Fetal BM-derived mesenchymal stem cells ... - Wiley Online Library

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DOI 10.1002/eji.200839070

Eur. J. Immunol. 2009. 39: 2840–2849

Fetal BM-derived mesenchymal stem cells promote the expansion of human Th17 cells, but inhibit the production of Th1 cells Zhenxing Guo1,2, Cuiling Zheng1,3, Zhenping Chen1,4, Dongsheng Gu1, Weiting Du1, Jing Ge1, Zhongchao Han1 and Renchi Yang1 1

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, P. R. China 2 Department of Hematology/Oncology, First Hospital of Tsinghua University, Beijing, P. R. China 3 Clinical Laboratory, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China 4 Hematology Center, Beijing Children’s Hospital Affiliated with Capital Medical University, Beijing, P. R. China Th type 17 (Th17) cells have been identified as a proinflammatory T-cell subset. Here, we investigated the regulation of human Th17 cells by fetal BM-derived mesenchymal stem cells (FBM-MSC). We cocultured FBM-MSC with human PBMC or CD41 T cells from healthy donors. FBM-MSC significantly suppressed the proliferation of CD41 T cells stimulated by PHA and recombinant IL-2. Significantly higher levels of IL-17 were observed in FBM-MSC cocultured with either PBMC or CD41 T cells than that in PBMC cultured alone or CD41 T cells cultured alone. Flow cytometry analysis showed that the percentage of Th17 cells in coculture of FBM-MSC and CD41 T cells was significantly higher than that in CD41 T-cell cultured alone. FBM-MSC did not express IL-17 protein. Consistent with the augmentation of Th17 cells, significantly higher levels of IL-6 and IL-1 were observed in coculture of FBMMSC and CD41 T cells than that in CD41 T-cell culture, while the levels of IL-23 were similar between FBM-MSC 1 PBMC coculture and PBMC alone, or FBM-MSC 1 CD41 T-cell and CD41 T-cell alone. The presence of FBM-MSC decreased the percentage of Th1 cells, but minimally affected the expansion of CD41CD251 T cells. In conclusion, our data demonstrate for the first time that FBM-MSC promote the expansion of Th17 cells and decrease IFN-c-producing Th1 cells. These data suggest that IL-6 and IL-1, instead of IL-23, may be partly involved in the expansion of Th17 cells.

Key words: Fetal BM-derived mesenchymal stem cells . IL-1 . IL-6 . Th1 . Th17

Introduction Mesenchymal stem cells (MSC) are multipotent stem cells found in many tissues in the body, such as BM, muscle, adipose, synovial membrane, amniotic fluid [1–4], umbilical cord, cord blood [5]

and fetal tissues [6]. Accumulating evidence has shown that MSC perform an immune regulatory function partly by suppressing the proliferation of T cells in vitro [7–9]. MSC-induced immunotolerance has been used therapeutically for reducing GVH disease and for modulation of autoimmune disorders [10, 11]. Classically, effector CD41 Th cells have been categorized into two subsets: Th1 and Th2. Th1 cells produce IFN-g, which is

Correspondence: Professor Renchi Yang e-mail: [email protected]

These authors contributed equally to this work.

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Immunomodulation

Figure 1. Immunophenotype of FBM-MSC. Flow cytometry analysis of FBM-MSC (passage 5) labeled with Ab against antigens: CD90, HLA-ABC, HLA-DR, CD34, CD45, CD29, CD13, CD14, CD166, CD31, CD133, CD105, CD44, CD73, CD117, CD54, CD49e and CD106. Empty profiles indicate fluorescence intensity of FBM-MSC labeled with isotype control Ab only; black profiles, positive reactivity with the indicated Ab. Histograms represent relative number of cells versus fluorescence intensity and the percentage of positive cells for each specific Ab (black profiles) is also annotated.

required for the clearance of certain intracellular pathogens; while Th2 cells produce IL-4, which is required for the clearance of parasites [12]. Recently, a new effector CD41 T-cell lineage, Th17, has been identified in an EAE model. Th17 cells are characterized by their production of a distinct profile of cytokines, including IL-17, IL-17F and IL-6, and are thought to be involved in inflammatory and autoimmune diseases [13, 14]. There have been many investigations in mouse Th17 cells, but the knowledge of human Th17 cells remains limited. So far little is known about the interaction of fetal BM-derived MSC (FBM-MSC) with immune cells, especially human Th17 cells. Therefore, in this study, we cocultured FBM-MSC with human PBMC or CD41 T cells and demonstrated for the first time


that FBM-MSC had the capability to promote the expansion of Th17 cells and that IL-6 and IL-1 might partly promote to the augmentation of Th17 cells. We also showed that FBM-MSC decreased the percentage of Th1 cells.

