CD39: A new surface marker of mouse regulatory γδ T cells
2013; Elsevier BV; Volume: 132; Issue: 6 Linguagem: Inglês
10.1016/j.jaci.2013.05.037
ISSN1097-6825
AutoresAtsushi Otsuka, Sho Hanakawa, Yoshiki Miyachi, Kenji Kabashima,
Tópico(s)Immunotherapy and Immune Responses
Resumoγδ T cells have many characteristic features and functions. Activated γδ T cells release cytokines, such as IFN-γ, IL-10, and IL-17.1Vantourout P. Hayday A. Six-of-the-best: unique contributions of gammadelta T cells to immunology.Nat Rev Immunol. 2013; 13: 88-100Crossref PubMed Scopus (808) Google Scholar A subset of γδ T cells that express CD27 produce IFN-γ, whereas IL-17 production is restricted to CD27− γδ T cells.2Ribot J.C. deBarros A. Pang D.J. Neves J.F. Peperzak V. Roberts S.J. et al.CD27 is a thymic determinant of the balance between interferon-gamma- and interleukin 17-producing gammadelta T cell subsets.Nat Immunol. 2009; 10: 427-436Crossref PubMed Scopus (439) Google Scholar In addition, a CD25+ CD122− γδ T subset produces IL-17.3Shibata K. Yamada H. Nakamura R. Sun X. Itsumi M. Yoshikai Y. Identification of CD25+ gamma delta T cells as fetal thymus-derived naturally occurring IL-17 producers.J Immunol. 2008; 181: 5940-5947PubMed Google Scholar However, a subset of γδ T cells can suppress T-cell and dendritic cell (DC) functions.4Peng G. Wang H.Y. Peng W. Kiniwa Y. Seo K.H. Wang R.F. Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by a unique toll-like receptor signaling pathway.Immunity. 2007; 27: 334-348Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar Consistently, adoptively transferred γδ T cells regulated graft-versus-host reactivity in mice.5Drobyski W.R. Vodanovic-Jankovic S. Klein J. Adoptively transferred gamma delta T cells indirectly regulate murine graft-versus-host reactivity following donor leukocyte infusion therapy in mice.J Immunol. 2000; 165: 1634-1640PubMed Google Scholar Therefore, the concept of regulatory γδ T cells exists; however, it has been difficult to evaluate the roles of regulatory γδ T cells because a marker of regulatory γδ T cells remains unclarified.In this study, we sought to identify a marker of these regulatory γδ T cells. Regulatory T (Treg) cells are well established as immunosuppressants. They express several markers, including CD25, forkhead box P3 (Foxp3), cytotoxic T-lymphocyte antigen 4 (CTLA-4), and CD39.6Borsellino G. Kleinewietfeld M. Di Mitri D. Sternjak A. Diamantini A. Giometto R. et al.Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression.Blood. 2007; 110: 1225-1232Crossref PubMed Scopus (910) Google Scholar We first analyzed the expression of these markers on γδ T cells in axillary and inguinal lymph nodes with mouse by using the FACSFortessa flow cytometric system (BD Bioscience, San Diego, Calif). We found that Thy1.2+ γδ TCR+ γδ T cells had a population of CD39+ (all antibodies for flow cytometry from eBioscience, San Diego, Calif) (Fig 1, A). CD39 is known for ectoenzyme that removes the nucleotide by hydrolytic cleavage and exhibits immunomodulatory influences on its proximal environment.6Borsellino G. Kleinewietfeld M. Di Mitri D. Sternjak A. Diamantini A. Giometto R. et al.Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression.Blood. 