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Regulation of gap junctions in melanoma and their impact on Melan-A/MART-1-specific CD8+ T lymphocyte emergence

Journal of Molecular Medicine volume 91, pages 1207–1220 (2013)Cite this article

Abstract

Gap junctions (GJs) enable intercellular communication between adjacent cells through channels of connexins. Using a three-dimensional construct, we previously showed that endothelial and tumor cells formed GJs, allowing melanoma-specific T lymphocytes to recognize and kill melanoma-derived endothelial cells. We demonstrate here on histological sections of melanoma biopsies that GJ formation occurs in vivo between tumor and endothelial cells and between T lymphocytes and target cells. We also show an in vitro increase of GJ formation in melanoma and endothelial cells following dacarbazin and interferon gamma (IFN-γ) treatment or hypoxic stress induction. Our data indicate that although connexin 43 (Cx43), the main GJ protein of the immune system, was localized at the immunological synapse between T lymphocyte and autologous melanoma cells, its over-expression or inhibition of GJs does not interfere with cytotoxic T lymphocyte (CTL) clone lytic function. In contrast, we showed that inhibition of GJs by oleamide during stimulation of resting PBMCs with Melan-A natural and analog peptides resulted in a decrease in antigen (Ag) specific CD8+ T lymphocyte induction. These Ag-specific CD8+ cells displayed paradoxically stronger reactivity as revealed by CD107a degranulation and IFN-γ secretion. These findings indicate that Cx43 does not affect lytic function of differentiated CTL, but reveal a major role for GJs in the regulation of antigen CD8+-naïve T lymphocyte activation.

Key message

  • GJ formation occurs in vivo between T lymphocytes and tumor cells

  • Cx43 localized at the immunological synapse between T and autologous melanoma cells

  • Inhibition of GJs resulted in a decrease in Ag-specific CD8+ T lymphocyte induction

  • A role for GJs in the regulation of antigen CD8+-naïve T lymphocyte activation

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References

  1. Trapani JA (2002) Tumor-mediated apoptosis of cancer-specific T lymphocytes—reversing the "kiss of death"? Cancer Cell 2:169–171

    PubMed  Article  CAS  Google Scholar 

  2. Chouaib S, Asselin-Paturel C, Mami-Chouaib F, Caignard A, Blay JY (1997) The host–tumor immune conflict: from immunosuppression to resistance and destruction. Immunol Today 18:493–497

    PubMed  Article  CAS  Google Scholar 

  3. Chouaib S (2003) Integrating the quality of the cytotoxic response and tumor susceptibility into the design of protective vaccines in tumor immunotherapy. J Clin Invest 111:595–597

    PubMed  CAS  Google Scholar 

  4. Willecke K, Eiberger J, Degen J, Eckardt D, Romualdi A, Guldenagel M, Deutsch U, Sohl G (2002) Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem 383:725–737

    PubMed  Article  CAS  Google Scholar 

  5. Goodenough DA, Goliger JA, Paul DL (1996) Connexins, connexons, and intercellular communication. Annu Rev Biochem 65:475–502

    PubMed  Article  CAS  Google Scholar 

  6. Veliz LP, Gonzalez FG, Duling BR, Saez JC, Boric MP (2008) Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo. Am J Physiol Heart Circ Physiol 295:H1056–H1066

    PubMed  Article  CAS  Google Scholar 

  7. Schajnovitz A, Itkin T, D'Uva G, Kalinkovich A, Golan K, Ludin A, Cohen D, Shulman Z, Avigdor A, Nagler A et al (2011) CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions. Nat Immunol 12:391–398

    PubMed  Article  CAS  Google Scholar 

  8. Oviedo-Orta E, Gasque P, Evans WH (2001) Immunoglobulin and cytokine expression in mixed lymphocyte cultures is reduced by disruption of gap junction intercellular communication. FASEB J 15:768–774

    PubMed  Article  CAS  Google Scholar 

  9. Neijssen J, Herberts C, Drijfhout JW, Reits E, Janssen L, Neefjes J (2005) Cross-presentation by intercellular peptide transfer through gap junctions. Nature 434:83–88

    PubMed  Article  CAS  Google Scholar 

  10. Benlalam H, Jalil A, Hasmim M, Pang B, Tamouza R, Mitterrand M, Godet Y, Lamerant N, Robert C, Avril MF et al (2009) Gap junction communication between autologous endothelial and tumor cells induce cross-recognition and elimination by specific CTL. J Immunol 182:2654–2664

