Laura Vinnenberg^a     Stefanie Bock^a     Petra Hundehege^a     Tobias Ruck^b     Sven G. Meuth^b 

^aDepartment of Neurology, Institute of Translational Neurology, University Hospital Muenster, Muenster, Germany, ^bDepartment of Neurology, University Hospital Duesseldorf, Duesseldorf, Germany

Key Words
Tregs • Ionchannels • CRAC • Kv1.3 • KCa3.1 • TRP • P2X • VRAC

Abstract
The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca²⁺ release-activated Ca²⁺, transient receptor potential, P2X, volume-regulated anion and K⁺ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.

 

 

Introduction

Regulatory T cells (Tregs) play a central role in maintaining self‐tolerance. They suppress unwanted or excessive immune reactions by limiting the activity of effector immune cells. Defects in Treg development, stability, or the suppressive mechanisms of Tregs are associated with several autoimmune diseases, including multiple sclerosis and rheumatoid arthritis [1–3]. For these diseases, Treg frequency is a well-characterized parameter, and fluctuation patterns due to disease phase and treatment regimen have been determined [4].
Also, a rare immune dysregulation disorder (polyendocrinopathy, enteropathy, X-linked (IPEX)) is linked to mutations in forkhead box protein 3 (FoxP3), the major transcription factor of Tregs [5]. Tregs have been highlighted as critical contributors to immune tolerance breakdown in autoimmunity and represent promising therapeutic targets. Hence, novel therapies aim to strengthen Treg generation and stability [6]. However, the regulation of Tregs has its downsides. An increased Treg expansion might support pathological events since immune cell action against tumor cells is suppressed [7]. To fully exploit the therapeutic potential of Tregs, a detailed understanding of molecular mechanisms in health and disease is essential.
FoxP3 is strongly expressed and considered the most prominent marker for Tregs [8]. Tregs belong to the CD4⁺ T cell compartment and are characterized by high CD25 and low CD127 expression in humans Treg. Thymus-derived (tTregs) and peripherally-induced Tregs (pTregs) can be distinguished, while further discrimination of Treg subpopulations is controversial [9]. tTregs have a high affinity for their antigen and are mainly involved in self-tolerance. pTregs develop from conventional T cells (Tconv) during an immune response, cooperatively contributing to immune homeostasis [10].
The suppressive mechanisms of Tregs have not been fully elucidated yet, however an interplay of different milieu adapting mechanisms is likely. On the one hand, Tregs inhibit effector immune cells by cell-cell contact via surface-bound receptors, e.g. cytotoxic T-lymphocyte antigen 4 (CTLA-4). On the other hand, Tregs release messenger substances, including the anti-inflammatory cytokines interleukin 10 (IL-10) and transforming growth factor-beta (TGF-β), in response to T cell receptor (TCR) stimulation [11]. Also, a cytotoxic effect by perforin and granzyme B secretion, disruption of metabolic processes, and competition for signals from antigen-presenting cells (APC) are discussed in this context [12–14].
Far less is known about regulatory mechanisms in Tregs themselves. The crucial role of ion channels for differentiation, maintenance, and activation immune cells has been strengthened in recent years. Ion channels participate in membrane potential regulation, calcium signaling pathways, and effector functions of immune cells [15, 16]. Furthermore, the membrane potential of CD4⁺ T cells is a subset-specific feature. Different subpopulations are characterized by specific membrane potentials that might be based on subset-specific ion channel expression or activity [17]. Understanding the distinct role of those ion channels might help to elucidate Treg regulatory mechanisms. Moreover, the dynamic pharmacological modulation of Treg differentiation and function by targeting ion channels might open new therapeutic avenues.
Therefore, the present review deals with the critical role of ion channels in controlling Treg lineage identity, regulation, and effector function. We summarize the literature focusing on gene-targeted mice and channel modulators. The role of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel in Ca²⁺ regulation and the significant contribution of the K⁺ channels Kv1.3 and KCa3.1 to the membrane potential are discussed. Moreover, we describe how transient receptor potential (TRP), anion (VRAC), and P2X channels affect Treg function.


