Introduction

CD4+CD25+ regulatory T cells (Tregs) are a subpopulation of thymus-derived T cells that express the forkhead box P3 (FoxP3) [1, 2]. Tregs help maintain immunological self-tolerance by producing immunoregulatory cytokines like IL-10 and transforming growth factor β (TGF-β), and can prevent autoimmunity and ameliorate graft-versus-host disease (GVHD) [3, 4]. However, clinical application of Tregs as a form of adoptive immunotherapy is hampered by the fact that Tregs are present in a relative low frequency in adult peripheral blood (APB) (1–2 %) [5]. In contrast to APB, umbilical cord blood (UCB) T cells contain a greater percentage of CD4+CD25+ subsets [6, 7] and are largely naïve and less likely to become CD4+CD25+ effector T cells upon stimulation. The skewing of UCB CD4+CD25+ T cells to a suppressor phenotype may contribute to the lessened severity of GVHD in UCB transplantation compared with adult bone marrow [8, 9].

Although IL-2 is generally used to expand Treg cells from APB [10, 11] or UCB [10], IL-15, another γ-chain signaling cytokine capable of enhancing NK and CD8+ T cells survival [12], has also been reported to augment Treg expansion [13, 14]. Our previous work showed that 4-day exposure to IL-15 had comparable effect on proliferation of CD4+CD25+ T cells as compared to IL-2 [15]. The present study examined the effect of IL-2 and IL-15 in conjunction with anti-CD3/CD28 beads on expansion of UCB and APB CD4+CD25+ T cells in a 2-week culture. We next compared FoxP3 expression and suppressive function of these expanded UCB and APB CD4+CD25+ T cells. Furthermore, we sought to determine the ability of IL-21 to reverse the suppression of responder proliferation induced by these expanded UCB and APB CD4+CD25+ T cells.

Methods

Magnetic cell sorting (MACS) purification of CD4+CD25+ T cells

UCB and APB samples were collected from normal full-term newborns umbilical cords following spontaneous vaginal deliveries and from healthy adult volunteers, respectively, according to guidelines established by the Human Subjects Protection Committee of the Chang Gung Memorial Hospital. Mononuclear cells (MNCs) were obtained via Ficoll–Hypaque (Amersham Bioscience, Piscataway, NJ, USA) density-gradient centrifugation. CD4+ cells were enriched with an isolation kit containing a cocktail of CD8, CD11b, CD16, CD19, CD36, and CD56 antibodies (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). The separation was performed with LS columns (Miltenyi Biotech), according to the manufacturer’s instructions. The CD4+ T cells (confirmed with TCR αβ staining) were further sorted to get CD25+ cell populations by positive selection with directly conjugated anti-CD25 magnetic microbeads (Miltenyi Biotec). The purity of CD4+CD25+ T cells was checked to be >95 % as determined by flow cytometry (FACSCalibur, Becton–Dickinson, San Jose, CA, USA).

Culturing APB and UCB CD4+CD25+ cells

MACS-purified APB and UCB CD4+CD25+ T cells at a concentration of 5 × 105 cells/ml in RPMI 1640 medium (Invitrogen-Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal calf serum in 24-well plates were then stimulated with Dynal beads coated with anti-CD3/CD28 (at beads: cell ratio of 4:1) (BioLegend, San Diego, CA, USA) for 7–14 days in the presence of IL-2 (500 IU/ml) or IL-15 (50 ng/ml) (both from Pepro Tech Inc., Rocky Hill, NJ, USA).

FoxP3 expression

For the intracellular staining of FoxP3, cells were incubated with PerCP-conjugated anti-CD4 (RPA-T4; BD PharMingen, San Diego, CA, USA) coupled with FITC-conjugated anti-CD25 (M-A251; BD PharMingen) and then fixed and permeabilized by freshly prepared fixation/permeabilization buffer (eBioscience) at 4 °C for 30 min. Subsequently, the cells were stained with anti-human FoxP3 (PCH101, eBioscience) or the isotype control antibodies. Cells were then analyzed on a FACSCalibur flow cytometer equipped with CellQuest software (BD Biosciences). The percentages of cells staining with each monoclonal antibody was determined by comparing each histogram with one from control cells stained with FITC- or PE-labeled isotype control antibodies.

