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International Immunology Advance Access originally published online on August 13, 2007
International Immunology 2007 19(10):1191-1199; doi:10.1093/intimm/dxm090
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Human CD4+ central and effector memory T cells produce IL-21: effect on cytokine-driven proliferation of CD4+ T cell subsets

Tadashi Onoda1,2, Mizanur Rahman1, Hidetoshi Nara1, Akemi Araki1, Koki Makabe3, Kouhei Tsumoto3, Izumi Kumagai3, Toshio Kudo4, Naoto Ishii5, Nobuyuki Tanaka5, Kazuo Sugamura5, Kiyoshi Hayasaka2 and Hironobu Asao1

1 Department of Immunology
2 Department of Pediatrics, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan
3 Department of Biomolecular Engineering, Graduate School of Engineering
4 Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
5 Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan

Correspondence to: H. Asao; E-mail: asao-h{at}med.id.yamagata-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
IL-21 regulates certain functions of T cells, B cells, NK cells and dendritic cells. Although activated CD4+ T cells produce IL-21, data identifying the specific CD4+ T cell subsets that produce IL-21 are conflicting. In a previous study, mouse IL-21 message was detected in TH2, whereas human IL-21 (hIL-21) message was found in both TH1 and follicular helper T cells. To identify the IL-21-secreting cell populations in human, we established a hybridoma cell line producing an anti-hIL-21 mAb. Intracellular hIL-21-staining experiments showed that hIL-21 was mainly expressed in activated CD4+ central memory T cells and in activated CD4+ effector memory T cells, but not in activated CD4+ naive T cells. Moreover, IL-21 was produced upon activation by some IFN-{gamma}-producing TH1-polarized cells and some IL-17-producing TH17-polarized cells, but not by IL-4-producing TH2-polarized cells. These results suggest that specific CD4+ T cell populations produce IL-21. In the functional analysis, we found that IL-21 significantly enhanced the cytokine-driven proliferation of CD4+ helper T cells synergistically with IL-7 and IL-15 without T cell activation stimuli. Taken together, IL-21 produced from CD4+ memory T cells may have a supportive role in the maintenance of CD4+ T cell subsets.

Keywords: homeostatic proliferation, TH1, TH17


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
IL-21 is a recently identified cytokine produced by activated CD4+ T cells (1). It shares structural homology with IL-2, IL-4 and IL-15 and binds to a cognate receptor, IL-21R, that belongs to the class I cytokine receptor superfamily (1, 2). The functional receptor complex for IL-21 consists of a unique subunit, IL-21R, and the common cytokine receptor {gamma}-chain ({gamma}c) (3, 4), qualifying the ligand as the sixth member of the {gamma}c-dependent cytokines; the others are IL-2, IL-4, IL-7, IL-9 and IL-15. Functionally, IL-21 is reported to regulate the proliferation of T cells (1, 5–8), the proliferation and differentiation of B cells (1, 9–12) and the activation and expansion of NK cells (13–18).

CD4+ T cells in the peripheral blood are mainly separated into two subsets, naive and memory cells. Memory T cells are also divided into two sub-populations, CD4+ central memory T (TCM) cells and CD4+ effector memory T (TEM) cells (19, 20). CD4+ TCM cells home to T cell areas of the secondary lymphoid organs and have few, if any, effector functions. Upon antigen stimulation, this subset principally produces IL-2 and quite low levels of IFN-{gamma} or IL-4; some time later, these cells proliferate and differentiate into effector cells. In contrast, CD4+ TEM cells migrate into inflamed peripheral tissues and provide immediate effector functions upon their activation, by producing massive amounts of IFN-{gamma} or IL-4. These memory cell sub-populations are distinguished by their expression of chemokine receptors and adhesion molecules, especially CCR7 and L-selectin (CD62L). These two molecules are involved in the ability of naive T cells to enter the lymph nodes and Peyer's patches through high endothelial venules. As with CD4+CD45RA+ naive T cells, human TCM cells express both CCR7 and CD62L. In contrast, human TEM cells lose CCR7 expression and show a heterogeneous pattern of CD62L expression. In terms of tissue distribution, TCM cells are found mainly in lymph nodes and tonsils, whereas TEM cells are largely found in the lung, liver and gut (21).

