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Keywords:

  • CD161;
  • RORC;
  • Th17

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

We have previously shown that human Th17 lymphocytes are characterized by the selective expression of IL-23 receptor (IL-23R), CCR6, CD161, and the transcription factor retinoic acid-related orphan receptor C (RORC), and originate from a CD161+CD4+ naïve T-cell precursor in response to the combined activity of IL-1β and IL-23. We show here that not only CD4+TCRαβ+, but also CD8+TCRαβ+, CD4CD8 TCRαβ+, and CD4CD8 TCRγδ+ circulating lymphocytes that produce IL-17 express the distinctive marker CD161 on their surface. In addition, we demonstrate that CD161 expression identifies CD8+ and CD4CD8 umbilical cord blood T cells that already express RORC and IL-23R mRNA and that can be induced to differentiate into IL-17-producing cells in the presence of IL-1β and IL-23. Finally, we provide evidence that umbilical cord blood naïve CD4+CD161 T cells, upon lentivirus-mediated transduction with RORC2 can acquire the ability to express IL-23R, IL-1RI, and CD161, as well as to produce IL-17. Taken together, these data allow to conclude that T-cell subsets able to produce IL-17, as well as precursors of IL-17-producing T cells, exhibit surface expression of CD161, and that this feature is at least in part RORC2-dependent.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Th lymphocytes have been functionally distinguished on the basis of their patterns of cytokine secretion in two main subsets, termed Th1 and Th2 lymphocytes 1, 2. Th1 cells produce high amount of IFN-γ and induce cell-mediated immunity and humoral response against intracellular pathogens, whereas Th2 cells produce IL-4 and stimulate humoral immunity against helminths. Recently, a third T-cell subset, known as Th17, was identified 3 in both mice and humans as a population of CD4+ effector T cells, producing the distinctive cytokines IL-17A and IL-17F. IL-17 family of cytokines include five members, designated IL-17A-F. IL-17A is disulfide-linked homodimeric glycoprotein, consisting of 155 amino acids 4, sharing great homology with IL-17F (55%). IL-17A and IL-17F can either exist as homodimers or as IL-17A-IL-17F heterodimers. The other IL-17 family members, IL-17B, IL-17C, and IL-17D, are produced by non-T-cell sources. Th17 cells play a critical role in the recruitment and activation of neutrophil granulocytes, both directly through CXCL8 production 5, and indirectly, by inducing the production of CSF and CXCL8 6 in tissue-resident cells. Moreover, Th17 lymphocytes are also able to stimulate production of CXCL chemokines and mucins, MUC5AC and MUC5B, in primary human bronchial epithelial cells in vitro7, and the expression of human β defensin-2 8 and CCL20 in lung epithelial cells 9. Accordingly, the main function of Th17 cells appears to be the clearance of extracellular pathogens during infections, but these cells also contribute to promote inflammation, suggesting the possibility that they could play a role in the pathogenesis of several autoimmune and chronic inflammatory diseases 10. Beyond the ability to produce the above-mentioned cytokines, human and murine Th17 cells also exhibit the expression of the transcription factor retinoic acid-related orphan receptor C (RORC), as well as of the surface IL-23 receptor (IL-23R) 11, 12, and of the chemokine receptor CCR6. More recently, another marker of human Th17 cells, the lectin receptor CD161, the human hortolog of murine NK1.1, was identified 13, 14.

Several studies have also demonstrated the existence of human CD4+ T cells able to produce both IL-17 and IFN-γ 11–13, which were named as Th17/Th1 cells 11, suggesting a possible common origin between human Th17 and Th1 cells. Accordingly, we have recently found that human Th17 cells originate from CD161+CD4+ naïve T-cell precursors, detectable in both umbilical cord blood (UCB) and thymus, in response to the combined activity of IL-1β and IL-23 13. We have also shown that TGF-β may play a favourable, but indirect, role in the process of human Th17 development by inhibiting the growth of Th1 cells 15. The finding that TGF-β is dispensable for Th17 differentiation and the possibility of its indirect role based on the suppression of Th1 and Th2 growth have recently been confirmed in mice 16, 17. It is worth noting that the combination of IL-1β and IL-23 is able to induce the differentiation not only of Th17, but also of Th17/Th1, and even of Th1 cells, suggesting that these three subpopulations can develop and coexist in the same microenviroment 18.

