ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis

Abstract

Lymphocytes mediate cytotoxicity by polarized release of the contents of cytotoxic granules toward their target cells. Here, we have studied the role of the calcium release-activated calcium channel ORAI1 in human lymphocyte cytotoxicity. Natural killer (NK) cells obtained from an ORAI1-deficient patient displayed defective store-operated Ca 2+ entry (SOCE) and severely defective cytotoxic granule exocytosis leading to impaired target cell lysis. Similar findings were obtained using NK cells from a stromal interaction molecule 1-deficient patient. The defect occurred at a late stage of the signaling process, because activation of leukocyte functional antigen (LFA)-1 and cytotoxic granule polarization were not impaired. Moreover, pharmacological inhibition of SOCE interfered with degranulation and target cell lysis by freshly isolated NK cells and CD8 + effector T cells from healthy donors. In addition to effects on lymphocyte cytotoxicity, synthesis of the chemokine macrophage inflammatory protein-1β and the cytokines TNF-α and IFN-γ on target cell recognition was impaired in ORAI1-deficient NK cells, as previously described for T cells. By contrast, NK cell cytokine production induced by combinations of IL-12, IL-15, and IL-18 was not impaired by ORAI1 deficiency. Taken together, these results identify a critical role for ORAI1-mediated Ca 2+ influx in granule exocytosis for lymphocyte cytotoxicity as well as for cytokine production induced by target cell recognition.

Mutations of the Ca2+-sensing Stromal Interaction Molecule STIM1 Regulate Ca2+ Influx by Altered Oligomerization of STIM1 and by Destabilization of the Ca2+ Channel Orai1

“…Both glycosylation sites are localized within the SAM domain, with the first site (Asn-131) immediately prior to the initial SAM domain ␣ helix (␣6) and the second site (Asn-171) localized at the begin- 2 . B, reaction scheme where OS(x) denotes one Orai1 tetramer with (x) STIM1 dimer bound with the corresponding reaction constants shown above the arrows.…”

“…The resulting reaction-diffusion system is studied by computer simulations based upon a two-dimensional Gillespie Monte Carlo Algorithm (25,26). Because CRAC channel activation is a local process (11), we model a geometry of 15 ϫ 15 grid cells, each describing an area of (0.2 m) 2 , which represents the cell plasma membrane and the ER membrane at the same time. For the diffusion of STIM1 monomers, STIM1 dimers, and Orai1, we assume periodic boundary conditions and only at the grid cell right in the middle of our geometry (Plasma Membrane Junction; see Fig.…”

“…The resulting inward Ca 2ϩ releaseactivated Ca 2ϩ current (I CRAC ) controls not only ubiquitous cellular functions, e.g. proliferation and apoptosis, but also cell specific functions such as cytokine production or target cell killing (1)(2)(3). The importance of I CRAC for the immune response is highlighted by the severe combined immunodeficiency in patients and mice with defective store operated Ca 2ϩ entry (SOCE) (4 -7).…”

Ion channels and anti-cancer immunity

“…Earlier studies indicated that elevated cytoplasmic levels of Ca 2þ are required for the polarization of cytotoxic granules to the killing synapse during contact between CTLs and their targets (reviewed in [35]). However, a recent study by MaulPavicic et al [36] clearly demonstrated that the cytotoxic granule polarization is not impaired by ORAI1 deficiency in human natural killer (NK) cells, whereas lytic granule exocytosis requires CRAC channels: degranulation is severely impaired in the absence of either ORAI1 or STIM1. Whether or not vesicle exocytosis is the only function depending on CRAC in CTLs, it was shown recently that functional CRAC is required for impeding engraftment of tumour cells and tumour growth in mice ( [37], see also below).…”

Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

“…Upon conjugation of NK cells with target cells, receptor activation triggers calcium influx into the NK cell which is required, but not sufficient, for NK cell degranulation. 31 Furthermore, Propidium Iodide (PI) rapidly diffuses through the perforin pore upon delivery of the lethal hit, when added to the assay medium. 32 These events can be monitored by imaging the cells in real time using time-lapse microscopy 32 and allows accurate quantification of the proportion of NK-tumor cell interactions that trigger calcium influx and result in a successful lethal hit.…”