Granule Calcium for Cell Killing

Sci. Signal.  01 Jan 2013:
Vol. 6, Issue 256, pp. ec3
DOI: 10.1126/scisignal.2003915

Exocytosis of cytolytic granules from cytotoxic T lymphocytes (CTLs) requires calcium (Ca2+) release from the endoplasmic reticulum (ER) and subsequent influx of Ca2+ through calcium release–activated calcium (CRAC) channels. However, this pathway of Ca2+ release, which can be triggered by inositol 1,4,5-trisphosphate (IP3), is not itself sufficient to trigger exocytosis. Davis et al. reported that the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes calcium stores from cytolytic granules by activating two-pore channels (TPCs) located on the granules. Treating primary human CTLs with a cell-permeable form of an NAADP precursor that is converted to NAADP in the cells triggered Ca2+ oscillations that were blocked by treating cells with an NAADP antagonist, by depleting Ca2+ from acidic organelles or from the pH-neutral ER, or by blocking calcium release from the ER with an IP3 receptor blocker. Stimulating the cells with a cell-permeable IP3 analog after the addition of the permeable NAADP precursor synergistically enhanced the intracellular Ca2+ signal compared with stimulation with the NAADP precursor alone or the IP3 analog followed by addition of the NAADP precursor. These observations are consistent with NAADP’s triggering release of Ca2+ from an acidic store that stimulates subsequent release of Ca2+ from the ER and amplification of the Ca2+ signal by activation of IP3 receptors. Binding of CTLs to antigen-presenting cells (APCs) activates the T cell receptor (TCR) and triggers an initial spike in Ca2+ release from intracellular stores followed by slower Ca2+ oscillations driven by Ca2+ influx. Treatment of cells with the NAADP antagonist or an IP3 receptor blocker reduced the amplitude of these oscillations. Treating cells with the NAADP antagonist or depleting intracellular calcium stores blocked cytolytic granule exocytosis in response to APC engagement and inhibited target cell killing. NAADP treatment stimulated exocytosis even in the absence of TCR activation by APCs. Increasing global Ca2+ concentrations in CTLs by treating them with an IP3 analog or a Ca2+ ionophore induced Ca2+ oscillations similar to those that followed TCR activation but did not stimulate exocytosis. NAADP activates TPCs, and knocking down TPC1 or TPC2 in Jurkat cells with small interfering RNAs reduced the amplitude of both NAADP- and TCR-induced Ca2+ oscillations. TPC1 and TPC2 were present on cytolytic granules and translocated to the immunological synapse with the granules upon TCR stimulation. These findings suggest a model in which NAADP-mediated activation of cytolytic granule–localized TPC channels promotes Ca2+ release from these acidic compartments, which triggers exocytosis and stimulates additional Ca2+ release from the ER to sustain exocytosis.

L. C. Davis, A. J. Morgan, J.-L. Chen, C. M. Snead, D. Bloor-Young, E. Shenderov, M. N. Stanton-Humphreys, S. J. Conway, G. C. Churchill, J. Parrington, V. Cerundolo, A. Galione, NAADP activates two-pore channels on T cell cytolytic granules to stimulate exocytosis and killing. Curr. Biol. 22, 2331–2337 (2012). [PubMed]