In this study, we provide evidence that CD161 is expressed not only by IL-17-producing CD4+TCRαβ+ cells, but also by CD8+TCRαβ+, CD4CD8TCRαβ+, and CD4CD8TCRγδ+, circulating lymphocytes, that are able to produce IL-17. Moreover, we demonstrate that UCB CD8+ and CD4CD8 T-cell populations exhibiting CD161, but not their CD161 counterparts, also express RORC and IL-23R mRNA and can acquire the ability to produce IL-17 in the presence of IL-1β and IL-23. Finally, we show that UCB CD4+CD161 T cells can acquire the expression of IL-23R, IL-1RI, and CD161 as well as the ability to produce IL-17 upon transduction with RORC2 viral vector, but not with the appropriate viral vector control.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

CD161 is a marker of human circulating T lymphocyte subsets showing the ability to produce IL-17

We have recently shown that IL-17-producing CD4+ lymphocytes isolated from the circulation of healthy subjects, as well as from the gut or skin of patients affected by Crohn's disease or psoriasis, respectively, express CD161 on their surface 13. It is worth noting that CD4+CD161+ lymphocytes can also include Th1, Th2, and Th0 cells, but IL-17-producing cells were never found in the CD161 fraction 13. The aim of this study was to establish whether CD161 is also correlated with IL-17 expression in other T-cell subsets. To address this point, we first evaluated CD161+, as well as CD161, circulating T lymphocytes from healthy subjects for TCRαβ, TCRγδ, CD4, and CD8 expression. The proportions of TCRαβ+ and TCRγδ+ cells in the CD161+ or the CD161 fraction was not significantly different (93.06%±0.40 and 5.84%±0.42 among CD161+ lymphocytes, and 89.91%±0.40 and 3.32%±0.23 among CD161 lymphocytes, respectively) (Fig. 1A, left part). As shown in Fig. 1A, three populations of TCRαβ+ cells, namely CD4+CD8, CD4CD8+, and CD4CD8 double negative, could be identified within both the CD161+ and the CD161 fraction. On the contrary, only CD4CD8 double negative cells were appreciable within both CD161+ and CD161 TCRγδ+ lymphocytes (Fig. 1A).

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Figure 1. IL-17-producing circulating T lymphocytes from healthy adults are contained within the CD161+ fraction. (A) Peripheral blood MNC were stained with anti-CD3, CD4, CD8, CD161, TCRαβ and TCRγδ, fluorochrome-conjugated mAb and analyzed by flow cytometry. In the left part, a representative dot plot showing TCRαβ and CD161 expression of CD3-gated cells. In the right part, columns show the percentage of the indicated cell population gated on the CD3+CD161+ (black columns) and the CD3+CD161 (white columns) gates (n=8 donors). (B) Representative flow cytometric dot plots showing CD45RA and CD45RO expression in all CD161+ and CD161 cell fractions. (C) RORC and IL-23R mRNA were evaluated in the indicated CD161+ (black columns), and CD161 (white columns) cell-sorted fractions, by RT-quantitative PCR. Data show mean±SE of mRNA levels found in eight healthy donors. (D) Evaluation by flow cytometry of IFN-γ-, IL-17-, and IL-22-producing cells among the indicated CD161+ (black columns), and CD161 (white columns) cell fractions. Data of the percentage of cells producing the indicated cytokine obtained in eight healthy donors. (E) TCRγδCD161+ and TCRγδCD161 cell fractions were stained with IL-17, IFN-γ, CD3, CD4, CD8, and TCRαβ mAb, whereas TCRγδ+ cell fractions were stained with IL-17, IFN-γ, CD3, CD4, CD8, and CD161 mAb and finally analyzed by flow cytometry. A representative experiment is depicted. Data show mean±SE (A, C, D). A standard two-tailed paired t-test was used for statistical analysis.

