An in vivo MAIT cell activation and rejuvenation system for liver cancer therapy

An in vivo MAIT cell activation and rejuvenation system for liver cancer therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article An in vivo MAIT cell activation and rejuvenation system for liver cancer therapy Yan-Ruide Li, Haochen Nan, Zhengyao Shao, Xinyuan Shen, Yuning Chen, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8518619/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Mucosal-associated invariant T (MAIT) cells are liver-enriched unconventional T cells with potent antitumor potential, yet in liver cancer they are numerically reduced and functionally exhausted, limiting their therapeutic efficacy. To overcome these limitations, we developed a MAIT cell activation and rejuvenation system (MARS), a biomimetic, liver-targeted nanoparticle platform designed for sustained in vivo delivery of the MR1-restricted MAIT agonist 5-OP-RU together with human IL-15. Ex vivo, MARS robustly expanded and activated MAIT cells from liver cancer patient peripheral blood and tumor tissues, enhancing effector cytokine production and cytotoxicity while limiting exhaustion. In vivo, MARS preferentially accumulated in the liver and induced durable MAIT cell activation, expansion, and potent tumor killing in human liver cancer xenograft models. Importantly, in a human myeloid cell–engrafted mouse model that recapitulates an immunosuppressive liver tumor microenvironment, MARS enabled MAIT cells to simultaneously eliminate liver tumor cells and MR1⁺ tumor-associated myeloid cells, resulting in effective tumor control and prolonged survival. Collectively, these findings establish MARS as a safe and effective in vivo MAIT cell engineering strategy that overcomes microenvironment-driven resistance and provides a promising immunotherapeutic approach for liver cancer. Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Biological sciences/Biotechnology/Nanobiotechnology Biological sciences/Cancer/Tumour immunology Mucosal-associated invariant T (MAIT) cell MAIT cell activation and rejuvenation system (MARS) liver cancer tumor microenvironment immunotherapy in vivo engineering 5-OP-RU IL-15 biomimetic nanoparticle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Mucosal-associated invariant T (MAIT) cells are a unique subset of unconventional αβ T cells with potent antimicrobial and antitumor activities 1–4 . These cells express a semi-invariant T-cell receptor (TCR) (Vα7.2-Jα33/12/20 in humans and Vα19-Jα33 in mice) that recognizes the monomorphic MHC class I-related molecule MR1 presenting vitamin B2 (riboflavin) metabolites such as 5-OP-RU 5–7 . Although MAIT cells comprise only about 1-5% of circulating T cells, they are highly enriched in the liver, where they can account for up to 30-45% of intrahepatic T cells in both healthy individuals and liver cancer patients 1,8,9 . This liver tropism is attributed to the preferential expression of chemokine receptors such as CCR6, CXCR6, and CXCR3 on MAIT cells, which guide their recruitment to the hepatic microenvironment 10,11 . Importantly, clinical and transcriptomic analyses have revealed that a higher MAIT cell gene signature is positively correlated with improved overall survival in hepatocellular carcinoma (HCC) patients, suggesting a potential protective and tumor-controlling role of MAIT cells in liver cancer 9 . However, MAIT cells within the liver tumor microenvironment (TME) of cancer patients often exhibit an exhausted and dysfunctional phenotype. Their infiltration into tumor tissues is generally limited, and those that do localize to tumors display impaired cytotoxicity and reduced effector function 9,12,13 . Human MAIT cells in HCC lesions frequently show increased expression of activation and exhaustion markers, including PD-1, CD25, and HLA-DR, reflecting a state of chronic stimulation coupled with functional impairment 9 . Recent studies have also highlighted that interactions with niche-residing CSF1R + PD-L1 + tumor-associated macrophages (TAMs) contribute to this dysfunctional state, acting as a key regulatory mechanism that restrains MAIT cell antitumor activity 9 . Collectively, these observations indicate that despite their intrinsic antitumor potential, MAIT cells in the liver TME are rendered ineffective by both cellular exhaustion and suppressive microenvironmental cues. Therefore, strategies aimed at activating or rejuvenating MAIT cells within the liver cancer TME are critical for restoring their cytotoxic function and enhancing their antitumor capacity. Microbial metabolites, such as 5-OP-RU, play a critical role in MAIT cell development, maturation, and functional competence, including their antitumor activity 5,14,15 . In vitro administration of 5-OP-RU can efficiently trigger MAIT cell-mediated killing of both MR1 + tumor cells and MR1 + TAMs, highlighting a dual capacity to target malignant cells and the immunosuppressive TME 16,17 . Moreover, in vivo co-administration of 5-OP-RU with CpG oligonucleotides has been shown to induce robust systemic expansion and activation of MAIT cells, leading to potent and broad antitumor immune responses in murine models of liver metastasis, HCC, lung metastasis, and subcutaneous tumors. 1 Despite these promising results, direct administration of 5-OP-RU faces significant limitations, including rapid degradation, short in vivo half-life, and the need for frequent dosing to maintain MAIT cell activation 18,19 . Additionally, systemic delivery may result in off-target effects or suboptimal localization within the TME. Therefore, there is a critical need to develop strategies that enable sustained, localized, and efficient in vivo activation of MAIT cells to fully harness their antitumor potential. To overcome these limitations and enhance MAIT cell-mediated antitumor immunity in liver cancer, we developed a MAIT cell activation and rejuvenation system (MARS), a biomimetic platform designed for targeted delivery to the liver and preferential engagement of liver-residing MAIT cells. MARS provides sustained release of 5-OP-RU to rejuvenate and activate MAIT cells, thereby restoring their cytotoxic capacity against liver tumor cells. In addition, the system delivers human IL-15 to promote MAIT cell survival, expansion, and sustained cytotoxic function. Through its biomimetic design and liver-localized immunostimulatory effects, MARS is expected to efficiently reprogram the liver TME, enhance MAIT cell activation and function, and elicit a potent, localized antitumor immune response in liver cancer, representing a promising strategy to overcome immunosuppression in the liver TME. Results Liver-resident MAIT cells exhibit reduced frequency and increased exhaustion in primary liver cancer patients To characterize the phenotype and functionality of MAIT cells in liver cancer, we performed single-cell RNA sequencing (scRNA-seq) and flow cytometry analyses on primary liver samples, including 31 liver cancer (LC) and 29 normal adjacent (NA) tissues (Fig.1a and Supplementary Table 1). The scRNA-seq analyses were conducted using two publicly available datasets containing matched LC and NA samples derived from the same patients (Supplementary Fig. 1a) 20,21 . In parallel, flow cytometry analyses were performed on in-house specimens from five LC and five patient-matched NA samples (Fig. 1a). The inclusion of paired LC and NA samples allowed us to directly compare MAIT cell phenotypes within the same individual, minimizing inter-patient variability and providing a more accurate assessment of disease-associated alterations. We first analyzed the scRNA-seq data of liver-resident T cells, including CD4 T, CD8 T, and MAIT cells (Fig.1b). MAIT cells were identified based on the expression of SLC4A10 , a specific marker distinguishing this subset from conventional T cells (Supplementary Fig. 1b) 22 . LC samples showed a decreased frequency of MAIT cells but an increased proportion of CD4 T cells compared to NA tissues (Figs. 1c and 1d). Comparative analyses of MAIT cells between LC and NA regions revealed that tumor-infiltrating MAIT cells exhibited enhanced proliferative, effector, memory, and exhaustion signatures, suggesting a state of activation and exhaustion driven by the liver TME (Fig. 1e), consistent with previous findings in HCC patients. 9 These results indicate that liver-resident MAIT cells in cancer exhibit a hyperactivated yet partially exhausted phenotype, reflecting their persistent engagement in the tumor immune response. Furthermore, when comparing CD4 T, CD8 T, and MAIT cells in both NA and LC samples, MAIT cells displayed the strongest effector and memory phenotypes, cytotoxic activity comparable to CD8 T cells but greater than CD4 T cells, the highest early exhaustion features, and lower terminal exhaustion compared to CD8 T cells but higher than CD4 T cells (Supplementary Fig. 1c). Consistent with these findings, heatmap analyses demonstrated that MAIT cells expressed significantly higher levels of natural killer (NK)–associated genes (e.g., KLRB1 , NCAM1 , NCR3 , and ZBTB16 ), effector-related genes ( CD69 , IL18R1 , and CD48 ), memory-associated genes ( GZMM , CCR5 , and FOS ), and cytotoxicity-related genes ( CYCS , SERPINB9 , PRF1 , and CTSN ) compared with CD4 and CD8 T cells (Fig. 1f). Pathway enrichment analyses further revealed that, in LC samples, MAIT cells showed significant upregulation of genes involved in NK cell–mediated cytotoxicity, antigen processing and presentation, TNF signaling, and Toll-like receptor signaling pathways compared with CD4 T cells (Fig. 1g). In addition, relative to CD8 T cells, MAIT cells exhibited enhanced enrichment of genes associated with the PD-1/PD-L1 immune checkpoint pathway, Th1/Th2 cell differentiation, TNF signaling, and NF-κB signaling pathways (Fig. 1g). Overall, these results indicate that MAIT cells in the liver TME possess strong innate-like effector and NK-mediated cytotoxic programs but simultaneously display features of activation-induced exhaustion. We next validated these findings by flow cytometry analysis of five paired in-house LC and NA liver samples (Supplementary Table 1). Consistent with the scRNA-seq results, LC tissues exhibited a significantly reduced frequency of MAIT cells compared with NA tissues, confirming numerical loss of MAIT cells in liver cancer (Figs. 1h-1j). Phenotypic characterization revealed that liver-resident MAIT cells from both LC and NA tissues, as well as MAIT cells in the peripheral blood of liver cancer patients, predominantly displayed a CD62L - CD45RO + effector memory phenotype (Fig. 1k). This phenotype was distinct from that of conventional T cells in the circulation, which comprised naïve (CD62L + CD45RO - ), central memory (CD62L + CD45RO + ), effector (CD62L - CD45RO - ), and effector memory subsets (Fig. 1k). In contrast, the majority of conventional T cells within the liver also exhibited an effector memory phenotype (Fig. 1k). These findings are consistent with previous reports identifying MAIT cells as a highly differentiated effector memory T cell subset 14,23,24 . We further examined the expression of exhaustion and cytotoxicity-associated markers in MAIT cells from LC and NA tissues. MAIT cells within LC samples expressed significantly higher levels of the inhibitory receptors PD-1 and LAG-3, accompanied by markedly reduced expression of cytotoxic effector molecules, including perforin and granzyme B (Figs. 1l and 1m). These data indicate that MAIT cells in liver cancer exhibit an exhaustion-associated phenotype coupled with impaired cytotoxic function. Collectively, these results demonstrate that MAIT cells represent a distinct T cell population within the liver, characterized by an effector memory phenotype and innate-like cytotoxic potential. However, in the context of liver cancer, MAIT cells appear to undergo chronic activation leading to functional exhaustion and diminished cytotoxic molecule production. These findings underscore the critical importance of MAIT cells in liver tumor immunity and highlight the need for therapeutic strategies aimed at reactivating and rejuvenating MAIT cell function to enhance antitumor responses in liver cancer. Engineering of MARS as a biomimetic platform delivering 5-OP-RU and IL-15 to activate MAIT cells We therefore developed a MAIT cell activation and rejuvenation system (MARS), a biomimetic, liver-targeted platform based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles that enables sustained delivery of the MAIT agonist 5-OP-RU and human IL-15 (Fig. 2a). 5-OP-RU specifically activates MAIT cells through TCR–MR1 recognition, while IL-15 enhances MAIT cell survival and granzyme B–dependent cytotoxicity 15,25,26 . Following systemic administration, MARS preferentially accumulates in the liver and is taken up by hepatic antigen-presenting cells, including myeloid cells, enabling prolonged MR1-restricted antigen presentation and localized MAIT cell activation (Fig. 2a). This strategy restores MAIT cell effector function and enables simultaneous targeting of liver tumor cells and immunosuppressive myeloid populations within the liver TME. MARS was fabricated by encapsulating the MAIT antigen precursors 5-amino-6-D-ribitylaminouracil (5-A-RU) and methylglyoxal (MGO) within PLGA nanoparticles, allowing in situ formation and sustained release of the active MAIT agonist 5-OP-RU 27 . To further recapitulate physiological MAIT cell activation, we engineered the nanoparticle surface to mimic cytokine trans-presentation by antigen-presenting cells. Specifically, IL-15 receptor α (IL-15Rα) was conjugated to the surface of MARS to bind and present human IL-15 as an IL-15/IL-15Rα complex, thereby enabling efficient IL-15 trans-presentation to MAIT cells expressing the IL-2/15 receptor β chain and common γ chain (Fig. 2a). This design allows coordinated delivery of MR1-restricted antigen and IL-15–dependent costimulatory signals, closely resembling natural APC-mediated MAIT cell activation in the liver. We first characterized the physicochemical properties of MARS to establish parameters critical for efficient MAIT cell activation and safe in vivo application. Dynamic light scattering analysis showed that nanoparticle size could be precisely controlled by modulating ultrasonication power, yielding uniform submicron particles optimized for systemic circulation and preferential hepatic accumulation, a prerequisite for effective engagement of liver-resident MAIT cells (Fig. 2b). Controlled release studies demonstrated formulation-dependent and sustained release of 5-OP-RU, with higher polymer-to-cargo ratios enabling prolonged antigen availability and supporting sustained MR1-restricted antigen presentation for continuous MAIT cell stimulation (Fig. 2c). Consistent with this, encapsulation efficiency decreased with reduced PLGA content, highlighting the importance of polymer composition in maintaining antigen stability and bioavailability (Fig. 2d). Scanning electron microscopy confirmed the spherical morphology and smooth surface of MARS nanoparticles (Fig. 2e), features associated with favorable biodistribution and cellular uptake, while elemental analysis verified successful incorporation of nitrogen- and sulfur-containing antigen components, confirming effective loading of the MAIT agonist (Fig. 2f). We next investigated whether MARS enables sustained MR1-restricted antigen presentation and functional engagement of MAIT cells in vitro . In the absence of continuous antigen availability, MR1 surface expression is known to rapidly decline, underscoring the need for sustained delivery of MAIT agonists 28,29 . To address this, MARS nanoparticles were co-cultured with CD14⁺ myeloid cells derived from healthy donor peripheral blood mononuclear cells (PBMCs), which serve as professional antigen-presenting cells (APCs). These APCs efficiently internalized MARS nanoparticles, with uptake detectable for up to two weeks and gradually declining by day 20 (Figs. 2g and 2h). Importantly, MARS-treated APCs exhibited enhanced and sustained surface expression of MR1 (Figs. 2g and 2h), indicating continuous release of 5-OP-RU from the nanoparticles and consistent with previous reports demonstrating that 5-OP-RU stabilizes and upregulates MR1 on APCs and tumor cells 30,31 . Functionally, MARS-loaded APCs maintained prolonged antigen presentation capacity, as evidenced by immunofluorescence imaging during MAIT/APC co-culture, which revealed progressively increased and stabilized cell–cell contacts over time (Fig. 2i). Quantitative analyses further demonstrated that these sustained interactions translated into enhanced MAIT cell activation (Fig. 2j). Together, these results indicate that MARS provides sustained 5-OP-RU release, promotes prolonged MR1 surface antigen availability, and thereby improves the efficiency and durability of MAIT cell engagement and activation. MARS demonstrates efficient and selective liver homing We next evaluated the in vivo homing behavior of MARS following systemic administration. Longitudinal whole-body fluorescence imaging revealed rapid and preferential accumulation of MARS nanoparticles in the liver, with sustained retention over an extended period, indicating efficient hepatic targeting (Figs. 2k-2n). This prolonged liver residence is critical for continuous local delivery of immunomodulatory cargos and effective engagement of liver-resident MAIT cells. Throughout the treatment period, no significant changes in body weight were observed, suggesting favorable tolerability and the absence of overt systemic toxicity (Fig. 2o). Consistent with the imaging data, tissue biodistribution analysis confirmed dominant localization of MARS within the liver, with minimal off-target accumulation in other major organs, including the lung, heart, spleen, and kidney (Figs. 2p and 2q). This selective biodistribution profile is consistent with the physicochemical properties of the biomimetic nanoparticles and supports their capacity for efficient hepatic uptake and retention 32 . Collectively, these results demonstrate that MARS achieves selective and sustained liver targeting with minimal systemic exposure, providing a favorable safety profile and establishing a strong foundation for localized MAIT cell activation and effective liver cancer immunotherapy in vivo . MARS induces superior MAIT cell activation and proliferation compared with conventional approaches We first evaluated the ability of MARS to induce MAIT cell proliferation and activation using PBMCs derived from liver cancer patients (Supplementary Table 1), in which MAIT cells comprised approximately 0.5–2% of total CD3⁺ T cells (Figs. 3a and 3b). Three stimulation strategies were compared: 5-OP-RU alone, 5-OP-RU combined with IL-15, and MARS (Fig. 3a). Notably, PBMCs contain abundant myeloid cells that function as APCs and mediate MR1-dependent presentation of 5-OP-RU to MAIT cells. Following a 15-day co-culture, all three conditions induced robust MAIT cell expansion and activation; however, MARS consistently elicited the strongest response, characterized by greater fold expansion and more rapid activation (Figs. 3b and 3c). MARS-stimulated MAIT cells exhibited marked upregulation of activation, effector, and memory-associated markers, including CD25, CD44, CD69, CD62L, and CD45RO (Figs. 3d and 3e). Importantly, compared with conventional T cells within the same cultures, MAIT cells expressed significantly higher levels of these markers, indicating selective and preferential activation of MAIT cells by MARS (Supplementary Fig. 2). We next assessed MARS-mediated activation in primary liver cancer tissues to more closely model liver-resident MAIT cells, which comprised approximately 10–30% of total viable cells, consistent with previous reports (Figs. 3f and 3g, and Supplementary Table 1) 9,13 . While all three stimulation strategies promoted MAIT cell expansion, MARS again demonstrated superior efficacy, as evidenced by greater MAIT cell enrichment, enhanced proliferation, and increased production of effector cytokines, including IFN-γ, TNF-α, IL-2, and IL-12, together with elevated expression of activation markers CD25 and CD69 (Figs. 3g-3j). Notably, sustained IL-15 delivery by MARS resulted in a reduced proportion of exhausted MAIT cells (PD-1⁺LAG-3⁺TIM-3⁺) and enhanced cytotoxic features, including increased granzyme B expression and CD107a degranulation (Figs. 3k and 3l). Collectively, these results demonstrate that MARS induces more robust, sustained, and MAIT cell–specific activation and expansion than conventional stimulation approaches, while simultaneously limiting exhaustion and enhancing cytotoxic function, highlighting its potential as an effective strategy to rejuvenate MAIT cells for liver cancer immunotherapy. MARS induces MAIT cell activation via TCR engagement and IL-15-dependent signaling We next sought to confirm that MARS-mediated MAIT cell activation is driven by coordinated TCR engagement and IL-15–dependent signaling. Liver-resident MAIT cells from liver cancer patients were stimulated using three approaches, including 5-OP-RU alone, 5-OP-RU combined with IL-15, or MARS, followed by cell sorting and analysis of downstream signaling pathways by western blot. All three stimulation strategies induced phosphorylation of key components of the MAIT TCR signaling cascade, including LCK, ZAP70, and PLCγ1, as well as activation of downstream transcription factors such as c-Jun and NF-κB (Figs. 3m and 3n). These findings confirm effective MR1-restricted TCR engagement by 5-OP-RU. Notably, MARS stimulation resulted in stronger and more sustained phosphorylation of these signaling molecules compared with soluble 5-OP-RU or 5-OP-RU plus IL-15, indicating enhanced and prolonged TCR signaling, likely due to sustained antigen availability (Figs. 3m and 3n). In addition to TCR signaling, both 5-OP-RU plus IL-15 and MARS robustly activated IL-15–dependent pathways, as evidenced by increased phosphorylation of JAK1, JAK3, STAT3, and STAT5. This activation was accompanied by upregulation of key transcriptional regulators associated with MAIT cell survival, proliferation, and effector differentiation, including c-Myc, c-Fos, and BCL2 (Fig. 3o and Supplementary Fig. 2) 33,34 . Importantly, MARS induced higher and more sustained activation of these IL-15–dependent signaling nodes compared with the soluble cytokine condition, consistent with prolonged IL-15 bioavailability (Fig. 3o and Supplementary Fig. 2). Collectively, these results demonstrate that MARS functions as a dual-signal platform that simultaneously delivers sustained MR1-restricted antigen stimulation and