Optogenetic control of T cells for immunomodulation Open Access

Despite a continued decline in mortality rates through 2022, cancer remains the second leading cause of death in the United States, with projections estimating over 2 million new cases and 618,120 deaths in 2025 [1]. While conventional treatments, such as chemotherapy, radiotherapy, and surgery, remain the foundation of oncology, growing attention has shifted toward emerging therapies that leverage novel technologies. These include gene therapy, stem cell therapy, photodynamic therapy, nanomedicine, targeted therapy, and immunotherapy, all of which hold promise as precision tumor treatments or complementary strategies to existing modalities [2]. Among these, immunotherapy has revolutionized cancer treatment by harnessing the host immune system, particularly T cells, to eradicate malignant cells [3]. Despite its success, immunotherapy still faces challenges in achieving precision, minimizing side effects, and optimizing efficacy [4]. To overcome these hurdles, researchers are exploring new technologies, with optogenetics standing out as one of the promising solutions. This innovative technology enables precise control over cellular functions through engineered light-sensitive pathways, offering new possibilities to improve the specificity and safety of immune cell-based therapies [5].

While the roadmap of optogenetics was first proposed by neuroscientists with the adoption of microbial channelrhodopsin-2 (ChR2) for optical control of neuronal membrane potential [6], adapting optogenetics from neuroscience to immunology presents unique challenges due to fundamental differences in signaling dynamics, cellular localization, and excitability between neurons and immune cells [7-9]. Unlike neurons, which rely on rapid changes in membrane potential for signal transmission, immune cells depend on intricate intracellular signaling pathways (Figure 1). This distinction has led to the development of non-opsin photosensitive modules that regulate protein–protein interactions and conformational changes, thereby enabling precise and reversible control over immune signaling pathways and cell functions. These innovations have significantly expanded the applications of optogenetics in immune cell modulation and therefore paved the way for next-generation immunotherapies.

To facilitate optogenetic control within living organisms, upconversion nanoparticles (UCNPs) [9-14] have been engineered to convert near-infrared (NIR) light into blue light, enabling deeper tissue penetration and activation of optogenetic tools. By enabling real-time, tunable regulation of T cell activity, optogenetics holds great promise for improving precision, safety, and efficacy of immunotherapies, helping to mitigate severe side effects such as cytokine release syndrome (CRS) [15]. The integration of optogenetics into cancer immunotherapy represents a significant leap forward in precision medicine. With the development of opsin-free, light-sensitive tools and sophisticated light delivery systems, optogenetics offers unprecedented control over immune cell functions, thereby setting the stage for next-generation cancer immunotherapies with enhanced efficacy and safety.

Calcium (Ca²⁺) signaling is fundamental for T cell activation, differentiation, and function. Upon T cell receptor (TCR) engagement, phospholipase Cγ1 (PLCγ1) is activated, leading to the production of inositol 1,4,5-trisphosphate (IP₃), which triggers Ca²⁺ release from the endoplasmic reticulum (ER). The depletion of ER Ca²⁺ store activates store-operated calcium entry (SOCE), where stromal interaction molecule proteins (STIM1/2) translocate to the plasma membrane and interact with ORAI1 channels, leading to sustained intracellular Ca²⁺ influx [16]. The prolonged Ca²⁺ elevation activates calcineurin, which in turn dephosphorylates nuclear factor of activated T cells (NFAT), enabling its translocation to drive the transcription of genes essential for T cell proliferation and immune responses (Figure 2A) [17,18]. Additionally, Ca²⁺ plays a crucial role in the interplays between T cells and antigen-presenting cells (APC) by regulating motility and cytoskeletal reorganization, further reinforcing immune network connectivity [19,20].

