Targeting CAR-T cell senescence through Mysm1-SSBP1 axis improves persistence and therapeutic efficacy

Targeting CAR-T cell senescence through Mysm1-SSBP1 axis improves persistence and therapeutic efficacy | 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 Targeting CAR-T cell senescence through Mysm1-SSBP1 axis improves persistence and therapeutic efficacy Lifen Gao, Songbo Zhao, Huimin Liu, Minghao Sui, Yuchan Xue, Baihui Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6947364/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Chimeric antigen receptor (CAR)-T cell immunotherapy has demonstrated remarkable success in treating hematological malignancies, yet its clinical efficacy remains limited, particularly against solid tumors. Emerging evidence implicates T cell senescence as a key immunosuppressive barrier in cancer immunotherapy. In this study, we engineered Mysm1-overexpressing CAR-T cells and identified Mysm1 augmentation significantly enhances cytotoxic function and anti-tumor activity across multiple preclinical models. Integrated transcriptomic and biochemical analyses revealed that Mysm1 sustains mitochondrial homeostasis in CAR-T cells by interacting with SSBP1. Mechanistically, MYSM1 catalyzed K48-linked deubiquitination of SSBP1, thereby preserving mitochondrial function and mitigating CAR-T cell senescence. This intervention resulted in prolonged persistence and sustained anti-tumor efficacy in both murine and human CAR-T cells. Our findings unveil a novel strategy to counteract CAR-T cell senescence and establish Mysm1 as a promising therapeutic target for enhancing CAR-T cell immunotherapy. Biological sciences/Immunology/Lymphocytes Health sciences/Diseases/Cancer/Cancer models CAR-T Mysm1 senescence anti-tumor SSBP1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Chimeric antigen receptor (CAR)-T cell immunotherapy is a novel and revolutionary approach to improve outcomes for patients with cancer. However, even for the appreciable quantities of successful CAR-T cell therapy 1 , current CAR-T therapies still face a major challenge with high relapse and low cure rate 2 , 3 . These challenges can be attributed to a variety of factors, including intrinsic T cell dysfunction, exhaustion, and impaired proliferation. Notable, senescence represents a critical factor that significantly contributes to the limited functionality of CAR-T cells. T cell senescence has been recognized to play an immunosuppressive role in cancer patients 4 . Despite growing interest in CAR-T cell therapies, the consequences of senescence on their long-term performance have not been systematically examined. Additionally, there is a critical need for developing approaches to counteract senescence in adoptive T cell therapies. Cell senescence, which defined as the irreversible loss of replicative potential, is initiated as a persistent DNA damage response to dysfunctional telomeres 5 , 6 . T cell senescence, which negatively affects T cell immunotherapy, is described as the degeneration of innate and adoptive immunity, characterized with an abnormal cell cycle arrest, loss of CD27 and CD28, and highly expressing CD57, TIGIT and killer cell lectin-like receptor subfamily G (KLRG1) 4 , 7 , 8 . The makers, CD57 and KLRG1, expressed by senescent T cells are indicative of their association with replicative senescence 9 . Interfering with the ligation of KLRG1 on T cells has shown enhanced proliferation capacity 4 . Senescent T cells are usually shown to expand in patients with chronic and persistent infections. In addition, the expression of p53, p21 and p16, considered as markers for senescence. It is reported that the number of senescent T cells, as well as memory T cells, in the body gradually increases with age 10 . We speculate that alleviating T cell senescence could improve persistence and effectiveness of CAR-T cells in vitro and in vivo. Currently, there is no effective and feasible way to alleviate the senescence of T cells. Mitochondrial dysfunction, operationally defined as a decreased respiratory capacity, impaired mitochondrial metabolism, and mitochondrial membrane potential (MMP), is a hallmark of cellular senescence 11 . Abnormal oxidative phosphorylation (OXPHOS) function is usually observed in various models of senescence, confirming the causal relationship between OXPHOS inefficiency and senescence. Mitochondrial dysfunction and lower OXPHOS have been found in stress-induced senescence, replicative senescence and senescence-triggered by telomere shortening 12 – 14 . Meanwhile, specific ligation of mitochondria from senescent cells could rescue many features of the senescent phenotype 15 . Moreover, mitochondrial dysfunction triggered by mitochondrial DNA (mtDNA) deleting and mutating can cause cellular senescence 16 . Therefore, maintaining mitochondrial homeostasis in cells is one of the effective strategies to improve cellular senescence. Numerous studies show that p53 regulates the expression of plenty of target genes involved in cell cycle arrest and senescence, and p53 plays a critical role in DNA damage response 17 , 18 . Emerging evidence shows that the activation of p53 stress response and abnormal cell differentiation are triggered by the deficiency of Myb-like, SWIRM, and MPN domains 1 (Mysm1), which is a histone 2A (H2A) deubiquitinase, a mark for epigenetic transcriptional repression and chromatin inaccessibility 19 – 21 . It has been reported that Mysm1 knockout caused embryonic and developmental aberration of hematopoietic stem cells 22 , 23 . Moreover, Mysm1-deletion leads to elevated expression of the senescence marker γ-H2AX, while p53 activation can rescue the proliferation defects and apoptosis phenotypes caused by Mysm1 loss 24 . More importantly, the Mysm1 functionally attenuates cellular senescence and the aging process by promoting DNA repair processes, and Mysm1 over-expression delays the aging process and increases lifespan in mice 25 . These findings suggest that Mysm1 may play a crucial role in regulating cellular senescence. However, the underlying mechanism of Mysm1 orchestrating cell senescence and its role in T cell fitness remains completely unknown. In this study, we engineered a second-generation CAR construct co-expressing Mysm1 via a T2A linker and demonstrated that Mysm1 overexpression significantly enhanced the persistence and anti-tumor efficacy of CAR-T cells in vivo. Mechanistically, Mysm1 ameliorated CAR-T cell senescence by improving mitochondrial function. Importantly, we found that Mysm1 catalyzed K48-linked deubiquitination of single-stranded DNA-binding protein 1 (SSBP1), which is essential for the regulation of mitochondrial homeostasis. Collectively, our findings reveal that Mysm1 regulates mitochondrial hemostasis by deubiquitinating SSBP1, thereby rejuvenating CAR-T cells to enhance persistence and anti-tumor efficacy. Results Mysm1 regulates T cell senescence Previous studies indicate that Mysm1 mitigates senescence and aging in mice 25 , yet its influence on T cell biology—particularly in the context of adoptive immunotherapy—remains unexplored. Initial experiments revealed that peripheral blood T cells isolated from three healthy donors exhibited a progressive decline in Mysm1 expression, which correlated with prolonged extended culture duration (Extended Data Fig. 1 a). In addition, RT-qPCR analysis showed an age-dependent decrease in Mysm1 expression (Extended Data Fig. 1 b). Similarly, splenic T cells from 24-month-old mice exhibited significantly lower Mysm1 protein levels compared to those from 1- and 12-month-old mice (Extended Data Fig. 1 c). These results suggest that Mysm1 expression might correlate with cellular senescence of T cells. Thus, we further investigated the relationship between Mysm1 and T cell senescence. Under optimized culture conditions, isolated primary T cells maintained proliferative capacity for over 14 days, with some cultures extending beyond 3 weeks 26 , 27 . Using CRISPR-Cas9 technology, we generated Mysm1-knockout T cells (T-MKO) on day 10 of culture and observed the emergence of a senescent phenotype (Extended Data Fig. 2 a). Notably, Mysm1 overexpression in 20-day cultured T cells (T-MOE) significantly attenuated multiple senescence-associated parameters, including the senescence-associated secretory phenotype (SASP) (Extended Data Fig. 2 ). These data establish Mysm1 as a potential key regulator of primary T cell senescence. Mysm1 alleviates CAR-T cell senescence and enhances CAR-T cell cytotoxicity To determine whether Mysm1 influences CAR-T cell senescence, the CAR-T cells targeting human CD19 and B7H3 were established (Extended Data Fig. 3 ). Subsequently, we performed RNA sequencing (RNA-seq) analysis on day 15 cultures of both conventional 19BBz CAR-T cells and Mysm1-overexpressing 19BBz CAR-T cells (19BBz-M) (Extended Data Fig. 4 a-c). As shown in Fig. 1 a and Extended Data Fig. 4 d-e, gene involved in cellular senescence, including SASP and senescence core signature genes, were significantly downregulated in 19BBz-M CAR-T cells, suggesting an obvious shift toward an alleviating senescence. Compared with untransduced T (UTD) and 19BBz CAR-T cells, the 19BBz-M CAR-T cells exhibited enhanced expression of CD28 and CD27 alongside reduced levels of TIGIT and CD57 (Fig. 1 b). Tumor suppressor p53 and cycle-dependent kinase (CDK) inhibitor p21 were activated in response to a persistent DNA damage response, and the upregulation of CDK4/6 inhibitor p16 prompts senescence 28 . Consistently, 19BBz-M CAR-T cells expressed lower γ-H2AX, p53, p21 and p16 on day 10 and 20 (Fig. 1 c). We subsequently generated a CAR-T cell variant expressing catalytically impaired Mysm1 (19BBz-Mmut) containing mutations in the JAMM/MPN + domain. 19BBz-Mmut CAR-T cells exhibited γ-H2AX expression levels comparable to conventional 19BBz CAR-T cells, but significantly higher than that of Mysm1-overexpressing 19BBz-M CAR-T cells (Fig. 1 d). Furthermore, Mysm1-deficient T cells exhibited elevated senescence-associated β-galactosidase (SA-β gal) activity, while Mysm1-overexpressing CAR-T cells showed significantly reduced SA-β gal staining and decreased expression of p16 and p21, as well as SASP factors (Fig. 1 e-g). To investigate whether Mysm1-mediated senescence alleviation extends to murine CAR-T cells, we engineered a second-generation CAR construct comprising of a human GPC3-targeting scFv, a murine CD8α-derived transmembrane domain, and intracellular signaling domains from murine 4-1BB and CD3ζ. (Extended Data Fig. 5 a-b). Consistent with our human CAR-T cell findings, Mysm1 overexpression significantly reduced senescence markers in murine CAR-T cells (Extended Data Fig. 5 c-f), demonstrating its conserved anti-senescence function across species. To model the tumor microenvironment (TME) in vitro, we cultured CAR-T cells with tumor cell culture supernatant-conditioned medium (TCM). Under TCM conditions, Mysm1 overexpression significantly attenuated CAR-T cell senescence (Fig. 1 h and Extended Data Fig. 6 ). To verify the effect of Mysm1 on CAR-T cell senescence in vivo, B lymphoma bearing mouse model was established (Fig. 1 i). As expected, obvious improvement of senescent phenotype in 19BBz-M CAR-T cell was observed (Fig. 1 j-n), indicating that Mysm1 could rejuvenate CAR-T cells even under TME conditions in vivo. To evaluate the cytotoxic efficacy of Mysm1-overexpressing CAR-T cells in vitro, we conducted flow cytometry-based co-culture assays using an established protocol with modifications 27 . Our results demonstrate that Mysm1 overexpression significantly enhanced the cytotoxic activity of human 19BBz CAR-T cells (but not UTD) against both Raji and Namalwa lymphoma cells in a dose-dependent manner (Extended Data Fig. 7 a). Primary B-cell lymphoma samples were isolated from treatment-naïve patients and 19BBz-M CAR-T cells exhibited significantly enhanced killing efficacy compared to conventional 19BBz CAR-T cells, as evidenced by a marked reduction in the survival of target cells (Extended Data Fig. 7 b). Consistent with these results, Mysm1 overexpression similarly potentiated the cytotoxic activity of B7H3-targeting CAR-T cells (Extended Data Fig. 8 ). Collectively, our findings demonstrate that Mysm1 significantly attenuates CAR-T cell senescence while concurrently enhancing their cytotoxic potential in vitro. Mysm1-overexpressed CAR-T cells mediate superior anti-tumor activity in vivo To further assess the anti-tumor effect of 19BBz-M CAR-T cells in vivo, we established an orthotopic B cell lymphoma model by intravenously ( i.v. ) injecting 5×10 5 luciferases (luc)-expressing namalwa cells (Na-luc) into B-NDG mice and four days later, mice were treated with CAR-T cells (Fig. 2 a). As illustrated in Fig. 2 b–d, 19BBz-M CAR-T cells showed superior anti-tumor efficacy, significantly suppressing tumor progression and prolonging overall survival compared to conventional 19BBz CAR-T cells. Notably, peripheral blood analysis revealed a higher quantity of CAR-T cells in the 19BBz-M group, suggesting enhanced in vivo expansion or survival capacity (Fig. 2 e). Moreover, 19BBz-M CAR-T cells exhibited upregulated expression of CD25 and CD127 in peripheral blood, concomitant with a significant reduction in CD19 + tumor cell burden in the liver, bone marrow, and peripheral blood compared to control groups (Fig. 2 f,g). To further assess Mysm1's broad impact on CAR-T cell functionality, the anti-tumor activity of CAR-T cells targeting GPC3 was analyzed in vivo (Fig. 2 h). Compared to GPCBBz CAR-T cells, GPCBBz-M CAR-T cells displayed significantly enhanced tumor suppression and improved survival outcomes in HepG2-bearing mice (Fig. 2 i-k). In addition, more GPCBBz-M CAR-T cell infiltration was found in tumor tissues (Fig. 2 l). Considering the immune deficiency in B-NDG mice, an immunocompetent mice tumor-bearing model was established to explore the effect of Mysm1 on CAR-T cell under TME. By subcutaneous injection of 1×10 5 4T1 expressing human GPC3 antigen (4T1-GPC3) into wild-type Balb/c mice, mouse CAR-T cell treatment was performed (Fig. 3 a). mGPCBBz-M CAR-T cells moderately controlled tumor progression and prolonged survival compared with mGPCBBz CAR-T cell (Fig. 3 b-e). We hypothesized that Mysm1 might enhance CAR-T cell cytotoxicity under TME conditions to potentiate the bystander anti-tumor effect. Accordingly, tumor tissues were collected 14 days post-treatment in a parallel experiment for analysis of immune cell infiltration within the TME. The flow cytometry gating strategy of this experiment is shown in Extended Data Fig. 9 . A significant increase of infiltrating CAR-T cells was detected in mGPCBBz-M infusion group in TME compared with that of controls (Fig. 3 f). Interestingly, Mysm1 in mGPCBBz CAR-T cell improved the infiltration of endogenous T cells (CD4 + T and CD8 + T cell) and NK cells (Fig. 3 g-i), suggesting that Mysm1 in CAR-T cells reprograms the immunosuppressive TME toward a more immunostimulatory condition. Overall, these results reveal that the Mysm1 overexpressed human and mouse CAR-T cells mediate an excellent anti-tumor efficacy and enhance infiltration of endogenous immune cells by remodeling TME. Mysm1 overexpression confers durable persistence The limited persistence of CAR-T cells continues to pose a significant challenge in achieving sustained therapeutic effects. Previous studies have demonstrated an age-dependent decline in Mysm1 expression levels in murine epidermal tissues 29 . Given its role in cellular maintenance, we speculate that Mysm1 may critically regulate CAR-T cell persistence. Therefore, a repetitive multi-round co-culture experiment was performed (Fig. 4 a). The results showed that 19BBz-M CAR-T cells continued to eliminate B-cell lymphoma cells significantly at the fourth round of co-culture (Fig. 4 b). In addition, CAR-T cells overexpressing Mysm1 showed the highest T-cell counts at the third and fourth round of co-cultures (Fig. 4 c,d). To determine whether Mysm1 enhances the sustained anti-tumor efficacy of CAR-T cells in vivo, CAR-T cells were intravenously injected into B-NDG mice followed by four sequential tumor challenges (Fig. 4 e). Mice treated with 19BBz CAR-T cells exhibited a significantly faster increase in tumor bioluminescence compared to the 19BBz-M CAR-T cell group (Fig. 4 f). It is well known that the persistence of CAR-T cells is tightly correlated with the durability of remission in mice 30 – 32 . Infusion of 19BBz-M CAR-T cells led to improved survival of tumor-bearing mice compared with 19BBz CAR-T cells (Fig. 4 g). Based on these findings, we hypothesize that 19BBz-M CAR-T cells would mediate sustained elimination of Na-luc cells, attributable to their enhanced proliferative capacity and resistance to apoptosis. As expected, quantitative analyses revealed that Mysm1 significantly inhibited apoptosis of CAR-T cells (Extended Data Fig. 10a-d), while enhancing their proliferative capacity (Extended Data Fig. 10e,f). In addition, longitudinal monitoring of circulating CAR-T cells by flow cytometry revealed sustained persistence of 19BBz-M CAR-T cells, with significantly higher absolute cell counts compared to the conventional 19BBz CAR-T cells at all timepoints through day 28 post-infusion (Fig. 4 h,i). This sustained persistence may be mediated through Mysm1-induced memory phenotype differentiation in CAR-T cells (Extended Data Fig. 11). Collectively, our findings demonstrate that Mysm1 overexpression significantly enhances CAR-T cell persistence in vitro and in vivo. Mysm1 mitigates CAR-T cell senescence through regulation of mitochondrial homeostasis Mitochondrial dysfunction, a hallmark of cellular senescence, is usually manifested decreased respiratory capacity, impaired mitochondrial metabolism, and MMP 11 . To date, little is known about the role of Mysm1 in mitochondrial function. To confirm the mechanism of T cell senescence induced by Mysm1 deletion, we turned our attention towards the role of mitochondrial hemostasis. Typically, senescent cells exhibit a hypercatabolic activity and produce abundant lactate, which correlates with the elevated glycolysis 33 . The RNA seq analysis indicated that Mysm1 downregulated glycolysis level of 19BBz CAR-T cells (Fig. 5 a). Furthermore, extracellular acidification rate (ECAR), an indicator of lactate production, was obviously decreased in 19BBz-M