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B Cell Chemoattractant CXCL13 Is Preferentially Expressed by Human Th17 Cell Clones¹

Rie Takagi^*, Takehiro Higashi^*, Kumiko Hashimoto^*, Kazuhisa Nakano^*,, Yosuke Mizuno, Yasushi Okazaki and Sho Matsushita^2,*

^* Department of Allergy and Immunology, Faculty of Medicine, Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama, and First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan

	Abstract

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Th 17 cells represent a novel subset of CD4⁺ T cells that have a protective effect against extracellular microbes, while they are also responsible for autoimmune disorders in mice. However, the protein expression profile of Th17 cells remains to be clarified. In this study, we report an effective method to establish human allo-reactive Th17 cell clones and demonstrate that human Th17, but not Th1 or Th2, cells express B cell chemoattractant CXCL13, by using DNA chips, RT-PCR, and ELISA. Such a pattern was also the case in Candida albicans-specific Th17 clones and synovial fluid specimens obtained from patients with rheumatoid arthritis. The biological implication of this finding is discussed.

	Introduction

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The Th cells that produce IL-17 (Th17) have been described as a distinct subset of effector cells that differentiate from naive T cells in response to IL-6 and TGFβ (1, 2). Recent studies have demonstrated that Th17 cells, rather than Th1 cells, play a pivotal role in the pathogenesis of autoimmune disease models, including experimental autoimmune encephalomyelitis and collagen-induced arthritis (3, 4, 5, 6). It is also interesting to note that Th17 cells developed to mediate protection against extracellular bacteria and fungi (7, 8). We herein report that human allo-reactive Th17 cell clones are effectively established in a primary limiting-dilution condition and that human Th17 cells, but not Th1 or Th2 cells, express B cell chemoattractant CXCL13.

	Materials and Methods

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Cytokines, Abs, and reagents

IL-1β, TNF-, IL-6, TGFβ, IL-12, IL-23, anti-IL-4 Ab, and anti-IFN- Ab were purchased from PeproTech; IL-4 and GM-CSF were purchased from Primmune. IL-17, IL-5, IL-4, IL-13, IFN-, and CXCL13 ELISA kits were purchased from R&D Systems.

Preparation of human monocyte-derived dendritic cells and T lymphocytes

Human CD14⁺ cells were isolated from PBMC by Ficoll-Paque (GE Healthcare) centrifugation and positive selection using CD14 MicroBeads (Miltenyi Biotec). These cells were further depleted with CD4⁺ T cell isolation kit (Miltenyi Biotec) and separated into CD45RO⁺ memory T cells and CD45RA⁺ naive T cells through the use of CD45RO MicroBeads (Miltenyi Biotec). The purity of these cells was >99% as assayed by a FACScan flow cytometer (BD Biosciences). The CD14⁺ cells were suspended in RPMI 1640 medium containing 10% FCS, 1% L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, 50 ng/ml IL-4 and GM-CSF and were cultured at 37°C in a humidified atmosphere with 5% CO₂. After 5 days, cells were harvested as immature monocyte-derived dendritic cells (Mo-DC).³ This study, which used the peripheral blood of healthy volunteers, was approved by the Saitama Medical University Ethics Committee.

MLR and cytokine measurement by ELISA

Immature Mo-DCs were further cocultured with HLA-DR-nonshared allogeneic CD4⁺ naive T cells to induce MLR. Alternatively, HLA-DR-nonshared PBMC of two donors (2 x 10³/individual donor/well of microculture plate 163118; Nunc) were cocultured to induce MLR. IL-1β (10 ng/ml), TNF- (10 ng/ml), IL-6 (20 ng/ml), and TGFβ (10 ng/ml) were added in these MLR cultures. After an 8-day culture, the wells where cells proliferated were typically 10% of all the culture wells. The proliferating wells were split into two wells of a 96-well flat-bottom culture plate (Falcon), followed by feeding with irradiated (45 Gy) PBMC used for MLR (1 x 10⁵/well) plus IL-23 (10 ng/ml). After 6 days of culture, the supernatant fluids were harvested to be subjected to IL-17, IL-5, IL-4, IL-13, and IFN- determination by ELISA. After an additional 24-h culture, typical Th1, Th2, and Th17 cells were cloned by limiting dilution at 0.3–1.0 T cells/well in the presence of irradiated PBMC. CXCL13 production by Th1, Th2, and Th17 cell clones in the frozen culture supernatant fluids was determined by ELISA.

