Chemoattractant CXCL13 Is Preferentially Expressed by Human Th17 Cell Clones

This information is current as Rie Takagi, Takehiro Higashi, Kumiko Hashimoto, Kazuhisa of September 24, 2021. Nakano, Yosuke Mizuno, Yasushi Okazaki and Sho Matsushita J Immunol 2008; 181:186-189; ; doi: 10.4049/jimmunol.181.1.186

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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 Matsushita2*

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. The Journal of Immunology, 2008, 181: 186–189. Downloaded from he Th cells that produce IL-17 (Th17) have been de- IU/ml penicillin, 50 ␮g/ml streptomycin, 50 ng/ml IL-4 and GM-CSF and scribed as a distinct subset of effector cells that differen- were cultured at 37°C in a humidified atmosphere with 5% CO2. After 5 tiate from naive T cells in response to IL-6 and TGF␤ (1, days, cells were harvested as immature -derived dendritic cells T (Mo-DC).3 This study, which used the peripheral blood of healthy volun- 2). Recent studies have demonstrated that Th17 cells, rather than teers, was approved by the Saitama Medical University Ethics Committee. Th1 cells, play a pivotal role in the pathogenesis of autoimmune MLR and measurement by ELISA disease models, including experimental autoimmune encephalo- http://www.jimmunol.org/ myelitis and collagen-induced arthritis (3–6). It is also interesting Immature Mo-DCs were further cocultured with HLA-DR-nonshared al- to note that Th17 cells developed to mediate protection against logeneic CD4ϩ naive T cells to induce MLR. Alternatively, HLA-DR- 3 extracellular bacteria and fungi (7, 8). We herein report that human nonshared PBMC of two donors (2 ϫ 10 /individual donor/well of micro- ␤ allo-reactive Th17 cell clones are effectively established in a pri- culture plate 163118; Nunc) were cocultured to induce MLR. IL-1 (10 ng/ml), TNF-␣ (10 ng/ml), IL-6 (20 ng/ml), and TGF␤ (10 ng/ml) were mary limiting-dilution condition and that human Th17 cells, but added in these MLR cultures. After an 8-day culture, the wells where cells not Th1 or Th2 cells, express B cell chemoattractant CXCL13. 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 (Fal- Materials and Methods con), followed by feeding with irradiated (45 Gy) PBMC used for MLR (1 ϫ 105/well) plus IL-23 (10 ng/ml). After 6 days of culture, the super- by guest on September 24, 2021 , Abs, and reagents natant fluids were harvested to be subjected to IL-17, IL-5, IL-4, IL-13, and ␥ IL-1␤, TNF-␣, IL-6, TGF␤, IL-12, IL-23, anti-IL-4 Ab, and anti-IFN-␥ Ab IFN- determination by ELISA. After an additional 24-h culture, typical were purchased from PeproTech; IL-4 and GM-CSF were purchased from Th1, Th2, and Th17 cells were cloned by limiting dilution at 0.3–1.0 T Primmune. IL-17, IL-5, IL-4, IL-13, IFN-␥, and CXCL13 ELISA kits were cells/well in the presence of irradiated PBMC. CXCL13 production by purchased from R&D Systems. Th1, Th2, and Th17 cell clones in the frozen culture supernatant fluids was determined by ELISA. Preparation of human monocyte-derived dendritic cells and T clones specific for Candida albicans 3 ϩ PBMC (5 ϫ 10 /microculture well) were cocultured with 100 ␮g/ml C. Human CD14 cells were isolated from PBMC by Ficoll-Paque (GE albicans Ag (Torii Pharmaceuticals). The determination of cytokine pro- Healthcare) centrifugation and positive selection using CD14 MicroBeads ϩ duction and limiting dilution was performed as described in the previous (Miltenyi Biotec). These cells were further depleted with CD4 T cell section. isolation kit (Miltenyi Biotec) and separated into CD45ROϩ memory T ϩ cells and CD45RA naive T cells through the use of CD45RO MicroBeads Collection of culture supernatants (Miltenyi Biotec). The purity of these cells was Ͼ99% as assayed by a FACScan flow cytometer (BD Biosciences). The CD14ϩ cells were sus- Allo-specific and C. albicans-specific T cells (3 ϫ 104/well) were stimu- pended in RPMI 1640 medium containing 10% FCS, 1% L-glutamine, 50 lated with irradiated allogenic PBMC and irradiated autologous PBMC (1.5 ϫ 105/well) plus C. albicans Ag (10 ␮g/ml), respectively, in a 96-well flat-bottom microculture plate. Alternatively, cloned T cells were stimu- *Department of and Immunology, Faculty of Medicine, †Division of Func- lated with PMA (10 ng/ml) plus ionomycin (1 ␮g/ml). The supernatant tional Genomics and Systems Medicine, Research Center for Genomic Medicine, fluid specimens were then harvested 16 and 48 h after the initiation of ‡ Saitama Medical University, Saitama, and First Department of Internal Medicine, culture, for IL-4 and the other cytokines, respectively. University of Occupational and Environmental Health, Kitakyushu, Japan Received for publication October 9, 2007. Accepted for publication April 30, 2008. DNA chip analysis The costs of publication of this article were defrayed in part by the payment of page The total RNA was extracted from M97.1, W10.1, and W110.1 cell clones charges. This article must therefore be hereby marked advertisement in accordance using RNeasy mini kit (Qiagen) according to the manufacturer’s instruc- with 18 U.S.C. Section 1734 solely to indicate this fact. tions. To minimize the contamination of signals due to irradiated PBMC 1 This work was supported in part by an Internal Research Grant from Saitama Med- which were added as APCs, the cell preparations were expanded by ical University and a Research Grant-In-Aid for Scientific Research by the Ministry anti-CD3 plus anti-CD28 Abs before RNA extraction. A gene expression 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 3 Abbreviations used in this paper: Mo-DC, monocyte-derived dendritic cells; OA, Allergy and Immunology, Faculty of Medicine, Saitama Medical University, 38 osteoarthritis; RA, rheumatoid arthritis. Morohongo, Moroyama, Saitama 350-0495, Japan. E-mail address: shomat@ saitama-med.ac.jp Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 187

