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Cutting Edge: Human Th17 Cells Are Identified as Bearing CCR2⁺CCR5^– Phenotype¹

Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi, Kodaira, Tokyo, Japan

	Abstract

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Recent reports have shown that IL-17-producing CD4⁺ T cells (Th17 cells) belong to a distinct helper T cell lineage and are critically involved in the pathogenesis of autoimmune diseases and allergies. However, the chemokine receptor profile of Th17 cells remains to be clarified. In this study, we report that human Th17 cells are identified as CCR2⁺CCR5^– memory CD4⁺ T cells. Analysis of PBMC from healthy donors showed that CCR2⁺ cells produce much larger amounts of IL-17 than CCR2^– cells, indicating the preferential expression of CCR2 on Th17 cells. Notably, CCR2⁺CCR5^– memory CD4⁺ T cells produced a large amount of IL-17 and little IFN-, whereas CCR2⁺CCR5⁺ cells reciprocally produced an enormous amount of IFN- but little IL-17. Moreover, a higher expression of T-bet was seen in the CCR5⁺ memory T cells. These results indicate that absence of CCR5 distinguishes human Th17 cells from Th1 cells.

	Introduction

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CD4⁺ Th cells are essential regulators of adaptive immune responses. Th cells have been classified as either Th1 or Th2 according to the cytokine production profile and functional properties. However, recent studies have demonstrated that IL-17-producing T cells, rather than Th1 cells, play a pivotal role in the pathogenesis of autoimmune disease models, including experimental autoimmune encephalomyelitis (EAE)³ (1, 2, 3, 4). IL-17 is a cytokine mainly produced by activated memory T cells and could recruit and expand neutrophils through induction of various chemokines and GM-CSF (5, 6, 7).

Numerous studies have provided evidence that IL-17-producing T cells belong to a distinct lineage of Th cells whose development is severely hampered in IL-23 knockout but not in IL-12 knockout mice (8, 9). Although IL-23 was initially thought to induce differentiation of the IL-17-producing cells, it now seems that IL-23 is not involved in differentiation but propagation of Th17 cells (10). In fact, recent studies have shown that a combination of TGF-1 plus IL-6 promotes the differentiation of Th17 cells in vitro (11, 12, 13). Differentiation of Th17 cells is prohibited by IFN- or IL-4 (11, 12, 13), further supporting the concept that Th17 cells comprise a distinct population cross-regulated by Th1 or Th2 cells. Notably, the independent nature of Th17 cells has been further highlighted by the recent discovery that the transcription factor RORt is critically involved in the development of Th17 cells (14).

During the critical process whereby naive CD4⁺ T cells differentiate, they acquire reciprocal sets of chemokine receptors (15), which would endow them a unique character of homing or migration to corresponding ligand chemokines. Namely, Th1 cells preferentially express CCR5 and CXCR3 and migrate to inflammatory milieu expressing the corresponding ligand chemokines, whereas Th2 cells express CCR4, CCR8, and CRTh2 indicative of a distinctive homing property (16, 17, 18, 19). It is conceivable that Th17 cells may also possess unique chemotactic and migratory property. However, chemokine receptor expression by Th17 cells has not been characterized yet, at least to our knowledge.

In this study, we attempted to identify chemokine receptor expression by human Th17 cells by examining cytokine production profiles of T cell subpopulation-bearing chemokine receptor(s) of interest (16, 20). We started by comparing CCR2⁺ and CCR2^– memory CD4⁺ T cells, because CCR2 and its ligand CCL2 were shown to be essential for development of EAE (21, 22). We found that only the CCR2⁺ subpopulation would produce IL-17. Further analysis has demonstrated that CCR5^– cells among the CCR2⁺CD4⁺ memory T cells produce IL-17, whereas a CCR5⁺ subpopulation produces IFN-. Thus, human Th17 cells are identified as uniquely bearing the CCR2⁺CCR5^– phenotype.

	Materials and Methods

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Reagents

Anti-CCR2-biotin mAb, anti-CCR5-FITC mAb, and goat anti-IL-23R polyclonal Abs were purchased from R&D Systems. Streptavidin-PE, anti-CD4-PC5 mAb, and anti-CD45RA-energy-coupled dye (ECD) mAb were obtained from Beckman Coulter, anti-CCR5-allophycocyanin mAb from BD Pharmingen, and donkey anti-goat IgG-FITC from Jackson ImmunoResearch Laboratories. Anti-T-bet mAb and isotype control Ab (mouse IgG1a) purchased from Santa Cruz Biotechnology were used for intracellular staining. RPMI 1640 medium (Invitrogen Life Technologies) was supplemented with 0.05 mM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, and 10% FBS.

