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* Institute of Immunology,
Department of Dermatology, and
Center for Natural Sciences and Medicine, Johannes Gutenberg University, Mainz, Germany
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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and CTLA-4 are the mediators of suppression (10, 11, 12), whereas others clearly excluded a function of these molecules (13, 14, 15). Contradictory results have been reported regarding the influence of Treg cells on Th2 cells. Depletion of CD25+ Treg cells from spleen cells in vitro was found to prevent the development of Th2 cells and simultaneously led to increased development of Th1 cells (16). Consequently, it has been proposed that CD25+ Treg cells bias the differentiation of CD4+ T cells toward Th2 cells. In addition, the finding that CD25+ Treg cells overexpress a subset of Th2-specific gene transcripts would fit the potential Th2-promoting properties of this T cell population (17). In contrast, it was very recently reported that the depletion of CD25+ Treg cells in vivo led to an increased development of Th2 cells (18). In line with these findings it was recently shown that CD25+ Treg cells suppress the differentiation and function of Th1 and Th2 cells in vitro and in vivo (19).
To clarify the role of CD25+ Treg cells in the development and stimulation of Th2 cells, we used highly purified conventional CD4+ T cells and CD25+ Treg cells in an IL-4-driven and Ag-specific (OVA323–339) or polyclonal Th2 developmental system. Our results clearly demonstrate that freshly isolated CD25+ Treg cells, on the one hand, strongly inhibit the IL-4-induced development of Th2 cells, but, on the other hand, have no influence on established Th2 cells. In contrast, preactivated CD25+ Treg cells (CD25+ preTreg) inhibit cytokine production and proliferation of established Th2 cells. In either case, inhibition of both conventional CD4+ T cells and Th2 cells is accompanied by a strongly enhanced expression of FoxP3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Mice of strains BALB/c and C57BL/6 were obtained from Charles River Laboratories (Sulzfeld, Germany) and bred in our own animal facility. Males and females were used at 6–12 wk of age. Mice transgenic for the OVA323–339-specific TCR-
(20) on a BALB/c genetic background were a gift from D. Y. Loh (Nippon Roche Research Center, Kamakura, Japan) through M. Kopf and were also bred in our own animal facility, identified by staining PBLs with the anti-TCR clonotype specific mAb KJ1-26; they were used at the age of 6–12 wk.
Cytokines, Abs, reagents, and Ags
Mouse rIL-4 was affinity purified using a column with anti-mIL-4 (11B11) mAb bound to Sepharose. Hybridoma cells producing anti-CD4 mAb GK1.5 were obtained from American Type Culture Collection (ATCC TIB 207; Manassas, VA). Anti-mIL-4 mAb 11B11 was a gift from Dr. W. Paul (National Institutes of Health, Bethesda, MD) (21). Anti-mIL-4 mAbs BVD4-1D11 and BVD6-24G2 were gifts from Dr. A. O’Garra (National Institute for Medical Research, Mill Hill, U.K.). PE-labeled rat anti-CD25 (7D4) was purchased from BD Biosciences (Heidelberg, Germany). In addition, the following mAbs were used: rat anti-MHC I (H-2b, AF6-88.5; H-2d, SF1-1.1) from BD Biosciences; rat anti-TCR clonotype KJ1-26 (22), anti-CD3 mAb 145-2C11 (23), anti-CD28 mAb 37.51 (24), and anti-IFN-
mAb XMG1.2 (25). If required, mAbs were affinity purified using protein G-Sepharose (Pharmacia Biotech, Freiburg, Germany) and coupled with FITC or biotin. Mitomycin C was purchased from Sigma-Aldrich (M 0503; St. Louis, MO). The antigenic OVA peptide (OVA323–339) was synthesized on an Applied Biosystems peptide synthesizer (Foster City, CA).