IL-15–mediated costimulatory signaling (Fig. 3p). By engaging both the MAIT TCR and IL-15 receptor pathways, MARS promotes robust MAIT cell activation, survival, and effector programming, providing a mechanistic basis for its superior capacity to rejuvenate MAIT cells within the liver TME. MARS enhances MAIT cell antitumor activity against liver cancer in vitro We next evaluated the cytotoxic activity of MARS-activated MAIT cells against human liver tumor cells in vitro (Fig. 4a). An in vitro tumor cell killing assay was performed using six human liver cancer cell lines representing distinct genetic backgrounds and tumor origins (Fig. 4b). Five parental tumor cell lines (HepG2, C3A, SNU423, SNU475, and SKHEP1) were engineered to express a firefly luciferase and enhanced green fluorescent protein dual reporter (FG), enabling quantitative assessment of tumor cell viability by both flow cytometry and luminescence assays (Fig. 4b). To directly assess MR1-dependent recognition, SKHEP1 cells were additionally engineered to overexpress MR1 (SKHEP1-MR1-FG). Baseline analysis confirmed heterogeneous MR1 expression across the tumor cell panel, reflecting the diversity of MR1 levels observed in human liver cancers (Figs. 4b and 4c). Liver cancer patient PBMC–derived MAIT cells were co-cultured with tumor cells under four conditions: no stimulation, 5-OP-RU alone, 5-OP-RU combined with IL-15, or MARS (Fig. 4a). During the first round of killing, all three stimulation strategies significantly enhanced MAIT cell–mediated cytotoxicity against all tumor cell lines compared with the unstimulated condition, with MARS consistently inducing the strongest tumor killing (Fig. 4d). These results indicate that MR1-restricted antigen presentation is sufficient to trigger MAIT cell recognition and cytotoxicity against liver tumor cells across a range of MR1 expression levels. Notably, SKHEP1-MR1-FG cells were killed more efficiently than parental SKHEP1-FG cells, demonstrating a positive correlation between MR1 expression and MAIT cell–mediated tumor killing (Fig. 4d). To assess the durability of MAIT cell cytotoxic function, we performed repeated tumor challenge assays, in which MAIT cells were exposed to fresh tumor cells for a third killing round. While MAIT cell cytotoxicity declined under soluble 5-OP-RU or 5-OP-RU plus IL-15 stimulation, MARS-treated MAIT cells maintained robust tumor-killing capacity (Fig. 4d). This sustained cytotoxicity was accompanied by significantly greater MAIT cell expansion (Fig. 4e), elevated production of effector cytokines (IFN-γ, TNF-α, and IL-2) (Fig. 4f), increased expression of the activation marker CD69, and enhanced production of cytotoxic molecules including perforin and granzyme B (Fig. 4g), indicating that MARS supports long-term MAIT cell functionality. We further evaluated MAIT cell cytotoxicity against primary liver tumor cells derived from liver cancer patients (Fig. 4h). These primary tumor cells expressed variable but generally high levels of MR1, supporting their susceptibility to MAIT cell–mediated recognition (Fig. 4i). Consistent with the cell line data, 5-OP-RU stimulation induced MAIT cell–mediated killing of primary tumor cells, while MARS significantly enhanced this effect (Fig. 4j). Importantly, primary liver tumor samples contained diverse immune cell populations within the liver TME, including immunosuppressive myeloid cells, T cells, B cells, and NK cells. Myeloid cells exhibited markedly higher MR1 expression compared with other immune subsets (Figs. 4k and 4l), and upon MARS activation, MAIT cells selectively and efficiently eliminated these MR1⁺ immunosuppressive myeloid cells while sparing other immune populations (Figs. 4m and 4n). These findings indicate that MARS not only enhances direct tumor cell killing but also enables MAIT cells to remodel the TME by selectively targeting MR1-expressing immunosuppressive cells. In conclusion, MARS confers robust, sustained, and MR1-dependent cytotoxic activity to MAIT cells against both liver tumor cells and immunosuppressive myeloid populations. By preserving MAIT cell effector function over repeated tumor encounters and selectively reshaping the TME, MARS establishes a powerful dual mechanism for enhancing MAIT cell–based immunotherapy in liver cancer. MARS enhances MAIT cell antitumor activity, persistence, and effector function against liver cancer in a xenograft mouse model We next evaluated the in vivo antitumor capacity of MAIT cells following activation by MARS. A human liver cancer xenograft model was established by intravenously injecting 5 × 10⁵ SKHEP1-MR1-FG cells into NSG mice, allowing preferential tumor seeding and growth within the liver, thereby mimicking the clinical features of liver cancer (Fig. 5a). Four therapeutic conditions were compared: adoptive transfer of MAIT cells alone, MAIT cells with a single intravenous dose of 5-OP-RU, MAIT cells with weekly intravenous doses of 5-OP-RU, and MAIT cells combined with intravenous administration of MARS (Fig. 5a). Bioluminescence imaging (BLI) revealed that adoptively transferred MAIT cells alone exerted minimal tumor control, likely due to insufficient endogenous antigen stimulation in vivo (Figs. 5b-5e). A single dose of 5-OP-RU significantly enhanced MAIT cell–mediated antitumor activity; however, repeated weekly administration of soluble 5-OP-RU did not further improve tumor control, likely reflecting its rapid degradation and short in vivo half-life (Figs. 5b-5e). In contrast, MARS treatment resulted in robust tumor elimination, as evidenced by near-complete loss of BLI signal in the liver and a marked improvement in overall survival (Figs. 5b-5e). Flow cytometric analysis of liver-resident cells confirmed a stepwise reduction in GFP⁺ tumor cells following MAIT cell transfer, further enhanced by 5-OP-RU treatment, and complete tumor clearance in the MARS-treated group (Fig. 5f). In parallel, human MAIT cells were readily detected in the liver following adoptive transfer. While MAIT cell persistence was limited in the absence of stimulation and only modestly improved by soluble 5-OP-RU, MARS treatment dramatically enhanced MAIT cell persistence in vivo , with detectable MAIT cells maintained in the liver for up to 60 days (Figs. 5g and 5h). Importantly, MARS-activated MAIT cells exhibited a highly functional phenotype, characterized by increased expression of activation markers CD25 and CD69 and reduced expression of exhaustion markers LAG-3 and TIM-3 (Figs. 5i, 5j, and Supplementary Fig. 4). These findings indicate that MARS provides sustained delivery of both 5-OP-RU and IL-15 in vivo , thereby promoting durable MAIT cell activation, persistence, and effector function within the liver. Overall, these results demonstrate that MARS markedly enhances the in vivo antitumor efficacy of MAIT cells by overcoming antigen limitation and functional exhaustion, leading to durable tumor clearance and prolonged survival in a clinically relevant liver cancer model. MARS reprograms the liver TME and sustains MAIT cell antitumor activity in a xenograft mouse model We next evaluated the capacity of MAIT cells to target both liver tumor cells and the immunosuppressive TME under conditions that more closely recapitulate the human liver cancer setting. To this end, we utilized NSG-SGM3 mice, which express human SCF, GM-CSF, and IL-3 and therefore support efficient engraftment and persistence of human myeloid cells, enabling the establishment of a humanized immunosuppressive TME 35,36 . Human CD14⁺ myeloid cells were administered weekly, resulting in sustained engraftment in vivo , including within the liver, where they functioned as key immunosuppressive components of the TME (Fig. 6a). On day 0, SKHEP1-FG liver tumor cells were intravenously injected to establish liver tumors (Fig. 6a). Importantly, these parental tumor cells expressed relatively low levels of MR1 (Fig. 4c), allowing us to directly assess whether MARS-mediated MAIT cell activation is sufficient to drive tumor killing in a complex TME without artificial MR1 overexpression. MAIT cells derived from the same donor as the myeloid cells were adoptively transferred, and MARS was administered intravenously (Fig. 6a). BLI confirmed successful tumor establishment in the liver, characterized by rapid tumor growth and reduced survival in control mice (Figs. 6c-6e). While adoptively transferred MAIT cells alone moderately suppressed tumor progression, MARS treatment significantly enhanced MAIT cell–mediated antitumor activity, as evidenced by markedly reduced tumor burden, prolonged survival, and diminished detection of GFP⁺ tumor cells in the liver (Figs. 6c-6e). These results demonstrate that MARS confers potent antitumor capacity to MAIT cells even within a highly immunosuppressive and humanized liver TME. Analysis of the liver microenvironment revealed robust engraftment of human myeloid cells (Figs. 6f and 6g). Notably, MAIT cell transfer led to a reduction in these myeloid populations, and this effect was further amplified by MARS treatment (Figs. 6f-6i). Phenotypic characterization showed that liver-infiltrating myeloid cells expressed high levels of immunosuppressive macrophage markers CD163 and CD206, as well as MR1, indicating that they represent MR1⁺ immunosuppressive targets within the TME (Fig. 6j). These findings suggest that MARS-activated MAIT cells selectively eliminate MR1-expressing immunosuppressive myeloid cells, thereby reshaping the TME in favor of antitumor immunity. Consistent with these cellular changes, serum cytokine analysis by ELISA revealed that MARS treatment significantly increased MAIT cell–associated effector cytokines, including IFN-γ and IL-2 (Fig. 6k), while reducing levels of proinflammatory myeloid-derived cytokines such as IL-6 and IL-1β (Fig. 6l). This cytokine profile is consistent with depletion of immunosuppressive myeloid cells and restoration of a more immunostimulatory liver environment. Finally, phenotypic analysis of liver-resident MAIT cells demonstrated that MARS activation induced a highly functional state, characterized by elevated expression of activation markers CD25 and CD69 and reduced expression of exhaustion markers TIM-3 and LAG-3 (Figs. 6m and 6n). Together, these data indicate that MARS not only enhances MAIT cell–mediated tumor killing but also sustains MAIT cell fitness and prevents functional exhaustion within the liver TME. Collectively, these results demonstrate that MARS enables MAIT cells to simultaneously target liver tumor cells and remodel an immunosuppressive humanized TME by selectively eliminating MR1⁺ myeloid cells, sustaining effector function, and promoting durable antitumor immunity in vivo . This dual targeting strategy positions MARS-activated MAIT cell therapy as a promising therapeutic approach for liver cancers that are resistant to conventional treatments due to a highly immunosuppressive TME. MARS demonstrates favorable safety profile in vivo We next evaluated the in vivo safety profile of MARS. We first assessed a mouse-adapted MARS formulation encapsulating 5-OP-RU and murine IL-15 in immunocompetent C57BL/6 mice (Fig. 7a). Following intravenous administration, mice remained healthy with no signs of morbidity or mortality for up to 40 days post-injection, indicating favorable tolerability. Immunophenotypic analysis revealed a transient enrichment of endogenous murine MAIT cells at approximately 10 days post-injection in the blood, liver, and spleen (Fig. 7b). Notably, given the substantially lower frequency of MAIT cells in mice compared with humans, this expansion was modest and less pronounced than that observed in humanized systems (Figs. 6f and 7b). Importantly, no significant alterations were detected in the frequency or phenotype of other immune populations, including conventional T cells, B cells, NK cells, or monocytes, indicating selective immune modulation by MARS (Fig. 7c). Comprehensive hematological analyses further demonstrated no significant differences in white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) levels, or hematocrit (HCT) values between MARS-treated and control mice, suggesting the absence of systemic toxicity or hematopoietic disruption (Fig. 7d). We next evaluated the safety of MARS in a humanized NSG mouse model receiving adoptive transfer of human MAIT cells (Fig. 7e). Following intravenous administration of MAIT cells and MARS, mice maintained stable body weight throughout the study period (Fig. 7f). Serum analyses revealed no elevation of organ damage–associated biomarkers, and histopathological examination showed no evidence of tissue injury or inflammation across major organs, including liver, lung, heart, spleen, and kidney, compared with untreated NSG controls (Figs. 7g and 7h). Collectively, these results demonstrate that MARS exhibits a favorable safety profile in both immunocompetent and humanized mouse models, supporting its translational potential as a targeted and well-tolerated MAIT cell–based immunotherapeutic strategy for liver cancer. Discussion MAIT cells have emerged as a key subset of unconventional T cells with potent antimicrobial, immunoregulatory, and antitumor functions. As a unique innate-like T cell population, MAIT cells are particularly enriched in the liver, accounting for a substantial proportion of intrahepatic lymphocytes. 11,37,38 Accumulating evidence indicates that MAIT cells play multifaceted roles in liver homeostasis and pathology. Studies characterizing their localization revealed that MAIT cells are predominantly concentrated in bile ducts, portal tracts, and hepatic sinusoids, rather than the parenchyma. 39 In chronic liver diseases of various etiologies, MAIT cells exhibit dynamic changes in frequency and function, often accumulating within fibrotic septa or showing signs of activation-induced apoptosis, suggesting their involvement in both tissue repair and inflammatory progression. 40,41 The role of MAIT cells in liver cancer remains controversial. Mengduan et al. reported that tumor-infiltrating MAIT cells in HCC express high levels of PD-1 and other exhaustion markers, with reduced abundance correlating with poor prognosis. 13 In contrast, Ruf et al. found that higher MAIT cell frequencies were associated with improved survival in HCC patients, and that checkpoint blockade targeting PD-1/PD-L1 could partially restore MAIT cell function, supporting their antitumor potential. 9 These seemingly conflicting observations may reflect the dual Th1- and Th17-like nature of MAIT cells, driven by co-expression of T-bet and RORγt. 42–46 Nonetheless, it is evident that MAIT cells become functionally impaired within the HCC TME (Fig. 1), emphasizing the importance of strategies to rejuvenate and re-activate MAIT cells for liver cancer therapy. We designed MARS to enable in vivo activation and rejuvenation of MAIT cells with several key advantages that directly address the major biological and therapeutic limitations of MAIT cell–based immunotherapy in liver cancer. First, MARS achieves preferential liver targeting and prolonged hepatic retention, allowing localized and sustained delivery of MAIT agonists within the liver TME (Figs. 2k-2q) 32,47 . This spatial control minimizes systemic exposure while maximizing engagement of liver-resident MAIT cells, which are uniquely enriched in this organ. Second, by providing sustained release of the MR1-restricted antigen 5-OP-RU, MARS maintains prolonged MR1 surface presentation on antigen-presenting cells, overcoming the intrinsic instability and short half-life of soluble MAIT antigens and enabling durable TCR-dependent MAIT cell activation (Figs. 2-5) 29,48 . Third, co-delivery of IL-15 supplies essential survival and costimulatory signals that promote MAIT cell expansion, persistence, and cytotoxic programming while limiting activation-induced exhaustion (Figs. 2-5) 49,50 . Fourth, by enabling MAIT cells to simultaneously eliminate MR1⁺ tumor cells and immunosuppressive myeloid populations, MARS effectively remodels the TME, overcoming a key mechanism of resistance in liver cancer immunotherapy (Fig. 6) 21,51–53 . Finally, MARS selectively activates MAIT cells without broadly perturbing other immune populations, resulting in a favorable safety profile in both immunocompetent and humanized models (Fig. 7) 54,55 . In this study, we employed a comprehensive and hierarchical set of experimental assays and disease-relevant models to systematically evaluate MARS-induced MAIT cell activation and antitumor efficacy. In vitro killing assays using diverse human liver cancer cell lines enabled precise dissection of MR1-dependent MAIT cell cytotoxicity across tumors with heterogeneous genetic backgrounds and MR1 expression levels (Figs. 4a-4g). Complementary analyses using primary liver cancer specimens further validated these findings in a clinically relevant context, demonstrating that MARS-enhanced MAIT cells effectively target patient-derived tumor cells while simultaneously eliminating MR1⁺ immunosuppressive myeloid populations within the native TME (Figs. 4h-4n) 5,17 . To assess in vivo efficacy and durability, we utilized a SKHEP1-MR1-FG human liver cancer xenograft model in NSG mice, which revealed that MARS enables sustained MAIT cell persistence, repeated tumor killing, and durable tumor clearance (Fig. 5). Importantly, the NSG-SGM3 myeloid cell–bearing xenograft model provided a stringent test of therapeutic performance under conditions that closely mimic the human immunosuppressive liver TME. In this setting, MARS not only preserved MAIT cell effector function but also reshaped the TME by selectively depleting MR1⁺ suppressive myeloid cells (Fig. 5). Together, these complementary models highlight the robustness, durability, and translational relevance of MARS as a strategy to unlock the full antitumor potential of MAIT cells in liver cancer. We acknowledge that the current experimental models, including the myeloid cell–bearing NSG-SGM3 system, do not fully recapitulate the complexity of the human liver TME, and that immunocompetent mouse models could, in principle, provide additional insights. However, substantial biological differences between murine and human MAIT cells limit the translational relevance of conventional mouse models for evaluating MAIT cell–based therapies. Murine MAIT cells differ markedly from their human counterparts in frequency, developmental pathways, tissue distribution, and functional programming 45,56,57 . In particular, MAIT cells constitute less than 1% of liver lymphocytes in commonly used mouse strains such as C57BL/6, whereas they represent approximately 30–45% of intrahepatic T cells in humans (Figs. 1h and 7b) 14,58–61 . This profound disparity reflects fundamental species-specific differences in microbial exposure, MR1-dependent thymic selection, and peripheral expansion, and results in limited effector capacity of endogenous murine MAIT cells. Moreover, murine MAIT cells exhibit distinct transcriptional and functional profiles, with reduced cytotoxic potential and altered cytokine responsiveness compared with human MAIT cells 22,37,62 . Consequently, immunocompetent mouse models may underestimate the therapeutic potential of MAIT cell–targeted strategies. In this context, the humanized models used in this study provide a more appropriate platform to evaluate MAIT cell activation, persistence, and antitumor function. These considerations further support the rationale that MARS is specifically optimized for human MAIT cell biology and may be particularly well suited for translational application in human liver cancer, where MAIT cells are abundant and functionally relevant. Future development of the MARS platform could further expand its therapeutic versatility and clinical impact. Beyond IL-15, incorporation of additional cytokines or cytokine variants, such as IL-7 to support MAIT cell homeostasis, IL-21 to enhance cytotoxic differentiation, or engineered cytokines with reduced systemic toxicity, may allow fine-tuning of MAIT cell activation states for specific disease contexts 34,63–65 . In addition, MARS could be adapted as a vehicle for in vivo MAIT cell engineering by delivering nucleic acid–based payloads, including mRNA encoding chimeric antigen receptors (CARs), dominant-negative inhibitory receptors, or transcriptional regulators 66,67 . Such an approach would enable transient or programmable reprogramming of endogenous MAIT cells directly in vivo , circumventing the need for ex vivo cell manipulation and adoptive transfer. Beyond liver cancer, the liver-targeted nature of MARS positions it as a promising platform for other liver-associated diseases characterized by immune dysregulation, including liver metastases, chronic viral hepatitis, nonalcoholic steatohepatitis, liver fibrosis, and autoimmune or inflammatory liver disorders 10,11,41 . Collectively, these future directions highlight the potential of MARS as a modular and broadly applicable in vivo immune engineering platform centered on MAIT cells. Methods Study approval This study complies with all relevant ethical regulations. All experiments involving primary liver cancer patient samples were approved by the Ronald Reagan UCLA Medical Center (IRB# IRB-25-0948). Animal studies were approved by the Division of Laboratory Animal Medicine at UCLA. Healthy donor PBMCs were provided by the UCLA/CFAR Virology Core Laboratory without identification information under federal and state regulations. Mice NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NOD- scid IL2Rg null , NSG), NOD.Cg- Prkdc scid Il2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NOD-scid IL2Rgnull-3/GM/SF, NSG-SGM3), and C57BL/6J (B6) mice were purchased from The Jackson Laboratory, and maintained in animal facilities of the UCLA in a temperature-controlled environment (68 °F to 79 °F) with a 12-hour light cycle. 