Ca²⁺ channels

CRAC channels
Calcium is known as an essential second messenger for the development and function of immune cells and is engaged in Treg differentiation. The depletion of intracellular calcium stores entails store-operated calcium entry (SOCE). The stromal interaction molecule (STIM1) is located in the membrane of the endoplasmic reticulum (ER) and senses intraluminal calcium. Under ER calcium depletion, STIM1 relocates to the plasma membrane and activates CRAC channels. The pore-forming subunit Orai, with the three known homologs Orai1, Orai2, and Orai3, mediates robust and sustained Ca²⁺ influx in human T cells.
Human T cell subtypes show small but consistent differences in their Ca²⁺ signatures. Interestingly, the most prominent SOCE response was detected for human Tregs [18, 19]. Human Tregs exhibit lower expression levels of Orai2 compared to Tconv [18]. Surprisingly, a correlation between low Orai2 expression levels and increased SOCE magnitude was independently confirmed: both downregulation of Orai2 in Jurkat T cells and murine genetic Orai2 deletion resulted in enhanced SOCE [20, 21]. In contrast, Orai1 deficiency led to plummeting SOCE and CRAC currents in mice [21]. However, the overall importance of Orai1 and Orai2 is controversial. It was observed that Orai1 and Orai2 compete for limited STIM1 molecules essential for activation under physiological conditions, and Orai2–STIM1 complexes conduct much smaller currents compared to Orai1–STIM1 complexes [22, 23]. Nevertheless, Treg functions of Orai1 and STIM1 deficient mice were only moderately impaired, indicating that the remaining Ca²⁺ influx, mediated by Orai2/3 and STIM2, can compensate for this defect [24]. Also, combined (but not individual) deletion of Orai1 and Orai2 was reflected in decreased Treg cell numbers in mice [21]. Moreover, STIM1/2 deletion resulted in impaired Treg frequencies and reduced the suppressive function [25, 26]. The abolished Ca²⁺ signal prevented differentiation into effector, tissue-resident, and follicular Tregs [26–28]. This defect was associated with both impaired IL-2 sensing and nuclear translocation of the transcription factors NFAT and NF-kB, both necessary for Treg development, stability and function [25, 27, 29, 30]. However, various other target genes, molecules, and pathways are also regulated by SOCE [31–33]. Deficiency of STIM1 and Orai1 in humans was associated with severe combined immune deficiency (SCID), autoimmunity, and reduced Treg numbers [34, 35].
These data highlight the essential role of the CRAC channel-controlled transcriptional network affecting Treg differentiation, expansion, and homeostasis. Yet, the underlying molecular mechanisms are not fully understood. Interestingly, inhibition of Calcineurin potentially blocked NFAT mediated T cell proliferation and FoxP3 expression in tTregs and pTregs. In contrast, the CRAC channel inhibitor ORAIci only affected pTregs. This way suggesting the existence of an independent Ca²⁺ -permissive channel in tTregs, that might compensate for abolished CRAC signaling [24]. Thus, the Treg Ca²⁺ signature might not be solely dependent on CRAC genes, which gives evidence for other yet unidentified channels such as TRPC3 or TRPM4 in Tregs [18].


K⁺ channels

K⁺ channels mediate continuous efflux of K⁺ ions and thus hyperpolarize the membrane potential. The best characterized K⁺ channels, predominantly controlling the membrane potential in Tregs, are the voltage-activated K⁺ channel Kv1.3 and the Ca²⁺ activated K⁺ channel KCa3.1 (or KCNN4, IKCa²⁺, SK4). The Kv1.3 channel is a homotetramer, and each α-subunit comprises six transmembrane domains (S1–S6). The voltage sensor in S4 detects depolarization, followed by a conformational change that opens the channel [36, 37]. In contrast, the tetrameric KCa3.1 channel is sensitive to intracellular Ca²⁺ concentration ([Ca²⁺]_i) changes [38]. Ca²⁺ responsivity is imparted by calmodulin, which is constitutively bound to the KCa3.1 channel. Upon Ca²⁺ binding, calmodulin induces conformational changes and evokes channel opening [39].
By regulating the membrane potential, both channels set the threshold for calcium influx and are involved in antigenic activation and proliferation of T cells [15, 40]. Interestingly, Kv1.3 and KCa3.1 are expressed at different levels in T cell subtypes and specific states of activation. This expression pattern indicates adaption mechanisms to highly specialized tasks in immune response regulation.
However, study results are controversial and partly contradictory. For example, high Kv1.3 channel activity has been implicated in the pathogenesis of autoimmune diseases, including multiple sclerosis, type-1 diabetes mellitus, and rheumatoid arthritis [41, 42]. Comparative expression analysis revealed that Tregs of MS patients express fewer Kv1.3 channels than naive cells, while this difference was not found in healthy donors [43]. Accordingly, modulation of Kv1.3 supports a protective channel function: upregulation resulted in boosted Treg proliferation, and experiments with the high-affinity Kv1.3 antagonist eplerenone revealed diminished TGF-β and IL-10 secretion in treated Tregs [44]. Furthermore, inhibition of Kv1.3 in human lymphocytes led to decreased Ca²⁺ entry with larger defects monitored in CD4⁺ than CD8⁺ T cells [45]. This indicates Kv1.3 as a selective target in CD4⁺ T cells.
In contrast to Kv1.3, inhibition of the KCa3.1 channel reduced the Ca²⁺ influx in CD4⁺ T cells to a lower extent than in CD8⁺ T cells [45]. KCa3.1 channel activity was shown to be necessary for TCR-stimulated Ca²⁺ influx, and differential expression patterns are known. Thus, only in the course of activation, T cells upregulate KCa3.1 channel expression [46]. Interestingly, dominant KCa3.1 expression was monitored in T helper 1 (Th1) cells, and their action is closely associated with autoimmunity [47, 48]. Genetic KCa3.1 depletion did not reduce the beneficial function of Tregs in mice. To this end, KCa3.1 antagonists have therapeutic potential for inhibiting autoimmune-inducing Th1, while KCa3.1 inhibition does not impair Treg function [45, 49].