While physiological Ca²⁺ signaling is tightly regulated for maintaining cellular homeostasis, direct elevation of cytosolic Ca²⁺ can induce T cell activation. Chemical agents such as ionomycin and thapsigargin, as well as agonists of Gq-coupled GPCRs, have been widely used to boost intracellular Ca²⁺ levels [21,22]. However, these compounds lack the spatiotemporal resolution and often result in systemic toxicity, limiting their utility in preclinical models [23,24]. Moreover, excessive or sustained Ca²⁺ influx can lead to T cell dysfunction, apoptosis, and exhaustion, ultimately impairing immune responses [25]. In contrast, by engineering light-sensitive ion channels or incorporating photosensory domains into Ca²⁺ release-activated Ca²⁺ (CRAC) channels, optogenetics allows for reversible control of Ca²⁺ influx with unprecedented spatiotemporal resolution. This technology enables fine-tuned regulation of T cell activation and function with high specificity, minimizing off-target effects and reducing the risk of cytotoxicity. Such precise control opens new avenues to optimize T cell responses for immunotherapeutic applications while preserving cell viability and function.

ChR2, a light-gated cation channel derived from Chlamydomonas reinhardtii, was the first optogenetic tool widely adopted for neuronal modulation [6]. Building on this foundation, Kim, et al. engineered a novel ChR2 variant, calcium translocating channelrhodopsin (CatCh), optimized for enhanced light sensitivity and increased Ca²⁺ permeability (Figure 2B) [26]. In a B16 melanoma murine model, local activation of CatCh-expressing CD8 cytotoxic T cells (Tc) resulted in robust T cell activation and significantly suppressed tumor growth when combined with peptide vaccination. Notably, localized photo-stimulation of CatCh also induced systematic anti-tumor effects, effectively controlling the progression of non-illuminated tumors at distal sites, highlighting its potential for treating metastatic cancers. Despite its promise in T cell regulation, ChR2 and subsequent variants exhibit inherent limitations, including non-selective ion permeability [27]. These drawbacks have prompted the development of opsin-free optogenetic systems for more precise and selective control of Ca²⁺ signaling in immune cells, enabling targeted immunomodulation without the complications associated with opsin-based approaches [7,9].

Key components of CRAC channels, particularly STIM1 and ORAI1, have been exploited to modulate intracellular Ca²⁺ influx and T cell activation. For instance, Ishii et al. have developed the Blue Light-Activated Ca²⁺ Channel Switch (BACCS), which is engineered by fusing the light-oxygen-voltage-sensing domain (LOV2) from Avena sativa with the STIM1 effector domain responsible for ORAI1 activation (Figure 2C) [28]. Upon blue light stimulation, a conformational change in the C-terminal Jα domain of LOV2 exposes the STIM1 fragment, allowing it to interact with ORAI1 and trigger Ca²⁺ influx, thereby activating NFAT-mediated gene expression. Another system, Opto-CRAC (Figure 2C) or light-operated Ca²⁺ channel actuators (LOCCa), utilizes wildtype or circularly permuted LOV2 (cpLOV2) domain fused to various cytosolic fragments of STIM1, allowing for light-controlled Ca²⁺ influx through activation of endogenous ORAI channels [10,11]. Notably, beyond its role in T cell activation, Opto-CRAC has been shown to sustain dendritic cell (DC) maturation and enhance antigen presentation, which in turn sensitizes T cells and amplifies anti-tumor immune responses in a B16-OVA murine melanoma model [11].

The precise control over Ca²⁺ signaling offered by optogenetic tools opens new possibilities for fine-tuning T cell responses in cancer immunotherapy. By enabling spatiotemporal modulation of T cell activation, these tools can enhance anti-tumor efficacy while reducing the risk of side effects, such as T cell exhaustion or CRS. Moreover, the ability to modulate not only T cells but also other immune cells, such as DCs, highlights the potential for optogenetics to orchestrate complex immune responses within the tumor microenvironment (TME).

TCR signaling is at the heart of adaptive immunity, enabling T cells to recognize and respond to specific antigens [29-31]. The intricate process is initiated when the α and β subunits of the TCR engage a peptide antigen presented by a major histocompatibility complex (MHC) molecule on the surface of APCs, such as DCs. This interaction triggers a cascade of signaling events, beginning with Lck-mediated phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within CD3 [32]. Phosphorylated ITAMs then serve as the docking sites for ZAP-70, which in turn phosphorylates the linker of activation of T cells (LAT), leading to the activation of downstream signaling pathways such as Ras/MAPK, PI3K/Akt, and Ca²⁺ signaling [33]. These pathways converge to activate key transcription factors such as NFAT, NF-κB, and AP-1, thereby driving cytokine production, T cell activation, proliferation, and differentiation (Figure 3A) [34].

