Regulation of IL-17 in Human CCR6+ Effector Memory T Cells Hong Liu and Christine Rohowsky-Kochan This information is current as J Immunol 2008; 180:7948-7957; ; of September 26, 2021. doi: 10.4049/jimmunol.180.12.7948 http://www.jimmunol.org/content/180/12/7948 Downloaded from References This article cites 25 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/180/12/7948.full#ref-list-1

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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

Regulation of IL-17 in Human CCR6؉ Effector Memory T Cells1

Hong Liu and Christine Rohowsky-Kochan2

IL-17–secreting T cells represent a distinct CD4؉ effector lineage (Th17) that appears to be essential in the pathogenesis of numerous inflammatory and autoimmune diseases. Although extensively studied in the murine system, human Th17 cells have not been well characterized. In this study, we identify CD4؉CD45RO؉CCR7؊CCR6؉ effector memory T cells as the principal IL-17-secreting T cells. Human Th17 cells have a unique profile because the majority coexpress TNF-␣ but not IL-6 and a minor subset express IL-17 with IL-22 or IL-17 and IFN-␥. We demonstrate that the that promote the differentiation of human naive T cells into IL-17-secreting cells regulate IL-17 production by memory T cells. IL-1␤ alone or in association with IL-23 and IL-6 markedly increase IL-17؉ CCR6؉ memory T cells and induce IL-17 production in CCR6؊ memory T cells. We ؉ also show that T cell activation induces Foxp3 expression in T cells and that the balance between the percentage of Foxp3 and Downloaded from IL-17؉ T cells is inversely influenced by the cytokine environment. These studies suggest that the cytokine environment may play a critical role in the expansion of memory T cells in chronic autoimmune diseases. The Journal of Immunology, 2008, 180: 7948–7957.

nterleukin-17 or IL-17A is the founding member of a family that for human naive CD4ϩ T cells, expression of the transcription ␥ ␤