CAR-T cells (Fig. 5 b). Evidence shows that disrupted mtDNA copy number and transcript levels result in impaired OXPHOS, elevated oxidative stress, and reduced lifespan. As the primary ATP-generating pathway, OXPHOS plays an indispensable role in maintaining mitochondrial function and regulating cellular senescence, which is critical for the proper operation of the immune system 34 . The heatmap analysis revealed significant activation of the OXPHOS signaling pathway in 19BBz-M CAR-T cells (Fig. 5 c). To further validate the role of Mysm1 in CAR-T cell metabolism, we quantitatively assessed mitochondrial respiration through cellular oxygen consumption rate (OCR) and spare respiratory capacity (SRC) measurements, which are signatures of memory-like T cells 34 . The results showed that overexpression of Mysm1 increased OCR and SRC in CAR-T cells (Fig. 5 d,e), further suggesting that 19BBz-M CAR-T cells were shifted towards the OXPHOS pathway. The decline of MMP is a landmark event in the early stage of mitochondrial dysfunction. The MMP was assessed in both wild-type and T-MKO cells using JC-1 staining. Quantitative analysis revealed a significant reduction in green fluorescence intensity, implying the Mysm1 deficiency decreased MMP of T cells (Fig. 5 f,g). On day 20, the total mtDNA copy number, critical for restoring OXPHOS, was significantly increased in 19BBz-M CAR-T cells than that in 19BBz CAR-T cells (Fig. 5 h). In addition, increased expression of TOM20, a sensor of oxidative stress, was observed in Mysm1-overexpressed CAR-T cells (Fig. 5 i). Next, we employed electron microscopy (EM) to assess the ultrastructural morphology of the CAR-T cells. Strikingly, 19BBz-M CAR-T cells displayed an elevated mitochondrial representation quantified by both a higher mitochondrial count per well and an expanded total mitochondrial area per well (Fig. 5 j), which is a signature of memory-like metabolic state for T cells 35 . To determine whether Mysm1 alleviates cellular senescence through regulation of mitochondrial homeostasis, we treated CAR-T cells with oligomycin (OM), an ATP synthase inhibitor that blocks OXPHOS. Analysis revealed that OM reversed Mysm1 overexpressed-mediated suppression of senescence markers including γ-H2AX, p53, SA-β gal, CD57, and KLRG1 (Fig. 5 k-n and Extended Data Fig. 12). Taken together, these results indicate that Mysm1 rejuvenates CAR-T cells by improving mitochondrial homeostasis and function. SSBP1 rescues cellular senescence in MKO CAR-T cells To explore the mechanism by which MYSM1 alleviates cellular senescence of CAR-T cells, we used LC-MS to identify the counterparts of Mysm1 in human T cells. Quantification of Mysm1-interacting proteins using LC-MS revealed that 151 proteins might specifically interact with Mysm1 in T cells (Extended Data Fig. 13a). We identified several mitochondrial regulators, including Gelsolin (GSN), Fragile X messenger ribonucleoprotein 1 (FMR1), TAR DNA-binding protein 43 (TDP43), and SSBP1. Co-IP assays revealed that Mysm1 binds GSN, FMR1, and SSBP1, but not TDP43 in T cells (Extended Data Fig. 13b). Notably, functional studies revealed a distinct role for SSBP1-, but not GSN and FMR1-, in regulating CAR-T cell senescence. (Fig. 6 a and Extended Data Fig. 13c-e). To further confirm the Mysm1-SSBP1 interaction, we performed Co-IP assays in HEK293T cells after ectopic expression of Mysm1 and Flag-SSBP1. Our data demonstrated that ectopic SSBP1was immunoprecipitated with Mysm1 and vice versa (Extended Data Fig. 14a,b). Moreover, we proved that endogenous Mysm1 could also bind to intrinsic SSBP1 in Jurkat cells, and reciprocally, Mysm1 was pulled down with SSBP1 (Extended Data Fig. 14c,d). To investigate the regulatory relationship between Mysm1 and SSBP1, we generated Jurkat cell lines overexpressing either MYSM1 or SSBP1. Our data demonstrated that Mysm1 overexpression elevated SSBP1 expression, whereas SSBP1 overexpression did not alter Mysm1 levels (Extended Data Fig. 14e,f), suggesting that Mysm1 operates upstream of SSBP1 in this regulatory cascade. To define the functional contribution of SSBP1 to Mysm1-mediated attenuation of CAR-T cell senescence, we generated Mysm1-knockout CAR-T cells with concurrent SSBP1 overexpression (19BBz-MKO-SOE). Strikingly, SSBP1 upregulation significantly rescued senescence-associated phenotypes in CAR-T cells induced by Mysm1 loss (Fig. 6 a-g and Extended Data Fig. 15a). The Na-luc mouse model was generated to systematically evaluate the in vivo role of SSBP1 in Mysm1-dependent regulation of CAR-T cell senescence (Fig. 6 h). The 19BB-MKO CAR-T cells exhibited significantly attenuated anti-tumor efficacy, whereas SSBP1 overexpression rescued the functional impairment (Fig. 6 i-k). Furthermore, evaluation of senescence-associated markers revealed that SSBP1-overexpression modulated 19BBz-MKO CAR-T cell aging phenotypes, significantly upregulating youthful markers (CD27, CD28) while downregulating senescence indicators (CD57, KLRG1) (Fig. 6 l-o and Extended Data Fig. 15e), suggesting that SSBP1 rescues senescence caused by Mysm1 deficiency. Collectively, our findings demonstrate that Mysm1 modulates cellular senescence and anti-tumor efficacy of CAR-T cells through physically interacting with SSBP1. Mysm1 rejuvenates CAR-T cells to enhance anti-tumor activity through hijacting SSBP1 Based on the above finding that Mysm1 alleviates CAR-T cell senescence through SSBP1 mediated regulation on mitochondrial function, we hypothesized that Mysm1 may restore mitochondrial homeostasis via deubiquitinating SSBP1, thereby attenuating cellular senescence and enhancing anti-tumor activity. As shown in Fig. 7 a, Mysm1-deficient mouse T cells exhibited decreased OXPHOS activity, which was restored by SSBP1 overexpression. For functional analysis of SSBP1 in Mysm1-deficient T cells, we generated mouse CAR-T cells with murine co-stimulators and human CD19 targeting capability. The results showed that, to some extent, SSBP1 overexpression rescued the MMP impairment and restored mtDNA copy numbers caused by Mysm1 deficiency in mouse 19BBz CAR-T cells (Fig. 7 b,c). SSBP1 upregulation significantly rescued Mysm1-deletion induced senescence-associated phenotypes in human CAR-T cells (Fig. 6 ) and mouse T cells (Fig. 7 d-f). In addition, SSBP1-overexpression enhanced mitochondrial representation in mouse 19BBz CAR-T cells, as evidenced by increased mitochondrial count and area per well, indicating its capacity to compensate for Mysm1 deficiency-induced mitochondrial impairment (Fig. 7 g,h). To comprehensively evaluate the impact of SSBP1 and Mysm1 on CAR-T cell anti-tumor efficacy, we established an immunocompetent mouse model using 4T1 cells engineered to express human CD19 (4T1-CD19) (Fig. 7 i). Mouse CAR-T cells specifically targeting human CD19 were adoptively transferred into wild-type Balb/c mice bearing 4T1-CD19 tumors. The results demonstrated that m19BBz-M CAR-T cells exhibited significantly enhanced tumor control and prolonged survival compared to conventional m19BBz CAR-T cells. Notably, genetic ablation of either Mysm1 or SSBP1 impaired anti-tumor efficacy of m19BBz CAR-T cells. Furthermore, SSBP1 overexpression rescued the compromised anti-tumor activity of Mysm1-deficient CAR-T cells (Fig. 7 j-m). In a parallel experimental cohort, tumor tissues were harvested at day 16 post-treatment for comprehensive analysis of CAR-T cell infiltration within the TME and the data revealed a significant increase in tumor-infiltrating m19BBz-M CAR-T cells and a moderate increase in m19BBz-MKO-SOE CAR-T cells within the TME compared to control groups (Fig. 7 n,o and Extended Data Fig. 15c). Collectively, our results demonstrate that Mysm1 enhances mitochondrial function depending on SSBP1, thereby attenuating CAR-T cell senescence and potentiating antitumor efficacy. Mysm1 mediates K48-linked deubiquitination of SSBP1 We next investigated the molecular mechanism by which Mysm1 regulates SSBP1. As a deubiquitinase, Mysm1 catalyzes the removal of ubiquitin from monoubiquitinated lysine K119 of H2A 36 . To date, no ubiquitin modification of SSBP1 has been reported. Thus, we investigated whether Mysm1 functions as a deubiquitinase targeting SSBP1. As shown in Fig. 8 a, Mysm1 expression in HEK293T cells significantly reduced SSBP1 ubiquitination ultimately resulting in increased SSBP1 protein abundance. Consistently, Mysm1-overexpression substantially attenuated SSBP1 ubiquitination in Jurkat-MKO-SKO cell line (Fig. 8 b). Ubiquitination primarily targets lysine residues on substrate proteins. In ubiquitin, the key lysine residues involved in chain formation include Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63 37 . To further determine which type of ubiquitin chain on SSBP1 was regulated by Mysm1, we conducted deubiquitination assays using a panel of ubiquitin variants, including linkage-specific isoforms and lysine mutants. As shown in Fig. 8 c, Mysm1 mainly removed K48-linked ubiquitin chains from SSBP1, resulting in a significant stabilization of SSBP1 protein levels. We next employed the K48R ubiquitin mutant in deubiquitination assays. This mutation abolished Mysm1-mediated deubiquitination of SSBP1, confirming that Mysm1 specifically cleaves K48-linked ubiquitin chains from SSBP1 (Fig. 8 d). Mysm1 comprises SANT, SWIRM, and catalytic JAMM/MPN domains. The JAMM/MPN domain in Mysm1 contains the essential catalytic residues responsible for its deubiquitinase activity 17 . We systematically deleted or mutagenized the JAMM/MPN domain of Mysm1 to assess its essential role in SSBP1 deubiquitination. Our results demonstrated that either JAMM/MPN domain deficiency or truncation in Mysm1 completely abolished SSBP1 deubiquitination, establishing this domain as essential for Mysm1's catalytic activity toward SSBP1 (Fig. 8 e,f). Under physiological conditions, Mysm1 primarily exhibits nuclear localization, where it interacts with chromatin and regulates gene expression. Confocal microscopy revealed that Mysm1 was localized in the nucleus, while SSBP1 showed cytoplasmic and nuclear localization (Fig. 8 g,h). To verify the subcellular localization of Mysm1 and SSBP1, we isolated cytoplasmic, mitochondrial, and nuclear fractions from human T cells, and the subcellular localization analysis revealed that Mysm1 exhibited predominant nuclear localization, with minor cytoplasmic distribution, while SSBP1 displayed both nuclear and mitochondrial localization (Extended Data Fig. 16). We sought to determine the subcellular localization of Mysm1-mediated deubiquitination of SSBP1 by transducing the plasmids into Jurkat cells. The results demonstrated that Mysm1-mediated deubiquitination of SSBP1 primarily localized in the nucleus (Fig. 8 i). Based on these findings, we propose that SSBP1 undergoes deubiquitination mainly in the nucleus, after which the deubiquitinated SSBP1 translocates to the mitochondria to exert its functional role. Discussion So far, there are no effective approaches to prevent or delay CAR-T cell senescence. Preventing CAR-T cell senescence induced by DNA damage response or antigen stimulation remains critical for improving CAR-T cell efficacy in tumors. Here, we engineered Mysm1-overexpressing CAR-T cells and identified that Mysm1 augmentation significantly enhances cytotoxicity and in vivo anti-tumor activity across multiple pre-clinical tumor models. Furthermore, we discovered that Mysm1 enhances mitochondrial homeostasis by catalyzing K48-linked deubiquitination of SSBP1, thereby stabilizing SSBP1 protein levels. This mechanism promotes prolonged persistence and sustained anti-tumor efficacy in both murine and human CAR-T cells. T cell senescence has been established as a key immunosuppressive mechanism in both aging individuals and cancer patients. The T cell compartment undergoes progressive differentiation, transitioning from naïve T cells to committed memory T cells and ultimately to senescent T cells 38 . During early life, the naïve T cell compartment gradually contracts while memory T cell populations expand, reaching a stable equilibrium in adulthood. However, after age 65, this balance shifts toward cellular senescence, marked by progressive accumulation of terminally differentiated CD28-negative T cells 39 . Developing strategies to prevent or reverse both replicative and premature T cell senescence is essential for extending healthspan and reducing cancer-related morbidity. Furthermore, targeting senescence in T cells from cancer patients represents a promising strategy to improve the efficacy of adoptive cell therapies. Our study reveals that Mysm1 overexpression in terminally differentiated CAR-T cells not only attenuates senescence-associated markers but also enhances their anti-tumor efficacy. As a key deubiquitinase, Mysm1 knockout has been reported to induce embryonic lethality and impair hematopoietic stem cell development. Mysm1 deficiency activates the p53 stress response pathway and induces aberrant cell differentiation. Furthermore, genetic ablation of Mysm1 results in decreased longevity in mice 18 . Notably, Mysm1 has been shown to functionally mitigate cellular senescence and delay aging by enhancing DNA repair mechanisms. Strikingly, Mysm1-overexpression extended lifespan and attenuated age-related decline in murine models 40 . The association between Mysm1 and T cells is currently unknown. Mitochondrial dysfunction, including diminished OXPHOS activity, is frequently detected in stress-induced senescence models. In addition, the role of Mysm1 in regulating mitochondrial biology has yet to be elucidated. Our findings reveal that Mysm1 deficiency promotes cellular senescence, while Mysm1-overexpression mitigates senescence in CAR-T cells by modulating mitochondrial function. Mysm1 predominantly localizes to the nucleus, though studies indicate it undergoes transient cytoplasmic accumulation in response to microbial infection 18 . In contrast to cytoplasmic Mysm1, nuclear Mysm1 exhibited remarkable stability and remained unaffected by de novo synthesis or degradation during infection. SSBP1 exhibits predominant mitochondrial localization, with lower nuclear expression and minimal detection in the cytosol (Uniprot and Go annotation). In this study, Mysm1-dependent deubiquitination of SSBP1 was detected mainly in nuclear fractions. Based on these findings, we propose a model wherein SSBP1 undergoes Mysm1-mediated deubiquitination in either the nucleus or cytoplasm, followed by translocation of the deubiquitinated form to mitochondria to perform its function. This mitochondrial accumulation of functional SSBP1 enhances mitochondrial homeostasis, ultimately attenuating CAR-T cell senescence. In vitro experiments demonstrated that T cells progressively developed senescent phenotypes with prolonged culture duration (Fig. 1 b). When cultured under optimized conditions, primary T cells maintained robust proliferation for at least 14 days, with a subset of cultures demonstrating continued expansion beyond 20 days 26 . 27 . Based on culture duration, we operationally defined T cells cultured for approximately 10 days as young cells and those maintained for ~ 20 days as senescent cells. Genetic ablation of Mysm1 at day 10 exacerbated T cell senescence, whereas Mysm1-overexpression at day 20 significantly attenuated senescent phenotypes (Extended Data Fig. 2 ), demonstrating that Mysm1 plays a critical role in sustaining CAR-T cell function throughout their lifespan. In summary, we demonstrate that Mysm1 alleviates CAR-T cell senescence by enhancing mitochondrial function, thereby boosting their anti-tumor efficacy. At the molecular level, Mysm1 sustains mitochondrial fitness by removing K48-linked ubiquitin chains from SSBP1, a mechanism critical for maintaining mitochondrial homeostasis (Fig. 9 ). Collectively, our findings demonstrate that Mysm1 maintains mitochondrial homeostasis through SSBP1 deubiquitination, thereby mitigating CAR-T cell senescence and potentiating their anti-tumor efficacy. However, the current study has several limitations that warrant further investigation. Firstly, the precise subcellular localization where Mysm1 mediates SSBP1 deubiquitination remains to be elucidated. Secondly, the mechanistic details underlying SSBP1 trafficking dynamics require more comprehensive characterization. Additionally, the exact deubiquitination sites on SSBP1 that are regulated by Mysm1 need to be precisely identified and mapped. Methods Cell lines and cell culture The following cell lines were cultured in RPMI 1640 (Gibco): Daudi, Namalwa and Jurkat cells which were cultured in RPMI1640 media were purchased from the American Type Culture Collection (Manassas, VA). LN229, A172, U251, HepG2, 4T1, HEK293T cells obtained from ATCC were cultured in DMEM (Gibco). Namalwa-luciferase (Na-luc), Daudi- luciferase (Daudi-luc) 4T1-FAP and 4T1-CD19 cell were constructed. DMEM and RPMI1640 media were supplemented with 10% fetal bovine serum (Gibco), 2 mM l -glutamine, 100 U ml – 1 penicillin and 100 µg ml – 1 streptomycin. The 4T1 expressing human CD19 (4T1-CD19) and GPC3 (4T1-GPC3) cell lines were constructed in our laboratory. All cells were cultured at 37°C in a humidified 5% CO 2 -containing atmosphere. All cell lines were mycoplasma free, and validated by flow cytometry for surface markers and functional readouts as needed. Generation of mouse and human CARs and lentivirus production CARs specific for human CD19, B7H3 and GPC3 were synthesized by Newhelix Biotech (Shanghai, China) as described in the below. Briefly, the human CD19-specfic scFv used for generating 19BBz CAR was derived from the high affinity antibody FMC63. The human GPC33 and B7-H3-specfic scFv used for generating B7BBz and GPCBBz were derived from antibodies MGA271 (WO 2021/207171 A1) and M11F1 (US 2010/0248359 A1) respectively. After codon optimization and synthesis, the scFv constructs were cloned in-frame into lentiviral vector pCDH-CMV-MCS-EF1-CopGFP or pLVX-EF1a-IRES-Puro that containing the hCD8 transmembrane domain, the h41BB, and the hCD3ζ-chain of the T-cell receptor complex. The mouse CAR cassette encoding the single-chain antibody targeting human CD19 (FMC63) or human GPC3, the mCD8 transmembrane domain, the m41BB and the mCD3ζ-chain. All studies involving human specimens were conducted in strict compliance with the ethical guidelines of Shandong Provincial Hospital (Ethics Approval