Multiple signaling events are required for NAADP synthesis by DUOX2 and formation of Ca ²⁺ microdomains to initiate T cell activation

T cell activation critically depends on the calcium ion (Ca²⁺)-mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate), which induces the formation of Ca²⁺ microdomains that initiate global Ca²⁺ signals. NAADP is produced in immune synapses in T cells by dual NADPH oxidase 2 (DUOX2). Here, we investigated the mechanisms that stimulate DUOX2 activity in T cells. DUOX2 activity was enhanced by a modest increase in intracellular Ca²⁺ concentration, similar to that induced by Ca²⁺ microdomains that arise in resting T cells through different T cell receptor (TCR)-independent mechanisms. In addition, DUOX2 was activated in vitro by phosphorylation of threonine-789 mediated by PKA Cβ or PKCθ, and genetic deficiency of PKA Cβ2 or PKCθ decreased NAADP-dependent Ca²⁺ microdomain formation in T cells. PKA Cβ2 was activated downstream of adenosine A_2A receptors, independently of the TCR. In contrast, PKCθ was activated by the tyrosine kinase LCK downstream of TCR stimulation. Inhibition of A_2A receptors or PKCθ to prevent full DUOX2 activation decreased the production of the proinflammatory cytokine IL-17 by effector T cells. Thus, full stimulation of NAADP signaling that is critical for T cell activation requires integration of multiple TCR-independent and -dependent signals with different spatiotemporal characteristics by DUOX2, a fine-tuning mechanism that could be relevant for inflammation.