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In order to better characterize all circulating CD161+ T-cell subsets, each cell population and their CD161 counterparts were sorted from the peripheral blood of healthy donors and the expression of CD45RA and CD45RO proteins and of IL-23R and RORC mRNA was analyzed by flow cytometry or by real-time (RT)-quantitative PCR, respectively. Moreover, the ability of the different subsets to produce IL-17, IL-22, and IFN-γ following stimulation with PMA plus ionomycin was assessed. All available CD161+ T-cell subsets showed a clear memory phenotype being characterized by the expression of CD45RO in the absence of CD45RA (Fig. 1B). As expected, CD161 T cells, which represent the majority of circulating T cells, were characterized by the expression of both CD45RO and CD45RA (Fig. 1B). As shown in Fig. 1C, all CD161+ T-cell subsets showed significantly higher IL-23R and RORC mRNA expression than CD161 ones. Accordingly, virtually all IL-17-producing cells were found in the CD161+ cell fraction (Fig. 1D and E). Moreover, CD161+ T-cell subsets showed higher frequencies of IL-22-producing cells, when compared with their CD161 counterparts (Fig. 1D). It is worth noting that the IFN-γ-producing cells were detectable in both CD161+ and CD161 T-cell fractions (Fig. 1D and E). Taken together, these observation allowed us to conclude that CD161 is expressed not only by CD4+CD8TCRαβ+, but also by CD4CD8+TCRαβ+, CD4CD8TCRαβ+, and CD4CD8TCRγδ+, IL-17-producing, circulating lymphocytes.

Although CD8+ T cells that produce IL-17, alone or in association with IFN-γ, have been recently observed in lesions of patients affected by psoriasis 19 or multiple sclerosis 20, as well as in the circulation of healthy subjects 21, our findings provide the first demonstration that among CD8+ T lymphocytes only those characterized by CD161 expression have the ability to produce IL-17. The same conclusion can be drawn with regard to CD4CD8TCRγδ+ lymphocytes. Although there is evidence that TCRγδ+ IL-17-producing cells are involved in the formation of tubercular granuloma in patients with pulmonary tuberculosis 22, their association with CD161 expression was not previously identified. Finally, the results of this study describe a population of IL-17-producing CD4CD8TCRαβ+CD161+ cells, very similar to those recently described in patients with systemic lupus erythematosus, involved in the pathophysiology of kidney damage in these patients 23.

IL-17-producing cells exclusively originate from naïve CD161+ T-cell precursors

In our previous study 13, we demonstrated that human Th17 cells originate from a naïve CD4+CD161+ T-cell precursor present in the UCB and thymus upon its in vitro stimulation in the presence of IL-1β plus IL-23. As we established the existence of CD161+ IL-17-producing CD4CD8+TCRαβ+, CD4CD8TCRαβ+, and CD4CD8TCRγδ+ lymphocytes, in the peripheral blood of adult healthy donors, we wondered if the same cell populations were also present as cell precursors in the UCB.

In addition to CD4+CD8TCRαβ+, flow cytometry analysis of UCB T cells revealed the presence of CD4CD8+TCRαβ+, CD4CD8TCRαβ+, and TCRγδ+CD4CD8, CD161+, T cells (0.55%±0.12 SE; 0.3%±0.02 SE; 0.08%±0.004 SE; and 0.04%±0.01 SE of CD3 lymphocytes, respectively). We then sorted CD4+CD8TCRαβ+, CD4CD8+TCRαβ+, and total CD4CD8 CD161+ lymphocytes as well as their CD161 counterparts. Due to the low frequency of CD3+CD4CD8 cells in UCB, the experiments on this subset have been performed independently of their TCR expression. All freshly isolated cell fractions were analyzed for CD45RA and CD45RO expression by flow cytometry and for RORC and IL-23R mRNA expression by RT-quantitative PCR. Both CD161+ and CD161 T-cell fraction showed a clear naïve phenotype being all composed of CD45RA+ cells (Fig. 2A). As shown in Fig. 2B, all CD161+ populations expressed higher RORC and IL-23R mRNA levels when compared with their CD161 counterparts. It is worth noting that the CD4CD8 T cells consistently exhibited the highest values of both RORC and IL-23R, a finding which remains unclear at the present time.

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Figure 2. CD161+ but not CD161 UCB T lymphocytes can be induced to produce IL-17. (A) Representative flow cytometric dot plots showing CD45RA and CD45RO expression in CD161+ and CD161 cell fractions. (B) RORC and IL-23R mRNA were evaluated in the different CD161+ (black columns), and CD161 (white columns) UCB cell populations by RT-PCR. Columns show mean±SE of mRNA levels found in five UCB preparations. (C) Evaluation by flow cytometry of IFN-γ-, IL-17-, and IFN-γ/IL-17-producing cells among the different CD161+ (black columns), and CD161 (white columns) cell fractions, after 1 wk of in vitro culture in the presence of IL-1β and IL-23. Columns show mean±SE of the percentage of cells producing the indicated cytokine obtained in five UCB preparations. (D) CD161+ and CD161 cell fractions were stained with anti-IL-17, IFN-γ, CD3, CD4, and CD8 mAb and finally analyzed by flow cytometry. One representative experiment, after 1 wk of in vitro culture in the presence of IL-1β and IL-23, is shown. A standard two-tailed paired t-test was used for statistical analysis.