of cytokines, comprising six members IL-17A-IL-17F. IL-17 factor ROR t and Th17 polarization were induced by IL-1 and http://www.jimmunol.org/ I is a proinflammatory cytokine that induces the production of IL-6 and suppressed by TGF-␤ and IL-12, whereas another group various inflammatory mediators including IL-6, IL-8, GM-CSF, showed that IL-1␤ and IL-23 were critical in the development of and PGE2 from fibroblasts, epithelial, and endothelial cells. IL-17 human Th17 cells (9, 10). Very little work has been done on the has been implicated in mediating protection against extracellular characterization of the IL-17-secreting memory T cells and the microbes and in playing a critical role in the pathogenesis of sev- factors that regulate IL-17 production. Initially IL-17 mRNA ex- eral inflammatory and autoimmune disorders. In both experimental pression was reported to be restricted to CD4ϩCD45ROϩ T cells autoimmune encephalomyelitis and type II collagen-induced ar- (11), although no protein data were shown. IL-17 expression in thritis, the animal models for multiple sclerosis and rheumatoid CD8ϩ memory T cells was shown to require costimulatory sig- ϩ ϩ arthritis, respectively, it was shown that IL-17-secreting CD4 nals from accessory cells because polyclonal activation of CD8 by guest on September 26, 2021 effector T cells are highly pathogenic and essential for the estab- CD45ROϩ T cells alone did not result in IL-17 production (12). lishment of organ-specific autoimmunity (1, 2). Recently, IL-17-secreting T cells were detected in both The IL-17-secreting Th cells (Th17 cells) have been described CD4ϩCD45ROϩCCR7ϩ central memory and CD4ϩCD45ROϩ in mice as a distinct subset of effector cells whose differentiation CCR7Ϫ effector memory T cell subsets, with central memory from naive T cells are promoted by IL-6 and TGF-␤ and require T cells having a 3-fold higher number than effector memory T the transcription factor ROR␥t (3–6). IFN-␥ and IL-4 inhibit the cells (13). Further analysis showed that the IL-17ϩ T cells ex- differentiation of naive T cells into Th17 cells, whereas IL-23 is pressed both CCR6 and CCR4 chemokine receptors. Moreover, necessary for the expansion and survival of IL-17-secreting mem- a number of studies have not been able to detect IL-17-produc- ory T cells (7, 8). In experimental autoimmune encephalomyelitis, ing T cells in the peripheral blood of healthy individuals only in IL-23 was found to be essential in the expansion of pathogenic, patients with inflammatory conditions (14, 15). The cellular autoreactive CD4ϩ T cells, which secreted IL-17A, IL-17F, IL-6, phenotype and regulation of human memory T cells secreting and TNF-␣ (1). IL-17 has not fully been elucidated. Since the identification of murine Th17 cells, extensive efforts In this study, we demonstrate that effector memory T cells that have been made to characterize the differentiation of naive CD4ϩ express the chemokine receptor CCR6 are the principal IL-17- IL-17-secreting T cells in humans. Recently, one group reported secreting cells and that IL-1␤ markedly enhances IL-17 production by increasing the percentage of CCR6ϩ IL-17ϩ T cells and by inducing IL-17 production in CCR6Ϫ memory T cells. Human Department of Neurology and Neurosciences, University of Medicine and Dentistry Th17 cells have a unique cytokine profile as the majority coexpress New Jersey-New Jersey Medical School, Newark, NJ 07103 TNF-␣ but not IL-6 and a minor subset expresses IL-17 with IL-22 Received for publication April 3, 2008. Accepted for publication April 3, 2008. or IL-17 and IFN-␥. Moreover, we show that T cell activation The costs of publication of this article were defrayed in part by the payment of page induces Foxp3 expression in T cells and that the balance between charges. This article must therefore be hereby marked advertisement in accordance ϩ ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. the percentage of Foxp3 and IL-17 T cells is inversely influ- enced by the cytokine environment. 1 This work was supported by Grant NS34245 from the National Institutes of Health (to C.R.-K.). 2 Address correspondence and reprint requests to Dr. Christine Rohowsky-Kochan, Materials and Methods University of Medicine and Dentistry New Jersey-New Jersey Medical School, 185 Isolation of T cell subsets South Orange Avenue, Newark, NJ 07103. E-mail address: [email protected] Research protocols were approved by the Institutional Review Board of Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 New Jersey Medical School in accordance with regulations mandated by www.jimmunol.org The Journal of Immunology 7949 the Department of Health and Human Services. Informed consent was ob- says (Applied Biosystems) and reagents were used according to the man- tained from each subject. PBMC were isolated by Ficoll-Hypaque gradient ufacturer’s instructions. Specific gene expression was normalized to the centrifugation of heparinized venous blood obtained from healthy individ- housekeeping genes GAPDH and HPRT-1. Expression of IL-17 mRNA uals. All isolations of T cell subsets were performed using magnetic beads levels was calculated by first determining the average threshold cycle (⌬Ct) and reagents from Miltenyi Biotec. CD4ϩ T cells were isolated from for each culture, which corresponded to the following: (average IL-17 PBMC by immunomagnetic depletion of non-Th cells (CD4ϩ T Cell Iso- threshold cycle Ϫ average HPRT-1 (or GAPDH) threshold cycle). Tripli- lation kit) according to the manufacturer’s instructions. Memory cate samples were used to calculate the average threshold cycle. The rep- CD45ROϩ T cells were purified from the CD4ϩ T cells using magnetic licate threshold cycle (⌬⌬Ct) was then calculated with the following for- Ͼ ⌬ Ϫ⌬ sorting with CD45RO beads, and the purity was 95% in all experiments. mula: ( Ctwith cytokine Ctwithout cytokine). IL-17 and RORC mRNA were Naive CD4ϩCD45RAϩ T cells were obtained by immunomagnetic deple- expressed as expression fold value (2Ϫ⌬⌬Ct). tion of CD45ROϩ T cells. For isolation of central and effector memory T cell subsets, CD4ϩ T cells were magnetically separated using CD45RA Statistical analysis microbeads and the CD45RAϪ fraction (CD45ROϩ) was labeled with FITC-conjugated CCR7 Ab (R&D Systems) followed by anti-FITC mi- Data were analyzed using the Statistical Package for Social Sciences soft- crobeads and passed through the magnetic sorting column. This yielded a ware program (SPSS). Data involving two groups only were analyzed us- population of purified central memory T cells (CD4ϩCD45ROϩCCR7ϩ) ing the paired Student’s t test, whereas data involving more than