Despite extensive research, the molecular mechanisms underlying signal transduction from the extracellular TCR domain to CD3 ITAM phosphorylation remain incompletely understood. One prevailing theory, known as the aggregation model, proposes that clustering of TCR-CD3 complexes upon antigen engagement brings associated Lck molecules into close proximity, thereby facilitating trans-autophosphorylation of adjacent receptors and amplifying signal propagation [35-37]. Experimental evidence supports this model, demonstrating that artificially induced aggregation of TCRs is sufficient to initiate TCR signaling in the absence of antigen engagement [38]. These findings suggest that receptor proximity and spatial organization play critical roles in TCR activation, providing a mechanistic framework for following optogenetic engineering.

Several optogenetic systems have been engineered to precisely modulate proximal TCR signaling in an antigen-independent manner. One notable example is the Opto-Ligand-TCR system, which employs a fusion protein comprising phytochrome interacting factor 6 (PIF6) and GFP attached to the ectodomain of the Vβ3 variable region of the TCR β subunit. This fusion protein is then paired with phytochrome B (PhyB) tetramers (PhyBt) that serve as the light-sensitive ligand [39]. Upon exposure to red light, PIF6 binds to PhyBt, leading to TCR clustering on the cell surface. This light-driven aggregation mimics natural antigen engagement and triggers robust TCR signaling, as indicated by a robust Ca²⁺ influx comparable with that observed during conventional anti-TCR antibody stimulation. This system highlights the precision and reversibility of optogenetic control in T cell activation, enabling antigen-independent modulation of TCR signaling with high spatiotemporal resolution. Building on the same concept, the OptoREACT system offers another strategy to regulate TCR signaling using a PIF6-coupled single-chain variable fragment (scFv) that binds to the TCR of T cells [40]. Upon red light stimulation, the scFv interacts with clustered PhyB, inducing receptor oligomerization and subsequent activation of downstream signaling pathways (Figure 3C). The Light-inducible T cell engager (LiTE) system further expands the toolkit for optogenetic TCR modulation, where a PIF6-coupled anti-TCR Fab fragment interacts with PhyB-coated surfaces under red light illumination [41]. This interaction effectively triggers NFAT-dependent gene expression and enhances CD8 T cell function. Importantly, the LiTE system enables both quantitative and qualitative modulation of T cell activation, providing fine-tuned control over T cell responses. A similar design was adopted in the SNAP-PIF^S-TCR system, where cells expressing surface-displayed PhyB_1–651 were utilized to activate the SNAP-PIF^S-TCR in Jurkat cells (Figure 3B) [42]. Beyond the proximal TCR complex, optogenetics has also been applied to downstream signaling components [43]. Dine and colleagues developed an optogenetic system to regulate the TCR signaling cascade by inducing the dimerization and clustering of key adaptor proteins. The iLID-SspB module was used to control the interaction between ZAP-70 and LAT, while the optoDroplet system was harnessed to facilitate clustering of ZAP-70:LAT heterodimers (Figure 3D) [43]. This light-inducible assembly effectively triggers Ca²⁺ signaling in Jurkat T cells, demonstrating the versatility of optogenetics in modulating not only receptor engagement but also the downstream signaling events that drive T cell activation.