No.: NSFC 2023 − 268). The human CAR lentivirus was produced as previous described 29 . Briefly, replication-defective lentiviral particles pseudotyped with VSV-G envelope were produced by transient transfection of HEK293T cells with 10 mg of the gene transfer constructs, 6.5 mg of Δ R, 3.5 mg of VSV-G and 2.5 mg of Rev. 12 hours later, the supernatant was replaced with fresh culture medium. And viral supernatants were harvested at 48 h and 72 h respectively. The mouse CAR lentivirus was obtained from Newhelix Biotech (Shanghai, China). CAR-T cell production Peripheral blood samples were obtained from several healthy donors. The peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare) and T cells were cultured in X-VIVO 15 (Lonza) medium supplemented with 50 IU mL − 1 IL-2 at a density of 1×10 6 cells mL − 1 , and activated with anti-CD3/CD28 beads (ThermoFisher Scientific). After 48 hours of activation, T cells were transduced with viral supernatants (MOI = 20). Post expansion, CAR-T cells were harvested and GFP expression was determined using flow cytometry. To generate mouse CAR-T cells, C57BL/6J mice was euthanized and spleen was isolated, then, spleen was grinded and mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque (GE healthcare) and T cells were obtained using sorting kit (Stemcell). T cells were stimulated with CD3/CD 28 activation beads (Miltenyi Biotec) at a 1:1 ratio (T cell: bead) for 48 hours in RPMI1640 medium supplemented with 10% fetal bovine serum, 50 IU mL − 1 IL-7 and 100 IU mL − 1 IL-15. After stimulating for 48 hours, activated T cells were transduced with lentiviruses (MOI = 100), and simultaneously supplemented with IL-7 and IL-15. Then, 12 hours later, T cells were harvested and cultured with RPMI1640 medium with IL-7 and IL-15. Flow cytometry The phenotype of primary human T cells and cell lines was determined using the following anti-human antibodies: B7-H3-PE/Cyanine7 (Biolegend, 351007), CD3-FITC (Biolegend, 300305), CD3-APC (Biolegend, 300411), CD25-APC (Biolegend, 302609), CD127-PE (BD, 557938), CD19-APC (BD, 561742), CD45RO-PE (Biohub, 78DA10076-100T), CD62L-PE (BD, 560966), CCR7-PE (BD, 561008), CD27-PE/Cyanine7 (Biolegend, 356411), CD28-APC (Biolegend, 302911), CD28-FITC (Biolegend, 302906), Ki67-APC (Biolegend, 350513), CD57-APC (Biolegend, 393305), KLRG1-PE (Biolegend, 368609), KLRG1-PE (BD, 568267), TIGIT-PC5.5 (Biolegend, 372717). The mouse T cells and immune cells was determined using the following anti-mouse antibodies: CD45-FITC (Biolegend, 103107), CD3-APC (Biolegend, 100236), CD4-PE/Cyanine7 (Biolegend, 100421), CD8-PC5.5 (Biolegend, 100737), NK1.1-PE (Biolegend, 108707), KLRG1-PE (Biolegend, 138407), TIGIT-APC (Biolegend, 156105). After staining, all cells were incubated at RT for 30 min, washed thrice with PBS, then analyzed on a flow cytometer (Cytoflex, Backman). For apoptotic cell analysis, T cells were stained with FITC-Annexin V and Propidium Iodide Kit (BD, 556547) according to the manufacturer’s instructions. For cell cycle arrest assay, CAR-T cells were detected using Cell Cycle Assay Kit (Elabscience, E-CK-A351) according to the manufacturer’s instructions. All samples were analyzed with FlowJo software (v10.8.1) and GraphPad Prism Software 8.01. Western blotting and Co-immunoprecipitation (co-IP) assay Protein was extracted as previously described. Briefly, equal amounts of proteins were resolved by RIPA buffer and separated using SDS-PAGE. Protein concentrations were determined using the BCA assay (Thermo Fisher Scientific). The following primary antibodies were used with dilution ratio of 1:500-1:10000 : anti-CD3ζ (Bioworld, P20963), anti-Mysm1 (Abcam, ab193081), anti-Actin (Abcam, ab7817), anti-p-γH2AX (Cell Signaling Technology (CST), 9718T), anti-p53 (CST, 30313), anti-p-p53 (CST, 82530), anti-p16 INK4A (CST, 92803), anti-p21 (CST, 2947), anti-Caspase3 (CST, 9662), anti-Bcl-2 (Bioworld, P10415), anti-Bcl-XL (Proteintech, 10783-AP), anti-Gelsolin (Proteintech, 11644-AP), anti-FMR1 (Proteintech, 13755-AP), anti-SSBP1 (OriGENE, TA382030), anti-Flag (Proteintech, 80801-2-RR), anti-VDAC1 (CST, 4866), anti-HA (CST, 3724), anti-Lamin A (OriGENE, TA803489). After blocking with 5% non-fat milk or bovine serum albumin, the blots were probed with primary antibodies. The bound antibodies were detected with an HRP-conjugated secondary antibody (1:5000) and visualized using a Tanon 4800 imaging system using super ECL detection reagent (YEASEN, 36208ES76). The nuclear, cytoplasmic and mitochondrial fraction were obtained using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027) and Cell Mitochondria Isolation Kit (Beyotime, C3601) according to the manufacturer’s protocol. For the whole cell lysate co-IP assay, after treatment, equal amounts of cells were harvested and incubated in Pierce IP lysis buffer (Thermo Scientific), supplemented with 1% protease inhibitor cocktail (YEASEN, 20123ES10), on ice for 20 min. After centrifugation for 15 min at 4℃, the supernatants were collected and incubated with primary IP antibodies at 4℃ for 12 h. After washed three times with ice-cold PBS, protein A/G magnetic beads (Selleckchem) was added and co-cultured at 4℃ for 2 h, then washed three times with ice-cold PBS. Finally, the samples were boiled in SDS loading buffer and analyzed by western blotting. CRISPR-Cas9 genome editing and overexpression of MYSM1 and SSBP1 in T and Jurkat cells Mysm1 and SSBP1 were deleted in T and Jurkat cells using CRISPR Cas9-mediated gene editing. Briefly, Mysm1 and SSBP1 single guide RNA (sgRNA) sequence were constructed in lentiCRISPR v2 (addgene) followed lentivirus packing using HEK293T cells. Then T or Jurkat cells were transfected using lentivirus and effect of knock out was confirmed by western blotting. The following sgRNA sequences were used: Mysm1 -forword-1: 5’-acgtagggtccgagacccat-3’; Mysm1 -reverse-1: 5’-ATGGGTCTCGGACCCTACGt-3’; Mysm1 -forword-2: 5’-GAGATGTTAATTGTATTGGA-3’; Mysm1 -reverse-2: 5’-TCCAATACAATTAACATCTC-3’; Mysm1 -forword-3: 5’-ttatggccggtcttctgatt-3’; Mysm1 -reverse-3: 5’-aatcagaagaccggccataa-3’; SSBP1 -forword: 5’-GGACCCTGTCTTGAGACAGG-3’; SSBP1 -reverse: 5’-CCTGTCTCAAGACAGGGTCC-3’; The lentiviral vectors of Mysm1 and 3×Flag tagged SSBP1 were created into a lentiviral vector pCDH-CMV-MCS-EF1-CopGFP. To create MPN + enzymatic domain-deleted mutant (ΔC) Mysm1 , 1-572 aa was retained and cloned into pCDH-CMV-MCS-EF1-CopGFP. Meanwhile, MPN + enzymatic domain-mutant (mut) Mysm1 was established and cloned into pCDH-CMV-MCS-EF1-CopGFP. The amino acid sequence of full-length MYSM1, mut MYSM1 and SSBP1 showed blow. qPCR and primers Total mRNA was extracted using TRIzol regent (TIANGEN, China) and analyzed by quantitative real-time PCR using the RevertAid First Strand cDNA Synthesis Kit(Thermo Fisher Scientific). Total DNA was extracted using Universal Genomic DNA Purification Mini Spin Kit (Beyotime, China). All reactions were performed with TaqMan Fast Universal PCR Master Mix (Vazyme, China) on a QuantStudioTM 3 Real-Time PCR Instrument (Applied Biosystems), using the following primer pairs. h Mysm1 -forward: 5’-ATCATGTTTAAGGGGACGTGCTGA-3’; h Mysm1 -reverse: 5’-TCCTGGCTGTCAGAAGTAATGAAT-3’; h p16 -forward: 5’-CTTCGGCTGACTGGCTGG-3’ h p16 -reverse: 5’-GCTGCCCATCATCATGACCT-3’ h p21 -forward: 5’-CTGTCACTGTCTTGTACCCTTGT-3’ h p21 -reverse: 5’-CCCAGCAGAGGAACCACTACTA-3’ h IL-1α -forward: 5’-CTTCTGGGAAACTCACGGCA-3’ h IL-1α -reverse: AGCACACCCAGTAGTCTTGC-3’ h IL-6 -forward: 5’-GTAGCCGCCCCACACAGA-3’ h IL-6 -reverse: 5’-CATGTCTCCTTTCTCAGGGCT-3’ h MMP9 -forward: 5’-GGACAAGCTCTTCGGCTTCT-3’ h MMP9 -reverse: 5’-TCGCTGGTACAGGTCGAGTA-3’ h TCF7 -forward: 5’-CTGGCTTCTACTCCCTGACCT-3’ h TCF7 -reverse: 5’-ACCAGAACCTAGCAT CAAGGA- 3 h β-actin -forward: 5’-GAAGAGCTACGAGCTGCCTGA-3’ h β-actin -reverse: 5’-CAGACAGCACTGTGTTGGCG-3’ hmtD- COX1 -forward: 5’-TCGCATCTGCTATAGTGGAG-3’ hmtD- COX1 -reverse: 5’-ATTATTCCGAAGCCTGGTAGG-3’ hmtD- Loop2 -forward: 5’-GGCTCTCAACTCCAGCATGT-3’ hmtD- Loop2 -reverse: 5’-AGGACGAGGGAGGCTACAAT-3’ h B2M -forward: 5’-CCAGCAGAGAATGGAAAGTCAA-3’ h B2M -reverse: 5’-TCTCTCTCCATTCTTCAGTAAGTCAACT-3’ Multi-rounds co-culture killing experiment Tumor cells and CAR-T cells were simultaneously seeded in 24-well plates at a concentration of 1×10 5 cells per well individually. 48 h later, cells were collected and T cells and tumor cells were measured by flow cytometry respectively. At the same time, 1×10 5 tumor cells were added to the parallel pales with mixture of CAR-T cells and residual tumor cells. One more 48 h later, cells were collected and measured by flow cytometry. For multiple rounds of co-culture, the CAR-T cells and residual tumor cells were record and total CAR-T cells per well were counted. SA-β gal assay Senescence-associated β-galactosidase (SA-β gal) staining was performed using Cell senescence Detection Kit (Dojindo, SG03) and CellEvent Senescence Green (Thermo Fisher, C10841) according to the manufacturer’s instructions. In senescence-inducing experiment, T or CAR-T cells were treated for 48 h with doxorubicin (2×10 7 M) and then analyzed using Cell senescence Detection Kit. Seahorse analysis Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured in XF media. Briefly, 1×10 6 purified CAR-T cells were seeded in a Seahorse Bioscience culture plate coated with Cell-Tak solution (Corning), and cultured in XF media (non-buffered X-VIVO 15 containing with 25 mM glucose, 2 mM glutamine and 1 mM pyruvate) under basal conditions and in response to 200 µM etomoxir (Tocris), 1 µM OM, 1.5 µM fluoro-carbonyl cyanide phenylhydrazone (FCCP) and 100 nM rotenone + 1 µM antimycin A. Basal, maximal OCR were measured by an XF96 Seahorse Extracellular Flux Analyzer (Agilent) following the manufacturer’s instruction. Measurement of mitochondrial membrane potential Mitochondrial membrane potential was estimated by measuring the fluorescence of free JC-1 monomers (green) and JC-1 aggregates in mitochondria (red) using mitochondrial membrane potential assay kit with JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide) (Beyotime, C2006) and MitoTracker Red (YEASEN, 40741ES50), according to the manufacturer’s protocol. And the results were expressed as the ratio of the aggregates/monomers of JC-1 in the percentage of control. Mitochondrial depolarization was indicated by a decrease in the polymer/monomer fluorescence intensity ratio. JC-1 staining was observed and photographed by the fluorescence microscope. In addition, CAR-T cells were loaded with 100 nM MitoTracker Red to measure mitochondrial membrane potential using a flow cytometry. Transmission electron microscopy CAR-T cells were fixed with 2.5% EM-grade glutaraldehyde and 2% EM-grade paraformaldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) at RT for 5 min. Following fixation, samples were washed in cocodylate buffer and post fixed in 1% osmium tetroxide. After extensive washing in H 2 O, T cells were rinsed with 0.1 M cacodylate buffer and incubated with 1% osmium tetroxide in H 2 O for 20 min. Cells were washed in H 2 O for 1 min four times and stained with 1% uranyl acetate in H 2 O for 14 min. Afterwards, cells were rinsed in H 2 O four times for 1 min each. Dehydration with an acetone series (50%, 70%, 90%, 2×100%) was performed for 45 s per step in the microwave. Cut sections were stained with uranyl acetate and lead citrate and then imaged using a JOEL 1200 EX transmission electron microscope equipped with an 8 MP ATMP digital camera (Advanced Microscopy Techniques). RNA-Seq analysis and LC-MS/MS assay Total RNA of CAR-T cells was extracted from CAR-T cells and messenger RNA libraries were prepared using RNA simple Total RNA Kit (DP419, TIANGEN). Paired-end libraries were synthesized using U-mRNAseq Library Prep Kit (AT4221, KAITAI-BIO) with Ribo off rRNA Depletion Kit (N407, Vazyme) following the RNA Sample Preparation Guide. Library construction and sequencing were performed by Sinotech Genomics (Shanghai, China). Briefly, raw reads were trimmed using fastp (v0.23.0), removing low-quality reads and removing reads with size inferior to 50 bp and poly A-containing mRNA molecules were purified using poly T oligo-attached magnetic beads. Paired-end sequence files were mapped to the reference genome using Hisat2 (Hierarchical Indexing for Spliced Alignment of Transcripts, V.2.0.5). The output SAM files were converted to binary alignment/map files and sorted using SAM tools (V.1.3.1). The LC-MC/MC analysis was performed by Shanghai Luming biological technology co., LTD (Shanghai, China). Briefly, Digested samples were separated by reverse phase C18 chromatography on nano-HPLC liquid phase system Easy-NLC1200, and the dried polypeptide samples were first re-dissolved in Nano-HPLC Buffer A, liquid A was 0.1% formic acid-aqueous solution, and liquid B was 0.1% formic acid-acetonitrile solution. The samples were then loaded by an automatic sampler and adsorbed to a Trap column, and separated on an Analysis column, 75 µm×150 mm at a flow rate of 300 nL/min. The samples were cleaned by mobile phase gradient with blank solvent for 30 min. The hydrolysates were separated by capillary high performance liquid chromatography and analyzed by Q-Exactive mass spectrometry (Thermo Scientific). The scanning range of parent ions was corrected by standard correction fluid at 300–1600 m/z, and the scanning mode of mass spectrometry was Data Dependent Aqcuisition (DDA). The 20 strongest fragment profiles (MS2 Scan) are collected after each full scan. Fragmentation was performed using high-energy collision dissociation (HCD, high energy) with an NCE energy of 28 and dynamic removal time of 25s. MS/MS spectra were searched using the MaxQuant against the Uniprot Mus Musculus database. Confocal imaging T or CAR-T cells were fixed by 4% paraformaldehyde for 30 min at room temperature. After washing, cells were blocked with 5% normal goat serum for 1 h and incubated with primary antibody at 4 ℃ overnight. The next day, cells were washed three times with PBS and incubated with 5 µg/mL Alexa Fluor 488 or 594 conjugated goat-anti-human (H + L) secondary antibody (Proteintech) for 1 h in the dark and then were stained with DAPI for 5 min. Imaging was acquired with a Leica DM6 confocal microscopy. The primary antibodies used were anti-TOM20 (Abclonal, A19403), anti-MYSM1 (Proteintech, 20078-1-AP) and anti-SSBP1 (OriGENE, TA382030). Clinical samples For clinical specimens, fresh tumor samples from patients with de-identified B lymphoma were obtained from Shandong Provincial Hospital Affiliated to Shandong First Medical University (Jinan, China). PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare), and B cell subsets were detected using anti-CD19 antibody. The samples were used, and the percentage of CD19-positive B cells was greater than 60%. In vitro killing experiment To validate the killing specificity of the constructed CAR-T cells, three killing ways were used. As previously described 27 , 5×10 5 target cells were co-incubated with different amounts of CAR-T cells in a total volume of 1 mL. After 18 h, the mixture of CAR-T cells and tumor cells were harvested and stained antibodies, then analyzed using flow cytometry, thus killing rates of CAR-T cells to targets were calculated according to changes of target cells. In addition, in vitro anti-target cells cytotoxicity of CAR-T cells was monitored in real-time using Smart cell real-time monitor (East China University of Science and Technology, Shanghai, six broad beans; CM100-α). Cell index correlates with the number of cells attached to the bottom of the plate. Briefly, ten thousands target cells were plated into specific 96-well plates. About five hours later, ten thousands CAR-T or control T cells were added to plates and co-cultured overnight. Then the growth curve of target cells was monitored by Smart cell real-time monitor. In vivo persistence experiment All animal experiments were performed on protocols approved by the Institutional Animal Care and Use Committee of Shandong First Medical University (Ethics Approval No.: NSFC. 2023 − 176). The in vivo persistence experiment was conducted using 5-6-week-old female B-NDG mice (NOD-Prkdc scid Il2rg tm1 ) (Biocytogen). The mice were intravenously injected with 5×10 5 T cells (UTD, 19BBz and 19BBz-M) suspended in 200 µL PBS with five to seven mice in each group. Mice were intravenously injected with 2×10 4 Na-luc cells on day 7, 12, 17, and 22 respectively. Peripheral blood was obtained every 5 days via venous blood collection, erythrocytes were lysed with red blood cell lysis buffer and mononuclear cells were stained with ant-CD3 antibody and analyzed by flow cytometry. In vivo bioluminescence imaging The D-luciferin (Beyotime) in PBS was used as a substrate for luciferase followed the manufacturer’s protocols. Tumor progression was monitored by bioluminescence imaging using an In Vivo Imaging System (IVIS) Spectrum Imaging System (PerkinElmer). Living Image V.4.5.5 (PerkinElmer) was used to acquire (and later quantify) the data 10 min after intraperitoneal injection of D-luciferin into animals that were anesthetized with 150 mg kg − 1 of 1% pentobarbital sodium (Sigma-Aldrich).The acquisition time ranged from 1 s to 1 min. Imaging settings were kept the same throughout the duration of the experiment. Tumor xenograft models The experiments were conducted at the Animal Experiment Center of Shandong First Medical University. Five to six-week-old B-NDG female mice were used in both lymphoma and hepatoma models. For the disseminated Na-luc model, 2–5×10 5 Na-luc were intravenously injected on day 0. 5×10 6 CAR-T cells were intravenously injected subsequently. For the local hepG2 model, mice were injected subcutaneously with 1×10 6 hepG2 cells, and on day 7 and 14, with 5×10 6 CAR-T cells, individually. Tumor burden was assessed weekly using bioluminescent IVIS Spectrum Imaging System. For the 4T1 model using Balb/c mice (HFK Bioscience), the mice were irradiated with 5 Gy on day − 2, and 1×10 5 4T1-CD19 (expressing human CD19) or 4T1-GPC3 (expressing human GPC3) cells were subcutaneously injected on day 0. 