two ϩ ϩ Ϫ groups were analyzed using one-way ANOVA and Tukey’s multiple com- and effector memory T cells (CD4 CD45RO CCR7 ). To obtain Ͻ CCR6ϩ/Ϫ effector and central memory T cells, we purified CD4ϩ T cells parison test. A value of p 0.01 was considered statistically significant. by magnetic sorting, stained the cells with a PerCP-conjugated CD4, PE- conjugated CD45RO, allophycocyanin-conjugated anti-CCR6, and FITC- Results conjugated anti-CCR7 and than sorted on a FACSVantage SE System (BD Ligation of the TCR and CD28 on CD4ϩ T cells induces the Biosciences). The purity of the sorted CCR6ϩ/Ϫ effector and central mem- ϩ secretion of IL-17 ory T cells was over 99%. CD8 T cells were isolated by positive selection Downloaded from using CD8 microbeads to Ͼ98% purity. The purity of each separated pop- Since several studies were not able to detect IL-17-secreting T ulation was assessed by immunofluorescence flow cytometry. cells in the peripheral blood of healthy individuals, we initially ϩ T cell cultures assessed the kinetics of IL-17 secretion by CD4 T cells activated with immobilized anti-CD3 and soluble CD28 Ab. IL-17 levels ϩ ϫ 5 Purified CD4 T cells (2 10 /well), resuspended in RPMI 1640 supple- were measured in the cell culture supernatants by ELISA. Cross- mented with 10% heat-inactivated FCS (HyClone Laboratories), penicillin, ϩ streptomycin, L-glutamine, and CD28 Ab (2 ␮g/ml; BD Pharmingen) were linking the TCR and CD28 receptor on CD4 T cells resulted in http://www.jimmunol.org/ cultured in 96-well U-bottom plates coated with anti-CD3 Ab (5 ␮g/ml; secretion of IL-17 with low levels of the cytokine detected in day BD Pharmingen) for 3 and 6 days. In some experiments, rIL-23 (50 ng/ml; 3 supernatants (mean Ϯ SEM, 726 Ϯ 254 pg/ml), which substan- ␤ R&D Systems), rIL-6 (40 ng/ml; BioSource International), rIL-1 (10 ng/ tially increased by day 6 (3357 Ϯ 1530 pg/ml) (Fig. 1a). Activa- ml; gift from Hoffmann-LaRoche), and rTGF-␤1 (5 ng/ml; R&D Systems) ϩ were added to the cultures either alone or in various combinations. Where tion of CD4 T cells with PMA and ionomycin induced optimal indicated cultures were supplemented with 5 ␮g/ml anti-IFN-␥ mAb (BD IL-17 production on day 3. IFN-␥ was also produced with similar ϩ Pharmingen) and 0.5 ␮g/ml of a polyclonal anti-IL-4 (R&D Systems). kinetics by the CD4 T cells activated with anti-CD3 and anti- Ϫ Supernatants were collected and stored at 80°C until levels of IL-17, CD28 as well as with PMA/ionomycin. No IL-17 was detected in ␥ IFN- , and IL-4 were determined by ELISA. the culture supernatants of the CD8ϩ T cells activated with anti- ELISA measurement of cytokine levels CD3 and anti-CD28, whereas PMA/ionomycin induced IL-17 se- by guest on September 26, 2021 cretion in the CD8ϩ T cells, although much less than in the CD4ϩ IL-17 was measured by ELISA using a plate-bound capture mAb (clone no. ϩ ␥ 41809, 2 ␮g/ml; R&D Systems) and a biotinylated polyclonal IL-17 de- T cells (Fig. 1b). CD8 T cells secreted IFN- regardless of which tection Ab (75 ng/ml; R&D Systems), with quantification by reference to activation method was used. Intracellular cytokine staining of the a rIL-17 standard (R&D Systems). IFN-␥ and IL-4 were quantitated using day 6 cultured cells confirmed the ELISA data and showed that the the OptEIA human IFN-␥ kit and IL-4 kit (BD Pharmingen) as per the CD4ϩ IL-17-secreting T cells were a distinct population from manufacturer’s instructions. The sensitivity of the ELISA was 7.8 pg/ml the CD4ϩ IFN-␥-secreting T cells, although a small percentage of for IL-17 and IL-4 and 4.7 pg/ml for IFN-␥. T cells secreted both IL-17 and IFN-␥ (Fig. 1c). No CD8ϩ IL-17- Intracellular cytokine staining secreting T cells were detected, although high levels of CD8ϩ ␥ Cultured cells were harvested on day 6, adjusted to 1 ϫ 106/ml and pulsed IFN- -secreting T cells were seen. with 20 ng/ml PMA (Sigma-Aldrich) and 100 ng/ml ionomycin (Sigma- ϩ ϩ Ϫ ϩ Aldrich) for 5 h. During the last 2 h, brefeldin A (Sigma-Aldrich) was CD4 CD45RO CCR7 CCR6 effector memory T cells are added to cultures to prevent protein secretion. Cells were stained for sur- the principal IL-17-secreting population face markers with fluorochrome-conjugated CD4, CD8, CD45RO, CCR6 ϩ (R&D Systems), and CCR7 (R&D Systems) Abs, fixed and permeabilized To show that CD4 memory T cells secrete IL-17, we first purified ϩ ϩ ϩ with 0.5% saponin (for Foxp3 staining methanol was used for permeabi- CD4 T cells into naive CD45RA and memory CD45RO T lization). Cells were incubated with a biotinylated IL-17 Ab (R&D Sys- cells and measured IL-17 production after TCR and CD28 cross- ␣ ϩ ϩ tems) plus streptavidin PE-Cy7 or with FITC-conjugated anti-TNF- or linking. IL-17 and IFN-␥ were both secreted by CD4 CD45RO PE-conjugated anti-IFN-␥, anti-IL-6, anti-IL-22, or anti-Foxp3 (Bioleg- T cells, whereas IFN-␥ but not IL-17 was detected in the super- end). All Abs with the exception of IL-17, CCR6, CCR7, and Foxp3 were ϩ ϩ obtained from BD Pharmingen. Data were analyzed on a FACSCalibur natants of the activated CD4 CD45RA T cells (Fig. 2a). PMA/ cytofluorometer using CellQuest software (BD Biosciences). ionomycin also stimulated production of IL-17 by CD4ϩ CD45ROϩ T cells but not by CD4ϩCD45RAϩ T cells (data not Real-time quantitative PCR shown). Intracytoplasmic immunofluorescence data confirmed that Total RNA was extracted from cells using the Absolutely RNA RT-PCR the IL-17ϩ T cells were of the CD4CD45RO phenotype (Fig. 2b). Miniprep kit (Stratagene) according to the manufacturer’s instructions. The Similar to the results with the unfractionated CD4ϩ T cells, the first strand of cDNA was obtained using Superscript First-Strand Synthesis ␥ System (Invitrogen) and transcripts were quantified by real-time quantita- IL-17-secreting memory T cells were distinct from the IFN- -se- tive PCR on an ABI Prism 7000 Sequence Detection System (Applied creting T cells, and a small subpopulation producing both IL-17 Biosystems). The IL-17 primers and probe were purchased from Applied and IFN-␥ was detected. Biosystems and the sequences are IL-17, sense 5Ј-ACAACCGATCCAC To further characterize which memory T cell subset secreted CTCACCTT-3Ј, antisense 5Ј-CTTTGCCTCCCAGATCACAGA-3Ј, and ϩ Ј Ј IL-17, we purified the memory T cells into central memory (CD4 probe 5 6FAM-CTCCACCGCAATGAGGACCCTGAG-TAMRA-3 . ϩ ϩ ϩ ϩ For RORC (Hs01076112_ml), GAPDH (Hs00266705_g1), and HPRT-1 CD45RO CCR7 ) and effector memory (CD4 CD45RO Ϫ (Hs99999909_ml) expression, predesigned TaqMan Gene Expression As- CCR7 ) T cells and measured IL-17 production following ligation 7950 HUMAN IL-17ϩ IN MEMORY T CELLS