Suppressive function assay

MACS-purified APB CD4+ T cells that were pulsed with 5 μM CFSE medium served as responder cells. The cells were activated with anti-CD3/anti-CD28 and cultured at a density of 1 × 105 responders/well in 96-well plates. The pre-expanded UCB or APB CD4+CD25+ T cells as suppressor cells were then added at the indicated ratios to the responder cultures (suppressor-to-responder cells ratio (S/R) 1:1) in a final volume of 200 μl. Three days later, the cells were collected and analyzed by flow cytometry. The percentages of divided CFSE+ cells in the co-culture were derived by comparing the proliferated CFSE+ cells in the culture of responder cells alone. In some experiments, interleukin-21 was added to the co-culture to see whether they could reverse the suppression.

Measurement of IFN-γ production

Secreted IFN-γ in culture supernatants was determined using a human IFN-γ ELISA kit (R&D systems) as recommended by the manufacturer’s instructions.

Statistical analysis

The paired t test was applied for analysis of the difference of responses before and after a treatment (calculated by SPSS 9.0 software). The data are presented as mean ± SE of mean. Groups being compared were considered significantly different if p was <0.05.

Results

Greater expansion of UCB CD4+CD25+ cells compared to their adult counterparts

We first examined the time course of the APB and UCB CD4+CD25+ cell expansion. Cells were stimulated with anti-CD3/CD28 beads for 14 days under the influence of IL-2 or IL-15. Figure 1 shows significant increases of both UCB and APB CD4+CD25+ cells during the 2-week culture in the presence of IL-2 or IL-15. When comparing their expansion ability, UCB CD4+CD25+ cells showed better expansion than their APB counterparts under the influence of IL-2 on Day 12 (41.1 ± 13.3 vs. 24.0 ± 5.5, p = 0.408) and on Day 14 (65.7 ± 16.8 vs. 34.4 ± 6.1, p = 0.085) despite the insignificant difference. Similar results were observed with IL-15. However, the expansion folds of UCB CD4+CD25+ cells stimulated with IL-15 were inferior to that of IL-2 on Day 12 (30.06 ± 7.71 vs. 41.13 ± 13.28, p = 0.602) and Day 14 (27.74 ± 8.46 vs. 65.66 ± 16.81, p = 0.140), respectively. APB CD4+CD25+ cells showed similar trends. Therefore, we harvested only APB and UCB CD4+CD25+ cells expanded with IL-2 for subsequent analyses.

Fig. 1
figure 1

IL-2 was superior to IL-15 to maintain the proliferation of APB and UCB CD4+CD25+ cells in 2-week culture. APB and UCB CD4+CD25+ cells were stimulated with anti-CD3/CD28 beads in the presence of IL-2 or IL-15. Cells were harvested on the indicated days. The cell number was determined by trypan blue exclusion. The fold of increase was defined as the cell number on indicated day over the number in the beginning of the culture. The data from 5 experiments are shown as mean ± SEM. *p < 0.05 and **p < 0.01 indicate cells under the influence of IL-2. ◆ p < 0.05 and ◆◆ p < 0.01 indicate cells under the influence of IL-15

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FoxP3 expression of APB and UCB CD4+CD25+ cells

We next examined FoxP3 expression in UCB and APB CD4+CD25+ cells during the 2-week culture in the presence of IL-2. Figure 2a shows the representative profile of FoxP3 staining of CD4+CD25+ cells isolated from UCB and APB. Figure 2b shows the percentage of FoxP3-expressing cells on Day 0, Day 7, and Day 14. The FoxP3 expression on Day 0 was higher in UCB CD4+CD25+ cells compared to adults (14.46 ± 2.15 vs. 4.13 ± 1.53 %, p = 0.009). The FoxP3-expressing cells significantly increased in UCB and APB during the culture, peaked around Day 7, and remained stationary on Day 14. Although FoxP3 expression was visibly elicited in APB CD4+CD25+ on day 7 compared to UCB counterparts (73.64 ± 1.17 % for UCB, 81.12 ± 2.33 % for APB, p = 0.047), it was comparable between these two cell populations on day 14 (84.04 ± 1.91 vs. 81.92 ± 1.85 %, p = 0.602).