Preparation of T cell populations (CD4+CD25– T cells and CD25+ Treg cells) and preactivation of CD25+ Treg cells
Conventional CD4+ T cells (GK1.5-FITC) and CD25+ Treg cells (7D4-PE) were isolated from spleen cells by positive selection using high gradient MACS (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions. The CD4-sort as well as the CD25-sort were performed twice. CD4+ T cells were subsequently depleted from CD25+ Treg cells using mAb PC61 and enriched >99%. They showed no proliferative response in the presence of Con A or soluble anti-CD3 mAb, indicating negligible contamination with accessory cells. CD25+-enriched Treg cells were additionally depleted from CD8+ T cells using anti-CD8 Dynabeads (Dynal Biotech, Hamburg, Germany), and the purity of the resulting CD25+ Treg cells was typically >95%. Preactivation of CD25+ Treg cells was performed using a combination of plate-bound anti-CD3 mAb (145-2C11, 3 µg/ml) and anti-CD28 mAb (37.51; 10 µg/ml). After 48 h the cells were harvested and used as preactivated CD25+ Treg cells (CD25+ preTreg) in coculture experiments with conventional CD4+ T cells and Th2 cells.
Assessment of Th2 development in the presence of CD25+ Treg cells
OVA323–339-specific.
Purified OVA-specific TCR transgenic (TCR-tg) CD4+ T cells (1 x 106/ml) were activated in the presence of mitomycin C-treated (60 µg/ml/107 cells, 30 min) A20 B-tumor cells (1 x 105/ml) and OVA323–339 (100 ng/ml) together with IL-4 (0.75–25 ng/ml) and anti-IFN- (XMG1.2; 10 µg/ml) mAb in the absence or the presence of TCR-tg CD25+ Treg cells (1 x 106/ml). A Th1-inducing effect of IL-12 could be excluded because A20 accessory cells cannot produce this cytokine (26, 27). After 5 days of priming, the resulting cells were restimulated by plate-bound anti-clonotype mAb (KJ1-26; 5 µg/ml), and cytokine content (IL-4, IL-5, IL-9, and IL-10) was assessed by ELISA after an additional 24 h.
Polyclonal.
Conventional CD4+ T cells (1 x 106/ml) from BALB/c mice were activated in the presence of mitomycin C-treated (60 µg/ml/107 cells, 30 min) A20 B-tumor cells (1 x 105/ml), and soluble anti-CD3 mAb (3 µg/ml) together with IL-4 (0.75–25 ng/ml) and anti-IFN- (XMG1.2; 10 µg/ml) in the absence or the presence of CD25+ Treg cells (1 x 106/ml) from C57BL/6 mice. After 5 days of priming, CD25+ Treg cells (1 x 106/ml) from C57BL/6 mice (H-2b) were depleted by MACS using anti-MHC I (H-2b) mAb. The remaining T cells from BALB/c mice (H-2d) were restimulated by plate-bound anti-CD3 mAb (145-C11; 5 µg/ml), and the contents of different cytokines (IL-4, IL-5, IL-9, and IL-10) was assessed by ELISA after an additional 24 h.
After restimulation, both Ag-specific and polyclonal, the resulting Th2 cells produced no or only minimal amounts (10–50 pg/ml) of IFN-. This was confirmed by FACS analyses via intracellular staining of IL-4 vs IFN- (data not shown).
Induction of Th2 development and coculture of Th2 cells and CD25+ Treg cells
TCR-tg Th2 cells were generated by stimulating purified TCR-tg CD4+ T cells (1 x 106/ml) in the presence of mitomycin C-treated (60 µg/ml/107 cells, 30 min) A20 B-tumor cells (1 x 105/ml) and OVA323–339 (100 ng/ml) together with IL-4 (7.5 ng/ml) and anti-IFN- mAb (XMG1.2; 10 µg/ml). After 5 days these Th2 cells were restimulated in the absence or the presence of freshly isolated or preactivated TCR-tg CD25+ Treg cells using mitomycin C-treated (60 µg/ml/107 cells, 30 min) A20 B-tumor cells (1 x 105/ml) and OVA323–339 (0.3, 1.0, and 3.0 µg/ml), and the production of IL-4 was assessed by ELISA after an additional 24 h.