The CD4+ T helper effector cells are divided into TH1, TH2 and IL-17-producing TH17 subsets. TH1 produce cytokines, including IFN-{gamma}, IL-2 and tumor necrosis factor-ß, and promote cell-mediated immunity. TH2 secrete ILs, including IL-4, IL-5, IL-6, IL-10 and IL-13, and regulate mainly B cell responses. TH17 were recently distinguished from other helper T cells; they are derived from TH precursor cells under the control of transforming growth factor-ß and IL-6 (22, 23).

Reports on IL-21 expression at the transcriptional level in TH subsets have given conflicting results. In one report, mouse IL-21 (mIL-21) was demonstrated to be a TH2 cytokine (24). In another study, on human IL-21 (hIL-21), a microarray analysis showed that hIL-21 mRNA was expressed in CD4+ TH1 and follicular helper T (TFH) cells, which promote B cell functions but have neither a TH1 nor TH2 phenotype (25). Moreover, these reports showed that IL-21 activated IFN-{gamma} production and up-regulated TH1-related genes in human T cells and NK cells (26, 27). These data support the idea that hIL-21 is a TH1-related cytokine. This interspecies discrepancy may arise from differences in the regulation of IL-21 transcripts between human and mouse. Finally, a finding of multiple instability elements in the 3' untranslated region of the IL-21 transcript indicated that the transcript is probably unstable, which might make obtaining reliable data on its expression pattern difficult (28). Therefore, we felt that a re-examination of the IL-21 expression profile at the protein level was warranted and might provide additional or corroborating data.

In this report, we demonstrate that, in humans, the IL-21 protein is produced mainly by activated CD4+ TCM cells and by some activated TEM cells polarized toward the TH1 or TH17 phenotype. This is the first demonstration that the activated CD4+ TCM cells produce cytokine other than IL-2 within several hours.

We also explored functional roles of IL-21, by studying its effects on the activity of two {gamma}c-dependent cytokines, IL-7 and IL-15, which play essential roles in the maintenance of both CD4+ and CD8+ memory T cells (29–32). These cytokines drive the proliferation of CD4+ TEM more effectively than that of TCM and have no effect on naive T cells (33, 34). We show that IL-21 significantly enhances IL-7- and IL-15-induced CD4+ T cell proliferation in the TCM and TEM cells, and in combination with these cytokines promotes proliferation in also in naive T cells.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Cytokines and antibodies
Recombinant human IL-2, IL-4, IL-7, IL-12, IL-15 and IFN-{gamma} (hIL-2, hIL-4, hIL-7, hIL-12, hIL-15 and hIFN-{gamma}) were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant hIL-21 was purchased from Biosource International (Camarillo, CA, USA). Antibodies to human CD3, CD28, IL-4, IL-12 and IFN-{gamma} (anti-CD3, anti-CD28, anti-IL-4, anti-IL-12 and anti-IFN-{gamma}), FITC-conjugated antibodies to human CD4, CD8, CD45RA, CD45RO, V{alpha}24, IL-2, IL-4 and IFN-{gamma} (FITC–anti-CD4, FITC–anti-CD8, FITC–anti-CD45RA, FITC–anti-CD45RO, FITC–anti-V{alpha}24, FITC–anti-IL-2, FITC–anti-IL-4 and FITC–anti-IFN-{gamma}), PE-conjugated antibodies to human CD45RO, CD62L and IL-4 (PE–anti-CD45RO, PE–anti-CD62L and PE–anti-IL-4), PE–cyanine 7 (PE–Cy7)-conjugated anti-human CCR7 antibody (PE–Cy7–anti-CCR7), PE-conjugated anti-mouse IgG antibody (PE–anti-mIgG) and allophycocyanin (APC)-conjugated anti-mouse IgG antibody (APC–anti-mIgG) were purchased from BD PharMingen (San Diego, CA, USA). PE-conjugated anti-human Foxp3 antibody (PE–anti-Foxp3) and PE-conjugated anti-human IL-17 antibody (PE–anti-IL-17) were purchased from eBioscience (Boston, MA, USA). HRP-conjugated anti-mouse IgG antibody (HRP–anti-mIgG) was purchased from Cell Signaling Technology (Beverly, MA, USA).