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It is worth noting that when cytokine production was assessed following stimulation with PMA plus ionomycin, none of the above-described T-cell subsets was able to produce IL-17 (data not shown). In order to evaluate the possibility to induce IL-17 production by the new identified CD161+ cell precursors, both CD161+ and CD161 cell fractions were stimulated in vitro with anti-CD3 plus anti-CD28 mAb in the presence or absence of IL-1β and IL-23. After 1 wk of in vitro culture, cells were re-stimulated with PMA plus ionomycin and intracelluar IL-17 and IFN-γ production was assessed. As shown in Fig. 2C and D, CD161+, but not CD161, T cells, acquired the ability to produce IL-17, alone or in association with IFN-γ, in response to the combined activity of IL-1β and IL-23. No IL-17-producing cells could be detected in the absence of both these cytokines as well as in the presence of other cytokines (IL-6 and IL-21) or combination of them (IL-1β plus IL-6, IL-23 plus IL-6, IL-1β plus IL-21, IL-6 plus IL-21, and IL-23 plus IL-21) (data not shown). In only two experiments we had the opportunity to sub-divide CD4CD8 T cells into the TCRαβ+ and TCRγδ+ fractions. Due to very low numbers of cells recovered, one experiment was directed to RT-PCR analysis of RORC and IL-23R mRNA expression, whereas in the other the ability of the two cell fractions to differentiate toward IL-17-producing cells was evaluated. Both TCRαβ+ and TCRγδ+CD4CD8CD161+ cells expressed higher RORC and IL-23R mRNA levels when compared with their CD161 cell counterparts and, accordingly they both differentiated in IL-17-producing cells when cultured in the presence of IL-1β plus IL-23 (data not shown).

The expression of RORC in CD161+ but not CD161 cells indicated a link between CD161 and RORC, and consequently between CD161 expression and IL-17 production. These observations led us to hypothesize that T cells characterized by the expression of CD161 possess the ability to differentiate into IL-17-producing cells under appropriate environmental conditions. This hypothesis is in keeping with our recent demonstration of the existence of Th1 clones, obtained from circulating CD4+CD161+ lymphocytes of healthy donors, that express RORC, whereas Th1 clones obtained from CD4+CD161 lymphocytes did not 24.

CD4+CD161 T cells transduction with a RORC2-viral vector results in induction of IL-17 and CD161

Previous studies demonstrated that transduction of PB naïve T cells with RORC2 resulted in upregulation of CD161 and IL-17 25. In order to further support the possible link between CD161 and RORC2 expression, as well as to investigate the nature of this link, UCB naïve CD4+ T cells were depleted of CD161+ cells and the remaining CD161 T cells were transduced with RORC2 or control lentivirus. The transduced cells were then evaluated for RORC, IL-23R, IL-1RI, and CD161 mRNA expression, as well as for the ability to express surface CD161 and produce IL-17.

As shown in Fig. 3A, CD4+CD161 cells transduced with RORC2 not only expressed high levels of RORC (as expected), but also exhibited higher IL-23R, IL-1RI, and CD161 mRNA levels when compared with either the empty vector-transduced or the untransduced cells. More importantly, when assessed at single-cell level, RORC2-transduced lymphocytes showed significantly higher proportions of CD161 and IL-17+ cells, when compared with both the untransduced and the empty vector-transduced cells (Fig. 3B and C). As shown in Fig. 3B and C, a few cells expressing CD161 were also present in untransduced, empty vector-transduced, and RORC2-transduced nerve grow factor receptor (NGFR) cells. One possible explanation is that freshly isolated CD161 cells contained some CD161 cell precursors that were CD161 at the time of cell sorting, but acquired its expression in vitro later on. Indeed, CD161+ cells isolated from untransduced and empty vector-transduced cell cultures on day 21 showed higher “natural” RORC expression when compared with their CD161 cell counterpart (data not shown). Interestingly, the acquisition of the ability to produce IL-17 by RORC2-transduced lymphocytes was independent of the addition in culture of IL-1β and IL-23, probably due to the higher levels of RORC2 expression induced by viral transduction in comparison with those present in ex vivo sorted CD4+CD161+UCB T lymphocytes. We also found that CD3+CD4CD8+ and CD3+CD4CD8, CD161 cells could be induced to express both IL-17 and CD161 upon their transduction with the viral vector containing RORC2 (data not shown). Finally, in order to strength the link between RORC and CD161, we cultured Th17 clones in the presence or in the absence of a synthetic RAR agonist, AM80, which has been shown to inhibit Th17 differentiation by suppressing RORC 26. In agreement with the previous results 26, RORC as well as IL-17 expression was significantly suppressed on Th17 clones at both mRNA and protein levels in the presence of AM80 when assessed 3 and 7 days after in vitro culturing (Fig. 3D and data not shown). More importantly, RORC downregulation was associated with a clear decrease of CD161 expression at both mRNA and protein levels (Fig. 3D). These observations lead us to conclude that the acquisition of the capacity to produce IL-17 and the ability to express CD161 are both a consequence of RORC expression. In any case, the possibility that CD161 expression can be also regulated by additional factors cannot be excluded. Indeed, our previous data have shown that CD4+ T cells expressing CD161 and low RORC mRNA levels can be present in the circulation of healthy donors 13, 24.