1×10 6 mouse CAR-T cells were intravenously injected. The tumor size of each mouse was measured every 2 days. The tumor size was calculated using the following formula: 4π/3×(tumor length/2)×(tumor width/2) 2 . The mice were humane manner when death was imminent or when the tumor size reached about 12 mm. In parallel mice experiment, the tumors were separated from body on day 16 and were digested with collagenase IV. The lysate was purified with red blood cell lysis buffer and cells were analyzed by flow cytometry. Statistical analysis The in vitro experiments were repeated at least three times for each group, and One-way ANOVA or Student’s t-test was used to compare quantitative data (mean ± SD) between the samples. A p -value < 0.05 indicated significance. For the in vivo experiments, the survival curves of mice were generated using the Kaplan–Meier method, and survival data were analyzed using the log-rank (Mantel-Cox) test. A p -value < 0.05 indicated significance. The obtained statistic was analyzed with Graphpad Prism 8.0.1. Declarations Acknowledgments We thank Professor Xiangbo Meng from Advanced Medical Research Institute, Shandong University for providing data analysis. The authors thank the support from Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong, Translational Medicine Core Facility of Advanced Medical Research Institute, Shandong University. Funding This work was supported by the Natural Science Foundation of Shandong Province (ZR2023QC179, ZR2024ZD11), National Natural Science Foundation of China (82271878, 82302067, 82370618, 82160124), and “Open Competition to Select the Best Candidates” Key Technology Program for Cell Therapy of NCTIB (NCTIB2023XB02006). Conflict of Interest The authors declare no conflict of interest. Author contributions SB. Z. performed the experiments, acquired and interpreted the data, and wrote the manuscript. HM. L. and MH. S. were involved in the construction of plasmid and lentiviral vectors. YC. X. and BH. W. contributed to the flow cytometry. TR. Z., H. D., XY. W. and H.G. participated in immunofluorescent staining and graphic plotting. 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Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Targeting CAR-T cell senescence through Mysm1-SSBP1 axis improves persistence and therapeutic efficacy Cite Share Download PDF Status: Under Review 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. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6947364","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":479416876,"identity":"566b60dc-6e9b-404c-b87a-b498d0676541","order_by":0,"name":"Lifen Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDACdsYHHx4w2DAwHAByeIjSwsxsOCOBIY10LYdJ0CLvzMzYkPDrvD3fjQTGB2/bGOTNCWkxPAzUkth3O3HmjQRmw7ltDIY7GwhpaeY//iCx53aCwY0ENmneNoYEgwMEtYBs6TlnD9TC/psoLfLMIL/8OMC4AWgLM1FaDEBaEhuSE2eeedgsOeechOEGgra0NzM2fPhjZ893PPnghzdlNvKEbQEpYGwDMRkbgIQEAfUgW0DqGP4QVjgKRsEoGAUjGAAAPoREGJWCPGwAAAAASUVORK5CYII=","orcid":"","institution":"School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Lifen","middleName":"","lastName":"Gao","suffix":""},{"id":479416877,"identity":"949512b9-fa7f-4cfd-92a4-8950dc07b4ff","order_by":1,"name":"Songbo Zhao","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University: Shandong Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Songbo","middleName":"","lastName":"Zhao","suffix":""},{"id":479416878,"identity":"e5304593-173c-496a-8aab-cfbd127541ab","order_by":2,"name":"Huimin Liu","email":"","orcid":"","institution":"School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Liu","suffix":""},{"id":479416879,"identity":"15c9f173-0b13-49cc-8f32-649ec6724837","order_by":3,"name":"Minghao Sui","email":"","orcid":"","institution":"Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Minghao","middleName":"","lastName":"Sui","suffix":""},{"id":479416880,"identity":"f507ee15-e824-41ee-9d61-022cf36be61d","order_by":4,"name":"Yuchan Xue","email":"","orcid":"","institution":"Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuchan","middleName":"","lastName":"Xue","suffix":""},{"id":479416881,"identity":"571c004c-16b9-42ad-9d7c-b414ac7dd7fb","order_by":5,"name":"Baihui Wang","email":"","orcid":"","institution":"Department of Gastroenterology, Qilu Hospital (Qingdao), Cheeloo College of Medicine, Shandong University, Qingdao, Shandong, China. Department of Gastroenterology, Shandong Provincial Hospital Affi","correspondingAuthor":false,"prefix":"","firstName":"Baihui","middleName":"","lastName":"Wang","suffix":""},{"id":479416882,"identity":"477b0a61-478b-4e2b-a09b-7b32ccf6da98","order_by":6,"name":"Tianru Zhang","email":"","orcid":"","institution":"Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tianru","middleName":"","lastName":"Zhang","suffix":""},{"id":479416883,"identity":"d3a33a71-7f53-443c-8951-6389dc2013df","order_by":7,"name":"Han Ding","email":"","orcid":"","institution":"Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Ding","suffix":""},{"id":479416884,"identity":"588e5e53-e2bf-43fd-a580-837df8526f02","order_by":8,"name":"Xinyu Wang","email":"","orcid":"","institution":"Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Wang","suffix":""},{"id":479416885,"identity":"ff67ce01-eabd-4695-9004-ca9141026c70","order_by":9,"name":"Jianni Qi","email":"","orcid":"","institution":"Department of Central Laboratory, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianni","middleName":"","lastName":"Qi","suffix":""},{"id":479416886,"identity":"f95f1190-1235-416e-8e5f-a89f61a9c872","order_by":10,"name":"Hong Guo","email":"","orcid":"","institution":"Shandong Desheng Biological Engineering Co. LTD","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Guo","suffix":""},{"id":479416887,"identity":"068648ef-7f2d-4c97-a32d-02c744ae0b65","order_by":11,"name":"Qiang Zhu","email":"","orcid":"","institution":"Department of Infectious Disease, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2025-06-22 02:45:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6947364/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6947364/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86262879,"identity":"698fc3be-0d76-45c7-9cf0-c0830e6229e2","added_by":"auto","created_at":"2025-07-08 15:04:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1006473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMysm1 alleviates CAR-T cell senescence.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Gene Set Enrichment Analysis (GSEA) and heatmap of the cellular senescence pathway in 19BBz versus 19BBz-M CAR-T cells. \u003cstrong\u003eb\u003c/strong\u003e, Expression of senescence associated markers CD27, CD28, TIGIT and CD57 on CAR-T cells co-cultured with tumor cells was detected at day 5, 10 and 15 using flow cytometry. \u003cstrong\u003ec\u003c/strong\u003e, Expression of senescence associated markers γ-H2AX, p-p53, p16 and p21 on CAR-T cells was detected by western blotting. \u003cstrong\u003ed\u003c/strong\u003e, γ-H2AX of 19BBz CAR-T cells with mutant Mysm1 was detected by western blotting. \u003cstrong\u003ee\u003c/strong\u003e, SA-β gal expression in T cells and CAR-T cells using flow cytometry. \u003cstrong\u003ef\u003c/strong\u003e, The expression of\u003cem\u003e p16\u003c/em\u003e and \u003cem\u003ep21\u003c/em\u003e was detected by RT-qPCR. \u003cstrong\u003eg\u003c/strong\u003e, SASP levels of CAR-T cells were assayed by RT-qPCR. \u003cstrong\u003eh\u003c/strong\u003e, CD27, CD28, CD57, KLRG1 and TIGIT expression in T cells and CAR-T cells treated with tumor cell culture supernatant-conditioned medium. Sup, supernatant. \u003cstrong\u003ei\u003c/strong\u003e, The scheme of experimental design. \u003cstrong\u003ej-n\u003c/strong\u003e. CD27 \u003cstrong\u003e(j)\u003c/strong\u003e, CD28 \u003cstrong\u003e(k)\u003c/strong\u003e, CD57 \u003cstrong\u003e(l)\u003c/strong\u003e, KLRG1\u003cstrong\u003e (m)\u003c/strong\u003e and TIGIT \u003cstrong\u003e(n)\u003c/strong\u003eexpression on CAR-T cells from Daudi bearing mice. Data are presented as mean ± SD from multiple independent experiments. ns means no significant difference, *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with indicated group by Student’s\u003cem\u003e t\u003c/em\u003e test and One-way ANOVA.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/e3da62a31dfdf9bb9ed01c31.png"},{"id":86262880,"identity":"14e8019d-57dc-4a0d-b0bf-90b85e5851f8","added_by":"auto","created_at":"2025-07-08 15:04:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":889818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMysm1 enhances anti-tumor activity of CAR-T cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, The scheme of experimental design. \u003cstrong\u003eb\u003c/strong\u003e, Tumor burden was monitored weekly \u003cem\u003evia\u003c/em\u003e bioluminescent imaging (n=6). \u003cstrong\u003ec\u003c/strong\u003e, The tumor burden of mice. \u003cstrong\u003ed\u003c/strong\u003e, Survival was calculated from the day of tumor cell inoculation until death. \u003cstrong\u003ee\u003c/strong\u003e, The peripheral blood CAR-T cell counts. \u003cstrong\u003ef\u003c/strong\u003e, The expression of CD25 and CD127 on the peripheral blood CAR-T cells. \u003cstrong\u003eg\u003c/strong\u003e, The CD19\u003csup\u003e+\u003c/sup\u003e tumor cells from liver, bone marrow and blood were analyzed on a flow cytometer. \u003cstrong\u003eh\u003c/strong\u003e, Experimental design for establishing the subcutaneous tumor-bearing models with HepG2 cells. \u003cstrong\u003ei\u003c/strong\u003e, Tumor images and weights from mice on day 25 post-engraftment. \u003cstrong\u003ej\u003c/strong\u003e, Tumor size was measured once every 2 days.\u003cstrong\u003e k\u003c/strong\u003e, The survival curve. \u003cstrong\u003el\u003c/strong\u003e, The CAR-T cells infiltrated in tumor tissues were measured. Data are presented as mean ± SD from multiple independent experiments. Statistical significance was determined by the log-rank (Mantel-Cox) test for survival curve and Student’s \u003cem\u003et\u003c/em\u003e test and One-way ANOVA for the comparison between the two groups. ns means no significant difference, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with indicated group.\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/d45c1e3611fe7db275fb5982.png"},{"id":86263755,"identity":"dc6a5863-7fa5-4263-a6ca-bd4ced1818c0","added_by":"auto","created_at":"2025-07-08 15:12:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1571543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMysm1 enhances anti-tumor activity of mouse CAR-T cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Experimental design for establishing subcutaneous tumor-bearing models. \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e, Tumor size was measured once every 2 days. \u003cstrong\u003ed\u003c/strong\u003e, Survival was calculated from the day of tumor cell inoculation until death. \u003cstrong\u003ee\u003c/strong\u003e, Images of tumor isolated from mice and tumor weight. \u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e, The CAR-T cells \u003cstrong\u003e(f)\u003c/strong\u003e, and intrinsic CD4\u003csup\u003e+\u003c/sup\u003e T cells \u003cstrong\u003e(g)\u003c/strong\u003e, CD8\u003csup\u003e+\u003c/sup\u003e T cells \u003cstrong\u003e(h)\u003c/strong\u003e and NK cells \u003cstrong\u003e(i)\u003c/strong\u003e infiltrated into tumor tssues were analyzed by flow cytometry. Statistical significance was determined by the log-rank (Mantel-Cox) test for survival curve and Student’s \u003cem\u003et\u003c/em\u003e test and One-way ANOVA for the comparison between the two groups. ns means no significant difference, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with indicated group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/12ba9af1a32178c752588f9f.png"},{"id":86263959,"identity":"dc60db4f-6af0-404e-afd7-b76029958677","added_by":"auto","created_at":"2025-07-08 15:20:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1126308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMysm1 improves CAR-T cell persistence.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Schematic representation of the multi-rounds co-culture experiment. \u003cstrong\u003eb\u003c/strong\u003e, The remaining tumor cells were recorded in the system. \u003cstrong\u003ec\u003c/strong\u003e, The CAR-T cells co-cultured with tumor cells were analyzed in the system. \u003cstrong\u003ed\u003c/strong\u003e, The overall CAR-T cells co-cultured with Raji cells were recorded. \u003cstrong\u003ee\u003c/strong\u003e, The scheme of experimental design. \u003cstrong\u003ef\u003c/strong\u003e, Tumor burden was monitored weekly via bioluminescent imaging (n=7). \u003cstrong\u003eg\u003c/strong\u003e, Survival was calculated from the day of tumor cell inoculation until death. \u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ei\u003c/strong\u003e, The CAR-T cells from blood were monitored every 7 days. Data are presented as mean ± SD from multiple independent experiments. Statistical significance was determined by the log-rank (Mantel-Cox) test for survival curve and Student’s \u003cem\u003et\u003c/em\u003etest and One-way ANOVA for the comparison between the two groups. ns means no significant difference, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 compared with indicated group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/095644e7331cd3603e53f30b.png"},{"id":86262889,"identity":"81ba20b8-0410-4313-8de8-a5fe3b0a7544","added_by":"auto","created_at":"2025-07-08 15:04:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2653944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMysm1 mitigates CAR-T cell senescence through regulation of mitochondrial homeostasis. a\u003c/strong\u003e, GSEA and heatmap analysis of glycolysis/gluconeogenesis pathway. \u003cstrong\u003eb\u003c/strong\u003e, ECAR of CAR-T cells at baseline and in response to oligomycin, FCCP, and rotenone. \u003cstrong\u003ec\u003c/strong\u003e, Heatmap analysis of OXPHOS pathway. \u003cstrong\u003ed\u003c/strong\u003e, OCR of CAR-T cells at baseline and in response to oligomycin, FCCP, and rotenone. \u003cstrong\u003ee\u003c/strong\u003e, Spare respiratory capacity (SRC). \u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003eg\u003c/strong\u003e, The MMP was assessed in both wild-type and T-MKO cells using JC-1 staining \u003cstrong\u003e(f)\u003c/strong\u003e and flow cytometry \u003cstrong\u003e(g)\u003c/strong\u003e. Scale bar, 50 μm. \u003cstrong\u003eh\u003c/strong\u003e, The relative total mtDNA copy number was assayed. \u003cstrong\u003ei\u003c/strong\u003e, TOM20 expression in CAR-T cells was assessed by immunofluorescence staining on day 20. Scale bar, 10 μm. \u003cstrong\u003ej\u003c/strong\u003e, Mitochondrial morphology and quantification in CAR-T cells were analyzed using transmission electron microscopy. Scale bar, 1 μm. \u003cstrong\u003ek\u003c/strong\u003e-\u003cstrong\u003en\u003c/strong\u003e. The senescence associated markers γ-H2AX, p-p53 \u003cstrong\u003e(k)\u003c/strong\u003e, β-gal \u003cstrong\u003e(l)\u003c/strong\u003e, CD57 \u003cstrong\u003e(m) \u003c/strong\u003eand KLRG1 \u003cstrong\u003e(n)\u003c/strong\u003e of CAR-T cells was analyzed. OM, oligomycin. Data are presented as mean ± SD from multiple independent experiments. ns means no significant difference, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 compared with indicated group by Student’s \u003cem\u003et\u003c/em\u003e test and One-way ANOVA.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/78deced3d4617819d5dc7cd0.png"},{"id":86262883,"identity":"6e89cfd2-4610-494f-a6c8-f9bba564f58d","added_by":"auto","created_at":"2025-07-08 15:04:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1049155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSSBP1 rescues cellular senescence in MKO CAR-T cells.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, The senescence associated markers of CAR-T cells were assayed by western blotting. \u003cstrong\u003eb\u003c/strong\u003e, \u003cem\u003ep16\u003c/em\u003e and \u003cem\u003ep21\u003c/em\u003emRNA expression was detected by RT-qPCR. \u003cstrong\u003ec\u003c/strong\u003e, Expression of\u003cem\u003e \u003c/em\u003eSASP was analyzed by RT-qPCR. \u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e. The CD27 \u003cstrong\u003e(d)\u003c/strong\u003e, CD28 \u003cstrong\u003e(e)\u003c/strong\u003e, CD57 \u003cstrong\u003e(f)\u003c/strong\u003e and KLRG1 \u003cstrong\u003e(g)\u003c/strong\u003e of CAR-T cells were assayed by flow cytometry. \u003cstrong\u003eh\u003c/strong\u003e, The scheme of experimental design. \u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e, Tumor burden was monitored weekly via bioluminescent imaging (n=6). \u003cstrong\u003ek\u003c/strong\u003e, Survival was calculated from the day of tumor cell inoculation until death. \u003cstrong\u003el\u003c/strong\u003e-\u003cstrong\u003eo\u003c/strong\u003e. The surface expression of CD27 \u003cstrong\u003e(l)\u003c/strong\u003e, CD28 \u003cstrong\u003e(m)\u003c/strong\u003e, CD57 \u003cstrong\u003e(n)\u003c/strong\u003e, and KLRG1 \u003cstrong\u003e(o)\u003c/strong\u003e on CAR-T cells was quantitatively assessed in vivo. Data are presented as mean ± SD from multiple independent experiments. Statistical significance was determined by the log-rank (Mantel-Cox) test for survival curve and One-way ANOVA for the comparison between the two groups. ns means no significant difference, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 compared with indicated group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/323fe58d3fa6ec3543de3937.png"},{"id":86264690,"identity":"793b8b2c-c01f-4e61-8392-f75d0619f650","added_by":"auto","created_at":"2025-07-08 15:28:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1461425,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMysm1 rejuvenates CAR-T cells to enhance anti-tumor activity through hijacting SSBP1. a\u003c/strong\u003e, OCR and SRC of human T cells in the absence of Mysm1 or together with SSBP1 overexpression. \u003cstrong\u003eb\u003c/strong\u003e, MitoTracker staining was performed in different human CAR-T cell populations. \u003cstrong\u003ec\u003c/strong\u003e, The relative total mtDNA copy number was measured in human CAR-T cells. \u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e. Senescent phenotypes in human Mysm1-deficiency CAR-T cells or together with SSBP1 overexpression. \u003cstrong\u003eg\u003c/strong\u003e, Mitochondrial morphology and quantification in human T cells were analyzed using transmission electron microscopy. Scale bar, 1 μm. \u003cstrong\u003eh\u003c/strong\u003e, Quantitative analysis of mitochondrial number and area.