Near-infrared photoactivatable control of Ca²⁺ signaling and optogenetic immunomodulation

Optogenetics is a technique that has been used to study nerve cells for several years. It involves genetically engineering these cells to produce proteins from light-sensitive bacteria, and results in nerve cells that will either send, or stop sending, nerve impulses when they are exposed to a particular color of light. Neuroscientists have learned a lot about brain circuits using the technique, and now researchers in many other fields are giving it a try.

There are, however, several challenges to using optogenetics in other types of cells. Nerve cells create a tiny electrical impulses when they are activated, which helps them quickly transmit messages. But other types of cells use more diverse means to communicate and transmit signals. This means that optogenetics techniques must be adapted. Additionally, many cells are located deep in the body and so getting the light to them can be difficult.

He, Zhang et al. have now developed an optogenetic system (termed “Opto-CRAC”) that can control immune cells buried deep in tissue. The action of immune cells can be tuned by controlling the flow of calcium ions through gate-like proteins in their membranes. He, Zhang et al. genetically engineered immune cells so that a calcium gate-controlling protein became light sensitive. When the cells were exposed to a blue light the calcium ion gates opened. When the light was turned off, the gates closed. More intense light caused more calcium to enter into the cells. Further experiments then revealed that exposing these engineered immune cells to blue light in the laboratory could trigger an immune response.

The next obstacle was getting light to immune cells in a live animal. So, He, Zhang et al. used specific nanoparticles that have been shown to help transmit light deep within tissue. In these experiments, mice were injected with the light-sensitive immune cells and the nanoparticles. Then, a near-infrared laser beam that can transmit into the tissues was pointed at the mice. This caused calcium channels to open in the engineered cells deep in the mice. Finally, further experiments were used to show that this light-based stimulation could boost an immune response to aid the killing of cancer cells. Other scientists will likely use the technique to help them study immune, heart, and other types of cells that use calcium to communicate.

Microbial opsin-based optogenetic technologies have been widely adopted to modulate neural activity (Fenno et al., 2011), but similar tools tailored for utilization in non-excitable tissues (e.g., the immune and hematopoietic system) are still limited. The application of channelrhodopsin (ChR)-based optogenetic tools is limited by the lack of ion selectivity and the inability to extend the spectral sensitivity into the near-infrared (NIR) range (Fenno et al., 2011). Here we present a tissue penetrable near infrared-stimulable optogenetic platform (termed 'Opto-CRAC') that can be used to reversibly photo-manipulate Ca²⁺ influx through one of the most Ca²⁺-selective ion channels, the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, which is abundantly present in most non-excitable cells (Hogan et al., 2010; Prakriya and Lewis, 2015). Our tool is based on the engineering of light sensitivity into the CRAC channel and its subsequent coupling to lanthanide-doped upconversion nanoparticles (UCNP), the latter of which act as nanotransducers to convert tissue penetrable NIR light into visible light emission (Shen et al., 2013; Chen et al., 2014). We demonstrate that Opto-CRAC tools can be applied to remotely control Ca²⁺ influx and generate repetitive Ca²⁺ oscillations, photo-tune Ca²⁺-dependent gene expression, and modulate a myriad of Ca²⁺-dependent activities in cells of the immune system, including effector T cell activation, macrophage-mediated inflammasome activation, dendritic cells (DC) maturation and antigen presentation. Our study set the stage for achieving the goal of remote optogenetic immunomodulation and spatiotemporal control over cellular immunotherapy in a wireless manner.