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Figure 3. RORC transduction induces naïve T lymphocytes to become CD161+ Th17 cells. (A) Expression of RORC, IL-23R, IL-1R1, and CD161 mRNA was evaluated by RT-PCR in untransduced, empty vector-transduced, and RORC2-transduced, CD4+CD161 T cells obtained from UCB after 3 wk of in vitro culture. (B) Evaluation by flow cytometry of IL-17-producing, and CD161-expressing cells in untransduced, empty vector transduced, and RORC2-transduced CD4+CD161 T cells obtained from UCB, after 3 wk of in vitro culture. (C) CD161 and ΔNGFR expression in one representative experiment is shown. (D) Expression of RORC and CD161 mRNA (left panel) and protein (right panel) was evaluated at different time points, by RT-PCR or flow cytometry, respectively, in two representative Th17 clones (top and bottom panel, respectively), cultured in the presence or absence of AM80. Data show mean±SE from three separate experiments (A, B, D). A standard two-tailed paired t-test was used for statistical analysis.

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Concluding remarks

In this study, we identified CD161 expression as a feature shared by all subsets of T lymphocytes capable of producing IL-17. Moreover, we showed that CD161+, but not CD161, CD3+CD4CD8+, and CD3+CD4CD8 lymphocytes present in UCB could be induced in vitro to become IL-17-producing cells in the presence of IL-1β and IL-23. Finally, we found that both CD161 surface expression and the capacity to produce IL-17 could be induced in CD161 UCB naïve T lymphocytes following transduction with a RORC2 viral vector.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Subjects

UCB samples were obtained from 20 donors. PB samples were obtained from 15 healthy volunteers. The procedures and all the experiments of the study were in accordance to the ethical standards of and approved by the Regional Committee on Human Experimentation.

Reagents

The culture media used were (i) RPMI 1640 (Seromed) supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 2×10−5 M 2-mercaptoethanol (2-ME; all from Invitrogen), and 10% FBS HyClone (Gibco Laboratories, Grand Island, NY, USA); (ii) DMEM (Sigma-Aldrich) supplemented or not with 10 mM HEPES (Sigma-Aldrich), or 1 μM sodium butyrate (Sigma-Aldrich). Unlabeled or fluorochrome-conjugated anti-TCRαβ, TCRγδ, CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD161, CD271 (NGFR) IFN-γ, and isotype-matched control mAb were purchased from BD Biosciences (San Jose, CA, USA). The fluorochrome-conjugated anti-IL-17, anti-IL-22, and anti-ROR-γ mAb were obtained from eBioscience (San Diego, CA, USA). PMA, ionomycin, brefeldin A, and AM80 were purchased from Sigma Chemical (St. Louis, MO). IL-1β, IL-6, and IL-23 were purchased from R&D Systems. IL-21 was purchased from Biosource (Camarillo, CA, USA).