Results Characterization of FBM-MSC FBM-MSC were spindle-shaped cells and had the capacity to adhere to plastic in culture. Immunophenotype analysis indicated that they had high expression of CD44, CD29, CD73, CD90, CD13, CD49e, CD105, CD166 and HLA-ABC, moderate

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Figure 2. Multilineage differentiation of FBM-MSC. Histochemical staining demonstrated the multilineage differentiation potential of FBM-MSC into adipocytes (A) by Oil Red O staining, osteoblasts (B) by von Kossa staining, and chondroblasts (C) by Alcian Blue staining. Original magnification: 400, scale bar: 50 mm. (D) Multilineage differentiation of FBM-MSC was assessed at the protein levels of the adipocyte-specific marker PPAR-g, osteoblast-specific marker osteocalcin and the chondrogenic-specific marker collagen II by flow cytometry. Undifferentiated FBMMSC were used as a control. Histograms represent relative number of cells versus fluorescence intensity and the positive rates of specific Ab (black profiles) are annotated.

FBM-MSC suppress the proliferation of CD41 T cells stimulated by PHA and recombinant IL-2 In the presence of PHA and recombinant IL-2 (rIL-2) (PHA1 rIL-2), 105 CD41 T cells were cultured with different ratios of FBM-MSC (CD41:FBM-MSC, 104:1, 103:1, 102:1, and 10:1). As shown in Fig. 3, FBM-MSC inhibited the proliferation of CD41 T cells at all these ratios (po0.05) and had the strongest inhibitory effect at the proportion of 10:1 (CD41:FBM-MSC). Therefore, the 10:1 optimum ratio was used for coculture. Figure 3. Inhibitory effect of FBM-MSC on CD41 T cells proliferation stimulated by PHA1rIL-2. The proliferation of 105 purified CD41 T cells in the presence of different numbers of FBM-MSC (104, 103, 102, 10 and 0) was evaluated by BrdU ELISA Kit. The percentage of BrdU-positive cells was obtained in four separate experiments from four different healthy donors. Error bars represent SD. p-Values were calculated with Student’s t-test. po0.01, po 0.05, MSC versus CD41 T cells, ‘‘Medium’’ refers to CD41 T cells alone.

expression of CD54 and CD106, and low expression of CD34, CD45, CD14, CD31, CD133, CD117 and HLA-DR (Fig. 1). They also demonstrated multilineage differentiation into mesodermtype cells such as osteoblasts, adipocytes and chondrocytes (Fig. 2). In addition, they retained a normal karyotype of 46, XX after several passages (data not shown).


FBM-MSC promote the expansion of Th17 cells Semi-quantitative RT-PCR analysis revealed that the mRNA expression of IL-17 was not detectable in freshly isolated PBMC but was elevated in PBMC stimulated by PHA1rIL-2 (Fig. 4A). As shown in Fig. 4B, compared with unstimulated cultures, treatment with PHA1rIL-2 increased the percentage of Th17 cells in CD41 T-cell cultures (0.7270.82% versus 2.3970.80%, po0.01) and in cocultures of FBM-MSC and CD41 T cells (FBM-MSC/CD41) (1.2570.90% versus 4.5371.27%, po0.01), as determined by intracellular cytokine analysis of IL-17. And therefore, in the following experiments all the cultures were stimulated by

