# The scramblase anoctamin 9 controls the immune response in lymphocytes

## TL;DR

ANO9, a protein involved in cell signaling, plays a key role in regulating immune responses in lymphocytes and may contribute to chronic kidney disease and inflammation.

## Contribution

This study reveals a novel role for ANO9 in modulating T cell activation and Ca2+ signaling, linking it to inflammatory diseases and kidney disorders.

## Key findings

- ANO9 knockout in Jurkat cells reduces T cell activation and Ca2+ signaling.
- T595A-Ano9 mutation in mice increases Ca2+ signaling and immune cell activation.
- ANO9 variants may contribute to chronic kidney disease and inflammation via altered PMCA expression.

## Abstract

We previously reported a loss of T cell activation following knockdown of the phospholipid scramblase and ion channel ANO9 (TMEM16J) in human Jurkat lymphocytes. We have now analyzed in detail the role of ANO9 in Jurkat ANO9 knockout cells and primary human pan-T cells. In addition, transgenic knockin mice carrying the T595A-Ano9 mutation were generated, as the human counterpart T604A-ANO9 and other variants had been shown to cause chronic kidney disease (CKD) and inflammation. Jurkat cells lacking expression of ANO9 demonstrated a loss of T-cell receptor-induced Ca2+ signaling and reduced expression of the plasma membrane Ca2+-ATPase (PMCA). Upregulation of PMCA-expression by vitamin D3 was abolished in the absence of ANO9. Activation of wild-type Jurkat cells by stimulation of CD3/CD28 receptors was potently inhibited by the putative ANO9-inhibitor flunisolide. Knockdown of ANO9 in primary human T cells reproduced the loss of activation demonstrated in Jurkat ANO9-knockout cells and caused a loss in store-operated Ca2+ entry (SOCE). Expression of Ano9 in mouse lymphocytes was low when compared to human, but was upregulated during activation of CD3 and CD28 receptors. Cells isolated from T595A-knockin mice exhibit upregulated Ca2+ signaling that caused pronounced depolarization of the membrane voltage, enhanced whole cell currents and an increase in IL-2 release and cell proliferation. Additional boosters of activation such as PMA/ phytohemagglutinin or concanavalin A were unable to further enhance activation in wild-type lymphocytes to the level observed in T595A-Ano9 cells. The data suggest that expression of the ANO9 variant T604A in lymphocytes and renal epithelial tissues may cause hyperinflammatory diseases and CKD by upregulated intracellular Ca2+ signaling due to augmented expression of PMCA.