Table I. Cytokine production by Th cell clonesa

Cytokine (pg/ml) Produced by MLR Cytokine (pg/ml) Produced by PMA/Ionomycin

Clone IFN␥ IL-4 IL-5 IL-13 IL-17 IFN␥ IL-4 IL-5 IL-13 IL-17

M97.1 5912 Ͻ20 Ͻ20 Ͻ20 Ͻ15 8790 60 140 88 90 W10.1 Ͻ40 1215 7428 1998 Ͻ15 230 2360 9320 3101 75 W110.1 Ͻ40 Ͻ20 Ͻ20 Ͻ20 2408 290 60 190 97 3744 M123.1 Ͻ40 Ͻ20 Ͻ20 Ͻ20 2115 245 40 160 90 3210

a Irradiated PBMC and Th cell clones were cocultured to induce MLR. Alternatively, cloned T cells were stimulated with PMA (10 ng/ml) plus ionomycin (1 ␮g/ml). Supernatant fluids were harvested 16 and 48 h after the initiation of culture, for IL-4 and other cytokines, respectively. ELISA was performed for quantitation.

analysis was performed using Affymetrix GeneChip according to the man- Results ␮ ufacturer’s instruction. In brief, 5 g of total RNA was reverse transcribed Establishment of human allo-reactive Th17 cell clones and second strand cDNA was obtained, then cRNA was synthesized by in vitro transcription, incorporating biotin-labeled nucleotide. Fragmented Previous reports have pointed out that IL-2 and IFN-␥ have an cRNA was hybridized on Affymetrix GeneChip mouse Genome 430 2.0, inhibitory effect on the development and maintenance of Th17 and stained by streptavidin PE. After scanning, the quantified data was analyzed by MAS5 normalization and visualized by Spotfire DecisionSite cells (11). We, therefore, used limiting dilution conditions as de- Downloaded from (Spotfire). scribed in Materials and Methods for the primary culture of T cells because such conditions are less likely to allow for cell-cell inter- RT-PCR actions that negatively affect the development of Th17 cells via a RNA was extracted using the TRIzol reagent (Invitrogen). First-strand bystander effect of IL-2 and IFN-␥. Furthermore, allo-reactive T cDNA synthesis was performed using the SuperScript ⌱⌱ RnaseHϪ reverse cells are mainly composed of naive CD4ϩ T cells (9, 12, 13), and transcriptase (Invitrogen), and cDNA was amplified using AmpliTaq Gold