6- to10-week-old mice were used for all experiments. All mice were bred and maintained under specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of UCLA, and all animal procedures were conducted in accordance with the animal care and use regulations of the Division of Laboratory Animal Medicine (DLAM) at UCLA. Given the nature of the liver cancer models, there were no restrictions on tumor size or burden, making direct inferences from external measures unfeasible. Since the tumor cells expressed luciferase, we established a radiance threshold of ≥ 10 10 photons/second per mouse as an upper surrogate limit. In addition, animals that experienced a 20% loss of their original body weight were euthanized. Experimental mice were randomly assigned to treatment groups to avoid statistically significant differences in the baseline tumor burden. Media and reagents Recombinant human IL-2, IL-7, and IL-15 were purchased from PeproTech. Fetal bovine serum (FBS), and β-mercaptoethanol (β-ME) were purchased from Sigma. Penicillin-streptomycin-glutamine (P/S/G), MEM nonessential amino acids (NEAA), HEPES buffer solution, and sodium pyruvate were purchased from Gibco. Normocin was purchased from InvivoGen. The RPMI 1640 cell culture medium and the DMEM cell culture medium were purchased from Thermo Fisher Scientific. The CryoStor Cell Cryopreservation Media CS10 was purchased from MilliporeSigma. The C10 medium was made of RPMI 1640 cell culture medium supplemented with FBS (10% v/v), P/S/G (1% v/v), NEAA (1% v/v), HEPES (10 mM), sodium pyruvate (1 mM), β-ME (50 μM), and Normocin (100 μg/ml). The D10 medium was made of DMEM supplemented with FBS (10% v/v), P/S/G (1% v/v), and Normocin (100 μg/ml). The R10 medium was made of RPMI 1640 supplemented with FBS (10% v/v), P/S/G (1% v/v), and Normocin (100 μg/ml). 5-Amino-4-D-ribitylaminouracil Dihydrochloride (90%) was purchased from TorontoResearchChemicals. Methylglyoxal (MGO) solution was purchased from Sigma. Human and mouse MR1/5-OP-RU tetramers were provided by NIH Tetramer Core Facility. Poly Lactic-co-Glycolic Acid (PLGA), 50:50 was purchased from Polysciences (cat. no. 23987). PRONOVA® UP VLVG (cat. no. 42000501-5G), Sodium carboxymethyl cellulose (cat. no. 419338), Ethylenediaminetetraacetic acid calcium disodium salt hydrate (cat. no. 340073), Calcium chloride (cat. no. C4901) and Acetic acid (cat. no. 338826) were purchased from sigma. Biotinylated human IL-15 R alpha (cat. no. ILA-H82F4) and human IL-15 (cat. no. IL5-H4117) were purchased from ACROBiosystems. MARS manufacturing The MARS was engineered to incorporate both the MAIT antigen precursors 5-A-RU and MGO for sustained generation of 5-OP-RU, as well as surface-presented IL-15. PLGA nanoparticles encapsulating 5-A-RU and MGO were prepared using a water-in-oil-in-water (W/O/W) double-emulsion solvent evaporation method. Briefly, an aqueous solution containing 5-A-RU and MGO was emulsified into an organic phase consisting of PLGA dissolved in an appropriate organic solvent to form the primary water-in-oil (W/O) emulsion. This emulsion was generated by probe ultrasonication at defined power outputs, expressed as a percentage of the maximum instrument output. The primary emulsion was subsequently added to an external aqueous phase and further sonicated to generate the secondary W/O/W emulsion. Organic solvent removal was achieved by evaporation under continuous stirring, resulting in nanoparticle solidification. Nanoparticles were collected by centrifugation, washed extensively with phosphate-buffered saline (PBS), and resuspended for subsequent surface functionalization. To enable physiological IL-15 trans-presentation, human IL-15 receptor α (IL-15Rα) was conjugated to the nanoparticle surface and used to load IL-15 as an IL-15/IL-15Rα complex. Briefly, recombinant human IL-15Rα protein was concentrated to 100 μL in PBS using an Amicon Ultra-2 centrifugal filter unit and reacted with Tetrazine-PEG5-NHS ester at a 1:5 molar ratio (protein:tetr azine) for 30 minutes at room temperature. Excess reagents were removed by desalting using a spin column, followed by five washes with PBS. The purified IL-15Rα–tetrazine conjugate was mixed with glycerol (1:1, v/v) and stored at −20 °C until use. Tetrazine-functionalized IL-15Rα was subsequently conjugated to the surface of PLGA nanoparticles via bioorthogonal chemistry, after which recombinant human IL-15 was loaded by incubation to form stable IL-15/IL-15Rα complexes on the nanoparticle surface. This dual-loading strategy enables MARS to provide sustained release of MR1-restricted MAIT antigen from the nanoparticle core while simultaneously delivering IL-15 through surface trans-presentation, thereby recapitulating key features of antigen-presenting cell–mediated MAIT cell activation. MARS size characterization The hydrodynamic diameter and size distribution of nanoparticles were measured by dynamic light scattering (DLS). Measurements were performed at room temperature, and reported values represent the average of at least three independent measurements. The influence of ultrasonication power output during emulsification on nanoparticle size was systematically evaluated. LC–MS analysis of drug release Release of 5-A-RU-PABC-Val-Cit-Fmoc and MGO from PLGA nanoparticles was quantified by liquid chromatography–mass spectrometry (LC–MS). Nanoparticles formulated with 5-A-RU-PABC-Val-Cit-Fmoc were incubated in PBS at 37 °C under gentle agitation. At predefined time points, samples were centrifuged to pellet nanoparticles, and the supernatants were collected for LC–MS analysis. Chromatographic separation was performed using a reverse-phase column with a water–organic solvent gradient containing a volatile modifier. Quantification of 5-A-RU-PABC-Val-Cit-Fmoc and MGO was achieved by comparison with calibration curves generated from freshly prepared standards. Cumulative release profiles were calculated based on the measured concentrations and reported as a percentage of the total encapsulated cargo. Immunofluorescence staining and imaging Cells were seeded on glass coverslips or imaging-compatible culture surfaces and cultured under indicated conditions. At designated time points, cells were fixed with 4% paraformaldehyde at room temperature, washed with PBS, and permeabilized with 0.1% Triton X-100 when intracellular staining was required. Non-specific binding was blocked using blocking buffer containing 1–5% bovine serum albumin (BSA) in PBS. Cells were incubated with primary antibodies against MAIT cell and antigen-presenting cell markers at manufacturer recommend dilutions, followed by incubation with fluorophore-conjugated secondary antibodies. Nuclei were counterstained with DAPI. After final washes, samples were mounted using antifade mounting medium and imaged using a confocal fluorescence microscope. Identical acquisition settings were used across conditions for quantitative comparison. For analysis of MAIT cell–APC interactions, representative fields were imaged, and conjugate formation or cell–cell contacts were quantified using image analysis software. Lentiviral vectors All lentiviral vectors used in this study were constructed from a parental vector pMNDW 68,69 . The 2A sequence derived from foot-and-mouth disease virus (F2A) was used to link the inserted genes to achieve co-expression. The Lenti/FG vector was constructed by inserting a synthetic bicistronic gene encoding Fluc-P2A-EGFP into the pMNDW 68,70 . The Lenti/MR1 vector was constructed by inserting a synthetic gene encoding human MR1 into the pMNDW. The synthetic gene fragments were obtained from GenScript and IDT. Lentiviruses were produced using human embryonic kidney 293T (HEK293T) cells (ATCC), following a standard transfection protocol using the Trans-IT-Lenti Transfection Reagent (Mirus Bio) and a centrifugation concentration protocol using the Amicon Ultra Centrifugal Filter Units, according to the manufacturer’s instructions (MilliporeSigma). Stable tumor cell lines Human liver cancer cell lines HEPG2, C3A, SNU423, SNU475, and SKHEP1 were purchased from the ATCC. The parental tumor cell lines were transduced with lentiviral vectors encoding the intended gene(s) to produce stable tumor cell lines overexpressing FG or human MR1. 72 hours post lentivector transduction, cells were subjected to flow cytometry sorting to isolate gene-engineered cells for generating stable cell lines. Six stable tumor cell lines were generated for this study, including HEPG2-FG, C3A-FG, SNU423-FG, SNU475-FG, SKHEP1-FG, and SKHEP1-MR1-FG cell lines. All tumor cell lines utilized in this study underwent short tandem repeat (STR) profiling, and the resulting profiles were compared to established databases to confirm accurate identification. Furthermore, the cell lines were regularly screened for mycoplasma contamination to preserve their integrity and authenticity. Liver and PBMC sample collection from liver cancer patients Primary liver cancer patient samples, including liver tumor, normal adjacent liver, and peripheral blood samples, were collected at the Ronald Reagan UCLA Medical Center from consented patients through an IRB-approved protocol (IRB-25-0948) and processed. Information regarding the patients' gender and age was not provided in this study to avoid including three or more indirect identifiers for the study participants. Patient gender was not considered in the study design and was determined based on self-reporting. PBMC collection from healthy donors Healthy donor PBMCs were provided by the UCLA/CFAR Virology Core Laboratory without identification information under federal and state regulations. PBMCs were cryopreserved in Cryostor CS10 (Sigma St. Louis, MO, USA) using CoolCell (BioCision, Larkspur, CA, UCA), and were frozen in liquid nitrogen for storage and to supply all experiments. Antibodies and flow cytometry Fluorochrome-conjugated antibodies specific for human CD3 (clone HIT3a, Pacific Blue, PE, or PE-Cy7-conjugated, 1:500), CD4 (clone OKT4, PE-Cy7, PerCP, or FITC-conjugated, 1:500), CD8 (clone SK1, PE, APC-Cy7, or APC-conjugated, 1:300), CD28 (clone CD28.2, APC, FITC, or Pacific Blue-conjugated, 1:200), CD45 (clone H130, PerCP, FITC or Pacific Blue-conjugated, 1:500), CD56 (clone QA18A21, APC-Cy7, APC, or PE-conjugated, 1:20), CD69 (clone FN50, PE-Cy7 or PerCP-conjugated, 1:50), MR1 (clone 26.5, PE-Cy7, FITC, or APC-conjugated,1:50), TCR Vα7.2 (clone 3C10, PE-Cy7, FITC, PE, or APC-conjugated,1:50), TCRαβ (clone I26, Pacific Blue or PE-Cy7-conjugated, 1:25), IFN-γ (clone B27, PE-Cy7-conjugated, 1:50), NKG2D (clone 1D11, PE-Cy7-conju gated, 1:50), DNAM-1 (clone 11A8, APC-conjugated, 1:50), NKp30 (clone P30-15, APC-conjugated, 1:50), NKp44 (clone P44-8, PE-Cy7-conjugated, 1:50), Granzyme B (clone QA16A02, APC-conjugated, 1:2,000 or 1:5,000), Perforin (clone dG9, PE-Cy7-conjugated, 1:50 or 1:100), IL-2 (clone MQ1-17H12, APC-Cy7-conjugated, 1:50), PD-1 (clone A17188A, FITC, APC, or PE-conjugated, 1:50), LAG-3 (clone 11C3C65, FITC, APC-Cy7, or PE-conjugated, 1:50), TIM-3 (clone A18087E, PE or APC-conjugated, 1:50), and CD45RO (clone UCHL1, PE-Cy7 or APC-Cy7-conjugated, 1:100) were purchased from BioLegend. Fluorochrome-conjugated antibodies specific for mouse CD45 (clone 30-F11, PerCP-conjugated, 1:200), CD3 (clone 17A2, APC-conjugated, 1:200), CD19 (clone 1D3/CD19, APC-Cy7-conjugated, 1:200), CD4 (clone GK1.5, FITC-conjugated, 1:200), CD8 (clone 53-6.7, PE-conjugated, 1:200), CD11b (clone M1/70, PE-Cy7-conjugated, 1:200), CD14 (clone M14-23, PE-conjugated, 1:200), and NK-1.1 (clone S17016D, FITC-conjugated, 1:200) were purchased from BioLegend. Fixable Viability Dye eFluor506 (e506, 1:500) was purchased from Affymetrix eBioscience. Mouse Fc Block (anti-mouse CD16/32) was purchased from BD Biosciences, and human Fc Receptor Blocking Solution (TrueStain FcX) was purchased from BioLegend. PE-conjugated human and mouse MR1/5-OP-RU Tetramer (1:500 dilution) was obtained from NIH Tetramer Core Facility. All flow cytometry staining was performed following standard protocols, as well as specific instructions provided by the manufacturer of a particular antibody. Stained cells were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotech), following the manufacturers’ instructions. FlowJo software version 9 (BD Biosciences) was used for data analysis. For intracellular cytokine staining, the cells were thawed and resuspended in C10 medium. Cells were stimulated with PMA (Calbiochem, cat. no. 524400; 50 ng/mL) and ionomycin (Calbiochem, cat. no. 407952.; 500 ng/mL) and incubated at 37°C for 2 hours. GolgiStop (BD Biosciences, car. No. 554724; 1.5 µL/mL) was then added to inhibit cytokine secretion, followed by an additional 4-hour incubation. Subsequently, intracellular staining was performed using the Cell Fixation/Permeabilization Kit (BD Biosciences, cat. no. 554714) according to the manufacturer’s instructions. Enzyme-linked immunosorbent cytokine assays (ELISAs) The ELISAs for detecting human cytokines were performed following a standard protocol from BD Biosciences. Supernatants from cell culture assays were collected and assayed to quantify human IFN-γ, IL-2, IL-12, and TNF-α. The capture and biotinylated pairs for detecting cytokines were purchased from BD Biosciences. The streptavidin-HRP conjugate was purchased from Invitrogen. Human cytokine standards were purchased from eBioscience. Tetramethylbenzidine substrate was purchased from KPL. The samples were analyzed for absorbance at 450 nm using an Infinite M1000 microplate reader (Tecan). Generation of MAIT cells from healthy donor or liver cancer patient PBMCs PBMCs from healthy donors or liver cancer patients were used to generate MAIT cells. Cells were enriched via a two-step MACS protocol, first stained with PE-conjugated MR1/5-OP-RU tetramer and then labeled with Anti-PE MicroBeads (Miltenyi Biotec) for magnetic separation. The sorted MAIT cells were co-cultured with irradiated autologous PBMCs at a 1:1 ratio in C10 medium supplemented with 5-OP-RU (50 nM) and human IL-7 and IL-15 (10 ng/mL each). Cultures were maintained for 2 weeks with periodic IL-7 and IL-15 cytokine supplementation. Expanded MAIT cells could be subsequently purified by FACS to isolate MR1/5-OP-RU tetramer + TCR Vα7.2 + CD3 + cells for downstream applications. In vitro MAIT cell stimulation assay MAIT cells were stimulated from either liver cancer patient PBMCs or tumor sample-derived single-cell suspensions under the indicated conditions. A total of 1 × 10 6 live cells were cultured in 1 mL C10 medium supplemented with human IL-7 and IL-15 (10 ng/mL each). Depending on the experimental setup, cultures were additionally treated with 5-OP-RU (100 nM), 5-OP-RU (100 nM) plus IL-15 (10 ng/mL), or MARS (1 x 10 6 nanoparticles). The frequency and activation phenotype of MAIT cells (identified as CD3 + MR1/5-OP-RU tetramer + TCR Vα7.2 + cells) were analyzed by flow cytometry throughout the assay. In vitro tumor cell killing assay Liver tumor cells (1 10 4 cells per well) were co-cultured with MAIT cells (at ratios indicated in the figures or figure legends) in Corning 96-well clear bottom black plates for 24 h in C10 medium. D-luciferin (150 mg/ml, Caliper Life Science) was added to cell cultures to quantify live tumor cells and luciferase activities were read out using an Infinite M1000 microplate reader (Tecan). Depending on the experimental condition, cultures were supplemented with 5-OP-RU (100 nM), 5-OP-RU (100 nM) plus IL-15 (10 ng/mL), or MARS (1 x 10 4 nanoparticles) as specified in the figure legends. In vitro assays using primary liver cancer patient samples In one assay, the primary liver cancer patient samples were analyzed for tumor cell phenotype and the TME composition using flow cytometry. Liver tumor cells were sorted using a Human Tumor Cell Isolation Kit (Miltenyi Biotec) and/or identified as CD45 - CD31 - FAP (fibroblast activation protein) - cells 71–73 , T cells were identified as CD45 + CD3 + cells, CD4 T cells were identified as CD4 + T cells, CD8 T cells were identified as CD8 + T cells, MAIT cells were identified as MR1/5-OP-RU tetramer + TCR Vα7.2 + T cells, B cells were identified as CD45 + CD19 + or CD45 + CD20 + cells, NK cells were identified as CD45 + CD56 + CD3 - cells, myeloid cells were identified as CD45 + CD11b + cells. Surface expression of MR1 on tumor or/and immune cells were also analyzed using flow cytometry. In another assay, the primary liver cancer patient samples were used to study tumor and TME cell killing by MAIT cells under various conditions. Patient samples (containing 1 x 10 5 cells) were directly co-cultured with MAIT cells (1 x 10 5 cells) in C10 medium in Corning 96-well Round Bottom Cell Culture plates for 24 hours. Depending on the experimental condition, cultures were supplemented with 5-OP-RU (100 nM), 5-OP-RU (100 nM) plus IL-15 (10 ng/mL), or MARS (1 x 10 5 nanoparticles) as specified in the figure legends. At the end of culture, cells were collected, and the tumor and TME cell targeting by MAIT cells was assessed using flow cytometry by quantifying live human tumor cells (identified as MR1/5-OP-RU tetramer - CD45 - cells), myeloid cells (identified as MR1/5-OP-RU tetramer - CD45 + CD11b + cells), T cells (identified as MR1/5-OP-RU tetramer - CD45 + CD3 + cells), B cells (identified as MR1/5-OP-RU tetramer - CD45 + CD19 + cells or MR1/5-OP-RU tetramer - CD45 + CD20 + cells), and NK cells (identified as MR1/5-OP-RU tetramer - CD45 + CD3 - CD56 + cells). A total of 3 primary liver cancer patient samples were included in this assay. Western blot Western blot analysis was performed to examine TCR- and IL-15–dependent signaling pathways in MAIT cells. MAIT cells from liver cancer patient samples were stimulated with soluble 5-OP-RU (100 nM), soluble 5-OP-RU (100 nM) in combination with IL-15 (10 ng/mL), or MARS (1 × 10 6 nanoparticles) for 15 minutes or 1 hour at 37 °C. Following stimulation, cells were immediately transferred into ice-cold PBS to terminate signaling and washed three times to remove residual antigen or cytokines. MAIT cells were then isolated by FACS based on CD3⁺TCR Vα7.2⁺ expression for subsequent protein extraction and western blot analysis. Total proteins were extracted using a RIPA lysis buffer (Thermo Fisher Scientific) containing 20 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Tritonx-100, and protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Protein concentration was measured using a Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher Scientific). Equal amounts of total protein were resolved on a 4–15% Mini-PROTEAN® TGX™ Precast Protein Gel (BIO-RAD) and then transferred to a polyvinylidene difluoride (PVDF) membrane by electrophoresis. The following antibodies were used to blot for the proteins of interest: anti-human p-LCK(Tyr394) (clone E5L3D, Cell Signaling Technology, CST), anti-human LCK (clone D88, CST), anti-human p-ZAP70 (Tyr319) (clone 65E4, CST), anti-human ZAP70 (clone D1C10E, CST), anti-human p-PLC gamma 1 (Tyr783) (clone D6M95, CST), anti-human PLC gamma 1 (clone D9H10, CST), anti-human p-c-Jun 1 (Ser73) (clone D47G9, CST), anti-human c-Jun (clone 60A8, CST), anti-human p-NF-kappaB p65 (Ser536) (clone 93H1, CST), anti-human NF-kappaB p65 (clone D14E12, CST), anti-human p-JAK1 (Tyr1034/1035) (clone D7N4Z1, CST), anti-human JAK1 (clone 6G4, CST), anti-human p-JAK3 (Tyr980/981) (clone D44E3, CST), anti-human JAK3 (clone D2E12, CST), anti-human p-STAT3 (Tyr705) (clone D3A7, CST), anti-human STAT3 (clone D7B12, CST), anti-human p-STAT5 (Tyr694) (clone D47E7, CST), anti-human STAT5 (clone D2O6Y, CST), anti-human C-Myc (clone D84C12, CST), anti-human C-Fos (clone 9F6, CST), anti-human BCL2 (clone D17C4, CST), and secondary anti-rabbit IgG (CST). b-Actin (clone D6A8, CST) was used as internal controls. Signals were visualized using a ChemiDoc ™ Imaging Systems (BIO-RAD). The data were analyzed using ImageJ (Version 1.53s). Single cell RNA sequencing (scRNA-seq) scRNA-seq was utilized to examine the gene profiles of primary liver cancer patient-derived MAIT cells. Data from Gene Expression Omnibus database (GSE149614), Genome Sequence Archive in BIG Data Center (HRA000069), and European Genome-phenome Archive (EGAS00001003449) were included for scRNA-seq analyses 20,21 . For cell clustering and annotation, the merged digital expression matrix generated by Cellranger was analyzed using an R package Seurat (v.4.0.0) following the guidelines 74–76 . Briefly, after filtering the low-quality cells, the expression matrix was normalized using NormalizeData function, followed by selecting variable features across datasets using FindVariableFeatures and SelectIntegrationFeatures functions. To correct the batch effect, FindIntegrrationAnchors and IntegrateData functions were used based on the selected feature genes. The corrected dataset was subjected to standard Seurat workflow for dimension reduction and clustering. In this study, clusters of therapeutic cells were manually merged and annotated based on gene signatures reported from Human Protein Atlas (proteinatlas.org) and previous studies 77–84 . AddModuleScore was used to calculate module scores of each list of gene signatures, and FeaturePlot function was used to visualize the expression of each signature in the UMAP plots. In vivo bioluminescence imaging (BLI) BLI was performed using a Spectral Advanced Molecular Imaging HTX system (Spectral Instrument Imaging). Live animal images were captured 5 minutes after intraperitoneal (i.p.) injection of D-Luciferin (1 mg per 100 μL PBS per mouse) to obtain total body bioluminescence. The imaging data were analyzed using AURA imaging software (version 3.2.0, Spectral Instrument Imaging). In vivo antitumor efficacy study of MAIT cells: SKHEP1-MR1-FG human liver cancer xenograft NSG mouse model Experimental design is shown in Fig. 5A. Briefly, on Day 0, NSG mice received i.v. inoculation of SKHEP1-MR1-FG human liver cancer cells (5 x 10 5 cells per mouse). On Day 5, the experimental mice received i.v. injection of Vehicle (100 μl PBS per mouse), or human MAIT cells (1 x 10 7 cells in 100 μl PBS per mouse). The MAIT cell-treated mice were divided into three groups: Group 1 received an intravenous injection of MARS (1 x 10 7 nanoparticles), Group 2 received a single dose of 5-OP-RU (1 µM in 200 µL PBS), and Group 3 received 5-OP-RU (1 µM in 200 µL PBS) once weekly. Over the experiment, mice were monitored for survival and their tumor loads were measured twice per week using BLI. At the end of the experiment, mice were euthanized, and their tissues were collected for analysis of MAIT cell phenotypes and functions by flow cytometry. In vivo antitumor and anti-TME efficacy study of MAIT cells: SKHEP1-FG human liver cancer xenograft NSG-SGM3 mouse model Experimental design is shown in Fig. 6A. Briefly, on Day -7 and -1, NSG-SGM3 mice received i.v. injection and healthy donor PBMC-derived CD14 + myeloid cells (5 x 10 6 cells per mouse) to establish a liver tumor microenvironment enriched with TAM-like cells. On Day 0, NSG-SGM3 mice received i.v. inoculation of SKHEP1- FG human liver cancer cells (5 x 10 5 cells per mouse). On Day 5, the experimental mice received i.v. injection of Vehicle (100 μl PBS per mouse), or human MAIT cells (1 x 10 7 cells in 100 μl PBS per mouse). The MAIT cell-treated mice were divided into two groups: Group 1 received an intravenous injection of MARS (1 x 10 7 nanoparticles), Group 2 received an intravenous injection