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Immunomodulation

PHA1rIL-2. In our coculture system, PBMC or CD41 T cells were cultured alone or cocultured with FBM-MSC at the ratio of 10:1 (PBMC or CD41 T:FBM-MSC) for 4 days. To our surprise, IL-17 mRNA expression was higher in FBM-MSC cocultured with PBMC (FBM-MSC/PBMC) compared with PBMC (relative value 1.2770.61 versus 0.5570.57; po0.01) (Fig. 4C). IL-17 transcript expression correlated with the secretion of IL-17 protein in cell cultures as assayed by ELISA:secretion in FBM-MSC/PBMC: 330.997313.38 pg/mL; FBM-MSC/CD41:388.457118.11 pg/mL was significantly higher than that in PBMC (122.927156.37 pg/ mL, po0.05) or CD41 T cells (142.71766.00 pg/mL, po0.01) (Fig. 4D). Furthermore, we found that the percentage of Th17 cells in FBM-MSC/CD41 was significantly higher than that in CD41 T-cell cultures (4.5371.27% versus 2.3970.80%, po0.01) (Fig. 4E-F). FBM-MSC by itself or stimulated by PHA1rIL-2 for 4 days lacked the expression of IL-17 as detected by quantitative RT-PCR (qRT-PCR), ELISA and flow cytometry (data not shown). The data therefore suggest that FBM-MSC promote the expansion of Th17 cells.

IL-6 might promote the augmentation of Th17 cells mediated by FBM-MSC In order to better understand the mechanisms of FBM-MSCmediated immunoregulatory action on human Th17 cells, we studied IL-6, an important cytokine to promote Th17-cell differentiation [15, 16]. By qRT-PCR analysis, we analyzed the expression of IL-6 in PBMC (relative value 0.3670.02) and FBM-MSC (relative value 2.1070.98). However, the production of IL-6 in FBM-MSC/PBMC (relative value 26.95714.47) was significantly higher than that in PBMC (po0.05) (Fig. 5A) or FBM-MSC (po0.05) (Fig. 5B). Furthermore, exogenous IL-6 increased the percentage of Th17 cells in CD41 T-cell cultures (po0.01) (Fig. 5C), while IL-6 Ab decreased the percentage of Th17 cells by intracellular cytokine analysis of IL-17 (anti-IL-6 Ab:3.2670.79%; without anti-IL-6 Ab:4.7171.35%, po0.05) in FBM-MSC/CD41 (Fig. 5D). At the same time, the simultaneous addition of IL-6 and TGF-b1 could revert the effect of IL-6 (po0.05), and TGF-b1 decreased the percentage of Th17 cells in the absence of IL-6 (po0.05) (Fig. 5C), consistent with the findings of Amadi-Obi et al. [17] and Acosta-Rodriguez [18]. Also, secretion of IL-6 in the supernatants of FBM-MSC was significantly lower than that in FBM-MSC/CD41 (109.65729.74 pg/mL versus 536.237 28.28 pg/mL, po0.01) (Fig. 5E). Taken together, our data suggest that FBM-MSC might promote the expansion of Th17 cells in part through the up-regulation of IL-6.

IL-1 might be involved in the expansion of Th17 cells mediated by FBM-MSC Similar to IL-6, IL-1 was reported to have a positive effect on the differentiation of Th17 cells [18]. Consequently, the present study


Figure 4. The expression level of IL-17 in different culture groups. (A) The expression of IL-17 transcripts in freshly isolated PBMC and PHA1rIL-2-stimulated PBMC was detected by semi-qRT-PCR. Data are representative of three independent experiments. ‘‘_’’ 5 freshly isolated PBMC, ‘‘1’’ 5 PHA1rIL-2-stimulated PBMC. (B) Intracellular cytokine analysis of the percentage of Th17 cells in CD41 T-cell culture or coculture of FBM-MSC/CD41 in the absence or presence of PHA1 rIL-2 (n 5 6), po0.01, ‘‘Control’’ 5 absence of PHA1rIL-2. (C) qRT-PCR analysis of IL-17 transcripts in PBMC and FBM-MSC/PBMC (n 5 7). (D) Detection of IL-17 secretion in PBMC and FBM-MSC/PBMC, or CD41 T-cell cultures and FBM-MSC/CD41 by ELISA (for PBMC, n 5 14; for CD41 T cells, n 5 6), po0.01, po0.05. (E and F) Intracellular cytokine analysis of Th17 cells in CD41 T-cell cultures and FBM-MSC/CD41 cocultures. (E) Representative data and the percentage of cells in the quadrants; (F) summary of data from six separate experiments, po0.01. Data show mean7SD. p-Values were calculated with Student’s t-test.