2007; 110: 1225-1232Crossref PubMed Scopus (910) Google Scholar We next analyzed other markers of γδ T cells and identified that CD39+ γδ T cells expressed CD25 but not CD27 or CD122 (Fig 1, A). In addition, CD39+ γδ T cells did not express programmed death-1 (Fig 1, A), Foxp3 (Fig 1, B), or CTLA-4 (data not shown). Because γδ T cells did not express Foxp3 or CTLA-4, we focused on the CD25+ and CD39+ γδ T-cell subset for further study.Next, we examined by using a T-cell proliferation assay whether CD25+ and CD39+ γδ T cells have an immunosuppressive activity like Treg cells. CD4+ CD25− T cells and DCs were sorted from splenocytes by using auto-MACS (Miltenyi Biotech, Bergisch Gladbach, Germany). CD39+ and CD39− γδ T cells were sorted from axillary and inguinal lymph nodes by using a FACSAria Ⅱ cell sorter (BD Bioscience). Then, CD4+ CD25− T cells (5 × 104 cells/well) were cultured with DCs (2 × 104 cells/well) with or without CD39+ γδ T cells (5, 2.5, 1 × 104 cells/well), CD39− γδ T cells (5, 2.5, 1 × 104 cells/well), or Treg cells (1 × 104 cells/well) in a 96-well plate. For the last 24 hours of incubation, the cells were pulsed with 0.5 μCi 3H-thymidine to measure cell proliferation. In contrast to CD39− γδ T cells, CD39+ γδ T cells suppressed T-cell proliferation in a dose-dependent manner (Fig 1, C). Because CD39− γδ T cells included a CD25+ γδ T-cell subset (Fig 1, A), we considered that CD39 is a candidate of a surface marker of regulatory γδ T cells. Intriguingly, CD39+ γδ T cells did not suppress T-cell proliferation in the absence of DCs using a plate coated with 1 μg/mL of anti-CD3 antibody, although Treg cells suppressed T-cell proliferation even in the absence of DCs (Fig 1, D). These results suggest that the suppressive activity of CD39+ γδ T cells depend on the presence of DCs.To identify what mediates the suppressive activity, we performed quantitative RT-PCR analysis. Total RNA from cells was isolated with an RNeasy Mini Kit (QIAGEN, Hilden, Germany). Quantitative RT-PCR with a LightCycler real-time PCR apparatus was performed by using SYBR Green I (Roche, Basel, Switzerland). The results were normalized to the glyceraldehyde-3-phosphate dehydrogenase mRNA level. Because γδ T cells produce IL-10, which acts as an immunosuppressive,1Vantourout P. Hayday A. Six-of-the-best: unique contributions of gammadelta T cells to immunology.Nat Rev Immunol. 2013; 13: 88-100Crossref PubMed Scopus (808) Google Scholar, 7Hao J. Wu X. Xia S. Li Z. Wen T. Zhao N. et al.Current progress in gammadelta T-cell biology.Cell Mol Immunol. 2010; 7: 409-413Crossref PubMed Scopus (18) Google Scholar we evaluated the IL-10 mRNA levels. The mRNA levels of IL-10 in CD39+ γδ T cells were significantly higher than in CD39− γδ T cells (Fig 1, E). However, the mRNA levels of IFN-γ in CD39+ γδ T cells were significantly lower than in CD39− γδ T cells. To address whether IL-10 in CD39+ γδ T cells is the main suppressive cytokine of regulatory γδ T cells, we examined a T-cell proliferation assay using IL-10 knockout mice (kindly provided by Dr K. Suto, Tokyo Medical University). Because CD39+ γδ T cells sorted from IL-10 knockout mice did not suppress T-cell proliferation (Fig 1, F), we concluded that IL-10 was the pivotal suppressive cytokine of CD39+ γδ T cells.In addition, we successfully induced CD39+ γδ T cells from CD39− γδ T cells under a condition of anti-CD3 stimulation and IL-2 for 10 days, which confirmed that almost all γδ T cells expressed CD39 (Fig 2, A). We next analyzed the surface marker of in vitro–induced CD39+ γδ T cells. CD39+ γδ T cells expressed CD27 and CD122 but did not express CD4, CD8, or Foxp3 (Fig 2, B). Therefore, naive CD39+ γδ T cells and in vitro–induced CD39+ γδ T cells showed a different expression of the same markers. Then, we addressed whether in vitro–induced CD39+ γδ T cells have a suppressive activity by using the proliferation assay. As naive CD39+ γδ T cells have a suppressive activity, as demonstrated in Fig 1, C, in vitro–induced CD39+ γδ T cells have a strong suppressive activity in vitro (Fig 2, C). Finally, we examined an in vivo immunosuppression assay by using a contact hypersensitivity reaction model, which is a prototype of skin allergic reaction.8Honda T. Egawa G. Grabbe S. Kabashima K. Update of immune events in the murine contact hypersensitivity model: toward the understanding of allergic contact dermatitis.J Invest Dermatol. 