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Open AccessArticle

Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing

1
Disciplinary Program of Immunology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, 8380453 Santiago, Chile
2
Millennium Institute on Immunology and Immunotherapy, Faculty of Medicine, University of Chile, 8380453 Santiago, Chile
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this manuscript.
Int. J. Mol. Sci. 2019, 20(18), 4509; https://doi.org/10.3390/ijms20184509
Received: 13 August 2019 / Revised: 3 September 2019 / Accepted: 6 September 2019 / Published: 12 September 2019
(This article belongs to the Special Issue Connexin-Based Channels in Inflammatory Processes)
Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at natural killer (NK) cell–tumor cell cytotoxic immunological synapse. In this report, we demonstrate the functional role of Cx43-GJs at the cytotoxic immunological synapse established between CTLs and melanoma cells during cytotoxicity. Using confocal microscopy, we evaluated Cx43 polarization to the contact site between CTLs isolated from pMEL-1 mice and B16F10 melanoma cells. We knocked down Cx43 expression in B16F10 cells and evaluated its role in the formation of functional GJs and the cytotoxic activity of CTLs, by calcein transfer and granzyme B activity assays, respectively. We found that Cx43 localizes at CTL/B16F10 intercellular contact sites via an antigen-dependent process. We also found that pMEL-1 CTLs but not wild-type naïve CD8+ T cells established functional GJs with B16F10 cells. Interestingly, we observed that Cx43-GJs were required for an efficient granzyme B activity in target B16F10 cells. Using an HLA-A2-restricted/MART-1-specific CD8+ T-cell clone, we confirmed these observations in human cells. Our results suggest that Cx43-channels are relevant components of cytotoxic immunological synapses and potentiate CTL-mediated tumor cell killing. View Full-Text
Keywords: connexin 43; gap junctions; melanoma; cytotoxic T lymphocyte; cytotoxic immunological synapse connexin 43; gap junctions; melanoma; cytotoxic T lymphocyte; cytotoxic immunological synapse
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MDPI and ACS Style

Hofmann, F.; Navarrete, M.; Álvarez, J.; Guerrero, I.; Gleisner, M.A.; Tittarelli, A.; Salazar-Onfray, F. Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing. Int. J. Mol. Sci. 2019, 20, 4509.

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  • Externally hosted supplementary file 1
    Doi: 10.5281/zenodo.3367468
    Description: Supplementary Figure 1. Phenotypic characterization of differentiated pMEL-1 cytotoxic T lymphocytes (CTLs) and isolated wild type naïve CD8+ T cells.
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