Vam6 upregulated by lactic acid inhibits anti-tumor effects of intratumoral iNKT cells via modulating AMPK/mTOR pathways

Vam6 upregulated by lactic acid inhibits anti-tumor effects of intratumoral iNKT cells via modulating AMPK/mTOR pathways | 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 Vam6 upregulated by lactic acid inhibits anti-tumor effects of intratumoral iNKT cells via modulating AMPK/mTOR pathways Huimin Zhang, Shiyu Bai, Qielan Wu, Shasha Zhu, Yuwei Zhang, Xuran Chen, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1990874/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Activation of mTORC1 is essential for anti-tumor function of iNKT cells. The mechanisms underlying impaired mTORC1 activation in intratumoral iNKT cells remain unclear. Here, we showed that lactic acid from tumor cells increased Vam6 expression in iNKT cells which led to impaired mTORC1 activation and IFN-γ production. Mechanistically, Vam6 in iNKT cells was essential for Rab7a-Vam6-AMPK complex formation and thus for recruitment of AMPK to lysosome to activate AMPK, a negative regulator of mTORC1. Additionally, Vam6 relieved inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation at mitochondria-lysosome contact site. Given the key roles of lactic acid-increased Vam6 in promoting AMPK activation in intratumoral iNKT cells, reducing Vam6 expression significantly enhanced the mTORC1 activation in intratumoral iNKT cells as well as their anti-tumor efficacy. Together, we propose Vam6 as a target for iNKT cell-based immunotherapy. Vam6 mTORC1 AMPK Rab7a-Vam6-AMPK complex iNKT cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Invariant natural killer T (iNKT) cells are innate-like T cells which express semi-invariant TCR and recognize lipid antigens presented by CD1d 1 . Upon activation, iNKT cells produce both Th1 and Th2 cytokines and mediate cytotoxicity as well. These cells bridge the innate immunity and adaptive immunity 2 , and are great candidates for immunotherapy against tumors, given their direct and indirect tumor killing effects and low risk of inducing cytokine storm and graft versus host disease 3 . However, dysfunction of intratumoral iNKT cells has been reported to hinder their anti-tumor effects, and that is attributed to impaired activation of mTORC1 4,5 . Although the lactic acid in tumor microenvironment has been shown to inhibit activation of mTORC1, the underlying mechanisms are largely unknown. Lysosomal proteins, lysosomal location of mTOR, and lysosome function are essential for mTORC1 activation 6 . AMPK that inhibits mTORC1 activation also locates at lysosome, and its activity is controlled by lysosome related proteins 7 . These findings indicate lysosome as a crucial hub for regulating mTORC1 pathway 8 . Vam6 (also named Vps39), a key member of the homotypic fusion and vacuole protein sorting complex that controls tethering and fusion of lysosome with intracellular compartments 9 , has been shown to regulate mTOR pathway in myoblast and senescent T cells 10 , 11 . However, opposite roles of Vam6 have been indicated by those two studies, and the molecular mechanisms that link Vam6 to mTOR pathway are still unknown. Whether and how Vam6 regulates mTORC1 activity and contributes to dysfunction of intratumoral iNKT cells remain to be explored. Here, we found that Vam6 promoted AMPK activation and consequently inhibited mTORC1 activation and IFN-γ production in iNKT cells via forming Rab7a-Vam6-AMPK complex and recruiting AMPK to lysosome. VDAC1 interacting with Rab7a at mitochondria-lysosome contact site interfered with Rab7a-Vam6-AMPK complex formation and inhibited AMPK activation, demonstrating a negative control of this signal pathway at mitochondria-lysosome contact site. Notably, Vam6 inhibited Rab7a -VDAC1 interaction and relieved the inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation. Our data propose dual roles of Vam6 in promoting AMPK activation. Moreover, we showed that lactic acid in tumor microenvironment increased Vam6 expression in iNKT cells, and that led to increased AMPK activation, diminished mTORC1 activation, and impaired IFN-γ production in intratumoral iNKT cells. Reducing Vam6 expression, on the contrary, successfully restored function of intratumoral iNKT cells and enhanced their anti-tumor efficacy in mouse tumor models. Our data suggest that Vam6 could be a target for iNKT cell-based anti-tumor immunotherapy. Results Vam6 upregulated by lactic acid inhibits function of intratumoral iNKT cells Previous study has reported dysfunction of intratumoral iNKT cells, and that is partially contributed to accumulation of lactic acid in tumor microenvironment 4 . Here, we found that intratumoral iNKT cells expressed higher level of Vam6 than splenic iNKT cells, irrespective of activation (Fig. 1a). The upregulation of Vam6 was caused by treatment of lactic acid (Fig. 1b), and knockdown of Ldha in tumor cells that led to less lactic acid production significantly reduced Vam6 expression in intratumoral iNKT cells (Fig. 1c). Next, we investigated whether the lactic acid-upregulated Vam6 inhibited iNKT cell function in tumors. Since Vam6 -/- homozygous mice were embryonically lethal 10 , we generated Vam6 +/- mice and confirmed reduction of Vam6 expression in these animals using splenic Vam6 +/- T cells (Fig. S1a). Reducing Vam6 expression in Vam6 +/- mice did not influence the frequencies of iNKT cells in thymuses, spleens, and livers (Fig. S1b and S1c). The absolute numbers of thymic and splenic iNKT cells were normal in Vam6 +/- mice, despite the slightly elevated numbers of hepatic iNKT cells (Fig. S1c). These results demonstrate normal iNKT cell development in Vam6 +/- mice. To study the influence of reduced Vam6 expression on iNKT cell function, we sorted iNKT cells from livers of Vam6 +/- mice and Vam6 +/+ mice, respectively, and activated them with CD1d-PBS57 tetramer in vitro . Upon activation, Vam6 +/- iNKT cells produced more IFN-g and IL-4 in supernatants, than Vam6 +/+ iNKT cells did (Fig. 1d). Additionally, Vam6 +/- iNKT cells expressed higher level of anti-apoptosis protein Bcl-2 (Fig. 1e), exhibited lower apoptosis (Fig. 1f) but higher proliferation (Fig. 1g). To exclude the possibility that the increased cytokine production in supernatants were due to enhanced cell proliferation, we measured the intracellular cytokine, 4 hours after injecting lipid antigen a-galactosylceramide (a-GC) into lethally irradiated iNKT-deficient J a 18 -/- mice that received CD45.2 + Vam6 +/- bone marrows mixed with CD45.1 + Vam6 +/+ bone marrows at a ratio of 1:1 (Fig. 1h). Consistently, reduced expression of Vam6 did not influence the development of iNKT cells in these chimeric mice, and CD45.2 + Vam6 +/- iNKT cells exhibited higher levels of IFN-γ production than CD45.1 + Vam6 +/+ iNKT cells did (Fig. 1i). These results also demonstrate the intrinsic role of Vam6 in inhibiting iNKT cell function in vivo . Moreover, we showed that reducing Vam6 expression restored IFN-γ production in lactic acid treated iNKT cells (Fig. 1j). In B16F10 tumors from those chimeric mice, intratumoral iNKT cells with reduced Vam6 expression produced more IFN-γ (Fig. 1k). Together, these data prove that the upregulation of Vam6 caused by lactic acid leads to iNKT cell dysfunction in tumors. Vam6 inhibits iNKT cell functions via modulating AMPK/mTOR pathways To study the role of Vam6 in controlling iNKT cell function, we performed RNA-seq experiments and found that reducing Vam6 expression altered gene expression profile in iNKT cells (Fig. 2a and 2b). 1712 genes were upregulated whereas 1728 genes were downregulated in Vam6 +/- iNKT cells. KEGG (Kyoto Encyclopedia of Genes and Genomespathway) analysis and GO (Gene Ontology) term analysis showed that the upregulated genes were enriched in cell cycle, cell activation, and metabolic processes, whereas the downregulated genes were enriched in cell death, autophagy, and AMPK pathway. The gene profile of Vam6 +/- iNKT cells was in line with their increased proliferation, survival, and function (Fig. 2c and 2d). It is well-known that cell growth and metabolic processes are promoted by mTORC1 12 whereas the autophagy is inhibited by mTORC1 13 . Additionally, AMPK is a negative regulator of mTORC1 14 , 15 . Therefore, our gene enrichment analysis implied alterations in AMPK/mTOR pathways in Vam6 +/- iNKT cells. Indeed, we found increased expression of genes related to mTORC1 pathway (Fig. 2e) and decreased expression of genes related to AMPK pathway (Fig. 2f) in these iNKT cells with reduced Vam6 expression. Next, we measured activation of mTORC1 and AMPK at protein levels, as indicated by the phosphorylation of S6 S235/236 and phosphorylation of AMPKa, respectively. iNKT cells were activated either in vivo by injecting a-GC into J a 18 -/- mice transplanted with CD45.2 + Vam6 +/- bone marrows and CD45.1 + Vam6 +/+ bone marrows or in vitro by anti-CD3 plus anti-CD28 stimulation (Fig. 3a-3d). We found that, in both conditions, reducing Vam6 expression elevated mTORC1 activation (Fig. 3a and 3c) and diminished AMPK activation (Fig. 3b and 3d) . Consistently, in tumors (Fig. 3e), Vam6 +/- iNKT cells displayed higher level of mTORC1 activation (Fig. 3f) and lower level of AMPK activation (Fig. 3g) than Vam6 +/+ iNKT cells after a-GC injection. Activation of mTORC1 has been previously reported to promote cytokine production in iNKT cells 5 . Notably, reducing Vam6 expression failed to enhance IFN-γ production in iNKT cells treated with mTORC1 inhibitor rapamycin, further confirming that reduced Vam6 increased iNKT cell function via promoting mTORC1 activation (Fig. 3h and 3i). AMPK is a well-known negative regulator of mTORC1 pathway 15 . Here, we showed that reducing AMPK activation via Compound C increased mTORC1 activation and IFN-γ production in CD1d-PBS57 tetramer-stimulated iNKT cells (Fig. 3j and 3k), confirming the role of AMPK in suppressing mTORC1 activation in iNKT cells. Together, our results indicate that Vam6 in iNKT cells inhibits mTORC1 activation and IFN-γ production via promoting AMPK activation. Vam6 promotes activation of AMPK via recruiting AMPK to lysosome Despite the reduced phosphorylation of AMPKa, Vam6 +/- iNKT cells expressed similar level of AMPKa protein as Vam6 +/+ cells did (Fig. 4a). Then, we investigated whether Vam6 promoted AMPKa phosphorylation via protein interactions. We performed co-immunoprecipitation experiments with expanded iNKT cells (Fig. 4b and Fig. S2a), in which reducing Vam6 expression (Figure. S2b) increased IFN-g production (Figure. S2c and S2d) and S6 S235/236 phosphorylation (Figure. S2e) whereas diminished AMPKa phosphorylation (Figure. S2f), as it did in fresh iNKT cells. We found that AMPKg3, Rab7a, and VDAC1 were co-immunoprecipitated with Vam6 (Fig. 4b), indicating interactions between these proteins. Rab7a is a marker protein for lysosome 16 . Recruiting AMPK to lysosome has been reported to be essential for AMPK activation 7 . Next, we investigated whether Vam6 regulated recruitment of AMPK to lysosome via protein interactions. Using in situ proximity ligation assay (PLA) approach, we showed that AMPKg interacted with Rab7a in activated iNKT cells, and reducing Vam6 expression impaired the Rab7a-AMPKg interaction, as indicated by reduced Rab7a-AMPKg PLA puncta numbers (Fig. 4c). Additionally, we proved that reducing Vam6 expression did not influence the protein level of Rab7a (Fig. 4d). In line with the lysosomal location of Rab7a, the Rab7a-AMPKg complex was colocalized with lysosome marker LAMP2, indicating lysosomal location of AMPKg-Rab7a interaction (Fig. 4e). In agreement with the reduced Rab7a-AMPKg interaction in activated Vam6 +/- iNKT cells, these cells had less amount of AMPK on lysosome in