Transwell experiments
Transwell experiments were performed using 24-well plates as described previously (28). Briefly, 1 x 106/ml CD4+ conventional TCR-tg T cells and TCR-tg CD25+ Treg cells were stimulated with mitomycin C-treated (60 µg/ml/107 cells, 30 min) A20 B-tumor cells (1 x 105/ml) in the presence of OVA323–339 (100 ng/ml), anti-IFN- (XMG1.2, 10 µg/ml), and IL-4 (7.5 ng/ml). In addition, 106/ml CD4+CD25+ T cells were directly added to cultures of conventional CD4+ T cells or were placed in Transwell chambers (Millicell, 0.4 µm pore size; Millipore, Bedford, MA) in the same well. After 5 days of culture (priming), the resulting T cell populations were restimulated using plate-bound mAb KJ1-26 (5 µg/ml), and IL-4 production was assessed 24 h later by ELISA.
Proliferation assays
The culture medium was IMDM (Life Technologies, Grand Island, NY) supplemented with 2 mM L-glutamine, 5 x 10–5 M 2-ME, 10 IU penicillin, 100 µg/ml streptomycin, and 5% FCS, inactivated at 56°C. TCR-tg Th2 cells (2 x 105/ml) were stimulated using flat-bottom, 96-well microplates in a total volume of 0.2 ml of culture medium in the presence or the absence of freshly isolated or preactivated CD25+ Treg cells (2 x 105/ml) together with mitomycin C-treated (60 µg/ml/107 cells, 30 min) A20 B-tumor cells (2 x 104/ml) and OVA323–339 as indicated in Fig. 4. After 96 h, [3H]TdR was added to the cultures, and after an additional 18 h, thymidine uptake was assessed by beta scintillation counting.
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Cytokines were assayed by specific two-site ELISA with reference standard curves using known amounts of the respective cytokine. For the detection of cytokines we used the following combinations of Abs: IL-4, BVD4-1D11 and BVD6-24G2 (29); IL-5, TRFK4 and TRFK5 (29); IL-9, D9302C12 and 229.8 (30); and IL-10, JES5-16E3 and JES5-2A5 (29).
mRNA detection
RNA was isolated using TRIzol (Invitrogen, Karlsruhe, Germany), and cDNA was synthesized with RevertAid Moloney murine leukemia virus reverse transcriptase following the recommendations of the supplier (MBI Fermentas, St. Leon-Rot, Germany). RT-PCR and real-time PCR were performed using the following oligonucleotides: FoxP3 forward, CAG CTG CCT ACA GTG CCC CTA G; FoxP3 reverse, CAT TTG CCA GCA GTG GGT AG; real-time-FoxP3 forward, CTT ATC CGA TGG GCC ATC CTG GAA G; real-time-FoxP3 reverse, TTC CAG GTG GCG GGG TTT CTG; IL-4 forward, GCA TGG TGG CTC AGT ACT ACG AGT A; IL-4 reverse, GAA TGT ACC AGG AGC CAT ATC CAC G; HGPRT forward, GTT GGA TAC AGG CCA GAC TTT GTT G; and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) reverse, GAG GGT AGG CTG GCC TAT AGG CT. Oligonucleotides were chosen to span at least one intron at the level of genomic DNA. Real-time analyses to quantify the expression of FoxP3, IL-4, and HGPRT mRNAs were performed on an iCycler (Bio-Rad, Munich, Germany) using the IQ SYBR Green Supermix (Bio-Rad). After normalization of the data according to the expression of HGPRT mRNA, relative expression levels of FoxP3 and IL-4 mRNAs were calculated.
CFSE staining
Either freshly isolated CD4+ T cells or Th2 cells were labeled with the vital dye CFSE (CFDASE; Molecular Probes, Leiden, The Netherlands). After washing cells twice in PBS (pH 7.4), they were incubated with 2.5 µM CFSE in PBS at 37°C in 5% CO2 for 4 min. To stop the staining reaction, IMDM + 10% FCS was added. The cells were then washed three times in IMDM and stimulated in the presence of 7.5 ng/ml IL-4 as described. The proliferation of the CFSE-labeled CD4+ T cells was analyzed by flow cytometry.