of Vehicle (100 μl PBS per mouse). Throughout the study, mice were given weekly i.v. injections of PBMC-derived CD14 + myeloid cells (5 × 10 6 cells per mouse) to sustain the humanized TME. Mice were monitored for survival and their tumor loads were measured twice per week using BLI. At the end of the experiment, mice were euthanized, and their tissues were collected for analysis of MAIT cell phenotypes and functions by flow cytometry. In vivo safety study of MARS in B6 mouse model Experimental design is shown in Fig. 7A. On Day 1, C57BL/6 (B6) mice were i.v. injected with MARS (1 x 10 7 nanoparticles) and monitored for potential toxicity and safety. On Days 10 and 40, mice were euthanized, and tissues including blood, spleen, and liver were collected for immunophenotypic analyses. Mouse immune cell populations were examined by flow cytometry and identified as follows: MAIT cells (CD45 + TCRβ + MR1/5-OP-RU tetramer + ), T cells (CD45 + TCRβ + ), B cells (CD45 + CD19+), NK cells (CD45 + NK1.1 + TCRβ − ), and monocytes (CD45 + CD11b + Ly6C + ). Peripheral blood samples were analyzed for hematological parameters, including white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT) levels. In vivo safety study of MARS in human MAIT cell xenograft NSG mouse model Experimental design is shown in Fig. 7E. On Day 0, NSG mice were i.v. injected with human MAIT cells (1 x 10 7 cells in 100 μl PBS per mouse). On Day 1, the experimental mice were i.v. injected with MARS (1 x 10 7 nanoparticles) and monitored for potential toxicity and safety. During the experiment, mouse body weights were recorded regularly to monitor general health status. On Days 1, 40, and 80, blood samples were collected for assessment of organ toxicity markers, including urea, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and glutamate dehydrogenase (GLDH), using ELISA assays. On Day 80, mice were euthanized, and multiple organs were harvested for histopathological analysis following established standard procedures 69,85 . Statistics Statistical data analysis was performed using GraphPad Prism 8 software (GraphPad). Student’s two-tailed t test was employed for pairwise comparisons. Ordinary one- or two-way ANOVA followed by Tukey’s or Dunnett’s multiple comparisons test was used for multiple comparisons. Log rank (Mantel-Cox) test adjusted for multiple comparisons was used for Meier survival curves analysis. Data are expressed as the mean ±SEM, unless otherwise indicated. In all figures and figure legends, n denotes the number of samples or animals utilized in the indicated experiments. A p-value of less than 0.05 was considered significant; ns indicates not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Declarations Acknowledgements We thank the University of California, Los Angeles (UCLA) animal facility for providing animal support; the UCLA Translational Pathology Core Laboratory (TPCL) for providing histology support; the UCLA Technology Centre for Genomics & Bioinformatics (TCGB) facility for providing RNA-seq services; the UCLA CFAR Virology Core for providing human cells; and the UCLA BSCRC Flow Cytometry Core Facility for cell sorting support. We thank NIH Tetramer Core Facility for providing the tetramers. This work was supported by a Partnering Opportunity for Discovery Stage Research Projects Award and a Partnering Opportunity for Translational Research Projects Awards from the California Institute for Regenerative Medicine (DISC2-11157, DISC2-13015, TRAN1-12250, and TRAN1-16050 to L.Y.), a Department of Defense CDMRP PRCRP Impact Award (CA200456 to L.Y.), a Department of Defense Kidney Cancer Research Program Award (KC230215 to L.Y.), a UCLA BSCRC Innovation Award (to L.Y.), and an Ablon Scholars Award (to L.Y.). L.Y. is a member of UCLA Parker Institute for Cancer Immunotherapy (PICI). Y.-R.L. is a postdoctoral fellow supported by a UCLA MIMG M. John Pickett Post-Doctoral Fellow Award, a CIRM-BSCRC Postdoctoral Fellowship, a UCLA Sydney Finegold Postdoctoral Award, a UCLA Chancellor’s Award for Postdoctoral Research, and a UCLA Goodman-Luskin Microbiome Center Collaborative Research Fellowship Award. AUTHOR CONTRIBUTIONS Y-R.L., H.N., Z.S., and X.S. designed the experiments, analyzed the data, and wrote the manuscript. L.Y. and S.L. conceived and oversaw the study. Y-R.L. H.N., Z.S., and X.S. performed all experiments, with assistance from Y.C., Y.Z., J.H., S.Y., and T.Y.. S.G. and V.G.A. provided the primary patient samples. DECLARATION OF INTERESTS L.Y. is a scientific advisor to AlzChem and Amberstone Biosciences, and a co-founder, stockholder, and advisory board member of Appia Bio. None of the declared companies contributed to or directed any of the research reported in this article. The remaining authors declare no competing interests. Data Availability Statement The data that support the findings of this study are available on request from the corresponding author. References Ruf, B. et al. Activating Mucosal-Associated Invariant T Cells Induces a Broad Antitumor Response. Cancer Immunol. Res. 9 , 1024–1034 (2021). Li, Y.-R. et al. 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None of the declared companies contributed to or directed any of the research reported in this article. The remaining authors declare no competing interests. Supplementary Files SupplementaryInformation.docx Supplementary Information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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08:11:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8518619/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8518619/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100416834,"identity":"02304499-9156-404d-9235-56916d283689","added_by":"auto","created_at":"2026-01-16 13:23:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5110530,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLiver-resident MAIT cells display reduced frequency and increased exhaustion in liver cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Experimental design to profile primary liver cancer patient samples using scRNA-seq and flow cytometry. 31 liver cancer samples (LC) and 29 normal adjacent liver samples (NA) were included for analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e Profiling liver-resident T cells in primary samples using scRNA-seq. \u003cstrong\u003eb\u003c/strong\u003e Combined UMAP showing the three major cell clusters. \u003cstrong\u003ec\u003c/strong\u003e Individual UMAP plots of the LC and NA samples. \u003cstrong\u003ed\u003c/strong\u003e Bar graphs showing the cell cluster proportions of the LC and NA samples. \u003cstrong\u003ee\u003c/strong\u003e Violin plots showing the expression distribution of the indicated gene signatures in the LC and NA samples. \u003cstrong\u003ef \u003c/strong\u003eDot plots showing the expression of representative signature genes. Color saturation reflects the average expression level of each gene, while dot size represents the percentage of cells in the cluster expressing that gene. \u003cstrong\u003eg\u003c/strong\u003e KEGG pathway analyses of genes upregulated in MAIT cells compared to CD4 or CD8 T cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003em\u003c/strong\u003e Profiling liver-resident MAIT cells in primary samples using flow cytometry. \u003cstrong\u003eh\u003c/strong\u003e FACS plots showing the presence of MAIT and conventional T (Tc) cells in matched LC and NA samples. \u003cstrong\u003ei\u003c/strong\u003e Quantification of MAIT percentage of total CD45\u003csup\u003e+\u003c/sup\u003e immune cells (n = 5). \u003cstrong\u003ej\u003c/strong\u003e Quantification of MAIT percentage of total CD3\u003csup\u003e+\u003c/sup\u003e T cells (n = 5). \u003cstrong\u003ek\u003c/strong\u003e FACS plots showing the MAIT subpopulations in the indicated liver and blood samples. Tn, naïve T; Tcm, century memory T; Tem, effector memory T; Te, effector T. \u003cstrong\u003el\u003c/strong\u003e FACS plots showing the PD-1 and LAG-3 expression, as well as Perforin and Granzyme B production in MAIT cells. iso, isotype staining. \u003cstrong\u003em\u003c/strong\u003e Quantification of\u003cstrong\u003e l\u003c/strong\u003e (n = 5).\u003c/p\u003e\n\u003cp\u003eRepresentative of 1 (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e) and 5 (\u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003em\u003c/strong\u003e) experiments. Data are presented as the mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, by Student’s \u003cem\u003et\u003c/em\u003e test (\u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e, and \u003cstrong\u003em\u003c/strong\u003e). P values shown in the violin plots were calculated using a two-tailed Wilcoxon rank-sum test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8518619/v1/0519e131737bef9c0ecdee89.png"},{"id":100417210,"identity":"142b453b-e670-414f-a2f6-50ddb82f9ce2","added_by":"auto","created_at":"2026-01-16 13:24:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5612758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenerate MARS as a biomimetic nanoparticle-based platform for liver-targeted delivery of 5-OP-RU and IL-15.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Schematic illustrating the working model of MARS. Following systemic administration, MARS preferentially homes to and accumulates in the liver, where the majority of nanoparticles are internalized by hepatic antigen-presenting cells (APCs). This uptake enables sustained MR1-restricted presentation of the MAIT agonist 5-OP-RU together with localized IL-15 delivery and trans-presentation. In parallel, a fraction of MARS directly engages MAIT cells through IL-15–mediated signaling. Coordinated TCR engagement and IL-15 stimulation result in robust MAIT cell activation, persistence, and cytotoxic programming, leading to enhanced targeting and elimination of MR1⁺ liver tumor cells and MR1⁺immunosuppressive myeloid cells within the tumor microenvironment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ef \u003c/strong\u003eStudy the physicochemical properties of MARS.\u003cstrong\u003e b\u003c/strong\u003e Size distributions of PLGA nanoparticles formulated with varying ultrasonication power output, (80%, 40%, and 20%), measured by dynamic light scattering (DLS), demonstrating tunable particle sizes across the submicron range.\u003cstrong\u003e c \u003c/strong\u003eCumulative release profiles of 5-OP-RU from PLGA nanoparticles at different PLGA-to-cargo ratios (30:1, 15:1, 5:1, and 2:1), showing formulation-dependent sustained release over 20 days (n = 4). \u003cstrong\u003ed\u003c/strong\u003e Encapsulation efficiency of 5-OP-RU as a function of PLGA content, indicating reduced loading efficiency at lower polymer ratios (n = 5).\u003cstrong\u003e e\u003c/strong\u003e Representative scanning electron microscopy (SEM) image of PLGA nanoparticles displaying uniform spherical morphology. Scale bars: 500 nm (left), 450 nm (right). \u003cstrong\u003ef\u003c/strong\u003eElemental composition analysis (C, O, N, S) of nanoparticles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003ej \u003c/strong\u003eStudy the sustained antigen presentation and MAIT-engaging properties of MARS.\u003cstrong\u003e g\u003c/strong\u003e Left: Confocal microscopy images showing intracellular uptake and MR1 presentation of MARS by APCs over time (days 1, 5, 14, and 20). The staining of nanoparticles (red), MR1 (green), and nuclei (DAPI, cyan) are shown. Scale bar: 8 μm. Right: Flow cytometry plots showing the percentage of nanoparticle-internalized APCs at corresponding time points. \u003cstrong\u003eh\u003c/strong\u003e Mean fluorescence intensity (MFI) analysis of intracellular PLGA signal decay and MR1 surface expression over time, indicating gradual nanoparticle degradation coupled with sustained MR1 presentation (n = 5). \u003cstrong\u003ei\u003c/strong\u003eRepresentative immunofluorescence images showing time-dependent enhancement of MAIT-APC interactions, consistent with increased MR1-restricted antigen presentation on APCs. The staining of CD3ε (green), CD14 (red), and nuclei (DAPI, cyan) are shown. \u003cstrong\u003ej \u003c/strong\u003eQuantification of CD3ε fluorescence intensity on MAIT cells (n = 10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ek-q\u003c/strong\u003eStudying the liver homing properties of MARS. \u003cstrong\u003ek\u003c/strong\u003e Longitudinal \u003cem\u003ein vivo\u003c/em\u003efluorescence imaging of Cy5-labeled MARS showing its preferential liver localization and prolonged retention.\u003cstrong\u003e l\u003c/strong\u003e Quantification of \u003cstrong\u003ek\u003c/strong\u003e (n = 3). \u003cstrong\u003em\u003c/strong\u003e Analysis of nanoparticle-associated signal area in mice over time (n = 3). \u003cstrong\u003en\u003c/strong\u003e Biodistribution analysis of fluorescence signal in liver versus other tissues at indicated time points (n = 3). \u003cstrong\u003eo\u003c/strong\u003e Body weight measurements over time (n = 3). \u003cstrong\u003ep\u003c/strong\u003e Tissue fluorescence imaging of major organs at day 10, confirming dominant MARS accumulation in the liver. \u003cstrong\u003eq\u003c/strong\u003eQuantification of \u003cstrong\u003ep\u003c/strong\u003e (n = 5).\u003c/p\u003e\n\u003cp\u003eRepresentative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, by one-way ANOVA (\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003e j\u003c/strong\u003e, and \u003cstrong\u003eq\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8518619/v1/47be5512d69b82ed746417fb.png"},{"id":100417312,"identity":"e046c091-d9b1-4b49-a5b5-9b1df3a4da58","added_by":"auto","created_at":"2026-01-16 13:24:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5769751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMARS induces MAIT cell activation via TCR engagement and IL-15-dependent signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e Studying the MARS effects on liver cancer patient PBMC-derived MAIT cells. \u003cstrong\u003ea\u003c/strong\u003eExperimental design. \u003cstrong\u003eb\u003c/strong\u003e FACS plots showing the enrichment of MAIT cells over time following stimulation by different methods. \u003cstrong\u003ec\u003c/strong\u003e Quantification of MAIT cell expansion folds and percentages over time following stimulation by different methods (n = 4). \u003cstrong\u003ed\u003c/strong\u003e FACS plots showing the expression of CD25 and CD62L on MAIT cells over time following MARS stimulation. \u003cstrong\u003ee\u003c/strong\u003eQuantification of expression of the indicated markers on MAIT cells over time following MARS stimulation (n = 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003el\u003c/strong\u003e Studying the MARS effects on patient liver tumor-derived MAIT cells. \u003cstrong\u003ef \u003c/strong\u003eExperimental design. \u003cstrong\u003eg\u003c/strong\u003e FACS plots showing the enrichment of MAIT cells 7 days following stimulation by different methods. \u003cstrong\u003eh\u003c/strong\u003e Quantification of MAIT cell expansion folds and percentages 7 days following stimulation by different methods (n = 4). \u003cstrong\u003ei\u003c/strong\u003e ELISA analyses of cytokine production in the culture supernatant 3 days following stimulation by different methods (n = 4). \u003cstrong\u003ej \u003c/strong\u003eFACS analyses of activation marker (i.e., CD25 and CD69) expression on MAIT cells with or without MARS activation (n = 4). \u003cstrong\u003ek\u003c/strong\u003e FACS analyses of exhausted MAIT cell percentage with or without MARS activation (n = 4). \u003cstrong\u003el\u003c/strong\u003e FACS analyses of cytotoxic molecules (i.e., Granzyme B and CD107a) production in MAIT cells with or without MARS activation (n = 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003em\u003c/strong\u003e-\u003cstrong\u003ep\u003c/strong\u003e Studying the TCR engagement and IL-15-dependent signaling in MAIT cells stimulated by MARS. \u003cstrong\u003em\u003c/strong\u003e Western blot analyses of key molecules involved in MAIT TCR engagement in MAIT cells following stimulation by different methods. \u003cstrong\u003en \u003c/strong\u003eQuantification of \u003cstrong\u003em\u003c/strong\u003e (n = 4). \u003cstrong\u003eo\u003c/strong\u003eWestern blot analyses of key molecules involved in IL-15-dependent signaling in MAIT cells following stimulation by different methods. \u003cstrong\u003ep\u003c/strong\u003e Schematics illustrating the mechanisms of MAIT cell activation by MARS.\u003c/p\u003e\n\u003cp\u003eRepresentative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, by Student’s\u003cem\u003e t\u003c/em\u003e test (\u003cstrong\u003ej\u003c/strong\u003e-\u003cstrong\u003el\u003c/strong\u003e), one-way ANOVA (\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eand \u003cstrong\u003en\u003c/strong\u003e), or two-way ANOVA (\u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8518619/v1/8fc42dc9adfaeaaa756d1d07.png"},{"id":100416840,"identity":"c97f303e-6153-4f47-b027-47303016247a","added_by":"auto","created_at":"2026-01-16 13:23:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3710082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMARS enhances MAIT cell antitumor activity against liver cancer \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e Studying MARS-enhanced MAIT cell antitumor activity using human liver cancer cell lines. \u003cstrong\u003ea\u003c/strong\u003e Experimental design. \u003cstrong\u003eb\u003c/strong\u003e Schematics showing the six human liver tumor cell lines utilized in this study. All were engineered to express the firefly luciferase (Fluc) and enhanced green fluorescent protein (EGFP) dual reporters (FG). \u003cstrong\u003ec\u003c/strong\u003e FACS detection of MR1 expression on the indicated tumor cells. \u003cstrong\u003ed\u003c/strong\u003e Tumor cell killing data at 24 h (n = 4). \u003cstrong\u003ee\u003c/strong\u003e MAIT cell expansion 24 hours after co-culture with SKHEP1-MR1-FG cells (n = 4). \u003cstrong\u003ef\u003c/strong\u003e ELISA analyses of effector cytokine production by MAIT cells 24 hours after co-culture with SKHEP1-MR1-FG cells (n = 4). \u003cstrong\u003eg\u003c/strong\u003e FACS analyses of activation marker (i.e., CD69) expression and cytotoxic molecule (i.e., Perforin and Granzyme B) production by MAIT cells 24 hours after co-culture with SKHEP1-MR1-FG cells (n = 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e Studying MARS-enhanced MAIT cell antitumor activity using primary liver cancer samples. \u003cstrong\u003eh \u003c/strong\u003eExperimental design. \u003cstrong\u003ei\u003c/strong\u003e FACS plots showing the MR1 expression on liver tumor cells from 3 primary patient samples. \u003cstrong\u003ej\u003c/strong\u003e Liver tumor cell killing data at 24 h (n = 4). \u003cstrong\u003ek\u003c/strong\u003e FACS plots showing the MR1 expression on liver TME cells from primary patient sample #1. \u003cstrong\u003el\u003c/strong\u003e Quantification of \u003cstrong\u003ek\u003c/strong\u003e (n = 3). \u003cstrong\u003em\u003c/strong\u003e Liver MR1\u003csup\u003e+\u003c/sup\u003e myeloid cell killing data at 24 h (n = 4). \u003cstrong\u003en\u003c/strong\u003e Immune cell (i.e., T, B, and NK) killing data at 24 h (n = 4).\u003c/p\u003e\n\u003cp\u003eRepresentative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, by one-way ANOVA (\u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e, and \u003cstrong\u003el\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e), or two-way ANOVA (\u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8518619/v1/34b8a482ec73cf754a43c232.png"},{"id":100416853,"identity":"3667eb55-f85e-4a72-b782-9aaa3d6ca60d","added_by":"auto","created_at":"2026-01-16 13:23:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4285312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMARS enhances MAIT cell antitumor activity, persistence, and effector function against liver cancer \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eExperimental design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e BLI images showing tumor loads in experimental mice over time. Data from three representative experimental mice are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003eQuantification of \u003cstrong\u003eb\u003c/strong\u003e (n = 7). Data from individual experimental mice are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed \u003c/strong\u003eBLI quantification displaying data from all experimental mice\u003cstrong\u003e \u003c/strong\u003e(n = 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003eKaplan-Meier survival curves of experimental mice over time (n = 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003eFACS analyses showing GFP⁺SKHEP1-MR1-FG liver tumor cells in mouse livers collected from the indicated groups at days 30 and 60 (n = 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003eFACS plots showing human MAIT cells in mouse livers collected from the indicated groups at days 30 and 60.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003eQuantification of \u003cstrong\u003eg \u003c/strong\u003e(n = 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei\u003c/strong\u003e FACS detection of the effector (i.e., CD25 and CD69) and exhaustion marker (i.e., LAG-3 and TIM-3) expression on MAIT cells in mouse livers collected from the indicated groups at day 30.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej \u003c/strong\u003eQuantification of \u003cstrong\u003ei \u003c/strong\u003e(n = 5).\u003c/p\u003e\n\u003cp\u003eRepresentative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, by one-way ANOVA (\u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e, and\u003cstrong\u003eg\u003c/strong\u003e), or log rank (Mantel-Cox) test adjusted for multiple comparisons (\u003cstrong\u003ee\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8518619/v1/e19c0e3a1eb8347c74490a27.png"},{"id":100417026,"identity":"a8555b80-c0df-4e66-b92e-d11952dd0438","added_by":"auto","created_at":"2026-01-16 13:24:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5053573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMARS reprograms the liver tumor microenvironment and sustains MAIT cell antitumor activity \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Experimental design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb \u003c/strong\u003eBLI images showing tumor loads in experimental mice over time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Quantification of \u003cstrong\u003eb\u003c/strong\u003e (n = 5-7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Kaplan-Meier survival curves of experimental mice over time (n = 5-7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e FACS analyses showing GFP⁺ SKHEP1-FG liver tumor cells in mouse livers collected from the indicated groups at day 20 (n = 3-5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e FACS plots showing GFP⁺ SKHEP1-FG liver tumor cells, human CD14\u003csup\u003e+\u003c/sup\u003e myeloid cells, and MAIT cells in mouse livers collected from the indicated groups at day 20.