T cell clones specific for Candida albicans

PBMC (5 x 10³/microculture well) were cocultured with 100 µg/ml C. albicans Ag (Torii Pharmaceuticals). The determination of cytokine production and limiting dilution was performed as described in the previous section.

Collection of culture supernatants

Allo-specific and C. albicans-specific T cells (3 x 10⁴/well) were stimulated with irradiated allogenic PBMC and irradiated autologous PBMC (1.5 x 10⁵/well) plus C. albicans Ag (10 µg/ml), respectively, in a 96-well flat-bottom microculture plate. Alternatively, cloned T cells were stimulated with PMA (10 ng/ml) plus ionomycin (1 µg/ml). The supernatant fluid specimens were then harvested 16 and 48 h after the initiation of culture, for IL-4 and the other cytokines, respectively.

DNA chip analysis

The total RNA was extracted from M97.1, W10.1, and W110.1 cell clones using RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. To minimize the contamination of signals due to irradiated PBMC which were added as APCs, the cell preparations were expanded by anti-CD3 plus anti-CD28 Abs before RNA extraction. A gene expression analysis was performed using Affymetrix GeneChip according to the manufacturer’s instruction. In brief, 5 µg of total RNA was reverse transcribed and second strand cDNA was obtained, then cRNA was synthesized by in vitro transcription, incorporating biotin-labeled nucleotide. Fragmented cRNA was hybridized on Affymetrix GeneChip mouse Genome 430 2.0, and stained by streptavidin PE. After scanning, the quantified data was analyzed by MAS5 normalization and visualized by Spotfire DecisionSite (Spotfire).

RT-PCR

RNA was extracted using the TRIzol reagent (Invitrogen). First-strand cDNA synthesis was performed using the SuperScript II RnaseH^– reverse transcriptase (Invitrogen), and cDNA was amplified using AmpliTaq Gold DNA polymerase (Applied Biosystems). The sense and antisense primers specific for β-actin gene were designed as described previously (9). The sense and antisense primer sequences specific for CXCL13 gene were 5'-TGTGTGTGTGGACCCTCAAG-3' and 5'-CAGAGCAGGGATAAGGGAAG-3', respectively. The transcripts were amplified by 30 cycles of the following: cDNA denaturation (30 s at 95°C), followed by 60 s of primer annealing at 57°C, and 60 s of extension at 72°C. The PCR products were subjected to electrophoresis on 1.5% agarose gel and then were visualized by ethidium bromide staining.

Synovial fluid specimens

Fourteen patients with rheumatoid arthritis (RA) and 13 control patients with osteoarthritis (OA)³ were diagnosed according to the American College of Rheumatology criteria (10). Synovial fluid specimens were collected during either diagnostic or therapeutic arthrocentesis of the knee (14 knees in 14 cases with RA or 13 knees in 13 cases with OA). All synovial samples were collected under sterile conditions, and the cellular components were removed immediately after centrifugation. The supernatants were stored at –30°C.

Statistics

Comparisons between the sets of two groups were performed using Student’s two-tailed t test.

	Results

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Establishment of human allo-reactive Th17 cell clones