\u003cstrong\u003e i\u003c/strong\u003e, Experimental design for establishing subcutaneous tumor-bearing models using non-immunodeficient mice. \u003cstrong\u003ej\u003c/strong\u003e,\u003cstrong\u003ek\u003c/strong\u003e, Tumor images and weights from mice on day 16 post-engraftment. \u003cstrong\u003el\u003c/strong\u003e, The tumor burden. \u003cstrong\u003em\u003c/strong\u003e, The survival curve of mice. \u003cstrong\u003en\u003c/strong\u003e,\u003cstrong\u003eo\u003c/strong\u003e, The CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CAR-T cell ratios and counts were analyzed by flow cytometry. Data are presented as mean ± SD from multiple independent experiments. Statistical significance was determined by the log-rank (Mantel-Cox) test for survival curve and One-way ANOVA for the comparison between the two groups. ns means no significant difference, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with indicated group. \u0026nbsp;\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/60d631cbfb8f1f3536175285.png"},{"id":86263757,"identity":"09d328c2-a04c-4ee7-bbbd-f8ae46148d2f","added_by":"auto","created_at":"2025-07-08 15:12:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1459693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMysm1 mediates K48-linked deubiquitination of SSBP1.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Mysm1 reduced SSBP1 ubiquitination in HEK293T cells. \u003cstrong\u003eb\u003c/strong\u003e, Mysm1-overexpression attenuated SSBP1 ubiquitination in Jurkat-MKO-SKO cells. \u003cstrong\u003ec\u003c/strong\u003e, Mysm1 removed K48-linked ubiquitin chains from SSBP1 in Jurkat cells. \u003cstrong\u003ed\u003c/strong\u003e, Mysm1 removed K48-linked ubiquitin chains from SSBP1 in Jurkat-MKO-SKO cells. \u003cstrong\u003ee\u003c/strong\u003e, The JAMM/MPN domain of Mysm1 was site-directed mutagenized to investigate its role in SSBP1 deubiquitination in Jurkat cells. \u003cstrong\u003ef\u003c/strong\u003e, The JAMM/MPN domain of Mysm1 was deleted (ΔC) (1-572aa) to investigate its role in SSBP1 deubiquitination. \u003cstrong\u003eg\u003c/strong\u003e, Subcellular localization of ectopically expressed Mysm1 and SSBP1 in HEK293T cells was analyzed by confocal microscopy. \u003cstrong\u003eh\u003c/strong\u003e, Confocal microscopy revealed the co-localization patterns of intrinsic Mysm1 and SSBP1 in human T cells. Scale bar, 5 μm. \u003cstrong\u003ei\u003c/strong\u003e, Mysm1-mediated deubiquitination of SSBP1 was detected in Jurkat cells.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/71c3377bcec169b64fa86762.png"},{"id":86262886,"identity":"c9b79993-7d5b-4330-94d0-a336ea594987","added_by":"auto","created_at":"2025-07-08 15:04:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":691741,"visible":true,"origin":"","legend":"\u003cp\u003eMysm1 deubiquitinates SSBP1 to maintain mitochondrial homeostasis, thereby attenuating CAR-T cell senescence and subsequently improving both persistence and antitumor efficacy.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/3bd1e7759041c4c9860ef4ef.png"},{"id":86264973,"identity":"7ea6c39a-ba04-491c-bad1-4b010e9b1ed4","added_by":"auto","created_at":"2025-07-08 15:37:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14801855,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/14027ce0-901e-4112-9bdf-72532fa515c6.pdf"},{"id":86262887,"identity":"27d899ef-8415-429f-9de7-10ce71d1e430","added_by":"auto","created_at":"2025-07-08 15:04:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7173478,"visible":true,"origin":"","legend":"Targeting CAR-T cell senescence through Mysm1-SSBP1 axis improves persistence and therapeutic efficacy","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6947364/v1/3aa99cf0ad759dae13536bbb.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Targeting CAR-T cell senescence through Mysm1-SSBP1 axis improves persistence and therapeutic efficacy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChimeric antigen receptor (CAR)-T cell immunotherapy is a novel and revolutionary approach to improve outcomes for patients with cancer. However, even for the appreciable quantities of successful CAR-T cell therapy\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, current CAR-T therapies still face a major challenge with high relapse and low cure rate\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These challenges can be attributed to a variety of factors, including intrinsic T cell dysfunction, exhaustion, and impaired proliferation. Notable, senescence represents a critical factor that significantly contributes to the limited functionality of CAR-T cells. T cell senescence has been recognized to play an immunosuppressive role in cancer patients\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Despite growing interest in CAR-T cell therapies, the consequences of senescence on their long-term performance have not been systematically examined. Additionally, there is a critical need for developing approaches to counteract senescence in adoptive T cell therapies.\u003c/p\u003e \u003cp\u003eCell senescence, which defined as the irreversible loss of replicative potential, is initiated as a persistent DNA damage response to dysfunctional telomeres\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. T cell senescence, which negatively affects T cell immunotherapy, is described as the degeneration of innate and adoptive immunity, characterized with an abnormal cell cycle arrest, loss of CD27 and CD28, and highly expressing CD57, TIGIT and killer cell lectin-like receptor subfamily G (KLRG1)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The makers, CD57 and KLRG1, expressed by senescent T cells are indicative of their association with replicative senescence\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Interfering with the ligation of KLRG1 on T cells has shown enhanced proliferation capacity\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Senescent T cells are usually shown to expand in patients with chronic and persistent infections. In addition, the expression of p53, p21 and p16, considered as markers for senescence. It is reported that the number of senescent T cells, as well as memory T cells, in the body gradually increases with age\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We speculate that alleviating T cell senescence could improve persistence and effectiveness of CAR-T cells in vitro and in vivo. Currently, there is no effective and feasible way to alleviate the senescence of T cells.\u003c/p\u003e \u003cp\u003eMitochondrial dysfunction, operationally defined as a decreased respiratory capacity, impaired mitochondrial metabolism, and mitochondrial membrane potential (MMP), is a hallmark of cellular senescence\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Abnormal oxidative phosphorylation (OXPHOS) function is usually observed in various models of senescence, confirming the causal relationship between OXPHOS inefficiency and senescence. Mitochondrial dysfunction and lower OXPHOS have been found in stress-induced senescence, replicative senescence and senescence-triggered by telomere shortening\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Meanwhile, specific ligation of mitochondria from senescent cells could rescue many features of the senescent phenotype\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Moreover, mitochondrial dysfunction triggered by mitochondrial DNA (mtDNA) deleting and mutating can cause cellular senescence\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Therefore, maintaining mitochondrial homeostasis in cells is one of the effective strategies to improve cellular senescence.\u003c/p\u003e \u003cp\u003eNumerous studies show that p53 regulates the expression of plenty of target genes involved in cell cycle arrest and senescence, and p53 plays a critical role in DNA damage response\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Emerging evidence shows that the activation of p53 stress response and abnormal cell differentiation are triggered by the deficiency of Myb-like, SWIRM, and MPN domains 1 (Mysm1), which is a histone 2A (H2A) deubiquitinase, a mark for epigenetic transcriptional repression and chromatin inaccessibility\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. It has been reported that Mysm1 knockout caused embryonic and developmental aberration of hematopoietic stem cells\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, Mysm1-deletion leads to elevated expression of the senescence marker γ-H2AX, while p53 activation can rescue the proliferation defects and apoptosis phenotypes caused by Mysm1 loss\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. More importantly, the Mysm1 functionally attenuates cellular senescence and the aging process by promoting DNA repair processes, and Mysm1 over-expression delays the aging process and increases lifespan in mice\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. These findings suggest that Mysm1 may play a crucial role in regulating cellular senescence. However, the underlying mechanism of Mysm1 orchestrating cell senescence and its role in T cell fitness remains completely unknown.\u003c/p\u003e \u003cp\u003eIn this study, we engineered a second-generation CAR construct co-expressing Mysm1 via a T2A linker and demonstrated that Mysm1 overexpression significantly enhanced the persistence and anti-tumor efficacy of CAR-T cells in vivo. Mechanistically, Mysm1 ameliorated CAR-T cell senescence by improving mitochondrial function. Importantly, we found that Mysm1 catalyzed K48-linked deubiquitination of single-stranded DNA-binding protein 1 (SSBP1), which is essential for the regulation of mitochondrial homeostasis. Collectively, our findings reveal that Mysm1 regulates mitochondrial hemostasis by deubiquitinating SSBP1, thereby rejuvenating CAR-T cells to enhance persistence and anti-tumor efficacy.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eMysm1 regulates T cell senescence\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies indicate that Mysm1 mitigates senescence and aging in mice \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, yet its influence on T cell biology\u0026mdash;particularly in the context of adoptive immunotherapy\u0026mdash;remains unexplored. Initial experiments revealed that peripheral blood T cells isolated from three healthy donors exhibited a progressive decline in Mysm1 expression, which correlated with prolonged extended culture duration (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In addition, RT-qPCR analysis showed an age-dependent decrease in Mysm1 expression (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Similarly, splenic T cells from 24-month-old mice exhibited significantly lower Mysm1 protein levels compared to those from 1- and 12-month-old mice (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). These results suggest that Mysm1 expression might correlate with cellular senescence of T cells.\u003c/p\u003e \u003cp\u003eThus, we further investigated the relationship between Mysm1 and T cell senescence. Under optimized culture conditions, isolated primary T cells maintained proliferative capacity for over 14 days, with some cultures extending beyond 3 weeks\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Using CRISPR-Cas9 technology, we generated Mysm1-knockout T cells (T-MKO) on day 10 of culture and observed the emergence of a senescent phenotype (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Notably, Mysm1 overexpression in 20-day cultured T cells (T-MOE) significantly attenuated multiple senescence-associated parameters, including the senescence-associated secretory phenotype (SASP) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These data establish Mysm1 as a potential key regulator of primary T cell senescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMysm1 alleviates CAR-T cell senescence and enhances CAR-T cell cytotoxicity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether Mysm1 influences CAR-T cell senescence, the CAR-T cells targeting human CD19 and B7H3 were established (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Subsequently, we performed RNA sequencing (RNA-seq) analysis on day 15 cultures of both conventional 19BBz CAR-T cells and Mysm1-overexpressing 19BBz CAR-T cells (19BBz-M) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e, gene involved in cellular senescence, including SASP and senescence core signature genes, were significantly downregulated in 19BBz-M CAR-T cells, suggesting an obvious shift toward an alleviating senescence. Compared with untransduced T (UTD) and 19BBz CAR-T cells, the 19BBz-M CAR-T cells exhibited enhanced expression of CD28 and CD27 alongside reduced levels of TIGIT and CD57 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Tumor suppressor p53 and cycle-dependent kinase (CDK) inhibitor p21 were activated in response to a persistent DNA damage response, and the upregulation of CDK4/6 inhibitor p16 prompts senescence\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Consistently, 19BBz-M CAR-T cells expressed lower γ-H2AX, p53, p21 and p16 on day 10 and 20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We subsequently generated a CAR-T cell variant expressing catalytically impaired Mysm1 (19BBz-Mmut) containing mutations in the JAMM/MPN\u003csup\u003e+\u003c/sup\u003e domain. 19BBz-Mmut CAR-T cells exhibited γ-H2AX expression levels comparable to conventional 19BBz CAR-T cells, but significantly higher than that of Mysm1-overexpressing 19BBz-M CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Furthermore, Mysm1-deficient T cells exhibited elevated senescence-associated β-galactosidase (SA-β gal) activity, while Mysm1-overexpressing CAR-T cells showed significantly reduced SA-β gal staining and decreased expression of p16 and p21, as well as SASP factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-g). To investigate whether Mysm1-mediated senescence alleviation extends to murine CAR-T cells, we engineered a second-generation CAR construct comprising of a human GPC3-targeting scFv, a murine CD8α-derived transmembrane domain, and intracellular signaling domains from murine 4-1BB and CD3ζ. (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). Consistent with our human CAR-T cell findings, Mysm1 overexpression significantly reduced senescence markers in murine CAR-T cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-f), demonstrating its conserved anti-senescence function across species.\u003c/p\u003e \u003cp\u003eTo model the tumor microenvironment (TME) in vitro, we cultured CAR-T cells with tumor cell culture supernatant-conditioned medium (TCM). Under TCM conditions, Mysm1 overexpression significantly attenuated CAR-T cell senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). To verify the effect of Mysm1 on CAR-T cell senescence in vivo, B lymphoma bearing mouse model was established (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). As expected, obvious improvement of senescent phenotype in 19BBz-M CAR-T cell was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej-n), indicating that Mysm1 could rejuvenate CAR-T cells even under TME conditions in vivo.\u003c/p\u003e \u003cp\u003eTo evaluate the cytotoxic efficacy of Mysm1-overexpressing CAR-T cells in vitro, we conducted flow cytometry-based co-culture assays using an established protocol with modifications\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Our results demonstrate that Mysm1 overexpression significantly enhanced the cytotoxic activity of human 19BBz CAR-T cells (but not UTD) against both Raji and Namalwa lymphoma cells in a dose-dependent manner (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Primary B-cell lymphoma samples were isolated from treatment-na\u0026iuml;ve patients and 19BBz-M CAR-T cells exhibited significantly enhanced killing efficacy compared to conventional 19BBz CAR-T cells, as evidenced by a marked reduction in the survival of target cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Consistent with these results, Mysm1 overexpression similarly potentiated the cytotoxic activity of B7H3-targeting CAR-T cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCollectively, our findings demonstrate that Mysm1 significantly attenuates CAR-T cell senescence while concurrently enhancing their cytotoxic potential in vitro.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMysm1-overexpressed CAR-T cells mediate superior anti-tumor activity in vivo\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further assess the anti-tumor effect of 19BBz-M CAR-T cells in vivo, we established an orthotopic B cell lymphoma model by intravenously (\u003cem\u003ei.v.\u003c/em\u003e) injecting 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e luciferases (luc)-expressing namalwa cells (Na-luc) into B-NDG mice and four days later, mice were treated with CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u0026ndash;d, 19BBz-M CAR-T cells showed superior anti-tumor efficacy, significantly suppressing tumor progression and prolonging overall survival compared to conventional 19BBz CAR-T cells. Notably, peripheral blood analysis revealed a higher quantity of CAR-T cells in the 19BBz-M group, suggesting enhanced in vivo expansion or survival capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Moreover, 19BBz-M CAR-T cells exhibited upregulated expression of CD25 and CD127 in peripheral blood, concomitant with a significant reduction in CD19\u003csup\u003e+\u003c/sup\u003e tumor cell burden in the liver, bone marrow, and peripheral blood compared to control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef,g). To further assess Mysm1's broad impact on CAR-T cell functionality, the anti-tumor activity of CAR-T cells targeting GPC3 was analyzed in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Compared to GPCBBz CAR-T cells, GPCBBz-M CAR-T cells displayed significantly enhanced tumor suppression and improved survival outcomes in HepG2-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei-k). In addition, more GPCBBz-M CAR-T cell infiltration was found in tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering the immune deficiency in B-NDG mice, an immunocompetent mice tumor-bearing model was established to explore the effect of Mysm1 on CAR-T cell under TME. By subcutaneous injection of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e 4T1 expressing human GPC3 antigen (4T1-GPC3) into wild-type Balb/c mice, mouse CAR-T cell treatment was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). mGPCBBz-M CAR-T cells moderately controlled tumor progression and prolonged survival compared with mGPCBBz CAR-T cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-e). We hypothesized that Mysm1 might enhance CAR-T cell cytotoxicity under TME conditions to potentiate the bystander anti-tumor effect. Accordingly, tumor tissues were collected 14 days post-treatment in a parallel experiment for analysis of immune cell infiltration within the TME. The flow cytometry gating strategy of this experiment is shown in Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. A significant increase of infiltrating CAR-T cells was detected in mGPCBBz-M infusion group in TME compared with that of controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Interestingly, Mysm1 in mGPCBBz CAR-T cell improved the infiltration of endogenous T cells (CD4\u003csup\u003e+\u003c/sup\u003e T and CD8\u003csup\u003e+\u003c/sup\u003e T cell) and NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-i), suggesting that Mysm1 in CAR-T cells reprograms the immunosuppressive TME toward a more immunostimulatory condition.