACACGGAGTACT-3′). The relative mRNA levels of specific genes were calculated by normalizing to the comparative threshold value of ACTB.

Analysis of pSTAT5 by flow cytometry

The procedure was followed according to the manufacturer’s instruction. Isolated PBMCs or CD8 memory T cells were cultured with IL-15 or with IL-15 plus IL-21 for 10 min, 30 min, and 7 days, followed by washing with Hanks’ buffer immediately. Cells were then stained for surface markers with anti-CD8 (-FITC or -TC) and anti-CD28 (-FITC, -PE, or -TC) for CD8 memory T cells and anti-CD45RA (-FITC or -PE) for PBMCs for 30 min on ice, followed by fixation in 2% paraformaldehyde for 10 min at room temperature and permeabilization in 90% methanol for 30 min on ice or overnight at –20°C. Cells were stained with anti-pSTAT3 (Y705)-PE for 30 min or with anti-pSTAT5 (Y694)-Alexa Fluor 488 for 1 h at room temperature and analyzed on a FACScan. Anti-pSTAT5 (Y694)-Alexa Fluor 488 was from BD Biosciences.

Statistical analysis

The differences of biological parameters among CD8 T cell subsets were analyzed by a two-tailed Student’s t-test. P < 0.05 were considered as significant.

RESULTS

IL-21 enhances IL-15-mediated proliferation of CD8 memory T cells in vitro

IL-21 is known to enhance proliferation of T cells that are activated by TCR engagement or homeostatic cytokines [13]. To determine whether IL-21 affects CD8 memory T cell proliferation induced by IL-15, we isolated CD8 memory T cells from human peripheral blood and cultured them with IL-15 alone or with IL-21 at different concentrations in vitro. There was no proliferation of resting CD8 memory T cells in the presence of IL-21 alone (Fig. 1A). However, CD8 memory T cells had a significantly greater increase in cell number with IL-21 + IL-15 than with IL-15 alone in all tested concentrations (Fig. 1A). We then analyzed IL-21 effects in more detail and found that the increase of proliferating memory cells in response to IL-21 occurred from Day 7 to Day 14, and no further increase in cell numbers occurred between Day 14 and Day 28 (Fig. 1B).

Figure 1.

Figure 1.

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IL-21 enhances IL-15-mediated CD8 memory T cell growth in a dose-dependent manner. (A) Growth of CD8 memory T cells cultured with IL-15 and IL-21 alone or in combination. CD8 memory T cells isolated from PBMCs of normal adult donors were cultured with IL-15 and IL-21 at concentrations indicated (ng/mL). Live cell numbers were counted by a standard trypan blue staining method after 7 days, and cell expansion (Fold Change) was calculated by dividing the live cell number at Day 7 over the initial seeding cell number at Day 0. Values represent the mean value ± sem obtained from independent experiments of five to 15 different donors. (B) Growth curves of CD8 memory T cells stimulated with IL-15 alone or IL-15 + IL-21. CD8 memory T cells isolated from PBMCs were stimulated with IL-15 alone or with either two concentrations of IL-21. Cells were counted at days indicated, and cell expansion (Fold Change) was calculated. Values represent the mean value ± sem obtained from independent experiments of nine different donors. Differences in cell count between IL-15 alone and IL-15 + IL-21 (low) have P< 0.05 for Days 7 and 28 and P < 0.01 for Days 14 and 21.

IL-21 induces IL-15-cultured CD8 memory T cells to enter cell division and undergo apoptosis through enhancing the signals of the STAT3 and STAT5 pathways