comparison with activated Vam6 +/+ iNKT cells (Fig. 4f). These data demonstrate that Vam6 promotes Rab7a-AMPKg interaction and recruits AMPK to lysosome. Notably, when AMPK in CD1d-PBS57 tetramer-stimulated iNKT cells was activated by AICAR, an AMP analog, the Rab7a-AMPKg PLA puncta numbers (Fig. 4g), the colocalization coefficient of AMPKg and LAMP2 (Fig. 4h), and the AMPKa phosphorylation (Fig. 4i) were all increased in a Vam6 dependent manner. As a consequence, the inhibitory effects of AICAR on mTORC1 activation (Fig. 4j) and on IFN-γ production (Fig. 4k) depended on Vam6 as well. Together, these data demonstrate an essential role of Vam6 in recruiting AMPK to lysosome and activating AMPK, and exclude the possibility of regulating AMPK activation via influencing AMP production. To understand how Vam6 regulated Rab7a-AMPKg interaction, we generated various truncated Vam6 based on previous study 17 , including the C terminal-deleted DCT, the middle fragment CLH-deleted DCLH, and the N terminal-deleted DCNH (Fig. S3a), and showed that Rab7a bound wide type Vam6 as well as DCT but not DCNH or DCLH, and AMPKg3 only bound wide type Vam6 (Fig. S3b). These results suggest that Rab7a binds Vam6 in a N terminal and CLH fragment dependent manner, and AMPK is dispensable for their interaction. Although DCT interacted with Rab7a, it failed to recruit AMPK, suggesting a key role of Vam6 C terminal in forming Rab7a-Vam6-AMPK complex. We could not exclude the possibility that AMPK might not directly bind Rab7a but indirectly interacts with Rab7a through Vam6. Vam6 relieves inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation Among the proteins co-immunoprecipitated with Vam6, we detected a mitochondrial protein VDAC1 (Fig. 4b). In activated iNKT cells, reducing Vam6 expression promoted VDAC1-Rab7a interaction, as indicated by increased puncta numbers of VDAC1-Rab7a PLA (Fig. 5a). Given the fact that VDAC1 and Rab7a are mitochondrial protein and lysosomal protein, respectively, the VDAC1-Rab7a interaction might occur at the contact site between lysosome and mitochondria 18 . Indeed, we found that VDAC1-Rab7a interaction sites were colocalized well with mitochondria probe Mitotracker Deep Red and with lysosome marker LAMP2 as well, confirming the existence of VDAC1-Rab7a interaction at mitochondrial-lysosome contact sites (Fig. 5b). In line with the increased VDAC1-Rab7a interaction in Vam6 +/- iNKT cells, these cells displayed elevated mitochondrial-lysosome contacts (Fig. S4a). Meanwhile, the expression levels of mitochondrial maker VDAC1 (Fig. S4b) and lysosomal markers LAMP2 (Fig. S4c) were not influenced by reduced Vam6 expression in iNKT cells, excluding the possible changes in organelle numbers. Next, we investigated whether VDAC1 at mitochondrial-lysosome contact sites regulated interactions between Vam6, Rab7a, and AMPKg. For this purpose, we generated Vdac1 +/- and Vdac1 -/- mice (Fig. S4d). Reducing VDAC1 expression in iNKT cells impaired mitochondrial-lysosome interaction without influencing the amounts of these two organelles (Fig. S4e-S4g). Notably, deleting one allele of Vdac1 significantly enhanced the Rab7a-AMPKg PLA puncta numbers (Fig. 5c), the Rab7a-Vam6 PLA puncta numbers (Fig. 5d), the Vam6-AMPKg3 PLA puncta numbers (Fig. 5e), and the AMPKa phosphorylation (Fig. 5f), whereas significantly reduced S6 S235/236 phosphorylation (Fig. 5g) and IFN-g production (Fig. 5h), although to a lesser extent than deleting two alleles (Fig. 5c-5h). Moreover, the increased Rab7a-AMPKg interaction (Fig. 5i), AMPK activation (Fig. 5j), and decreased mTOR activation (Fig. 5k), IFN-g production (Fig. 5l), in Vdac1 +/- iNKT cells were recovered by reducing Vam6 expression. These results demonstrate that mitochondrial protein VDAC1 inhibits AMPK activation via interfering with Rab7a-Vam6-AMPK complex formation. Given the inhibitory effects of Vam6 on VDAC1-Rab7a interaction as well as lysosome-mitochondria contact, our study imply that Vam6 relieves the inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation and AMPK activation. In line with the increased Vam6 expression in intratumoral iNKT cells, we further confirmed that these cells showed reduced mTORC1 activation (Fig. S5a), increased AMPK activation (Fig. S5b), elevated Rab7a-AMPKg interaction (Fig. S5c), reduced VDAC1-Rab7a interaction (Fig. S5d), and lower lysosome-mitochondria contact (Fig. S5e) than splenic iNKT cells, after activation. Vam6 +/− iNKT cells exhibits enhanced anti-tumor efficacy To study the anti-tumor efficacy of Vam6 +/− iNKT in vivo , we transferred expanded Vam6 +/− iNKT cells and expanded Vam6 +/+ iNKT cells into wide type mice bearing subcutaneous B16F10 tumor cells, respectively (Fig. 6a). The expanded Vam6 +/- iNKT cells exhibited better anti-tumor efficacy than expanded Vam6 +/+ iNKT cells, as indicated by lower tumor weight and smaller tumor size (Fig. 6b-6d). IFN-g production has been previously shown to be important for anti-tumor efficacy of iNKT cells 5 . To investigate the IFN-g production in transferred intratumoral iNKT cells, we transferred expanded Vam6 +/− iNKT cells and expanded Vam6 +/+ iNKT cells into J a 18 -/- mice bearing subcutaneous B16F10 tumor cells, respectively, and injected these mice with a-GC (Fig. 6e). The expanded intratumoral Vam6 +/− iNKT cells produced more IFN-g and showed higher level of S6 phosphorylation than intratumoral expanded Vam6 +/+ iNKT cells, in response to a-GC injection (Fig. 6f). Additionally, we tested the anti-tumor effect of expanded Vam6 +/− iNKT cells with tumor metastasis models. We respectively transferred expanded Vam6 +/− iNKT cells and expanded Vam6 +/+ iNKT cells into mice that were intravenously injected with B16F10 tumor cells one day before (Fig. 6g). Again, transfer of expanded Vam6 +/- iNKT cells better inhibited lung metastasis (Fig. 6h and 6i) and prolonged mouse survival (Fig. 6j) than transfer of expanded Vam6 +/+ iNKT cell. Taken together, our data confirm that Vam6 +/- iNKT cells exhibit enhanced anti-tumor efficacy. Discussion In this study, we demonstrate that reduction of Vam6 expression impairs AMPK activation and thus increases mTORC1 activation in iNKT cells. Recruitment of AMPK to lysosome is essential for its activation, and that is mediated through protein interactions between AMPK, AXIN, LKB1, and LAMTOR1 7 . In addition to these proteins, our results indicate that Vam6 plays an essential role in recruiting AMPK to lysosome and activating AMPK via promoting formation of Rab7a-Vam6-AMPK complex at lysosome. On the other hand, mitochondrial protein VDAC1 at mitochondria-lysosome contact site interferes with this complex formation via protein-protein interaction, and this inhibitory effect of VDAC1 is relieved by Vam6. It is rational that this de-suppressive effect of Vam6 would help to stabilize the Rab7a-Vam6-AMPK complex, in addition to the adaptor protein function of Vam6 in complex formation. Here, our results reveal that the mitochondria-lysosome contact site serves as a platform for blocking AMPK activation in iNKT cells via VDAC1-mediated inhibition of Rab7a-Vam6-AMPK complex formation. Although mitochondria-lysosome contact has been identified for years, the molecules tethering and regulating the contact as well as its contribution to cell fate and functional determination remain unclear. In yeasts, the mitochondria-vacuole contact is mediated by interaction between Vam6, Ypt7 (Rab7a homologue), and Tom40 19 . In contrary to its role in yeasts, Vam6 in iNKT cells serves as a negative regulator that interferes with VDAC1-Rab7a interaction and mitochondria-lysosome interaction. Our data demonstrate an essential role of VDAC1 in tethering mitochondria and lysosome in iNKT cells. Mitochondrial protein VDAC1 has been shown to control the calcium transport at mitochondria-lysosome contact site 20 . In addition to favoring the molecule transport between mitochondria and lysosome, our study indicates that VDAC1 also modulates activity of AMPK-mTORC1 pathways at the contact site between these two organelles. In line with our results, lysosomal Rab7a has been previously reported to maintain mitochondria-lysosome contact in both mammalian cells 21 and yeasts 19 . A previous study focusing on Rab7a GTPase-activating protein TBC1D15 suggests that active Rab7a promotes mitochondria-lysosome contact 18 . Active Rab7a but not inactive Rab7a exhibits lysosomal location 22 . Despite the discrepancies in Vam6’s potential guanidine exchange factor (GEF) activity toward Rab7a 2 3 , 24 , we found that reduction of Vam6 increased VDAC1-Rab7a interaction at lysosome and promoted mitochondria-lysosome contact. It is rational that Vam6 inhibits the VDAC1-Rab7a interaction and mitochondria-lysosome contact independently of its GEF activity, but through protein-protein interaction. Our findings that Vam6 promotes AMPK activation and diminishes mTORC1 activation in iNKT cells are in line with a recent study on Vam6 knockdown myoblasts 10 . On the other hand, another study on senescent conventional T cells demonstrates sustained activation of mTORC1 in a lysosomal function independent but Vam6-controlled late endosome dependent manner 11 . In the same study, the contribution of Vam6 to mTORC1 activation in young T cells differs at different time windows 11 . Additionally, Vam6 in fission yeasts has been reported to activate mTORC1 through activating Gtr1-Gtr2 25 . Whether the senescent T cells and the fission yeasts share similar mechanisms in controlling mTORC1 activation remain to be revealed. It is possible that mTORC1 pathway is regulated by different mechanisms depending on cell type, spatial and temporal distribution of mTOR and Vam6, and other pathways involved. Notably, iNKT cells are good candidates for anti-tumor immunotherapy 3 , 5 . Impaired activation of mTORC1 caused by accumulation of lactic acid hinders anti-tumor function of intratumoral iNKT cells 4 . Here, we demonstrate that lactic acid inhibits mTORC1 activation in tumor infiltrating iNKT cells through elevating expression of Vam6. Despite the mechanisms controlling Vam6 expression remain unclear, our findings that reducing Vam6 expression in iNKT leads to enhanced iNKT cell function and exhibits augmented anti-tumor efficacy shed light on future gene editing in iNKT cell-based immunotherapy against tumors. Material And Methods Mice Vam6 +/− , Vdac1 +/− , and Vdac1 −/− mice were generated using CRISPR/Cas9. V a 14 Tg mice were gifts from Dr. Albert Bendelac. Mice used in our experiments were 6-12 weeks old and cohoused littermates, and were on C57BL/6J background and maintained under specific pathogen-free conditions. To activate iNKT cells in vivo , mice were intraperitoneally injected with 2 mg a-GC for 4 hours. To generate chimeric mice, bone marrow cells isolated from CD45.1 Vam6 +/+ mice and CD45.2 Vam6 +/ - mice were mixed at 1:1 ratio (1 × 10 6 ) and then were intravenously injected into irradiated J a 18 -/- recipient mice (10 Gy) for 7 weeks. Animal procedures were approved by the Animal Care and Use Committee of University of Science and Technology of China, and all experiments were performed in accordance with the approved guidelines. Mouse tumor models Mice were subcutaneously injected with B16F10 tumor cells (5 × 10 5 ) on day 0. Recipient mice were then paratumorally injected with expanded Vam6 +/+ iNKT cells or expanded Vam6 +/- iNKT cells (5 × 10 6 ) on day 12. Tumor size was measured by vernier caliper every 2 days and tumor volume was calculated as length × width 2 × 0.52. To investigate the function of intratumoral iNKT cells, J a 18 -/- chimeric mice were intraperitoneally administered with PBS or 2 mg a-GC on day 14. In lung metastasis models, B16F10 cells (5 × 10 4 ) were intravenously injected