Establishment of a hybridoma producing an anti-hIL-21 mAb
Preparation of the recombinant His/Myc-tagged hIL-21 was described previously (35). BALB/cJ mice were immunized intra-peritoneally with purified recombinant His/Myc-tagged hIL-21 at 50 µg per mouse in an emulsion with CFA (Difco Laboratories, Detroit, MI, USA). Two weeks after the priming immunization, we re-administered the antigens with IFA (Difco Laboratories) and repeated the immunization every 2 weeks until an increase in antibody titer in the immunized mouse serum was confirmed by indirect ELISA. The sensitized spleen cells were fused with mouse myeloma cells, P3-X63-Ag8-6.5.3 (36), in the presence of PEG4000 (Sigma–Aldrich, St Louis, MO, USA). The fused cells were cultured in IMDM (Iscove's Modified Dulbecco's Medium, Gibco–Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS), HAT (hypoxanthine, aminopterin and thymidine mixed solution, Dainippon Sumitomo Pharma, Co., Ltd., Osaka Japan) and 10% Origen Hybridoma Cloning Factor (IGEN International, Inc., Gaithersburg, MD, USA). After screening the produced antibodies by ELISA, one hybridoma (4BG1) was isolated and re-cloned twice by the limiting dilution method. We confirmed the specificity of 4BG1 against hIL-21 as follows. The hIL-21 expression vector, pEFneohIL-21 (M. Rahman and H. Asao, submitted for publication) or the control empty vector, pEFneo (37), were transiently transfected into 293T cells by the calcium–phosphate transfection method. hIL-21 expressed in 293T cells was detected by flow cytometry using 4BG1 in an intracellular staining experiment, whereas empty vector transfected cells had only the background signals (Fig. 1A). Next, the supernatants of pEFneohIL-21 or empty vector transfected 293T cells were subjected to SDS–PAGE and immunoblot assay. 4BG1 clearly detected single band ~16 kDa molecular mass only in the supernatants of pEFneohIL-21-transfected 293T cells but not in that of empty vector (Fig. 1B). The subclass of the 4BG1 mAb was IgG2b ({kappa}), which was determined using the Mouse Monoclonal Antibody Isotyping Kit (GE Healthcare Bio-Sciences, Uppsala, Sweden). Purified 4BG1 was biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL, USA) following the manufacturer's instructions. All experiments were performed in accordance with the guidelines of the Laboratory Animal Center of Yamagata University Faculty of Medicine and were approved by the animal experiment committee of Yamagata University Faculty of Medicine.


Figure 1
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  		Fig. 1. Activated CD4+ T cells express hIL-21. (A) Flow cytometry analysis of 293T cells transiently transfected with the expression vector for hIL-21 (right panel) or control empty vector (left panel). Twenty hours after the transfection, the cells were cultured in the presence of brefeldin A for 4 h more and then stained with 4BG1 (open histograms) or isotype-matched control mAb (filled histograms) as first antibodies and with PE–anti-mIgG as a second antibody. Percentages in the gated population are indicated. The data are representative of three independent experiments. (B) Ten microliters of the supernatants of 293T cells transfected with pEFneohIL-21 (lane 2) or with the empty vector (lane 1) were analyzed by immunoblot assay using 4BG1 and HRP–anti-mIgG. Ten nanograms of human recombinant IL-21 was loaded as a positive control (lane 3). The position of hIL-21 is indicated by an arrow. (C) PBMCs depleted of CD14+ and cultured with (right panels) or without PMA and ionomycin (left panels) for 5 h. Cells were stained with 4BG1/PE–anti-mIgG and FITC–anti-CD4 (upper panels) or FITC–anti-CD8 (lower panels). Quadrant percentages are indicated. These flow cytometry profiles are representative of samples taken from three healthy volunteers. CD4+CD14– cells were isolated from PBMCs and cultured with the combinations of 20 ng ml–1 PMA and 1 µM ionomycin as indicated for 5 h (D) or cultured with 20 ng ml–1 PMA and 1 µM ionomycin for 0–6 h (E). Cells were stained with 4BG1/APC–anti-mIgG. Representative data are shown from three healthy volunteers.