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg \u003c/strong\u003eand\u003cstrong\u003e h\u003c/strong\u003e, FACS analyses showing the percentage of human CD14\u003csup\u003e+\u003c/sup\u003e myeloid (\u003cstrong\u003eg\u003c/strong\u003e) and MAIT (\u003cstrong\u003eh\u003c/strong\u003e) cells in mouse livers collected from the indicated groups at day 20 (n = 3-5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei \u003c/strong\u003ePie charts showing the proportions of human CD14\u003csup\u003e+\u003c/sup\u003e myeloid and MAIT cells (n = 3-5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej\u003c/strong\u003e FACS plots showing the expression of human CD163, CD206, and MR1 on myeloid cells collected from experimental mice in Vehicle group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ek\u003c/strong\u003e and \u003cstrong\u003el\u003c/strong\u003e, ELISA analyses immune-activating cytokines (IFN-γ and IL-2; \u003cstrong\u003ek\u003c/strong\u003e), and immunosuppressive TME cytokines (IL-6 and IL-1β; \u003cstrong\u003el\u003c/strong\u003e) in mouse serum at day 20 (n = 3-5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003em \u003c/strong\u003eFACS detection of the effector (CD25 and CD69) and exhaustion marker (LAG-3 and TIM-3) expression on MAIT cells in mouse livers with or without MARS at day 20.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003en \u003c/strong\u003eQuantification of \u003cstrong\u003em \u003c/strong\u003e(n = 5).\u003c/p\u003e\n\u003cp\u003eRepresentative of 2 experiments. Data are presented as the mean ± SEM. ns, not significant, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, by Student’s \u003cem\u003et\u003c/em\u003e test (\u003cstrong\u003en\u003c/strong\u003e), one-way ANOVA (\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e g\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e, and\u003cstrong\u003e l\u003c/strong\u003e), or log rank (Mantel-Cox) test adjusted for multiple comparisons (\u003cstrong\u003ed\u003c/strong\u003e).\u003cstrong\u003e\u003cbr\u003e\n\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8518619/v1/6a0d160d44af990c0a6ae5a3.png"},{"id":100417055,"identity":"c0d1d504-254c-41f4-a5ee-801e72b3872d","added_by":"auto","created_at":"2026-01-16 13:24:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3636267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMARS demonstrated high safety profile \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e Studying the \u003cem\u003ein vivo\u003c/em\u003e safety of MARS in C57BL/6 mice. \u003cstrong\u003ea\u003c/strong\u003e Experimental design. \u003cstrong\u003eb\u003c/strong\u003eFACS analysis of MAIT cell percentages among total T cells in mice with or without MARS injection across the indicated tissues and time points (n = 5). Mouse MAIT cells were defined as mouse MR1/5-OP-RU tetramer⁺TCRβ⁺ cells. \u003cstrong\u003ec\u003c/strong\u003e FACS analysis of other immune cell percentages among total leukocytes in mice with or without MARS injection across the indicated tissues and time points (n = 5). \u003cstrong\u003ed\u003c/strong\u003e Blood panel analyses, including white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT) levels in mice with or without MARS injection across the indicated time points (n = 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e Studying the \u003cem\u003ein vivo\u003c/em\u003e safety of human MAIT cells and MARS in NSG mice. \u003cstrong\u003ee\u003c/strong\u003e Experimental design. \u003cstrong\u003ef \u003c/strong\u003eBody weight changes of NSG mice monitored over 80 days following MAIT cell and MARS injections (n = 5). \u003cstrong\u003eg\u003c/strong\u003e Heatmap showing the organ damage markers measured in blood samples at days 1, 40, and 80. Data collected from five mice were averaged and normalized to values obtained from blank NSG mice. \u003cstrong\u003eh\u003c/strong\u003e Pathological assessment of major organs collected from experimental mice at day 80.\u003c/p\u003e\n\u003cp\u003eRepresentative of 3 (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e) and 1 (\u003cstrong\u003eh\u003c/strong\u003e) experiments. Data are presented as the mean ± SEM. ns, not significant, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, by one-way ANOVA (\u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8518619/v1/0c232cfd4ed280085b2c962e.png"},{"id":102298091,"identity":"d7dfd338-aeac-42ad-95b3-1a679adfb0a1","added_by":"auto","created_at":"2026-02-10 10:30:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30304836,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8518619/v1/de3aede3-adab-47aa-8432-1cdf0ea9786d.pdf"},{"id":100416368,"identity":"67558c03-fc71-4f5f-83fb-643062d72853","added_by":"auto","created_at":"2026-01-16 13:23:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2896834,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8518619/v1/6b8819924cf3267de3ff354e.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nL.Y. is a scientific advisor to AlzChem and Amberstone Biosciences, and a co-founder, stockholder, and advisory board member of Appia Bio. None of the declared companies contributed to or directed any of the research reported in this article. The remaining authors declare no competing interests.","formattedTitle":"An in vivo MAIT cell activation and rejuvenation system for liver cancer therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMucosal-associated invariant T (MAIT) cells are a unique subset of unconventional \u0026alpha;\u0026beta; T cells with potent antimicrobial and antitumor activities\u003csup\u003e1\u0026ndash;4\u003c/sup\u003e. These cells express a semi-invariant T-cell receptor (TCR) (V\u0026alpha;7.2-J\u0026alpha;33/12/20 in humans and V\u0026alpha;19-J\u0026alpha;33 in mice) that recognizes the monomorphic MHC class I-related molecule MR1 presenting vitamin B2 (riboflavin) metabolites such as 5-OP-RU\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e. Although MAIT cells comprise only about 1-5% of circulating T cells, they are highly enriched in the liver, where they can account for up to 30-45% of intrahepatic T cells in both healthy individuals and liver cancer patients\u003csup\u003e1,8,9\u003c/sup\u003e. This liver tropism is attributed to the preferential expression of chemokine receptors such as CCR6, CXCR6, and CXCR3 on MAIT cells, which guide their recruitment to the hepatic microenvironment\u003csup\u003e10,11\u003c/sup\u003e. Importantly, clinical and transcriptomic analyses have revealed that a higher MAIT cell gene signature is positively correlated with improved overall survival in hepatocellular carcinoma (HCC) patients, suggesting a potential protective and tumor-controlling role of MAIT cells in liver cancer\u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHowever, MAIT cells within the liver tumor microenvironment (TME) of cancer patients often exhibit an exhausted and dysfunctional phenotype. Their infiltration into tumor tissues is generally limited, and those that do localize to tumors display impaired cytotoxicity and reduced effector function\u003csup\u003e9,12,13\u003c/sup\u003e. Human MAIT cells in HCC lesions frequently show increased expression of activation and exhaustion markers, including PD-1, CD25, and HLA-DR, reflecting a state of chronic stimulation coupled with functional impairment\u003csup\u003e9\u003c/sup\u003e. Recent studies have also highlighted that interactions with niche-residing CSF1R\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e tumor-associated macrophages (TAMs) contribute to this dysfunctional state, acting as a key regulatory mechanism that restrains MAIT cell antitumor activity\u003csup\u003e9\u003c/sup\u003e. Collectively, these observations indicate that despite their intrinsic antitumor potential, MAIT cells in the liver TME are rendered ineffective by both cellular exhaustion and suppressive microenvironmental cues. Therefore, strategies aimed at activating or rejuvenating MAIT cells within the liver cancer TME are critical for restoring their cytotoxic function and enhancing their antitumor capacity.\u003c/p\u003e\n\u003cp\u003eMicrobial metabolites, such as 5-OP-RU, play a critical role in MAIT cell development, maturation, and functional competence, including their antitumor activity\u003csup\u003e5,14,15\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e administration of 5-OP-RU can efficiently trigger MAIT cell-mediated killing of both MR1\u003csup\u003e+\u0026nbsp;\u003c/sup\u003etumor cells and MR1\u003csup\u003e+\u003c/sup\u003e TAMs, highlighting a dual capacity to target malignant cells and the immunosuppressive TME\u003csup\u003e16,17\u003c/sup\u003e. Moreover, \u003cem\u003ein vivo\u003c/em\u003e co-administration of 5-OP-RU with CpG oligonucleotides has been shown to induce robust systemic expansion and activation of MAIT cells, leading to potent and broad antitumor immune responses in murine models of liver metastasis, HCC, lung metastasis, and subcutaneous tumors.\u003csup\u003e1\u003c/sup\u003e Despite these promising results, direct administration of 5-OP-RU faces significant limitations, including rapid degradation, short \u003cem\u003ein vivo\u003c/em\u003e half-life, and the need for frequent dosing to maintain MAIT cell activation\u003csup\u003e18,19\u003c/sup\u003e. Additionally, systemic delivery may result in off-target effects or suboptimal localization within the TME. Therefore, there is a critical need to develop strategies that enable sustained, localized, and efficient \u003cem\u003ein vivo\u003c/em\u003e activation of MAIT cells to fully harness their antitumor potential.\u003c/p\u003e\n\u003cp\u003eTo overcome these limitations and enhance MAIT cell-mediated antitumor immunity in liver cancer, we developed a MAIT cell activation and rejuvenation system (MARS), a biomimetic platform designed for targeted delivery to the liver and preferential engagement of liver-residing MAIT cells. MARS provides sustained release of 5-OP-RU to rejuvenate and activate MAIT cells, thereby restoring their cytotoxic capacity against liver tumor cells. In addition, the system delivers human IL-15 to promote MAIT cell survival, expansion, and sustained cytotoxic function. Through its biomimetic design and liver-localized immunostimulatory effects, MARS is expected to efficiently reprogram the liver TME, enhance MAIT cell activation and function, and elicit a potent, localized antitumor immune response in liver cancer, representing a promising strategy to overcome immunosuppression in the liver TME.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eLiver-resident MAIT cells exhibit reduced frequency and increased exhaustion in primary liver cancer patients\u003c/p\u003e\n\u003cp\u003eTo characterize the phenotype and functionality of MAIT cells in liver cancer, we performed single-cell RNA sequencing (scRNA-seq) and flow cytometry analyses on primary liver samples, including 31 liver cancer (LC) and 29 normal adjacent (NA) tissues (Fig.1a and Supplementary Table 1). The scRNA-seq analyses were conducted using two publicly available datasets containing matched LC and NA samples derived from the same patients (Supplementary Fig.\u0026nbsp;1a)\u003csup\u003e20,21\u003c/sup\u003e. In parallel, flow cytometry analyses were performed on in-house specimens from five LC and five patient-matched NA samples (Fig. 1a). The inclusion of paired LC and NA samples allowed us to directly compare MAIT cell phenotypes within the same individual, minimizing inter-patient variability and providing a more accurate assessment of disease-associated alterations.\u003c/p\u003e\n\u003cp\u003eWe first analyzed the scRNA-seq data of liver-resident T cells, including CD4 T, CD8 T, and MAIT cells (Fig.1b). MAIT cells were identified based on the expression of \u003cem\u003eSLC4A10\u003c/em\u003e, a specific marker distinguishing this subset from conventional T cells (Supplementary Fig.\u0026nbsp;1b)\u003csup\u003e22\u003c/sup\u003e. LC samples showed a decreased frequency of MAIT cells but an increased proportion of CD4 T cells compared to NA tissues (Figs. 1c and 1d). Comparative analyses of MAIT cells between LC and NA regions revealed that tumor-infiltrating MAIT cells exhibited enhanced proliferative, effector, memory, and exhaustion signatures, suggesting a state of activation and exhaustion driven by the liver TME (Fig. 1e), consistent with previous findings in HCC patients.\u003csup\u003e9\u003c/sup\u003e These results indicate that liver-resident MAIT cells in cancer exhibit a hyperactivated yet partially exhausted phenotype, reflecting their persistent engagement in the tumor immune response.\u003c/p\u003e\n\u003cp\u003eFurthermore, when comparing CD4 T, CD8 T, and MAIT cells in both NA and LC samples, MAIT cells displayed the strongest effector and memory phenotypes, cytotoxic activity comparable to CD8 T cells but greater than CD4 T cells, the highest early exhaustion features, and lower terminal exhaustion compared to CD8 T cells but higher than CD4 T cells (Supplementary Fig.\u0026nbsp;1c). Consistent with these findings, heatmap analyses demonstrated that MAIT cells expressed significantly higher levels of natural killer (NK)\u0026ndash;associated genes (e.g., \u003cem\u003eKLRB1\u003c/em\u003e, \u003cem\u003eNCAM1\u003c/em\u003e, \u003cem\u003eNCR3\u003c/em\u003e, and \u003cem\u003eZBTB16\u003c/em\u003e), effector-related genes (\u003cem\u003eCD69\u003c/em\u003e, \u003cem\u003eIL18R1\u003c/em\u003e, and \u003cem\u003eCD48\u003c/em\u003e), memory-associated genes (\u003cem\u003eGZMM\u003c/em\u003e, \u003cem\u003eCCR5\u003c/em\u003e, and \u003cem\u003eFOS\u003c/em\u003e), and cytotoxicity-related genes (\u003cem\u003eCYCS\u003c/em\u003e, \u003cem\u003eSERPINB9\u003c/em\u003e, \u003cem\u003ePRF1\u003c/em\u003e, and \u003cem\u003eCTSN\u003c/em\u003e) compared with CD4 and CD8 T cells (Fig. 1f). Pathway enrichment analyses further revealed that, in LC samples, MAIT cells showed significant upregulation of genes involved in NK cell\u0026ndash;mediated cytotoxicity, antigen processing and presentation, TNF signaling, and Toll-like receptor signaling pathways compared with CD4 T cells (Fig. 1g). In addition, relative to CD8 T cells, MAIT cells exhibited enhanced enrichment of genes associated with the PD-1/PD-L1 immune checkpoint pathway, Th1/Th2 cell differentiation, TNF signaling, and NF-\u0026kappa;B signaling pathways (Fig. 1g). Overall, these results indicate that MAIT cells in the liver TME possess strong innate-like effector and NK-mediated cytotoxic programs but simultaneously display features of activation-induced exhaustion.\u003c/p\u003e\n\u003cp\u003eWe next validated these findings by flow cytometry analysis of five paired in-house LC and NA liver samples (Supplementary Table 1). Consistent with the scRNA-seq results, LC tissues exhibited a significantly reduced frequency of MAIT cells compared with NA tissues, confirming numerical loss of MAIT cells in liver cancer (Figs. 1h-1j). Phenotypic characterization revealed that liver-resident MAIT cells from both LC and NA tissues, as well as MAIT cells in the peripheral blood of liver cancer patients, predominantly displayed a CD62L\u003csup\u003e-\u003c/sup\u003eCD45RO\u003csup\u003e+\u003c/sup\u003e effector memory phenotype (Fig. 1k). This phenotype was distinct from that of conventional T cells in the circulation, which comprised na\u0026iuml;ve (CD62L\u003csup\u003e+\u003c/sup\u003eCD45RO\u003csup\u003e-\u003c/sup\u003e), central memory (CD62L\u003csup\u003e+\u003c/sup\u003eCD45RO\u003csup\u003e+\u003c/sup\u003e), effector (CD62L\u003csup\u003e-\u003c/sup\u003eCD45RO\u003csup\u003e-\u003c/sup\u003e), and effector memory subsets (Fig. 1k). In contrast, the majority of conventional T cells within the liver also exhibited an effector memory phenotype (Fig. 1k). These findings are consistent with previous reports identifying MAIT cells as a highly differentiated effector memory T cell subset\u003csup\u003e14,23,24\u003c/sup\u003e. We further examined the expression of exhaustion and cytotoxicity-associated markers in MAIT cells from LC and NA tissues. MAIT cells within LC samples expressed significantly higher levels of the inhibitory receptors PD-1 and LAG-3, accompanied by markedly reduced expression of cytotoxic effector molecules, including perforin and granzyme B (Figs. 1l and 1m). These data indicate that MAIT cells in liver cancer exhibit an exhaustion-associated phenotype coupled with impaired cytotoxic function.\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that MAIT cells represent a distinct T cell population within the liver, characterized by an effector memory phenotype and innate-like cytotoxic potential. However, in the context of liver cancer, MAIT cells appear to undergo chronic activation leading to functional exhaustion and diminished cytotoxic molecule production. These findings underscore the critical importance of MAIT cells in liver tumor immunity and highlight the need for therapeutic strategies aimed at reactivating and rejuvenating MAIT cell function to enhance antitumor responses in liver cancer.\u003c/p\u003e\n\n\u003cp\u003eEngineering of MARS as a biomimetic platform delivering 5-OP-RU and IL-15 to activate MAIT cells\u003c/p\u003e\n\u003cp\u003eWe therefore developed\u0026nbsp;a MAIT cell activation and rejuvenation system (MARS), a biomimetic, liver-targeted platform based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles that enables sustained delivery of the MAIT agonist 5-OP-RU and human IL-15 (Fig. 2a). 5-OP-RU specifically activates MAIT cells through TCR\u0026ndash;MR1 recognition, while IL-15 enhances MAIT cell survival and granzyme B\u0026ndash;dependent cytotoxicity\u003csup\u003e15,25,26\u003c/sup\u003e. Following systemic administration, MARS preferentially accumulates in the liver and is taken up by hepatic antigen-presenting cells, including myeloid cells, enabling prolonged MR1-restricted antigen presentation and localized MAIT cell activation (Fig. 2a). This strategy restores MAIT cell effector function and enables simultaneous targeting of liver tumor cells and immunosuppressive myeloid populations within the liver TME.\u003c/p\u003e\n\u003cp\u003eMARS was fabricated by encapsulating the MAIT antigen precursors 5-amino-6-D-ribitylaminouracil (5-A-RU) and methylglyoxal (MGO) within PLGA nanoparticles, allowing \u003cem\u003ein situ\u003c/em\u003e formation and sustained release of the active MAIT agonist 5-OP-RU\u003csup\u003e27\u003c/sup\u003e. To further recapitulate physiological MAIT cell activation, we engineered the nanoparticle surface to mimic cytokine trans-presentation by antigen-presenting cells. Specifically, IL-15 receptor \u0026alpha; (IL-15R\u0026alpha;) was conjugated to the surface of MARS to bind and present human IL-15 as an IL-15/IL-15R\u0026alpha; complex, thereby enabling efficient IL-15 trans-presentation to MAIT cells expressing the IL-2/15 receptor \u0026beta; chain and common \u0026gamma; chain (Fig. 2a). This design allows coordinated delivery of MR1-restricted antigen and IL-15\u0026ndash;dependent costimulatory signals, closely resembling natural APC-mediated MAIT cell activation in the liver.\u003c/p\u003e\n\u003cp\u003eWe first characterized the physicochemical properties of MARS to establish parameters critical for efficient MAIT cell activation and safe \u003cem\u003ein vivo\u003c/em\u003e application. Dynamic light scattering analysis showed that nanoparticle size could be precisely controlled by modulating ultrasonication power, yielding uniform submicron particles optimized for systemic circulation and preferential hepatic accumulation, a prerequisite for effective engagement of liver-resident MAIT cells (Fig. 2b). Controlled release studies demonstrated formulation-dependent and sustained release of 5-OP-RU, with higher polymer-to-cargo ratios enabling prolonged antigen availability and supporting sustained MR1-restricted antigen presentation for continuous MAIT cell stimulation (Fig. 2c). Consistent with this, encapsulation efficiency decreased with reduced PLGA content, highlighting the importance of polymer composition in maintaining antigen stability and bioavailability (Fig. 2d). Scanning electron microscopy confirmed the spherical morphology and smooth surface of MARS nanoparticles (Fig. 2e), features associated with favorable biodistribution and cellular uptake, while elemental analysis verified successful incorporation of nitrogen- and sulfur-containing antigen components, confirming effective loading of the MAIT agonist (Fig. 2f).\u003c/p\u003e\n\u003cp\u003eWe next investigated whether MARS enables sustained MR1-restricted antigen presentation and functional engagement of MAIT cells \u003cem\u003ein vitro\u003c/em\u003e. In the absence of continuous antigen availability, MR1 surface expression is known to rapidly decline, underscoring the need for sustained delivery of MAIT agonists\u003csup\u003e28,29\u003c/sup\u003e. To address this, MARS nanoparticles were co-cultured with CD14⁺\u0026nbsp;myeloid cells derived from healthy donor peripheral blood mononuclear cells (PBMCs), which serve as professional antigen-presenting cells (APCs). These APCs efficiently internalized MARS nanoparticles, with uptake detectable for up to two weeks and gradually declining by day 20 (Figs. 2g and 2h). Importantly, MARS-treated APCs exhibited enhanced and sustained surface expression of MR1 (Figs. 2g and 2h), indicating continuous release of 5-OP-RU from the nanoparticles and consistent with previous reports demonstrating that 5-OP-RU stabilizes and upregulates MR1 on APCs and tumor cells\u003csup\u003e30,31\u003c/sup\u003e. Functionally, MARS-loaded APCs maintained prolonged antigen presentation capacity, as evidenced by immunofluorescence imaging during MAIT/APC co-culture, which revealed progressively increased and stabilized cell\u0026ndash;cell contacts over time (Fig. 2i). Quantitative analyses further demonstrated that these sustained interactions translated into enhanced MAIT cell activation (Fig. 2j). Together, these results indicate that MARS provides sustained 5-OP-RU release, promotes prolonged MR1 surface antigen availability, and thereby improves the efficiency and durability of MAIT cell engagement and activation.