into mice on day 0. After 24 hours, expanded Vam6 +/ − iNKT cells or expanded Vam6 +/ + iNKT cells (1 × 10 6 ) were intraperitoneally injected into these B16F10-transfered mice. On day 19 or 21, tumor-bearing mice were sacrificed for analysis of lung metastasis. Tumor-bearing animals were euthanized if they exhibited signs of distress or the tumor reached a diameter of 1.60 cm. Cell stimulation and expansion To measure the cytokine production in supernatants, iNKT cells from livers of Vam6 +/+ or Vam6 +/- mice were isolated as TCRb + CD1d-PBS57 tetramer + cells by a BD influx cell sorter (BD FACSAriaIII). Cells were stimulated with or without plate-coated 2 mg/mL CD1d-PBS57 tetramer for 48 hours, and cytokines in supernatants were measured using cytometric bead array kit (BD, 558296 and BD, 558298). To measure the intracellular cytokines, activation of AMPK and mTORC1, cell proliferation and apoptosis, magnetic beads (Miltenyi Biotec)-enriched splenic iNKT cells were stimulated with plate-coated 4 mg/mL anti-CD3 plus 4 mg /mL anti-CD28 antibodies for 4 hours, or with 2 mg/mL CD1d-PBS57 tetramer for 18 hours, or with 1 mg/mL anti-CD3 plus 1 mg /mL anti-CD28 antibodies for 18 hours in vitro . To inhibit or activate AMPK, Compound C (Selleck, S7840) or 200 mM AICAR (Sigma, A9978) was added to cells in the last 30 minutes. To inhibit mTORC1, 100 mM rapamycin (Sigma, 53123-88-9) was added to cells in the last 30 minutes. To expand iNKT cells in vitro , spleen cells from V a 14 Tg/Vam6 +/ − mice or V a 14 Tg/Vam6 +/ + mice were stimulated with 100 ng/mL a-GC for 3 days in the presence of 200 IU/mL IL-2, and then were cultured for another 4 to 7 days in the presence of IL-2, the purity of iNKT cells was about 80%. Sequence alignment and analysis cDNA library was sequenced using the Illumina sequencing platform (NovaSeq6000). The size of the library was ~300 bp, and both ends of the library were sequenced to a length of 100 bp. The raw reads were cleaned by removing adaptor sequences, short sequences (length < 35 bp), low-quality bases (quality < 20). and ambiguous sequences (i.e., reads with more than two unknown bases ‘N’). We used Hisat2（version:2.0.4）to map the cleaned RNA-seq reads to the mouse mm10 genome, with two mismatches, two gaps, and one multihit allowed. After genome mapping, Stringtie（version:1.3.0）was used to quantify gene expression. The gene expression value was normalized by FPKM and adjusted by a geometric algorithm. Cell transfection To knock down Vam6 in NIH-3T3 cells, we generated lentiviruses carrying the sh Vam6 sequences (sigma, TRCN0000120-894). The lentivirus-containing media were collected and added to NIH-3T3 cells for 48 hours to knock down Vam6 in the presence of 6 mg/mL Polybrene (Sigma-Aldrich, TR-1003-G). To restore expression of Vam6 or its truncated mutants in Vam6 knockdown NIH-3T3 cells, lentiviruses carrying m Cherry-Vam6, m Cherry- DCT, m Cherry- DCLH, m Cherry- DCNH, and mCheery as control were packaged in 293T cells and were used to transduce target genes, respectively. PLA After surface staining of CD1d-PBS57 tetramer, enriched iNKT cells were fixed, permeabilized, and blocked, followed by intracellular staining with primary antibodies against Tom20 (CST, 42406s, 1:200) and LAMP2 (Thermo Fisher, MA1-205, 1:200), against VDAC1 (Abcam, ab154856, 1:200) and Rab7a (Sigma, R8779, 1:200), against AMPKg (Invitrogen, PA5-36314, 1:200) and Rab7a (Sigma, R8779, 1:200), against Vam6 (Thermo Fisher, PA5-21104, 1:200) and Rab7a (Sigma, R8779, 1:200), and against Vam6 (Thermo Fisher, PA5-21104, 1:200) and AMPKg3 (Invitrogen, MA5-31868, 1:200), respectively. As negative controls, cells were stained with rabbit IgG (Invitrogen, 31235) and mouse IgG2b (Invitrogen, 02-6300), or stained with rabbit IgG (Invitrogen, 31235) and mouse IgG1 (biolegend, 400166). After washing, cells were stained with PLA detection reagents according to the manufacturer’s instructions (Duolink, Sigma). For colocalization analysis, cells were incubated with 500 nM Mitotracker Deep Red (ThermoFisher, 22426) for 30 minutes at 37 ℃ before surface staining, or stained with antibody against LAMP2 (eBioscience, 53-1072-82, 1:100) after PLA staining. PLA puncta in CD1d-PBS57 tetramer + cells were detected by confocal microscope (ZEISS LSM980) with a 40/63/100 × oil objective or ImageStreamX imaging flow cytometry (Merck Millipore) with 40 × magnification. Images were analyzed with ImageJ software (Fiji) or with IDEAS software. Antibodies and flow cytometry After blocking with anti-CD16/32 (Biolegend, 101302, 1:500), cells were stained with antibodies against surface molecules. For intracellular staining, cells were fixed and permeabilized with foxp3 staining buffer kit (eBioscience, 00-5523-00) after surface staining, followed by staining with antibodies against intracellular molecules. Fluorophore-conjugated or unconjugated antibodies included anti-TCRb (Biolegend, 109222, 1:200), anti-IFN-g (Biolegend, 505842, 1:100), anti-IL-4 (Biolegend, 504119, 1:100), anti-CD45.1 (Biolegend, 110-708, 1:200), anti-CD45.2 (Biolegend, 109828, 1:200), anti-B220 (Biolegend, 103224, 1:200), anti-Bcl2 (Biolegend, 633508, 1:200), anti-p-S6 S235/236 (CST, 4803S, 1:200), anti-AMPKa (Abcam, ab32047, 1:100), anti-p-AMPKa (Invitrogen, 44-1150G, 1:200), anti-Vam6 (Thermo Fisher, PA5-21104, 1:200), anti-VDAC1 (Abcam, ab154856, 1:200), anti-Rab7a (NewEast, 21069, 1:200), anti-LAMP2 (Invitrogen, A15449, 1:200), and anti-Ki67 (BD Pharmingen, 556026, 1:200). Apoptosis was measured using Annexin V apoptosis detection kit with PI (Biolegend, 640914, 1:20). CD1d-PBS57 tetramer was provided by the NIH Tetramer Core Facility. Secondary antibodies included donkey-anti-rabbit IgG-PE (Biolegend, 406421) and goat-anti-rabbit IgG-FITC (Jackson Immunoresearch, 111-095-003). Isotype controls included BV510 rat IgG1 (Biolegend, 400435), BV421 rat IgG1 (Biolegend, 400429), PE mouse IgG1 (Biolegend, 400113), rabbit IgG (Invitrogen, 31235), Alexa Fluor 488 rabbit IgG (CST, 2975S), and APC rat IgG2a (eBioscience, 17-4321-81). Samples were acquired with a BD FACSVerse flow cytometry, and data were analyzed with FlowJo software (TreeStar). Co-immunoprecipitation and immunoblotting Antibodies were incubated with Dynabeads protein G (Invitrogen, 10004D) at 4 °C for at least 30 minutes. Cells were lysed in NP-40 buffer (Beyotime, P0013F) supplemented with protease inhibitor cocktail (Thermo Fisher, 87786). Cell lysate was incubated with antibody-coated beads at 4 °C overnight. Antibodies used for immunoblotting and co-immunoprecipitation included anti-Vam6 (Thermo fisher, PA5-21104), anti-VDAC1 (Abcam, ab154856), anti-Rab7a (NewEast, 21069), anti-AMPKg (Invitrogen, PA5-36314), anti-mCherry (Invitrogen, PA5-34974), anti-b-actin (Proteintech, 66009-1-Ig), and rabbit IgG isotype ctrl (Invitrogen, 31235). HRP-conjugated secondary antibodies included mouse-anti-rabbit IgG light chain (CST, 93702S/ Jackson ImmunoResearch, 211-032-171), goat-anti-mouse IgG (H+L) (Proteintech, SA00001-1), and goat-anti-rabbit IgG (H+L) (Proteintech, SA00001-2). Antibodies and isotype controls used in co-immunoprecipitation experiments were at 1 mg/mL, and antibodies for immunoblotting were used at 1:1000 dilution unless other described. Statistical analysis Statistical analyses were performed with two-tailed unpaired Student’s t-test, Mann-Whitney test, Wilcoxon matched-pairs signed rank test, two-way ANOVA, and log-rank (Mantel-Cox) test, using GraphPad Prism software. Colocalization analyses were performed with ImageJ software (Fiji). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 were considered statistically significant. ns, not significant. Declarations Author contributions SY. B., QL. W., XR. C., and J. P. performed experiments, T. Y. and LF. X. generated knockout mice, SS. Z., YW. Z., SH. L., D. X., CX.T., L. W., SC. F, X. L., AM. J., D. L., JJ. H., YH. X., T. X., ZG. T., and RB. Z. provided materials, developed methods, or discussed experiments. SY. B., QL. W., HM. Z., and L. B. conceived the idea, designed the experiments, and wrote the manuscript. Acknowledgements We thank NIH Tetramer Core Facility for providing CD1d-PBS57 tetramer. This work was supported by National Key R&D Program of China 2021YFC2300600, National Natural Science Foundation of China 91942310, 91954122, 81771671, 82071736, 82101912, Fundamental Research Funds for the Central Universities WK9110000149 and WK9100000001, the University Synergy Innovation Program of Anhui Province GXXT-2021-066, and the Natural Science Foundation of Hefei 2021024. Competing Interests statement The authors declare no competing financial interests. References Godfrey, D. I., MacDonald, H. R., Kronenberg, M., Smyth, M. J. & Van Kaer, L. NKT cells: what's in a name? Nat Rev Immunol 4 , 231-237, doi:10.1038/nri1309 (2004). Bendelac, A., Savage, P. B. & Teyton, L. The Biology of NKT Cells. Annual Review of Immunology 25 , 297-336, doi:10.1146/annurev.immunol.25.022106.141711 (2007). Heczey, A. et al. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy. 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New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion. J Cell Biol 151 , 551-562, doi:10.1083/jcb.151.3.551 (2000). Valbuena, N., Guan, K. L. & Moreno, S. The Vam6 and Gtr1-Gtr2 pathway activates TORC1 in response to amino acids in fission yeast. J Cell Sci 125 , 1920-1928, doi:10.1242/jcs.094219 (2012). Additional Declarations (Not answered) Supplementary Files Fig.S1.tif Fig.S2.tif Fig.S3.tif Fig.S4.tif Fig.S5.tif Supplementaryfigurelegends.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-1990874","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":132346110,"identity":"4e068302-ae57-46b9-bcd5-a23ee57aa6cc","order_by":0,"name":"Huimin 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of Science and Technology of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Bai","suffix":""}],"badges":[],"createdAt":"2022-08-23 15:52:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1990874/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1990874/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25897754,"identity":"b0760427-72e9-4df4-81fb-14b984cc1d62","added_by":"auto","created_at":"2022-08-31 16:23:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1254827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased Vam6 in intratumoral iNKT cells leads to impaired IFN-g production.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eVam6 expression in splenic (SP) and intratumoral (IT) iNKT cells from MC38 tumor-bearing mice, 4 hours post a-GC injection. n = 7 mice for each group.\u003cstrong\u003e b \u003c/strong\u003eVam6 expression\u003cstrong\u003e \u003c/strong\u003ein splenic iNKT cells activated with or without plate-coated anti-CD3 plus anti-CD28 for 4 hours, in the presence or absence of 5 mM lactic acid. n = 10 replicates for each group.\u003cstrong\u003e c\u003c/strong\u003e Vam6 expression in intratumoral iNKT cells from \u003cem\u003eLdha\u003c/em\u003e knockdown B16F10 tumors or NTC B16F10 tumors, 4 hours post a-GC injection\u003cem\u003e.