Cell preparation

Peripheral blood was obtained from healthy human volunteers (24–42 years of age) from whom informed consent was obtained. The Ethics Committee of the National Center of Neurology and Psychiatry approved the study. PBMC were freshly isolated by density gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences). To purify whole T cells or memory CD4⁺ T cells from PBMC, we used a Pan T cell isolation kit II or Memory CD4⁺ T cell isolation kit (Miltenyi Biotec), respectively. Briefly, PBMC were labeled with a mixture of biotin-conjugated mAbs directed against either non-T or non-memory CD4⁺ T cells and then reacted with magnetic microbead-conjugated anti-biotin mAbs. The magnetically labeled non-T or non-memory CD4⁺ T cells were depleted with autoMACS (Miltenyi Biotec), which yielded >95% purity of whole T cells or memory CD4⁺ T cells as assessed by flow cytometry for the proportion of CD3⁺ cells or CD4⁺CD45RA^– cells.

To further separate the purified cells according to CCR2 or CCR5 expression, they were labeled with anti-CCR2-biotin, streptavidin-PE, and anti-PE microbeads (Miltenyi Biotec) or anti-CCR5-FITC and anti-FITC microbeads (Miltenyi Biotec). The magnetically labeled cells were separated into positive (CCR2⁺ and CCR5⁺) and negative (CCR2^– and CCR5^–) fractions with autoMACS (>99% purity of CCR2 ^–or CCR5^– cells and >90% purity of CCR2⁺ or CCR5⁺ cells). To obtain CCR2⁺CCR5^– and CCR2⁺CCR5⁺ memory CD4⁺ T cells, CCR2⁺ memory CD4⁺ T cells were labeled with anti-CCR5-allophycocyanin and separated into CCR2⁺CCR5^– (>80% purity) and CCR2⁺CCR5⁺ cells (>95% purity) by flow cytometric cell sorter Epics Altra (Beckman Coulter).

Cell culture and cytokine measurement by ELISA

Purified T cell populations were resuspended at 5 x 10⁵/ml and stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) in 96-well U-bottom plates for 24 h. The concentrations of IFN- and IL-17 in the supernatants were measured by using a Human IFN- ELISA Set (BD Pharmingen) and a Human IL-17 DuoSet (R&D Systems).

Flow cytometric analysis of chemokine receptors

To evaluate the expression of chemokine receptors, purified memory CD4⁺ T cells were stained with anti-CD4-PC5, anti-CD45RA-ECD, anti-CCR5-FITC and PE-conjugated mAbs against anti-CCR2-biotin were analyzed with Epics flow cytometry (Beckman Coulter). To examine the expression of IL-23R, memory CD4⁺ T cells were stained with goat anti-IL-23R and anti-goat-IgG-FITC and were analyzed with a FACSCalibur (BD Pharmingen).

Intracellular staining of T-bet

Purified memory CD4⁺ T cells were first stained with biotin-conjugated anti-CCR2, streptavidin-PE, and allophycocyanin-CCR5, then fixed in PBS containing 2% paraformaldehyde and permeabilized with 0.1% saponin solution. Subsequently, the cells were stained with FITC-anti-T-bet. Mouse IgG1a was used as an isotype control.

Statistics

An unpaired Student’s t test or one-way ANOVA was used for statistical analysis. We considered p < 0.01 as significant.

	Results and Discussion

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Both Th17 cells and Th1 cells are enriched in CCR2⁺CD4⁺ memory T cells