2013; 133: 303-315Crossref PubMed Scopus (267) Google Scholar We sensitized mice with 25 μL of 0.5% dinitrofluorobenzene (Nacalai Tesque, Kyoto, Japan) in acetone/olive oil (4:1) on shaved abdominal skin. Five days later, a total of 4 × 103 cells of isolated CD4+ T cells or the in vitro–induced CD39+ γδ T cells in 20 μL PBS were subcutaneously injected into the ventral surface of each ear. Ear thickness was measured before and at the indicated time points after elicitation with 20 μL of 0.3% dinitrofluorobenzene. Consistent with the in vitro findings, the injection of in vitro–induced CD39+ γδ T cells significantly suppressed ear swelling, whereas the injection of CD4+ T cells rather promoted ear swelling (Fig 2, D).Fig 2The expression and suppressive activity of in vitro–induced CD39+ γδ T cells. A, The expression of CD39 on γδ T cells before (left panel) and after (right panel) in vitro stimulation. B, The expression of CD4, CD8, CD27, CD122, Foxp3, and CD25 on CD39+γδ T cells. C, The proliferative activity with in vitro–induced CD39+ γδ T cells. D, The time course of the contact hypersensitivity reaction with adoptive cell transfer of CD4+ or CD39+ γδ T cells. All data are presented as the mean ± SD (n = 5) and are representative of 3 experiments. *P < .05 (Wilcoxon signed-rank test) versus corresponding groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In conclusion, we have identified a candidate of a new surface marker of mouse regulatory γδ T cells, which have immunosuppressive activity possibly via IL-10. In addition, we demonstrated that CD39+ γδ T cells were induced from CD39− γδ T cells in vitro and have a strong suppressive activity both in vitro and in vivo. From a clinical perspective, our results may prove relevant to adoptive immunotherapy of regulatory γδ T cells to allergic skin reactions. γδ T cells have many characteristic features and functions. Activated γδ T cells release cytokines, such as IFN-γ, IL-10, and IL-17.1Vantourout P. Hayday A. Six-of-the-best: unique contributions of gammadelta T cells to immunology.Nat Rev Immunol. 2013; 13: 88-100Crossref PubMed Scopus (808) Google Scholar A subset of γδ T cells that express CD27 produce IFN-γ, whereas IL-17 production is restricted to CD27− γδ T cells.2Ribot J.C. deBarros A. Pang D.J. Neves J.F. Peperzak V. Roberts S.J. et al.CD27 is a thymic determinant of the balance between interferon-gamma- and interleukin 17-producing gammadelta T cell subsets.Nat Immunol. 2009; 10: 427-436Crossref PubMed Scopus (439) Google Scholar In addition, a CD25+ CD122− γδ T subset produces IL-17.3Shibata K. Yamada H. Nakamura R. Sun X. Itsumi M. Yoshikai Y. Identification of CD25+ gamma delta T cells as fetal thymus-derived naturally occurring IL-17 producers.J Immunol. 2008; 181: 5940-5947PubMed Google Scholar However, a subset of γδ T cells can suppress T-cell and dendritic cell (DC) functions.4Peng G. Wang H.Y. Peng W. Kiniwa Y. Seo K.H. Wang R.F. Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by a unique toll-like receptor signaling pathway.Immunity. 