FACS-based cell sort
After coculture, CFSE-labeled CD4+ T cells or CFSE-labeled Th2 cells were washed in PBS plus 4% FCS and isolated using a cell sorter (FACSvantage SE and CellQuest Pro; BD Biosciences), with exclusion of dead cells by propidium iodide incorporation. The purity of the sorted cells was always >99%.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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It has been shown that CD25+ Treg cells isolated from mice expressing a transgenic TCR that recognizes OVA323–339 (TCR-tg CD25+ Treg cells) strongly suppress the Ag-specific proliferation of conventional TCR-tg CD4+ T cells (31). When freshly isolated TCR-tg CD4+ T cells were activated by Ag (OVA323–339) under Th2-inducing conditions, i.e., in the presence of IL-4- and anti-IFN--specific mAb, the addition of TCR-tg CD25+ Treg cells (ratio of 1:1) led to a pronounced inhibition of Th2 development, which is reflected by a strongly reduced secondary production of IL-4 (Fig. 1A), IL-5, IL-9, and IL-10 (Table I). In this context it should be mentioned that none of these Th2-type cytokines could be detected in the supernatant of activated CD25+ Treg cells. The inhibitory capacity of the freshly isolated TCR-tg CD25+ Treg cells is contact dependent, because the physical separation of conventional CD4+ T cells and CD25+ Treg cells in Transwell experiments completely abrogated the differentiation-inhibiting properties of the CD25+ Treg cells (Fig. 1B). These findings indicate that CD25+ Treg cells inhibit Th2 differentiation by the same contact-dependent suppressive mechanism that has been observed with respect to the suppression of freshly isolated conventional CD4+ T cells and Th1 cells (5, 32). As mentioned above, it has been suggested that the suppressive capacity of CD25+ Treg cells might be mediated by IL-10, membrane-bound TGF-, or CTLA-4 (10, 11, 33). However, convincing data demonstrated the opposite (13, 14, 15). In addition, we found that activation of CD25+ Treg cells did not induce the production of detectable amounts of TGF- or IL-10 (data not shown). Thus, it is rather unlikely that the suppressive mechanism of CD25+ Treg cells is based on IL-10 and TGF-, as has been described for Tr1 cells and Th3 cells, respectively (34, 35).
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mAb. Restimulation of the purified conventional CD4+ T cells revealed that they also produce strongly reduced amounts of IL-4 when the CD25+ Treg cells were removed after 5 days of coculture. This result unequivocally demonstrates that Th2 development can be profoundly inhibited by CD25+ Treg cells (Fig. 1C). CD25+ Treg cells strongly suppress the proliferation of conventional CD4+ T cells. Hence, their suppressive capacity for the development of Th2 cells may simply be based on this proliferation-inhibiting property. Therefore, the same CD4+ T cells that had been used for the experiment illustrated in Fig. 1C were quantified after depletion from CD25+ Treg cells on day 5 of priming. Fig. 1D clearly shows that in the presence of IL-4, there is no significant difference in the number of CD4+ cells primed in the presence or the absence of CD25+ Treg cells. This result is confirmed by measuring proliferation using CSFE-labeled CD4+ T cells (see Fig. 5A). In addition, an intensive proliferation of CD4+ T cells is obviously not a prerequisite for Th2 differentiation because it has been shown by Richter et al. (36) that the development of IL-4-producing cells does not depend on cell proliferation. These authors have demonstrated that primary activation in the presence of IL-4 led within 42 h to the development of CD4+ T cells that produce IL-4 upon restimulation, also within the population of cells that had not yet divided but had entered the S phase of the first cell cycle.
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It has been demonstrated in humans that cocultivation of conventional CD4+ T cells with peripheral CD25+ Treg cells resulted in the conveyance of suppressive properties from CD25+ Treg cells to the conventional CD4+ T cells (13). In a similar approach, conventional murine CD4+ T cells were cocultivated for 5 days with CD25+ Treg cells under Th2-priming conditions. The resulting CD4+ T cells, termed CD4sup, were subsequently depleted from these Treg cells and finally stimulated alone or in combination with freshly isolated conventional CD4+ T cells. Fig. 2 shows that CD4+sup exerted a comparatively low proliferation and had only a very limited suppressive capacity regarding the proliferation of the coactivated conventional CD4+ T cells. In contrast, CD25+ Treg cells that were used as a positive control strongly suppressed the proliferation of conventional CD4+ T cells. Thus, CD4+sup had an anergic phenotype with respect to proliferation and cytokine production, but they had not gained profound suppressive properties. Nevertheless, preliminary data revealed that activation of CD4sup in the presence of IL-4 overrode their anergic state and gave rise to the development of Th2 cells (data not shown).