IL-15 and IL-21 involve the JAK/STAT signaling pathway. IL-15 signals through STAT5, and IL-21 signals mainly through STAT1 and STAT3 (and to a lesser extent, STAT5) [13]. To examine the mechanism of IL-21 enhancement of IL-15-mediated proliferation of CD8 memory T cells, we analyzed pSTAT3 and pSTAT5. Similar levels of pSTAT5 were found in IL-15- or IL-15 + IL-21-treated CD8 memory T cells, whereas significantly higher levels of pSTAT3 were found in IL-15 + IL-21- than in IL-15-treated CD8 memory T cells (Fig. 2A). This suggests that the effect of IL-21 on IL-15-mediated proliferation of CD8 memory T cells is not a simple additive effect to IL-15 signaling (pSTAT5) but rather through a distinct signal (pSTAT3) of IL-21. To investigate whether the increased numbers of CD8 memory T cells in the presence of IL-21 were a result of an increase in cell division and/or a decrease in apoptosis, we analyzed the cell division profiles of CD8 memory T cells in the presence or absence of IL-21 using a cell division tracking dye, CFSE. At Day 7 of culture, significantly more cells underwent cell divisions (Fig. 2B) in the presence of IL-15 + IL-21 than in the presence of IL-15 alone. Interestingly, although there was no significant difference in apoptosis in the presence or absence of IL-21 at Day 7 of culture, a significant increase of apoptotic cells was observed at Day 14 and Day 21 of culture in the presence of IL-21 (Fig. 3). Thus, IL-21 increased IL-15-mediated homeostatic proliferation of CD8 memory T cells at an early expansion stage and induces more apoptosis at the late stage of culture.

Figure 2.

Figure 2.

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IL-21 induces pSTAT3 and promotes more CD8 memory T cells to enter cell division. (A) pSTAT3 and pSTAT5 in CD8 memory T cells upon stimulation with IL-15 (50 ng/mL) in the absence or presence of IL-21 (25 ng/mL). pSTAT3 and pSTAT5 were determined by specific antibodies against pSTAT3 and pSTAT5 and represented as the MFI. Values represent the average MFI ± sem, obtained from experiments for cells isolated from eight adult donors; **, P < 0.01; ***, P < 0.001. (B) Cell division profiles of CD8 memory T cells cultured with IL-15 alone or IL-15 + IL-21. Isolated CD8 memory T cells were labeled with CFSE and cultured with IL-15 alone or with IL-15 + IL-21. Representative histograms of CFSE profiles of cultured CD8 memory T cells from one of eight adult donors are presented. Values represent the mean percentage of undivided cells of eight donors with P = 0.0002.

Figure 3.

Figure 3.

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IL-21 induces more cell death at a late stage of IL-15-mediated culture. Isolated CD8 memory T cells were cultured with IL-15 (50 ng/ml) alone or with IL-15 (50 ng/ml) plus IL-21 (25 ng/ml). A representative dot-plot of cells stained for Annexin-V (ANXA-V) and 7-AAD after culture from one of six different adult donors presented. Values represent the mean percentage, P≤ 0.05 for Days 14 and 21.

IL-21 selectively enhances proliferation of CD28+ CD8 memory T cells

It has been reported that IL-21 prevents loss of CD28 expression in IL-15-cultured, naïve CD8 T cells [19]. To investigate whether IL-21 also prevents the loss of CD28 expression in CD8 memory T cells, we analyzed the number of CD28+ and CD28– CD8 memory T cells during IL-15 culture. No significant changes in the total number of CD28– CD8 memory T cells were observed in the presence or absence of IL-21 (Fig. 4A). In contrast, a significant increase of CD28+ CD8 memory T cells was observed in the presence of IL-21 (Fig. 4A). This suggested that IL-21 may preferentially enhance proliferation of CD28+ over CD28– CD8 memory T cells. As the purified CD8 memory T cells from normal adult donors contain various percentages of CD28– cells that may complicate their proliferative response, we therefore isolated CD28+ and CD28– CD8 memory T cells by cell sort and cultured them separately. Again, no obvious difference in number for CD28– cells was found in the presence or absence of IL-21, but a significant increase of CD28+ cells was observed in IL-15 + IL-21 as compared with IL-15 alone (Fig. 4B).

Figure 4.

Figure 4.