These innovative optogenetic systems offer powerful tools for precisely controlling TCR signaling, with significant implications for cancer immunotherapy. By enabling antigen-independent T cell activation with high spatiotemporal resolution, optogenetics can help overcome key challenges in T cell therapies, such as antigen escape and efficacy loss [44]. The reversibility and tunability of these light-controlled systems also provide a unique advantage, allowing dynamic regulation of T cell activity based on therapeutic needs.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the landscape of cancer immunotherapy, achieving remarkable success in treating hematological immunotherapy [45-47]. By genetically engineering T cells to express synthetic receptors that recognize tumor-associated antigens (TAAs), CAR T-cell therapy empowers a targeted and robust immune response against cancer cells. Despite these groundbreaking achievements, several critical challenges continue to limit the broader application and safety of CAR T-cell therapies [15,48,49]. One of the most significant concerns is the on-target, off-tumor toxicity, where engineered T cells inadvertently attack normal tissues that express low levels of a target antigen, leading to potentially severe collateral damage [49]. Additionally, CAR T-cell therapy is often associated with severe immune-related adverse effects, such as CRS and neurotoxicity, both of which can escalate to life-threatening complications [50]. Another critical limitation of conventional CAR T-cell therapy is the lack of spatial and temporal control over T-cell activity post-infusion [9]. Once administered, CAR T cells remain continuously active, leaving clinicians with limited options to regulate or halt their function in real time, ultimately exacerbating toxicities and hindering treatment management. Addressing these limitations is crucial for enhancing the safety and efficacy of CAR.

At the core of CAR T-cell therapy is the modular receptor design composed of four main components: an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and intracellular signaling domains (Figure 4A) [9]. This modular architecture allows for flexible customization, enabling the fine-tuning of CAR configuration through combining different protein domains to optimize antigen recognition, receptor stability, and signaling strength [51]. The adaptability of this modular design has also paved the way for the integration of optogenetic tools for spatiotemporal control over T-cell activation. One particularly promising strategy refers to the split-CAR system, in which the intracellular domains, such as 4–1BB or CD3ζ, are divided into two separate fragments, each fused to a light-inducible dimerization component. Upon light stimulation, these split halves reassemble into a functional CAR, enabling reversible and precise control over CAR T-cell activity [9].

A pioneering example of this concept is the light-switchable anti-CD19 CAR T-cells (LiCAR-T) developed by Nguyen and Huang et al., which relies on the iLiD-sspB heterodimerization modules to control functional CAR assembly (Figure 4B) [52]. In a C57BL/6 J mouse model of CD19-expressing melanoma, LiCAR T-cells effectively suppressed tumor growth in the presence of light. Importantly, LiCAR T-cell therapy exhibited enhanced safety and reduced severe side effects, as evidenced by attenuated B-cell aplasia and decreased IL-6 secretion, mitigating the risk of CRS. Another compelling feature of the LiCAR T-cell system is its spatial precision. In vivo experiments confirmed that LiCAR T-cells exerted anti-tumor effects exclusively within the illuminated area, ensuring high spatial control and significantly reducing the risk of on-target, off-tumor toxicity.

The split-CAR strategy has also been adapted in other optogenetic designs, including LOV2-Zdk-based CAR and an alternative iLID-sspB-based CAR system (Figure 4C) [53,54]. Both studies demonstrated that reducing the duration of CAR T-cell activation could effectively mitigate CAR T-cell exhaustion – a major challenge in CAR T-cell therapy [53,54]. Since sustained CAR signaling is a key driver of T-cell dysfunction and exhaustion, pulsed light activation at an optimal frequency could enhance CAR T-cell persistence and improve therapeutic efficacy. These findings underscore the potential of optogenetic strategies to fine-tune CAR T-cell responses, balancing potent anti-tumor activity with reduced toxicity and improved long-term performance.

While split-CAR systems focus on light-inducible receptor reassembly, an alternative optogenetic strategy aims to regulate CAR gene expression, offering another layer of control. Huang et al. developed the Light-Inducible Nuclear Translocation and Dimerization (LINTAD) system, which integrates the cryptochrome 2 (CRY2) and cryptochrome-interacting basic-helix-loop-helix (CIB1) heterodimerization pair with the LOV2-based light-inducible nuclear localization signal (biLINuS) for controlled CAR gene expression under light exposure (Figure 4D) [55]. The LINTAD system successfully demonstrated light-dependent tumor cell killing both in vitro and in vivo. However, a significant limitation of this approach is the inability to deactivate CAR activity once induced. This lack of flexibility reduces the ability to finely regulate CAR-T cell dynamics post-induction, potentially increasing the risk of prolonged activity and related adverse effects.