    PubMed  Article  CAS  Google Scholar 

  11. Saccheri F, Pozzi C, Avogadri F, Barozzi S, Faretta M, Fusi P, Rescigno M (2010) Bacteria-induced gap junctions in tumors favor antigen cross-presentation and antitumor immunity. Sci Transl Med 2:44–57

    Article  Google Scholar 

  12. Bopp T, Becker C, Klein M, Klein-Hessling S, Palmetshofer A, Serfling E, Heib V, Becker M, Kubach J, Schmitt S et al (2007) Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression. J Exp Med 204:1303–1310

    PubMed  Article  CAS  Google Scholar 

  13. Elgueta R, Tobar JA, Shoji KF, De Calisto J, Kalergis AM, Bono MR, Rosemblatt M, Saez JC (2009) Gap junctions at the dendritic cell–T cell interface are key elements for antigen-dependent T cell activation. J Immunol 183:277–284

    PubMed  Article  CAS  Google Scholar 

  14. Oviedo-Orta E, Perreau M, Evans WH, Potolicchio I (2010) Control of the proliferation of activated CD4+ T cells by connexins. J Leukoc Biol 88:79–86

    PubMed  Article  CAS  Google Scholar 

  15. Mendoza-Naranjo A, Bouma G, Pereda C, Ramirez M, Webb KF, Tittarelli A, Lopez MN, Kalergis AM, Thrasher AJ, Becker DL et al (2011) Functional gap junctions accumulate at the immunological synapse and contribute to T cell activation. J Immunol 187:3121–3132

    PubMed  Article  CAS  Google Scholar 

  16. Meslin F, Thiery J, Richon C, Jalil A, Chouaib S (2007) Granzyme B-induced cell death involves induction of p53 tumor suppressor gene and its activation in tumor target cells. J Biol Chem 282:32991–32999

    PubMed  Article  CAS  Google Scholar 

  17. Hansen MB, Nielsen SE, Berg K (1989) Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 119:203–210

    PubMed  Article  CAS  Google Scholar 

  18. Le Floc'h A, Jalil A, Vergnon I, Le Maux CB, Lazar V, Bismuth G, Chouaib S, Mami-Chouaib F (2007) Alpha E beta 7 integrin interaction with E-cadherin promotes antitumor CTL activity by triggering lytic granule polarization and exocytosis. J Exp Med 204:559–570

    PubMed  Article  Google Scholar 

  19. Harris AL (2002) Hypoxia—a key regulatory factor in tumour growth. Nat Rev Cancer 2:38–47

    PubMed  Article  CAS  Google Scholar 

  20. Guan X, Cravatt BF, Ehring GR, Hall JE, Boger DL, Lerner RA, Gilula NB (1997) The sleep-inducing lipid oleamide deconvolutes gap junction communication and calcium wave transmission in glial cells. J Cell Biol 139:1785–1792

    PubMed  Article  CAS  Google Scholar 

  21. Evans WH, Boitano S (2001) Connexin mimetic peptides: specific inhibitors of gap-junctional intercellular communication. Biochem Soc Trans 29:606–612

    PubMed  Article  Google Scholar 

  22. Ciovacco WA, Goldberg CG, Taylor AF, Lemieux JM, Horowitz MC, Donahue HJ, Kacena MA (2009) The role of gap junctions in megakaryocyte-mediated osteoblast proliferation and differentiation. Bone 44:80–86

    PubMed  Article  CAS  Google Scholar 

  23. Boger DL, Patterson JE, Guan X, Cravatt BF, Lerner RA, Gilula NB (1998) Chemical requirements for inhibition of gap junction communication by the biologically active lipid oleamide. Proc Natl Acad Sci U S A 95:4810–4815

    PubMed  Article  CAS  Google Scholar 

  24. Saito-Katsuragi M, Asada H, Niizeki H, Katoh F, Masuzawa M, Tsutsumi M, Kuniyasu H, Ito A, Nojima H, Miyagawa S (2007) Role for connexin 26 in metastasis of human malignant melanoma: communication between melanoma and endothelial cells via connexin 26. Cancer 110:1162–1172

    PubMed  Article  CAS  Google Scholar 

  25. Nardin A, Wong WC, Tow C, Molina TJ, Tissier F, Audebourg A, Garcette M, Caignard A, Avril MF, Abastado JP et al (2011) Dacarbazine promotes stromal remodeling and lymphocyte infiltration in cutaneous melanoma lesions. J Invest Dermatol 131:1896–1905

    PubMed  Article  CAS  Google Scholar 

  26. Eugenin EA, Branes MC, Berman JW, Saez JC (2003) TNF-alpha plus IFN-gamma induce connexin43 expression and formation of gap junctions between human monocytes/macrophages that enhance physiological responses. J Immunol 170:1320–1328