Other channels

P2X purinoreceptor channels
The P2X family of ionotropic, adenosine triphosphate (ATP) gated cation channels comprises seven subunits (P2X1–7), and, interestingly, P2X7 is expressed by most cells of the immune system [50]. Binding of extracellular ATP causes a conformational change in P2X purinoreceptor channels and allows the flow of Ca²⁺, Na⁺, and K⁺ ions [51, 52]. While the inwardly rectifying current flows, the membrane depolarizes and induces additional Ca²⁺ influx via voltage gated Ca²⁺ channel and thus, NFAT activation and IL-2 synthesis in T cells [53]. T cells respond to ATP secreted by other cells, and, also, an autocrine secretion of ATP via pannexin-1 hemichannels occurs [54, 55]. This control mechanism might ensure Ca²⁺ signaling by fortifying weak TCR signals [56].
The contribution of P2X7 to Treg stability and function was investigated using (ant)agonists and knockout mice. P2rx7, the gene encoding the P2X7 receptor, is a Treg signature gene, and it has been postulated that P2X7 triggers ATP-mediated cell death in Tregs [57, 58]. Increased P2X7 stimulation with BzATP of Tregs was shown to diminish FoxP3 expression but increased levels of Th17 specific transcription factors [59]. By mediating Treg instability and conversion to Th17 cells, ATP/P2X7 signaling appeared to be proinflammatory [60]. Furthermore, the potential to activate the inflammasome was reported [61]. Compared to Tconv, Tregs produced substantially lower amounts of ATP after TCR stimulation, and the ectonucleotidases CD39 and CD73 converted ATP to adenosine, a suppressor of leukocyte activation [54, 62]. In contrast, ATP also revealed anti-inflammatory properties in some experiments. The suppressive activity of Tregs was enhanced under ATP exposure, and P2X7 receptor inhibition diminished suppressor function [63]. In T cells, P2X1 and P2X4 were implicated in Ca²⁺ influx, but only a few experiments investigated their role in Treg function [55]. P2X1 inhibition of Tregs with NF-449 did not affect activation or suppressive capacity [64].
The influence of P2X channels on the developmental and immunosuppressive program of Tregs needs further investigation to uncover the underlying molecular mechanisms and to unveil the connections between different study outcomes. Although examining all isoforms (P2X1–7) seems worthwhile, a specific role of P2X7 in Treg is likely.