FIGURE 1. Activated CD4ϩ T cells secrete IL-17. Purified CD4ϩ (2 ϫ 105/well) or CD8ϩ T cells were activated with immobilized CD3 and soluble CD28 mAbs (3 and 6 days) or PMA and ionomycin (3 days). IL-17 and IFN-␥ levels (in picograms per milliliter) measured by ELISA in cul- ture supernatants from CD4ϩ T cells (a) and CD8ϩ T cells (b) are shown. Downloaded from Data from two representative donors are shown (right) as are the mean Ϯ SEM (inset) of all donors tested for CD4 (n ϭ 5 individuals) and CD8 (n ϭ 6 individuals). Supernatants from CD8ϩ T cells were collected http://www.jimmunol.org/ only on day 6. c, Intracytoplasmic staining for IL-17 and IFN-␥ was per- formed on the activated CD4ϩ and CD8ϩ T cells following restimulation with PMA/ionomycin for5hinthe presence of brefeldin A. Analysis was performed on a FACScan. Quadrant plot analysis shows the percentage of IL-17ϩ and IFN-␥ϩ CD4 and CD8 T cells. by guest on September 26, 2021

of the TCR and CD28 receptor. IL-17 was secreted predominantly secreting cells compared with the CCR7ϩ central memory T cells. by the CD4ϩCD45ROϩCCR7Ϫ effector memory T cells; although We than examined CCR7 expression on all memory CD45ROϩ very little amounts of IL-17 were produced by the CD4ϩCD45ROϩ IL-17ϩ T cells. Analysis of the IL-17ϩ T cells showed that 77.3% CCR7ϩ central memory T cells (Fig. 2c). IFN-␥ was also secreted of the IL-17-secreting cells were CD45ROϩCCR7Ϫ, whereas predominantly by the effector memory T cells with low levels of ϳ22.7% of the IL-17 producers were CD45ROϩ and expressed IFN-␥ secreted by the central memory T cells. Intracellular cyto- low levels of CCR7 (Fig. 2e). To ascertain whether expression of kine staining was performed to corroborate the protein data and the the chemokine receptor CCR6 was associated with IL-17 secre- results from one of five representative donors are shown in Fig. 2d. tion, we activated purified CD4ϩCD45ROϩ T cells and analyzed In the effector memory subpopulation, there was more than a IL-17 secretion in CCR6ϩ/Ϫ effector memory T cells. As illus- 4-fold increase (4.3 was median fold increase) in IL-17ϩ T cells as trated in Fig. 2f, the IL-17-producing T cells (13.3%) were pre- compared with the central memory T cell subset (Fig. 2d). The dominantly present in the CD4ϩCD45ROϩCCR7ϪCCR6ϩ subset majority of the effector memory T cells secreted only IL-17; how- with the low percentage of IL-17ϩ T cells (1.2%) in the CCR6Ϫ ever, a proportion (13%) of the IL-17ϩ T cells secreted both IL-17 fraction probably due to contaminating CCR7lowCCR6ϩ cells. The and IFN-␥. Because CCR7 may be expressed at low levels on majority of the CCR6ϩ subset secreted only IL-17 but no IFN-␥, effector memory T cells, we examined CCR7 expression on IL- but a subpopulation (3.9%) secreted both IL-17 and IFN-␥.In 17ϩ T cells. We activated purified CD4ϩCD45ROϩ T cells and contrast, IFN-␥-producing T cells were detected in both the analyzed IL-17 in the CCR7ϩ/Ϫ memory T cells. As shown in Fig. CCR6ϩ (21.9%) and CCR6Ϫ (44.4%) subset with the majority 2e, the CCR7Ϫ effector memory T cells had twice as many IL-17- being found in the CCR6Ϫ fraction. The Journal of Immunology 7951 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. CD4ϩCD45ROϩCCR7ϪCCR6ϩ effector memory T cells secrete IL-17. a, IL-17 and IFN-␥ levels in supernatants from purified CD4ϩ CD45ROϩ and CD4ϩCD45RAϩ T cells activated with immobilized CD3 and soluble CD28 mAbs as described in Fig. 1 are shown. Mean cytokine levels Ϯ SEM (n ϭ 7 individuals) (inset) and two representative donors are shown. b, Intracellular staining analysis performed on activated CD4ϩCD45ROϩ and CD4ϩCD45RAϩ T cells depicts the percentage of IL-17ϩ and IFN-␥ϩ T cells. Dot analysis of one of seven representative donors is shown. c and d, Magnetic bead purified CD4ϩCD45ROϩCCR7Ϫ and CD4ϩCD45ROϩCCR7ϩ T cells were activated with CD3 and CD28 mAbs and IL-17 and IFN-␥ levels were measured by ELISA (c) and intracellular staining (d). Mean cytokine levels Ϯ SEM (n ϭ 4 individuals) (inset) and two representative 7952 HUMAN IL-17ϩ IN MEMORY T CELLS

FIGURE 3. Neutralization of IFN-␥ and IL-4 does not affect IL-17 se- cretion by memory T cells. Cross-linking of the TCR and CD28 receptor on CD4ϩ T cells was performed in the presence and absence of IFN-␥ and IL-4 mAbs. IL-17 was measured in the culture supernatants by ELISA (a) and by intracellular immunofluorescence techniques (b). ELISA data rep- resent the mean Ϯ SEM (n ϭ 6 individuals) and the flow cytometric anal- ysis represents one of six donors.