Previous reports have pointed out that IL-2 and IFN- have an inhibitory effect on the development and maintenance of Th17 cells (11). We, therefore, used limiting dilution conditions as described in Materials and Methods for the primary culture of T cells because such conditions are less likely to allow for cell-cell interactions that negatively affect the development of Th17 cells via a bystander effect of IL-2 and IFN-. Furthermore, allo-reactive T cells are mainly composed of naive CD4⁺ T cells (9, 12, 13), and these T cells exist in the peripheral blood at a higher clonal frequency in comparison to naive CD4⁺ T cells specific for exogenous Ags. We, therefore, attempted to establish Th17 clones by HLA-DR-nonshared MLR, in the presence of IL-1β, TNF-, IL-6, and TGFβ. For the establishment of allo-reactive Th1 and Th2 cell clones, IL-12 plus anti-IL-4 Ab and IL-4 plus anti-IFN- Ab were used, respectively. Typical Th1, Th2, and Th17 cell clones were established. Among them, clones M97.1, W10.1, W110.1, and M123.1 as shown in Table I were used for further analysis. They exhibited typical cytokine production patterns when stimulated by MLR, i.e., in an Ag-specific manner. However, when they were stimulated by PMA plus ionomycin, i.e., in an Ag-nonspecific manner, minor contaminating signals were thus detected. They are likely to be attributable to irradiated PBMC which were added to the culture as APCs.

The differences in the gene expression among three cell lines were compared by using the Affymetrix GeneChip system. Because contaminating signals were detected as shown in Table I, the T cell preparations were further expanded by anti-CD3 plus anti-CD28 Abs before RNA extraction. The normalized expression data were visualized in Fig. 1. The CXCL13 expression as indicated by the arrows was high in Th17 cells, whereas that in Th1 and Th2 cells was none and trace, respectively. Table II shows typical Th17-specific transcriptomes. CCL20 (14), IL-26 (14), RORC (human analog of RORgt) (15), and CXCL13 were preferentially expressed by Th17 clone W110.1. In contrast, none of the three clones expressed CCR6 (16, 17) at high levels. We next performed a RT-PCR analysis for CCR6. Indeed, CCR6 was expressed by Th17, but not by either Th1 or Th2 (data not shown). This discrepancy might be attributable to the probe for CCR6 loaded on the DNA chip.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. The expression profiling of three different cell lines. The expression level (normalized value by MAS5) of each spot was plotted for W110.1 vs M97.1 (A) and W110 vs W10.1 (B). M97.1, W10.1, and W110.1 indicate Th1, Th2, and Th17 cells, respectively. The arrow indicates CXCL13.

To confirm the results of transcriptome analysis, we next examined the expression patterns of CXCL13 mRNA by the Th cell clones, using RT-PCR. As shown in Fig. 2, the Th17 cells exhibited a marked expression of CXCL13 mRNA, whereas none of the Th1 and Th2 cells did. The Th2 cells exhibited a very faint band when much volume was loaded (data not shown).

View larger version (29K): [in this window] [in a new window]   FIGURE 2. The CXCL13 mRNA expression levels on the Th cell clones evaluated by using RT-PCR analysis. M97.1 and W10.1 indicate Th1 and Th2 cell clones, respectively. W110.1 and M123.1 indicate Th17 cell clones.

To correlate the cytokine production profile and CXCL13 production in Th cell populations, we compared concentrations of CXCL13 in culture supernatant fluids using allo-reactive Th1, Th2, and Th17 cells. As shown in Fig. 3, none of the Th1 cells exhibited CXCL13 production, whereas all Th17 cells exhibited a marked production of CXCL13. Although most of Th2 cells exhibited a small expression of CXCL13, minor proportion of Th2 cells expressed >200 pg/ml CXCL13. To confirm that the correlation of IL-17 and CXCL13 is not limited to allo-reactive Th cells, we performed the same analysis using Th17 cell clones specific for C. albicans Ag. As shown in Fig. 4, a clear correlation of IL-17 and CXCL13 was observed, indicating that the Th17 cell clones differentiated in physiological conditions carry the same property. To confirm the reproducibility of these results and to rule out any donor-to-donor variance, we established Th1, Th2, and Th17 clones specific for C. albicans, by using PBMC from nine healthy individuals. As shown in Table III, CXCL13 was preferentially produced from Th17 cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Production of CXCL13 by Th1, Th2, and Th17 cell clones. Each symbol represents one clone and was plotted for allo-reactive Th1, Th2, and Th17 cell clones. *, p < 0.05; **, p < 0.01; N.S., not significant.

