Experimental Model for Studying the Involvement of Regulatory Cytotoxic T Cells in Bone Resorption

  • Giacomina BrunettiEmail author
  • Angela Oranger
  • Silvia Colucci
  • Maria Grano
Part of the Methods in Molecular Biology book series (MIMB, volume 1186)


T cells may affect the activity of osteoclasts, the bone resorbing cells. In particular, recently authors focalize their attention on CD8+ T cells, demonstrating that murine pre-osteoclasts can recruit naïve CD8+ T cells and induce them to differentiate in regulatory cytotoxic T cells (TcREG), which in turn may suppress osteoclast formation and activity. Thus, here we describe the methodological approach needed to obtain TcREG and to evaluate TcREG effects on murine osteoclast activity and formation in an in vitro experimental model.

Key words

T cells Osteoclastogenesis FoxP3+CD8+ T cells TRAP staining Resorption assay 


  1. 1.
    Mori G, D’Amelio P, Faccio R, Brunetti G (2013) The interplay between the bone and the immune system. Clin Dev Immunol 2013(720504):1–9CrossRefGoogle Scholar
  2. 2.
    Boyle WJ, Simonet WS, Lacey DL (2003) Osteoclast differentiation and activation. Nature 423:337–342PubMedCrossRefGoogle Scholar
  3. 3.
    Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, Capparelli C, Li J, Elliott R, McCabe S, Wong T, Campagnuolo G, Moran E, Bogoch ER, Van G, Nguyen LT, Ohashi PS, Lacey DL, Fish E, Boyle WJ, Penninger JM (1999) Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 402:304–309PubMedCrossRefGoogle Scholar
  4. 4.
    Colucci S, Brunetti G, Cantatore FP, Oranger A, Mori G, Quarta L, Cirulli N, Mancini L, Corrado A, Grassi FR, Grano M (2007) Lymphocytes and synovial fluid fibroblasts support osteoclastogenesis through RANKL, TNFalpha, and IL-7 in an in vitro model derived from human psoriatic arthritis. J Pathol 212:47–55PubMedCrossRefGoogle Scholar
  5. 5.
    Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, Ellen RP, Penninger JM (2000) Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest 106:R59–R67PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Brunetti G, Colucci S, Pignataro P, Coricciati M, Mori G, Cirulli N, Zallone A, Grassi FR, Grano M (2005) T cells support osteoclastogenesis in an in vitro model derived from human periodontitis patients. J Periodontol 76:1675–1680PubMedCrossRefGoogle Scholar
  7. 7.
    Colucci S, Brunetti G, Rizzi R, Zonno A, Mori G, Colaianni G, Del Prete D, Faccio R, Liso A, Capalbo S, Liso V, Zallone A, Grano M (2004) T cells support osteoclastogenesis in an in vitro model derived from human multiple myeloma bone disease: the role of the OPG/TRAIL interaction. Blood 104:3722–3730PubMedCrossRefGoogle Scholar
  8. 8.
    Roato I, Grano M, Brunetti G, Colucci S, Mussa A, Bertetto O, Ferracini R (2005) Mechanisms of spontaneous osteoclastogenesis in cancer with bone involvement. FASEB J 19:228–230PubMedGoogle Scholar
  9. 9.
    Faienza MF, Brunetti G, Colucci S, Piacente L, Ciccarelli M, Giordani L, Del Vecchio GC, D’Amore M, Albanese L, Cavallo L, Grano M (2009) Osteoclastogenesis in children with 21-hydroxylase deficiency on long term glucocorticoid therapy: the role of RANKL/OPG imbalance. J Clin Endocrinol Metab 94:2269–2276PubMedCrossRefGoogle Scholar
  10. 10.
    Choi Y, Woo KM, Ko SH, Lee YJ, Park SJ, Kim HM, Kwon BS (2001) Osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8(+) T cells. Eur J Immunol 31:2179–2188PubMedCrossRefGoogle Scholar
  11. 11.
    Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and function of CD4 + CD25+ regulatory T cells. Nat Immunol 4:330–336PubMedCrossRefGoogle Scholar
  12. 12.
    Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057–1061PubMedCrossRefGoogle Scholar
  13. 13.
    Zaiss MM, Axmann R, Zwerina J, Polzer K, Gückel E, Skapenko A, Schulze-Koops H, Horwood N, Cope A, Schett G (2007) Treg cells suppress osteoclast formation: a new link between the immune system and bone. Arthritis Rheum 56:4104–4112PubMedCrossRefGoogle Scholar
  14. 14.
    Buchwald ZS, Kiesel JR, DiPaolo R, Pagadala MS, Aurora R (2012) Osteoclast activated FoxP3+ CD8+ T-cells suppress bone resorption in vitro. PLoS One 7:e38199PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Sharabi A, Mozes E (2008) The suppression of murine lupus by a tolerogenic peptide involves foxp3-expressing CD8 cells that are required for the optimal induction and function of foxp3-expressing CD4 cells. J Immunol 181:3243–3251PubMedCrossRefGoogle Scholar
  16. 16.
    Kiniwa Y, Miyahara Y, Wang HY, Peng W, Peng G, Wheeler TM, Thompson TC, Old LJ, Wang RF (2007) CD8+ Foxp3+ regulatory T cells mediate immunosuppression in prostate cancer. Clin Cancer Res 13:6947–6958PubMedCrossRefGoogle Scholar
  17. 17.
    Singh RP, La Cava A, Wong M, Ebling F, Hahn BH (2007) CD8+ T cell mediated suppression of autoimmunity in a murine lupus model of peptide induced immune tolerance depends on Foxp3 expression. J Immunol 178:7649–7657PubMedCrossRefGoogle Scholar
  18. 18.
    Kiesel J, Miller C, Abu-Amer Y, Aurora R (2007) Systems level analysis of osteoclastogenesis reveals intrinsic and extrinsic regulatory interactions. Dev Dyn 236:2181–2197PubMedCrossRefGoogle Scholar
  19. 19.
    Kiesel JR, Buchwald ZS, Aurora R (2009) Cross-presentation by osteoclasts induces FoxP3 in CD8+ T cells. J Immunol 182:5477–5487PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Giacomina Brunetti
    • 1
    • 2
    Email author
  • Angela Oranger
    • 1
    • 2
  • Silvia Colucci
    • 1
    • 2
  • Maria Grano
    • 1
    • 2
  1. 1.Section of Human Anatomy and Histology, Department of Basic Medical Sciences, Neurosciences, and Sense OrgansUniversity of BariBariItaly
  2. 2.PoliclinicoBariItaly

Personalised recommendations