    PubMed  CAS  Google Scholar 

  27. Heinrich M, Oberbach A, Schlichting N, Stolzenburg JU, Neuhaus J (2011) Cytokine effects on gap junction communication and connexin expression in human bladder smooth muscle cells and suburothelial myofibroblasts. PLoS One 6:e20792

    PubMed  Article  CAS  Google Scholar 

  28. Lim PK, Bliss SA, Patel SA, Taborga M, Dave MA, Gregory LA, Greco SJ, Bryan M, Patel PS, Rameshwar P (2011) Gap junction-mediated import of microRNA from bone marrow stromal cells can elicit cell cycle quiescence in breast cancer cells. Cancer Res 71:1550–1560

    PubMed  Article  CAS  Google Scholar 

  29. Sirnes S, Bruun J, Kolberg M, Kjenseth A, Lind GE, Svindland A, Brech A, Nesbakken A, Lothe RA, Leithe E et al (2012) Connexin43 acts as a colorectal cancer tumor suppressor and predicts disease outcome. Int J Cancer 131:570–581

    PubMed  Article  CAS  Google Scholar 

  30. Klee P, Allagnat F, Pontes H, Cederroth M, Charollais A, Caille D, Britan A, Haefliger JA, Meda P (2011) Connexins protect mouse pancreatic beta cells against apoptosis. J Clin Invest 121:4870–4879

    PubMed  Article  CAS  Google Scholar 

  31. Oviedo-Orta E, Howard Evans W (2004) Gap junctions and connexin-mediated communication in the immune system. Biochim Biophys Acta 1662:102–112

    PubMed  Article  CAS  Google Scholar 

  32. Cancelas JA, Koevoet WL, de Koning AE, Mayen AE, Rombouts EJ, Ploemacher RE (2000) Connexin-43 gap junctions are involved in multiconnexin-expressing stromal support of hemopoietic progenitors and stem cells. Blood 96:498–505

    PubMed  CAS  Google Scholar 

  33. Kuczma M, Lee JR, Kraj P (2011) Connexin 43 signaling enhances the generation of Foxp3+ regulatory T cells. J Immunol 187:248–257

    PubMed  Article  CAS  Google Scholar 

  34. Endong L, Shijie J, Sonobe Y, Di M, Hua L, Kawanokuchi J, Mizuno T, Suzumura A (2011) The gap-junction inhibitor carbenoxolone suppresses the differentiation of Th17 cells through inhibition of IL-23 expression in antigen presenting cells. J Neuroimmunol 240–241:58–64

    PubMed  Article  Google Scholar 

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Acknowledgments

We thank Nadine Gervois and Nathalie Labarrière for the HLA-A2/Melan-A tetramer gift as well as Lisa Bain for manuscript editing.

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Author notes

  1. Houssem Benlalam

    Present address: Inserm U892, CRCNA, Université de Nantes, 44000, Nantes, France

Affiliations

  1. Institut National de la Santé et de la Recherche Médicale (INSERM U753), Institut Fédératif de Recherche 54 (IFR54), Institut Gustave Roussy, 39 Rue Camille Desmoulins, 94805, Villejuif, France

    Houssem Benlalam, Thibault Carré, Abdelali Jalil, Zaeem Noman, Andrés Tittarelli & Salem Chouaib

  2. Department of Pathology, Translational Research Laboratory and Biobank, Institut de Cancérologie Gustave Roussy, 94800, Villejuif, France

    Bernard Caillou & Philippe Vielh

  3. Département de Médecine, Institut Gustave Roussy, 94800, Villejuif, France

    Caroline Robert

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Correspondence to Salem Chouaib.

Additional information

Houssem Benlalam and Thibault Carré contributed equally to this work.

Electronic supplementary material

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Cx43 localizes at the immunological synapse. Intracellular trafficking of Cx43-GFP to the contact area between Cx43-GFP transfected M4T and autologous CTL was analyzed by confocal microscopy (AVI 5,082 kb)

Cx43 localizes at the immunological synapse and gives rise to functional GJs. Calcein was transferred from CTL to M4T cells after contact as shown by confocal microscopy (AVI 66,204 kb)

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Benlalam, H., Carré, T., Jalil, A. et al. Regulation of gap junctions in melanoma and their impact on Melan-A/MART-1-specific CD8+ T lymphocyte emergence. J Mol Med 91, 1207–1220 (2013). https://doi.org/10.1007/s00109-013-1058-5

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  • DOI: https://doi.org/10.1007/s00109-013-1058-5

Keywords

  • Melanoma
  • Cytotoxic T cell
  • Antigen presentation
  • Gap junction
  • Connexin 43