View larger version (10K): [in this window] [in a new window]   FIGURE 4. Production of CXCL13 by Th17 cell clones specific for C. albicans Ag. N.S., not significant.

To further confirm the association between Th17 and CXCL13, not only under physiological conditions but also in Th17-associated human disease, synovial fluid specimens from patients with RA were collected and analyzed for IL-17 and CXCL13. The synovial fluid specimens from patients with OA were analyzed as controls. As shown in Table IV, CXCL13 exhibited markedly higher levels in RA, in comparison to OA (p < 0.01). Furthermore, a statistically significant correlation between IL-17 and CXCL13 was observed by using Spearman’s rank correlation (p < 0.01).

	Discussion

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Because studies by others (18) have shown controversial effects of TGFβ and IL-6 on human Th17 cells, we checked the efficiency for the establishment and maintenance of human Th17 cells. Indeed, TGFβ exhibited an inhibitory effect on the establishment of Th17 (by 50%; data not shown), whereas the addition of IL-6 exhibited an enhancing effect (data not shown). We also tried adding IL-23, not only for maintenance but also for the establishment of Th17, and we found that IL-23 increased the efficiency of the establishment of Th17 (14).

B cells in mice lacking CXCR5 do not migrate from the T cell-rich zone into B cell follicles in the spleen or the Peyer’s patches (19). Therefore, the ligand for CXCR5, which has now been identified as CXCL13 (20), is essential for B cell migration within a specific anatomical compartment. Not only Th cells but also B cells are reported to play pivotal roles for the development of murine autoimmune diseases. For example, collagen-induced arthritis, where Th17 cells contribute much to the development of arthritis (3, 4, 5, 6), can be adoptively transferred by an autoantibody alone (21). Because CXCL13 is one of the powerful B cell chemoattractants, it is highly likely that Th17 cells can effectively and closely interact with B cells for the production of autoantibodies. In the case of protective immunity to extracellular microorganisms, such an interaction should allow B cells to produce protective Abs that can help in terminating the infection. Indeed, Acosta-Rodriguez et al. (18) reported that Th17 cells carry Ig class-switching activity. However, the effect of Th17 cells on IgG subclasses remains obscure. Further studies regarding such Th17-B cell interaction both in vitro and in vivo are currently underway.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by an Internal Research Grant from Saitama Medical University and a Research Grant-In-Aid for Scientific Research by the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan.

² Address correspondence and reprint requests to Dr. Sho Matsushita, Department of Allergy and Immunology, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Saitama 350-0495, Japan. E-mail address: shomat{at}saitama-med.ac.jp

³ Abbreviations used in this paper: Mo-DC, monocyte-derived dendritic cells; OA, osteoarthritis; RA, rheumatoid arthritis.

Received for publication October 9, 2007. Accepted for publication April 30, 2008.