these T cells exist in the peripheral blood at a higher clonal fre- http://www.jimmunol.org/ DNA polymerase (Applied Biosystems). The sense and antisense primers ϩ specific for ␤-actin gene were designed as described previously (9). The quency in comparison to naive CD4 T cells specific for exoge- sense and antisense primer sequences specific for CXCL13 gene were 5Ј- nous Ags. We, therefore, attempted to establish Th17 clones by TGTGTGTGTGGACCCTCAAG-3Ј and 5Ј-CAGAGCAGGGATAAGG HLA-DR-nonshared MLR, in the presence of IL-1␤, TNF-␣, IL-6, GAAG-3Ј, respectively. The transcripts were amplified by 30 cycles of the and TGF␤. For the establishment of allo-reactive Th1 and Th2 cell following: cDNA denaturation (30 s at 95°C), followed by 60 s of primer clones, IL-12 plus anti-IL-4 Ab and IL-4 plus anti-IFN-␥ Ab were 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 used, respectively. Typical Th1, Th2, and Th17 cell clones were by ethidium bromide staining. established. Among them, clones M97.1, W10.1, W110.1, and M123.1 as shown in Table I were used for further analysis. They Synovial fluid specimens exhibited typical cytokine production patterns when stimulated by by guest on September 24, 2021 Fourteen patients with rheumatoid arthritis (RA) and 13 control patients MLR, i.e., in an Ag-specific manner. However, when they were with osteoarthritis (OA)3 were diagnosed according to the American Col- stimulated by PMA plus ionomycin, i.e., in an Ag-nonspecific lege of Rheumatology criteria (10). Synovial fluid specimens were col- manner, minor contaminating signals were thus detected. They are lected 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 likely to be attributable to irradiated PBMC which were added to samples were collected under sterile conditions, and the cellular compo- the culture as APCs. nents were removed immediately after centrifugation. The supernatants were stored at Ϫ30°C. Transcriptome analysis Statistics The differences in the gene expression among three cell lines were compared by using the Affymetrix GeneChip system. Because Comparisons between the sets of two groups were performed using Stu- dent’s two-tailed t test. 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 ex- pressed by Th17 clone W110.1. In contrast, none of the three

Table II. Th17-specific transcriptomesa

DNA Microarray M97.1 W10.1 W110.1

CCR6 133 56 147 IL-23R␣ N.E. N.E. N.E. CCL20 18 8 7757 FIGURE 1. The expression profiling of three different cell lines. The IL-26 2839 1000 11412 expression level (normalized value by MAS5) of each spot was plotted for RORC 10 17 1548 W110.1 vs M97.1 (A) and W110 vs W10.1 (B). M97.1, W10.1, and CXCL13 4 5667 62003 W110.1 indicate Th1, Th2, and Th17 cells, respectively. The arrow indi- a A gene expression analysis was performed using Affymetrix GeneChip, as de- cates CXCL13. scribed in Materials and Methods. N.E., Not evaluated. 188 HUMAN Th17 CLONES EXPRESS CXCL13