The TME presents a significant barrier to effective cancer immunotherapy, often suppressing immune responses and facilitating tumor progression [56]. Tumors often evade immune surveillance by exploiting various immunosuppressive mechanisms, such as recruiting regulatory T cells and myeloid-derived suppressor cells. They also engage immune checkpoint pathways, notably programmed cell death protein 1 (PD-1) and its ligand PD-L1, whose interaction suppresses T cell activation and diminishes immune responses [57-59]. Overcoming these barriers and reshaping the TME to support robust T cell responses has become a focal point in the development of next-generation cancer therapies.

One promising approach to reshape the TME involves the induction of immunogenic cell death (ICD), which triggers the release of danger-associated molecular patterns (DAMPs) that activate APCs, ultimately promoting tumor-specific immune responses [60]. The light‐induced non‐apoptotic tools (LiPOPs) have been developed to precisely control two forms of ICD, necroptosis and pyroptosis, through light stimulation [61]. LiPOP1 induces necroptosis by fusing the N-terminal domain of mixed lineage kinase domain-like protein (MLKL-NT) residues 1–125 to CRY2, enabling blue light-induced MLKL oligomerization and subsequent membrane rupture (Figure 5B). LiPOP2 induces pyroptosis via the gasdermin D (GSDMD) executor. LiPOP2a uses the LOV2 trap and release of protein (LOVTRAP) system to release GSDMD N-terminal fragment (GSDMD-NT) upon blue light exposure, while LiPOP2b employs a cpLOV2-based design to uncage GSDMD-NT, both leading to pore formation, cell swelling, and lysis (Figure 5B). These optogenetic tools offer non-invasive, spatiotemporal control over cell death pathways, allowing for localized induction of ICD. By promoting lymphocyte infiltration and activating the TME, LiPOPs represent a powerful approach to augment cancer immunotherapy.

DCs play a pivotal role in orchestrating immune responses by capturing, processing, and presenting antigens to T cells [62]. Enhancing DC function can significantly improve anti-tumor immunity, and activation of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway represents one of the most effective strategies. This activation triggers the production of type I interferons and pro-inflammatory cytokines, which can boost both innate and adaptive immune responses [63,64]. To harness this pathway, Dou et al. developed light-inducible SMOC-like repeats (LiSmore), an optogenetic system that enables precise activation of the STING pathway in DCs [65]. LiSmore fuses the STING C-terminal tail (CTT) repeats to CRY2clust, facilitating blue light-induced STING clustering (Figure 5A) [65]. Upon light exposure, LiSmore effectively activates CD8⁺ T cells, promotes solid tumor clearance, and overcomes checkpoint blockade resistance in lung carcinoma models [65]. Similarly, OptoSTING provides an alternative optogenetic method to modulate the STING pathway [66]. By utilizing a nanobody-fused CRY2 to target fluorescent protein-tagged STING, OptoSTING enables light-induced STING clustering and activation (Figure 5A) [66]. These tools allow researchers to precisely modulate innate immune responses, amplifying anti-tumor immunity while minimizing systemic inflammation.

The immunosuppressive TME often imposes significant metabolic constraints on T cell recruitment and effector function, hindering effective anti-tumor responses [56]. To address this challenge, OptoMito-On, a genetically encoded, light-activated proton pump “Mac” embedded in the mitochondrial inner membrane, has been engineered [67]. Upon light stimulation, OptoMito-On enhanced mitochondrial ATP production in activated CD8⁺ T cells (Figure 5C), leading to increased oxidative phosphorylation, improved T cell migration, and elevated granzyme B expression [67]. This study demonstrates the potential of metabolic reprogramming to boost tumor-infiltrating lymphocyte (TIL) function, thus offering a promising avenue to counteract the metabolic suppression imposed by the TME and improve the efficacy of cancer immunotherapy [67].

