Abstract
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.
Highlights
Gap junctions (GJs) are clusters of intercellular channels found at the plasma membrane that allow direct communication between the cytoplasm of adjacenT cells
We and others have demonstrated that connexin 43 (Cx43) channels accumulate at the immunological synapses during dendritic cells (DCs)-T cell and DC-natural killer (NK) cell interactions [15,16,17] and at the NK cell-tumor cell cytotoxic immunological synapse [17,18], allowing DC-mediated T- and NK-cell activation and NK cell-mediated target tumor cell killing, respectively
Given that the cytotoxic immunological synapses formed by NK cells and cytotoxic T lymphocytes (CTLs) share some structural features [19], in this report we aimed to examine whether Cx43 channels have a role in CTL-target tumor cell immunological synapses
Summary
Gap junctions (GJs) are clusters of intercellular channels found at the plasma membrane that allow direct communication between the cytoplasm of adjacenT cells. Each GJ channel is formed by two hexameric hemichannels, known as connexons, one provided by each of the two contacting cells. Each hemichannel is composed of six transmembrane proteins called connexins (Cx) [1]. Once functional GJs are established, these channels allow the exchange of small molecules (up to ≈1.4 nm) between the cytoplasm of adjacenT cells, including adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), inositol triphosphate (IP3), Ca2+, glutamate, microRNAs, and small peptides [2]. The Cx gene family is composed of 21 different members in humans and 20 in mice, where different isoforms are expressed in a tissue-specific manner and determine channel properties. Cx43 is expressed almost ubiquitously and it is the main Cx member expressed in the immune system cells [3,4,5]
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