Previous reports on the CCR2 requirement for development of EAE (21, 22) prompted us to compare the cytokine-producing ability of CCR2⁺ and CCR2^– cells isolated from whole T cells. The results showed that CCR2⁺ cells produced a larger amount of IFN- and IL-17 as compared with CCR2^– cells, whereas unseparated whole T cells showed intermediate values (Fig. 1A, upper panels). This indicates that CCR2⁺ cells contain the vast majority of Th1 and Th17 cells. We next separated the whole T cells into CCR5⁺ and CCR5^– populations to compare the cytokine profile. Although CCR5⁺ cells produced a larger amount of IFN- as compared with CCR5^– or the whole T cells (Fig. 1A, lower panels), production of IL-17 did not increase after enrichment for CCR5⁺ cells. These results suggest that Th17 cells may be selectively enriched in CCR2⁺, but not in CCR5⁺ populations. However, because the CCR2⁺ T cell preparation also contains CD8⁺ T cells and T cells, capable of producing IL-17 (23, 24, 25), it remained possible that the major source of IL-17 could be CD8⁺ T or T cells. Therefore, we next needed to assess the production of IFN- and IL-17 from purified CD4⁺ T cells. Preparatory experiments showed that CCR2⁺ or CCR5⁺CD4⁺ populations are mainly confined to the CD45RA^– memory T cell population (Fig. 1B). Consequently, we decided to use memory CD4⁺ T cells that could be obtained after deleting CD8⁺, , and naive CD4⁺ T cells for further analysis. Analysis of the purified memory CD4⁺ T cells has also demonstrated that the CCR2⁺ population produced a significantly larger amount of both IFN- and IL-17 compared to the CCR2^– population, with the values of unseparated cells being intermediate (Fig. 1C). These results strongly indicate that Th17 cells as well as Th1 cells are enriched in CCR2⁺CD4⁺ memory T cells. However, since Th1 and Th17 cells are thought to belong to distinct T cell lineages, we speculated that they might be further divided into two subpopulations based on expression of chemokine receptors.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Th17 cells are enriched in CCR2+CD4+CD45RA– cells. A, IL-17-producing cells are selectively enriched in the CCR2+, but not in a CCR5+ population. From PBMC of healthy donors, whole T cells were purified magnetically by negative selection using a Pan T cell isolation kit II with autoMACS. The purity of the cells was generally >98%, as determined by FACS analysis. Purified CCR2+ and CCR2– T cells (or CCR5+ and CCR5– T cells) were stimulated with PMA and ionomycin for 24 h before the supernatants were collected. The IFN- and IL-17 protein in each supernatant was measured using ELISA. Results are expressed as mean ± SD of a representative of five independent experiments. B, Chemokine receptor (CCR2 or CCR5) expressing T cells are largely confined to CD45RA– memory T cells. PBMC from healthy subjects were stained with anti-CCR2 (PE), anti-CCR5 (FITC), anti-CD4 (PC5), and anti-CD45RA (ECD) and analyzed after being gated for CD4. Shown is a representative of five individual data sets. C, Th17 cells are enriched in CCR2+CD4+CD45RA– cells. Memory CD4+ T cells were purified by a memory CD4+ T cell isolation kit with autoMACS. CCR2+ and CCR2– T cells were further isolated by anti-CCR2-biotin, streptavidin-PE, anti-PE microbeads, and autoMACS. Purified cells were stimulated with PMA and ionomycin for 24 h before collecting supernatants. Results are expressed as mean ± SD of a representative of five independent experiments. *, p < 0.01.

CCR2⁺CCR5^– memory CD4⁺ T cells predominantly produce IL-17 but not IFN-

Simultaneous staining of CCR2 and CCR5 showed that the CCR2⁺ memory T cell population could be divided into CCR5^– (CCR2 single positive (SP)) and CCR5⁺ (CCR2 and CCR5 double positive (DP)) subpopulations (Fig. 2). Since CCR5 is reported to be expressed predominantly on Th1 cells (16, 17, 18), we hypothesized that SP and DP cells might correspond to Th17 and Th1 cells, respectively. To correlate cytokine production profile and chemokine receptor expression in T cell populations, we first thought of staining total unseparated T cells to detect intracellular cytokines as well as surface CCR; however, the cell activation process required for intracellular cytokine staining was found to down-regulate CCR2 and CCR5 significantly (data not shown), as reported previously (26). To accurately correlate the expression of CCR2 or CCR5 with the cytokine profile ex vivo, we decided to first isolate SP and DP cells from memory CCR2⁺ T cells by using a flow cytometric cell sorter and stimulate them with PMA and ionomycin. We then measured IFN- and IL-17 in the supernatant (Fig. 3A). Remarkably, the sorted T cell subpopulations exhibited different cytokine production patterns: SP cells produced a large amount of IL-17 and a small amount of IFN-, whereas DP cells produced a small amount of IL-17 and a large amount of IFN- (Fig. 3B). These results suggest that Th17 cells are largely confined to SP cells, whereas DP cells contain a majority of Th1 cells.

View larger version (33K): [in this window] [in a new window]   FIGURE 2. CCR2+ memory CD4+ T cells consist of CCR5+ and CCR5– subsets. Purified memory CD4+ T cells were stained with anti-CCR2-biotin and streptavidin-PE as well as anti-CCR5-FITC. The separation of positive and negative populations was determined by histogram plots.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. CCR2+CCR5– (SP) and CCR2+CCR5+ (DP) cells correspond to Th17 and Th1 cells, respectively. SP and DP subsets were sorted from memory CD4+ T cells by flow cytometry and stimulated with PMA/ionomycin. A, A representative of five individual data sets showing the purity of the sorted cells. B, SP and DP cells were stimulated with PMA/ionomycin for 24 h. Then the amounts of IFN- and IL-17 in the supernatant were measured using ELISA. Results are expressed as mean ± SD of a representative of five independent experiments. *, p < 0.01