\u003c/em\u003e NTC, none target control cells transfected with scramble shRNA. n = 6-10 mice for each group. \u003cstrong\u003ed \u003c/strong\u003eSupernatant IFN-g and IL-4 produced by\u003cem\u003e \u003c/em\u003esorted hepatic iNKT cells from\u003cem\u003e Vam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Vam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e mice, stimulated with or without plate-coated CD1d-PBS57 tetramer for 48 hours. n = 11-12 mice for each group. ND, not detected. \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e Bcl-2\u003csup\u003e \u003c/sup\u003eexpression level (\u003cstrong\u003ee\u003c/strong\u003e, n = 9 samples for each group), frequencies of apoptotic cells (\u003cstrong\u003ef\u003c/strong\u003e, n = 12 samples for each group), and frequencies of Ki67\u003csup\u003e+\u003c/sup\u003e cells (\u003cstrong\u003eg\u003c/strong\u003e, n = 12 samples for each group) in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Vam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with or without CD1d-PBS57 tetramer for 18 hours. \u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e Experimental procedure (\u003cstrong\u003eh\u003c/strong\u003e) and IFN-g production (\u003cstrong\u003ei\u003c/strong\u003e) in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+ \u003c/sup\u003eiNKT cells\u003csup\u003e \u003c/sup\u003eand \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/- \u003c/sup\u003eiNKT cells from spleens of chimeric mice, 4 hours after a-GC or PBS injection. n = 10 mice for each group. \u003cstrong\u003ej \u003c/strong\u003eIFN-g production in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e and \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e iNKT cells activated by plate-coated anti-CD3 plus anti-CD28 for 18 hours, in the presence or absence of 10 mM lactic acid. n = 6 replicates for each group. \u003cstrong\u003ek\u003c/strong\u003e Experimental procedure and IFN-g production in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+ \u003c/sup\u003eiNKT cells and\u003cem\u003e Vam6\u003c/em\u003e\u003csup\u003e+/- \u003c/sup\u003eiNKT cells from B16F10 tumors, 4 hours post a-GC injection. n = 7 mice. Data are shown as mean±SEM (\u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003ej)\u003c/strong\u003e and pooled from two (\u003cstrong\u003ej\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e), three (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e), or four (\u003cstrong\u003ei\u003c/strong\u003e) independent experiments. Data were analyzed by two-tailed Mann-Whitney tests (\u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003ej\u003c/strong\u003e) and two-tailed\u003cstrong\u003e \u003c/strong\u003eWilcoxon matched-pairs signed rank tests (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, and \u003cstrong\u003ek\u003c/strong\u003e). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. See also Fig S1.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/58376b69516459a09a753ace.png"},{"id":25897756,"identity":"8def151c-cd02-42c9-b9c8-827ebeb4fec7","added_by":"auto","created_at":"2022-08-31 16:23:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1060001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emRNA-seq data reveal the altered AMPK/mTORC1 signaling in \u003cem\u003eVam6\u003c/em\u003e\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e iNKT cells. \u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Heatmap displaying 3440 significantly dysregulated genes in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells in comparison with \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e iNKT cells after stimulation with CD1d-PBS57 tetramer for 4 hours. The significance threshold is fold change ≥ 2 and FDR ≤ 0.05. \u003cstrong\u003eb\u003c/strong\u003e Correlation plot\u0026nbsp;displaying expression of genes in\u0026nbsp;\u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells and in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e iNKT cells. \u003cstrong\u003ec\u003c/strong\u003e\u0026nbsp;KEGG pathway analysis showing pathways downregulated and upregulated in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells. Bars represent the number of differentially expressed genes contributing to each gene set. \u003cstrong\u003ed\u003c/strong\u003e GO term analysis showing pathways upregulated in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells. \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e Heatmaps showing expression of genes involved in mTORC1 pathway (\u003cstrong\u003ee\u003c/strong\u003e) and AMPK pathway (\u003cstrong\u003ef\u003c/strong\u003e) in\u003cem\u003e Vam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells and in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e iNKT cells.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/82999c29f4e96c37d0ff8a91.png"},{"id":25897760,"identity":"8d125743-c97c-44b2-90b7-eb7b2faa1546","added_by":"auto","created_at":"2022-08-31 16:23:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":624649,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced Vam6 expression promotes mTORC1 activation and IFNg production via suppressing AMPK activation.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eb\u003c/strong\u003e Phosphorylation of S6\u003csup\u003eS235/S236\u003c/sup\u003e (\u003cstrong\u003ea\u003c/strong\u003e) and phosphorylation of AMPKa (\u003cstrong\u003eb\u003c/strong\u003e) in CD45.1\u003csup\u003e+\u003c/sup\u003e \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+ \u003c/sup\u003eand CD45.2\u003csup\u003e+\u003c/sup\u003e \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/- \u003c/sup\u003eiNKT cells in\u003cstrong\u003e \u003c/strong\u003emixed bone marrow chimeras, as described in Fig.1\u003cstrong\u003eh\u003c/strong\u003e. n = 10 mice for each group. \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e Phosphorylation of S6\u003csup\u003eS235/S236\u003c/sup\u003e (\u003cstrong\u003ec\u003c/strong\u003e) and phosphorylation of AMPKa (\u003cstrong\u003ed\u003c/strong\u003e)\u003csup\u003e \u003c/sup\u003ein \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with anti-CD3 plus anti-CD28 for 4 hours. n = 9-12 replicates for each group. \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e Experimental procedure (\u003cstrong\u003ee\u003c/strong\u003e), phosphorylation of S6\u003csup\u003eS235/S236\u003c/sup\u003e (\u003cstrong\u003ef\u003c/strong\u003e), and phosphorylation of AMPKa (\u003cstrong\u003eg\u003c/strong\u003e) in CD45.1\u003csup\u003e+\u003c/sup\u003e \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+ \u003c/sup\u003eand CD45.2\u003csup\u003e+\u003c/sup\u003e \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/- \u003c/sup\u003eiNKT cells from B16F10 tumors in\u003cstrong\u003e \u003c/strong\u003emixed bone marrow chimeras. n = 7-8 mice for each group. \u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ei \u003c/strong\u003ePhosphorylation of S6\u003csup\u003eS235/S236\u003c/sup\u003e (\u003cstrong\u003eh\u003c/strong\u003e) and IFN-g production (\u003cstrong\u003ei\u003c/strong\u003e) in splenic \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with CD1d-PBS57 tetramer for 18 hours in the presence or absence of rapamycin. n = 12 replicates for each group. \u003cstrong\u003ej\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e Phosphorylation of S6\u003csup\u003eS235/S236\u003c/sup\u003e (\u003cstrong\u003ej\u003c/strong\u003e) and IFN-g production (\u003cstrong\u003ek\u003c/strong\u003e) in splenic iNKT cells stimulated with or without CD1d-PBS57 tetramer for 18 hours in the presence or absence of Compound C. n = 12 replicates for each group. Data are shown as mean ± SEM (\u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e) and pooled from two (\u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e), three (\u003cstrong\u003ec \u003c/strong\u003eand\u003cstrong\u003e h\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e), or four (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e) independent experiments. Data were analyzed by two-tailed Mann-Whitney tests (\u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ek)\u003c/strong\u003e and two-tailed\u003cstrong\u003e \u003c/strong\u003eWilcoxon matched-pairs signed rank tests (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. ns, not significant. \u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/50165335a832e98d9f8e9a9c.png"},{"id":25898481,"identity":"1f5d3674-be94-459c-9688-d6ca2f68f271","added_by":"auto","created_at":"2022-08-31 16:28:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1455968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVam6 promotes Rab7a-AMPKg interaction and recruits AMPK to lysosome.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Protein level of AMPKa in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with CD1d-PBS57 tetramer. n = 12 replicates for each group. \u003cstrong\u003eb \u003c/strong\u003eCo-immunoprecipitation of Vam6 with AMPKg3, VDAC1, and Rab7a in expanded iNKT cells. \u003cstrong\u003ec \u003c/strong\u003eRepresentative Rab7a-AMPKg PLA images (left) and PLA punctum number per cell (right) in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with anti-CD3 plus anti-CD28 for 4 hours. n = 60-65 cells for each group. Scale bar, 3 µm. \u003cstrong\u003ed\u003c/strong\u003e Protein level of Rab7a in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with anti-CD3 plus anti-CD28 for 4 hours. n = 9 replicates for each group. \u003cstrong\u003ee\u003c/strong\u003e Representative confocal image showing colocaliztion of Rab7a-AMPKg PLA puncta and lysosomal LAMP2 in iNKT cells stimulated with CD1d-PBS57 tetramer for 4 hours. Scale bar, 3 µm.\u003cstrong\u003e f\u003c/strong\u003e Colocalization of AMPKg and LAMP2 in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with CD1d-PBS57 tetramer for 4 hours. Scale bar, 3 µm. \u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e Rab7a-AMPKg PLA puncta (\u003cstrong\u003eg\u003c/strong\u003e, n = 41-46 cells for each group) and colocalization of AMPKg and LAMP2 (\u003cstrong\u003eh\u003c/strong\u003e, n = 49-53 cells for each group) in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with CD1d-PBS57 tetramer for 4 hours in the presence or absence of AICAR. Scale bar, 3 µm. \u003cstrong\u003ei-k\u003c/strong\u003e Phosphorylation of AMPKa (\u003cstrong\u003ei\u003c/strong\u003e), phosphorylation of S6\u003csup\u003eS235/S236\u003c/sup\u003e (\u003cstrong\u003ej\u003c/strong\u003e), and IFN-g production (\u003cstrong\u003ek\u003c/strong\u003e) in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with \u0026nbsp;CD1d-PBS57 tetramer in the presence or absence of AICAR. n = 9-12 replicates for each group. Data are shown as mean ± SEM (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e, and \u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e) and pooled from three (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, and\u003cstrong\u003e j\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e) or four (\u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e) independent experiments. Data were analyzed by two-tailed\u003cstrong\u003e \u003c/strong\u003eunpaired Student’s\u003cstrong\u003e \u003c/strong\u003et test (\u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e) and two-tailed Mann-Whitney tests (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e, and \u003cstrong\u003ei\u003c/strong\u003e-\u003cstrong\u003ek)\u003c/strong\u003e. **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. ns, not significant. See also Fig S2 and Fig S3.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/5a432f140d2892800c840885.png"},{"id":25897757,"identity":"8431d8a8-cc34-4e98-b568-a5bc0d156806","added_by":"auto","created_at":"2022-08-31 16:23:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1021219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVDAC1 at mitochondria\u003cem\u003e-\u003c/em\u003elysosome contact site interferes with Vam6- Rab7a-AMPKg complex formation and AMPK activation.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eVDAC1-Rab7a PLA puncta in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Vam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with anti-CD3 plus anti-CD28 for 4 hours. n = 62-66 cells for each group. Scale bar, 3 mm.