Following antigen presentation, T cell receptor (TCR) engagement triggers a cascade of signaling events in T lymphocytes that elicit the influx of extracellular Ca²⁺ through the CRAC channel, a classic example of store operated Ca²⁺ entry (SOCE) (Hogan et al., 2010; Prakriya and Lewis, 2015). The molecular choreography of SOCE is mainly coordinated by two proteins that are located in distinct cellular compartments: (i) ORAI1, a four-pass transmembrane protein that constitutes the CRAC channel pore-forming subunit in the plasma membrane (PM); and (ii) the stromal interaction molecule 1 (STIM1), an ER-resident Ca²⁺ sensor protein that is responsible for sensing ER Ca²⁺ depletion and directly gating ORAI1 channels through its cytosolic domain (STIM1-CT). Store depletion induced Ca²⁺ influx through CRAC channels further activates calcineurin, a downstream Ca²⁺-dependent phosphatase that dephosphorylates the master transcriptional regulator NFAT (nuclear factor of activated T cells) and subsequently causes NFAT nuclear translocation (Müller and Rao, 2010). In the presence of the co-stimulatory pathway, which activates the activator protein 1 (AP-1), NFAT cooperates with AP-1 to turn on genes (e.g., IL-2 and IFN-γ) that are characteristic of a productive immune response (Müller and Rao, 2010). To enable light control over the Ca²⁺/NFAT pathway, we set out to install light sensitivity into STIM1 by fusing a handful of STIM1-CT fragments with the genetically-encoded photoswitch LOV2 (light, oxygen, voltage) domain (residues 404–546) of Avena sativa phototropin 1 (Christie et al., 1999; Harper, 2003; Yao et al., 2008; Wu et al., 2009) (Figure 1a and Figure 1—figure supplement 1). When expressed alone, these STIM1-CT fragments are capable of eliciting varying degrees of constitutive activation of ORAI1 channels to mediate Ca²⁺ entry from the extracellular space to the cytosol (Yuan et al., 2009; Park et al., 2009; Zhou et al., 2010a; Soboloff et al., 2012). In the dark, the C-terminal Jα helix docks to the LOV2 domain (Harper, 2003; Yao et al., 2008; Wu et al., 2009) and keeps the ORAI1-activating STIM1-CT fragments quiescent. Upon blue light illumination, photoexcitation generates a covalent adduct between LOV2 residue C450 and the cofactor FMN (Figure 1—figure supplement 1d), thereby promoting the undocking and unwinding of the Jα helix to expose the STIM1-CT fragments. Unleashed STIM1-CT fragments further move toward the plasma membrane to directly engage and activate ORAI1 Ca²⁺ channels (Figure 1a,b).

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We first created a series of Opto-CRAC constructs by varying the length of STIM1-CT fragments, introducing mutations into the LOV2 domain and optimizing the linker between these two moieties (Figure 1—figure supplement 1a). After an initial screen of approximately 100 constructs using NFAT nuclear translocation and Ca²⁺ influx as readouts, we decided to use the LOV2-STIM1_336-486 chimera (designated as 'LOVSoc') in our following experiments because it showed no discernible dark activity and exhibited the highest dynamic range in terms of evoking light-inducible Ca²⁺ influx (Figure 1—figure supplement 1a,b). When expressed as an mCherry-tagged fusion protein in HEK293-ORAI1 stable cells, LOVSoc underwent rapid translocation between the cytosol and the PM in response to blue light illumination (t_1/2,on = 6.8 ± 2.3 s; t_1/2,off = 28.7 ± 6.5 s; Figure 1b and Video 1). This process could be readily reversed by switching the light off, and could be repeated multiple times without significant loss in the magnitude of response. The light-dependent association between LOVSoc and ORAI1 or ORAI1 C-terminus (ORAI1-CT) was further confirmed by a pulldown assay using purified recombinant proteins and by coimmunoprecipitation assays (Figure 1—figure supplement 2). In mammalian cells expressing LOVSoc, the degree of Ca²⁺ influx could be tuned by varying the light power densities (Figure 1—figure supplement 3a). After photostimulation for 1 min with a power density of 40 μW/mm² at 470 nm, LOVSoc triggered significant yet varied elevation of cytosolic Ca²⁺ concentrations to approximately 500–800 nM in a dozen of mammalian cell types derived from various non-excitable tissues (Figure 1—figure supplement 3b), likely owing to the varied endogenous levels of ORAI proteins among the tested cells. A Light-triggered global Ca²⁺ influx and oscillations in HeLa or HEK293T cells expressing mCherry-LOVSoc could be monitored in real-time by either Fura-2 (Figure 1—figure supplement 3c) or genetically-encoded Ca²⁺ indicators (GECIs), including GCaMP6 (Figure 1c and Videos 2,3) (Chen et al., 2013), R-CaMP2 (Figure 1—figure supplement 3d) (Inoue et al., 2015), and R-GECO1.2 (Figure 1d and Figure 1—figure supplement 3e) (Wu et al., 2013). Notably, localized light stimulation can be applied to achieve local activation of Ca²⁺ influx at a defined spatial resolution (Figure 1—figure supplement 4 and Video 4), thereby providing a new approach to dissect the effect of Ca²⁺ microdomains in various biological processes (Parekh, 2008). Depending on the kinetic properties of the Ca²⁺ indicators used, the half-life time of the cytosolic Ca²⁺ rise in response to light stimulation ranged from 23 s to 36 s. After switching off the light, the cytosolic Ca²⁺ signal decayed with a half-life time of approximately 25–35 s (Figure 1—figure supplement 3f). These values are largely in agreement with the time scale of SOCE under physiological stimulation (Hogan et al., 2010; Prakriya and Lewis, 2015; Soboloff et al., 2012). We further measured the photo-activated currents by whole-cell recording in HEK293 cells stably expressing ORAI1 (Figure 1e). Following light stimulation, HEK293 cell transfected with LOVSoc developed a typical inward rectifying current, which is characteristic of the CRAC channel and distinct from the greater outward currents of non-selective cation channels such as TRPC (Prakriya and Lewis, 2015). Substitution of the most abundant extracellular cation Na⁺ by a non-permeant ion NMDG⁺ did not alter the amplitude or overall shape of the CRAC current, implying that Na⁺ has negligible contribution to LOVSoc- mediated photoactivatable Ca²⁺-selective CRAC currents.

