Cytolytic granules supply Ca²⁺ for their own exocytosis via NAADP and resident two-pore channels

Abstract

When cytotoxic T-lymphocytes (CTLs) kill infected or cancerous cells they secrete cytolytic proteins (perforin and granzymes) into the target cell. These “death factors” are pre-stored in cytolytic granules within the CTL until an increase in the intracellular Ca²⁺ drives granule exocytosis. However, not all sources of Ca²⁺ stimulate exocytosis: we have recently demonstrated that it is the cytolytic granules themselves that are the source of the Ca²⁺ that most efficiently drives their own exocytosis; release of Ca²⁺ from the granules is only activated by the Ca²⁺-mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) that acts upon target two-pore channels (TPCs) present on the granules. That NAADP is a unique stimulus of exocytosis may be of fundamental importance not only to immunology but to cell biology in general.

The role of acidic Ca²⁺ stores in immune cells is, at present, poorly understood but our recent findings indicate that they may be crucially important for fighting infection. One powerful mechanism that cytotoxic T lymphocytes (CTLs) use to kill virus-infected or tumorigenic target cells is the exocytosis of cytolytic proteins (e.g., granzymes and perforin) at the immunological synapse formed at the contact interface between the CTL and its target cell.^1,2 These “death factors” are pre-stored in specialized secretory lysosomes (termed cytolytic granules) within the CTL until an increase in intracellular Ca²⁺ concentration drives granule exocytosis.³

A trigger for CTL stimulation is the activation of its T-cell receptor (TCR) by antigen-MHC complexes on the target cell resulting in a biphasic elevation of Ca²⁺ i.e., an initial release of Ca²⁺ from intracellular stores followed by Ca²⁺ entry across the plasma membrane. Because intracellular Ca²⁺ release in T-cells can make a relatively small contribution to the global Ca²⁺ signal, Ca²⁺ influx has understandably garnered more attention and the store-operated Ca²⁺ influx pathway [Stim/Orai, that is regulated by the Ca²⁺-filling state of the endoplasmic reticulum, (ER)] has been implicated in supporting granule exocytosis and target-cell killing.

Nonetheless, Ca²⁺ release from intracellular stores may assume a greater importance than merely “tuning” Ca²⁺ influx. A theme in Ca²⁺ signaling is that not all sources or patterns of intracellular Ca²⁺ are equivalent and Ca²⁺ channels can differentially couple to cellular processes.^1,4 In CTLs, the exocytosis of cytolytic factors is clearly Ca²⁺-dependent; however, although store-operated Ca²⁺ influx across the plasma membrane is a necessary component for the exocytosis of cytolytic proteins, it is not a sufficient stimulus per se because it requires the additional activation of protein kinases.^5,6 We therefore hypothesized that a different Ca²⁺ channel family couples more directly to exocytosis.

Although the ER is the largest and the best-characterized Ca²⁺ store, acidic organelles (e.g., endo-lysosomes) are emerging as important Ca²⁺ stores but ones using a different second messenger pathway to the familiar Ins(1,4,5)P₃: acidic Ca²⁺ stores are preferentially mobilized by the Ca²⁺-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) that activates two-pore channels (TPCs).^7-10 We tested whether this pathway is important for granule exocytosis and cell killing in CTLs.¹¹

Using pharmacological and genetic approaches, we first showed that this acidic Ca²⁺ store/NAADP/TPC pathway was present and contributed to TCR-stimulated Ca²⁺ signals.¹¹ More remarkably, NAADP-induced Ca²⁺ release could drive exocytosis of cytolytic granules whereas the Ins(1,4,5)P₃/Orai system or ionomycin (Ca²⁺ ionophore) were ineffective i.e., there was a selectivity for the acidic Ca²⁺ store pathway and NAADP.¹¹ A further twist in the tale was that the targets for NAADP, TPCs, were on the cytolytic granules themselves, suggesting that the granules served a dual function: they contributed to as well as responded to Ca²⁺ signals to efficiently drive their own exocytosis.¹¹ We hypothesize that privileged local Ca²⁺ nanodomains around TPCs on the acidic Ca²⁺ stores are sensed by the neighboring exocytotic machinery and this locally high Ca²⁺ is what distinguishes NAADP from the other stimuli.

In summary, TCR activation recruits NAADP to activate target TPC channels resident on the exocytotic granules themselves, and TPCs consequently translocate toward the immunological synapse. Therefore, these granules store and release the Ca²⁺ for their own exocytosis and deliver Ca²⁺ in an “autocrine” fashion via TPCs, presumably acting in local perigranular Ca²⁺ nanodomains (Fig. 1). That NAADP is a unique stimulus of exocytosis may be of fundamental importance not only to immune cell function but may impact on stimulus-secretion coupling in wider cellular contexts.

Figure 1. Activation of the T-cell receptor (TCR) by contact with the target cell results in Ca²⁺ signals in the CTL: (1) the second messenger, NAADP, is synthesized; (2) NAADP activates target TPCs (two-pore channels) on the acidic cytolytic granules themselves; (3) TPCs generate local Ca²⁺ domains around the granules that act in concert with the ER/Ca²⁺ influx pathway to evoke exocytosis (4).

Davis LC, Morgan AJ, Chen JL, Snead CM, Bloor-Young D, Shenderov E, et al. NAADP activates two-pore channels on T cell cytolytic granules to stimulate exocytosis and killing. Curr Biol. 2012;22:2331–7. doi: 10.1016/j.cub.2012.10.035.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.