 
Intracellular cytokine staining and flow cytometry analysis
PBMCs were obtained from four healthy volunteers by the density gradient centrifugation method using Ficoll-PaqueTM Plus (GE Healthcare Bio-Sciences). Monocytes were removed from the PBMCs with BDTM IMag anti-hCD14 particles (BD Bioscience) and the CD4+CD14– T cells, hereafter referred to as CD4+ T cells, were isolated from the PBMCs using BDTM IMag anti-hCD4 particles (BD Bioscience). The isolated CD4+ T cells were suspended in RPMI 1640 medium supplemented with 10% FBS. Cells were stimulated with 20 ng ml–1 phorbol myristate acetate (PMA) (Sigma) and 1 µM ionomycin (Sigma) for 5 h. Brefeldin A (Sigma) was added to the cells at 10 ng ml–1 to block cytokine release. For intracellular cytokine staining, the cells were fixed and permeabilized with BD Cytofix/Cytoperm solution (BD PharMingen) for 15 min on ice and incubated with 4BG1 or biotinylated-4BG1 in BD Perm/Wash solution (BD PharMingen) on ice for 60 min. After the cells were washed twice, the optimal concentration of PE–anti-mIgG, APC–anti-mIgG or APC-labeled streptavidin (BD PharMingen) was added and the cells were incubated on ice for 30 min. Cells were washed and blocked with normal mouse serum (DakoCytomation, Glostrup, Denmark) for 15 min and then incubated with fluorescent dye-labeled antibodies against cell-surface molecules or other cytokines. Finally, the cells were analyzed by FACSCalibur or FACSAria (BD Biosciences). Numbers in each quadrant indicate the percentage of cells in the designated gates.

In vitro TH differentiation
CD45RA+CD45RO– naive T cells were sorted with FACSAria from the CD4+ T cells. These sorted CD4+ naive T cells were cultured with 2 µg ml–1 immobilized anti-CD3, 2 µg ml–1 anti-CD28 and 1 nM hIL-2 for 7–10 days in the presence of, for the TH1-skewing condition, 1 ng ml–1 hIL-12 and 10 µg ml–1 neutralizing anti-IL-4 or, for the TH2-skewing condition, 50 ng ml–1 hIL-4, 10 µg ml–1 neutralizing anti-IL-12 and 10 µg ml–1 neutralizing anti-IFN-{gamma}. These media were replaced with fresh media supplemented with only 10% FBS and 1 nM hIL-2, 4 days after the priming and every 3–4 days thereafter until the cells were analyzed.

Carboxyfluorescein diacetate succinimidyl ester labeling of CD4+ T cells and CD4+ T cell proliferation assay
CD4+ T cells were incubated with the FITC–anti-CD45RA and PE–anti-CD62L on ice for 30 min. CD4+CD45RA+CD62L+ naive T cells, CD4+CD45RA–CD62L+ TCM cells and CD4+CD45RA–CD62L– TEM cells were sorted into separate groups. The sorted cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) using the CellTraceTM cell proliferation kit (Molecular Probes, Eugene, OR, USA), following the manufacturer's instructions. The labeled cells were cultured for 7 days with various combinations of exogenous IL-7, IL-15 and IL-21. The cells were harvested and the amount of cell division was determined.