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To confer more flexibility to the Opto-CRAC system with varied optical sensitivity, we explored the use of co-expression, membrane tethering or fusion strategies to generate five more variants of Opto-CRAC (Figure 1—figure supplement 5). We used either an internal ribosome entry site (IRES)-based bicistronic vector or a self-cleaving 2A peptide strategy (de Felipe et al., 2006) to enable the coexpression of ORAI1 and LOVSoc in the same cell with a single vector. Compared to LOVSoc alone, both co-expression systems resulted in ~1.4-fold increase in Ca²⁺ response (Figure 1—figure supplement 5a). Tethering LOVSoc to the plasma membrane (PM) with an N-terminal PM-targeting sequence derived from the Src kinase Lyn (Inoue et al., 2005) (Lyn11-LOVSoc) expedited the photoactivation process by 3.5-fold (Figure 1—figure supplement 5b), presumably owing to its increased local concentration and much closer proximity to the ORAI1 channels. By contrast, a concatemeric form of LOVSoc with two copies covalently connected in a single polypeptide or its fusion to ORAI1 substantially slowed down photoactivatable Ca²⁺ influx (Figure 1—figure supplement 5c). Collectively, we have created a set of Opto-CRAC constructs that meet the varying needs on sensitivity and photoactivation kinetics (Figure 1—figure supplement 5d,e).

We next asked if we could manipulate the light pulse to generate diverse temporal patterns of Ca²⁺ signals to tune the degree of NFAT activation, which would be reflected in the efficiency of NFAT nuclear translocation and NFAT-dependent luciferase expression. We applied a fixed light pulse of 30 s while varying the interpulse intervals from 0.5 to 4 min to generate Ca²⁺ oscillation patterns with defined temporal resolution (Figure 1d and Figure 1—figure supplement 6) and compared the levels of NFAT activation in HeLa cells. As shown in Figure 1f prolonged interpulse interval was largely accompanied by a decrease in the nuclear accumulation of NFAT. This observation agrees well with previous reports showing that higher Ca²⁺ oscillation frequencies, or faster repetitive Ca²⁺ pulses, tend to increase the ability to activate NFAT (Lewis et al., 1998). Thus, we have demonstrated that the engineered Opto-CRAC tools are able to achieve remote and photo-tunable activation of NFAT in mammalian cells (Figure 1f and Video 5). We further confirmed the NFAT-dependent gene expression in HeLa cells transfected with an NFAT-driven luciferase (NFAT-Luc) reporter construct. In the presence of the co-stimulatory pathway (mimicked by the addition of the pleiotropic PKC activator PMA), light illumination led to a robust increase in luciferase gene expression (Figure 2a). A decrease in the light pulse frequency also caused a reduction in the efficiency of Ca²⁺/NFAT-driven luciferase expression (Figure 1f). To obviate the use of carcinogenic PMA to photo-trigger gene expression, we also introduced a synthetic 5’ transcription regulatory region upstream of gene Ins1 (Stanley et al., 2012), which contains a furin cleavage site that allows insulin processing in non-beta cells such as HEK293 cells (Shifrin et al., 2001). The 5’ region is composed of three Ca²⁺-responsive elements in cis, including 2–3 copies of serum response elements (SRE), cAMP response elements (CRE) and NFAT response elements with a minimal promoter. Upon light stimulation, we observed a robust production of insulin in cells transfected with LOVSoc, but not in those without LOVSoc expression (Figure 2a).