\u003c/p\u003e\n\n\u003cp\u003eMARS demonstrates efficient and selective liver homing\u003c/p\u003e\n\u003cp\u003eWe next evaluated the \u003cem\u003ein vivo\u003c/em\u003e homing behavior of MARS following systemic administration. Longitudinal whole-body fluorescence imaging revealed rapid and preferential accumulation of MARS nanoparticles in the liver, with sustained retention over an extended period, indicating efficient hepatic targeting (Figs. 2k-2n). This prolonged liver residence is critical for continuous local delivery of immunomodulatory cargos and effective engagement of liver-resident MAIT cells.\u003c/p\u003e\n\u003cp\u003eThroughout the treatment period, no significant changes in body weight were observed, suggesting favorable tolerability and the absence of overt systemic toxicity (Fig. 2o). Consistent with the imaging data, tissue biodistribution analysis confirmed dominant localization of MARS within the liver, with minimal off-target accumulation in other major organs, including the lung, heart, spleen, and kidney (Figs. 2p and 2q). This selective biodistribution profile is consistent with the physicochemical properties of the biomimetic nanoparticles and supports their capacity for efficient hepatic uptake and retention\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that MARS achieves selective and sustained liver targeting with minimal systemic exposure, providing a favorable safety profile and establishing a strong foundation for localized MAIT cell activation and effective liver cancer immunotherapy \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\n\u003cp\u003eMARS induces superior MAIT cell activation and proliferation compared with conventional approaches\u003c/p\u003e\n\u003cp\u003eWe first evaluated the ability of MARS to induce MAIT cell proliferation and activation using PBMCs derived from liver cancer patients (Supplementary Table 1), in which MAIT cells comprised approximately 0.5\u0026ndash;2% of total CD3⁺\u0026nbsp;T cells (Figs. 3a and 3b). Three stimulation strategies were compared: 5-OP-RU alone, 5-OP-RU combined with IL-15, and MARS (Fig. 3a). Notably, PBMCs contain abundant myeloid cells that function as APCs and mediate MR1-dependent presentation of 5-OP-RU to MAIT cells.\u003c/p\u003e\n\u003cp\u003eFollowing a 15-day co-culture, all three conditions induced robust MAIT cell expansion and activation; however, MARS consistently elicited the strongest response, characterized by greater fold expansion and more rapid activation (Figs. 3b and 3c). MARS-stimulated MAIT cells exhibited marked upregulation of activation, effector, and memory-associated markers, including CD25, CD44, CD69, CD62L, and CD45RO (Figs. 3d and 3e). Importantly, compared with conventional T cells within the same cultures, MAIT cells expressed significantly higher levels of these markers, indicating selective and preferential activation of MAIT cells by MARS (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003eWe next assessed MARS-mediated activation in primary liver cancer tissues to more closely model liver-resident MAIT cells, which comprised approximately 10\u0026ndash;30% of total viable cells, consistent with previous reports (Figs. 3f and 3g, and Supplementary Table 1)\u003csup\u003e9,13\u003c/sup\u003e. While all three stimulation strategies promoted MAIT cell expansion, MARS again demonstrated superior efficacy, as evidenced by greater MAIT cell enrichment, enhanced proliferation, and increased production of effector cytokines, including IFN-\u0026gamma;, TNF-\u0026alpha;, IL-2, and IL-12, together with elevated expression of activation markers CD25 and CD69 (Figs. 3g-3j). Notably, sustained IL-15 delivery by MARS resulted in a reduced proportion of exhausted MAIT cells (PD-1⁺LAG-3⁺TIM-3⁺) and enhanced cytotoxic features, including increased granzyme B expression and CD107a degranulation (Figs. 3k and 3l).\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that MARS induces more robust, sustained, and MAIT cell\u0026ndash;specific activation and expansion than conventional stimulation approaches, while simultaneously limiting exhaustion and enhancing cytotoxic function, highlighting its potential as an effective strategy to rejuvenate MAIT cells for liver cancer immunotherapy.\u003c/p\u003e\n\n\u003cp\u003eMARS induces MAIT cell activation via TCR engagement and IL-15-dependent signaling\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;We next sought to confirm that MARS-mediated MAIT cell activation is driven by coordinated TCR engagement and IL-15\u0026ndash;dependent signaling. Liver-resident MAIT cells from liver cancer patients were stimulated using three approaches, including 5-OP-RU alone, 5-OP-RU combined with IL-15, or MARS, followed by cell sorting and analysis of downstream signaling pathways by western blot.\u003c/p\u003e\n\u003cp\u003eAll three stimulation strategies induced phosphorylation of key components of the MAIT TCR signaling cascade, including LCK, ZAP70, and PLC\u0026gamma;1, as well as activation of downstream transcription factors such as c-Jun and NF-\u0026kappa;B (Figs. 3m and 3n). These findings confirm effective MR1-restricted TCR engagement by 5-OP-RU. Notably, MARS stimulation resulted in stronger and more sustained phosphorylation of these signaling molecules compared with soluble 5-OP-RU or 5-OP-RU plus IL-15, indicating enhanced and prolonged TCR signaling, likely due to sustained antigen availability (Figs. 3m and 3n).\u003c/p\u003e\n\u003cp\u003eIn addition to TCR signaling, both 5-OP-RU plus IL-15 and MARS robustly activated IL-15\u0026ndash;dependent pathways, as evidenced by increased phosphorylation of JAK1, JAK3, STAT3, and STAT5. This activation was accompanied by upregulation of key transcriptional regulators associated with MAIT cell survival, proliferation, and effector differentiation, including c-Myc, c-Fos, and BCL2 (Fig. 3o and Supplementary Fig.\u0026nbsp;2)\u003csup\u003e33,34\u003c/sup\u003e. Importantly, MARS induced higher and more sustained activation of these IL-15\u0026ndash;dependent signaling nodes compared with the soluble cytokine condition, consistent with prolonged IL-15 bioavailability (Fig. 3o and Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that MARS functions as a dual-signal platform that simultaneously delivers sustained MR1-restricted antigen stimulation and IL-15\u0026ndash;mediated costimulatory signaling (Fig. 3p). By engaging both the MAIT TCR and IL-15 receptor pathways, MARS promotes robust MAIT cell activation, survival, and effector programming, providing a mechanistic basis for its superior capacity to rejuvenate MAIT cells within the liver TME.\u003c/p\u003e\n\n\u003cp\u003eMARS enhances MAIT cell antitumor activity against liver cancer \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe next evaluated the cytotoxic activity of MARS-activated MAIT cells against human liver tumor cells \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e(Fig. 4a). An \u003cem\u003ein vitro\u003c/em\u003e tumor cell killing assay was performed using six human liver cancer cell lines representing distinct genetic backgrounds and tumor origins (Fig. 4b). Five parental tumor cell lines (HepG2, C3A, SNU423, SNU475, and SKHEP1) were engineered to express a firefly luciferase and enhanced green fluorescent protein dual reporter (FG), enabling quantitative assessment of tumor cell viability by both flow cytometry and luminescence assays (Fig. 4b). To directly assess MR1-dependent recognition, SKHEP1 cells were additionally engineered to overexpress MR1 (SKHEP1-MR1-FG). Baseline analysis confirmed heterogeneous MR1 expression across the tumor cell panel, reflecting the diversity of MR1 levels observed in human liver cancers (Figs. 4b and 4c).\u003c/p\u003e\n\u003cp\u003eLiver cancer patient PBMC\u0026ndash;derived MAIT cells were co-cultured with tumor cells under four conditions: no stimulation, 5-OP-RU alone, 5-OP-RU combined with IL-15, or MARS (Fig. 4a). During the first round of killing, all three stimulation strategies significantly enhanced MAIT cell\u0026ndash;mediated cytotoxicity against all tumor cell lines compared with the unstimulated condition, with MARS consistently inducing the strongest tumor killing (Fig. 4d). These results indicate that MR1-restricted antigen presentation is sufficient to trigger MAIT cell recognition and cytotoxicity against liver tumor cells across a range of MR1 expression levels. Notably, SKHEP1-MR1-FG cells were killed more efficiently than parental SKHEP1-FG cells, demonstrating a positive correlation between MR1 expression and MAIT cell\u0026ndash;mediated tumor killing (Fig. 4d).\u003c/p\u003e\n\u003cp\u003eTo assess the durability of MAIT cell cytotoxic function, we performed repeated tumor challenge assays, in which MAIT cells were exposed to fresh tumor cells for a third killing round. While MAIT cell cytotoxicity declined under soluble 5-OP-RU or 5-OP-RU plus IL-15 stimulation, MARS-treated MAIT cells maintained robust tumor-killing capacity (Fig. 4d). This sustained cytotoxicity was accompanied by significantly greater MAIT cell expansion (Fig. 4e), elevated production of effector cytokines (IFN-\u0026gamma;, TNF-\u0026alpha;, and IL-2) (Fig. 4f), increased expression of the activation marker CD69, and enhanced production of cytotoxic molecules including perforin and granzyme B (Fig. 4g), indicating that MARS supports long-term MAIT cell functionality.\u003c/p\u003e\n\u003cp\u003eWe further evaluated MAIT cell cytotoxicity against primary liver tumor cells derived from liver cancer patients (Fig. 4h). These primary tumor cells expressed variable but generally high levels of MR1, supporting their susceptibility to MAIT cell\u0026ndash;mediated recognition (Fig. 4i). Consistent with the cell line data, 5-OP-RU stimulation induced MAIT cell\u0026ndash;mediated killing of primary tumor cells, while MARS significantly enhanced this effect (Fig. 4j). Importantly, primary liver tumor samples contained diverse immune cell populations within the liver TME, including immunosuppressive myeloid cells, T cells, B cells, and NK cells. Myeloid cells exhibited markedly higher MR1 expression compared with other immune subsets (Figs. 4k and 4l), and upon MARS activation, MAIT cells selectively and efficiently eliminated these MR1⁺\u0026nbsp;immunosuppressive myeloid cells while sparing other immune populations (Figs. 4m and 4n). These findings indicate that MARS not only enhances direct tumor cell killing but also enables MAIT cells to remodel the TME by selectively targeting MR1-expressing immunosuppressive cells.\u003c/p\u003e\n\u003cp\u003eIn conclusion, MARS confers robust, sustained, and MR1-dependent cytotoxic activity to MAIT cells against both liver tumor cells and immunosuppressive myeloid populations. By preserving MAIT cell effector function over repeated tumor encounters and selectively reshaping the TME, MARS establishes a powerful dual mechanism for enhancing MAIT cell\u0026ndash;based immunotherapy in liver cancer.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\n\u003cp\u003eMARS enhances MAIT cell antitumor activity, persistence, and effector function against liver cancer in a xenograft mouse model\u003c/p\u003e\n\u003cp\u003eWe next evaluated the \u003cem\u003ein vivo\u003c/em\u003e antitumor capacity of MAIT cells following activation by MARS. A human liver cancer xenograft model was established by intravenously injecting 5 \u0026times; 10⁵ SKHEP1-MR1-FG cells into NSG mice, allowing preferential tumor seeding and growth within the liver, thereby mimicking the clinical features of liver cancer (Fig. 5a). Four therapeutic conditions were compared: adoptive transfer of MAIT cells alone, MAIT cells with a single intravenous dose of 5-OP-RU, MAIT cells with weekly intravenous doses of 5-OP-RU, and MAIT cells combined with intravenous administration of MARS (Fig. 5a).\u003c/p\u003e\n\u003cp\u003eBioluminescence imaging (BLI) revealed that adoptively transferred MAIT cells alone exerted minimal tumor control, likely due to insufficient endogenous antigen stimulation \u003cem\u003ein vivo\u003c/em\u003e (Figs. 5b-5e). A single dose of 5-OP-RU significantly enhanced MAIT cell\u0026ndash;mediated antitumor activity; however, repeated weekly administration of soluble 5-OP-RU did not further improve tumor control, likely reflecting its rapid degradation and short \u003cem\u003ein vivo\u003c/em\u003e half-life (Figs. 5b-5e). In contrast, MARS treatment resulted in robust tumor elimination, as evidenced by near-complete loss of BLI signal in the liver and a marked improvement in overall survival (Figs. 5b-5e).\u003c/p\u003e\n\u003cp\u003eFlow cytometric analysis of liver-resident cells confirmed a stepwise reduction in GFP⁺\u0026nbsp;tumor cells following MAIT cell transfer, further enhanced by 5-OP-RU treatment, and complete tumor clearance in the MARS-treated group (Fig. 5f). In parallel, human MAIT cells were readily detected in the liver following adoptive transfer. While MAIT cell persistence was limited in the absence of stimulation and only modestly improved by soluble 5-OP-RU, MARS treatment dramatically enhanced MAIT cell persistence \u003cem\u003ein vivo\u003c/em\u003e, with detectable MAIT cells maintained in the liver for up to 60 days (Figs. 5g and 5h). Importantly, MARS-activated MAIT cells exhibited a highly functional phenotype, characterized by increased expression of activation markers CD25 and CD69 and reduced expression of exhaustion markers LAG-3 and TIM-3 (Figs. 5i, 5j, and Supplementary Fig.\u0026nbsp;4). These findings indicate that MARS provides sustained delivery of both 5-OP-RU and IL-15 \u003cem\u003ein vivo\u003c/em\u003e, thereby promoting durable MAIT cell activation, persistence, and effector function within the liver.\u003c/p\u003e\n\u003cp\u003eOverall, these results demonstrate that MARS markedly enhances the \u003cem\u003ein vivo\u003c/em\u003e antitumor efficacy of MAIT cells by overcoming antigen limitation and functional exhaustion, leading to durable tumor clearance and prolonged survival in a clinically relevant liver cancer model.\u003c/p\u003e\n\n\u003cp\u003eMARS reprograms the liver TME and sustains MAIT cell antitumor activity in a xenograft mouse model\u003c/p\u003e\n\u003cp\u003eWe next evaluated the capacity of MAIT cells to target both liver tumor cells and the immunosuppressive TME under conditions that more closely recapitulate the human liver cancer setting. To this end, we utilized NSG-SGM3 mice, which express human SCF, GM-CSF, and IL-3 and therefore support efficient engraftment and persistence of human myeloid cells, enabling the establishment of a humanized immunosuppressive TME\u003csup\u003e35,36\u003c/sup\u003e. Human CD14⁺\u0026nbsp;myeloid cells were administered weekly, resulting in sustained engraftment \u003cem\u003ein vivo\u003c/em\u003e, including within the liver, where they functioned as key immunosuppressive components of the TME (Fig. 6a). On day 0, SKHEP1-FG liver tumor cells were intravenously injected to establish liver tumors (Fig. 6a). Importantly, these parental tumor cells expressed relatively low levels of MR1 (Fig. 4c), allowing us to directly assess whether MARS-mediated MAIT cell activation is sufficient to drive tumor killing in a complex TME without artificial MR1 overexpression. MAIT cells derived from the same donor as the myeloid cells were adoptively transferred, and MARS was administered intravenously (Fig. 6a).\u003c/p\u003e\n\u003cp\u003eBLI confirmed successful tumor establishment in the liver, characterized by rapid tumor growth and reduced survival in control mice (Figs. 6c-6e). While adoptively transferred MAIT cells alone moderately suppressed tumor progression, MARS treatment significantly enhanced MAIT cell\u0026ndash;mediated antitumor activity, as evidenced by markedly reduced tumor burden, prolonged survival, and diminished detection of GFP⁺\u0026nbsp;tumor cells in the liver (Figs. 6c-6e). These results demonstrate that MARS confers potent antitumor capacity to MAIT cells even within a highly immunosuppressive and humanized liver TME.\u003c/p\u003e\n\u003cp\u003eAnalysis of the liver microenvironment revealed robust engraftment of human myeloid cells (Figs. 6f and 6g). Notably, MAIT cell transfer led to a reduction in these myeloid populations, and this effect was further amplified by MARS treatment (Figs. 6f-6i). Phenotypic characterization showed that liver-infiltrating myeloid cells expressed high levels of immunosuppressive macrophage markers CD163 and CD206, as well as MR1, indicating that they represent MR1⁺\u0026nbsp;immunosuppressive targets within the TME (Fig. 6j). These findings suggest that MARS-activated MAIT cells selectively eliminate MR1-expressing immunosuppressive myeloid cells, thereby reshaping the TME in favor of antitumor immunity.\u003c/p\u003e\n\u003cp\u003eConsistent with these cellular changes, serum cytokine analysis by ELISA revealed that MARS treatment significantly increased MAIT cell\u0026ndash;associated effector cytokines, including IFN-\u0026gamma; and IL-2 (Fig. 6k), while reducing levels of proinflammatory myeloid-derived cytokines such as IL-6 and IL-1\u0026beta; (Fig. 6l). This cytokine profile is consistent with depletion of immunosuppressive myeloid cells and restoration of a more immunostimulatory liver environment. Finally, phenotypic analysis of liver-resident MAIT cells demonstrated that MARS activation induced a highly functional state, characterized by elevated expression of activation markers CD25 and CD69 and reduced expression of exhaustion markers TIM-3 and LAG-3 (Figs. 6m and 6n). Together, these data indicate that MARS not only enhances MAIT cell\u0026ndash;mediated tumor killing but also sustains MAIT cell fitness and prevents functional exhaustion within the liver TME.\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that MARS enables MAIT cells to simultaneously target liver tumor cells and remodel an immunosuppressive humanized TME by selectively eliminating MR1⁺\u0026nbsp;myeloid cells, sustaining effector function, and promoting durable antitumor immunity \u003cem\u003ein vivo\u003c/em\u003e. This dual targeting strategy positions MARS-activated MAIT cell therapy as a promising therapeutic approach for liver cancers that are resistant to conventional treatments due to a highly immunosuppressive TME.\u003c/p\u003e\n\n\u003cp\u003eMARS demonstrates favorable safety profile \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe next evaluated the \u003cem\u003ein vivo\u003c/em\u003e safety profile of MARS. We first assessed a mouse-adapted MARS formulation encapsulating 5-OP-RU and murine IL-15 in immunocompetent C57BL/6 mice (Fig. 7a). Following intravenous administration, mice remained healthy with no signs of morbidity or mortality for up to 40 days post-injection, indicating favorable tolerability. Immunophenotypic analysis revealed a transient enrichment of endogenous murine MAIT cells at approximately 10 days post-injection in the blood, liver, and spleen (Fig. 7b). Notably, given the substantially lower frequency of MAIT cells in mice compared with humans, this expansion was modest and less pronounced than that observed in humanized systems (Figs. 6f and 7b). Importantly, no significant alterations were detected in the frequency or phenotype of other immune populations, including conventional T cells, B cells, NK cells, or monocytes, indicating selective immune modulation by MARS (Fig. 7c). Comprehensive hematological analyses further demonstrated no significant differences in white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) levels, or hematocrit (HCT) values between MARS-treated and control mice, suggesting the absence of systemic toxicity or hematopoietic disruption (Fig. 7d).\u003c/p\u003e\n\u003cp\u003eWe next evaluated the safety of MARS in a humanized NSG mouse model receiving adoptive transfer of human MAIT cells (Fig. 7e). Following intravenous administration of MAIT cells and MARS, mice maintained stable body weight throughout the study period (Fig. 7f). Serum analyses revealed no elevation of organ damage\u0026ndash;associated biomarkers, and histopathological examination showed no evidence of tissue injury or inflammation across major organs, including liver, lung, heart, spleen, and kidney, compared with untreated NSG controls (Figs. 7g and 7h).\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that MARS exhibits a favorable safety profile in both immunocompetent and humanized mouse models, supporting its translational potential as a targeted and well-tolerated MAIT cell\u0026ndash;based immunotherapeutic strategy for liver cancer.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMAIT cells have emerged as a key subset of unconventional T cells with potent antimicrobial, immunoregulatory, and antitumor functions. As a unique innate-like T cell population, MAIT cells are particularly enriched in the liver, accounting for a substantial proportion of intrahepatic lymphocytes.\u003csup\u003e11,37,38\u003c/sup\u003e Accumulating evidence indicates that MAIT cells play multifaceted roles in liver homeostasis and pathology. Studies characterizing their localization revealed that MAIT cells are predominantly concentrated in bile ducts, portal tracts, and hepatic sinusoids, rather than the parenchyma.\u003csup\u003e39\u003c/sup\u003e In chronic liver diseases of various etiologies, MAIT cells exhibit dynamic changes in frequency and function, often accumulating within fibrotic septa or showing signs of activation-induced apoptosis, suggesting their involvement in both tissue repair and inflammatory progression.\u003csup\u003e40,41\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe role of MAIT cells in liver cancer remains controversial. Mengduan et al. reported that tumor-infiltrating MAIT cells in HCC express high levels of PD-1 and other exhaustion markers, with reduced abundance correlating with poor prognosis.\u003csup\u003e13\u003c/sup\u003e In contrast, Ruf et al. found that higher MAIT cell frequencies were associated with improved survival in HCC patients, and that checkpoint blockade targeting PD-1/PD-L1 could partially restore MAIT cell function, supporting their antitumor potential.\u003csup\u003e9\u003c/sup\u003e These seemingly conflicting observations may reflect the dual Th1- and Th17-like nature of MAIT cells, driven by co-expression of T-bet and ROR\u0026gamma;t.\u003csup\u003e42\u0026ndash;46\u003c/sup\u003e Nonetheless, it is evident that MAIT cells become functionally impaired within the HCC TME (Fig. 1), emphasizing the importance of strategies to rejuvenate and re-activate MAIT cells for liver cancer therapy.