\u003cstrong\u003e b \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003econfocal images showing colocalization between VDAC1-Rab7a PLA puncta, Mitotracker deep red, and lysosomal LAMP2 in \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Vam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with CD1d-PBS57 tetramer for 4 hours. Scale bar, 3 mm.\u003cstrong\u003e c\u003c/strong\u003e-\u003cstrong\u003ee \u003c/strong\u003eRab7a-AMPKg PLA puncta (\u003cstrong\u003ec\u003c/strong\u003e, n = 46-56 cells for each group), Rab7a-Vam6 PLA puncta (\u003cstrong\u003ed\u003c/strong\u003e, n = 45-49 cells for each group), and Vam6-AMPKg PLA puncta (\u003cstrong\u003ee\u003c/strong\u003e, n = 33-37 cells for each group) in \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e, and \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e iNKT cells stimulated with CD1d-PBS57 tetramer for 4 hours. Scale bar, 3 mm. \u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003eh \u003c/strong\u003ePhosphorylation of AMPKa\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ef\u003c/strong\u003e, n = 16 replicates for each group), phosphorylation of S6\u003csup\u003eS235/S236\u003c/sup\u003e (\u003cstrong\u003eg\u003c/strong\u003e, n = 12 replicates for each group), and IFN-g production (\u003cstrong\u003eh\u003c/strong\u003e, n = 12 replicates for each group) in \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e, and \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e iNKT cells stimulated with CD1d-PBS57 tetramer for 18 hours. \u003cstrong\u003ei \u003c/strong\u003eRab7a-AMPKg PLA puncta in \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e, and \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with CD1d-PBS57 tetramer for 4 hours. n = 44-51 cells for each group. Scale bar, 3 mm. \u003cstrong\u003ej\u003c/strong\u003e-\u003cstrong\u003el \u003c/strong\u003ePhosphorylation of AMPKa\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ej\u003c/strong\u003e, n = 16 replicates for each group), phosphorylation of S6\u003csup\u003eS235/S236\u003c/sup\u003e (\u003cstrong\u003ek\u003c/strong\u003e, n = 16 replicates for each group), and IFN-g production (\u003cstrong\u003el\u003c/strong\u003e, n = 16 replicates for each group) in \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e, and\u003cem\u003e Vdac1\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells stimulated with CD1d-PBS57 tetramer for 18 hours. Data are shown as means ± SEM (\u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003el)\u003c/strong\u003e and pooled from two (\u003cstrong\u003ee\u003c/strong\u003e), three (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e, and\u003cstrong\u003e g-i\u003c/strong\u003e), or four (\u003cstrong\u003ef \u003c/strong\u003eand\u003cstrong\u003e j-l\u003c/strong\u003e) independent experiments. Data were analyzed by two-tailed\u003cstrong\u003e \u003c/strong\u003eunpaired Student’s\u003cstrong\u003e \u003c/strong\u003et test (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e i\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eand two-tailed Mann-Whitney tests (\u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003eh \u003c/strong\u003eand\u003cstrong\u003e j-l\u003c/strong\u003e). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ****P \u0026lt; 0.0001. See also Fig S4 and Fig S5.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/503b88b4281905e8c1fa6c68.png"},{"id":25898484,"identity":"1f5b5756-007b-485d-97b4-9cfd0f28004c","added_by":"auto","created_at":"2022-08-31 16:28:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2430768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduction of Vam6 expression enhances anti-tumor efficacy of expanded iNKT cells.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Experimental procedure for \u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e. \u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e Representative image of B16F10 tumors (\u003cstrong\u003eb\u003c/strong\u003e), tumor weight on day 20 (\u003cstrong\u003ec\u003c/strong\u003e), and tumor size (\u003cstrong\u003ed\u003c/strong\u003e) in wide type mice receiving PBS (n = 9 mice), expanded\u003cem\u003e Vam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e iNKT cells (n = 9 mice), and expanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells (n = 10 mice), respectively. Red arrow in \u003cstrong\u003ed\u003c/strong\u003e indicates time point of paratumoral injection. \u003cstrong\u003ee\u003c/strong\u003e Experimental procedure for \u003cstrong\u003ef\u003c/strong\u003e. \u003cstrong\u003ef\u003c/strong\u003e IFN-g\u003csup\u003e \u003c/sup\u003eproduction and phosphorylation of S6\u003csup\u003eS235/S236 \u003c/sup\u003ein transferred expanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e iNKT cells\u003csup\u003e \u003c/sup\u003eand\u003csup\u003e \u003c/sup\u003eexpanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/- \u003c/sup\u003eiNKT cells from tumors of \u003cem\u003eJa18\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice, after PBS or a-GC injection on Day 14. n = 1-2 mice for PBS groups, n = 3-5 mice for a-GC groups. \u003cstrong\u003eg\u003c/strong\u003e Experimental procedure for \u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ej\u003c/strong\u003e. \u003cstrong\u003eh\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e Representative image of lungs with melanoma foci (\u003cstrong\u003eh\u003c/strong\u003e) and numbers of melanoma foci in lungs (\u003cstrong\u003ei\u003c/strong\u003e) from mice injected with B16F10 cells (i.v.) and received PBS, expanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eiNKT cells, and expanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells, respectively. n = 7 mice for each group. \u003cstrong\u003ej\u003c/strong\u003e Survival rates of mice injected with B16F10 cells (i.v.) and received PBS, expanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eiNKT cells, and expanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells, respectively. n = 16 mice for PBS, n = 12 mice for \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e iNKT cells, n = 12 mice for\u003cem\u003e Vam6\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e iNKT cells.\u003cstrong\u003e \u003c/strong\u003eData are shown as mean ± SEM (\u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e, and\u003cstrong\u003e i\u003c/strong\u003e) and pooled from one (\u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e) or two (\u003cstrong\u003ef \u003c/strong\u003eand\u003cstrong\u003e i\u003c/strong\u003e-\u003cstrong\u003ej\u003c/strong\u003e) independent experiments. Two-tailed Mann-Whitney tests were applied in \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e, and\u003cstrong\u003e i\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eTwo-way ANOVA was applied in \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eand Log-rank (Mantel-Cox) test\u003cstrong\u003e \u003c/strong\u003ewas applied in\u003cstrong\u003e j\u003c/strong\u003e. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. \u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/dd8a3950221d98469f628734.png"},{"id":26742366,"identity":"7456a39b-9d21-44de-a880-e261eb343089","added_by":"auto","created_at":"2022-09-21 01:45:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2467025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/45cbb548-53b6-40f2-b2d4-fafc5f10cf16.pdf"},{"id":25898482,"identity":"58de4ec3-47aa-4838-a9c2-c1681c12f134","added_by":"auto","created_at":"2022-08-31 16:28:55","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1109348,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.tif","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/78f1f52c7908a004acf1da31.tif"},{"id":25898859,"identity":"1ad31eae-ee8f-4df9-b46c-cd81a1a3e7df","added_by":"auto","created_at":"2022-08-31 16:33:55","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":953788,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S2.tif","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/59779e454e99bd992f73199f.tif"},{"id":25897762,"identity":"8c8dcb6b-5b31-45ba-ba3e-b6d44d39cddb","added_by":"auto","created_at":"2022-08-31 16:23:55","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":923676,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S3.tif","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/c8250fb98bfa30188afb228e.tif"},{"id":25897765,"identity":"1cab3c1d-cd9b-4c74-9621-b9b3bd876d19","added_by":"auto","created_at":"2022-08-31 16:23:56","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":972558,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S4.tif","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/4156f7b787c19baa4b6d508b.tif"},{"id":25897763,"identity":"9c12465a-5103-427f-ba7a-64aaa55ef90a","added_by":"auto","created_at":"2022-08-31 16:23:55","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2369868,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S5.tif","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/1f5c5eecec55e30ad5a62634.tif"},{"id":25897764,"identity":"4753689b-1de5-4880-b71f-3ea29ec68a8e","added_by":"auto","created_at":"2022-08-31 16:23:55","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":6494796,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-1990874/v1/922db442766bc04d37e22c15.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Vam6 upregulated by lactic acid inhibits anti-tumor effects of intratumoral iNKT cells via modulating AMPK/mTOR pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInvariant natural killer T (iNKT) cells are innate-like T cells which express semi-invariant TCR and recognize lipid antigens presented by CD1d\u003csup\u003e1\u003c/sup\u003e. Upon activation, iNKT cells produce both Th1 and Th2 cytokines and mediate cytotoxicity as well. These cells bridge the innate immunity and adaptive immunity\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and are great candidates for immunotherapy against tumors, given their direct and indirect tumor killing effects and low risk of inducing cytokine storm and graft versus host disease\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, dysfunction of intratumoral iNKT cells has been reported to hinder their anti-tumor effects, and that is attributed to impaired activation of mTORC1\u003csup\u003e4,5\u003c/sup\u003e. Although the lactic acid in tumor microenvironment has been shown to inhibit activation of mTORC1, the underlying mechanisms are largely unknown.\u003c/p\u003e \u003cp\u003eLysosomal proteins, lysosomal location of mTOR, and lysosome function are essential for mTORC1 activation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. AMPK that inhibits mTORC1 activation also locates at lysosome, and its activity is controlled by lysosome related proteins\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. These findings indicate lysosome as a crucial hub for regulating mTORC1 pathway\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Vam6 (also named Vps39), a key member of the homotypic fusion and vacuole protein sorting complex that controls tethering and fusion of lysosome with intracellular compartments\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, has been shown to regulate mTOR pathway in myoblast and senescent T cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, opposite roles of Vam6 have been indicated by those two studies, and the molecular mechanisms that link Vam6 to mTOR pathway are still unknown. Whether and how Vam6 regulates mTORC1 activity and contributes to dysfunction of intratumoral iNKT cells remain to be explored.