All human specimens were obtained under informed consent. The protocol for the human research has been approved by the Ethics Committee of Yamagata University Faculty of Medicine.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
hIL-21 is expressed in activated CD4+ T cells
CD4+ T cells were reported to express IL-21 mRNA in human peripheral blood (1). We first tried to detect hIL-21 protein expression in peripheral lymphocytes with our new mAb, 4BG1. PBMCs isolated from healthy volunteers were stimulated with PMA and ionomycin in the presence of brefeldin A, as described in Methods. Cells were stained with 4BG1 intracellularly and with FITC–anti-CD4 or FITC–anti-CD8. hIL-21 was detected only in small population of the activated CD4+ T cells, and not in unstimulated cells, activated CD4– cells or activated CD8+ T cells (Fig. 1C). Thus, hIL-21 was expressed only by CD4+ T cells and not by other lymphocyte populations.

Then, we searched an optimal stimulatory condition for hIL-21 production from CD4+ T cells. A combination of PMA and ionomycin induced IL-21 production efficiently but either ionomycin or PMA alone could not (Fig. 1D). Previous report showed that ionomycin alone induced significant amount of mIL-21 mRNA (38). We confirmed that ionomycin stimulation alone could activate transcription of hIL-21 gene (M. Rahman and H. Asao, submitted for publication), however, could not produce detectable level of hIL-21 protein. Next, we performed the kinetics study of hIL-21 expression. IL-21-positive CD4+ T cells appeared 2 h after stimulation with PMA and ionomycin and reached to the maximal level at around 3–4 h after stimulation (Fig. 1E). Longer stimulation than 6 h did not increase the population of IL-21-producing cells (data not shown). So we determined to stimulate cells with PMA and ionomycin for 5 h for further experiments.

hIL-21 is expressed in activated CD4+ TCM and TEM
CD4+ T cells are categorized according to their functions and cytokine production profiles. Therefore, to understand the functional roles of IL-21, it was very important to identify the hIL-21-expressing CD4+ T cell sub-populations. CD4+ T cells are mainly divided into the CD45RA+ naive T cells and the CD45RO+ memory T cells. First, we investigated which populations expressed hIL-21. CD45RA+CD45RO– naive T cells and CD45RA–CD45RO+ memory T cells were sorted from peripheral CD4+ T cells as indicated (Fig. 2A), and then stimulated with PMA and ionomycin. We found hIL-21 only in activated CD4+CD45RA–CD45RO+ memory T cells and not in activated CD4+ naive T cells or resting CD4+ memory T cells (Fig. 2B). Peripheral CD45RO+ memory T cells are further subdivided into CD62L+CCR7+ TCM and CD62L–CCR7– TEM subsets. We next analyzed the hIL-21 expression in these sub-populations. CD4+CD45RA– memory T cells were analyzed for the expression of CD62L and CCR7, and then CD62L+CCR7+ TCM and CD62L–CCR7– TEM cells were sorted (Fig. 3A). Purity of these sorted cells was 94.0 and 99.1%, respectively, as indicated. These sorted cells were stimulated with PMA and ionomycin. Interestingly, both memory cell populations produced IL-21 similarly (Fig. 3B). IL-2 has been thought to be the main cytokine produced by TCM cells. We compared the kinetics of IL-21 and IL-2 production in both memory cells. IL-21 expression patterns of TCM and TEM were very similar to that of IL-2; however, the number of IL-2-producing cells of each memory cells was much higher than that of IL-21 (Fig. 3C). These results demonstrate that not only TEM but also TCM can produce IL-21 within 4 h after activation.


Figure 2
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  		Fig. 2. Activated CD4+ memory T cells express hIL-21. (A) CD4+CD14– T cells from PBMCs were isolated and stained with FITC–anti-CD45RA and PE–anti-CD45RO (CD4+ T cells). CD45RA+ naive T cells (naive T cells) or CD45RO+ memory T cells (memory T cells) were sorted from CD4+ T cells as indicated. Purity of these sorted cells was indicated. These sorted cells were stimulated with or without PMA and ionomycin for 5 h and stained with biotinylated-4BG1 and APC-labeled streptavidin. IL-21 expression profiles of these cells were shown (B). These profiles are representative of samples taken from three healthy volunteers.