\u003c/p\u003e \u003cp\u003eOverall, these results reveal that the Mysm1 overexpressed human and mouse CAR-T cells mediate an excellent anti-tumor efficacy and enhance infiltration of endogenous immune cells by remodeling TME.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMysm1 overexpression confers durable persistence\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe limited persistence of CAR-T cells continues to pose a significant challenge in achieving sustained therapeutic effects. Previous studies have demonstrated an age-dependent decline in Mysm1 expression levels in murine epidermal tissues\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Given its role in cellular maintenance, we speculate that Mysm1 may critically regulate CAR-T cell persistence. Therefore, a repetitive multi-round co-culture experiment was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The results showed that 19BBz-M CAR-T cells continued to eliminate B-cell lymphoma cells significantly at the fourth round of co-culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In addition, CAR-T cells overexpressing Mysm1 showed the highest T-cell counts at the third and fourth round of co-cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,d). To determine whether Mysm1 enhances the sustained anti-tumor efficacy of CAR-T cells in vivo, CAR-T cells were intravenously injected into B-NDG mice followed by four sequential tumor challenges (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Mice treated with 19BBz CAR-T cells exhibited a significantly faster increase in tumor bioluminescence compared to the 19BBz-M CAR-T cell group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). It is well known that the persistence of CAR-T cells is tightly correlated with the durability of remission in mice\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Infusion of 19BBz-M CAR-T cells led to improved survival of tumor-bearing mice compared with 19BBz CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eBased on these findings, we hypothesize that 19BBz-M CAR-T cells would mediate sustained elimination of Na-luc cells, attributable to their enhanced proliferative capacity and resistance to apoptosis. As expected, quantitative analyses revealed that Mysm1 significantly inhibited apoptosis of CAR-T cells (Extended Data Fig.\u0026nbsp;10a-d), while enhancing their proliferative capacity (Extended Data Fig.\u0026nbsp;10e,f). In addition, longitudinal monitoring of circulating CAR-T cells by flow cytometry revealed sustained persistence of 19BBz-M CAR-T cells, with significantly higher absolute cell counts compared to the conventional 19BBz CAR-T cells at all timepoints through day 28 post-infusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh,i). This sustained persistence may be mediated through Mysm1-induced memory phenotype differentiation in CAR-T cells (Extended Data Fig.\u0026nbsp;11). Collectively, our findings demonstrate that Mysm1 overexpression significantly enhances CAR-T cell persistence in vitro and in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMysm1 mitigates CAR-T cell senescence through regulation of mitochondrial homeostasis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMitochondrial dysfunction, a hallmark of cellular senescence, is usually manifested decreased respiratory capacity, impaired mitochondrial metabolism, and MMP\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. To date, little is known about the role of Mysm1 in mitochondrial function. To confirm the mechanism of T cell senescence induced by Mysm1 deletion, we turned our attention towards the role of mitochondrial hemostasis. Typically, senescent cells exhibit a hypercatabolic activity and produce abundant lactate, which correlates with the elevated glycolysis\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The RNA seq analysis indicated that Mysm1 downregulated glycolysis level of 19BBz CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Furthermore, extracellular acidification rate (ECAR), an indicator of lactate production, was obviously decreased in 19BBz-M CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Evidence shows that disrupted mtDNA copy number and transcript levels result in impaired OXPHOS, elevated oxidative stress, and reduced lifespan. As the primary ATP-generating pathway, OXPHOS plays an indispensable role in maintaining mitochondrial function and regulating cellular senescence, which is critical for the proper operation of the immune system\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The heatmap analysis revealed significant activation of the OXPHOS signaling pathway in 19BBz-M CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). To further validate the role of Mysm1 in CAR-T cell metabolism, we quantitatively assessed mitochondrial respiration through cellular oxygen consumption rate (OCR) and spare respiratory capacity (SRC) measurements, which are signatures of memory-like T cells\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The results showed that overexpression of Mysm1 increased OCR and SRC in CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed,e), further suggesting that 19BBz-M CAR-T cells were shifted towards the OXPHOS pathway.\u003c/p\u003e \u003cp\u003eThe decline of MMP is a landmark event in the early stage of mitochondrial dysfunction. The MMP was assessed in both wild-type and T-MKO cells using JC-1 staining. Quantitative analysis revealed a significant reduction in green fluorescence intensity, implying the Mysm1 deficiency decreased MMP of T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef,g). On day 20, the total mtDNA copy number, critical for restoring OXPHOS, was significantly increased in 19BBz-M CAR-T cells than that in 19BBz CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). In addition, increased expression of TOM20, a sensor of oxidative stress, was observed in Mysm1-overexpressed CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). Next, we employed electron microscopy (EM) to assess the ultrastructural morphology of the CAR-T cells. Strikingly, 19BBz-M CAR-T cells displayed an elevated mitochondrial representation quantified by both a higher mitochondrial count per well and an expanded total mitochondrial area per well (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej), which is a signature of memory-like metabolic state for T cells\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. To determine whether Mysm1 alleviates cellular senescence through regulation of mitochondrial homeostasis, we treated CAR-T cells with oligomycin (OM), an ATP synthase inhibitor that blocks OXPHOS. Analysis revealed that OM reversed Mysm1 overexpressed-mediated suppression of senescence markers including γ-H2AX, p53, SA-β gal, CD57, and KLRG1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek-n and Extended Data Fig.\u0026nbsp;12).\u003c/p\u003e \u003cp\u003eTaken together, these results indicate that Mysm1 rejuvenates CAR-T cells by improving mitochondrial homeostasis and function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSSBP1 rescues cellular senescence in MKO CAR-T cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the mechanism by which MYSM1 alleviates cellular senescence of CAR-T cells, we used LC-MS to identify the counterparts of Mysm1 in human T cells. Quantification of Mysm1-interacting proteins using LC-MS revealed that 151 proteins might specifically interact with Mysm1 in T cells (Extended Data Fig.\u0026nbsp;13a). We identified several mitochondrial regulators, including Gelsolin (GSN), Fragile X messenger ribonucleoprotein 1 (FMR1), TAR DNA-binding protein 43 (TDP43), and SSBP1. Co-IP assays revealed that Mysm1 binds GSN, FMR1, and SSBP1, but not TDP43 in T cells (Extended Data Fig.\u0026nbsp;13b). Notably, functional studies revealed a distinct role for SSBP1-, but not GSN and FMR1-, in regulating CAR-T cell senescence. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;13c-e). To further confirm the Mysm1-SSBP1 interaction, we performed Co-IP assays in HEK293T cells after ectopic expression of Mysm1 and Flag-SSBP1. Our data demonstrated that ectopic SSBP1was immunoprecipitated with Mysm1 and vice versa (Extended Data Fig.\u0026nbsp;14a,b). Moreover, we proved that endogenous Mysm1 could also bind to intrinsic SSBP1 in Jurkat cells, and reciprocally, Mysm1 was pulled down with SSBP1 (Extended Data Fig.\u0026nbsp;14c,d). To investigate the regulatory relationship between Mysm1 and SSBP1, we generated Jurkat cell lines overexpressing either MYSM1 or SSBP1. Our data demonstrated that Mysm1 overexpression elevated SSBP1 expression, whereas SSBP1 overexpression did not alter Mysm1 levels (Extended Data Fig.\u0026nbsp;14e,f), suggesting that Mysm1 operates upstream of SSBP1 in this regulatory cascade.\u003c/p\u003e \u003cp\u003eTo define the functional contribution of SSBP1 to Mysm1-mediated attenuation of CAR-T cell senescence, we generated Mysm1-knockout CAR-T cells with concurrent SSBP1 overexpression (19BBz-MKO-SOE). Strikingly, SSBP1 upregulation significantly rescued senescence-associated phenotypes in CAR-T cells induced by Mysm1 loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-g and Extended Data Fig.\u0026nbsp;15a). The Na-luc mouse model was generated to systematically evaluate the in vivo role of SSBP1 in Mysm1-dependent regulation of CAR-T cell senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). The 19BB-MKO CAR-T cells exhibited significantly attenuated anti-tumor efficacy, whereas SSBP1 overexpression rescued the functional impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei-k). Furthermore, evaluation of senescence-associated markers revealed that SSBP1-overexpression modulated 19BBz-MKO CAR-T cell aging phenotypes, significantly upregulating youthful markers (CD27, CD28) while downregulating senescence indicators (CD57, KLRG1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003el-o and Extended Data Fig.\u0026nbsp;15e), suggesting that SSBP1 rescues senescence caused by Mysm1 deficiency.\u003c/p\u003e \u003cp\u003eCollectively, our findings demonstrate that Mysm1 modulates cellular senescence and anti-tumor efficacy of CAR-T cells through physically interacting with SSBP1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMysm1 rejuvenates CAR-T cells to enhance anti-tumor activity through hijacting SSBP1\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the above finding that Mysm1 alleviates CAR-T cell senescence through SSBP1 mediated regulation on mitochondrial function, we hypothesized that Mysm1 may restore mitochondrial homeostasis via deubiquitinating SSBP1, thereby attenuating cellular senescence and enhancing anti-tumor activity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, Mysm1-deficient mouse T cells exhibited decreased OXPHOS activity, which was restored by SSBP1 overexpression. For functional analysis of SSBP1 in Mysm1-deficient T cells, we generated mouse CAR-T cells with murine co-stimulators and human CD19 targeting capability. The results showed that, to some extent, SSBP1 overexpression rescued the MMP impairment and restored mtDNA copy numbers caused by Mysm1 deficiency in mouse 19BBz CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb,c). SSBP1 upregulation significantly rescued Mysm1-deletion induced senescence-associated phenotypes in human CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) and mouse T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed-f). In addition, SSBP1-overexpression enhanced mitochondrial representation in mouse 19BBz CAR-T cells, as evidenced by increased mitochondrial count and area per well, indicating its capacity to compensate for Mysm1 deficiency-induced mitochondrial impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg,h).\u003c/p\u003e \u003cp\u003eTo comprehensively evaluate the impact of SSBP1 and Mysm1 on CAR-T cell anti-tumor efficacy, we established an immunocompetent mouse model using 4T1 cells engineered to express human CD19 (4T1-CD19) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). Mouse CAR-T cells specifically targeting human CD19 were adoptively transferred into wild-type Balb/c mice bearing 4T1-CD19 tumors. The results demonstrated that m19BBz-M CAR-T cells exhibited significantly enhanced tumor control and prolonged survival compared to conventional m19BBz CAR-T cells. Notably, genetic ablation of either Mysm1 or SSBP1 impaired anti-tumor efficacy of m19BBz CAR-T cells. Furthermore, SSBP1 overexpression rescued the compromised anti-tumor activity of Mysm1-deficient CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej-m). In a parallel experimental cohort, tumor tissues were harvested at day 16 post-treatment for comprehensive analysis of CAR-T cell infiltration within the TME and the data revealed a significant increase in tumor-infiltrating m19BBz-M CAR-T cells and a moderate increase in m19BBz-MKO-SOE CAR-T cells within the TME compared to control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003en,o and Extended Data Fig.\u0026nbsp;15c).\u003c/p\u003e \u003cp\u003eCollectively, our results demonstrate that Mysm1 enhances mitochondrial function depending on SSBP1, thereby attenuating CAR-T cell senescence and potentiating antitumor efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMysm1 mediates K48-linked deubiquitination of SSBP1\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next investigated the molecular mechanism by which Mysm1 regulates SSBP1. As a deubiquitinase, Mysm1 catalyzes the removal of ubiquitin from monoubiquitinated lysine K119 of H2A\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. To date, no ubiquitin modification of SSBP1 has been reported. Thus, we investigated whether Mysm1 functions as a deubiquitinase targeting SSBP1. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, Mysm1 expression in HEK293T cells significantly reduced SSBP1 ubiquitination ultimately resulting in increased SSBP1 protein abundance. Consistently, Mysm1-overexpression substantially attenuated SSBP1 ubiquitination in Jurkat-MKO-SKO cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eUbiquitination primarily targets lysine residues on substrate proteins. In ubiquitin, the key lysine residues involved in chain formation include Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63\u003csup\u003e37\u003c/sup\u003e. To further determine which type of ubiquitin chain on SSBP1 was regulated by Mysm1, we conducted deubiquitination assays using a panel of ubiquitin variants, including linkage-specific isoforms and lysine mutants. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, Mysm1 mainly removed K48-linked ubiquitin chains from SSBP1, resulting in a significant stabilization of SSBP1 protein levels. We next employed the K48R ubiquitin mutant in deubiquitination assays. This mutation abolished Mysm1-mediated deubiquitination of SSBP1, confirming that Mysm1 specifically cleaves K48-linked ubiquitin chains from SSBP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). Mysm1 comprises SANT, SWIRM, and catalytic JAMM/MPN domains. The JAMM/MPN domain in Mysm1 contains the essential catalytic residues responsible for its deubiquitinase activity\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We systematically deleted or mutagenized the JAMM/MPN domain of Mysm1 to assess its essential role in SSBP1 deubiquitination. Our results demonstrated that either JAMM/MPN domain deficiency or truncation in Mysm1 completely abolished SSBP1 deubiquitination, establishing this domain as essential for Mysm1's catalytic activity toward SSBP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee,f).\u003c/p\u003e \u003cp\u003eUnder physiological conditions, Mysm1 primarily exhibits nuclear localization, where it interacts with chromatin and regulates gene expression. Confocal microscopy revealed that Mysm1 was localized in the nucleus, while SSBP1 showed cytoplasmic and nuclear localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg,h). To verify the subcellular localization of Mysm1 and SSBP1, we isolated cytoplasmic, mitochondrial, and nuclear fractions from human T cells, and the subcellular localization analysis revealed that Mysm1 exhibited predominant nuclear localization, with minor cytoplasmic distribution, while SSBP1 displayed both nuclear and mitochondrial localization (Extended Data Fig.\u0026nbsp;16). We sought to determine the subcellular localization of Mysm1-mediated deubiquitination of SSBP1 by transducing the plasmids into Jurkat cells. The results demonstrated that Mysm1-mediated deubiquitination of SSBP1 primarily localized in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ei). Based on these findings, we propose that SSBP1 undergoes deubiquitination mainly in the nucleus, after which the deubiquitinated SSBP1 translocates to the mitochondria to exert its functional role.