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. clones expressed CCR6 (16, 17) at high levels. We next performed a RT-PCR analysis for CCR6. Indeed, CCR6 was expressed by FIGURE 4. Production of CXCL13 by Th17 cell clones specific for C. Th17, but not by either Th1 or Th2 (data not shown). This dis- albicans Ag. N.S., not significant. crepancy might be attributable to the probe for CCR6 loaded on Downloaded from the DNA chip. clones specific for C. albicans, by using PBMC from nine healthy RT-PCR analysis individuals. As shown in Table III, CXCL13 was preferentially produced from Th17 cells. To confirm the results of transcriptome analysis, we next examined the expression patterns of CXCL13 mRNA by the Th cell clones, CXCL13 in synovial fluids using RT-PCR. As shown in Fig. 2, the Th17 cells exhibited a To further confirm the association between Th17 and CXCL13, not http://www.jimmunol.org/ marked expression of CXCL13 mRNA, whereas none of the Th1 only under physiological conditions but also in Th17-associated and Th2 cells did. The Th2 cells exhibited a very faint band when human disease, synovial fluid specimens from patients with RA much volume was loaded (data not shown). were collected and analyzed for IL-17 and CXCL13. The synovial Production of CXCL13 by Th1, Th2, and Th17 cell clones fluid specimens from patients with OA were analyzed as controls. As shown in Table IV, CXCL13 exhibited markedly higher levels To correlate the cytokine production profile and CXCL13 produc- in RA, in comparison to OA ( p Ͻ 0.01). Furthermore, a statisti- tion in Th cell populations, we compared concentrations of cally significant correlation between IL-17 and CXCL13 was ob- CXCL13 in culture supernatant fluids using allo-reactive Th1, served by using Spearman’s rank correlation ( p Ͻ 0.01).

Th2, and Th17 cells. As shown in Fig. 3, none of the Th1 cells by guest on September 24, 2021 exhibited CXCL13 production, whereas all Th17 cells exhibited a Discussion marked production of CXCL13. Although most of Th2 cells ex- In the present study, allo-reactive Th17 cell clones were effectively hibited a small expression of CXCL13, minor proportion of Th2 established in a primary limiting-dilution condition, which will cells expressed Ͼ200 pg/ml CXCL13. To confirm that the corre- allow us to investigate various conditions for the induction of Th lation of IL-17 and CXCL13 is not limited to allo-reactive Th cells, subtypes. Furthermore, it was of note that human Th17 cells, but we performed the same analysis using Th17 cell clones specific for not Th1 or Th2 cells, expressed B cell chemoattractant CXCL13 C. albicans Ag. As shown in Fig. 4, a clear correlation of IL-17 by using DNA chips, RT-PCR, and ELISA. This should highlight and CXCL13 was observed, indicating that the Th17 cell clones a new aspect of Th17 biology, i.e., the involvement of B cells in differentiated in physiological conditions carry the same property. protective to extracellular microorganisms and in To confirm the reproducibility of these results and to rule out any . donor-to-donor variance, we established Th1, Th2, and Th17 Because studies by others (18) have shown controversial effects of TGF␤ and IL-6 on human Th17 cells, we checked the efficiency

Table III. Production of CXCL13 from Candida-specific Th17 and non-Th17 cellsa

CXCL13 Production (pg/ml) From

Donor Th1 Th2 Th17

170Ϯ 30 210 Ϯ 65 3750 Ϯ 1420 2 Ͻ50 80 Ϯ 25 1512 Ϯ 390 385Ϯ 35 180 Ϯ 70 5460 Ϯ 2100 460Ϯ 15 300 Ϯ 110 3420 Ϯ 960 5 110 Ϯ 25 155 Ϯ 65 4030 Ϯ 525 6 Ͻ50 80 Ϯ 40 980 Ϯ 475 7 130 Ϯ 50 490 Ϯ 240 8640 Ϯ 1950 8 Ͻ50 220 Ϯ 45 2590 Ϯ 700 990Ϯ 20 280 Ϯ 120 4270 Ϯ 1080 FIGURE 3. Production of CXCL13 by Th1, Th2, and Th17 cell clones. a Th1, Th2, and Th17 cell clones were established from PBMC of nine healthy Each symbol represents one clone and was plotted for allo-reactive Th1, individuals. Each value was calculated from CXCL13 concentrations derived from 3 .p Ͻ 0.01; N.S., not significant. to 15 T cell clones. Mean Ϯ SD ,ءء ;p Ͻ 0.05 ,ء .Th2, and Th17 cell clones The Journal of Immunology 189

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