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In order to confirm light-inducible gene expression in a more physiologically relevant system, we retrovirally transduced the mCherry-tagged LOVSoc construct into naïve CD4⁺ T cells isolated from mice (Figure 2b and Figure 2—figure supplement 1). We then compared the expression levels of two signature genes that are characteristic of activated CD4⁺ T cells (IL-2 and IFN-γ), in the presence or absence of light illumination, using qRT-PCR and ELISA (Figure 2b). Again, in the presence of PMA, light stimulation faithfully mimicked ionomycin-induced effects on the Ca²⁺/NFAT pathway and remarkably boosted the cytokine production by over 15–30 fold in CD4⁺ T cells transduced with mCh-LOVSoc. By contrast, control cells transduced with the mock retrovirus failed to exhibit light-dependent production of cytokines (Figure 2—figure supplement 1). In addition to its well-established role in driving effector T cell activation, intracellular Ca²⁺ immobilization in macrophage is critical for the activation of the NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) inflammasome (Murakami et al., 2012; Lee et al., 2012; Horng, 2014), which is accompanied by the release of processed caspase-1 (p20 subunit) and the proinflammatory cytokine IL-1β into culture supernatants (Figure 2c). Following photostimulation at 5 or 50 μW/mm², we observed a notable light intensity-dependent boost in the production of IL-1β and processed caspase -1 (p20 subunit) in lipopolysaccharide (LPS)-primed THP1-derived macrophages in the presence of a commonly used inflammasome inducer nigericin (Figure 2c), thus confirming the feasibility of harnessing the power of light to amplify macrophage-mediated inflammatory responses ex vivo. In aggregate, light-induced activation of the Opto-CRAC channel can generate both global and local Ca²⁺ signals and subsequently cause hallmark physiological responses in both model cellular systems (e.g., HeLa or HEK293 cells) and rodent or human cells of the immune system.

One fundamental roadblock that hampers the application of optogenetic tools in vivo is their inability to stimulate deep within tissues without the use of invasive indwelling fiber optic probes. In order to seek the possibility of controlling the Ca²⁺/NFAT pathway using light in the deep tissue penetrating near-infrared range, we explored the use of lanthanide-doped upconversion nanoparticles (UCNPs) as the NIR light transducer (Sun et al., 2015; Gnach et al., 2015; Wu et al., 2015). Our UCNPs proved to be highly photostable, and their unique upconversion (NIR excitation and emission at visible light range) properties make them an ideal for the remote photoactivation of Opto-CRAC channel activities (Wu et al., 2009; Ostrowski et al., 2012). In order to match the absorption window of LOV2, we chose mono-dispersed 40-nm β-NaYF₄: Yb, Tm@β-NaYF₄ UCNPs (Figure 3—figure supplement 1) that exhibit bright blue emission upon 980 nm CW laser irradiation. When excited at 980 nm, the synthesized UCNPs displayed a sharp emission peak centered around 470 nm (Figure 3a). Like direct blue light illumination, UCNPs were able to cause photoactivation of recombinant LOV2 proteins, as reflected by the absorbance changes following NIR light stimulation and the subsequent recovery to the dark state (Figure 3b). This finding clearly validates the feasibility of shifting the spectral sensitivity toward the NIR window to activate LOV2-based optogenetic tools.