	References

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1. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, B. Stockinger. 2006. TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24: 179-189. [Medline]
2. Mangan, P. R., L. E. Harrington, D. B. O'Quinn, W. S. Helms, D. C. Bullard, C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, C. T. Weaver. 2006. Transforming growth factor-β induces development of the T(H)17 lineage. Nature 441: 231-234. [Medline]
3. Cua, D. J., J. Sherlock, Y. Chen, C. A. Murphy, B. Joyce, B. Seymour, L. Lucian, W. To, S. Kwan, T. Churakova, et al 2003. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421: 744-748. [Medline]
4. Murphy, C. A., C. L. Langrish, Y. Chen, W. Blumenschein, T. McClanahan, R. A. Kastelein, J. D. Sedgwick, D. J. Cua. 2003. Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J. Exp. Med. 198: 1951-1957. [Abstract/Free Full Text]
5. Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham, J. D. Sedgwick, T. McClanahan, R. A. Kastelein, D. J. Cua. 2005. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201: 233-240. [Abstract/Free Full Text]
6. Korn, T., E. Bettelli, W. Gao, A. Awasthi, A. Jager, T. B. Strom, M. Oukka, V. K. Kuchroo. 2007. IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells. Nature 448: 484-487. [Medline]
7. Ye, P., F. H. Rodriguez, S. Kanaly, K. L. Stocking, J. Schurr, P. Schwarzenberger, P. Oliver, W. Huang, P. Zhang, J. Zhang, et al 2001. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. J. Exp. Med. 194: 519-527. [Abstract/Free Full Text]
8. Happel, K. I., M. Zheng, E. Young, L. J. Quinton, E. Lockhart, A. J. Ramsay, J. E. Shellito, J. R. Schurr, G. J. Bagby, S. Nelson, J. K. Kolls. 2003. Cutting edge: roles of Toll-like receptor 4 and IL-23 in IL-17 expression in response to Klebsiella pneumoniae infection. J. Immunol. 170: 4432-4436. [Abstract/Free Full Text]
9. Wakui, M., K. Nakano, S. Matsushita. 2007. Notch ligand mRNA levels of human APCs predict Th1/Th2-promoting activities. Biochem. Biophys. Res. Commun. 358: 596-601. [Medline]
10. Arnett, F. C., S. M. Edworthy, D. A. Bloch, D. J. McShane, J. F. Fries, N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, et al 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31: 315-324. [Medline]
11. Laurence, A., C. M. Tato, T. S. Davidson, Y. Kanno, Z. Chen, Z. Yao, R. B. Blank, F. Meylan, R. Siegel, L. Hennighausen, et al 2007. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity 26: 371-381. [Medline]
12. de Jong, E. C., P. L. Vieira, P. Kalinski, J. H. Schuitemaker, Y. Tanaka, E. A. Wierenga, M. Yazdanbakhsh, M. L. Kapsenberg. 2002. Microbial compounds selectively induce Th1 cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse th cell-polarizing signals. J. Immunol. 168: 1704-1709. [Abstract/Free Full Text]
13. Matsushita, S., T. Liu, M. Wakui, Y. Uemura. 2005. Adjuvants that enhance Th2 or Tr responses. Allergol. Int. 54: 507-513.
14. Wilson, N. J., K. Boniface, J. R. Chan, B. S. McKenzie, W. M. Blumenschein, J. D. Mattson, B. Basham, K. Smith, T. Chen, F. Morel, et al 2007. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat. Immunol. 8: 950-957. [Medline]
15. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, D. J. Cua, D. R. Littman. 2006. The orphan nuclear receptor RORt directs the differentiation program of proinflammatory IL-17⁺ T helper cells. Cell 126: 1121-1133. [Medline]
16. Annunziato, F., L. Cosmi, V. Santarlasci, L. Maggi, F. Liotta, B. Mazzinghi, E. Parente, L. Fili, S. Ferri, F. Frosali, et al 2007. Phenotypic and functional features of human Th17 cells. J. Exp. Med. 204: 1849-1861. [Abstract/Free Full Text]
17. Acosta-Rodriguez, E. V., L. Rivino, J. Geginat, D. Jarrossay, M. Gattorno, A. Lanzavecchia, F. Sallusto, G. Napolitani. 2007. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat. Immunol. 8: 639-646. [Medline]
18. Acosta-Rodriguez, E. V., G. Napolitani, A. Lanzavecchia, F. Sallusto. 2007. Interleukins 1β and 6 but not transforming growth factor-β are essential for the differentiation of interleukin 17-producing human T helper cells. Nat. Immunol. 8: 942-949. [Medline]
19. Forster, R., A. E. Mattis, E. Kremmer, E. Wolf, G. Brem, M. Lipp. 1996. A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen. Cell 87: 1037-1047. [Medline]
20. Gunn, M. D., V. N. Ngo, K. M. Ansel, E. H. Ekland, J. G. Cyster, L. T. Williams. 1998. A B-cell-homing chemokine made in lymphoid follicles activates Burkitt’s lymphoma receptor-1. Nature 391: 799-803. [Medline]
21. Terato, K., K. A. Hasty, R. A. Reife, M. A. Cremer, A. H. Kang, J. M. Stuart. 1992. Induction of arthritis with monoclonal antibodies to collagen. J. Immunol. 148: 2103-2108. [Abstract]

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