\u003c/p\u003e \u003cp\u003eHere, we found that Vam6 promoted AMPK activation and consequently inhibited mTORC1 activation and IFN-γ production in iNKT cells via forming Rab7a-Vam6-AMPK complex and recruiting AMPK to lysosome. VDAC1 interacting with Rab7a at mitochondria-lysosome contact site interfered with Rab7a-Vam6-AMPK complex formation and inhibited AMPK activation, demonstrating a negative control of this signal pathway at mitochondria-lysosome contact site. Notably, Vam6 inhibited Rab7a -VDAC1 interaction and relieved the inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation. Our data propose dual roles of Vam6 in promoting AMPK activation. Moreover, we showed that lactic acid in tumor microenvironment increased Vam6 expression in iNKT cells, and that led to increased AMPK activation, diminished mTORC1 activation, and impaired IFN-γ production in intratumoral iNKT cells. Reducing Vam6 expression, on the contrary, successfully restored function of intratumoral iNKT cells and enhanced their anti-tumor efficacy in mouse tumor models. Our data suggest that Vam6 could be a target for iNKT cell-based anti-tumor immunotherapy.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eVam6 upregulated by lactic acid inhibits function of intratumoral iNKT cells \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious study has reported dysfunction of intratumoral iNKT cells, and that is partially contributed to accumulation of lactic acid in tumor microenvironment\u003csup\u003e4\u003c/sup\u003e. Here, we found that intratumoral iNKT cells expressed higher level of Vam6 than splenic iNKT cells, irrespective of activation (Fig. 1a). The upregulation of Vam6 was caused by treatment of lactic acid (Fig. 1b), and knockdown of \u003cem\u003eLdha\u003c/em\u003e in tumor cells that led to less lactic acid production significantly reduced Vam6 expression in intratumoral iNKT cells (Fig. 1c). Next, we investigated whether the lactic acid-upregulated Vam6 inhibited iNKT cell function in tumors. Since \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003ehomozygous mice were embryonically lethal\u003csup\u003e10\u003c/sup\u003e, we generated \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003emice and confirmed reduction of Vam6 expression in these animals using splenic \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eT cells (Fig. S1a). Reducing Vam6 expression in \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003emice did not influence the frequencies of iNKT cells in thymuses, spleens, and livers (Fig. S1b and S1c). The absolute numbers of thymic and splenic iNKT cells were normal in \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e mice, despite the slightly elevated numbers of hepatic iNKT cells (Fig. S1c). These results demonstrate normal iNKT cell development in \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e mice. To study the influence of reduced Vam6 expression on iNKT cell function, we sorted iNKT cells from livers of\u003cem\u003e Vam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e mice and \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e mice, respectively, and activated them with CD1d-PBS57 tetramer\u003cem\u003e in vitro\u003c/em\u003e. Upon activation, \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells produced more IFN-g and IL-4 in supernatants, than \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/+ \u003c/sup\u003e\u003c/em\u003eiNKT cells did (Fig. 1d). Additionally,\u003cem\u003e Vam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells expressed higher level of anti-apoptosis protein Bcl-2 (Fig. 1e), exhibited lower apoptosis (Fig. 1f) but higher proliferation (Fig. 1g). To exclude the possibility that the increased cytokine production in supernatants were due to enhanced cell proliferation, we measured the intracellular cytokine, 4 hours after injecting lipid antigen a-galactosylceramide (a-GC) into lethally irradiated iNKT-deficient \u003cem\u003eJ\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e18\u003csup\u003e-/- \u003c/sup\u003e\u003c/em\u003emice that received CD45.2\u003cem\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e Vam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e bone marrows mixed with CD45.1\u003cem\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e bone marrows at a ratio of 1:1 (Fig. 1h). Consistently, reduced expression of Vam6 did not influence the development of iNKT cells in these chimeric mice, and CD45.2\u003cem\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e Vam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells exhibited higher levels of IFN-\u0026gamma; production than CD45.1\u003cem\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/+ \u003c/sup\u003e\u003c/em\u003eiNKT cells did (Fig. 1i). These results also demonstrate the intrinsic role of Vam6 in inhibiting iNKT cell function\u003cem\u003e in vivo\u003c/em\u003e. Moreover, we showed that reducing Vam6 expression restored IFN-\u0026gamma; production in lactic acid treated iNKT cells (Fig. 1j). In B16F10 tumors from those chimeric mice, intratumoral iNKT cells with reduced Vam6 expression produced more IFN-\u0026gamma; (Fig. 1k). Together, these data prove that the upregulation of Vam6 caused by lactic acid leads to iNKT cell dysfunction in tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVam6 inhibits iNKT cell functions via modulating AMPK/mTOR pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the role of Vam6 in controlling iNKT cell function, we performed RNA-seq experiments and found that reducing Vam6 expression altered gene expression profile in iNKT cells (Fig. 2a and 2b). 1712 genes were upregulated whereas 1728 genes were downregulated in \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells. KEGG (Kyoto Encyclopedia of Genes and Genomespathway) analysis and GO (Gene Ontology) term analysis showed that the upregulated genes were enriched in cell cycle, cell activation, and metabolic processes, whereas the downregulated genes were enriched in cell death, autophagy, and AMPK pathway. The gene profile of \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells was in line with their increased proliferation, survival, and function (Fig. 2c and 2d). It is well-known that cell growth and metabolic processes are promoted by mTORC1\u003csup\u003e12\u003c/sup\u003e whereas the autophagy is inhibited by mTORC1\u003csup\u003e13\u003c/sup\u003e. Additionally, AMPK is a negative regulator of mTORC1\u003csup\u003e14\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e15\u003c/sup\u003e. Therefore, our gene enrichment analysis implied alterations in AMPK/mTOR pathways in \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells. Indeed, we found increased expression of genes related to mTORC1 pathway (Fig. 2e) and decreased expression of genes related to AMPK pathway (Fig. 2f) in these iNKT cells with reduced Vam6 expression.\u003c/p\u003e\n\u003cp\u003eNext, we measured activation of mTORC1 and AMPK at protein levels, as indicated by the phosphorylation of S6\u003csup\u003eS235/236\u003c/sup\u003e and phosphorylation of AMPKa, respectively. iNKT cells were activated either \u003cem\u003ein vivo\u003c/em\u003e by injecting a-GC into \u003cem\u003eJ\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e18\u003csup\u003e-/- \u003c/sup\u003e\u003c/em\u003emice transplanted with CD45.2\u003cem\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e Vam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e bone marrows and CD45.1\u003cem\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e bone marrows or \u003cem\u003ein vitro\u003c/em\u003e by anti-CD3 plus anti-CD28 stimulation (Fig. 3a-3d). We found that, in both conditions, reducing Vam6 expression elevated mTORC1 activation (Fig. 3a and 3c) and diminished AMPK activation (Fig. 3b and 3d)\u003cem\u003e. \u003c/em\u003eConsistently, in tumors (Fig. 3e), \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells displayed higher level of mTORC1 activation (Fig. 3f) and lower level of AMPK activation (Fig. 3g) than \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e iNKT cells after a-GC injection. Activation of mTORC1 has been previously reported to promote cytokine production in iNKT cells\u003csup\u003e5\u003c/sup\u003e. Notably, reducing Vam6 expression failed to enhance IFN-\u0026gamma; production in iNKT cells treated with mTORC1 inhibitor rapamycin, further confirming that reduced Vam6 increased iNKT cell function via promoting mTORC1 activation (Fig. 3h and 3i). AMPK is a well-known negative regulator of mTORC1 pathway\u003csup\u003e15\u003c/sup\u003e. Here, we showed that reducing AMPK activation via Compound C increased mTORC1 activation and IFN-\u0026gamma; production in CD1d-PBS57 tetramer-stimulated iNKT cells (Fig. 3j and 3k), confirming the role of AMPK in suppressing mTORC1 activation in iNKT cells. Together, our results indicate that Vam6 in iNKT cells inhibits mTORC1 activation and IFN-\u0026gamma; production via promoting AMPK activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVam6 promotes activation of AMPK via recruiting AMPK to lysosome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDespite the reduced phosphorylation of AMPKa, \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells expressed similar level of AMPKa protein as \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e cells did (Fig. 4a). Then, we investigated whether Vam6 promoted AMPKa phosphorylation via protein interactions. We performed co-immunoprecipitation experiments with expanded iNKT cells (Fig. 4b and Fig. S2a), in which reducing Vam6 expression (Figure. S2b) increased IFN-g production (Figure. S2c and S2d) and S6\u003csup\u003eS235/236 \u003c/sup\u003ephosphorylation (Figure. S2e) whereas diminished AMPKa phosphorylation (Figure. S2f), as it did in fresh iNKT cells. We found that AMPKg3, Rab7a, and VDAC1 were co-immunoprecipitated with Vam6 (Fig. 4b), indicating interactions between these proteins. Rab7a is a marker protein for lysosome\u003csup\u003e16\u003c/sup\u003e. Recruiting AMPK to lysosome has been reported to be essential for AMPK activation\u003csup\u003e7\u003c/sup\u003e. Next, we investigated whether Vam6 regulated recruitment of AMPK to lysosome via protein interactions. Using \u003cem\u003ein situ\u003c/em\u003e proximity ligation assay (PLA) approach, we showed that AMPKg interacted with Rab7a in activated iNKT cells, and reducing Vam6 expression impaired the Rab7a-AMPKg interaction, as indicated by reduced Rab7a-AMPKg PLA puncta numbers (Fig. 4c). Additionally, we proved that reducing Vam6 expression did not influence the protein level of Rab7a (Fig. 4d). In line with the lysosomal location of Rab7a, the Rab7a-AMPKg complex was colocalized with lysosome marker LAMP2, indicating lysosomal location of AMPKg-Rab7a interaction (Fig. 4e). In agreement with the reduced Rab7a-AMPKg interaction in activated \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells, these cells had less amount of AMPK on lysosome in comparison with activated \u003cem\u003eVam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e iNKT\u003cem\u003e\u003csup\u003e \u003c/sup\u003e\u003c/em\u003ecells (Fig. 4f). These data demonstrate that Vam6 promotes Rab7a-AMPKg interaction and recruits AMPK to lysosome. Notably, when AMPK in CD1d-PBS57 tetramer-stimulated iNKT\u003cem\u003e\u003csup\u003e \u003c/sup\u003e\u003c/em\u003ecells was activated by AICAR, an AMP analog, the Rab7a-AMPKg PLA puncta numbers (Fig. 4g), the colocalization coefficient of AMPKg and LAMP2 (Fig. 4h), and the AMPKa phosphorylation (Fig. 4i) were all increased in a Vam6 dependent manner. As a consequence, the inhibitory effects of AICAR on mTORC1 activation (Fig. 4j) and on IFN-\u0026gamma; production (Fig. 4k) depended on Vam6 as well. Together, these data demonstrate an essential role of Vam6 in recruiting AMPK to lysosome and activating AMPK, and exclude the possibility of regulating AMPK activation via influencing AMP production.