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In order to effectively and specifically illuminate the LOV2-based optogenetic construct in a cellular context, we first developed streptavidin-conjugated UCNPs, then engineered a genetically-encoded streptavidin-binding tag (StrepTag) into the second extracellular loop of the ORAI1 Ca²⁺ channel (mCh-ORAI1^StrepTag, Figure 3c) and assessed its capability to recruit streptavidin-conjugated UCNPs (UCNPs-Stv, Figure 3—figure supplement 1). In HeLa cells expressing mCh-ORAI1-StrepTag, we detected remarkable local accumulation of UCNPs-Stv on the plasma membrane (Figure 3c), confirming the cell-specific targeting of functionalized nanoparticles. To examine whether UCNPs-transduced blue light is sufficient to trigger the opening of Opto-CRAC channels, we monitored cytosolic Ca²⁺ changes using GCaMP6s in HeLa cells co-expressing LOVSoc, mCh-ORAI1-StrepTag and GCaMP6s following NIR light stimulation (980 nm). Within 20 s, transfected HeLa cells exhibited a significant increase in GCaMP6s fluorescence, indicating a rapid rise in the intracellular Ca²⁺ concentration that was evoked by NIR light (Figure 3d). This was further confirmed by using a red-emitting Ca²⁺ indicator R-GECO1.2, which enabled recording of reversible Ca²⁺ fluctuation cycles and circumvented the complications associated with potential direct activation of LOVSoc by the green light source used to excite GCaMP6 signals (Figure 3e). This increase was found to be caused by Ca²⁺ influx through NIR-to-blue activated Opto-CRAC channels because cells incubated with the control NIR-to-green UCNPs (β-NaYF₄: Yb, 2% Er @ β-NaYF₄; emission maxima at 510 nm) did not show discernible changes in the GCaMP6s signal upon stimulation with the same NIR light (Figure 3—figure supplement 2). We then employed NIR light to remotely activate the downstream effector NFAT at the cellular level, and observed NFAT nuclear translocation (Figure 3d), as well as NFAT-dependent IFN-γ production in CD4⁺ T lymphocytes (Figure 3f). Next, we sought to demonstrate the potential application of NIR-triggered activation of the Opto-CRAC system in vivo. We performed a proof-of-principle experiment by implanting NFAT-Luc/LOVSoc expressing HeLa cells pre-incubated with UCNPs-Stv subcutaneously in the flanks of mice. The implanted site was irradiated by a 980-nm CW laser outside the body (Figure 3e) without noticeable heat production (Figure 3—figure supplement 3a,b) or severe damage to local tissues (Figure 3—figure supplement 3c). Luciferase-catalyzed bioluminescence was readily detected after NIR irradiation, whereas no discernible background activation was observed in the negative controls where LOVSoc expression and/or NIR light were absent (Figure 3g).

To explore the application of the NIR Opto-CRAC system in a more disease-relevant context, we set out to combine the use of our optogenetic system with DC-mediated immunotherapy in the B16-OVA mouse model of melanoma (Briles and Kornfeld, 1978; Fidler, 1975), in which ovalbumin (OVA) (Falo et al., 1995; Mayordomo et al., 1995) is used as a surrogate tumor antigen (Figure 4a). Dendritic cells, which provide the essential link between the innate and adaptive immune responses, are adept at capturing tumor antigens and cross-presenting these antigens to T cells in tumor draining lymph nodes (dLNs), thereby sensitizing and generating tumor-specific cytotoxic lymphocytes (CTLs) to cause tumor regression or rejection (Palucka and Banchereau, 2012). One of the major challenges of DC vaccination-based immunotherapy is how to efficiently maintain the maturational status of DCs. Pharmacological agents (e.g., ionomycin) or signaling pathways controlling intracellular Ca²⁺ mobilization have been reported to facilitate immature dendritic cell maturation through up-regulation of co-stimulatory molecules CD80 or CD86, major histocompatibility complex (MHC) class I and class II, as well as the chemokine receptor CCR7 (Félix et al., 2013; Matzner et al., 2008; Hsu et al., 2001; Koski et al., 1999; Czerniecki, 1997). We hypothesize that photoactivatable Ca²⁺ influx in DCs will lead to similar phenotypic changes to expedite and sustain DC maturation and promote antigen presentation, thereby maximally sensitizing T lymphocytes toward tumor antigens to boost anti-tumor immune response. To quickly test this in vitro, we transduced bone marrow-derived DCs (BMDCs) with retroviruses encoding both LOVSoc and ORAI1^StrepTag (termed 'Opto-CRAC DCs'), pulsed cells with a mixture of OVAp (₂₅₇SIINFEKL₂₆₄) and UCNPs-Stv nanoparticles. NIR light stimulation resulted in approximately 2–8 fold increase in the surface expression of MHC-I/II, CD86, and CCR7 (Figure 4b), which are characteristic of matured DCs that are capable of homing to dLNs to interact with T cells to modulate adaptive immune response (Palucka and Banchereau, 2012). We next used ex vivo cross-presentation assay to examine how CD8 T cells from OT-1 Rag1^-/- mice respond to the OVA antigen presented by DCs. The isolated OT-1 CD8 T cells, bearing transgenic T cell receptors that specifically recognize processed OVA peptides (Clarke et al., 2000; Hogquist et al., 1994), were co-cultured with Opto-CRAC DCs in the presence of OVAp and UCNPs-Stv. After NIR stimulation, co-cultured OT-1 CD8 T cells exhibited over 2-fold increase in both proliferation (Figure 4c) and IFN-γ release (Figure 4d), clearly attesting to the feasibility of using the NIR-stimulable Opto-CRAC system to expand and photo-prime antigen-specific T cells.