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In an additional approach, TCR-tg CD25+ Treg cells were preactivated by stimulation with a combination of anti-CD3 and anti-CD28 mAb for 2 days. Such preactivated CD25+ Treg cells (CD25+ preTreg cells) were subsequently coactivated with conventional CD4+ T cells under Th2-inducing conditions. Fig. 3A clearly illustrates that CD25+ preTreg cells exhibit a strong inhibitory potency on the development of Th2 cells that was at least comparable to that of freshly isolated CD25+ Treg cells. These data corroborate findings that demonstrated that preactivated CD25+ Treg cells retained their suppressive potency even after a vigorous polyclonal stimulus (3, 4).
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The proliferative response, just as their cytokine production, of activated Th2 cells are of comparatively rapid kinetics. In addition, it is conceivable that rather low amounts of endogenous IL-4 can induce considerable proliferation of activated Th2 cells. Thus, high concentrations of OVA323–339 (1–3 µg/ml) that induce the production of low, but significant, amounts of IL-4 even in the presence of CD25+ Treg cells might override the potential suppressive capacity of CD25+ Treg cells or CD25+ preTreg cells regarding the proliferation of Th2 cells. Therefore, Th2 cells were activated with low amounts of OVA323–339 (27–729 ng/ml) to investigate whether CD25+ Treg cells or CD25+ preTreg cells can inhibit the proliferation of activated Th2 cells. Fig. 4 clearly demonstrates that, in analogy to cytokine production of Th2 cells, freshly isolated CD25+ Treg cells could not inhibit the proliferation of Ag-activated Th2 cells. In contrast, CD25+ preTreg cells significantly reduced the proliferation of the coactivated Th2 cells. These results confirm previous data showing that the suppressive potency of CD25+ preTreg cells exceeds that of freshly isolated CD25+ Treg cells (4, 32). In addition, these findings strongly support and emphasize in vivo data demonstrating that the elimination of CD25+ Treg cells profoundly increased the Th2 response of BALB/c mice to Leishmania major, leading to an exacerbated infection (18). The authors assumed that the suppression of the immediate L. major-induced production of IL-4 that ultimately leads to the development of Th2 cells was the pivotal impact of the CD25+ Treg cells in this context (37). Our results are in agreement with this assumption and clearly demonstrate, in addition, that CD25+ preTreg cells not only can inhibit IL-4 production, but also can directly suppress the development of Th2 cells even in the presence of high amounts of IL-4. Finally, it was reported very recently that Tr1 cells or CD4+ T cells engineered to produce IL-10 can also inhibit Th2 cell activation in vivo via the production of IL-10 (38, 39). This implies that at least two populations of regulatory T cells, Tr1 cells and CD25+ Treg cells, can participate in the suppression of Th2-mediated disorders.
CD25+ Treg induce the expression of FoxP3 in naive CD4+ T cells and Th2 cells
It has recently been well documented that FoxP3, a transcription factor of the forkhead family, is strongly expressed in CD25+ Tregs and is required for both their development and function, but the underlying molecular mechanisms are still elusive (40, 41, 42). Furthermore, it has been reported that upon overexpression in CD4+ T cells, FoxP3 attenuates activation-induced cytokine production and proliferation (40, 43). Transcriptional repression by FoxP3 is probably achieved via binding to a consensus sequence adjacent to NFAT sites critical for cytokine gene transcription (43). Therefore, FoxP3 appeared to be a candidate able to suppress conventional CD4+ T cells in the presence of IL-4 and Th2 cells upon contact with CD25+ Treg.
