Gap Junction Intercellular Communications Regulate NK Cell Activation and Modulate NK Cytotoxic Capacity

Abstract

Gap junctions (GJs) mediate intercellular communication between adjacent cells. Previously, we showed that connexin 43 (Cx43), the main GJ protein in the immune system, mediates Ag transfer between human dendritic cells (DCs) and is recruited to the immunological synapse during T cell priming. This crosstalk contributed to T cell activation, intracellular Ca2+ responses, and cytokine release. However, the role of GJs in NK cell activation by DCs and NK cell–mediated cytotoxicity against tumor cells remains unknown. In this study, we found polarization of Cx43 at the NK/DC and NK/tumor cell-contact sites, accompanied by the formation of functional GJs between NK/DCs and NK/tumor cells, respectively. Cx43–GJ-mediated intercellular communication (GJIC) between human NK and DCs was bidirectional. Blockage of Cx43-GJIC inhibited NK cell activation, though it affected neither the phenotype nor the function of DCs. Cx43 knockdown or inhibition using mimetic peptides greatly reduced CD69 and CD25 expression and IFN-γ release by DC-stimulated NK cells. Moreover, blocking Cx43 strongly inhibited the NK cell–mediated tumor cell lysis associated with inhibition of granzyme B activity and Ca2+ influx. Our data identify a novel and active role for Cx43-GJIC in human NK cell activation and antitumor effector functions that may be important for the design of new immune therapeutic strategies.

The Selective Degradation of Synaptic Connexin 43 Protein by Hypoxia-induced Autophagy Impairs Natural Killer Cell-mediated Tumor Cell Killing

“…Ectopic Overexpression of Cx43 Enhances NK-mediated Tumor Cell Killing by Stabilizing the Immune Synapse-It has been reported that the inhibition of Cx43 channels strongly decreased NK-mediated tumor cell lysis (10). In keeping with this, we investigated whether the overexpression of Cx43 in the M4T melanoma cell model influences their susceptibility to NK cell-mediated lysis.…”

“…Autophagy was inhibited by addition of 50 M hydroxychloroquine (HCQ) or 5 mM 3-methyladenine (both from Sigma-Aldrich). Cx43 channels were inhibited using Cx43-mimetic peptides (300 M, gap27, SRPTEKTIFII, Synprosis) (10,24). A scrambled peptide (TFEPIRISITK) was used as a control.…”

“…Cells were analyzed with a Zeiss LSM-510 Meta laser-scanning confocal microscope (Carl Zeiss), and images were captured with a ϫ63 oil objective lens. The recruitment of Cx43 to the cell-to-cell contact site (immune synapses) was quantified using ImageJ (National Institutes of Health) software as described previously (10). Cx43-LC3 colocalization was analyzed using Imaris software.…”

“…Because Cxs have short half-lives, their turnover regulation and trafficking critically impact the control of GJmediated intercellular communication (5). Connexin 43 (Cx43), the main GJ protein found in immune cells (6), has been shown recently to accumulate at the immunological synapse and allows GJ-mediated intercellular communication between effector immune and tumor target cells (7)(8)(9)(10). These observations suggest that loss of Cx43 expression during tumor progression may represent a mechanism by which cancer cells evade immune control by the local microenvironment (11).…”

Cell-Cell Communication in the Tumor Microenvironment, Carcinogenesis, and Anticancer Treatment

“…NKT cells string together characteristics of innate and adaptive immunity [150], reviewed in [151]: they activate receptors and express inhibiting receptors that sense the presence of the MHC class I molecules expressed on all healthy cells [152], reviewed in [151]. Cx43 regulates NKT activation: knockdown reduced CD69 and CD25 expression and also the IFN-γ secretion usually released by NKT induced through human dendritic cells and blocking the Cx43-suppressed NKT-mediated tumor cell lysis [153]. The αv-containing Integrins are known to be essential for fibrosis [154], a pharmacological blockade of the αv-subunit has been shown to attenuate liver and lung fibrosis in mice treated with a novel small molecule, (CWHM 12) [155].…”