2007; 27: 334-348Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar Consistently, adoptively transferred γδ T cells regulated graft-versus-host reactivity in mice.5Drobyski W.R. Vodanovic-Jankovic S. Klein J. Adoptively transferred gamma delta T cells indirectly regulate murine graft-versus-host reactivity following donor leukocyte infusion therapy in mice.J Immunol. 2000; 165: 1634-1640PubMed Google Scholar Therefore, the concept of regulatory γδ T cells exists; however, it has been difficult to evaluate the roles of regulatory γδ T cells because a marker of regulatory γδ T cells remains unclarified. In this study, we sought to identify a marker of these regulatory γδ T cells. Regulatory T (Treg) cells are well established as immunosuppressants. They express several markers, including CD25, forkhead box P3 (Foxp3), cytotoxic T-lymphocyte antigen 4 (CTLA-4), and CD39.6Borsellino G. Kleinewietfeld M. Di Mitri D. Sternjak A. Diamantini A. Giometto R. et al.Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression.Blood. 2007; 110: 1225-1232Crossref PubMed Scopus (910) Google Scholar We first analyzed the expression of these markers on γδ T cells in axillary and inguinal lymph nodes with mouse by using the FACSFortessa flow cytometric system (BD Bioscience, San Diego, Calif). We found that Thy1.2+ γδ TCR+ γδ T cells had a population of CD39+ (all antibodies for flow cytometry from eBioscience, San Diego, Calif) (Fig 1, A). CD39 is known for ectoenzyme that removes the nucleotide by hydrolytic cleavage and exhibits immunomodulatory influences on its proximal environment.6Borsellino G. Kleinewietfeld M. Di Mitri D. Sternjak A. Diamantini A. Giometto R. et al.Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression.Blood. 2007; 110: 1225-1232Crossref PubMed Scopus (910) Google Scholar We next analyzed other markers of γδ T cells and identified that CD39+ γδ T cells expressed CD25 but not CD27 or CD122 (Fig 1, A). In addition, CD39+ γδ T cells did not express programmed death-1 (Fig 1, A), Foxp3 (Fig 1, B), or CTLA-4 (data not shown). Because γδ T cells did not express Foxp3 or CTLA-4, we focused on the CD25+ and CD39+ γδ T-cell subset for further study. Next, we examined by using a T-cell proliferation assay whether CD25+ and CD39+ γδ T cells have an immunosuppressive activity like Treg cells. CD4+ CD25− T cells and DCs were sorted from splenocytes by using auto-MACS (Miltenyi Biotech, Bergisch Gladbach, Germany). CD39+ and CD39− γδ T cells were sorted from axillary and inguinal lymph nodes by using a FACSAria Ⅱ cell sorter (BD Bioscience). Then, CD4+ CD25− T cells (5 × 104 cells/well) were cultured with DCs (2 × 104 cells/well) with or without CD39+ γδ T cells (5, 2.5, 1 × 104 cells/well), CD39− γδ T cells (5, 2.5, 1 × 104 cells/well), or Treg cells (1 × 104 cells/well) in a 96-well plate. For the last 24 hours of incubation, the cells were pulsed with 0.5 μCi 3H-thymidine to measure cell proliferation. In contrast to CD39− γδ T cells, CD39+ γδ T cells suppressed T-cell proliferation in a dose-dependent manner (Fig 1, C). Because CD39− γδ T cells included a CD25+ γδ T-cell subset (Fig 1, A), we considered that CD39 is a candidate of a surface marker of regulatory γδ T cells. Intriguingly, CD39+ γδ T cells did not suppress T-cell proliferation in the absence of DCs using a plate coated with 1 μg/mL of anti-CD3 antibody, although Treg cells suppressed T-cell proliferation even in the absence of DCs (Fig 1, D). These results suggest that the suppressive activity of CD39+ γδ T cells depend on the presence of DCs. To identify what mediates the suppressive activity, we performed quantitative RT-PCR analysis. Total RNA from cells was isolated with an RNeasy Mini Kit (QIAGEN, Hilden, Germany). Quantitative RT-PCR with a LightCycler real-time PCR apparatus was performed by using SYBR Green I (Roche, Basel, Switzerland). The results were normalized to the glyceraldehyde-3-phosphate dehydrogenase mRNA level. Because γδ T cells produce IL-10, which acts as an immunosuppressive,1Vantourout P. Hayday A. Six-of-the-best: unique contributions of gammadelta T cells to immunology.Nat Rev Immunol. 