Aquaporins are a family of ubiquitously expressed transmembrane water channels implicated in a broad range of physiological functions. We have previously reported that Aquaporin 4 (AQP4) is expressed on T cells and that treatment with a small molecule AQP4 inhibitor significantly delays T cell mediated heart allograft rejection. The goal of this study was to identify mechanisms of prolonged graft survival following AQP4 blockade.

WT or AQP4^-/- T cells were activated with anti-CD3/CD28 antibodies in vitro with or without small molecule inhibitor of AQP4, AER-270, and complementary approaches of flow cytometry, luciferase assays, phosphoflow, calcium flux and confocal imaging were utilized to interrogate the function of AQP4 in T cell activation.

We found that stimulation of murine WT T cells + AER-270 or AQP4^-/- T cells resulted in decreased upregulation of T cell activation markers CD25 and CD44, and reduced cell volume when compared to control WT T cells. In addition, the absence or inhibition of AQP4 also limited IL-2 production by T cells following stimulation. Intact AQP4 was also required for optimal IFNγ production by T cells activated under Th1 polarizing conditions. Analogously, AQP4 inhibition significantly limited the ability of human T cells to proliferate following TCR stimulation. We then examined the role of AQP4 in TCR signal transduction and found that AQP4 was required for nuclear translocation of NF-κB and NFAT and for optimal calcium flux following TCR cross-linking leading us to examine TCR proximal phosphorylation events.  Either the absence or inhibition of AQP4 impaired TCR signal transduction as early as phosphorylation of Lck (pY394), ZAP70 (pY319) and LAT (p171). As AQP4 was previously shown to associate with the actin cytoskeleton in astrocytes and skeletal muscle cells, we next tested its involvement in TCR polarization/capping. We found that the absence or inhibition of AQP4 resulted in significantly impaired TCR capping as well as dysfunctional post-TCR stimulation actin remodeling kinetics.

Our findings reveal novel mechanisms underlying AQP4 importance in optimal T lymphocyte activation and identify AQP4 as a potential therapeutic target for preventing TCR-mediated T cell activation in solid organ transplantation and other diseases.