Calcium signalling in T cells

Abstract

Calcium (Ca²⁺) signalling is of paramount importance to immunity. Regulated increases in cytosolic and organellar Ca²⁺ concentrations in lymphocytes control complex and crucial effector functions such as metabolism, proliferation, differentiation, antibody and cytokine secretion and cytotoxicity. Altered Ca²⁺ regulation in lymphocytes leads to various autoimmune, inflammatory and immunodeficiency syndromes. Several types of plasma membrane and organellar Ca²⁺-permeable channels are functional in T cells. They contribute highly localized spatial and temporal Ca²⁺ microdomains that are required for achieving functional specificity. While the mechanistic details of these Ca²⁺ microdomains are only beginning to emerge, it is evident that through crosstalk, synergy and feedback mechanisms, they fine-tune T cell signalling to match complex immune responses. In this article, we review the expression and function of various Ca²⁺-permeable channels in the plasma membrane, endoplasmic reticulum, mitochondria and endolysosomes of T cells and their role in shaping immunity and the pathogenesis of immune-mediated diseases.

Figure 1 |. Calcium signalling in T cells.

Stimulation of the T cell receptor (TCR) by specific antigens leads to activation of phospholipase Cγ1 (PLCγ1), the production of inositol-1,4,5-trisphosphate (IP₃) and Ca²⁺ release from endoplasmic reticulum (ER) Ca²⁺ stores via IP₃ receptor (IP₃R) channels. The decrease in Ca²⁺ levels within the ER lumen is sensed by low affinity EF-hands of stromal interaction molecule 1 (STIM1) and STIM2. STIM proteins gain an extended conformation to trap and activate ORAI1 proteins at the plasma membrane (PM) and induce store operated Ca²⁺ entry (SOCE). SOCE activates Ca²⁺–calmodulin and its target enzymes and transcription factors, most notably nuclear factor for activated T cells (NFAT) isoforms. Other PM channels are involved in mediating Ca²⁺ signals during T cell activation and include non-selective transient receptor potential (TRP) channels, purinergic ionotropic receptors (P2RX) and CaV channels. Ca²⁺ release by IP₃R (and Ca²⁺ entry through PM channels) is transferred into mitochondria through the mitochondrial Ca²⁺ uniporter (MCU) at highly specialized membrane contact sites termed mitochondria-associated membranes (MAMs), which effectively couple TCR ligation to enhanced bioenergetics and ATP production required for clonal expansion and secretion of cytokines. Sources of Ca²⁺ uptake into endolysosomes remain incompletely understood but contributions from the ER and PM are likely. Endolysosomal Ca²⁺ release controls vesicular fusion, trafficking and secretion of cargo and replenishment of exhausted signalling molecules at the PM of activated T cells. Cytoplasmic, ER and mitochondrial Ca²⁺ homeostasis are maintained by the actions of transporters and pumps, including the PM Ca²⁺ ATPase (PMCA), the sarcoplasmic/ER Ca²⁺ ATPase (SERCA) and the mitochondrial Na⁺/Ca²⁺/Li⁺ exchanger (NCLX). LAT, linker for activation of T cells; ZAP70, ζ-chain-associated protein kinase of 70 kDa.

Figure 2 |. ORAI channels: major players in T cell activation.