evaluated the cytokine secretion of IL-1 in the culture supernatants by ELISA. Significantly higher levels of IL-1 were observed in the supernatants of FBM-MSC/CD41 cocultures than that in CD41 T-cell cultures (11 654.1971605.15 pg/mL versus 9147.237 1499.29 pg/mL, po0.01) (Fig. 6A). However, FBM-MSC alone www.eji-journal.eu

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Figure 6. The expression level of IL-1 in different culture groups. (A) Compared with FBM-MSC, the detection of IL-1 secretion in CD41 T-cell cultures and FBM-MSC/CD41 cocultures by ELISA (n 5 6), po0.01. (B) Intracellular cytokine analysis of the percentage of Th17 cells in FBM-MSC/CD41 cocultures in the absence or presence of antiIL-1 Ab (n 5 4), po0.05. Data show mean7SD. p-Values were calculated with Student’s t-test.

Figure 5. The expression level of IL-6 in different culture groups. (A) qRT-PCR analysis of IL-6 transcripts in PBMC and FBM-MSC/PBMC (n 5 3). (B) The different expression level of IL-6 transcripts of FBMMSC, compared with PBMC and FBM-MSC/PBMC, po0.05. (C) Intracellular cytokine analysis of the percentage of Th17 cells in CD41 T-cell cultures in the presence of various combinations of cytokines (n 5 4), po0.01, po0.05. ‘‘Control’’ 5 with PHA1rIL-2. (D) Intracellular cytokine analysis of the percentage of Th17 cells in FBM-MSC/CD41 in the absence or presence of anti-IL-6 Ab (n 5 4), po0.05. (E) Detection of IL-6 secretion in FBM-MSC and FBM-MSC/CD41 by ELISA (n 5 4), po0.01. Data show mean7SD. p-Values were calculated with Student’s t-test.

did not secrete IL-1 protein by ELISA (Fig. 6A). Furthermore, in FBM-MSC/CD41, anti-IL-1 Ab decreased the percentage of Th17 cells by intracellular cytokine analysis of IL-17 (anti-IL-1 Ab:3.2970.21%; without anti-IL-1 Ab:4.0070.12%, po0.05) (Fig. 6B). These data indicate that IL-1 might be involved in the expansion of Th17 cells mediated by FBM-MSC.

Figure 7. The expression level of IL-23 detected by qRT-PCR and ELISA analysis. (A) qRT-PCR analysis of IL-23 transcripts in PBMC and FBMMSC/PBMC (n 5 6) (p 5 0.826). (B) Detection of IL-23 secretion in CD41 T-cell cultures and FBM-MSC/CD41 cocultures by ELISA (n 5 6) (p 5 0.065), NS denotes no significant statistical difference. Data show mean7SD. p-Values were calculated with Student’s t-test.

MSC/PBMC (relative value 2.1470.93) (p 5 0.826) (Fig. 7A). In addition, IL-23 transcripts of FBM-MSC were much lower, in comparison with PBMC or FBM-MSC/PBMC (data not shown). ELISA analysis showed that the concentration of IL-23 in the supernatants between FBM-MSC/CD41 (1469.207160.03 pg/ mL) and CD41 T-cell cultures (1346.587176.56 pg/mL) was not significantly different (p 5 0.065) (Fig. 7B). In addition, FBM-MSC alone did not secrete IL-23 protein by ELISA (data not shown). These results suggest that IL-23 might not be involved in the expansion of Th17 cells mediated by FBM-MSC.