 

Figure 3
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  		Fig. 3. Comparison of IL-21 expression in activated CD4+ TCM and TEM.. (A) CD4+CD14– T cells from PBMCs were isolated and stained with FITC–anti-CD45RA, PE–anti-CD62L and PE–Cy7–anti-CCR7. Expression profile of CD45RA and CD62L (CD4+ T cells) and CCR7 and CD62L of CD45RA– cells was shown (CD45RA– cells). CD62L+CCR7+ TCM cells and CD62L–CCR7– TEM cells were sorted (sorted TCM and sorted TEM, respectively). Gated cells and quadrant percentages are indicated. (B) These sorted cells were cultured with (PMA + ionomycin) or without (unstimulated) PMA and ionomycin for 5 h. Cells were stained with biotinylated-4BG1 and APC-labeled streptavidin. Quadrant percentages are indicated. These profiles are representative of samples taken from three healthy volunteers. (C) CD62L+CD45RA– TCM cells (solid bars) and CD62L–CD45RA– TEM cells (open bars) were sorted and cultured with PMA and ionomycin for 0–5 h. Cells were stained with the combination of 4BG1/APC–anti-mIgG (left panel) or with FITC–anti-IL-2 (right panel). These were the representatives from three healthy volunteers.

 
hIL-21 is expressed in a part of activated TH1- and TH17-polarized cells
We conducted a further analysis of the memory cells that expressed IL-21. Once these CD4+ memory T cells are activated, they secrete cytokines, including IFN-{gamma}, IL-4, IL-17 and IL-2. Since the set of cytokines expressed by these activated cells categorizes the cells' function, we examined the cytokine production profiles of activated human CD4+ T cells to learn which polarized CD4+ T cells produce IL-21. Intracellular double-staining experiments showed that hIL-21 was co-expressed in some IFN-{gamma}-producing cells (~6.1%), some IL-17-producing cells (~10.8%) and some IL-2-producing cells (~4.3%), but it was not co-expressed in IL-4-producing cells (Fig. 4A). Next, we induced naive CD4+ T cells to differentiate into TH1 or TH2 in vitro to confirm their IL-21 production. CD45RA+CD45RO– naive CD4+ T cells were sorted and cultured in TH1- or TH2-skewing conditions as described in Methods. Three percent of the IFN-{gamma}-producing TH1 expressed IL-21, and few IL-4-producing TH2, if any, expressed it (Fig. 4B). Taken together, these results show the existence of two hIL-21-producing CD4+ memory T cell populations: one, a TH1 sub-population that produces IFN-{gamma} and the other, a TH17 sub-population that produces IL-17.


Figure 4
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  		Fig. 4. Co-expression of IL-21 and IFN-{gamma} or IL-17 but not IL-4. CD4+CD14– cells from PBMCs were isolated and cultured with or without PMA and ionomycin for 5 h. Cells were stained with 4BG1/PE–anti-mIgG and FITC–anti-IFN-{gamma}, FITC–anti-IL-4 or FITC–anti-IL-2 or 4BG1/APC–anti-mIgG and PE–anti-IL-17 (A), with 4BG1/APC–anti-mIgG and PE–anti-Foxp3 (C) or with 4BG1/PE–anti-mIgG and FITC–anti-V{alpha}24 (D), as indicated. CD45RA+CD45RO– naive CD4+ T cells from PBMCs were isolated and cultured under TH1- or TH2-skewing conditions as described in Methods. Cells were re-stimulated with PMA and ionomycin for 5 h and then stained with 4BG1/APC–anti-mIgG and FITC–anti-IFN-{gamma}, to label TH1, or PE–anti-IL-4, to label TH2 (B). Quadrant percentages are indicated. Representative results for samples from three healthy volunteers are shown.