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To further validate the immunomodulatory function in vivo, we injected UCNPs-Stv/OVA loaded Opto-CRAC DCs to the B16-OVA murine model of melanoma (Falo et al., 1995; Mayordomo et al., 1995), in which the B16 tumor cells bearing the OVA antigen could be readily recognized by OT-1 CD8 T cells to elicit anti-tumor immune responses (Matzner et al., 2008; Hsu et al., 2001). We next adoptively transferred CFSE-abled, OVA-specific OT-I CD8 T cells into the B16-OVA mice and examined their in vivo activation and phenotypic profiles following photoactivatable DC maturation. Compared to the control group shielded from NIR, the proliferation of CD8 T cells was substantially up-regulated after light stimulation, by judging from decreased CFSE staining due to proliferative dilution and increased population of OT-1 CD8T cells in tumor draining LNs and spleens (Figure 4e). To assess the functional consequence of immunosensitization of tumor cells toward Opto-CRAC DC-activated immune response, we monitored the tumor growth in mouse melanoma models generated by either subcutaneous or i.v. injection of B16-OVA melanoma cells (Figure 4a). NIR light stimulation significantly suppressed the tumor growth with diminished tumor volume (Figure 4f) or reduced numbers of tumor foci in the lungs (Figure 4g). Both our ex vivo and in vivo data converge to support the conclusion that NIR-stimulable Opto-CRAC DC can robustly enhance tumor cell susceptibility to CTL-mediated killing, thereby improving antigen-specific immune responses to selectively destruct tumor cells. By acting as a genetically-encoded 'photoactivatable adjuvant', the Opto-CRAC system may hold high potential for its future use in cancer immunotherapy.

In the present study, we described an NIR-stimulable optogenetic platform based on engineered CRAC channels and lanthanide-doped upconversion nanoparticles. Depending on the pulse and intensity of light input, the photosensitive module, LOVSoc, can reversibly generate both sustained and oscillatory Ca²⁺ signals. The magnitude and kinetics of photo-activated Ca²⁺ influx largely mimic the physiological responses following engagement of immunoreceptors or ligand binding to its cognate membrane receptors that leads to store depletion (Prakriya and Lewis, 2015). Ectopic expression of a single component of LOVSoc at endogenous levels of ORAI is sufficient to elicit strong intracellular Ca²⁺ elevation in a dozen of cell types derived from a wide range of human or rodent tissues. Most critically, light-generated Ca²⁺ signals can further lead to hallmark physiological responses in cells of the immune system. The sensitivity and photoactivation kinetics of this system can be further tuned by tethering LOVSoc to PM or through co-expression and fusion with ORAI1. When paired with deep tissue-penetrant and NIR-stimulable UCNPs, we have successfully demonstrated the potential application of our Opto-CRAC system to drive Ca²⁺-dependent gene expression and to photo-modulate immune response both in vivo and ex vivo. Compared to other existing optical tools, our Opto-CRAC system that has several distinctive features: First, complementary to the existing ChR-based tools that exhibit less stringent ion selectivity and tend to perturb intracellular pH due to high proton permeability, our Opto-CRAC system is engineered from a bona fide Ca²⁺ channel that is regarded as one of the most Ca²⁺-selective ion channels. Although the unitary conductance of CRAC channel is estimated to be low (<10 fS in 2 mM extracellular Ca²⁺ in T cells; compared to 4–10 pS for voltage-gated Ca²⁺ channels) (Prakriya and Lewis, 2015; Zweifach and Lewis, 1993), sustained Ca²⁺ influx (up to minutes) through native ORAI1 channels is sufficient to activate downstream effectors. The high Ca²⁺ selectivity (P_Ca/P_Na: >1000) and its small unitary conductance is speculated to reduce the energy requirement of pumping out Na⁺ during sustained Ca²⁺ entry, thereby enhancing the specificity of downstream effector function (Prakriya and Lewis, 2015). Second, the Opto-CRAC tool has a relatively small size (<900 bp, compared to >2.2 kb of ChR) and is thus compatible with almost all existing viral vectors used for in vivo gene delivery. Indeed, we have successfully used retroviral and lentiviral expression systems to deliver Opto-CRAC into primary T cells, macrophages and dendritic cells. Its potential delivery into excitable tissues (e.g., muscle, heart and brain) using adeno-associated viruses remains to be tested in follow-on studies. Third, the tunable and relatively slow kinetics make it most suitable for interrogating Ca²⁺-modulated functions in non-excitable cell types, such as cells in the endocrine, immune and hematopoietic system. We find that our system may find broad use in adoptive cell transfer experiments or adoptive immunotherapies, which are widely used in both basic research and the clinic settings (Palucka and Banchereau, 2012; Restifo et al., 2012). Fourth, in conjunction with upconversion nano-transducers, the light harvesting window can be shifted to the NIR region where deep tissue penetration and remote stimulation are feasible. Results from our in vivo studies clearly indicate that the Opto-CRAC channel and its downstream effectors can be remotely activated using NIR light, thereby paving the way for its future applications in more (patho)physiologically-relevant mouse models, or ultimately, in cancer immunotherapies with improved spatiotemporal control over engineered therapeutic T cells or DCs to reduce off-tumor cross-reaction and mitigate toxicity (Morgan et al., 2010). Given the spatial and temporal accuracy of NIR light, it is also possible to use guided NIR light to confine localized blue light generation, thus avoiding the photoactivation of off-target regions. Lastly, but critically, the lanthanide-doped UCNPs can be applied to activate other optogenetic tools that are dependent on blue light-absorbing cofactors (e.g., ChR2 and CRY2). We anticipate that the flexible adaptability of our novel approach will lead to new opportunities to fine-tune Ca²⁺-dependent immune responses and interrogate other light-controllable cellular processes while minimally interfering with the host’s physiology.