To investigate this hypothesis, we performed coculture experiments using CFSE-labeled conventional CD4+ T cells in combination with either unlabeled CD25+ Treg cells or unlabeled conventional CD4+ T cells in a ratio of 1:1 under Th2-inducing conditions. CD4+ T cells cultured with CD25+ Treg cells in the presence of IL-4 showed only slightly reduced proliferation compared with CD4+ T cells activated without Treg (Fig. 5A, day 3 of coculture; see also Fig. 1D). To analyze the expression of FoxP3, we FACS-sorted the CFSE-labeled CD4+ T cells corresponding to M1 and M2 in Fig. 5A and conducted RT-PCR. As shown in Fig. 5B, FoxP3 was indeed expressed in CD4+ T cells that had been costimulated with CD25+ Treg cells, whereas the expression of IL-4 mRNA was abrogated. However, a low basal level of FoxP3 mRNA was also detectable in CD4+ T cells used as a reference. Using this experimental approach, we analyzed the expression of FoxP3 by real-time PCR in CFSE-labeled CD4+ T cells that had been costimulated with either freshly isolated CD25+ Treg or CD25+ preTreg cells. As detailed in Fig. 5C, the expression of FoxP3 was even more pronounced in CD4+ T cells that had been cocultured with CD25+ preTreg cells. Nevertheless, such cocultivated CD4+ T cells only expressed 
40% of the FoxP3 mRNA level as CD25+ Treg cells (see Figs. 5C and 6A).
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Using retroviral transduction or transgenic mice, several groups recently reported that CD4+CD25– T cells overexpressing FoxP3 acquire suppressive activity (40, 41, 42). However, it should be noted that such genetically engineered T cells are not as efficient as naturally occurring CD25+ Treg cells, although CD4+CD25– T cells from FoxP3 transgenic mice produced even more FoxP3 mRNA than CD25+ Treg cells from their littermate controls (41). Against this background we propose that under physiological conditions the induction of FoxP3 expression in CD4+ T cells upon contact with activated CD25+ Treg might be involved in the suppression of cytokine production and the proliferation of the former population, as has been demonstrated using conventional CD4+ T cells (40, 43). Obviously, as shown in Fig. 2, such conditions are insufficient to confer profound suppressive properties to cocultivated conventional CD4+ T cells as has been shown in humans (13, 44).
In conclusion, we could clearly demonstrate that CD25+ preTreg cells can profoundly inhibit the differentiation, cytokine production, and proliferation of Th2 cells and simultaneously induce a strong expression of FoxP3 in such cells. Thus, at least CD25+ preTreg cells represent a new and promising therapeutic option for the causative treatment of Th2-mediated diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Edgar Schmitt, Institute of Immunology, Hochhaus am Augustusplatz, D-55101 Mainz, Germany. E-mail address: eschmitt{at}mail.uni-mainz.de
3 Abbreviations used in this paper: Treg, T regulatory; tg, transgenic; HGPRT, hypoxanthine-guanine phosphoribosyltransferase.
Received for publication October 20, 2003. Accepted for publication April 23, 2004.
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References |
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E7 identifies unique subsets of CD25+ as well as CD25– regulatory T cells. Proc. Natl. Acad. Sci. USA 99:13031.. J. Exp. Med. 194:629.1 production and responsiveness. J. Exp. Med. 196:237. and IL-4, and is inhibited by IFN-. J. Immunol. 153:3989.[Abstract]
-secreting Th3 regulatory cells. Immunol. Rev. 182:207.[Medline]
4V8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6:541.[Medline]
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P. Yu, R. K. Gregg, J. J. Bell, J. S. Ellis, R. Divekar, H.-H. Lee, R. Jain, H. Waldner, J. C. Hardaway, M. Collins, et al. Specific T Regulatory Cells Display Broad Suppressive Functions against Experimental Allergic Encephalomyelitis upon Activation with Cognate Antigen J. Immunol., June 1, 2005; 174(11): 6772 - 6780. [Abstract] [Full Text] [PDF]
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C. T. Duthoit, D. J. Mekala, R. S. Alli, and T. L. Geiger Uncoupling of IL-2 Signaling from Cell Cycle Progression in Naive CD4+ T Cells by Regulatory CD4+CD25+ T Lymphocytes J. Immunol., January 1, 2005; 174(1): 155 - 163. [Abstract] [Full Text] [PDF]
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) or together with freshly isolated TCR-tg CD25+ Treg cells (2 x 104/ml; 
) or preactivated TCR-tg CD25+ Treg cells (2 x 104/ml; 
) in the presence of the indicated concentrations of OVA323–339 and A20 cells (2 x 103/ml) as APC. Proliferation was assessed after 4 days. Shown are the means of two experiments performed in triplicate ± SD.