T-cell recovery and expansion

Mononuclear cell (MNC) suspensions were obtained from PB and UCB by centrifugation on Ficoll–Hypaque gradient. TCRγδ+ T cells were obtained from PBMC by immunomagnetic cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) and further divided into CD161+ and CD161 by using the FACSAria (BD Biosciences). On the other hand, TCRγδ cells obtained from PBMC were divided into CD161+ and CD161 cells by immunomagnetic cell sorting (Miltenyi Biotec). TCRαβ+CD3+CD4+, CD3+CD8+, and CD3+CD4CD8 T cells were then obtained from both CD161+ and CD161 fractions by using FACSAria. Each T-cell population was finally analyzed by quantitative RT-PCR for gene expression and evaluated for cytokine production after polyclonal stimulation. MNC obtained from UCB were divided into CD161+ and CD161 cells by immunomagnetic cell sorting (Miltenyi Biotec). Both CD161+ and CD161 cell populations from UCB were stained with fluorochrome-conjugated anti-CD3, anti-CD4, anti-CD8 mAb, and then separated by using FACSAria (BD Biosciences) in the following subpopulations: CD3+CD4+, CD3+CD8+, and CD3+CD4CD8 Each T-cell population was finally analyzed by quantitative RT-PCR for gene expression, and also in vitro cultured for 1 wk with anti-CD3 (5 μg/mL) plus anti-CD28 (5 μg/mL), mAb in the absence or presence of IL-1β (10 ng/mL), IL-6 (2 ng/mL), IL-21 (50 ng/mL), IL-23 (20 ng/mL), or combinations of them. On day 7, T cells were stimulated for intracellular cytokines detection.

Two Th17 clones derived from PB of healthy subjects were assessed for RORC and CD161 expression at both mRNA and protein level at different time points upon in vitro culture in the absence or in the presence of the synthetic RAR agonist, AM80 (100 nmol/L).

Flow cytometry analysis of cytokine production

To perform flow cytometry analysis of intracellular cytokines, 1×106 cells were stimulated with PMA (10 ng/mL) plus ionomycin (1 μM) for 6 h, the last four in the presence of brefeldin A (5 μg/mL). After stimulation, cells were washed twice with PBS, pH 7.2, fixed 15 min with formaldehyde (2% in PBS, pH 7.2), washed twice with 0.5% BSA in PBS, pH 7.2, permeabilized with PBS, pH 7.2, containing 0.5% BSA and 0.5% saponin, and then incubated for 15 min at room temperature with the specific mAb. Cells were then washed and analyzed on a BDLSR II flow cytometry using the FACSDiva software (Becton Dickinson). The area of positivity was determined using an isotype-matched mAb, a total of 104 events for each sample were acquired 24.

RNA isolation, cDNA synthesis, and RT-quantitative PCR

Total RNA was extracted by using the RNeasy Micro kit (QIAGEN) and treated with DNase I to eliminate possible genomic DNA contamination. Taq-Man RT-PCR was performed as described elsewhere 13. Primers and probes used were purchased from Applied Biosystems.

Lentivirus trasduction

pCCL EF1α NGFR h RORC2 is a third-generation bidirectional lentiviral vector with the transgene RORC2 under EF1α (Human Elongator factor 1α) promoter, which includes a truncated version of the ΔNGFR as reporter gene under control of minimal CMV (cytomegalovirus) promoter. pCCL EF1α NGFR is the same vector without the transgene RORC2 25.

UCB CD3+CD4+CD161 cells were activated with anti-CD3, anti-CD28, and IL-2 (100 U/mL, Eurocetus, Italy). After 16 h, pCCL EF1α NGFR or pCCL EF1α NGFR hRORC2 lentivirus was added at a multiplicity of infection of five. Transduced T cells were purified after 1 wk with MACSelect LNGFR MicroBeads (Miltenyi Biotec), purities greater than 98% being consistently achieved, and then expanded with IL2 (100 U/mL) for other 2 wk. After this period of time, each T-cell population was analyzed by quantitative RT-PCR for gene expression and evaluated for cytokine production after polyclonal stimulation.

Statistical analysis

A standard two-tailed paired t-test was used for statistical analysis; p-values of 0.05 or less were considered significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

The experiments reported in this article have been supported by funds of the Italian Ministry of Education (PRIN), the Italian Ministry of Health (Strategic Project 2008), the Ente Cassa di Risparmio, Florence, Italy, EU Projects SENS-IT-IV (FP6-LSBH-CT-2006-018861), and INNOCHEM (FP6-LSHB-CT-2005-518167), Associazione Italiana Ricerca sul Cancro (AIRC) and the Canadian Institutes for Health Research (to M. K. L., MOP-93793). M. K. L. holds a Canada Research Chair in Transplantation. S. Q. C. holds a MSFHR Senior Graduate Studentship award, and a CIHR/UBC Skin Research Trainee award.

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

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  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References
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