*Purpose: Store-operated/calcium release activated calcium (CRAC) channels are essential for the signaling of immune cells via calcineurin, a calcium-calmodulin dependent serine-threonine phosphatase. The central importance of this pathway is reflected by the high immunosuppressive potency of calcineurin inhibitors (CNI). However, CNIs have multiple toxicities, and development of an alternative strategy to block calcineurin activity is of considerable clinical value. We reasoned that blockade of CRAC channels in normal human T cells is an effective strategy to prevent plenary T cell activation, and that such blockade should be observable at the pre-translational level.

*Methods: STIM and Orai proteins are prototypic CRAC channels and BTP2, a cell-permeable pyrazole, is a blocker of STIM1 and Orai1 dependent calcium entry in a number of cell lines. Cell lines and normal cells differ in signaling requirements. We therefore established in-vitro cultures using normal human peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers, and investigated the effect of BTP2 on the activation of PBMC by the polyclonal mitogen phytohemagglutinin (PHA). We used RT-qPCR assays to evaluate the effect of BTP2 on the expression of a hypothesis based panel of mRNAs encoding immunoregulatory proteins implicated in the anti-allograft repertoire

*Results: Activation of normal human PBMC with PHA and blockade of CRAC channels with BTP2 had differential effects on gene expression in PBMC (Table).

Among the CRAC channels, PHA activation induced Orai3 down regulation was reversed by BTP2; and activation-dependent decrease in CD103 mRNA was blocked by BTP2. In contrast, PHA activation dependent increases in the levels of mRNA for IL-2, CD28, and granzyme B were blocked by BTP2 whereas activation induced increases in mRNA for IFNy or IFNy induced IP10 and MIG were not significantly inhibited by BTP2.

*Conclusions: Neither PHA- activation of normal human PBMC nor blockade of CRAC channels with BTP2 has a monotonous impact on the expression of mRNAs encoding immunoregulatory proteins. Importantly, BTP2, via its impact on Orai3, may facilitate trafficking of immune T cells to the allograft via chemoattractants IP10/MIG and donor antigen education by preserving CD103-E cadherin interactions while blocking clonal expansion by reducing IL-2 and CD28 and cytotoxicity by reducing granzyme B – all conducive to tipping the balance from an allograft destructive immune response to a protective one including tolerance.

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