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IL-21 preferentially enhances IL-15-mediated growth of CD28+ CD8 memory T cells. (A) Growth curves of CD28+ and CD28– CD8 memory T cells stimulated with IL-15 alone or IL-15 + IL-21. Total CD8 memory T cells were isolated and cultured. The live cell numbers were determined by trypan blue staining under a regular light microscope. CD28+ and CD28– CD8 memory T cells in the culture were determined by FACS analysis, and the values were then calculated and represent the mean ± sem obtained from results of nine adult donors. Differences in cell count for CD28+ cells under each condition have P< 0.05 for Day 7 and P < 0.01 for Days 14, 21, and 28. (B) Comparison of growth of separately cultured CD28+ and CD28– CD8 memory T cells. CD28+ and CD28– CD8 memory T cells were isolated from total CD8 memory T cells by cell sort and cultured separately with IL-15 alone or with IL-21. At the indicated days, the cells were harvested and counted. Values represent the mean value ± sem (n = 13 for Day 7; n = 5 for Day 14); *, P < 0.05; ***, P < 0.001.

To investigate the mechanism of the differential response of CD28+ and CD28– CD8 memory T cells to IL-21, we compared the expressions of cytokine receptors and their signaling pathways in response to IL-15 stimulation, with or without IL-21. IL21RA and IL15RA (mRNA) were expressed significantly higher in freshly isolated CD28+ T cells than in CD28– CD8 T cells, but there were no significant differences in expression of IL2RB and IL2RG between these two subsets (Fig. 5A). After culture, the mRNA levels of all receptors were down-regulated to similar levels in CD28+ and CD28– cells, regardless of the presence or absence of IL-21 (data not shown). Interestingly, although the mRNA levels were similar, a higher protein level of the γc was found on the surface of CD28+ cells than on the CD28– counterparts before and after stimulation (Fig. 5B). Furthermore, a higher surface expression of γc in both subsets was found in the presence of IL-21 than in the absence of it (Fig. 5B). To determine the consequence of the different levels of receptor expression, we examined levels of pSTAT5 and pSTAT3, which are involved in signaling through these receptors. Higher levels of pSTAT5 were found in CD28+ than in CD28– cells upon stimulation with IL-15 alone and IL-15 in combination with IL-21 (MFI were 55 ± 7.5 and 38 ± 4 for CD28+ and CD28– cells after 10 min of IL-15 exposure; n = 7; P = 0.003; Fig. 5C). Higher levels of pSTAT3 were also found in CD28+ cells over their CD28– counterpart but to a lesser extent (data not shown). Together, these findings provide a potential mechanism for the preferential expansion of CD28+ CD8 memory T cells in response to IL-15 and IL-21 stimulation.

Figure 5.

Figure 5.

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CD28+ CD8 memory T cells express higher levels of IL-15R and IL-21R and exhibit stronger STAT5 signaling than their CD28– counterparts. (A) CD28+ and CD28– CD8 memory (CD45RA–) T cells were isolated by cell sort and cultured under IL-15 alone or with IL-21. (A) The mRNA levels of the receptors (IL15RA, IL21R, IL2RB, and IL2RG) in freshly isolated CD28+ and CD28– CD8 memory T cells were determined by qRT-PCR and normalized to ACTB. Values represent the mean value ± sem obtained from results of one of six different human donors. (B) Surface protein expression of CD132 (Common γc) in CD28+ and CD28– CD8 memory T cells. Values represent the average MFI ± sem obtained from experiments for cells isolated from five different human donors for Day 0 and 14 donors for Day 7. (C) pSTAT5 in CD28+ and CD28– CD8 memory T cells after IL-15 and IL-21 exposure. pSTAT5 was determined using the MFI of fluorescent-labeled antibodies against pSTAT5 molecules and normalized to unstimulated cells. Values represent the average MFI ± sem obtained from experiments for cells isolated from eight different adult donors at 10 and 30 min after treatment. Throughout the figure: *, P < 0.05; ***, P< 0.001.

IL-21 reduces IL-15-induced CD28 down-regulation in CD8 memory T cells

We have observed that loss of CD28 expression in IL-15 cultured CD8 memory T cells, particularly in those most rapidly dividing cells [6], and it is also known that IL-21 can sustain CD28 expression in CD8 naïve T cells [16]. To determine whether IL-21 is able to sustain CD28 expression in rapidly dividing CD28+ CD8 memory T cells, we isolated CD28+ CD8 T cells and cultured them with IL-15 in the presence or absence of IL-21. After 14 days of culture, fewer CD28– cells were generated among the extensively dividing cells (more than five cell divisions) in the presence of IL-21 than in the absence of IL-21 (Fig. 6A), and the ratios of CD28+:CD28– cells were approximately threefold higher after culture with IL-21 than without IL-21 (Fig. 6B). Furthermore, CD28 expression in CD8 memory T cells was higher in the presence of IL-21 than in the absence of IL-21 at the surface protein (Fig. 6C) and mRNA levels (Fig. 6D). These findings suggested that IL-21 is capable of reducing the loss of CD28 expression in IL-15-induced, actively dividing CD8 memory T cells.