ORAI proteins form highly Ca²⁺ selective homo-hexameric and hetero-hexameric channels in the plasma membrane (PM). On T cell receptor (TCR) ligation and subsequent inositol-1,4,5-trisphosphate (IP₃)-mediated endoplasmic reticulum (ER) Ca²⁺ store depletion, stromal interaction molecule (STIM) proteins move to ER–PM junctions and physically interact with ORAI channels, causing their activation. Mammals express three ORAI proteins (ORAI1–ORAI3) encoded by three separate genes and the major isoform mediating store operated Ca²⁺ entry (SOCE) in most cells including T cells is ORAI1. ORAI1 exists in two isoforms ORAI1α (long) and ORAI1β (short) due to alternative translation-initiation of Orai1 mRNA^,. ORAI1α and ORAI1β do not seem to form hetero-hexamers. ORAI1β has not been studied in T cells. ORAI2 can mediate residual SOCE in T cells from Orai1^−/− mice and was proposed to negatively modulate ORAI1 activity through formation of ORAI1–ORAI2 heteromeric associations. ORAI3, which unlike ORAI1, is resistant to inhibitory oxidation and is upregulated in effector T cells. The exclusively mammalian ORAI1α and ORAI3 proteins were shown to form heteromeric channels that are activated independently of store depletion in other cell types, but their existence in T cells is unknown. Ca²⁺ entry through ORAI channels activates calcineurin and subsequent nuclear translocation of isoforms of nuclear factor for activated T cells (NFAT). ORAI1α physically associates through its N-terminus with the Ca²⁺-activated adenylyl cyclase 8 (AC8), thus connecting Ca²⁺ microdomains through ORAI1α to cAMP production, activation of protein kinase A (PKA) and the transcription factor cAMP-responsive-element-binding protein (CREB). Ca²⁺ entry through heteromeric ORAI1α–ORAI3 channels was proposed to regulate the AKT pathway, although the exact mechanisms remain unknown. AP-1, activator protein-1; CaM, calmodulin; CaMK-II, Ca²⁺-calmodulin-dependent kinase II; MEF2, myocyte-specific enhancer factor 2; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor-κB; PI3K, phosphoinositide 3-kinase.

Figure 3 |. TRP channels: regulators of T cell Ca²⁺ signalling.

The transient receptor potential melastatin 4 (TRPM4) is a Ca²⁺-activated Na+ selective channel that is a potent regulator of ORAI-mediated Ca²⁺ entry in T cells. TRPM4 mediates its action through Na+ entry, causing plasma membrane depolarization, which limits the driving force for Ca²⁺ and inhibits Ca²⁺ entry through ORAI channels. As such, during Ca²⁺ responses in T cells, Ca²⁺ activation by TRPM4 can shape the magnitude of sustained Ca²⁺ signals (plateaus) as well as the frequency and amplitude of oscillatory Ca²⁺ responses and therefore determine which downstream transcription factors and gene programmes are activated. An opposing role is played by Ca²⁺- and voltage-dependent K+ channels (KCa and Kv), which maintain hyperpolarized Vm to support Ca²⁺ entry. The Mg²⁺- and Ca²⁺-permeable channel-enzyme TRPM7 is one of few reported Ca²⁺ channels with a crucial role in T cell development and homeostasis and this function appears to be mediated by Ca²⁺ entry through the channel domain. The kinase domain of TRPM7 regulates ORAI1 signalling and coordinates antigen receptor signalling termination in lymphocytes, likely through phosphorylation of phospholipase Cγ (PLCγ) isoforms. TRPM2 channels are non-selective Ca²⁺-conducting channels which are activated by hydrogen peroxide (H2O2) and through the cytosolic second messengers nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP ribose (cADPR). TRPM2 has a more established role in neutrophils and was proposed to support T cell Ca²⁺ signalling in high oxidant inflammatory milieu.

Figure 4 |. P2RX receptors: amplifiers of T cell receptor-mediated Ca²⁺ signalling through paracrine and autocrine ATP.

Purinergic ionotropic receptors (P2RX), including P2RX1, P2RX4 and P2RX7 are trimeric ionotropic non-selective Ca²⁺-conducting channels that are activated by direct binding of extracellular ATP. Mitochondrial ATP production is enhanced in activated T cells by Ca²⁺ transfer from ORAI- and IP₃R-generated Ca²⁺ microdomains to the mitochondrial matrix via the mitochondrial Ca²⁺ uniporter (MCU). ATP is then exported outside T cells by the pannexin 1 hemichannels and activates P2RX receptors to cause further Ca²⁺ entry and mitochondrial ATP production. In this regard, P2RX receptor signalling acts as a Ca²⁺ signalling enhancer for nuclear factor for activated T cells (NFAT)-mediated transcription. P2RX7 was shown to promote T helper 17 (TH17) cell differentiation and inflammation by promoting retinoic acid receptor-related orphan receptor γt (RORγt) while inhibiting forkhead box P3 (FOXP3) transcription to suppress regulatory T (Treg) cells. However, Ca²⁺ signals through ORAI1 are required for Treg cell development (for both thymic Treg cells and induced Treg cells in peripheral lymphoid organs).

Figure 5 |. CaV channels: modulators of T cell Ca²⁺ signalling.