TRP channels
The superfamily of TRP channels comprises integral membrane proteins that function as ion channels. TRP channels are widely expressed non-selective cation permeable channels acting as polymodal sensors and are involved in many physiological and pathological pathways. Based on their protein homology and domain structure, six TRP subfamilies were classified in mammals, including TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin) [65, 66]. The knowledge of TRP functions in immune cells has been continuously expanded in recent years. For example, TRPM4 mediated V_m depolarization prevents Ca²⁺ overload in response to SOCE [67].
Moreover, the TRP channel contribution to cytokine production was detected [68, 69]. Although the knowledge on TRP channel function in immune cells is growing, further detailed research is required.
The specified role of TRP channels in subpopulations such as Tregs is of interest. Genetic Trpm4^-/- in mice did not alter the number of Tregs, expression levels of activation markers (CD25, CD69), or cytokine production [70]. In contrast, initial evidence for an important role of TRPC5 in the suppressive function of Tregs in autoimmunity was provided. Treg mediated TRPC5 activity was identified as a mechanism potentially contributing to suppression of experimental autoimmune encephalomyelitis in knockout mice lacking gangliotetraose gangliosides [71–73]. Also, the TRPM7 channel was identified as a promising target in inflammatory disorders, e.g., graft-versus-host disease (GvHD). TRPM7 is a bifunctional protein operating as a cation channel and serine-threonine kinase [74]. Trpm7^-/- mice are embryonically lethal, but modulating the enzymatic activity might harbor pharmacological potential [75]. Inactivation of its catalytic activity via K1646R mutation in mice did not affect differentiation of Tregs but reduced differentiation of proinflammatory Th17 [76]. To this end, identifying TRPM7 kinase substrates and inhibitors represents a promising strategy to treat inflammatory disorders.

Volume-regulated anion channel (VRAC)
To compensate intra- and extracellular osmotic variability, cells adapt their cell volume. Within this process, the volume-regulated anion channel (VRAC) was identified as a key player. VRAC is ubiquitously expressed in vertebrate cells, nearly inactive under resting conditions, and activated by hypotonic swelling. VRAC mediated Cl^- efflux provokes the release of osmotically absorbed water and thereby counteracts swelling [77–79]. Using a genome-wide screening of small interfering RNA, the leucine-rich repeat-containing protein 8A (LRRC8A alias SWELL1) was identified as an essential, potentially pore-forming component of the heterooligomeric VRAC [80, 81]. Characterization revealed increased expression of LRRC8A on T cells compared to other immune cells, and Lrrc8a^-/- mice gave evidence for the special functioning of LRRC8A in Tregs. While the number of Tconv was reduced, Treg number and density were increased [82]. However, the molecular mechanism leading to this remarkable difference was not uncovered so far.


Conclusion

The study of ion homeostasis in Tregs and its significance for their protective function has received rising attention in recent years (Fig. 1). The participation of CRAC, Kv1.3, KCa3.1, and VRAC channels in Treg signaling have already been reported. Nevertheless, the detailed mechanisms and differentiation from other immune cells are not fully understood. Most studies have focused on CRAC mediated Ca²⁺ signaling, while there is growing evidence for a specialized contribution of voltage-dependent Ca²⁺ and K⁺ channels and the Ca²⁺ extrusion pump PMCA [83, 84]. Next to the already elucidated channels, Tregs are named as further candidates in several reviews dealing with ion channels, known channelopathies, and associated diseases in all immune cells (Table 1) [85, 86]. However, in comparison to conventional T cells (~-47mV), Tregs reveal a hyperpolarized membrane potential around -70 mV, suggesting additional or altered ion fluxes [11]. As the resting membrane potential of a cell is due to the outward diffusion of potassium ions, the closer examination of voltage- and calcium-gated K2P channels (e.g., TRESK and TASK subfamilies) in Tregs could be worthwhile [87]. Accordingly, an in-silico model of human T cell electrophysiological behavior takes the contribution of further ion channels into account and highlights their impact on physiological and pathological conditions [88]. In this context, the importance of locally restricted ion channels and redistribution depending on the activation status was reported [89–91]. Hence, ion channel modulation in Tregs has a substantial therapeutic potential for autoimmune diseases. Identifying ion channel distribution patterns and potent (ant)agonists is of outstanding relevance for further studies.
Last but not least, little attention has been paid to H⁺ mediated regulation of pH and the roles of Mg²⁺ and Zn²⁺ as cofactors and second messengers. This indicates that research on the impact of diverse ion channels on Treg function is just at a starting point, and major challenges remain. Also, understanding specific ion channel expression frequencies and patterns in Tregs represents a powerful approach to gain new insights on immunological tolerance and pathophysiological participation in immune diseases.

Acknowledgements

Author Contributions
S.M. and P.H. devised the main concept and structure of the review. L.V. collected relevant papers to summarize, interpret and discuss the current state of research regarding the impact of diverse ion channels on regulatory T cell functions. L.V. wrote the manuscript with support of critical input from S.M. and P.H. All authors critically revised the paper and certified the final version of the paper.

Funding
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, GRK 2515, “Chemical biology of ion channels”).

 

Disclosure Statement

The authors have no conflicts of interest to declare.

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