\u003c/p\u003e\n\u003cp\u003eWe designed MARS to enable \u003cem\u003ein vivo\u003c/em\u003e activation and rejuvenation of MAIT cells with several key advantages that directly address the major biological and therapeutic limitations of MAIT cell\u0026ndash;based immunotherapy in liver cancer. First, MARS achieves preferential liver targeting and prolonged hepatic retention, allowing localized and sustained delivery of MAIT agonists within the liver TME (Figs. 2k-2q)\u003csup\u003e32,47\u003c/sup\u003e. This spatial control minimizes systemic exposure while maximizing engagement of liver-resident MAIT cells, which are uniquely enriched in this organ. Second, by providing sustained release of the MR1-restricted antigen 5-OP-RU, MARS maintains prolonged MR1 surface presentation on antigen-presenting cells, overcoming the intrinsic instability and short half-life of soluble MAIT antigens and enabling durable TCR-dependent MAIT cell activation (Figs. 2-5)\u003csup\u003e29,48\u003c/sup\u003e. Third, co-delivery of IL-15 supplies essential survival and costimulatory signals that promote MAIT cell expansion, persistence, and cytotoxic programming while limiting activation-induced exhaustion (Figs. 2-5)\u003csup\u003e49,50\u003c/sup\u003e. Fourth, by enabling MAIT cells to simultaneously eliminate MR1⁺\u0026nbsp;tumor cells and immunosuppressive myeloid populations, MARS effectively remodels the TME, overcoming a key mechanism of resistance in liver cancer immunotherapy (Fig. 6)\u003csup\u003e21,51\u0026ndash;53\u003c/sup\u003e. Finally, MARS selectively activates MAIT cells without broadly perturbing other immune populations, resulting in a favorable safety profile in both immunocompetent and humanized models (Fig. 7)\u003csup\u003e54,55\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, we employed a comprehensive and hierarchical set of experimental assays and disease-relevant models to systematically evaluate MARS-induced MAIT cell activation and antitumor efficacy. \u003cem\u003eIn vitro\u003c/em\u003e killing assays using diverse human liver cancer cell lines enabled precise dissection of MR1-dependent MAIT cell cytotoxicity across tumors with heterogeneous genetic backgrounds and MR1 expression levels (Figs. 4a-4g). Complementary analyses using primary liver cancer specimens further validated these findings in a clinically relevant context, demonstrating that MARS-enhanced MAIT cells effectively target patient-derived tumor cells while simultaneously eliminating MR1⁺\u0026nbsp;immunosuppressive myeloid populations within the native TME (Figs. 4h-4n)\u003csup\u003e5,17\u003c/sup\u003e. To assess \u003cem\u003ein vivo\u003c/em\u003e efficacy and durability, we utilized a SKHEP1-MR1-FG human liver cancer xenograft model in NSG mice, which revealed that MARS enables sustained MAIT cell persistence, repeated tumor killing, and durable tumor clearance (Fig. 5). Importantly, the NSG-SGM3 myeloid cell\u0026ndash;bearing xenograft model provided a stringent test of therapeutic performance under conditions that closely mimic the human immunosuppressive liver TME. In this setting, MARS not only preserved MAIT cell effector function but also reshaped the TME by selectively depleting MR1⁺\u0026nbsp;suppressive myeloid cells (Fig. 5). Together, these complementary models highlight the robustness, durability, and translational relevance of MARS as a strategy to unlock the full antitumor potential of MAIT cells in liver cancer.\u003c/p\u003e\n\u003cp\u003eWe acknowledge that the current experimental models, including the myeloid cell\u0026ndash;bearing NSG-SGM3 system, do not fully recapitulate the complexity of the human liver TME, and that immunocompetent mouse models could, in principle, provide additional insights. However, substantial biological differences between murine and human MAIT cells limit the translational relevance of conventional mouse models for evaluating MAIT cell\u0026ndash;based therapies. Murine MAIT cells differ markedly from their human counterparts in frequency, developmental pathways, tissue distribution, and functional programming\u003csup\u003e45,56,57\u003c/sup\u003e. In particular, MAIT cells constitute less than 1% of liver lymphocytes in commonly used mouse strains such as C57BL/6, whereas they represent approximately 30\u0026ndash;45% of intrahepatic T cells in humans (Figs. 1h and 7b)\u003csup\u003e14,58\u0026ndash;61\u003c/sup\u003e. This profound disparity reflects fundamental species-specific differences in microbial exposure, MR1-dependent thymic selection, and peripheral expansion, and results in limited effector capacity of endogenous murine MAIT cells. Moreover, murine MAIT cells exhibit distinct transcriptional and functional profiles, with reduced cytotoxic potential and altered cytokine responsiveness compared with human MAIT cells\u003csup\u003e22,37,62\u003c/sup\u003e. Consequently, immunocompetent mouse models may underestimate the therapeutic potential of MAIT cell\u0026ndash;targeted strategies. In this context, the humanized models used in this study provide a more appropriate platform to evaluate MAIT cell activation, persistence, and antitumor function. These considerations further support the rationale that MARS is specifically optimized for human MAIT cell biology and may be particularly well suited for translational application in human liver cancer, where MAIT cells are abundant and functionally relevant.\u003c/p\u003e\n\u003cp\u003eFuture development of the MARS platform could further expand its therapeutic versatility and clinical impact. Beyond IL-15, incorporation of additional cytokines or cytokine variants, such as IL-7 to support MAIT cell homeostasis, IL-21 to enhance cytotoxic differentiation, or engineered cytokines with reduced systemic toxicity, may allow fine-tuning of MAIT cell activation states for specific disease contexts\u003csup\u003e34,63\u0026ndash;65\u003c/sup\u003e. In addition, MARS could be adapted as a vehicle for \u003cem\u003ein vivo\u003c/em\u003e MAIT cell engineering by delivering nucleic acid\u0026ndash;based payloads, including mRNA encoding chimeric antigen receptors (CARs), dominant-negative inhibitory receptors, or transcriptional regulators\u003csup\u003e66,67\u003c/sup\u003e. Such an approach would enable transient or programmable reprogramming of endogenous MAIT cells directly \u003cem\u003ein vivo\u003c/em\u003e, circumventing the need for \u003cem\u003eex vivo\u003c/em\u003e cell manipulation and adoptive transfer. Beyond liver cancer, the liver-targeted nature of MARS positions it as a promising platform for other liver-associated diseases characterized by immune dysregulation, including liver metastases, chronic viral hepatitis, nonalcoholic steatohepatitis, liver fibrosis, and autoimmune or inflammatory liver disorders\u003csup\u003e10,11,41\u003c/sup\u003e. Collectively, these future directions highlight the potential of MARS as a modular and broadly applicable \u003cem\u003ein vivo\u003c/em\u003e immune engineering platform centered on MAIT cells.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eStudy approval\u003c/p\u003e\n\u003cp\u003eThis study complies with all relevant ethical regulations. All experiments involving primary liver cancer patient samples were approved by the Ronald Reagan UCLA Medical Center (IRB#\u0026nbsp;IRB-25-0948). Animal studies were approved by\u0026nbsp;the Division of Laboratory Animal Medicine at UCLA.\u0026nbsp;Healthy donor PBMCs were provided by the UCLA/CFAR Virology Core Laboratory without identification information under federal and state regulations.\u003c/p\u003e\n\n\u003cp\u003eMice\u003c/p\u003e\n\u003cp\u003eNOD.Cg-\u003cem\u003ePrkdc\u003csup\u003escid\u003c/sup\u003e Il2rg\u003csup\u003etm1Wjl\u003c/sup\u003e\u003c/em\u003e/SzJ\u0026nbsp;(NOD-\u003cem\u003escid\u003c/em\u003e IL2Rg\u003csup\u003enull\u003c/sup\u003e, NSG),\u0026nbsp;NOD.Cg-\u003cem\u003ePrkdc\u003csup\u003escid\u003c/sup\u003e Il2rg\u003csup\u003etm1Wjl\u003c/sup\u003e\u003c/em\u003e Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NOD-scid IL2Rgnull-3/GM/SF, NSG-SGM3), and C57BL/6J (B6)\u0026nbsp;mice were purchased from The Jackson Laboratory, and maintained in animal facilities of the UCLA in a temperature-controlled environment (68\u0026thinsp;\u0026deg;F to 79\u0026thinsp;\u0026deg;F) with a 12-hour light cycle. 6- to10-week-old mice were used for all experiments. All mice were bred and maintained under specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of UCLA, and all animal procedures were conducted in accordance with the animal care and use regulations of the Division of Laboratory Animal Medicine (DLAM) at UCLA. Given the nature of the liver cancer models, there were no restrictions on tumor size or burden, making direct inferences from external measures unfeasible. Since the tumor cells expressed luciferase, we established a radiance threshold of \u0026ge; 10\u003csup\u003e10\u003c/sup\u003e photons/second per mouse as an upper surrogate limit. In addition, animals that experienced a 20% loss of their original body weight were euthanized. Experimental mice were randomly assigned to treatment groups to avoid statistically significant differences in the baseline tumor burden.\u003c/p\u003e\n\n\u003cp\u003eMedia and reagents\u003c/p\u003e\n\u003cp\u003eRecombinant human IL-2, IL-7, and IL-15 were purchased from PeproTech. Fetal bovine serum (FBS), and \u0026beta;-mercaptoethanol (\u0026beta;-ME) were purchased from Sigma. Penicillin-streptomycin-glutamine (P/S/G), MEM nonessential amino acids (NEAA), HEPES buffer solution, and sodium pyruvate were purchased from Gibco. Normocin was purchased from InvivoGen. The RPMI 1640 cell culture medium and the DMEM cell culture medium were purchased from Thermo Fisher Scientific. The CryoStor Cell Cryopreservation Media CS10 was purchased from MilliporeSigma.\u003c/p\u003e\n\u003cp\u003eThe C10 medium was made of RPMI 1640 cell culture medium supplemented with FBS (10% v/v), P/S/G (1% v/v), NEAA (1% v/v), HEPES (10 mM), sodium pyruvate (1 mM), \u0026beta;-ME (50 \u0026mu;M), and Normocin (100 \u0026mu;g/ml). The D10 medium was made of DMEM supplemented with FBS (10% v/v), P/S/G (1% v/v), and Normocin (100 \u0026mu;g/ml). The R10 medium was made of RPMI 1640 supplemented with FBS (10% v/v), P/S/G (1% v/v), and Normocin (100 \u0026mu;g/ml).\u003c/p\u003e\n\u003cp\u003e5-Amino-4-D-ribitylaminouracil Dihydrochloride (90%) was purchased from TorontoResearchChemicals. Methylglyoxal (MGO) solution was purchased from Sigma. Human and mouse MR1/5-OP-RU tetramers were provided by NIH Tetramer Core Facility.\u0026nbsp;Poly Lactic-co-Glycolic Acid (PLGA), 50:50 was purchased from Polysciences (cat. no. 23987). PRONOVA\u0026reg; UP VLVG (cat. no. 42000501-5G), Sodium carboxymethyl cellulose (cat. no. 419338), Ethylenediaminetetraacetic acid calcium disodium salt hydrate (cat. no. 340073), Calcium chloride (cat. no. C4901) and Acetic acid (cat. no. 338826) were purchased from sigma. Biotinylated human IL-15 R alpha (cat. no. ILA-H82F4) and human IL-15 (cat. no. IL5-H4117) were purchased from ACROBiosystems.\u003c/p\u003e\n\n\u003cp\u003eMARS manufacturing\u003c/p\u003e\n\u003cp\u003eThe MARS was engineered to incorporate both the MAIT antigen precursors 5-A-RU and MGO for sustained generation of 5-OP-RU, as well as surface-presented IL-15. PLGA nanoparticles encapsulating 5-A-RU and MGO were prepared using a water-in-oil-in-water (W/O/W) double-emulsion solvent evaporation method. Briefly, an aqueous solution containing 5-A-RU and MGO was emulsified into an organic phase consisting of PLGA dissolved in an appropriate organic solvent to form the primary water-in-oil (W/O) emulsion. This emulsion was generated by probe ultrasonication at defined power outputs, expressed as a percentage of the maximum instrument output. The primary emulsion was subsequently added to an external aqueous phase and further sonicated to generate the secondary W/O/W emulsion. Organic solvent removal was achieved by evaporation under continuous stirring, resulting in nanoparticle solidification. Nanoparticles were collected by centrifugation, washed extensively with phosphate-buffered saline (PBS), and resuspended for subsequent surface functionalization.\u003c/p\u003e\n\u003cp\u003eTo enable physiological IL-15 trans-presentation, human IL-15 receptor \u0026alpha; (IL-15R\u0026alpha;) was conjugated to the nanoparticle surface and used to load IL-15 as an IL-15/IL-15R\u0026alpha; complex. Briefly, recombinant human IL-15R\u0026alpha; protein was concentrated to 100\u0026thinsp;\u0026mu;L in PBS using an Amicon Ultra-2 centrifugal filter unit and reacted with Tetrazine-PEG5-NHS ester at a 1:5 molar ratio (protein:tetr azine) for 30\u0026thinsp;minutes at room temperature. Excess reagents were removed by desalting using a spin column, followed by five washes with PBS. The purified IL-15R\u0026alpha;\u0026ndash;tetrazine conjugate was mixed with glycerol (1:1, v/v) and stored at \u0026minus;20\u0026thinsp;\u0026deg;C until use. Tetrazine-functionalized IL-15R\u0026alpha; was subsequently conjugated to the surface of PLGA nanoparticles via bioorthogonal chemistry, after which recombinant human IL-15 was loaded by incubation to form stable IL-15/IL-15R\u0026alpha; complexes on the nanoparticle surface.\u003c/p\u003e\n\u003cp\u003eThis dual-loading strategy enables MARS to provide sustained release of MR1-restricted MAIT antigen from the nanoparticle core while simultaneously delivering IL-15 through surface trans-presentation, thereby recapitulating key features of antigen-presenting cell\u0026ndash;mediated MAIT cell activation.\u003c/p\u003e\n\n\u003cp\u003eMARS size characterization\u003c/p\u003e\n\u003cp\u003eThe hydrodynamic diameter and size distribution of nanoparticles were measured by dynamic light scattering (DLS). Measurements were performed at room temperature, and reported values represent the average of at least three independent measurements. The influence of ultrasonication power output during emulsification on nanoparticle size was systematically evaluated.\u003c/p\u003e\n\n\u003cp\u003eLC\u0026ndash;MS analysis of drug release\u003c/p\u003e\n\u003cp\u003eRelease of 5-A-RU-PABC-Val-Cit-Fmoc and MGO from PLGA nanoparticles was quantified by liquid chromatography\u0026ndash;mass spectrometry (LC\u0026ndash;MS). Nanoparticles formulated with 5-A-RU-PABC-Val-Cit-Fmoc were incubated in PBS at 37 \u0026deg;C under gentle agitation. At predefined time points, samples were centrifuged to pellet nanoparticles, and the supernatants were collected for LC\u0026ndash;MS analysis. Chromatographic separation was performed using a reverse-phase column with a water\u0026ndash;organic solvent gradient containing a volatile modifier. Quantification of 5-A-RU-PABC-Val-Cit-Fmoc and MGO was achieved by comparison with calibration curves generated from freshly prepared standards. Cumulative release profiles were calculated based on the measured concentrations and reported as a percentage of the total encapsulated cargo.\u003c/p\u003e\n\n\u003cp\u003eImmunofluorescence staining and imaging\u003c/p\u003e\n\u003cp\u003eCells were seeded on glass coverslips or imaging-compatible culture surfaces and cultured under indicated conditions. At designated time points, cells were fixed with 4% paraformaldehyde at room temperature, washed with PBS, and permeabilized with 0.1% Triton X-100 when intracellular staining was required. Non-specific binding was blocked using blocking buffer containing 1\u0026ndash;5% bovine serum albumin (BSA) in PBS. Cells were incubated with primary antibodies against MAIT cell and antigen-presenting cell markers at manufacturer recommend dilutions, followed by incubation with fluorophore-conjugated secondary antibodies. Nuclei were counterstained with DAPI. After final washes, samples were mounted using antifade mounting medium and imaged using a confocal fluorescence microscope. Identical acquisition settings were used across conditions for quantitative comparison. For analysis of MAIT cell\u0026ndash;APC interactions, representative fields were imaged, and conjugate formation or cell\u0026ndash;cell contacts were quantified using image analysis software.\u003c/p\u003e\n\n\u003cp\u003eLentiviral vectors\u003c/p\u003e\n\u003cp\u003eAll lentiviral vectors used in this study were constructed from a parental vector pMNDW\u003csup\u003e68,69\u003c/sup\u003e.\u0026nbsp;The 2A sequence derived from foot-and-mouth disease virus (F2A) was used to link the inserted genes to achieve co-expression. The Lenti/FG vector was constructed by inserting a synthetic bicistronic gene encoding Fluc-P2A-EGFP into the pMNDW\u003csup\u003e68,70\u003c/sup\u003e. The Lenti/MR1 vector was constructed by inserting a synthetic gene encoding human MR1 into the pMNDW. The synthetic gene fragments were obtained from GenScript and IDT. Lentiviruses were produced using human embryonic kidney 293T (HEK293T) cells (ATCC), following a standard transfection protocol using the Trans-IT-Lenti Transfection Reagent (Mirus Bio) and a centrifugation concentration protocol using the Amicon Ultra Centrifugal Filter Units, according to the manufacturer\u0026rsquo;s instructions (MilliporeSigma).\u003c/p\u003e\n\n\u003cp\u003eStable tumor cell lines\u003c/p\u003e\n\u003cp\u003eHuman liver cancer cell lines HEPG2, C3A, SNU423, SNU475, and SKHEP1 were purchased from the ATCC. The parental tumor cell lines were transduced with lentiviral vectors encoding the intended gene(s) to produce stable tumor cell lines overexpressing FG or human MR1. 72 hours post lentivector transduction, cells were subjected to flow cytometry sorting to isolate gene-engineered cells for generating stable cell lines. Six stable tumor cell lines were generated for this study, including HEPG2-FG, C3A-FG, SNU423-FG, SNU475-FG, SKHEP1-FG, and SKHEP1-MR1-FG cell lines. All tumor cell lines utilized in this study underwent short tandem repeat (STR) profiling, and the resulting profiles were compared to established databases to confirm accurate identification. Furthermore, the cell lines were regularly screened for mycoplasma contamination to preserve their integrity and authenticity.\u003c/p\u003e\n\n\u003cp\u003eLiver and PBMC sample collection from liver cancer patients\u003c/p\u003e\n\u003cp\u003ePrimary liver cancer patient samples, including liver tumor, normal adjacent liver, and peripheral blood samples, were collected at the Ronald Reagan UCLA Medical Center from consented patients through an IRB-approved protocol (IRB-25-0948) and processed. Information regarding the patients\u0026apos; gender and age was not provided in this study to avoid including three or more indirect identifiers for the study participants. Patient gender was not considered in the study design and was determined based on self-reporting.\u003c/p\u003e\n\n\u003cp\u003ePBMC collection from healthy donors\u003c/p\u003e\n\u003cp\u003eHealthy donor PBMCs were provided by the UCLA/CFAR Virology Core Laboratory without identification information under federal and state regulations. PBMCs were cryopreserved in Cryostor CS10 (Sigma St. Louis, MO, USA) using CoolCell (BioCision, Larkspur, CA, UCA), and were frozen in liquid nitrogen for storage and to supply all experiments.\u003c/p\u003e\n\n\u003cp\u003eAntibodies and flow cytometry\u003c/p\u003e\n\u003cp\u003eFluorochrome-conjugated antibodies specific for human CD3 (clone HIT3a, Pacific Blue, PE, or PE-Cy7-conjugated, 1:500), CD4 (clone OKT4, PE-Cy7, PerCP, or FITC-conjugated, 1:500), CD8 (clone SK1, PE, APC-Cy7, or APC-conjugated, 1:300), CD28 (clone CD28.2, APC, FITC, or Pacific Blue-conjugated, 1:200), CD45 (clone H130, PerCP, FITC or Pacific Blue-conjugated, 1:500), CD56 (clone QA18A21, APC-Cy7, APC, or PE-conjugated, 1:20), CD69 (clone FN50, PE-Cy7 or PerCP-conjugated, 1:50), MR1 (clone 26.5, PE-Cy7, FITC, or APC-conjugated,1:50), TCR V\u0026alpha;7.2 (clone 3C10, PE-Cy7, FITC, PE, or APC-conjugated,1:50), TCR\u0026alpha;\u0026beta; (clone I26, Pacific Blue or PE-Cy7-conjugated, 1:25), IFN-\u0026gamma; (clone B27, PE-Cy7-conjugated, 1:50), NKG2D (clone 1D11, PE-Cy7-conju gated, 1:50), DNAM-1 (clone 11A8, APC-conjugated, 1:50), NKp30 (clone P30-15, APC-conjugated, 1:50), NKp44 (clone P44-8, PE-Cy7-conjugated, 1:50), Granzyme B (clone QA16A02, APC-conjugated, 1:2,000 or 1:5,000), Perforin (clone dG9, PE-Cy7-conjugated, 1:50 or 1:100), IL-2 (clone MQ1-17H12, APC-Cy7-conjugated, 1:50), PD-1 (clone A17188A, FITC, APC, or PE-conjugated, 1:50), LAG-3 (clone 11C3C65, FITC, APC-Cy7, or PE-conjugated, 1:50), TIM-3 (clone A18087E, PE or APC-conjugated, 1:50), and CD45RO (clone UCHL1, PE-Cy7 or APC-Cy7-conjugated, 1:100) were purchased from BioLegend.\u0026nbsp;Fluorochrome-conjugated antibodies specific for mouse CD45 (clone 30-F11, PerCP-conjugated, 1:200), CD3 (clone 17A2, APC-conjugated, 1:200), CD19 (clone 1D3/CD19, APC-Cy7-conjugated, 1:200), CD4 (clone GK1.5, FITC-conjugated, 1:200), CD8 (clone 53-6.7, PE-conjugated, 1:200), CD11b (clone M1/70, PE-Cy7-conjugated, 1:200), CD14 (clone M14-23, PE-conjugated, 1:200), and NK-1.1 (clone S17016D, FITC-conjugated, 1:200) were purchased from BioLegend. Fixable Viability Dye eFluor506 (e506, 1:500) was purchased from Affymetrix eBioscience. Mouse Fc Block (anti-mouse CD16/32) was purchased from BD Biosciences, and human Fc Receptor Blocking Solution (TrueStain FcX) was purchased from BioLegend. PE-conjugated human and mouse MR1/5-OP-RU Tetramer (1:500 dilution) was obtained from NIH Tetramer Core Facility. All flow cytometry staining was performed following standard protocols, as well as specific instructions provided by the manufacturer of a particular antibody. Stained cells were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotech), following the manufacturers\u0026rsquo; instructions. FlowJo software version 9 (BD Biosciences) was used for data analysis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;For intracellular cytokine staining, the cells were thawed and resuspended in C10 medium. Cells were stimulated with PMA (Calbiochem, cat. no. 524400; 50 ng/mL) and ionomycin (Calbiochem, cat. no. 407952.; 500 ng/mL) and incubated at 37\u0026deg;C for 2 hours. GolgiStop (BD Biosciences, car. No. 554724; 1.5 \u0026micro;L/mL) was then added to inhibit cytokine secretion, followed by an additional 4-hour incubation. Subsequently, intracellular staining was performed using the Cell Fixation/Permeabilization Kit (BD Biosciences, cat. no. 554714) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\n\u003cp\u003eEnzyme-linked immunosorbent cytokine assays (ELISAs)\u003c/p\u003e\n\u003cp\u003eThe ELISAs for detecting human cytokines were performed following a standard protocol from BD Biosciences. Supernatants from cell culture assays were collected and assayed to quantify human IFN-\u0026gamma;, IL-2, IL-12, and TNF-\u0026alpha;. The capture and biotinylated pairs for detecting cytokines were purchased from BD Biosciences. The streptavidin-HRP conjugate was purchased from Invitrogen. Human cytokine standards were purchased from eBioscience. Tetramethylbenzidine substrate was purchased from KPL. The samples were analyzed for absorbance at 450 nm using an Infinite M1000 microplate reader (Tecan).