Finally, we assessed whether SP and DP cells are distinctive in expression of transcription factor T-bet and IL-23R. T-bet is an essential transcription factor for Th1 differentiation (27), whereas it was reported to be redundant for Th17 cells (3, 8, 9, 11, 14). IL-23 has been shown to play a pivotal role in the survival and expansion of Th17 cells (2, 10). Magnetically purified memory CD4⁺ T cells were first stained with biotin-conjugated CCR2, streptavidin-PE, and allophycocyanin-CCR5, and then were intracellularly stained with FITC-anti-T-bet or were stained with goat anti-IL-23R Ab and anti-goat IgG-FITC. We compared T-bet expression in SP vs DP cells by evaluating the mean fluorescence intensity (MFI) (Fig. 4, A and B). T-bet was significantly expressed by SP as well as CCR2^–CCR5^– double-negative (DN) cells, but its expression was much higher in DP cells and CCR2^–CCR5⁺ cells, suggesting that Th1 cells may be confined to CCR5⁺ populations. On the other hand, the frequency of IL-23R⁺ cells was highest in the SP fraction, compared with the others (Fig. 4, C and D). Given the distinguished ability to produce IL-17 as well as higher IL-23R and lower T-bet expression, we propose that Th17 cells are confined to SP cells, whereas Th1 cells are either DP or CCR2^–CCR5⁺. It has recently been reported that T-bet directly regulates the transcription of IL-23R in mice (28). It is possible that weak expression of IL-23R by non-Th17 cells (DP, DN, and CCR2^–CCR5⁺) may result from baseline activation of T-bet.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. T-bet expression of memory CD4+ T cells. A, Histogram plots of T-bet expression within memory CD4+ T cells. SP, DP, DN (CCR2–CCR5–), and CCR2–CCR5+ T cells were stained with anti-T-bet or isotype control Ab. Shown is a representative of five individual data sets. B, The MFI of each histogram plot. Data for the MFI of T-bet expression subtracted by that of control Ab are calculated and shown as bar graphs with error bars showing the SD of four individual data sets. C, Histogram plots of IL-23R expression within memory CD4+ T cells. The cells were stained with goat anti-IL-23R polyclonal Ab and anti-goat-FITC Ab. A representative of seven individual data sets is shown. *, p < 0.01. D, The frequency of IL-23R-positive cells of each histogram plot. Data are shown as bar graphs with error bars showing the SD of seven individual data sets. *, p < 0.01.

Using freshly isolated healthy human lymphocytes, we showed here that CCR2⁺CCR5^– memory T cells would produce a large amount of IL-17 but not IFN-, whereas CCR2^– memory T cells produced IFN-, but not IL-17. Although we presented the data obtained after stimulation with PMA/ionomycin, polyclonal stimulation by anti-CD3/CD28 also gave similar results (data not shown). Moreover, when we stimulated CCR2⁺CD4⁺ memory T cells by IFN-, IL-4, IL-2, or IL-23 in addition to PMA/ionomycin, IL-17 production was not changed (data not shown).

The frequency of Th17 cells among this subset is an important issue to be investigated. By using the ELISPOT assay, we found that 200 spots of IL-17-producing cells could be detected among 1 x 10⁵ memory CCR2⁺CD4⁺ T cells (0.2%), whereas the numbers of IFN--producing cells were about 5-fold higher (1.0%). Although this needs to be systematically verified, the lower frequency of IL-17-producing cells is consistent with the lower value of IL-17 than IFN- in supernatants detected by ELISA.

The unique chemokine receptor expression pattern of Th17 cells provides a basis for their recruitment to specialized inflammatory conditions in vivo, which should be relevant for understanding the pathogenesis of autoimmune diseases.

	Acknowledgments

 
We thank Hiromi Yamaguchi for excellent technical assistance.

	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 by a Grant-in-Aid for Scientific Research from the Japan Society (S) to T.Y. and Grant-in-Aid for Young Scientists (Start-up) to T.A. for the Promotion of Science and Research Grants from the Ministry of Health, Labour and Welfare of Japan.

² Address correspondence and reprint requests to Dr. Takashi Yamamura, Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. E-mail address: yamamura{at}ncnp.go.jp

³ Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; DN, double negative; DP, double positive; SP, single positive; MFI, mean fluorescence intensity.

Received for publication January 26, 2007. Accepted for publication April 23, 2007.

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Top Abstract Introduction Materials and Methods Results and Discussion Disclosures References

 

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