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSo far, there are no effective approaches to prevent or delay CAR-T cell senescence. Preventing CAR-T cell senescence induced by DNA damage response or antigen stimulation remains critical for improving CAR-T cell efficacy in tumors. Here, we engineered Mysm1-overexpressing CAR-T cells and identified that Mysm1 augmentation significantly enhances cytotoxicity and in vivo anti-tumor activity across multiple pre-clinical tumor models. Furthermore, we discovered that Mysm1 enhances mitochondrial homeostasis by catalyzing K48-linked deubiquitination of SSBP1, thereby stabilizing SSBP1 protein levels. This mechanism promotes prolonged persistence and sustained anti-tumor efficacy in both murine and human CAR-T cells.\u003c/p\u003e \u003cp\u003eT cell senescence has been established as a key immunosuppressive mechanism in both aging individuals and cancer patients. The T cell compartment undergoes progressive differentiation, transitioning from naïve T cells to committed memory T cells and ultimately to senescent T cells\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. During early life, the naïve T cell compartment gradually contracts while memory T cell populations expand, reaching a stable equilibrium in adulthood. However, after age 65, this balance shifts toward cellular senescence, marked by progressive accumulation of terminally differentiated CD28-negative T cells\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Developing strategies to prevent or reverse both replicative and premature T cell senescence is essential for extending healthspan and reducing cancer-related morbidity. Furthermore, targeting senescence in T cells from cancer patients represents a promising strategy to improve the efficacy of adoptive cell therapies. Our study reveals that Mysm1 overexpression in terminally differentiated CAR-T cells not only attenuates senescence-associated markers but also enhances their anti-tumor efficacy.\u003c/p\u003e \u003cp\u003eAs a key deubiquitinase, Mysm1 knockout has been reported to induce embryonic lethality and impair hematopoietic stem cell development. Mysm1 deficiency activates the p53 stress response pathway and induces aberrant cell differentiation. Furthermore, genetic ablation of Mysm1 results in decreased longevity in mice\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Notably, Mysm1 has been shown to functionally mitigate cellular senescence and delay aging by enhancing DNA repair mechanisms. Strikingly, Mysm1-overexpression extended lifespan and attenuated age-related decline in murine models\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The association between Mysm1 and T cells is currently unknown. Mitochondrial dysfunction, including diminished OXPHOS activity, is frequently detected in stress-induced senescence models. In addition, the role of Mysm1 in regulating mitochondrial biology has yet to be elucidated. Our findings reveal that Mysm1 deficiency promotes cellular senescence, while Mysm1-overexpression mitigates senescence in CAR-T cells by modulating mitochondrial function.\u003c/p\u003e \u003cp\u003eMysm1 predominantly localizes to the nucleus, though studies indicate it undergoes transient cytoplasmic accumulation in response to microbial infection\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In contrast to cytoplasmic Mysm1, nuclear Mysm1 exhibited remarkable stability and remained unaffected by de novo synthesis or degradation during infection. SSBP1 exhibits predominant mitochondrial localization, with lower nuclear expression and minimal detection in the cytosol (Uniprot and Go annotation). In this study, Mysm1-dependent deubiquitination of SSBP1 was detected mainly in nuclear fractions. Based on these findings, we propose a model wherein SSBP1 undergoes Mysm1-mediated deubiquitination in either the nucleus or cytoplasm, followed by translocation of the deubiquitinated form to mitochondria to perform its function. This mitochondrial accumulation of functional SSBP1 enhances mitochondrial homeostasis, ultimately attenuating CAR-T cell senescence.\u003c/p\u003e \u003cp\u003eIn vitro experiments demonstrated that T cells progressively developed senescent phenotypes with prolonged culture duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). When cultured under optimized conditions, primary T cells maintained robust proliferation for at least 14 days, with a subset of cultures demonstrating continued expansion beyond 20 days\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e.\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Based on culture duration, we operationally defined T cells cultured for approximately 10 days as young cells and those maintained for ~ 20 days as senescent cells. Genetic ablation of Mysm1 at day 10 exacerbated T cell senescence, whereas Mysm1-overexpression at day 20 significantly attenuated senescent phenotypes (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), demonstrating that Mysm1 plays a critical role in sustaining CAR-T cell function throughout their lifespan.\u003c/p\u003e \u003cp\u003eIn summary, we demonstrate that Mysm1 alleviates CAR-T cell senescence by enhancing mitochondrial function, thereby boosting their anti-tumor efficacy. At the molecular level, Mysm1 sustains mitochondrial fitness by removing K48-linked ubiquitin chains from SSBP1, a mechanism critical for maintaining mitochondrial homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Collectively, our findings demonstrate that Mysm1 maintains mitochondrial homeostasis through SSBP1 deubiquitination, thereby mitigating CAR-T cell senescence and potentiating their anti-tumor efficacy. However, the current study has several limitations that warrant further investigation. Firstly, the precise subcellular localization where Mysm1 mediates SSBP1 deubiquitination remains to be elucidated. Secondly, the mechanistic details underlying SSBP1 trafficking dynamics require more comprehensive characterization. Additionally, the exact deubiquitination sites on SSBP1 that are regulated by Mysm1 need to be precisely identified and mapped.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eCell lines and cell culture\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe following cell lines were cultured in RPMI 1640 (Gibco): Daudi, Namalwa and Jurkat cells which were cultured in RPMI1640 media were purchased from the American Type Culture Collection (Manassas, VA). LN229, A172, U251, HepG2, 4T1, HEK293T cells obtained from ATCC were cultured in DMEM (Gibco). Namalwa-luciferase (Na-luc), Daudi- luciferase (Daudi-luc) 4T1-FAP and 4T1-CD19 cell were constructed. DMEM and RPMI1640 media were supplemented with 10% fetal bovine serum (Gibco), 2 mM \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-glutamine, 100 U ml\u003csup\u003e–\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e penicillin and 100 µg ml\u003csup\u003e–\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e streptomycin. The 4T1 expressing human CD19 (4T1-CD19) and GPC3 (4T1-GPC3) cell lines were constructed in our laboratory. All cells were cultured at 37°C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e-containing atmosphere. All cell lines were mycoplasma free, and validated by flow cytometry for surface markers and functional readouts as needed.\u003c/p\u003e\u003cp\u003e \u003cb\u003eGeneration of mouse and human CARs and lentivirus production\u003c/b\u003e \u003c/p\u003e\u003cp\u003eCARs specific for human CD19, B7H3 and GPC3 were synthesized by Newhelix Biotech (Shanghai, China) as described in the below. Briefly, the human CD19-specfic scFv used for generating 19BBz CAR was derived from the high affinity antibody FMC63. The human GPC33 and B7-H3-specfic scFv used for generating B7BBz and GPCBBz were derived from antibodies MGA271 (WO 2021/207171 A1) and M11F1 (US 2010/0248359 A1) respectively. After codon optimization and synthesis, the scFv constructs were cloned in-frame into lentiviral vector pCDH-CMV-MCS-EF1-CopGFP or pLVX-EF1a-IRES-Puro that containing the hCD8 transmembrane domain, the h41BB, and the hCD3ζ-chain of the T-cell receptor complex. The mouse CAR cassette encoding the single-chain antibody targeting human CD19 (FMC63) or human GPC3, the mCD8 transmembrane domain, the m41BB and the mCD3ζ-chain.\u003c/p\u003e\u003cp\u003e All studies involving human specimens were conducted in strict compliance with the ethical guidelines of Shandong Provincial Hospital (Ethics Approval No.: NSFC 2023 − 268).\u003c/p\u003e\u003cp\u003eThe human CAR lentivirus was produced as previous described\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Briefly, replication-defective lentiviral particles pseudotyped with VSV-G envelope were produced by transient transfection of HEK293T cells with 10 mg of the gene transfer constructs, 6.5 mg of \u003cb\u003eΔ\u003c/b\u003eR, 3.5 mg of VSV-G and 2.5 mg of Rev. 12 hours later, the supernatant was replaced with fresh culture medium. And viral supernatants were harvested at 48 h and 72 h respectively. The mouse CAR lentivirus was obtained from Newhelix Biotech (Shanghai, China).\u003c/p\u003e\u003cp\u003e \u003cb\u003eCAR-T cell production\u003c/b\u003e \u003c/p\u003e\u003cp\u003ePeripheral blood samples were obtained from several healthy donors. The peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare) and T cells were cultured in X-VIVO 15 (Lonza) medium supplemented with 50 IU mL\u003csup\u003e− 1\u003c/sup\u003e IL-2 at a density of 1×10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e− 1\u003c/sup\u003e, and activated with anti-CD3/CD28 beads (ThermoFisher Scientific). After 48 hours of activation, T cells were transduced with viral supernatants (MOI = 20). Post expansion, CAR-T cells were harvested and GFP expression was determined using flow cytometry.\u003c/p\u003e\u003cp\u003eTo generate mouse CAR-T cells, C57BL/6J mice was euthanized and spleen was isolated, then, spleen was grinded and mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque (GE healthcare) and T cells were obtained using sorting kit (Stemcell). T cells were stimulated with CD3/CD 28 activation beads (Miltenyi Biotec) at a 1:1 ratio (T cell: bead) for 48 hours in RPMI1640 medium supplemented with 10% fetal bovine serum, 50 IU mL\u003csup\u003e− 1\u003c/sup\u003e IL-7 and 100 IU mL\u003csup\u003e− 1\u003c/sup\u003e IL-15. After stimulating for 48 hours, activated T cells were transduced with lentiviruses (MOI = 100), and simultaneously supplemented with IL-7 and IL-15. Then, 12 hours later, T cells were harvested and cultured with RPMI1640 medium with IL-7 and IL-15.\u003c/p\u003e\u003cp\u003e \u003cb\u003eFlow cytometry\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe phenotype of primary human T cells and cell lines was determined using the following anti-human antibodies: B7-H3-PE/Cyanine7 (Biolegend, 351007), CD3-FITC (Biolegend, 300305), CD3-APC (Biolegend, 300411), CD25-APC (Biolegend, 302609), CD127-PE (BD, 557938), CD19-APC (BD, 561742), CD45RO-PE (Biohub, 78DA10076-100T), CD62L-PE (BD, 560966), CCR7-PE (BD, 561008), CD27-PE/Cyanine7 (Biolegend, 356411), CD28-APC (Biolegend, 302911), CD28-FITC (Biolegend, 302906), Ki67-APC (Biolegend, 350513), CD57-APC (Biolegend, 393305), KLRG1-PE (Biolegend, 368609), KLRG1-PE (BD, 568267), TIGIT-PC5.5 (Biolegend, 372717).\u003c/p\u003e\u003cp\u003eThe mouse T cells and immune cells was determined using the following anti-mouse antibodies: CD45-FITC (Biolegend, 103107), CD3-APC (Biolegend, 100236), CD4-PE/Cyanine7 (Biolegend, 100421), CD8-PC5.5 (Biolegend, 100737), NK1.1-PE (Biolegend, 108707), KLRG1-PE (Biolegend, 138407), TIGIT-APC (Biolegend, 156105).\u003c/p\u003e\u003cp\u003eAfter staining, all cells were incubated at RT for 30 min, washed thrice with PBS, then analyzed on a flow cytometer (Cytoflex, Backman). For apoptotic cell analysis, T cells were stained with FITC-Annexin V and Propidium Iodide Kit (BD, 556547) according to the manufacturer’s instructions. For cell cycle arrest assay, CAR-T cells were detected using Cell Cycle Assay Kit (Elabscience, E-CK-A351) according to the manufacturer’s instructions. All samples were analyzed with FlowJo software (v10.8.1) and GraphPad Prism Software 8.01.\u003c/p\u003e\u003cp\u003e \u003cb\u003eWestern blotting and Co-immunoprecipitation (co-IP) assay\u003c/b\u003e \u003c/p\u003e\u003cp\u003eProtein was extracted as previously described. Briefly, equal amounts of proteins were resolved by RIPA buffer and separated using SDS-PAGE. Protein concentrations were determined using the BCA assay (Thermo Fisher Scientific). The following primary antibodies were used with dilution ratio of 1:500-1:10000 : anti-CD3ζ (Bioworld, P20963), anti-Mysm1 (Abcam, ab193081), anti-Actin (Abcam, ab7817), anti-p-γH2AX (Cell Signaling Technology (CST), 9718T), anti-p53 (CST, 30313), anti-p-p53 (CST, 82530), anti-p16\u003csup\u003eINK4A\u003c/sup\u003e(CST, 92803), anti-p21 (CST, 2947), anti-Caspase3 (CST, 9662), anti-Bcl-2 (Bioworld, P10415), anti-Bcl-XL (Proteintech, 10783-AP), anti-Gelsolin (Proteintech, 11644-AP), anti-FMR1 (Proteintech, 13755-AP), anti-SSBP1 (OriGENE, TA382030), anti-Flag (Proteintech, 80801-2-RR), anti-VDAC1 (CST, 4866), anti-HA (CST, 3724), anti-Lamin A (OriGENE, TA803489). After blocking with 5% non-fat milk or bovine serum albumin, the blots were probed with primary antibodies. The bound antibodies were detected with an HRP-conjugated secondary antibody (1:5000) and visualized using a Tanon 4800 imaging system using super ECL detection reagent (YEASEN, 36208ES76). The nuclear, cytoplasmic and mitochondrial fraction were obtained using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027) and Cell Mitochondria Isolation Kit (Beyotime, C3601) according to the manufacturer’s protocol.\u003c/p\u003e\u003cp\u003eFor the whole cell lysate co-IP assay, after treatment, equal amounts of cells were harvested and incubated in Pierce IP lysis buffer (Thermo Scientific), supplemented with 1% protease inhibitor cocktail (YEASEN, 20123ES10), on ice for 20 min. After centrifugation for 15 min at 4℃, the supernatants were collected and incubated with primary IP antibodies at 4℃ for 12 h. After washed three times with ice-cold PBS, protein A/G magnetic beads (Selleckchem) was added and co-cultured at 4℃ for 2 h, then washed three times with ice-cold PBS. Finally, the samples were boiled in SDS loading buffer and analyzed by western blotting.\u003c/p\u003e\u003cp\u003e \u003cb\u003eCRISPR-Cas9 genome editing and overexpression of\u003c/b\u003e \u003cb\u003eMYSM1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eSSBP1\u003c/b\u003e \u003cb\u003ein T and Jurkat cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003e \u003cem\u003eMysm1\u003c/em\u003e and \u003cem\u003eSSBP1\u003c/em\u003e were deleted in T and Jurkat cells using CRISPR Cas9-mediated gene editing. Briefly, \u003cem\u003eMysm1\u003c/em\u003e and \u003cem\u003eSSBP1\u003c/em\u003e single guide RNA (sgRNA) sequence were constructed in lentiCRISPR v2 (addgene) followed lentivirus packing using HEK293T cells. Then T or Jurkat cells were transfected using lentivirus and effect of knock out was confirmed by western blotting. The following sgRNA sequences were used:\u003c/p\u003e\u003cp\u003e \u003cem\u003eMysm1\u003c/em\u003e-forword-1: 5’-acgtagggtccgagacccat-3’;\u003c/p\u003e\u003cp\u003e \u003cem\u003eMysm1\u003c/em\u003e-reverse-1: 5’-ATGGGTCTCGGACCCTACGt-3’;\u003c/p\u003e\u003cp\u003e \u003cem\u003eMysm1\u003c/em\u003e-forword-2: 5’-GAGATGTTAATTGTATTGGA-3’;\u003c/p\u003e\u003cp\u003e \u003cem\u003eMysm1\u003c/em\u003e-reverse-2: 5’-TCCAATACAATTAACATCTC-3’;\u003c/p\u003e\u003cp\u003e \u003cem\u003eMysm1\u003c/em\u003e-forword-3: 5’-ttatggccggtcttctgatt-3’;\u003c/p\u003e\u003cp\u003e \u003cem\u003eMysm1\u003c/em\u003e-reverse-3: 5’-aatcagaagaccggccataa-3’;\u003c/p\u003e\u003cp\u003e \u003cem\u003eSSBP1\u003c/em\u003e-forword: 5’-GGACCCTGTCTTGAGACAGG-3’;\u003c/p\u003e\u003cp\u003e \u003cem\u003eSSBP1\u003c/em\u003e-reverse: 5’-CCTGTCTCAAGACAGGGTCC-3’;\u003c/p\u003e\u003cp\u003eThe lentiviral vectors of \u003cem\u003eMysm1\u003c/em\u003e and 3×Flag tagged \u003cem\u003eSSBP1\u003c/em\u003e were created into a lentiviral vector pCDH-CMV-MCS-EF1-CopGFP. To create MPN\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e enzymatic domain-deleted mutant (ΔC) \u003cem\u003eMysm1\u003c/em\u003e, 1-572 aa was retained and cloned into pCDH-CMV-MCS-EF1-CopGFP. Meanwhile, MPN\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e enzymatic domain-mutant (mut) \u003cem\u003eMysm1\u003c/em\u003e was established and cloned into pCDH-CMV-MCS-EF1-CopGFP. The amino acid sequence of full-length MYSM1, mut MYSM1 and SSBP1 showed blow.\u003c/p\u003e\u003cp\u003e \u003cb\u003eqPCR and primers\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTotal mRNA was extracted using TRIzol regent (TIANGEN, China) and analyzed by quantitative real-time PCR using the RevertAid First Strand cDNA Synthesis Kit(Thermo Fisher Scientific). Total DNA was extracted using Universal Genomic DNA Purification Mini Spin Kit (Beyotime, China). All reactions were performed with TaqMan Fast Universal PCR Master Mix (Vazyme, China) on a QuantStudioTM 3 Real-Time PCR Instrument (Applied Biosystems), using the following primer pairs.