Fig. 2
figure 2

FoxP3 expression was comparable between APB and UCB CD4+CD25+ cells during 2-week culture. a APB and UCB CD4+CD25+ cells were stimulated with anti-CD3/CD28 beads in the presence of IL-2. IL-2 was supplemented every 2–3 days. Cells were harvested on the indicated days. The harvested cells were stained with PerCP-conjugated anti-CD4 and FITC-conjugated anti-CD25 antibodies followed by permeabilization and staining with PE-conjugated anti-FoxP3 antibody. Cells were then analyzed by FACSCalibur equipped with CellQuest software. One representative sample of five experiments is shown. b The percentages of FoxP3-expressing cells of five experiments are shown as mean ± SEM. **p < 0.01, compared with Day 0

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Higher suppression activity of expanded UCB CD4+CD25+ T cells than the APB counterparts

To examine the regulatory function of expanded APB and UCB CD4+CD25+ T cells, CD4+CD25+ cells (suppressor cells) were co-cultured with CFSE-labeled allogeneic adult CD4+ T cells (responder cells) that were stimulated to proliferate by anti-CD3/CD28 beads. Without CD4+CD25+ T cells either from UCB or APB, responder cells proliferated vigorously (Fig. 3a). The suppression was observed when the CD4+CD25+ T cells were added to the culture. Greater degree of inhibition on responder proliferation was observed with addition of UCB CD4+CD25+ cells compared to corresponding APB at S/R ratio of 1/1 (39.67 ± 9.04 vs. 20.03 ± 3.83, p = 0.03) and 1/2 (15.46 ± 6.17 vs. 6.44 ± 1.60, p = 0.144), respectively (Fig. 4). Similar to that observed with the proliferation of responder cells, IFN-γ production of allogeneic responder CD4+ T cells (927.35 ± 297.89 pg/ml) was significantly suppressed by UCB CD4+CD25+ cells (281.32 ± 124.39 pg/ml, p = 0.033) (Fig. 3b).

Fig. 3
figure 3

Expanded UCB CD4+CD25+ cells showed stronger suppression ability than their APB counterparts. a The enriched allogeneic APB CD4+ T cells as responder cells were labeled with CFSE and activated with anti-CD3/CD28 beads. The pre-expanded APB or UCB CD4+CD25+ cells were then added at the indicated ratios to the cultures of responder cells for 3 days. In the end of culture, cells were harvested and analyzed by flow cytometry. The proportion with negative CFSE staining shown on the left of the histogram is CD4+CD25+ cells. One representative sample of five experiments is shown. b Extra group of the experiment to show that IL-21 was added to the co-culture. The supernatants were collected for IFN-γ measurement using ELISA. The data from eight experiments are shown as mean ± SEM. *p < 0.05

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Fig. 4
figure 4

IL-21 reversed the proliferation of responder cells which were co-cultured with APB or UCB CD4+CD25+ cells. IL-21 was added to the co-culture of CFSE-labeled responder and APB or UCB CD4+CD25+ cells. Three days later, cells were harvested and analyzed by flow cytometry. The inhibition percentage was defined as the decreased percentage of proliferating cells versus that of control proliferating cells. The data from eight experiments are shown as mean ± SEM. *p < 0.05 and **p < 0.01

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Reversal of suppressive function by IL-21

Previous studies have shown that IL-21 may counteract the suppressive activities of CD4+CD25+ cells. IL-21 was therefore added in the S/R co-culture to see the effect of this cytokine on responder proliferation and IFN-γ production. As shown in Fig. 4, IL-21 counteracts the suppressive function of APB and UCB CD4+CD25+ cells. Interestingly, the Treg-mediated suppression of IFN-γ production by UCB Treg was not reversed by IL-21(data not shown).