FBM-MSC decrease the production of Th1 cells IL-23 might not be involved in the expansion of Th17 cells mediated by FBM-MSC qRT-PCR analysis demonstrated no significant difference in IL-23 expression between PBMC (relative value 2.2671.99) and FBM-


The expression of IFN-g transcripts was detected by qRT-PCR in both FBM-MSC/PBMC and PBMC. The expression of IFN-g in FBM-MSC/PBMC was lower, without significant difference, than that in PBMC (relative value 0.8971.11 versus 4.0876.34,

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Immunomodulation

Figure 8. The expression level of IFN-g in different culture groups. (A) qRT-PCR analysis of IFN-g transcripts in PBMC and FBM-MSC/PBMC (n 5 7). (B) Detection of IFN-g secretion in CD41 T-cell cultures and FBM-MSC/CD41 cocultures by ELISA (n 5 6), po0.01. (C) Intracellular cytokine analysis of Th1 cells in CD41 T-cell cultures and FBM-MSC/CD41 cocultures (n 5 6), po0.01. Data show mean7SD. p-Values were calculated with Student’s t-test.

Discussion

Figure 9. Flow cytometry analysis of CD41CD251 in CD41 T-cell cultures and FBM-MSC/CD41 cocultures. Data are representative of six separate experiments (p 5 0.052). Data show mean7SD. p-Values were calculated with Student’s t-test. NS denotes no significant statistical difference.

p 5 0.213) (Fig. 8A). Consistent with the RNA data, secretion of IFN-g as assayed by ELISA was significantly lower in FBM-MSC/CD41 cocultures than that in CD41 T-cell cultures (81.44736.32 pg/mL versus 131.03751.44 pg/mL, po0.01) (Fig. 8B). In addition, we detected the production of IFN-g in CD41 T cells by intracellular cytokine analysis. Interestingly, we found that the percentage of IFN-g-producing CD41 T cells (Th1) was decreased in FBM-MSC/CD41 cocultures compared with CD41 T-cell cultures (4.8770.64% versus 7.0071.15%, po0.01) (Fig. 8C). These findings indicate that FBM-MSC have a suppressive effect on Th1 cells.

Limited role of FBM-MSC in the expansion of CD41 CD251 T cells Similar percentage levels of CD41CD251 T cells were found in CD41 T-cell cultures (66.61718.14%) and cocultures of FBMMSC/CD41 (84.7573.31%) by flow cytometry analysis (p 5 0.052) (Fig. 9). Our data therefore suggest that FBM-MSC might play a negligible role in the expansion of CD41CD251 T cells.


Many studies in vitro and in vivo have highlighted the immunoregulatory properties of MSC. MSC have been shown to suppress the proliferation of T cells, B cells and NK cells induced by allogeneic PBMC as well as inhibit the maturation of DC [8, 19–21]. In the present study, we have examined the immunoregulatory functions of FBM-MSC by coculturing them with human PBMC or CD41 T cells, and demonstrated for the first time that FBM-MSC were capable of promoting the expansion of Th17 cells. Although suppressive activities of MSC on the proliferation of lymphocytes have been reported in previous studies, the effect of FBM-MSC on the proliferation of human CD41 T cells is still unknown. We found significant suppression by FBM-MSC on the proliferation of CD41 T cells, even at the ratio of 104:1 (CD41:FBM-MSC). MSC might inhibit T-cell proliferation through ‘‘cell to cell’’ contact and/or soluble molecules [8, 19, 22, 23]. IL-17 has pleiotropic activities, inducing the expression of proinflammatory cytokines (such as IL-6 and TNF), chemokines (such as MCP-1, KC and MIP-2) and matrix metalloproteases, leading to tissue destruction [24]. Evidences suggest that IL-17 is associated with several human autoimmunity diseases, such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematous [25, 26]. Therefore, IL-17 may have therapeutic importance for autoimmune diseases. MSC treatment is considered an attractive novel modality for the modulation of autoimmune diseases. Their immunosuppressive properties have already been applied to the clinical setting with preliminary clinical trials showing safety-encouraging efficacy [10, 27–29]. Based on immunosuppressive function and clinical efficacy of MSC, we hypothesized that FBM-MSC might suppress the function of Th17 cells. To our surprise, by qRT-PCR analysis, we found that the expression level of IL-17 in FBM-MSC/PBMC was significantly higher than that in PBMC. The increased IL-17 RNA transcripts were supported by higher protein secretion as evaluated by ELISA assay. We further confirmed that the increased