 
hIL-21 is not expressed in CD4+Foxp3+ regulatory T cells
Besides the naive and memory T cells, CD4+ lymphocytes also include regulatory T cells (Tregs) and NKT cells. CD4+ Tregs express CD25 and Foxp3, allowing them to be readily identified. We examined the hIL-21 expression in Tregs following stimulation with PMA and ionomycin. Figure 4(C) shows that the activated CD4+ Foxp3– cells, but not the Foxp3+ Tregs expressed hIL-21. On the other hand, there are very small numbers of NKT cells in human peripheral blood. Since a part of human NKT cells also display the CD4+ phenotype, we tested whether the IL-21-producing cells express V{alpha}24 or not. IL-21-producing CD4+ cells were all V{alpha}24– cells (Fig. 4D). These data indicate that the major hIL-21-producing cell type in human peripheral blood is the conventional CD4+ memory T cell.

hIL-21 enhances the cytokine-driven proliferation of CD4+ T cell subsets
Previous report demonstrated that IL-21 had a supportive effect on activated CD4+ T cell proliferation (1). These CD4+ T cells constitutively express both the IL-21 receptor and {gamma}c before activation (data not shown). This prompted us to presume some functional roles of IL-21 on these not activated CD4+ T cells. Two {gamma}c cytokine members, IL-7 and IL-15, are thought to be important factors to maintain the homeostasis of CD4+ memory T cells. So we investigated the effects of another {gamma}c cytokine, IL-21, on the proliferation of CD4+ T cells without T cell activation stimuli. The naive, TCM and TEM CD4+ T cell subsets were sorted from healthy donors and labeled with CFSE as described in Methods. These cells were cultured in various combinations of exogenous homeostatic cytokines, IL-7 and IL-15, and IL-21 for 7 days (Fig. 5). IL-21 alone did not affect cell growth in any CD4+ T cell subset without T cell activation. Surprisingly, the combination of IL-21 with IL-7, IL-15 or with IL-15 and IL-7 significantly enhanced the proliferation of CD4+ TEM and TCM cells and also CD4+ naive T cells. These results suggest that hIL-21 may have a supportive role on cytokine-driven CD4+ T cell proliferation.


Figure 5
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  		Fig. 5. IL-21 significantly enhances the cytokine-driven proliferation of CD4+ T cell subsets. CD4+CD45RA+CD62L+ naive T cells, CD4+CD45RA–CD62L+ TCM cells and CD4+CD45RA–CD62L– TEM cells were sorted and labeled with CFSE. Labeled cells were cultured for 7 days in combinations of IL-7, IL-15 and IL-21 as indicated. The profiles of viable CFSE-positive cells were analyzed by flow cytometry. Representative results for samples from three healthy volunteers are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
IL-21 regulates certain functions of T cells, B cells and NK cells. Furthermore, considerable evidence implicates IL-21 in various diseases, including autoimmune diseases, allergies and tumorigenesis (39). However, details of hIL-21-producing cells and the effects of secreted hIL-21 on CD4+ T cells have been largely unknown. In this study, we clarify that hIL-21 is mainly expressed in activated CD4+ TCM cells, and in some TEM cells belonging to the TH1 or TH17 subsets, in the peripheral blood. Furthermore, we show that hIL-21 plays a supportive role in enhancing proliferation of CD4+ T cells without antigen stimulation ex vivo.

Chtanova et al. (25) reported hIL-21 mRNA in tonsil CD4+ TFH cells expressing CXCR5, which belong to the memory T cell subsets and migrate into B cell follicles in response to B cell-attracting chemokine 1. All the tonsil CXCR5+ T cells were in the activated state (40). CXCR5+ T cells in peripheral blood also belong to the CD4+ memory T cell subset; however, in contrast to tonsillar cells, these cells are in the resting state and migrate only weakly in response to chemokines. In addition, the migration and immune responses of CXCR5+ T cells depend on the expression of CCR7, which is co-expressed on virtually all circulating CXCR5+ T cells (41). In agreement with the previous study, we obtained data showing that the IL-21-producing CCR7+ TCM cells also expressed CXCR5 (data not shown). Our findings may suggest that some resting CD4+CCR7+ TCM cells migrate to lymphoid organs, e.g. the tonsil, and, after antigen exposure, become IL-21-producing TFH cells.