Discussion

In the present study, we examined the ability of IL-2/IL-15 to maintain APB and UCB CD4+CD25+ cell expansion in a 2-week culture. The results demonstrate that UCB CD4+CD25+ cells expanded better than their APB counterparts. In addition, IL-2 was superior to IL-15 in maintaining the expanded UCB and APB CD4+CD25+ cells. Although FoxP3 expression of expanded CD4+CD25+ cells was comparable between UCB and APB, expanded UCB CD4+CD25+ cells showed greater suppressive function in allogeneic mixed lymphocyte reaction (MLR) compared to their APB counterparts. IL-21, however, counteracted the suppressive function of expanded UCB CD4+CD25+ cells to inhibit proliferation but not IFN-γ production of responder cells.

Despite statistical insignificance between the expansion of APB and UCB CD4+CD25+ cells with IL-2 or IL-15, we observed UCB CD4+CD25+ cells expanded more than APB counterparts in each of the 5 experiments. Consistent with several recent studies [7, 16], we also demonstrated that UCB CD4+CD25+ cells could be expanded to a greater extent compared to their adult counterparts. We showed a superior capability of IL-2 compared to IL-15 in expanding CD4+CD25+ cells in a long-term culture. Several studies including ours suggest that IL-15 supports regulatory T cell expansion in the short-term culture [13–15, 17]. Wuest et al., however, showed that IL-2, but not IL-7 or IL-15, was capable of inducing Treg suppressive function [18]. Our finding supports previous protocols using IL-2 and anti-CD3/CD28 antibody-coated beads to expand Treg cells for adoptive immunotherapy [10, 11].

We observed a comparable enhancement of FoxP3 expression of CD4+CD25+ cells following 2-week culture between UCB and APB. Although FoxP3 has been demonstrated to be the most reliable markers for regulatory T cells [19, 20]), up-regulation of Foxp3 expression was also reported from activated T cells [21] or pre-activated Treg cells [22] and may not fully reflect the regulatory capacity of the expanded UCB and APB CD4+CD25+ T cells.

MLR experiments revealed that the expanded UCB CD4+CD25+ T cells inhibited the allogeneic CD4+ cell proliferation to a greater extent compared to their adult counterparts. The suppressive effect of UCB CD4+CD25+ T cells was also demonstrated in the decreased IFN-γ production of the responder cells. Our finding confirms a recent study showing that expanded UCB CD4+CD25+ Tregs also showed enhanced suppressive function compared to their adult counterparts.

IL-21, a cytokine produced by activated CD4+ T cells, blocks the differentiation of transforming growth factor-beta 1-induced regulatory T cells and renders CD4+ T cells resistant to the suppressive effects of regulatory T cells [23, 24]. We observed that IL-21 counteracted the suppressive effect of the expanded UCB CD4+CD25+ cells on responder cell proliferation, but had no effect on IFN-γ production of responders. The ability of IL-21 to inhibit IFN-γ production in developing CD4+ T cells has been reported [25–27]. Moreover, Attridge et al. [28] proposed recently that IL-2 and IFN-γ production was suppressed when CD4+CD25− T cells were cultured in the presence of IL-21.

Taken together, we demonstrated differential suppressive function between UCB and APB CD4+CD25+ T cells stimulated with IL-2 or IL-15. The greater regulatory function of UCB CD4+CD25+ T cells may contribute to the decreased severity of GVHD observed following UCB transplantation. UCB CD4+CD25+ T cells may serve as an optimal starting population for further studies, aiming at a larger scale of Treg expansion using IL-2 or IL-15. These expanded UCB Treg cells can then be applied for adoptive immunotherapy to alleviate the severity of GVHD following UCB transplantation.

Conclusions

In this study, UCB CD4+CD25+ T cells expand more than APB CD4+CD25+ T cells after 2-week culture. Furthermore, the expanded UCB CD4+CD25+ T cells demonstrate superior suppressive ability to the APB counterparts. From the results, UCB is an ideal source for Treg cells.