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IL-17 was produced by Th17 cells. However, IL-17 was not expressed by FBM-MSC. Therefore, we provided evidence for the first time that FBM-MSC promoted the expansion of Th17 cells, a T-cell subset that induces severe autoimmune disease. We then explored the potential mechanisms of FBM-MSC promotion of Th17-cell expansion in the coculture system. Recent reports proposed that IL-6 was an important cytokine for promoting the differentiation of Th17 cells [15, 16]. Thus, we postulated that IL-6 signal might be involved in the expansion of Th17 cells mediated by FBM-MSC. Our data demonstrated that IL-6 might play an important role in the augmentation of Th17 cells. The expression of IL-6 in FBM-MSC/PBMC was much higher than that in FBM-MSC or PBMC, which was in agreement with recent data showing that up-regulation of IL-6 was observed in the supernatants of a MSC/PBMC coculture model [8]. Furthermore, blocking studies showed that the expansion of Th17 cells could be partly inhibited by IL-6 specific Ab. Based on the recent known evidence [15, 16], we considered that IL-6 might be partly involved in the expansion of Th17 cells mediated by FBM-MSC. In addition, Sallusto and coworkers [18] suggested that IL-1 was essential for the differentiation of human Th17 cells. In our study, the protein level of IL-1 in cocultures of FBMMSC/CD41 was higher than that in CD41 T-cell cultures. Similar to anti-IL-6 Ab, the expansion of Th17 cells could be partly inhibited by anti-IL-1 Ab. This, to some extent, confirmed the importance of IL-1 in the augmentation of Th17 cells and suggested that IL-1 might possibly be involved in the mechanisms for the expansion of Th17 cells mediated by FBM-MSC. As for IL-23, controversial effects were reported on driving the differentiation of human Th17 cells. Both Wilson et al. [30] and Chen et al. [31] described that IL-23 alone was more effective in promoting human Th17-cell differentiation. In contrast, Sallusto and coworkers [18] showed that IL-23 alone poorly induced the production of Th17 cells. In the present study, there was no statistical difference in the expression of IL-23 between FBMMSC/PBMC and PBMC, or FBM-MSC/CD41 and CD41 T-cell cultures, suggesting that IL-23 might not play a role in the MSCmediated augmentation of Th17 cells. We also investigated the effects of FBM-MSC on Th1 and CD41 CD251 T cells. We confirmed that FBM-MSC significantly suppressed the expansion of Th1 cells, consistent with Aggarwal and Pittenger’s results [8]. In contrast, we found that FBM-MSC might have a limited effect on the expansion of CD41CD251 T cells. In conclusion, our data suggest for the first time that FBM-MSC are able to promote the expansion of Th17 cells, decrease the percentage of Th1 cells, but play a negligible role in expanding CD41CD251 T cells. Our data also indicate that FBM-MSC might promote the expansion of Th17 cells partly through increasing expression of IL-6 and IL-1, but without the involvement of IL-23. Further exploration into the potential pathways and other mechanisms in addition to IL-6 and IL-1 are needed. Given the highly proinflammatory and severe autoimmune-inducing nature of Th17 cells, the mechanism underlying the immunoregulatory function of FBM-MSC remains to be fully elucidated.