Figure 6.

Figure 6.

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IL-21 reduces IL-15-induced down-regulation of CD28 expression in CD8 memory T cells. (A) Analysis of cell division and CD28 expression in cultured CD8 memory T cells under IL-15 alone or IL-15 + IL-21. Isolated CD8 memory T cells were labeled with CFSE and cultured with IL-15 alone or with IL-15 + IL-21 for 14 days. CFSE and CD28 profiles were collected using flow cytometry and analyzed by FlowJo. A representative dot-plot of the CD28 versus CFSE profile was presented from four different donors. Dotted lines indicate cell division. (B) Quantitative presentation of the ratio of CD28+ over CD28– in fast-dividing (≥5 cell divisions) and slow/no-dividing (0–4 cell divisions). (C) MFI of the surface CD28 expression for cells that have undergone little (0–4) or extensive (5 or more) divisions. Values represent the mean value ± sem obtained from experiments for cells isolated from four different human donors. (D) CD28 mRNA levels of CD8 memory T cells cultured with IL-15 alone or IL-15 + IL-21. The data were derived from qRT-PCR analysis and normalized to ACTB and normalized further to a Day 0 value. Values represent the mean value ± sem obtained from experiments for cells isolated from six different human donors for Days 0 and 7 or five donors for Day 14. C and D use the same legend as in B, the hatched bar in D represents freshly isolated cells with no stimulation. Throughout the figure: *, P< 0.05; **, P < 0.01: †, P < 0.06.

IL-15- and/or IL-21-induced proliferation of CD28+ CD8 memory T cells is comparable between young and old donors

Homeostatic proliferation is essential for the maintenance of memory T cells and becomes increasingly important with age, particularly in older individuals, when thymic output is decreased significantly. To understand whether the ability of IL-21 to enhance IL-15-mediated proliferation changes with aging, we isolated CD28+ CD8 memory T cells from young (age ≤30; n = 6) and old (age ≥70; n = 4) adults and compared their growth response to IL-15 in the presence or absence of IL-21 for 14 days. CD8 memory T cells (CD45RA–CD28+) had similar rates of growth between young and old donors, regardless of the presence or absence of IL-21 (Fig. 7). This finding suggests that the homeostatic cytokine-induced proliferation of CD28+ CD8 memory T cells is well-maintained throughout the adult life.

Figure 7.

Figure 7.

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IL-15- and IL-21-induced proliferation of CD28+ CD8 memory T cells is stable through adult age. CD28+ CD8 memory T cells were isolated from old donors (age ≥70; n = 4) and young donors (age ≤30; n = 6) by cell sort and cultured with IL-15 (50 ng/mL) alone or with IL-21 (25 ng/mL) for 14 days. Live cell numbers were counted with trypan blue staining, and cell expansion (Fold Change) was calculated by dividing the cell count after culture over the initial seeding cell count. Values represent mean ± sem.

DISCUSSION

Consistent with the previous reports that IL-21 enhances the antigen-induced proliferation of naive and memory T cells [14], our findings showed that IL-21 enhances IL-15-mediated growth of CD8 memory T cells. Interestingly, we observed that IL-21 selectively enhances proliferation of CD28+ CD8 memory T cells over their CD28– counterparts. Further investigation revealed that these CD28+ CD8 memory T cells express higher levels of IL21R and IL15RA mRNA and higher levels of surface expression of the γc than their CD28– counterparts. Moreover, CD28+ cells delivered much stronger signals upon exposure to IL-15 and IL-21 than did their CD28– counterparts, as demonstrated by the higher levels of pSTAT5. These findings reveal the differences in homeostatic maintenance between CD28+ and CD28– CD8 memory T cells. Whether such differences are limited to the levels of receptor expression and STAT5 signaling or involve additional, specific downstream events requires further study. Considering the critical importance of homeostasis in CD8 T cell function, it is essential to understand how CD28+ and CD28– CD8 memory T cells are maintained by cytokines, particularly the interaction between positive and negative regulating factors, and how age alters the balance of their maintenance.