2013; 13: 88-100Crossref PubMed Scopus (808) Google Scholar, 7Hao J. Wu X. Xia S. Li Z. Wen T. Zhao N. et al.Current progress in gammadelta T-cell biology.Cell Mol Immunol. 2010; 7: 409-413Crossref PubMed Scopus (18) Google Scholar we evaluated the IL-10 mRNA levels. The mRNA levels of IL-10 in CD39+ γδ T cells were significantly higher than in CD39− γδ T cells (Fig 1, E). However, the mRNA levels of IFN-γ in CD39+ γδ T cells were significantly lower than in CD39− γδ T cells. To address whether IL-10 in CD39+ γδ T cells is the main suppressive cytokine of regulatory γδ T cells, we examined a T-cell proliferation assay using IL-10 knockout mice (kindly provided by Dr K. Suto, Tokyo Medical University). Because CD39+ γδ T cells sorted from IL-10 knockout mice did not suppress T-cell proliferation (Fig 1, F), we concluded that IL-10 was the pivotal suppressive cytokine of CD39+ γδ T cells. In addition, we successfully induced CD39+ γδ T cells from CD39− γδ T cells under a condition of anti-CD3 stimulation and IL-2 for 10 days, which confirmed that almost all γδ T cells expressed CD39 (Fig 2, A). We next analyzed the surface marker of in vitro–induced CD39+ γδ T cells. CD39+ γδ T cells expressed CD27 and CD122 but did not express CD4, CD8, or Foxp3 (Fig 2, B). Therefore, naive CD39+ γδ T cells and in vitro–induced CD39+ γδ T cells showed a different expression of the same markers. Then, we addressed whether in vitro–induced CD39+ γδ T cells have a suppressive activity by using the proliferation assay. As naive CD39+ γδ T cells have a suppressive activity, as demonstrated in Fig 1, C, in vitro–induced CD39+ γδ T cells have a strong suppressive activity in vitro (Fig 2, C). Finally, we examined an in vivo immunosuppression assay by using a contact hypersensitivity reaction model, which is a prototype of skin allergic reaction.8Honda T. Egawa G. Grabbe S. Kabashima K. Update of immune events in the murine contact hypersensitivity model: toward the understanding of allergic contact dermatitis.J Invest Dermatol. 2013; 133: 303-315Crossref PubMed Scopus (267) Google Scholar We sensitized mice with 25 μL of 0.5% dinitrofluorobenzene (Nacalai Tesque, Kyoto, Japan) in acetone/olive oil (4:1) on shaved abdominal skin. Five days later, a total of 4 × 103 cells of isolated CD4+ T cells or the in vitro–induced CD39+ γδ T cells in 20 μL PBS were subcutaneously injected into the ventral surface of each ear. Ear thickness was measured before and at the indicated time points after elicitation with 20 μL of 0.3% dinitrofluorobenzene. Consistent with the in vitro findings, the injection of in vitro–induced CD39+ γδ T cells significantly suppressed ear swelling, whereas the injection of CD4+ T cells rather promoted ear swelling (Fig 2, D). In conclusion, we have identified a candidate of a new surface marker of mouse regulatory γδ T cells, which have immunosuppressive activity possibly via IL-10. In addition, we demonstrated that CD39+ γδ T cells were induced from CD39− γδ T cells in vitro and have a strong suppressive activity both in vitro and in vivo. From a clinical perspective, our results may prove relevant to adoptive immunotherapy of regulatory γδ T cells to allergic skin reactions. We thank Ms Hiromi Doi for technical assistance.
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