FIGURE 4. IL-17 is coexpressed with TNF-␣, IL-22 but not with IL-6. Neutralizing IFN-␥ and IL-4 does not effect IL-17 secretion by Activated CD4ϩ T cells were stained by intracellular techniques performed memory T cells following restimulation with PMA/ionomycin for5hinthepresence of brefeldin A as described in Fig. 1. Cells were assessed for IL-17, TNF-␣, To determine whether IFN-␥ or IL-4 had an effect on the produc- ϩ ϩ Downloaded from ϩ ϩ IL-22, and IL-6 on gated CD4 CD45RO T cells. a, Quadrant data for tion of IL-17 by memory T cells, we activated CD4 CD45RO T IL-17, TNF-␣, IL-22, and IL-6 are shown for one of four representative cells in the presence and absence of IFN-␥ and IL-4 Abs. Levels individuals. b, Intracellular analysis of coexpression of IL-17 with IFN-␥ of IL-17 (2123 Ϯ 711 pg/ml) in cultures with anti-IFN-␥ and anti- or IL-22 in gated populations of CD4ϩCD45ROϩCCR7ϪCCR6ϩ and CD4ϩ IL-4 were not significantly different from levels (2080 Ϯ 849 pg/ CD45ROϩCCR7ϪCCR6Ϫ T cells. Data are representative of one of two ml) in cultures without the Abs (Fig. 3a). IL-4 was not detected in donors. any of the cultures. Likewise, there was no difference in the num- http://www.jimmunol.org/ ber of IL-17-secreting T cells as measured by intracellular cyto- kine analysis in the cultures activated in the presence or absence of tants. Ligation of the TCR and CD28 on CD4ϩCD45ROϩ T cells anti-IFN-␥ and anti-IL-4 (Fig. 3b). in the absence of exogenous cytokines resulted in the induction of IL-17 (median value, 2231 pg/ml), which significantly increased IL-17 is coexpressed with TNF-␣ and to a lesser extent with when either IL-23 (4550 pg/ml, p Ͻ 0.01) or IL-1␤ (5485 pg/ml, IL-22 in memory T cells p Ͻ 0.02) alone were added to the cell cultures (Fig. 5a). Addition In mice, Th17 cells have been reported to secrete IL-17, TNF-␣, of IL-6 alone caused a modest, although not significant increase in IL-22, and IL-6 (1). To determine whether the same is true in IL-17 (2989 pg/ml). However, when IL-6 was added together with humans, we examined the expression of IL-17, IL-22, IL-6, IL-23, a significant increase in IL-17 levels (4422 pg/ml, p Ͻ by guest on September 26, 2021 TNF-␣, and IFN-␥ in memory T cells. We observed that only 0.004) was observed. The greatest effect on IL-17 secretion was 33% of the total IL-17-secreting cells coexpressed IL-22 and observed when IL-1␤ was added in combination with either IL-23 that the majority of IL-17-secreting T cells (67%) did not pro- (11,726 pg/ml, p Ͻ 0.004) or IL-6 (8358 pg/ml, p Ͻ 0.004). In duce IL-22 (Fig. 4a). Coexpression of IL-22 with IL-17 was contrast, addition of TGF-␤ alone inhibited, albeit not signifi- very similar to that of IFN-␥ and IL-17. In contrast, TNF-␣ was cantly, IL-17 levels (1185 pg/ml). The inhibitory effect of TGF-␤ ϩ associated with the majority of IL-17 T cells (70%). A pop- on IL-17 production was significantly counteracted in the presence ulation of IL-22-secreting T cells that did not produce IL-17 of IL-1␤ alone (3607 pg/ml, p Ͻ 0.03), IL-23 together with IL-6 was also detected. IL-6 production was not detected following (3461 pg/ml, p ϭ 0.05), or the combination of IL-1␤, IL-6, and TCR and CD28 receptor ligation in any of the individuals IL-23 (4098 pg/ml, p Ͻ 0.04). tested. Similar results were obtained when we examined IL-22 We then examined whether these cytokines had an effect on ϩ ϩ Ϫ ϩ expression in the CD4 CD45RO CCR7 CCR6 T cells (Fig. IL-17 mRNA expression by real-time PCR. Similar to the effect ϩ 4b). A small subset (3.6%) of CCR6 effector memory T cells observed on protein levels, IL-17 mRNA expression was increased produced both IL-17 and IL-22, although the majority of by IL-23, IL-6, and IL-1␤ alone, whereas IL-23 and IL-1␤ together ϩ ϩ CCR6 IL-17 effector memory T cells (15%) did not secrete markedly augmented IL-17 transcription (Fig. 5b). Addition to the ϩ IL-22. Likewise, 4% of the CCR6 effector memory T cells cell cultures of IL-6 and IL-23, IL-6 and IL-1␤, IL-6, IL-23, and secreted IL-17 and IFN-␥, whereas 13% secreted only IL-17. TGF-␤, or IL-6, IL-1␤, IL-23, and TGF-␤ also enhanced IL-17 mRNA expression. No effect on IL-17 transcription was detected ␤ IL-1 and to a lesser extent IL-6 and IL-23 up-regulate IL-17 in the presence of TGF-␤ alone. We than assessed the expres- expression in effector memory T cells sion of RORC, the human ortholog of mouse ROR␥t, a tran- To determine whether certain cytokines regulate IL-17 expression scription factor that is involved in Th17 differentiation, in these of memory T cells, we activated purified CD4ϩCD45ROϩ T cells cultures. RORC mRNA expression was detected in the acti- with anti-CD3 and anti-CD28 in the presence of IL-23, IL-1␤, vated CD4 memory T cells (Fig. 5c). Addition of IL-1␤ with IL-6, or TGF-␤ and measured IL-17 levels in the culture superna- either IL-23 or IL-6 to the cultures markedly augmented IL-17

individuals (inset) are shown in c. Data from one representative experiment (n ϭ 5) are depicted for the intracellular analysis. The CD4ϩCD45ROϩCCR7Ϫ T cells for donor 1 and donor 2 have 4.5% and 9% residual CCR7ϩ cells, respectively, whereas the CD4ϩCD45ROϩCCR7ϩ T cells were Ͼ85% pure. e, Intracellular staining of activated CD4ϩCD45ROϩ T cells was performed and CCR7 expression was assessed on gated CD45ROϩ and IL-17ϩ T cells. f, Activated CD4ϩCD45ROϩ T cells were analyzed for CCR7 and CCR6 expression and the percentage of IL-17- and IFN-␥-secreting cells is illustrated. Data represent one of three independent experiments. The Journal of Immunology 7953 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 7954 HUMAN IL-17ϩ IN MEMORY T CELLS mRNA expression but had no affect on RORC mRNA expres- fold increase, respectively, over levels in the absence of exog- sion. Unexpectedly, expression of RORC mRNA was increased enous cytokines. No IL-17 was secreted by the CCR6ϩ/Ϫ cen- in the presence of TGF-␤. tral memory T cells in the absence or presence of IL-1␤ and To further examine the role of these individual cytokines on IL-23. IL-17 secretion, we performed intracellular cytokine analysis of CCR6ϩ/Ϫ effector memory T cells activated in the presence and ␤ absence of IL-1 , IL-6, and IL-23. We ascertained the effect of Ligation of the TCR and CD28 leads to Foxp3 expression these cytokines on three populations of memory T cells: those ϩ ␥ Several studies have reported that TCR ligation of human CD4 secreting only IL-17, those secreting IL-17 plus IFN- , and those Ϫ secreting IL-17 plus IL-22. As expected, in the absence of exog- CD25 T cells induces expression of Foxp3 (16–18). We in- enously added cytokines, we detected CCR6ϩ IL-17ϩ (11.7%), vestigated whether Foxp3-expressing T cells were present in ϩ CCR6ϩ IL-17ϩ IFN-␥ϩ (3.6%), and CCR6ϩ IL-17ϩ IL-22ϩ our cultures along with IL-17 T cells and whether IL-1␤, IL-6, ϩ (3.4%) T cells (Fig. 5d). Activation with IL-1␤ led to a 3-fold IL-23, and TGF-␤ affectedFoxp3 expression. We activated CD4 ϩ Ϫ increase in CCR6ϩ IL-17ϩ T cells (33.7%) and a 4-fold augmen- CD45RO CD25 T cells with anti-CD3 and anti-CD28 in the tation of CCR6ϩ IL-17ϩ IFN-␥ϩ (13.9%) and CCR6ϩ IL-17ϩ presence or absence of these cytokines and assessed the per- ϩ ϩ IL-22ϩ (13.5%) T cells. When IL-23 or IL-6 was added alone centage IL-17 T cells and Foxp3 T cells using intracellular during activation, there was a doubling in the percentage of all cytokine staining and flow cytometry. Cell cultures were gated three IL-17-secreting subsets. The addition of exogenous IL-23 or on surface expression of CD45RO and examined for intracel-