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Author details

1. Lian He

   Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, United States

   Contribution

   LH, GH, YZho, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Yuanwei Zhang, Guolin Ma and Peng Tan

   Competing interests

   The author declares that no competing interests exist.

2. Yuanwei Zhang

   Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   YZha, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Lian He, Guolin Ma and Peng Tan

   Competing interests

   The author declares that no competing interests exist.

3. Guolin Ma

   Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, United States

   Contribution

   GM, YH, PGH, Conception and design, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Lian He, Yuanwei Zhang and Peng Tan

   Competing interests

   The author declares that no competing interests exist.

4. Peng Tan

   Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, United States

   Contribution

   PT, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Lian He, Yuanwei Zhang and Guolin Ma

   Competing interests

   The author declares that no competing interests exist.

5. Zhanjun Li

   Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   ZL, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The author declares that no competing interests exist.

6. Shengbing Zang

   Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, United States

   Contribution

   SZ, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The author declares that no competing interests exist.

7. Xiang Wu

   Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   XW, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The author declares that no competing interests exist.

8. Ji Jing

   Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, United States

   Contribution

   JJ, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The author declares that no competing interests exist.

9. Shaohai Fang

   Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, United States

   Contribution

   SF, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The author declares that no competing interests exist.

10. Lijuan Zhou

    Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, China

    Contribution

    LZ, Conception and design, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The author declares that no competing interests exist.

11. Youjun Wang

    Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, China

    Contribution

    YW, Conception and design, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The author declares that no competing interests exist.

12. Yun Huang

    Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, United States

    Contribution

    YH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    The author declares that no competing interests exist.

13. Patrick G Hogan

    Division of Signaling and Gene Expression, La Jolla Institute for Allergy and Immunology, La Jolla, United States

    Contribution

    GM, YH, PGH, Conception and design, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The author declares that no competing interests exist.

14. Gang Han

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

    Contribution

    LH, GH, YZho, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    Gang.Han@umassmed.edu

    Competing interests

    The author declares that no competing interests exist.

15. Yubin Zhou

    1. Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, United States
    2. Department of Medical Physiology, College of Medicine, Texas A&M University Health Science Center, Temple, United States

    Contribution

    LH, GH, YZho, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    yzhou@ibt.tamhsc.edu

    Competing interests

    The author declares that no competing interests exist.

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