\u003c/p\u003e\u003cp\u003eh\u003cem\u003eMysm1\u003c/em\u003e-forward: 5’-ATCATGTTTAAGGGGACGTGCTGA-3’;\u003c/p\u003e\u003cp\u003eh\u003cem\u003eMysm1\u003c/em\u003e-reverse: 5’-TCCTGGCTGTCAGAAGTAATGAAT-3’;\u003c/p\u003e\u003cp\u003eh\u003cem\u003ep16\u003c/em\u003e-forward: 5’-CTTCGGCTGACTGGCTGG-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003ep16\u003c/em\u003e-reverse: 5’-GCTGCCCATCATCATGACCT-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003ep21\u003c/em\u003e-forward: 5’-CTGTCACTGTCTTGTACCCTTGT-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003ep21\u003c/em\u003e-reverse: 5’-CCCAGCAGAGGAACCACTACTA-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eIL-1α\u003c/em\u003e-forward: 5’-CTTCTGGGAAACTCACGGCA-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eIL-1α\u003c/em\u003e-reverse: AGCACACCCAGTAGTCTTGC-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eIL-6\u003c/em\u003e-forward: 5’-GTAGCCGCCCCACACAGA-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eIL-6\u003c/em\u003e-reverse: 5’-CATGTCTCCTTTCTCAGGGCT-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eMMP9\u003c/em\u003e-forward: 5’-GGACAAGCTCTTCGGCTTCT-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eMMP9\u003c/em\u003e-reverse: 5’-TCGCTGGTACAGGTCGAGTA-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eTCF7\u003c/em\u003e-forward: 5’-CTGGCTTCTACTCCCTGACCT-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eTCF7\u003c/em\u003e-reverse: 5’-ACCAGAACCTAGCAT CAAGGA- 3\u003c/p\u003e\u003cp\u003eh\u003cem\u003eβ-actin\u003c/em\u003e-forward: 5’-GAAGAGCTACGAGCTGCCTGA-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eβ-actin\u003c/em\u003e-reverse: 5’-CAGACAGCACTGTGTTGGCG-3’\u003c/p\u003e\u003cp\u003ehmtD-\u003cem\u003eCOX1\u003c/em\u003e-forward: 5’-TCGCATCTGCTATAGTGGAG-3’ \u003c/p\u003e\u003cp\u003ehmtD-\u003cem\u003eCOX1\u003c/em\u003e-reverse: 5’-ATTATTCCGAAGCCTGGTAGG-3’\u003c/p\u003e\u003cp\u003ehmtD-\u003cem\u003eLoop2\u003c/em\u003e-forward: 5’-GGCTCTCAACTCCAGCATGT-3’\u003c/p\u003e\u003cp\u003ehmtD-\u003cem\u003eLoop2\u003c/em\u003e-reverse: 5’-AGGACGAGGGAGGCTACAAT-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eB2M\u003c/em\u003e-forward: 5’-CCAGCAGAGAATGGAAAGTCAA-3’\u003c/p\u003e\u003cp\u003eh\u003cem\u003eB2M\u003c/em\u003e-reverse: 5’-TCTCTCTCCATTCTTCAGTAAGTCAACT-3’\u003c/p\u003e\u003cp\u003e \u003cb\u003eMulti-rounds co-culture killing experiment\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTumor cells and CAR-T cells were simultaneously seeded in 24-well plates at a concentration of 1×10\u003csup\u003e5\u003c/sup\u003e cells per well individually. 48 h later, cells were collected and T cells and tumor cells were measured by flow cytometry respectively. At the same time, 1×10\u003csup\u003e5\u003c/sup\u003e tumor cells were added to the parallel pales with mixture of CAR-T cells and residual tumor cells. One more 48 h later, cells were collected and measured by flow cytometry. For multiple rounds of co-culture, the CAR-T cells and residual tumor cells were record and total CAR-T cells per well were counted.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSA-β gal assay\u003c/b\u003e \u003c/p\u003e\u003cp\u003eSenescence-associated β-galactosidase (SA-β gal) staining was performed using Cell senescence Detection Kit (Dojindo, SG03) and CellEvent Senescence Green (Thermo Fisher, C10841) according to the manufacturer’s instructions. In senescence-inducing experiment, T or CAR-T cells were treated for 48 h with doxorubicin (2×10\u003csup\u003e7\u003c/sup\u003e M) and then analyzed using Cell senescence Detection Kit.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSeahorse analysis\u003c/b\u003e \u003c/p\u003e\u003cp\u003eOxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured in XF media. Briefly, 1×10\u003csup\u003e6\u003c/sup\u003e purified CAR-T cells were seeded in a Seahorse Bioscience culture plate coated with Cell-Tak solution (Corning), and cultured in XF media (non-buffered X-VIVO 15 containing with 25 mM glucose, 2 mM glutamine and 1 mM pyruvate) under basal conditions and in response to 200 µM etomoxir (Tocris), 1 µM OM, 1.5 µM fluoro-carbonyl cyanide phenylhydrazone (FCCP) and 100 nM rotenone + 1 µM antimycin A. Basal, maximal OCR were measured by an XF96 Seahorse Extracellular Flux Analyzer (Agilent) following the manufacturer’s instruction.\u003c/p\u003e\u003cp\u003e \u003cb\u003eMeasurement of mitochondrial membrane potential\u003c/b\u003e \u003c/p\u003e\u003cp\u003eMitochondrial membrane potential was estimated by measuring the fluorescence of free JC-1 monomers (green) and JC-1 aggregates in mitochondria (red) using mitochondrial membrane potential assay kit with JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide) (Beyotime, C2006) and MitoTracker Red (YEASEN, 40741ES50), according to the manufacturer’s protocol. And the results were expressed as the ratio of the aggregates/monomers of JC-1 in the percentage of control. Mitochondrial depolarization was indicated by a decrease in the polymer/monomer fluorescence intensity ratio. JC-1 staining was observed and photographed by the fluorescence microscope. In addition, CAR-T cells were loaded with 100 nM MitoTracker Red to measure mitochondrial membrane potential using a flow cytometry.\u003c/p\u003e\u003cp\u003e \u003cb\u003eTransmission electron microscopy\u003c/b\u003e \u003c/p\u003e\u003cp\u003eCAR-T cells were fixed with 2.5% EM-grade glutaraldehyde and 2% EM-grade paraformaldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) at RT for 5 min. Following fixation, samples were washed in cocodylate buffer and post fixed in 1% osmium tetroxide. After extensive washing in H\u003csub\u003e2\u003c/sub\u003eO, T cells were rinsed with 0.1 M cacodylate buffer and incubated with 1% osmium tetroxide in H\u003csub\u003e2\u003c/sub\u003eO for 20 min. Cells were washed in H\u003csub\u003e2\u003c/sub\u003eO for 1 min four times and stained with 1% uranyl acetate in H\u003csub\u003e2\u003c/sub\u003eO for 14 min. Afterwards, cells were rinsed in H\u003csub\u003e2\u003c/sub\u003eO four times for 1 min each. Dehydration with an acetone series (50%, 70%, 90%, 2×100%) was performed for 45 s per step in the microwave. Cut sections were stained with uranyl acetate and lead citrate and then imaged using a JOEL 1200 EX transmission electron microscope equipped with an 8 MP ATMP digital camera (Advanced Microscopy Techniques).\u003c/p\u003e\u003cp\u003e \u003cb\u003eRNA-Seq analysis and LC-MS/MS assay\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTotal RNA of CAR-T cells was extracted from CAR-T cells and messenger RNA libraries were prepared using RNA simple Total RNA Kit (DP419, TIANGEN). Paired-end libraries were synthesized using U-mRNAseq Library Prep Kit (AT4221, KAITAI-BIO) with Ribo off rRNA Depletion Kit (N407, Vazyme) following the RNA Sample Preparation Guide. Library construction and sequencing were performed by Sinotech Genomics (Shanghai, China). Briefly, raw reads were trimmed using fastp (v0.23.0), removing low-quality reads and removing reads with size inferior to 50 bp and poly A-containing mRNA molecules were purified using poly T oligo-attached magnetic beads. Paired-end sequence files were mapped to the reference genome using Hisat2 (Hierarchical Indexing for Spliced Alignment of Transcripts, V.2.0.5). The output SAM files were converted to binary alignment/map files and sorted using SAM tools (V.1.3.1).\u003c/p\u003e\u003cp\u003eThe LC-MC/MC analysis was performed by Shanghai Luming biological technology co., LTD (Shanghai, China). Briefly, Digested samples were separated by reverse phase C18 chromatography on nano-HPLC liquid phase system Easy-NLC1200, and the dried polypeptide samples were first re-dissolved in Nano-HPLC Buffer A, liquid A was 0.1% formic acid-aqueous solution, and liquid B was 0.1% formic acid-acetonitrile solution. The samples were then loaded by an automatic sampler and adsorbed to a Trap column, and separated on an Analysis column, 75 µm×150 mm at a flow rate of 300 nL/min. The samples were cleaned by mobile phase gradient with blank solvent for 30 min. The hydrolysates were separated by capillary high performance liquid chromatography and analyzed by Q-Exactive mass spectrometry (Thermo Scientific). The scanning range of parent ions was corrected by standard correction fluid at 300–1600 m/z, and the scanning mode of mass spectrometry was Data Dependent Aqcuisition (DDA). The 20 strongest fragment profiles (MS2 Scan) are collected after each full scan. Fragmentation was performed using high-energy collision dissociation (HCD, high energy) with an NCE energy of 28 and dynamic removal time of 25s. MS/MS spectra were searched using the MaxQuant against the Uniprot Mus Musculus database.\u003c/p\u003e\u003cp\u003e \u003cb\u003eConfocal imaging\u003c/b\u003e \u003c/p\u003e\u003cp\u003eT or CAR-T cells were fixed by 4% paraformaldehyde for 30 min at room temperature. After washing, cells were blocked with 5% normal goat serum for 1 h and incubated with primary antibody at 4 ℃ overnight. The next day, cells were washed three times with PBS and incubated with 5 µg/mL Alexa Fluor 488 or 594 conjugated goat-anti-human (H + L) secondary antibody (Proteintech) for 1 h in the dark and then were stained with DAPI for 5 min. Imaging was acquired with a Leica DM6 confocal microscopy. The primary antibodies used were anti-TOM20 (Abclonal, A19403), anti-MYSM1 (Proteintech, 20078-1-AP) and anti-SSBP1 (OriGENE, TA382030).\u003c/p\u003e\u003cp\u003e \u003cb\u003eClinical samples\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor clinical specimens, fresh tumor samples from patients with de-identified B lymphoma were obtained from Shandong Provincial Hospital Affiliated to Shandong First Medical University (Jinan, China). PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare), and B cell subsets were detected using anti-CD19 antibody. The samples were used, and the percentage of CD19-positive B cells was greater than 60%.\u003c/p\u003e\u003cp\u003e \u003cb\u003eIn vitro killing experiment\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTo validate the killing specificity of the constructed CAR-T cells, three killing ways were used. As previously described\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, 5×10\u003csup\u003e5\u003c/sup\u003e target cells were co-incubated with different amounts of CAR-T cells in a total volume of 1 mL. After 18 h, the mixture of CAR-T cells and tumor cells were harvested and stained antibodies, then analyzed using flow cytometry, thus killing rates of CAR-T cells to targets were calculated according to changes of target cells.\u003c/p\u003e\u003cp\u003eIn addition, in vitro anti-target cells cytotoxicity of CAR-T cells was monitored in real-time using Smart cell real-time monitor (East China University of Science and Technology, Shanghai, six broad beans; CM100-α). Cell index correlates with the number of cells attached to the bottom of the plate. Briefly, ten thousands target cells were plated into specific 96-well plates. About five hours later, ten thousands CAR-T or control T cells were added to plates and co-cultured overnight. Then the growth curve of target cells was monitored by Smart cell real-time monitor.\u003c/p\u003e\u003cp\u003e \u003cb\u003eIn vivo persistence experiment\u003c/b\u003e \u003c/p\u003e\u003cp\u003e All animal experiments were performed on protocols approved by the Institutional Animal Care and Use Committee of Shandong First Medical University (Ethics Approval No.: NSFC. 2023 − 176). The in vivo persistence experiment was conducted using 5-6-week-old female B-NDG mice (NOD-Prkdc\u003csup\u003escid\u003c/sup\u003e Il2rg\u003csup\u003etm1\u003c/sup\u003e) (Biocytogen). The mice were intravenously injected with 5×10\u003csup\u003e5\u003c/sup\u003e T cells (UTD, 19BBz and 19BBz-M) suspended in 200 µL PBS with five to seven mice in each group. Mice were intravenously injected with 2×10\u003csup\u003e4\u003c/sup\u003e Na-luc cells on day 7, 12, 17, and 22 respectively. Peripheral blood was obtained every 5 days via venous blood collection, erythrocytes were lysed with red blood cell lysis buffer and mononuclear cells were stained with ant-CD3 antibody and analyzed by flow cytometry.\u003c/p\u003e\u003cp\u003e \u003cb\u003eIn vivo bioluminescence imaging\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe D-luciferin (Beyotime) in PBS was used as a substrate for luciferase followed the manufacturer’s protocols. Tumor progression was monitored by bioluminescence imaging using an In Vivo Imaging System (IVIS) Spectrum Imaging System (PerkinElmer). Living Image V.4.5.5 (PerkinElmer) was used to acquire (and later quantify) the data 10 min after intraperitoneal injection of D-luciferin into animals that were anesthetized with 150 mg kg\u003csup\u003e− 1\u003c/sup\u003e of 1% pentobarbital sodium (Sigma-Aldrich).The acquisition time ranged from 1 s to 1 min. Imaging settings were kept the same throughout the duration of the experiment.\u003c/p\u003e\u003cp\u003e \u003cb\u003eTumor xenograft models\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe experiments were conducted at the Animal Experiment Center of Shandong First Medical University. Five to six-week-old B-NDG female mice were used in both lymphoma and hepatoma models. For the disseminated Na-luc model, 2–5×10\u003csup\u003e5\u003c/sup\u003e Na-luc were intravenously injected on day 0. 5×10\u003csup\u003e6\u003c/sup\u003e CAR-T cells were intravenously injected subsequently. For the local hepG2 model, mice were injected subcutaneously with 1×10\u003csup\u003e6\u003c/sup\u003e hepG2 cells, and on day 7 and 14, with 5×10\u003csup\u003e6\u003c/sup\u003e CAR-T cells, individually. Tumor burden was assessed weekly using bioluminescent IVIS Spectrum Imaging System.\u003c/p\u003e\u003cp\u003eFor the 4T1 model using Balb/c mice (HFK Bioscience), the mice were irradiated with 5 Gy on day − 2, and 1×10\u003csup\u003e5\u003c/sup\u003e 4T1-CD19 (expressing human CD19) or 4T1-GPC3 (expressing human GPC3) cells were subcutaneously injected on day 0. 1×10\u003csup\u003e6\u003c/sup\u003e mouse CAR-T cells were intravenously injected. The tumor size of each mouse was measured every 2 days. The tumor size was calculated using the following formula: 4π/3×(tumor length/2)×(tumor width/2)\u003csup\u003e2\u003c/sup\u003e. The mice were humane manner when death was imminent or when the tumor size reached about 12 mm. In parallel mice experiment, the tumors were separated from body on day 16 and were digested with collagenase IV. The lysate was purified with red blood cell lysis buffer and cells were analyzed by flow cytometry.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe in vitro experiments were repeated at least three times for each group, and One-way ANOVA or Student’s \u003cem\u003et-test\u003c/em\u003e was used to compare quantitative data (mean ± SD) between the samples. A \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 indicated significance. For the in vivo experiments, the survival curves of mice were generated using the Kaplan–Meier method, and survival data were analyzed using the log-rank (Mantel-Cox) test. A \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 indicated significance. The obtained statistic was analyzed with Graphpad Prism 8.0.1.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eWe thank Professor Xiangbo Meng from Advanced Medical Research Institute, Shandong University for providing data analysis. The authors thank the support from Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong, Translational Medicine Core Facility of Advanced Medical Research Institute, Shandong University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Shandong Province (ZR2023QC179, ZR2024ZD11), National Natural Science Foundation of China (82271878, 82302067, 82370618, 82160124), and “Open Competition to Select the Best Candidates” Key Technology Program for Cell Therapy of NCTIB (NCTIB2023XB02006). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eSB. Z. performed the experiments, acquired and interpreted the data, and wrote the manuscript. HM. L. and MH. S. were involved in the construction of plasmid and lentiviral vectors. YC. X. and BH. W. contributed to the flow cytometry. TR. Z., H. D., XY. W. and H.G. participated in immunofluorescent staining and graphic plotting. JN. Q. contributed to the animal studies partly. Q. Z. and LF. G. designed the project, assisted in the experiments, provided funding support and finalized the manuscript. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePark JH. et al. CD19-targeted CAR T-cell therapeutics for hematologic malignancies: Interpreting clinical outcomes to date. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 3312\u0026ndash;3320 (2016). \u003c/li\u003e\n\u003cli\u003eFraietta JA. et al. 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T cell replicative senescence in human aging. \u003cem\u003eCurr Pharm Des\u003c/em\u003e. \u003cstrong\u003e19\u003c/strong\u003e, 1680\u0026ndash;98 (2013). \u003c/li\u003e\n\u003cli\u003eMou D. et al. CD28 negative T cells: is their loss our gain? \u003cem\u003eAm J Transplant\u003c/em\u003e. \u003cstrong\u003e14\u003c/strong\u003e, 2460\u0026ndash;6 (2014). \u003c/li\u003e\n\u003cli\u003eKroeger C. et al. Interaction of Deubiquitinase 2A-DUB/MYSM1 with DNA Repair and Replication Factors. \u003cem\u003eInt J Mol Sci\u003c/em\u003e. \u003cstrong\u003e21\u003c/strong\u003e, 3762 (2020). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CAR-T, Mysm1, senescence, anti-tumor, SSBP1","lastPublishedDoi":"10.21203/rs.3.rs-6947364/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6947364/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChimeric antigen receptor (CAR)-T cell immunotherapy has demonstrated remarkable success in treating hematological malignancies, yet its clinical efficacy remains limited, particularly against solid tumors. Emerging evidence implicates T cell senescence as a key immunosuppressive barrier in cancer immunotherapy. In this study, we engineered Mysm1-overexpressing CAR-T cells and identified Mysm1 augmentation significantly enhances cytotoxic function and anti-tumor activity across multiple preclinical models. Integrated transcriptomic and biochemical analyses revealed that Mysm1 sustains mitochondrial homeostasis in CAR-T cells by interacting with SSBP1. Mechanistically, MYSM1 catalyzed K48-linked deubiquitination of SSBP1, thereby preserving mitochondrial function and mitigating CAR-T cell senescence. This intervention resulted in prolonged persistence and sustained anti-tumor efficacy in both murine and human CAR-T cells. Our findings unveil a novel strategy to counteract CAR-T cell senescence and establish Mysm1 as a promising therapeutic target for enhancing CAR-T cell immunotherapy.\u003c/p\u003e","manuscriptTitle":"Targeting CAR-T cell senescence through Mysm1-SSBP1 axis improves persistence and therapeutic efficacy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-08 15:04:53","doi":"10.21203/rs.3.rs-6947364/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"08a80976-0edf-42ec-bff6-7c5ec1d4fca6","owner":[],"postedDate":"July 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":50904814,"name":"Biological sciences/Immunology/Lymphocytes"},{"id":50904815,"name":"Health sciences/Diseases/Cancer/Cancer models"}],"tags":[],"updatedAt":"2025-07-08T15:04:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-08 15:04:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6947364","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6947364","identity":"rs-6947364","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}