\u003c/p\u003e\n\n\u003cp\u003eGeneration of MAIT cells from healthy donor or liver cancer patient PBMCs\u003c/p\u003e\n\u003cp\u003ePBMCs from healthy donors or liver cancer patients were used to generate MAIT cells. Cells were enriched via a two-step MACS protocol, first stained with PE-conjugated MR1/5-OP-RU tetramer and then labeled with Anti-PE MicroBeads (Miltenyi Biotec) for magnetic separation. The sorted MAIT cells were co-cultured with irradiated autologous PBMCs at a 1:1 ratio in C10 medium supplemented with 5-OP-RU (50 nM) and human IL-7 and IL-15 (10 ng/mL each). Cultures were maintained for 2 weeks with periodic IL-7 and IL-15 cytokine supplementation. Expanded MAIT cells could be subsequently purified by FACS to isolate MR1/5-OP-RU tetramer\u003csup\u003e+\u003c/sup\u003eTCR V\u0026alpha;7.2\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e cells for downstream applications.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e MAIT cell stimulation assay\u003c/p\u003e\n\u003cp\u003eMAIT cells were stimulated from either liver cancer patient PBMCs or tumor sample-derived single-cell suspensions under the indicated conditions. A total of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e live cells were cultured in 1 mL C10 medium supplemented with human IL-7 and IL-15 (10 ng/mL each). Depending on the experimental setup, cultures were additionally treated with 5-OP-RU (100 nM), 5-OP-RU (100 nM) plus IL-15 (10 ng/mL), or MARS (1 x 10\u003csup\u003e6\u003c/sup\u003e nanoparticles). The frequency and activation phenotype of MAIT cells (identified as CD3\u003csup\u003e+\u003c/sup\u003eMR1/5-OP-RU tetramer\u003csup\u003e+\u003c/sup\u003eTCR V\u0026alpha;7.2\u003csup\u003e+\u003c/sup\u003e cells) were analyzed by flow cytometry throughout the assay.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e tumor cell killing assay\u003c/p\u003e\n\u003cp\u003eLiver tumor cells (1\u0026nbsp;\u003cimg width=\"11\" height=\"17\" src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1768563400.png\" alt=\"image\"\u003e\u0026nbsp;10\u003csup\u003e4\u003c/sup\u003e cells per well) were co-cultured with MAIT cells (at ratios indicated in the figures or figure legends) in Corning 96-well clear bottom black plates for 24 h in C10 medium. D-luciferin (150 mg/ml, Caliper Life Science) was added to cell cultures to quantify live tumor cells and luciferase activities were read out using an Infinite M1000 microplate reader (Tecan). Depending on the experimental condition, cultures were supplemented with 5-OP-RU (100 nM), 5-OP-RU (100 nM) plus IL-15 (10 ng/mL), or MARS (1 x 10\u003csup\u003e4\u003c/sup\u003e nanoparticles) as specified in the figure legends.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e assays using primary liver cancer patient samples\u003c/p\u003e\n\u003cp\u003eIn one assay, the primary liver cancer patient samples were analyzed for tumor cell phenotype and the TME composition using flow cytometry. Liver tumor cells were sorted using a Human Tumor Cell Isolation Kit (Miltenyi Biotec) and/or identified as CD45\u003csup\u003e-\u003c/sup\u003eCD31\u003csup\u003e-\u003c/sup\u003eFAP (fibroblast activation protein)\u003csup\u003e-\u003c/sup\u003e cells\u003csup\u003e71\u0026ndash;73\u003c/sup\u003e, T cells were identified as CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e cells, CD4 T cells were identified as CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8 T cells were identified as CD8\u003csup\u003e+\u003c/sup\u003e T cells, MAIT cells were identified as MR1/5-OP-RU tetramer\u003csup\u003e+\u003c/sup\u003eTCR V\u0026alpha;7.2\u003csup\u003e+\u003c/sup\u003e T cells, B cells were identified as CD45\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e or CD45\u003csup\u003e+\u003c/sup\u003eCD20\u003csup\u003e+\u003c/sup\u003e cells, NK cells were identified as CD45\u003csup\u003e+\u003c/sup\u003eCD56\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e-\u003c/sup\u003e cells, myeloid cells were identified as CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e cells. Surface expression of MR1 on tumor or/and immune cells were also analyzed using flow cytometry.\u003c/p\u003e\n\u003cp\u003eIn another assay, the primary liver cancer patient samples were used to study tumor and TME cell killing by MAIT cells under various conditions. Patient samples (containing 1 x 10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells) were directly co-cultured with MAIT cells (1 x 10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells) in C10 medium in Corning 96-well Round Bottom Cell Culture plates for 24 hours. Depending on the experimental condition, cultures were supplemented with 5-OP-RU (100 nM), 5-OP-RU (100 nM) plus IL-15 (10 ng/mL), or MARS (1 x 10\u003csup\u003e5\u003c/sup\u003e nanoparticles) as specified in the figure legends. At the end of culture, cells were collected, and the tumor and TME cell targeting by MAIT cells was assessed using flow cytometry by quantifying live human tumor cells (identified as MR1/5-OP-RU tetramer\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e-\u0026nbsp;\u003c/sup\u003ecells), myeloid cells (identified as MR1/5-OP-RU tetramer\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e cells), T cells (identified as MR1/5-OP-RU tetramer\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e cells), B cells (identified as MR1/5-OP-RU tetramer\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e cells or MR1/5-OP-RU tetramer\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD20\u003csup\u003e+\u003c/sup\u003e cells), and NK cells (identified as MR1/5-OP-RU tetramer\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e-\u003c/sup\u003eCD56\u003csup\u003e+\u003c/sup\u003e cells). A total of 3 primary liver cancer patient samples were included in this assay.\u003c/p\u003e\n\n\u003cp\u003eWestern blot\u003c/p\u003e\n\u003cp\u003eWestern blot analysis was performed to examine TCR- and IL-15\u0026ndash;dependent signaling pathways in MAIT cells. MAIT cells from liver cancer patient samples were stimulated with soluble 5-OP-RU (100 nM), soluble 5-OP-RU (100 nM) in combination with IL-15 (10 ng/mL), or MARS (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e nanoparticles) for 15 minutes or 1 hour at 37\u0026thinsp;\u0026deg;C. Following stimulation, cells were immediately transferred into ice-cold PBS to terminate signaling and washed three times to remove residual antigen or cytokines. MAIT cells were then isolated by FACS based on CD3⁺TCR V\u0026alpha;7.2⁺\u0026nbsp;expression for subsequent protein extraction and western blot analysis.\u003c/p\u003e\n\u003cp\u003eTotal proteins were extracted using a RIPA lysis buffer (Thermo Fisher Scientific) containing 20 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Tritonx-100, and protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Protein concentration was measured using a Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher Scientific). Equal amounts of total protein were resolved on a 4\u0026ndash;15% Mini-PROTEAN\u0026reg; TGX\u0026trade; Precast Protein Gel (BIO-RAD) and then transferred to a polyvinylidene difluoride (PVDF) membrane by electrophoresis. The following antibodies were used to blot for the proteins of interest: anti-human p-LCK(Tyr394) (clone E5L3D, Cell Signaling Technology, CST), anti-human LCK (clone D88, CST), anti-human p-ZAP70 (Tyr319) (clone 65E4, CST), anti-human ZAP70 (clone D1C10E, CST), anti-human p-PLC gamma 1 (Tyr783) (clone D6M95, CST), anti-human PLC gamma 1 (clone D9H10, CST), anti-human p-c-Jun 1 (Ser73) (clone D47G9, CST), anti-human c-Jun (clone 60A8, CST), anti-human p-NF-kappaB p65 (Ser536) (clone 93H1, CST), anti-human NF-kappaB p65 (clone D14E12, CST), anti-human p-JAK1 (Tyr1034/1035) (clone D7N4Z1, CST), anti-human JAK1 (clone 6G4, CST), anti-human p-JAK3 (Tyr980/981) (clone D44E3, CST), anti-human JAK3 (clone D2E12, CST), anti-human p-STAT3 (Tyr705) (clone D3A7, CST), anti-human STAT3 (clone D7B12, CST), anti-human p-STAT5 (Tyr694) (clone D47E7, CST), anti-human STAT5 (clone D2O6Y, CST), anti-human C-Myc (clone D84C12, CST), anti-human C-Fos (clone 9F6, CST), anti-human BCL2 (clone D17C4, CST), and secondary anti-rabbit IgG (CST).\u0026nbsp;b-Actin (clone D6A8, CST) was used as internal controls. Signals were visualized using a ChemiDoc\u003csup\u003e\u0026trade;\u003c/sup\u003e Imaging Systems (BIO-RAD). The data were analyzed using ImageJ (Version 1.53s).\u003c/p\u003e\n\n\u003cp\u003eSingle cell RNA sequencing (scRNA-seq)\u003c/p\u003e\n\u003cp\u003escRNA-seq was utilized to examine the gene profiles of primary liver cancer patient-derived MAIT cells. Data from Gene Expression Omnibus database (GSE149614), Genome Sequence Archive in BIG Data Center (HRA000069), and European Genome-phenome Archive (EGAS00001003449) were included for scRNA-seq analyses\u003csup\u003e20,21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor cell clustering and annotation, the merged digital expression matrix generated by Cellranger was analyzed using an R package Seurat (v.4.0.0) following the guidelines\u003csup\u003e74\u0026ndash;76\u003c/sup\u003e.\u0026nbsp;Briefly, after filtering the low-quality cells, the expression matrix was normalized using NormalizeData function, followed by selecting variable features across datasets using FindVariableFeatures and SelectIntegrationFeatures functions. To correct the batch effect, FindIntegrrationAnchors and IntegrateData functions were used based on the selected feature genes. The corrected dataset was subjected to standard Seurat workflow for dimension reduction and clustering. In this study, clusters of therapeutic cells were manually merged and annotated based on gene signatures reported from Human Protein Atlas (proteinatlas.org) and previous studies\u003csup\u003e77\u0026ndash;84\u003c/sup\u003e. AddModuleScore was used to calculate module scores of each list of gene signatures, and FeaturePlot function was used to visualize the expression of each signature in the UMAP plots.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e bioluminescence imaging (BLI)\u003c/p\u003e\n\u003cp\u003eBLI was performed using a Spectral Advanced Molecular Imaging HTX system (Spectral Instrument Imaging). Live animal images were captured 5 minutes after intraperitoneal (i.p.) injection of D-Luciferin (1 mg per 100\u0026nbsp;\u0026mu;L PBS per mouse) to obtain total body bioluminescence. The imaging data were analyzed using AURA imaging software (version 3.2.0, Spectral Instrument Imaging).\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e antitumor efficacy study of MAIT cells: SKHEP1-MR1-FG human liver cancer xenograft NSG mouse model\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperimental design is shown in Fig. 5A. Briefly,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eon Day 0,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNSG mice received i.v. inoculation of SKHEP1-MR1-FG human liver cancer cells (5 x 10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells per mouse). On Day 5, the experimental mice received i.v. injection of Vehicle (100 \u0026mu;l PBS per mouse), or human MAIT cells (1 x 10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003ecells in 100 \u0026mu;l PBS per mouse). The MAIT cell-treated mice were divided into three groups: Group 1 received an intravenous injection of MARS (1 x 10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003enanoparticles), Group 2 received a single dose of 5-OP-RU (1 \u0026micro;M in 200 \u0026micro;L PBS), and Group 3 received 5-OP-RU (1 \u0026micro;M in 200 \u0026micro;L PBS) once weekly. Over the experiment, mice were monitored for survival and their tumor loads were measured twice per week using BLI. At the end of the experiment, mice were euthanized, and their tissues were collected for analysis of MAIT cell phenotypes and functions by flow cytometry.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e antitumor and anti-TME efficacy study of MAIT cells: SKHEP1-FG human liver cancer xenograft NSG-SGM3 mouse model\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperimental design is shown in Fig. 6A. Briefly,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eon Day -7 and -1, NSG-SGM3 mice received i.v. injection and healthy donor PBMC-derived CD14\u003csup\u003e+\u003c/sup\u003e myeloid cells (5 x 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ecells per mouse) to establish a liver tumor microenvironment enriched with TAM-like cells. On Day 0,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNSG-SGM3 mice received i.v. inoculation of SKHEP1- FG human liver cancer cells (5 x 10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells per mouse). On Day 5, the experimental mice received i.v. injection of Vehicle (100 \u0026mu;l PBS per mouse), or human MAIT cells (1 x 10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003ecells in 100 \u0026mu;l PBS per mouse). The MAIT cell-treated mice were divided into two groups: Group 1 received an intravenous injection of MARS (1 x 10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003enanoparticles), Group 2 received an intravenous injection of Vehicle (100 \u0026mu;l PBS per mouse). Throughout the study, mice were given weekly i.v. injections of PBMC-derived CD14\u003csup\u003e+\u003c/sup\u003e myeloid cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mouse) to sustain the humanized TME. Mice were monitored for survival and their tumor loads were measured twice per week using BLI. At the end of the experiment, mice were euthanized, and their tissues were collected for analysis of MAIT cell phenotypes and functions by flow cytometry.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e safety study of MARS in B6 mouse model\u003c/p\u003e\n\u003cp\u003eExperimental design is shown in Fig. 7A. On Day 1, C57BL/6 (B6) mice were i.v. injected with MARS (1 x 10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003enanoparticles) and monitored for potential toxicity and safety. On Days 10 and 40, mice were euthanized, and tissues including blood, spleen, and liver were collected for immunophenotypic analyses. Mouse immune cell populations were examined by flow cytometry and identified as follows: MAIT cells (CD45\u003csup\u003e+\u003c/sup\u003eTCR\u0026beta;\u003csup\u003e+\u003c/sup\u003eMR1/5-OP-RU tetramer\u003csup\u003e+\u003c/sup\u003e), T cells (CD45\u003csup\u003e+\u003c/sup\u003eTCR\u0026beta;\u003csup\u003e+\u003c/sup\u003e), B cells (CD45\u003csup\u003e+\u003c/sup\u003eCD19+), NK cells (CD45\u003csup\u003e+\u003c/sup\u003eNK1.1\u003csup\u003e+\u003c/sup\u003eTCR\u0026beta;\u003csup\u003e\u0026minus;\u003c/sup\u003e), and monocytes (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e). Peripheral blood samples were analyzed for hematological parameters, including white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT) levels.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e safety study of MARS in human MAIT cell xenograft NSG mouse model\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperimental design is shown in Fig. 7E. On Day 0, NSG mice were i.v. injected with human MAIT cells (1 x 10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003ecells in 100 \u0026mu;l PBS per mouse). On Day 1, the experimental mice were i.v. injected with MARS (1 x 10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003enanoparticles) and monitored for potential toxicity and safety. During the experiment, mouse body weights were recorded regularly to monitor general health status. On Days 1, 40, and 80, blood samples were collected for assessment of organ toxicity markers, including urea, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and glutamate dehydrogenase (GLDH), using ELISA assays. On Day 80, mice were euthanized, and multiple organs were harvested for histopathological analysis following established standard procedures\u003csup\u003e69,85\u003c/sup\u003e.\u003c/p\u003e\n\n\u003cp\u003eStatistics\u003c/p\u003e\n\u003cp\u003eStatistical data analysis was performed using GraphPad Prism 8 software (GraphPad). Student\u0026rsquo;s two-tailed t test was employed for pairwise comparisons. Ordinary one- or two-way ANOVA followed by Tukey\u0026rsquo;s or Dunnett\u0026rsquo;s multiple comparisons test was used for multiple comparisons. Log rank (Mantel-Cox) test adjusted for multiple comparisons was used for Meier survival curves analysis. Data are expressed as the mean \u0026plusmn;SEM, unless otherwise indicated. In all figures and figure legends, n denotes the number of samples or animals utilized in the indicated experiments. A p-value of less than 0.05 was considered significant; ns indicates not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the University of California, Los Angeles (UCLA) animal facility for providing animal support; the UCLA Translational Pathology Core Laboratory (TPCL) for providing histology support; the UCLA Technology Centre for Genomics \u0026amp; Bioinformatics (TCGB) facility for providing RNA-seq services; the UCLA CFAR Virology Core for providing human cells; and the UCLA BSCRC Flow Cytometry Core Facility for cell sorting support. We thank NIH Tetramer Core Facility for providing the tetramers. This work was supported by a Partnering Opportunity for Discovery Stage Research Projects Award and a Partnering Opportunity for Translational Research Projects Awards from the California Institute for Regenerative Medicine (DISC2-11157, DISC2-13015, TRAN1-12250, and TRAN1-16050 to L.Y.), a Department of Defense CDMRP PRCRP Impact Award (CA200456 to L.Y.), a Department of Defense Kidney Cancer Research Program Award (KC230215 to L.Y.), a UCLA BSCRC Innovation Award (to L.Y.), and an Ablon Scholars Award (to L.Y.). L.Y. is a member of UCLA Parker Institute for Cancer Immunotherapy (PICI). Y.-R.L. is a postdoctoral fellow supported by a UCLA MIMG M. John Pickett Post-Doctoral Fellow Award, a CIRM-BSCRC Postdoctoral Fellowship, a UCLA Sydney Finegold Postdoctoral Award, a UCLA Chancellor\u0026rsquo;s Award for Postdoctoral Research, and a UCLA Goodman-Luskin Microbiome Center Collaborative Research Fellowship Award.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY-R.L., H.N., Z.S., and X.S. designed the experiments, analyzed the data, and wrote the manuscript. L.Y. and S.L. conceived and oversaw the study. Y-R.L. H.N., Z.S., and X.S. performed all experiments, with assistance from Y.C., Y.Z., J.H., S.Y., and T.Y.. S.G. and V.G.A. provided the primary patient samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.Y. is a scientific advisor to AlzChem and Amberstone Biosciences, and a co-founder, stockholder, and advisory board member of Appia Bio. None of the declared companies contributed to or directed any of the research reported in this article. The remaining authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRuf, B. \u003cem\u003eet al.\u003c/em\u003e Activating Mucosal-Associated Invariant T Cells Induces a Broad Antitumor Response. \u003cem\u003eCancer Immunol. Res.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1024\u0026ndash;1034 (2021).\u003c/li\u003e\n\u003cli\u003eLi, Y.-R. \u003cem\u003eet al.\u003c/em\u003e Mucosal-associated invariant T cells for cancer immunotherapy. \u003cem\u003eMol. 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Commun.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1248 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mucosal-associated invariant T (MAIT) cell, MAIT cell activation and rejuvenation system (MARS), liver cancer, tumor microenvironment, immunotherapy, in vivo engineering, 5-OP-RU, IL-15, biomimetic nanoparticle","lastPublishedDoi":"10.21203/rs.3.rs-8518619/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8518619/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Mucosal-associated invariant T (MAIT) cells are liver-enriched unconventional T cells with potent antitumor potential, yet in liver cancer they are numerically reduced and functionally exhausted, limiting their therapeutic efficacy. To overcome these limitations, we developed a MAIT cell activation and rejuvenation system (MARS), a biomimetic, liver-targeted nanoparticle platform designed for sustained in vivo delivery of the MR1-restricted MAIT agonist 5-OP-RU together with human IL-15. Ex vivo, MARS robustly expanded and activated MAIT cells from liver cancer patient peripheral blood and tumor tissues, enhancing effector cytokine production and cytotoxicity while limiting exhaustion. In vivo, MARS preferentially accumulated in the liver and induced durable MAIT cell activation, expansion, and potent tumor killing in human liver cancer xenograft models. Importantly, in a human myeloid cell–engrafted mouse model that recapitulates an immunosuppressive liver tumor microenvironment, MARS enabled MAIT cells to simultaneously eliminate liver tumor cells and MR1⁺ tumor-associated myeloid cells, resulting in effective tumor control and prolonged survival. Collectively, these findings establish MARS as a safe and effective in vivo MAIT cell engineering strategy that overcomes microenvironment-driven resistance and provides a promising immunotherapeutic approach for liver cancer.","manuscriptTitle":"An in vivo MAIT cell activation and rejuvenation system for liver cancer therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 12:14:12","doi":"10.21203/rs.3.rs-8518619/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"562bf2b0-fb6a-40d8-be39-8527fc698b09","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61090653,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":61090654,"name":"Biological sciences/Biotechnology/Nanobiotechnology"},{"id":61090655,"name":"Biological sciences/Cancer/Tumour immunology"}],"tags":[],"updatedAt":"2026-02-10T09:17:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 12:14:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8518619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8518619","identity":"rs-8518619","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}