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Materials and methods Isolation and identification of FBM-MSC The use of fetal BM for the purpose of research was approved by the Medical Ethics Review Board of the Chinese Academy of Medical Sciences and Peking Union Medical College. Pregnant women gave written informed consent. Fetal BM was obtained from fetuses at 15 wk gestation, whose tissues would otherwise be discarded. BM was collected from fetal long bones and the mononuclear cell fraction was isolated by using Ficoll-Paque gradient centrifugation (specific gravity 1.077 g/mL; Hao Yang Biological Manufacture, Tianjin, China). The isolation and identification of FBM-MSC were performed as described previously [32]. Briefly, human FBM-MSC were separated by their adherence on plastic flasks, while the non-adherent cells were discarded after 3 days. The cells were cultured in DF12 (Gibco Life Technologies, Paisley, UK) culture medium containing 40% MCDB-201 (Sigma, St. Louis, USA), 2% FCS (Hyclone, UT, USA), 1 ITS (Gibco Life Technologies), 108 M dexamethasone (Sigma), 50 U/mL penicillin/streptomycin, 10 ng/mL human basic-fibroblast growth factor, 10 ng/mL human EGF (Peprotech, London, UK) and 2 mM L-glutamine (Gibco Life Technologies). At about 80–85% confluency, the adherent cells were detached by treatment with 0.125% trypsin and 0.1% EDTA (Sigma) and replated at a 1:3 dilution under the same culture conditions. At passage 5, the cells were analyzed for the surface markers: CD90, HLA-ABC, HLA-DR, CD45, CD34, CD29, CD13, CD14, CD166, CD31, CD133, CD105, CD44, CD73, CD117, CD54, CD49e (all from BD PharMingen, USA), and CD106 (Ancell, Bayport, MN, USA). To evaluate mutilineage differentiation capacity, FBM-MSC (passage 5) were seeded into 6-well plates and induced into adipocytes and osteoblasts, while the cell pellet of FBM-MSC was cultured in chondrogenic medium for chondrogenesis, according to the previous description [33]. Undifferentiated FBM-MSC under growth conditions were used as control. The differentiation medium was changed every 3–4 days. After 21 days, they were analyzed by Oil Red-O staining, von Kossa staining and Alcian Blue staining to demonstrate neutral lipid vacuoles in adipocytes, calcium deposition in osteocytes and extracellular matrix mucopolysaccharides of chondrogenic cells, respectively. In addition, the expression of adipocyte-specific marker PPAR-g, osteoblast-specific marker osteocalcin and cartilage-specific marker collagen II (all from Santa Cruz Biotechnology, California, USA) in the differentiated and undifferentiated FBM-MSC were assessed by flow cytometry.

Cells isolation and coculture PBMC were obtained from healthy adult donors after written, informed consent was obtained from all subjects. PBMC were isolated from heparinized venous blood by Ficoll-Paque gradient centrifugation. CD41 T cells were purified by

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anti-CD4 mAb-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions, and the purity was consistently 498%. PBMC and CD41 T cells, in the coculture medium, were cultured alone or cocultured with FBM-MSC at the ratio of 10:1(PBMC or CD41: FBM-MSC). The coculture medium was composed of IMDM (Gibco Life Technologies), 20% fetal equine serum (Keji, Tianjin, China), 50 U/mL penicillin/streptomycin, 55 mM 2-mercaptoethanol (Gibco Life Technologies). PBMC or CD41 T cells in the absence or presence of FBM-MSC were stimulated with 5 mg/mL PHA (Roche, Penzberg, Germany) and 5 ng/mL of rIL-2 (Peprotech) for 4 days at 371C, with 5% CO2. In some experiments, CD41 T cells were directly cultured with FBM-MSC in the absence of PHA1rIL-2. In another series of experiments, we added exogenous 200 IU/mL IL-6 with or without 100 IU/mL TGF-b1 (Peprotech) in PHA1rIL-2 stimulated CD41 T cells cultured alone, or 10 mg/mL antiIL-6 Ab (eBioscience, CA, USA) or 10 mg/mL anti-IL-1 Ab (R&D Systems) in PHA1rIL-2 stimulated FBM-MSC/CD41 coculture.

Immunomodulation

IL-23p19 b-actin

Sense Anti-sense Sense Anti-sense

50 -TGTGGAGATGGCTGTGAC-30 , 50 -GGACTGAGGCTTGGAATC-30 ; 50 -GGCACCCAGCACAATGAAG-30 , 50 -CGTCATACTCCTGCTTGCTG-30 .

To control the specificity of amplification products, a melting curve analysis was performed. The quantity of target mRNA expression was normalized to b-actin mRNA expression, as shown by the equation Relative quantity (%) 5 2DDCt 100%.

Cytokine quantification by ELISA The concentrations of IL-17, IL-6, IL-1, IL-23 and IFN-g in the supernatants were assayed by ELISA using specific kits according to the manufacturer’s instructions (R&D Systems). The experiments were performed in duplicate to ensure the reproducibility.