Cytokines capable of regulating CD28 expression in T cells have been reported previously, including those down-regulators, IL-2, IL-15, and TNF-α [5, 6, 20, 21], and up-regulators, such as IL-12 and IL-21 [16, 22]. Alves et al. [16] show that IL-21 can prevent loss of CD28 expression completely in IL-15-stimulated, naïve CD8 T cells. Our findings here suggest that IL-21 is capable of reducing, but not completely preventing, the loss of CD28 expression in actively dividing CD8 memory T cells under IL-15 stimulation. Whether this difference is a result of the intrinsic difference between naïve and memory CD8 T cells or extrinsic differences such as the length of culture time (7 days of naïve cells vs. 14 days of memory cells) and/or concentrations of cytokines used in our system is unknown currently. Our preliminary results suggest that maintenance of CD28 by IL-21 is more effective in naïve CD8 T cells than in memory CD8 T cells. However, further studies will be needed to clarify this and more importantly, to understand the mechanism of sustaining CD28 expression by IL-21.

The effects of IL-21 on CD28+ CD8 T cells reported here show that IL-21 has an ability not only to enhance the proliferation preferentially but also to maintain CD28 expression of CD28+ CD8 memory T cells. It is, therefore, not surprising that there is a significant decrease in proliferation of CD8 T cells from older donors in response to IL-15 + IL-21 stimulation, as they have fewer CD28+ CD8 T cells. Interestingly, there was no obvious decrease in proliferation of the CD28+ CD8 memory T cells (CD45RA–CD28+) in response to IL-15 + IL-21 with age. This suggests that there is no age-associated decrease of homeostatic proliferation of the CD28+ CD8 memory T cells induced by IL-15 + IL-21. Therefore, the reported age-related decline in the proliferative response of T cells may reside largely in the CD28– T cell compartment with potential defects in the expression of receptors for IL-15 and IL-21, in the signaling strength of these receptors, and in their downstream targets. Indeed, Fulop et al. [23] observe a higher level of basal pSTAT5 in the T cells but no obvious increase after stimulation with IL-6 from the elderly compared with the young, indicating age-associated defects in cytokine signaling in T cells. Whether defects on the cell surface and downstream events exist and how these declined functions were induced with age will require further studies.

In summary, we have demonstrated that IL-21 preferentially enhances IL-15-mediated proliferation of CD28+ CD8 memory T cells. The basis for such preference is the differential expression of the IL-15R and IL-21R and the consequential enhanced STAT5 signaling in CD28+ CD8 memory T cells upon exposure to IL-15 and IL-21. Furthermore, our results also show that IL-21 is capable of reducing the loss of CD28 expression in actively dividing CD8 memory T cells. Finally, the ability of IL-21 + IL-15 to mediate homeostatic proliferation of CD28+ CD8 memory T cells is well-maintained with age. Together, these findings suggest that IL-21 is a critical cytokine in CD8 memory T cell homeostasis. A better understanding of the mechanisms underlying the IL-21 effect will not only shed light on the cytokine-mediated homeostasis of CD8 memory T cells but also may open new means to reduce the accumulation of CD28– CD8 T cells with aging.

Acknowledgments

This work was supported by the Intramural Research Program of the NIA and National Cancer Institute, National Institutes of Health. We thank Dr. Richard Hodes for critical reading of the manuscript. We thank Christa Morris of the Flow Cytometry Unit for data collection, the NIA Apheresis Unit for collecting blood samples, and Ana Lustig for proofreading the manuscript.

Footnotes

Abbreviations: γc=γ-chain, 7-AAD=7-amino-actinomycin, ACTB=β-actin, APC=allophycocyanin, h=human, MFI=mean fluorescent intensity, NIA=National Institute on Aging, pSTAT=STAT phorphorylation, qRT-PCR=quantitative RT-PCR, TC=tricolor

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