Here, we showed that IL-21 enhanced IL-7- and IL-15-driven proliferation of CD4+ TCM cells, TEM cells and naive cells effectively, without antigen stimulation ex vivo. Immune responses to viral infections are known to induce transient lymphopenia occasionally. To maintain the homeostasis of immune system, quick recovery of the reduced number of lymphocytes is required. In lymphoid tissues, IL-21 may support effective proliferation of not only activated T cells in expansion phase but also bystander cells including memory and naive CD4+ T cells cooperatively with IL-7 and IL-15 to expand the reduced peripheral T cell pool quickly.

Zeng et al. (42) showed that mIL-21 enhanced cytokine-driven proliferation of mouse CD8+ T cells but not of CD4+ T cells. The reason of this discrepancy between human and mouse is unknown. Many species differences of the function of IL-21 between human and mouse are reported. IL-21 may not be required for the homeostatic maintenance of CD4+ T cells in mouse.

Human NKT cells display the CD4+ or CD4–CD8– phenotype. These cells co-express the NK receptor, NK1.1/NKRP1A (CD161), and a semi-invariant TCR, which consists of a V{alpha}24J{alpha}18 chain combined with variable TCR Vß11 chains (43–45). CD4+ NKT cells also express memory T cell markers such as CD45RO, CD62L and CCR7 (46, 47) and produce both TH1 and TH2 cytokines (48, 49). Recently, mouse and human NKT cells were shown to express IL-21 mRNA efficiently after stimulation and suppress IgE responses (50). The authors also mentioned that main IL-21 producer cells in mouse are NKT cells, while NKT and conventional CD4+ T cells in human. We detected hIL-21 protein expression only in the conventional CD4+ T cells in the human peripheral blood. We could analyze only a small number of NKT cells from PBMCs, and then the IL-21 protein-producing capacity of human NKT cells should be further elucidated at the tissue level also.

Our finding that a part of IL-17-producing cells can express IL-21 simultaneously may suggest that IL-21 produced from TH17 is involved in inflammatory and autoimmune diseases in humans. It is a quite interesting issue to clarify how the IL-21, which is produced from TH17 controls immune responses. In mouse, IL-21 is reported to possess pro-inflammatory properties (51). Furthermore, pre-treatment with IL-21 before the induction of mouse experimental autoimmune encephalomyelitis (EAE) exacerbates the disease (52). These reports strongly suggested that mIL-21 cooperates with IL-17 for EAE development and progression of many inflammatory diseases. However, very recently, Fröhlich et al. (53) reported that mIL-21 is integral to the TH2 but not to the TH1 or TH17 responses. There are still conflicting reports in the function of mIL-21; moreover, a lot of species differences between human and mIL-21 are reported. Further studies are needed to elucidate the role of IL-21 in immune response and diseases in human.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 17659139); grant-in-aid from the 21st Century Center of Excellence Program (F03) of the Japan Society for the Promotion of Science.


   Acknowledgements
 
We thank Kinya Nagata and Kohtaro Tada for materials and critical comments and Hiroyuki Kumagai and Sachiko Horie for their critical review of the manuscript.


   Abbreviations
 
APC, allophycocyanin
CFSE, carboxyfluorescein diacetate succinimidyl ester
{gamma}c, common cytokine receptor {gamma}-chain
EAE, experimental autoimmune encephalomyelitis
FBS, fetal bovine serum
hIL-21, human IL-21
mIL-21, mouse IL-21
PMA, phorbol myristate acetate
TCM, central memory T
TEM, effector memory T
TFH, follicular helper T
Treg, regulatory T cell

   Notes
 
Transmitting editor: M. Miyasaka

Received 3 April 2007, accepted 11 July 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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