Intracellular cytokine analysis by flow cytometry Proliferation assay For proliferation studies, different numbers of human FBM-MSC (104, 103, 102, 10/well) were plated in quadruplicate into 96-well plates and were irradiated (50 Gy) after adherence. As described elsewhere [21], a total of 105 purified human CD41 T cells were added to each well in the presence of the PHA1rIL-2. On day 4, BrdU was added. About 18 h later, BrdU uptake was quantified by cell proliferation ELISA through the use of a BrdU Kit (Roche). The percentage of BrdU-positive cells was expressed as mean7SD, reflecting the growth characteristics of CD41 T cells.

Semi-quantitative and quantitative RT-PCR analysis Total RNA of PBMC and FBM-MSC was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). All RNA samples were DNA-free. The reverse transcription reactions were performed using the Superscript First-Strand Synthesis System (Invitrogen Life Technologies) according to the manufacturer’s instructions. IL-17 gene amplification was performed by semi-quantitative RT-PCR analysis using 36 cycles. Aliquots were run on 1.2% agarose gels and stained with ethidium bromide. qRT-PCR was performed with the QuantiTectTM SYBR Green RT-PCR Kit (Applied-Biosystems, Foster City, CA, USA) and an ABI 7500 Sequence Detection System (Applied-Biosystems). PCR primer sequences were as follows IL-17 IFN-g IL-6

Sense Anti-sense Sense Anti-sense Sense Anti-sense

50 -AACCGATCCACCTCACCTTG-30 , 50 -GTAGTCCACGTTCCCATCAG-30 ; 50 -GAGTGTGGAGACCATCAAGGAAG-30 , 50 -TGAGTTCATGTATTGCTTTG-30 ; 50 -TGTGAAAGCAGCAAAGAGGC-30 , 50 -TGGGTCAGGGGTGGTTATT-30 ;


After 4 days of culture, CD41 T cells were harvested and restimulated for another 5 h with 25 ng/mL PMA and 1 mg/mL ionomycin in the presence of Golgistop, Brefeldin A (10 mg/mL) (Sigma). Intracellular cytokines were analyzed by flow cytometry as described previously [34, 35]. For CD41 T cells, one part was labeled with APC conjugated anti-CD4 mAb (BD Biosciences) and PEconjugated anti-CD25 mAb (R&D Systems) and another part, after labeled with APC-conjugated anti-CD4 mAb, was fixed, permeabilized and stained with FITC-conjugated anti-IFN-g (BD PharMingen) and PE-conjugated anti-IL-17 (eBioscience) mAb at room temperature in the dark for 30 min. Gating was on the CD41 cells. Cells were initially gated by forward scatter/side scatter; secondary gates were set on the basis of staining with isotypic control mAb. At least 20 000 cells were collected and analyzed by CellQuest software. Data were displayed as dot plots of IFN-g FITC versus IL-17 PE. We defined the CD41 T-cell population as follows: Th1, IFN-g positive and IL-17 negative; Th17, IFN-g negative and IL-17 positive.

Statistical analysis Data were represented as mean7SD. Statistical differences between the two groups were evaluated by Student’s t-test analysis using SPSS software. po0.05 was considered statistically significant.

Acknowledgements: The authors would like to thank Prof. ManChiu Poon (Department of Medical Oncology, University of Calgary, Canada) and Dr. Lisa Schelde (Department of Pathology, School of medicine, Emory University, USA) for critical review of the manuscript. This work was supported in part by grants from the National Natural Science Foundation of China (30670900),

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Zhenxing Guo et al.

Ministry of Education of China (20060023038) and Ministry of Health of China (200802031).

Eur. J. Immunol. 2009. 39: 2840–2849

generation of pathogenic effector TH17 and regulatory T cells. Nature 2006. 441: 235–238. 16 Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M. and

Conflict of interest: The authors declare no financial or commercial conflict of interest.

Stockinger, B., TGF-b in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006. 24: 179–189. 17 Amadi-Obi, A., Yu, C. R., Liu, X., Mahdi, R. M., Clarke, G. L., Nussenblatt, R. B., Gery, I. et al., TH17 cells contribute to uveitis and scleritis and are

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