The ubiquitous water channels aquaporins mediate a variety of immune functions. We previously demonstrated that aquaporin 4 (AQP4) is expressed by CD4 and CD8 T cells and that targeted blockade of AQP4 with small molecule inhibitor AER-270/271 significantly prolongs survival of heart allografts in two robust models of rejection. The goal of this study was to determine the effects of AQP4 blockade on T cells.

Administration of AER-271 into naïve non-transplanted mice (250 [mu]g i.p. every 6 h on d. 0-4) decreased numbers of both CD4 and CD8 T cells, but not B cells, in peripheral blood compared to untreated controls. Despite marked reduction in circulating T cell numbers (>90%), the CaspGLOW staining showed <10 % of apoptotic cells. Furthermore, the frequencies and numbers of CD4 and CD8 T cells in the spleen and the lymph nodes were not significantly affected by AQP4 blockade, suggesting that the lack of circulating T cells is not due to systemic depletion but rather to altered T cell trafficking.

The effect of AER-271 treatment was transient, with circulating T cell numbers restored to pretreatment levels after 3 weeks. AER-271 treated animals were able to reject a heart allograft transplanted 24 days after treatment cessation (MST 6 d., n=4, comparable to untreated controls).

We next tested the effects of AQP4 blockade on Sphingosine-1 Phosphate Receptor 1 (S1PR1), a known mediator of T cell trafficking between circulation and secondary lymphoid organs. S1PR1 mRNA expression was reduced in isolated mouse spleen T cells within 3h of in vitro Incubation with 0.25[mu]M AER-270. No change in S1PR1 mRNA expression was seen in AER-270 treated B cells. In addition, chemokine receptor CCR5 mRNA expression was not affected by 3h AQP4 blockade. To determine if the decreased S1PR1 mRNA expression translated into altered cell trafficking, we tested the ability of AER270 treated T cells to migrate toward the S1PR1 ligand, Sphingosine-1 Phosphate (S1P) in a transwell system. AER-270 reduced T cell chemotaxis toward S1P in a dose dependent manner.

Our data suggest that while well-studied S1PR1 superagonist FTY720 causes receptor internalization, AER-270/271 treatment results in altered S1PR1 transcription thus changing T cell trafficking and prolonging allograft survival. Therefore, AQP4 blockade may be an attractive therapeutic strategy in transplantation and other immune-mediated diseases.

CITATION INFORMATION: Nicosia M., Fan R., Miyairi S., Beavers A., Farr G., McGuirk P., Pelletier M., Valujskikh A. Aquaporin 4 Blockade Alters T Cell Trafficking through a Novel Mechanism of S1PR1 Regulation Am J Transplant. 2017;17 (suppl 3).

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