\u003c/p\u003e\n\u003cp\u003eTo understand how Vam6 regulated Rab7a-AMPKg interaction, we generated various truncated Vam6 based on previous study\u003csup\u003e17\u003c/sup\u003e, including the C terminal-deleted DCT, the middle fragment CLH-deleted DCLH, and the N terminal-deleted DCNH (Fig. S3a), and showed that Rab7a bound wide type Vam6 as well as DCT but not DCNH or DCLH, and AMPKg3 only bound wide type Vam6 (Fig. S3b). These results suggest that Rab7a binds Vam6 in a N terminal and CLH fragment dependent manner, and AMPK is dispensable for their interaction. Although DCT interacted with Rab7a, it failed to recruit AMPK, suggesting a key role of Vam6 C terminal in forming Rab7a-Vam6-AMPK complex. We could not exclude the possibility that AMPK might not directly bind Rab7a but indirectly interacts with Rab7a through Vam6. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVam6 relieves inhibitory effect of \u003c/strong\u003e\u003cstrong\u003eVDAC1 on Rab7a-Vam6-AMPK complex formation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the proteins co-immunoprecipitated with Vam6, we detected a mitochondrial protein VDAC1 (Fig. 4b). In activated iNKT cells, reducing Vam6 expression promoted VDAC1-Rab7a interaction, as indicated by increased puncta numbers of VDAC1-Rab7a PLA (Fig. 5a). Given the fact that VDAC1 and Rab7a are mitochondrial protein and lysosomal protein, respectively, the VDAC1-Rab7a interaction might occur at the contact site between lysosome and mitochondria\u003csup\u003e18\u003c/sup\u003e. Indeed, we found that VDAC1-Rab7a interaction sites were colocalized well with mitochondria probe Mitotracker Deep Red and with lysosome marker LAMP2 as well, confirming the existence of VDAC1-Rab7a interaction at mitochondrial-lysosome contact sites (Fig. 5b). In line with the increased VDAC1-Rab7a interaction in\u003cem\u003e Vam6\u003c/em\u003e\u003cem\u003e\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells, these cells displayed elevated mitochondrial-lysosome contacts (Fig. S4a). Meanwhile, the expression levels of mitochondrial maker VDAC1 (Fig. S4b) and lysosomal markers LAMP2 (Fig. S4c) were not influenced by reduced Vam6 expression in iNKT cells, excluding the possible changes in organelle numbers. Next, we investigated whether VDAC1 at mitochondrial-lysosome contact sites regulated interactions between Vam6, Rab7a, and AMPKg. For this purpose, we generated \u003cem\u003eVdac1\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e and\u003cem\u003e Vdac1\u003csup\u003e-/-\u003c/sup\u003e \u003c/em\u003emice (Fig. S4d). Reducing VDAC1 expression in iNKT cells impaired mitochondrial-lysosome interaction without influencing the amounts of these two organelles (Fig. S4e-S4g). Notably, deleting one allele of \u003cem\u003eVdac1\u003c/em\u003e significantly enhanced the Rab7a-AMPKg PLA puncta numbers (Fig. 5c), the Rab7a-Vam6 PLA puncta numbers (Fig. 5d), the Vam6-AMPKg3 PLA puncta numbers (Fig. 5e), and the AMPKa phosphorylation (Fig. 5f), whereas significantly reduced S6\u003csup\u003eS235/236 \u003c/sup\u003ephosphorylation (Fig. 5g) and IFN-g production (Fig. 5h), although to a lesser extent than deleting two alleles (Fig. 5c-5h). Moreover, the increased Rab7a-AMPKg interaction (Fig. 5i), AMPK activation (Fig. 5j), and decreased mTOR activation (Fig. 5k), IFN-g production (Fig. 5l), in \u003cem\u003eVdac1\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells were recovered by reducing Vam6 expression. These results demonstrate that mitochondrial protein VDAC1 inhibits AMPK activation via interfering with Rab7a-Vam6-AMPK complex formation. Given the inhibitory effects of Vam6 on VDAC1-Rab7a interaction as well as lysosome-mitochondria contact, our study imply that Vam6 relieves the inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation and AMPK activation.\u003c/p\u003e\n\u003cp\u003eIn line with the increased Vam6 expression in intratumoral iNKT cells, we further confirmed that these cells showed reduced mTORC1 activation (Fig. S5a), increased AMPK activation (Fig. S5b), elevated Rab7a-AMPKg interaction (Fig. S5c), reduced VDAC1-Rab7a interaction (Fig. S5d), and lower lysosome-mitochondria contact (Fig. S5e) than splenic iNKT cells, after activation. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVam6\u003csup\u003e+/\u0026minus;\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e iNKT cells exhibits\u003c/strong\u003e\u003cstrong\u003e enhanced anti-tumor efficacy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the anti-tumor efficacy of \u003cem\u003eVam6\u003csup\u003e+/\u0026minus;\u003c/sup\u003e\u003c/em\u003e iNKT \u003cem\u003ein vivo\u003c/em\u003e, we transferred expanded \u003cem\u003eVam6\u003csup\u003e+/\u0026minus;\u003c/sup\u003e\u003c/em\u003e iNKT cells and expanded \u003cem\u003eVam6\u003csup\u003e+/+ \u003c/sup\u003e\u003c/em\u003eiNKT cells into wide type\u003cem\u003e\u003csup\u003e \u003c/sup\u003e\u003c/em\u003emice bearing subcutaneous B16F10 tumor cells, respectively (Fig. 6a). The expanded \u003cem\u003eVam6\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells exhibited better anti-tumor efficacy than expanded \u003cem\u003eVam6\u003csup\u003e+/+ \u003c/sup\u003e\u003c/em\u003eiNKT cells, as indicated by lower tumor weight and smaller tumor size (Fig. 6b-6d). IFN-g production has been previously shown to be important for anti-tumor efficacy of iNKT cells\u003csup\u003e5\u003c/sup\u003e. To investigate the IFN-g production in transferred intratumoral iNKT cells, we transferred expanded \u003cem\u003eVam6\u003csup\u003e+/\u0026minus;\u003c/sup\u003e\u003c/em\u003e iNKT cells and expanded \u003cem\u003eVam6\u003csup\u003e+/+ \u003c/sup\u003e\u003c/em\u003eiNKT cells into \u003cem\u003eJ\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e18\u003c/em\u003e\u003csup\u003e-/-\u003cem\u003e \u003c/em\u003e\u003c/sup\u003emice bearing subcutaneous B16F10 tumor cells, respectively, and injected these mice with a-GC (Fig. 6e). The expanded intratumoral \u003cem\u003eVam6\u003csup\u003e+/\u0026minus;\u003c/sup\u003e\u003c/em\u003e iNKT cells produced more IFN-g and showed higher level of S6 phosphorylation than intratumoral expanded \u003cem\u003eVam6\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e iNKT cells, in response to a-GC injection (Fig. 6f). Additionally, we tested the anti-tumor effect of expanded \u003cem\u003eVam6\u003csup\u003e+/\u0026minus;\u003c/sup\u003e\u003c/em\u003e iNKT cells with tumor metastasis models. We respectively transferred expanded \u003cem\u003eVam6\u003csup\u003e+/\u0026minus;\u003c/sup\u003e\u003c/em\u003e iNKT cells and expanded \u003cem\u003eVam6\u003csup\u003e+/+ \u003c/sup\u003e\u003c/em\u003eiNKT cells into mice that were intravenously injected with B16F10 tumor cells one day before (Fig. 6g). Again, transfer of expanded Vam6\u003csup\u003e+/- \u003c/sup\u003eiNKT cells better inhibited lung metastasis (Fig. 6h and 6i) and prolonged mouse survival (Fig. 6j) than transfer of expanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e iNKT cell. Taken together, our data confirm that\u003cem\u003e Vam6\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e iNKT cells exhibit enhanced anti-tumor efficacy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrate that reduction of Vam6 expression impairs AMPK activation and thus increases mTORC1 activation in iNKT cells. Recruitment of AMPK to lysosome is essential for its activation, and that is mediated through protein interactions between AMPK, AXIN, LKB1, and LAMTOR1\u003csup\u003e7\u003c/sup\u003e. In addition to these proteins, our results indicate that Vam6 plays an essential role in recruiting AMPK to lysosome and activating AMPK via promoting formation of Rab7a-Vam6-AMPK complex at lysosome. On the other hand, mitochondrial protein VDAC1 at mitochondria-lysosome contact site interferes with this complex formation via protein-protein interaction, and this inhibitory effect of VDAC1 is relieved by Vam6. It is rational that this de-suppressive effect of Vam6 would help to stabilize the Rab7a-Vam6-AMPK complex, in addition to the adaptor protein function of Vam6 in complex formation.\u003c/p\u003e \u003cp\u003eHere, our results reveal that the mitochondria-lysosome contact site serves as a platform for blocking AMPK activation in iNKT cells via VDAC1-mediated inhibition of Rab7a-Vam6-AMPK complex formation. Although mitochondria-lysosome contact has been identified for years, the molecules tethering and regulating the contact as well as its contribution to cell fate and functional determination remain unclear. In yeasts, the mitochondria-vacuole contact is mediated by interaction between Vam6, Ypt7 (Rab7a homologue), and Tom40\u003csup\u003e19\u003c/sup\u003e. In contrary to its role in yeasts, Vam6 in iNKT cells serves as a negative regulator that interferes with VDAC1-Rab7a interaction and mitochondria-lysosome interaction. Our data demonstrate an essential role of VDAC1 in tethering mitochondria and lysosome in iNKT cells. Mitochondrial protein VDAC1 has been shown to control the calcium transport at mitochondria-lysosome contact site\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In addition to favoring the molecule transport between mitochondria and lysosome, our study indicates that VDAC1 also modulates activity of AMPK-mTORC1 pathways at the contact site between these two organelles. In line with our results, lysosomal Rab7a has been previously reported to maintain mitochondria-lysosome contact in both mammalian cells\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and yeasts\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. A previous study focusing on Rab7a GTPase-activating protein TBC1D15 suggests that active Rab7a promotes mitochondria-lysosome contact\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Active Rab7a but not inactive Rab7a exhibits lysosomal location\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Despite the discrepancies in Vam6\u0026rsquo;s potential guanidine exchange factor (GEF) activity toward Rab7a\u003csup\u003e2\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, we found that reduction of Vam6 increased VDAC1-Rab7a interaction at lysosome and promoted mitochondria-lysosome contact. It is rational that Vam6 inhibits the VDAC1-Rab7a interaction and mitochondria-lysosome contact independently of its GEF activity, but through protein-protein interaction.\u003c/p\u003e \u003cp\u003eOur findings that Vam6 promotes AMPK activation and diminishes mTORC1 activation in iNKT cells are in line with a recent study on \u003cem\u003eVam6\u003c/em\u003e knockdown myoblasts\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. On the other hand, another study on senescent conventional T cells demonstrates sustained activation of mTORC1 in a lysosomal function independent but Vam6-controlled late endosome dependent manner\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In the same study, the contribution of Vam6 to mTORC1 activation in young T cells differs at different time windows\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Additionally, Vam6 in fission yeasts has been reported to activate mTORC1 through activating Gtr1-Gtr2\u003csup\u003e25\u003c/sup\u003e. Whether the senescent T cells and the fission yeasts share similar mechanisms in controlling mTORC1 activation remain to be revealed. It is possible that mTORC1 pathway is regulated by different mechanisms depending on cell type, spatial and temporal distribution of mTOR and Vam6, and other pathways involved.