IL-6 either alone or together, with IL-1␤ did not further enhance lular expression of IL-17 and Foxp3. In the absence of exoge- Downloaded from ϩ the number of IL-17-secreting cells over the number detected with nous cytokines, we detected a low percentage of CD4 ϩ ϩ ϩ IL-1␤ alone. IL-1␤ and IL-6 slightly increased the percentage of CD45RO IL-17 T cells (1.7%) and a high number of CD4 ϩ ϩ total CCR6ϩ T cells, whereas IL-23 slightly decreased the per- CD45RO Foxp3 T cells (11.9%) following cross-linking of centage (data not shown). A slight decrease in the percentages of the TCR and CD28 receptor (Fig. 6). Addition of either IL-23 ϩ CCR6ϩ IFN-␥-secreting T cells was seen in the presence of all of or IL-1␤ alone led to a doubling in the percentage of IL-17 T ϩ these cytokines, whereas no effect was seen in the percentages cells but a decrease in the percentage of Foxp3 T cells, http://www.jimmunol.org/ of CCR6ϩ IL-22ϩ T cells. whereas IL-6 had no effect on IL-17 expression but similarly Surprisingly, in the presence of these three cytokines, IL-17- decreased Foxp3 expression. In contrast, activation with TGF-␤ ϩ producing T cells were seen in the CCR6Ϫ population. In the ab- resulted in a slight decrease in IL-17 T cells (1.3%) and an ϩ sence of exogenously added cytokines, very low numbers of increase in Foxp3 T cells (16.0%). Activation with IL-1␤ and CCR6Ϫ IL-17ϩ (1.4%), CCR6Ϫ IL-17ϩ IFN-␥ϩ (1.1%), and IL-23 together resulted in the greatest effect on the expression CCR6Ϫ IL-17ϩ IL-22ϩ (0.5%) T cells were observed (Fig. 5d). of both IL-17 and Foxp3 with a marked increase in IL-17-se- Activation in the presence of IL-1␤ induced a 10-fold increase in creting cells (11.4%) and a concomitant decrease in Foxp3 ex- CCR6Ϫ IL-17ϩ (13.8%), CCR6Ϫ IL-17ϩ IFN-␥ (9.7%) and pression from (5.3%). Similar but to a lesser extent were the CCR6Ϫ IL-17ϩ IL-22ϩ (5.2%) memory T cell subsets. Addition of effects of activating with either IL-23 and IL-6 or IL-1␤ and by guest on September 26, 2021 IL-23 resulted in almost a 6-fold enhancement in the number of all IL-6. The addition of exogenous TGF-␤ with IL-1␤, IL-6, and three subsets, whereas IL-6 led to a 3-fold increase. The addition IL-23 either alone or in combination led to similar results, of exogenous IL-6 or IL-23 with IL-1␤ resulted in a slight increase which were an increase in IL-17 and decrease in Foxp3 in the percentage of CCR6Ϫ IL-17ϩ-secreting T cells compared expression. with the addition of IL-1␤ alone. IL-1␤ and IL-6 slightly decreased To further investigate the relationship between IL-17 and Foxp3 ϩ the percentage of total CCR6Ϫ T cells, whereas IL-23 slightly expression, we compared the percentage of IL-17 T cells and the ϩ increased the percentage (data not shown). No effect was observed percentage of Foxp3 cells upon activation under the different on the frequency of CCR6Ϫ IFN-␥ϩ T cells, although the fre- culture conditions. We observed that TGF-␤ promotes the great- quency of CCR6Ϫ IL-22ϩ T cells was slightly increased in the est increase in percentage of Foxp3ϩ cells and decrease in per- presence of these cytokines. centage of IL-17ϩ T cells (Fig. 7). Addition of IL-1␤, IL-6, or We than sorted CD4ϩCD45ROϩ T cells based on the expres- IL-23 alone or of IL-6 with IL-23 to TGF-␤ steadily decreases sion of CCR6 and CCR7, activated them with anti-CD3 and the percentage of Foxp3ϩ cells with a slight increase in IL-17ϩ anti-CD28 with or without IL-1␤ and IL-23, and measured T cells. The percentage of Foxp3ϩ cells continues to steadily de- IL-17 levels. In the presence of IL-1␤ and IL-23, there was a cline in the presence of IL-1␤, IL-6, IL-23 alone or IL-6 with marked increase in IL-17 secretion in both the CCR6ϩ and either IL-1␤ and IL-23, and under these conditions the percentage CCR6Ϫ effector T cells (Fig. 5e). The CCR6ϩ effector memory of IL-17ϩ T cells and of Foxp3ϩ cells are very similar. The highest T cells secreted 2222 pg/ml IL-17, whereas the CCR6Ϫ effector percentage of IL-17ϩ T cells is observed upon addition of IL-1␤ memory T cells produced 327 pg/ml, which was a 9- and 17- and IL-23 to the cultures, which induces the greatest drop in the

FIGURE 5. Regulation of IL-17 expression in effector memory T cells. Purified CD4ϩCD45ROϩ T cells were activated by CD3 and CD28 ligation in the presence of IFN-␥ and IL-4 mAbs and IL-1␤, IL-6, IL-23, and TGF-␤ alone or in various combinations for 6 days. a, IL-17 and IFN-␥ levels were measured by ELISA and median values from n ϭ 6 separate experiments are depicted. a, p Ͻ 0.02; b, p Ͻ 0.004 compared with no cytokine; c, p Ͻ 0.04; and d, p ϭ 0.05 compared with TGF-␤ alone. b, IL-17 mRNA expression in the T cell cultures activated in the presence of various cytokines was assessed by real-time PCR and one of three representative experiments are shown. Real-time PCR results are expressed as 2Ϫ⌬⌬Ct normalized to HPRT-1. c, RORC (f) and IL-17 (o) mRNA expression in the T cell cultures activated in the presence of various cytokines was determined by real-time PCR and is shown. d, Activated CD4ϩ CD45ROϩ T cells from a were subsequently stained for intracellular IL-17, IFN-␥, and IL-22. Flow cytometric analysis was performed on gated CCR6ϩ and CCR6Ϫ effector memory T cells. e, Sorted CCR6ϩ/Ϫ effector memory T cells were activated with anti-CD3 and anti-CD28 in the presence and absence of IL-1␤ and IL-23, and IL-17 levels were measured by ELISA. The fold increase in IL-17 levels in the cultures with IL-1␤ and IL-23 was compared with the cultures without these cytokines. Data from one of two representative donors are shown. The Journal of Immunology 7955 Downloaded from