Based on knockout of the β3 and β4 regulatory subunits of L-type Ca²⁺ channels which causes decreased expression of CaV1.2 and CaV1.1 at the plasma membrane and decreased T cell receptor (TCR)-activated Ca²⁺ entry and nuclear factor for activated T cells (NFAT) activity, CaV1.2 and CaV1.1 were proposed to play a role in TCR-mediated Ca²⁺ signalling. Although no CaV1.2 currents were reported in T cells, there is evidence of spontaneous CaV1.2 currents from channel clusters at hyperpolarized membrane potentials, as low as –90 mV, in smooth muscle cells^,. In this case, CaV1.2 activity can be sensitized by protein kinase A (PKA) and protein kinase C (PKC)-mediated phosphorylation; both enzymes can be activated downstream of store operated Ca²⁺ entry (SOCE). One intriguing hypothesis in T cells is the existence of a conformational coupling between CaV1.1 and ryanodine receptors (RYRs), similar to skeletal muscle. Voltage-activated CaV1.4 was proposed to support SOCE by mediating constitutive Ca²⁺ activity to help refill the ER Ca²⁺ stores and sustain CRAC channel activity, but these studies remain highly controversial. Voltage-activated whole-cell T-type Ca²⁺ currents mediated by CaV3.1 were recorded in T cells, generating window currents between –65 and –25mV, which are within T cell resting membrane potential. CaV3.1-mediated Ca²⁺ entry operates independently of SOCE and provides Ca²⁺ microdomains that synergize with SOCE to support NFAT activity. CaV3.1 activity is required for the expression of GM-CSF and RORγt.

Figure 6 |. Organellar Ca²⁺ channels: initiators and master orchestrators of Ca²⁺ signalling microdomains during T cell activation.

The role of inositol-1,4,5-trisphosphate (IP₃) in initiating and sustaining T cell Ca²⁺ signalling is well established. Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP ribose (cADPR) were proposed to synergize with IP₃ during T cell Ca²⁺ signalling, but their role requires further clarification. For instance, the identity of the NAADP-producing enzyme downstream TCR ligation and the accessory molecule required for NAADP action on type 1 ryanodine receptors (RYR1) remain obscure. Nevertheless, according to this model, NAADP produced during the first few seconds of T cell receptor (TCR) stimulation was proposed to represent the crucial initial trigger for T cell Ca²⁺ signalling, preceding IP₃-mediated ER Ca²⁺ release. NAADP would activate RYR1 and cause endoplasmic reticulum (ER) Ca²⁺ release, which would synergize with IP₃ to cause further Ca²⁺ release via IP₃ receptor (IP₃R). Subsequent production of cADPR and activation of RYR2 and RYR3 by cADPR would serve to sustain Ca²⁺ signalling for extended periods of time. Through a positive feedback loop, Ca²⁺ entry through ORAI channels might activate RYR isoforms to maintain store depletion and store operated Ca²⁺ entry (SOCE). Mitochondrial Ca²⁺ uptake through the mitochondrial Ca²⁺ uniporter (MCU) at the vicinity of ORAI channels maintains CRAC channel activity by relieving its Ca²⁺-dependent inhibition (CDI). Mitochondria can also buffer Ca²⁺ near plasma membrane Ca²⁺ ATPase (PMCA) pumps, thus preventing PMCA modulation, inhibiting Ca²⁺ extrusion to the outside and maintaining cytosolic Ca²⁺ signalling during T cell activation. Ca²⁺ shuttling to the outside of mitochondria by the activity of the Na+/Ca²⁺/Li+ exchanger (NCLX) provides Ca²⁺ microdomains to the sarcoplasmic/endoplasmic Ca²⁺ ATPase (SERCA) pump and serves to replenish ER Ca²⁺ levels by these mobile organelles, thus sustaining SOCE and lymphocyte activation. NAADP-mediated activation of lysosomal two pore channels (TPC) is important for secretion of cytolytic granules by cytotoxic T cells. The endolysosomal TRP mucolipin (TRPML) channels are crucial to endolysosomal function, such as endocytosis, exocytosis and autophagy, and are likely important, during T cell activation, for endocytosis of exhausted membrane proteins and replenishment of these proteins through exocytosis at the immune synapse. Purinergic ionotropic receptor P2RX4 located in endolysosomal membranes was proposed to be important for vesicular fusion, but its role in T cell endolysosomal function remains unknown. CICR, Ca²⁺-induced Ca²⁺ release.