Collectively, optogenetics presents a transformative strategy for reshaping the TME and modulating innate immune responses. Tools such as LiPOP, LiSmore, OptoSTING, and OptoMito-On showcase the versatility of optogenetics in precisely modulating cell death pathways, amplifying innate immune signaling, and reprogramming T cell metabolism. These technologies not only strengthen anti-tumor immunity but also provide novel strategies to optimize combination therapies, potentially improving outcomes for patients with otherwise resistant tumors.

Optogenetics modulation of T cells is emerging as a groundbreaking approach in precision immunomodulation, offering real-time, reversible, and spatially precise control over immune responses. By leveraging light-responsive proteins, this technology allows for fine-tuning of key aspects of T cell-based immunotherapy, including antigen recognition, activation, proliferation, and cytotoxicity, thereby improving treatment efficacy while minimizing off-target effects (Table 1).

Regardless of the opportunities brought by these optogenetic tools for T cell immunomodulation, several barriers hinder their immediate clinical adoption. One major concern is phototoxicity, which can result from extended or intense light exposure in both experimental and therapeutic contexts. In addition, the introduction of exogenous optogenetic components carries the risk of unintended interactions with host proteins, potentially disrupting normal signaling pathways. These components may also provoke immune responses due to their immunogenic nature. Furthermore, incomplete caging or mislocalization of optogenetic modules may lead to basal activation and off-target effects, which might compromise the spatiotemporal precision of these tools. Addressing these limitations will be critical for fully realizing the therapeutic potential of optogenetics in cancer immunotherapy.

For successful clinical translation, rigorous preclinical testing is essential to evaluate the safety, efficacy, and long-term effects on immune responses, particularly in mitigating risks such as CRS. Innovations in light delivery systems, such as UCNPs, are crucial for overcoming the limited penetration depth of light, which currently restricts the application of optogenetics to superficial tissues [9-14,68]. UCNPs can convert NIR light, which penetrates deeper into tissues, into blue light to activate optogenetic tools. By enabling deep tissue penetration and precise light delivery, these systems have the potential to expand the use of optogenetic immunotherapy to deep-seated tumors. However, biocompatibility, biodistribution, and long-term safety of these nanoparticles still need thorough evaluation. Gene delivery presents another major challenge. Although adeno-associated viruses (AAVs) have been clinically validated, directing them to specific cell and tissue subsets remains challenging, and the size of many existing optogenetic tools exceeds AAV’s limited packaging capacity. Therefore, the development of safe, efficient, and targeted delivery methods is critical for introducing photo-responsive proteins into immune cells and ensuring spatially confined activation at tumor sites.

In summary, optogenetic immunotherapy represents a promising frontier in cancer treatment. With its potential for personalized, controlled, and effective cancer treatment, optogenetics offers a path toward safer and more targeted immunotherapies, opening new possibilities for patients with resistant or hard-to-treat tumors. Though challenges remain, ongoing advancements in light delivery systems, gene editing technologies, and immune engineering are steadily bringing optogenetic immunotherapy closer to clinical application, offering safer, more effective, and personalized therapeutic interventions for cancer patients.

The authors declare no financial or commercial competing interest.

We gratefully acknowledge grant support from the National Institutes of Health [R21CA277257, R21AI174606, R01GM144986, and R01CA232017, to Y.Z.], the Leukemia & Lymphoma Society (to Y.Z.), the Cancer Prevention and Research Institute of Texas [RP250468 to Y.Z.] and the Welch Foundation [BE-1913-20220331 to Y.Z.].

Y.Z. and T.L.: Conceptualization. B.M. and S.L.: Resources. B.M., S.L, P.C., Z.Y., T.L., and Y.Z.: Writing—original draft. T.L. and Y.Z.: Writing—review & editing.

Figures were created using BioRender.com.

CRS

cytokine release syndrome

ChR2

channelrhodopsin-2

NFAT

nuclear factor of activated T cells

NIR

near-infrared

TME

tumor microenvironment

UCNPs

upconversion nanoparticles