FIGURE 6. CD3 and CD28 cross-linking induces Foxp3 expression. CD4ϩCD45ROϩ T cells were activated in the presence of IFN-␥ and IL-4 mAbs and IL-1␤, IL-6, IL-23, and TGF-␤ alone or in various combinations for 6 days similar to culture conditions described in Fig. 5. Cells were examined for intracellular expression of IL-17 and Foxp3. Histogram analysis illustrates the percentage of CD4ϩCD45ROϩ IL-17ϩ and Foxp3ϩ T cells. Results are representative of one of four individuals tested. http://www.jimmunol.org/ percentage of Foxp3ϩ cells. The cytokines that promote the great- Foxp3ϩ cells and the percentage of IL-17ϩ T cells is inversely est increase in IL-17 cause the greatest decrease in Foxp3 expres- influenced by the cytokine environment. sion and conversely the cytokine that induces the greatest in- crease in Foxp3 promotes the greatest decrease in the ϩ Discussion percentage of IL-17 T cells. No association between the per- In the present study, we have characterized the phenotype, cyto- ␥ϩ ϩ centage of IFN- T cells and Foxp3 cells under any of these kine profile, and the factors regulating human memory T cells

␥ ϩ ϩ Ϫ by guest on September 26, 2021 culture conditions was observed. The percentage of IFN- -ex- secreting IL-17. We have identified the CD4 CD45RO CCR7 pressing T cells always greatly exceeded the percentage of ϩ ϩ CCR6 effector memory T cell as the principal IL-17-secreting Foxp3 T cells. Thus, the balance between the percentage of T cell. In contrast, IFN-␥-secreting T cells were found in both CCR6ϩ/Ϫ effector memory subsets with the majority being in the CCR6Ϫ fraction. Although IL-17 mRNA expression had been ini- tially reported in human memory T cells, no protein data had been shown. During the preparation of this study, a report was published showing the human CD4ϩ memory T cells secrete IL-17 (19). Recently, it has been reported that CCR6 expression identifies hu- man IL-17-secreting memory T cells and that both central and effector memory T cells produce IL-17 with a higher percentage of IL-17ϩ T cells detected within the central memory subset (13). Our data are partially in agreement with this study because we found that only CCR6ϩ effector memory T cells secreted IL-17 and the low percentage of IL-17-secreting T cells detected in the CCR6ϩ central memory population represent cells with low levels of CCR7 expression. Effector memory T cells have been described as not expressing CCR7 or expressing very low levels of this che- mokine receptor. In mice, Th17 cells have been reported to secrete IL-17, TNF-␣, IL-22, and IL-6 (1, 20). Cytometric single-cell analysis of the cy- tokines produced by human memory IL-17ϩ T cells revealed co- expression of TNF-␣ in the majority of IL-17-secreting T cells, whereas IL-22 was only coexpressed in a small subset of the IL- 17ϩ T cells. Similar to the results with IL-22, IFN-␥ was coex-

