Aquaporin 4 (AQP4) belongs to the family of integral transmembrane proteins highly permeable to water. We have previously reported that blocking AQP4 with small molecule inhibitor AER270/271 during cold ischemia storage (CIS) and for 5 days posttransplant significantly prolonged survival of BALB/c heart allografts subjected to 8 h of CIS and transplanted into B6 recipients (MST of 25 d, n=24 vs. 6 d, n=5). At the time of rejection, the frequencies of donor-reactive T cells were significantly lower in AQP4 inhibitor treated than in saline treated recipients (500 vs. 2500 IFNγ spots/1×10⁶ splenocytes, P<0.001).The goal of the current study was to test whether the activation and functions of alloreactive T cells may be directly affected by AER270. Real-time RT-PCR analysis showed AQP4 expression in naïve and effector/memory CD8, but not CD4 T cells from naïve B6 mice. CFSE-labeled naïve T lymphocytes were stimulated in vitro for 72 h with anti-CD3/anti-CD28 mAb. AER270 inhibited CFSE dilution in a dose dependent manner with >75% inhibition achieved at 0.25 [mu]M. To assess how AQP4 blockade affects effector functions of previously primed T cells, spleen cells from B6 recipients of BALB/c skin allografts were tested in a 24 h recall IFNγ ELISPOT assay in response to donor BALB/c alloantigens. AER270 blocked IFNγ production by effector/memory alloreactive T cells (>80% inhibition at 0.25 [mu]M). The decreased IFNɣ secretion was not due to direct reagent cytotoxicity, as the numbers of live cells were not reduced by AER270. To test whether the effects of AQP4 inhibition in vivo are limited to alloreactive T cell responses, we immunized B6 mice with ovalbumin (OVA) in CFA s.c. and treated with AER271, a prodrug converted into AER270 by liver phosphatases, on d 0-5 after immunization. AER271 treatment significantly reduced the activation of OVA-specific T cells in the draining lymph nodes at d. 10 post immunization compared to control group. This is the first evidence that AQP4 water channel is expressed in T cells and that blocking AQP4 inhibits activation and functions of both naïve and effector/memory T cells. Our results suggest that AQP4 can be a promising therapeutic target to diminish the detrimental effects of alloreactive T cells in transplantation.

CITATION INFORMATION: Ayasoufi K, Kohei N, Farr G, McGuirk P, Pelletier M, Fairchild R, Valujskikh A. Aquaporin 4 Blockade Is a Novel Strategy to Prevent T Cell Activation and Effector Functions. Am J Transplant. 2016;16 (suppl 3).

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