ϩ ϩ pressed with IL-17 in a small subset of memory T cells. We con- FIGURE 7. Relationship between the percentage of IL-17 and Foxp3 ϩ ϩ ␥ϩ ϩ sistently observed three subpopulations of memory T cells: IL-17 T cells. A comparison of the percentage for IL-17 , IFN- , and Foxp3 ϩ ϩ ϩ ␤ memory T cells, IL-17 and IL-22 memory T cells, and IL-17 T cells in the cultures activated in the presence of IL-1 , IL-6, IL-23, or ϩ TGF-␤ was made. The percentage of IL-17ϩ T cells vs that of Foxp3ϩ T and IFN-␥ memory T cells. In fact, it is possible that the IL- ϩ ϩ ϩ cells (top) and the percentage of IFN-␥ϩ T cells vs Foxp3ϩ T cells (bottom) 17 IL-22 cells are IFN-␥ . Recently, it has been shown that are plotted. Results are representative of one of four individuals tested. most of the human, naive T cells activated in the presence of IL-1␤ 7956 HUMAN IL-17ϩ IN MEMORY T CELLS and IL-6 secrete IL-17 and TNF-␣ and approximately one-half Another potential mechanism by which these cytokines regulate secrete IL-17 and IL-22 (9). Our observations of memory T cells IL-17 secretion is by modulating Foxp3 expression. We have ob- secreting the same cytokines suggest that these subpopulations served that cross-linking of the TCR and CD28 coreceptor on CD4ϩ represent stable phenotypes of long-lived memory T cells. Human CD45ROϩCD25Ϫ T cells results in the expression of Foxp3ϩ as Th17 and IFN-␥-producing Th17 cells have been detected in the well as IL-17ϩ T cells and that IL-1␤, IL-6, IL-23, or TGF-␤ gut of subjects with Crohn’s disease and in the blood of healthy concomitantly modulated Foxp3 and IL-17 expression. IL-1␤, individuals (15, 19). IL-6 was not induced following TCR and IL-6, and IL-23 either alone or in various combinations up-regu- CD28 cross-linking in our experiments. Whether murine Th17 ac- lated IL-17 and down-regulated Foxp3 expression, whereas TGF-␤ tually secrete IL-6 is unclear because the initial studies showing modestly decreased IL-17 and up-regulated Foxp3 expression. that IL-23 expands a population of IL-17, TNF, and IL-6 cells Moreover, we found that the balance between the percentage of were performed by gene expression analysis and may have con- Foxp3ϩ and IL-17ϩ T cells is inversely influenced by these cyto- tained contaminating IL-6-secreting dendritic cells (1). kines. It has been shown that TCR stimulation of human CD4ϩ In a number of studies, IL-17-producing T cells were not de- CD25Ϫ T cells results in expression of Foxp3 and that these tected in the peripheral blood of healthy individuals only in pa- Foxp3ϩ cells have regulatory function (16). Other studies reported tients with inflammatory conditions (11, 14). We have consistently that the induced Foxp3 expression did not correlate with suppres- detected IL-17 after TCR and CD28 ligation of CD4ϩ T cells from sive function (18, 23). The Foxp3 mAb used in our experiments is healthy subjects. We observed that peak secretion of IL-17 occurs clone 259D, which has been shown to specifically bind to the on day 6 following TCR and CD28 receptor ligation of CD4ϩ T Foxp3 transcription factor (23). Further studies will determine cells and on day 3 for PMA/ionomycin activation. In previous whether the Foxp3-expressing T cells detected in our culture sys- Downloaded from studies, IL-17 levels were only measured early, between 24 and tem are “truly” T regulatory cells. 72 h, after CD3/CD28 cross-linking and not at later time points Regulation of established Th17 effector memory cells might be (11, 14, 21, 22). One explanation for the failure to detect IL-17ϩ of relevance in autoimmune diseases where the amounts of IL-17 T cells in peripheral blood of healthy individuals could be that may determine the degree of tissue damage. Our study suggests IL-17 levels were measured at early and not at later times follow- that there are a number of mechanisms by which IL-17 secretion ϩ ing activation. Whether activated CD8 T cells secrete IL-17 was by memory T cells may be enhanced and perpetuated. IL-17 in- http://www.jimmunol.org/ unclear because IL-17 mRNA was found to be highly expressed in duces the production of a number of proinflammatory cytokines CD8ϩ T cells activated with PMA/ionomycin but no protein data such as TNF, IL-1␤, and IL-6 that may act in an autocrine loop and were available (12). Another study showed that CD8ϩ T cells se- enhance IL-17 secretion of memory cells. IL-1␤ with the assis- creted very low levels of IL-17 (22). In our experiments, IL-17 was tance of IL-23 or IL-6 enhances IL-17 secretion by increasing the not detected by cross-linking of CD3 and CD28 on CD8ϩ T cells, frequency of CCR6ϩ memory T cells as well as inducing IL-17 although IFN-␥ was produced by these cells. PMA and ionomycin secretion from CCR6Ϫ memory T cells. Moreover, these cytokines induced IL-17 production by CD8ϩ T cells; however, the levels down-regulate expression of Foxp3, which results in increased were ϳ100-fold less than that secreted by CD4ϩ T cells. IL-17 levels. The coexistence of IL-17ϩ and IL-22ϩ and IL-17ϩ Our study is the first to characterize the cytokines and the mech- and IFN-␥ϩ memory T cells suggests that these cells may be in- by guest on September 26, 2021 anisms used to regulate IL-17-secreting memory T cells. We dem- volved in tissue inflammation and damage. We propose that there onstrate that the cytokines that promote the differentiation of hu- is one memory T cell subset that is IL-17ϩ IL-22ϩ IFN-␥ϩ and man naive T cells into IL-17-secreting cells regulate IL-17 that this population plays a critical role in autoimmunity. During production by memory T cells. IL-1␤, IL-23, and to a lesser extent acute experimental autoimmune encephalomyelitis, it has been IL-6 up-regulated IL-17 secretion with an additive effect seen shown that ϳ50% of the T cells infiltrating the CNS coexpress when IL-1␤ is added together with IL-23 or IL-6. IL-23 with IL-6 IL-17 and IFN-␥ (24). Recently, one study reported that numerous enhanced IL-17 production but the effect was similar to that seen CD45ROϩ cells immunopositive for IL-17 or IL-22 were found in with IL-23 alone. In contrast, TGF-␤ inhibited IL-17 when added multiple sclerosis lesions (25). Further studies characterizing the alone or together with either IL-1␤, IL-23, or IL-6. The regulatory role of the IL-17 subsets and the Foxp3-expressing cells in human effects of the cytokines were seen both at the protein level and at autoimmune conditions are needed. mRNA transcription. In contrast to IL-17, RORC expression was not affected by these cytokines, suggesting that IL-17 expression in Acknowledgments memory T cells is likely regulated by other transcription factors. We thank Tammy Galenkamp for performing the cell sorting. Cytometric single-cell analysis of CCR6ϩ/Ϫ memory T cells re- vealed that the one of the mechanisms by which these cytokines ϩ Disclosures augment IL-17 secretion is by increasing the number of CCR6 ϩ The authors have no financial conflict of interest. IL-17 memory T cells. IL-1␤ had the greatest effect, whereas both IL-23 and IL-6 had more modest effects. Moreover, these cytokines induced IL-17 secretion in CCR6Ϫ memory T cells with References IL-1␤ displaying the strongest effect followed by IL-23 and IL-6. 1. Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham, J. D. Sedgwick, T. McClanahan, R. A. Kastelein, and D. J. Cua. 2005. IL-23 Whether IL-1␤, IL-6, and IL-23 induce IL-17 production in mem- drives a pathogenic T cell population that induces autoimmune inflammation. ory T cells that are truly CCR6Ϫ or whether they act on memory J. Exp. Med. 201: 233–240. T cells that have down-regulated CCR6 is not certain. Our results 2. Murphy, C. A., C. L. Langrish, Y. Chen, W. Blumenschein, T. McClanahan, Ϫ R. A. Kastelein, J. D. Sedgwick, and D. J. Cua. 2003. Divergent pro- and anti- suggest that in the presence of IL-1␤ and IL-6, CCR6 acquire the inflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. capacity to produce IL-17 and that IL-23 may function in down- J. Exp. Med. 198: 1951–1957. 3. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, regulating CCR6 expression. Furthermore, IL-17 and IL-22 as well and V. K. Kuchroo. 2006. 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