\u003c/p\u003e \u003cp\u003eNotably, iNKT cells are good candidates for anti-tumor immunotherapy\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Impaired activation of mTORC1 caused by accumulation of lactic acid hinders anti-tumor function of intratumoral iNKT cells\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Here, we demonstrate that lactic acid inhibits mTORC1 activation in tumor infiltrating iNKT cells through elevating expression of Vam6. Despite the mechanisms controlling Vam6 expression remain unclear, our findings that reducing Vam6 expression in iNKT leads to enhanced iNKT cell function and exhibits augmented anti-tumor efficacy shed light on future gene editing in iNKT cell-based immunotherapy against tumors.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e, \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e, and \u003cem\u003eVdac1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were generated using CRISPR/Cas9. \u003cem\u003eV\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e14 Tg\u003c/em\u003e mice were gifts from Dr. Albert Bendelac. Mice used in our experiments were 6-12 weeks old and cohoused littermates, and were on C57BL/6J background and maintained under specific pathogen-free conditions. To activate iNKT cells \u003cem\u003ein vivo\u003c/em\u003e, mice were intraperitoneally injected with 2\u0026nbsp;mg\u0026nbsp;a-GC for 4 hours.\u0026nbsp;To generate chimeric mice, bone marrow cells isolated from CD45.1 \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e mice and CD45.2 \u003cem\u003eVam6\u003csup\u003e+/\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e mice were mixed at 1:1 ratio (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) and then were intravenously injected into irradiated \u003cem\u003eJ\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e18\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e recipient mice (10 Gy) for 7 weeks. Animal procedures were approved by the Animal Care and Use Committee of University of Science and Technology of China, and all experiments were performed in accordance with the approved guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse tumor models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were subcutaneously injected with B16F10 tumor cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) on day 0. Recipient mice were then paratumorally injected with expanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e iNKT cells or expanded \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/-\u0026nbsp;\u003c/sup\u003eiNKT cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) on day 12. Tumor size was measured by vernier caliper every 2 days and tumor volume was calculated as length \u0026times; width\u003csup\u003e2\u003c/sup\u003e \u0026times; 0.52. To investigate the function of intratumoral iNKT cells, \u003cem\u003eJ\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e18\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e chimeric\u0026nbsp;mice were intraperitoneally administered with PBS or 2\u0026nbsp;mg\u0026nbsp;a-GC on day 14.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eIn lung metastasis models, B16F10 cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were intravenously injected into mice on day 0. After 24 hours, expanded \u003cem\u003eVam6\u003csup\u003e+/\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/em\u003e iNKT cells or expanded \u003cem\u003eVam6\u003csup\u003e+/\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e iNKT cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) were intraperitoneally injected into these B16F10-transfered mice. On day 19 or 21, tumor-bearing mice were sacrificed for analysis of lung metastasis. Tumor-bearing animals were euthanized if they exhibited signs of distress or the tumor reached a diameter of 1.60 cm.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell stimulation and expansion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo measure the cytokine production in supernatants, iNKT cells from livers of \u003cem\u003eVam6\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e or \u003cem\u003eVam6\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice were isolated as TCRb\u003csup\u003e+\u003c/sup\u003e CD1d-PBS57 tetramer\u003csup\u003e+\u003c/sup\u003e cells by a BD influx cell sorter (BD FACSAriaIII). Cells\u0026nbsp;were stimulated with or without plate-coated 2\u0026nbsp;mg/mL CD1d-PBS57 tetramer for 48 hours, and cytokines in supernatants were measured using cytometric bead array kit (BD, 558296 and BD, 558298). To\u0026nbsp;measure the intracellular cytokines, activation of AMPK and mTORC1, cell proliferation and apoptosis, magnetic beads (Miltenyi Biotec)-enriched splenic iNKT cells were stimulated with plate-coated 4\u0026nbsp;mg/mL anti-CD3 plus 4\u0026nbsp;mg /mL anti-CD28 antibodies for 4 hours, or with 2\u0026nbsp;mg/mL\u0026nbsp;CD1d-PBS57 tetramer for 18 hours, or with 1\u0026nbsp;mg/mL anti-CD3 plus 1\u0026nbsp;mg /mL anti-CD28 antibodies for 18 hours\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e.\u0026nbsp;To inhibit or activate AMPK, Compound C (Selleck, S7840) or 200\u0026nbsp;mM AICAR (Sigma, A9978) was added to cells in the last 30 minutes. To inhibit mTORC1, 100\u0026nbsp;mM rapamycin (Sigma, 53123-88-9) was added to cells in the last 30 minutes.\u0026nbsp;To expand iNKT cells \u003cem\u003ein vitro\u003c/em\u003e, spleen cells from \u003cem\u003eV\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e14 Tg/Vam6\u003csup\u003e+/\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/em\u003e mice or\u003cem\u003e\u0026nbsp;V\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e14 Tg/Vam6\u003csup\u003e+/\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e mice\u0026nbsp;were stimulated with 100 ng/mL\u0026nbsp;a-GC for 3 days in the presence of 200 IU/mL IL-2, and then were cultured for another 4 to 7 days in the presence of IL-2, the purity of iNKT cells was about 80%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence alignment and analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ecDNA library was sequenced using the Illumina sequencing platform (NovaSeq6000). The size of the library was ~300 bp, and both ends of the library were sequenced to a length of 100 bp. The raw reads were cleaned by removing adaptor sequences, short sequences (length \u0026lt; 35 bp), low-quality bases (quality \u0026lt; 20). and ambiguous sequences (i.e., reads with more than two unknown bases \u0026lsquo;N\u0026rsquo;). We used Hisat2（version:2.0.4）to map the cleaned RNA-seq reads to the mouse mm10 genome, with two mismatches, two gaps, and one multihit allowed. After genome mapping, Stringtie（version:1.3.0）was used to quantify gene expression. The gene expression value was normalized by FPKM and adjusted by a geometric algorithm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo knock down \u003cem\u003eVam6\u0026nbsp;\u003c/em\u003ein\u003cem\u003e\u0026nbsp;\u003c/em\u003eNIH-3T3 cells, we generated lentiviruses carrying the sh\u003cem\u003eVam6\u003c/em\u003e sequences\u003cem\u003e\u0026nbsp;\u003c/em\u003e(sigma, TRCN0000120-894). The lentivirus-containing media were collected and added to NIH-3T3 cells for 48 hours to knock down \u003cem\u003eVam6\u003c/em\u003e in the presence of 6\u0026nbsp;mg/mL Polybrene (Sigma-Aldrich, TR-1003-G). To restore expression of Vam6 or its truncated mutants in \u003cem\u003eVam6\u003c/em\u003e knockdown NIH-3T3 cells, lentiviruses carrying m\u003cem\u003eCherry-Vam6,\u0026nbsp;\u003c/em\u003em\u003cem\u003eCherry-\u003c/em\u003eDCT, m\u003cem\u003eCherry-\u003c/em\u003eDCLH, m\u003cem\u003eCherry-\u003c/em\u003eDCNH, and \u003cem\u003emCheery\u003c/em\u003e as control were packaged in 293T cells and were used to transduce target genes, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePLA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter surface staining of CD1d-PBS57 tetramer, enriched iNKT cells were fixed, permeabilized, and blocked, followed by intracellular staining with primary antibodies against Tom20 (CST, 42406s, 1:200) and LAMP2 (Thermo Fisher, MA1-205, 1:200), against VDAC1 (Abcam, ab154856, 1:200) and Rab7a (Sigma, R8779, 1:200), against AMPKg\u0026nbsp;(Invitrogen, PA5-36314, 1:200) and Rab7a (Sigma, R8779, 1:200), against Vam6 (Thermo Fisher, PA5-21104, 1:200) and Rab7a (Sigma, R8779, 1:200), and against Vam6 (Thermo Fisher, PA5-21104, 1:200) and AMPKg3\u0026nbsp;(Invitrogen, MA5-31868, 1:200), respectively. As negative controls, cells were stained with rabbit IgG (Invitrogen, 31235) and mouse IgG2b (Invitrogen, 02-6300), or stained with rabbit IgG (Invitrogen, 31235) and mouse IgG1 (biolegend, 400166). After washing, cells were stained with PLA detection reagents according to the manufacturer\u0026rsquo;s instructions (Duolink, Sigma). For colocalization analysis, cells were incubated with 500 nM Mitotracker Deep Red (ThermoFisher, 22426) for 30 minutes at 37 ℃\u0026nbsp;before surface staining, or stained with antibody against LAMP2 (eBioscience, 53-1072-82, 1:100) after PLA staining. PLA puncta in CD1d-PBS57 tetramer\u003csup\u003e+\u003c/sup\u003e cells were detected by confocal microscope (ZEISS LSM980) with a 40/63/100 \u0026times; oil objective or ImageStreamX imaging flow cytometry (Merck Millipore) with 40 \u0026times; magnification. Images were analyzed with ImageJ software (Fiji) or with IDEAS software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibodies and flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter blocking with anti-CD16/32 (Biolegend, 101302, 1:500), cells were stained with antibodies against surface molecules. For intracellular staining, cells were fixed and permeabilized with foxp3 staining buffer kit (eBioscience, 00-5523-00) after surface staining, followed by staining with antibodies against intracellular molecules. Fluorophore-conjugated or unconjugated antibodies included anti-TCRb\u0026nbsp;(Biolegend, 109222, 1:200), anti-IFN-g\u0026nbsp;(Biolegend, 505842, 1:100), anti-IL-4 (Biolegend, 504119, 1:100), anti-CD45.1 (Biolegend, 110-708, 1:200), anti-CD45.2 (Biolegend, 109828, 1:200), anti-B220 (Biolegend, 103224, 1:200), anti-Bcl2 (Biolegend, 633508, 1:200), anti-p-S6\u003csup\u003eS235/236\u003c/sup\u003e (CST, 4803S, 1:200), anti-AMPKa\u0026nbsp;(Abcam, ab32047, 1:100), anti-p-AMPKa\u0026nbsp;(Invitrogen, 44-1150G, 1:200), anti-Vam6 (Thermo Fisher, PA5-21104, 1:200), anti-VDAC1 (Abcam, ab154856, 1:200), anti-Rab7a (NewEast, 21069, 1:200), anti-LAMP2 (Invitrogen, A15449, 1:200), and anti-Ki67 (BD Pharmingen, 556026, 1:200). Apoptosis was measured using Annexin V apoptosis detection kit with PI (Biolegend, 640914, 1:20). CD1d-PBS57 tetramer was provided by the NIH Tetramer Core Facility. Secondary antibodies included donkey-anti-rabbit IgG-PE (Biolegend, 406421) and goat-anti-rabbit IgG-FITC (Jackson Immunoresearch, 111-095-003). Isotype controls included BV510 rat IgG1 (Biolegend, 400435), BV421 rat IgG1 (Biolegend, 400429), PE mouse IgG1 (Biolegend, 400113), rabbit IgG (Invitrogen, 31235), Alexa Fluor 488 rabbit IgG\u0026nbsp;(CST, 2975S), and APC rat IgG2a (eBioscience, 17-4321-81). Samples were acquired with a BD FACSVerse flow cytometry, and data were analyzed with FlowJo software (TreeStar).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation and immunoblotting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibodies were incubated with Dynabeads protein G (Invitrogen, 10004D) at 4 \u0026deg;C for at least 30 minutes. Cells were lysed in NP-40 buffer (Beyotime, P0013F) supplemented with protease inhibitor cocktail (Thermo Fisher, 87786). Cell lysate was incubated with antibody-coated beads at 4 \u0026deg;C overnight. Antibodies used for immunoblotting and co-immunoprecipitation included anti-Vam6 (Thermo fisher, PA5-21104), anti-VDAC1 (Abcam, ab154856), anti-Rab7a (NewEast, 21069), anti-AMPKg\u0026nbsp;(Invitrogen, PA5-36314), anti-mCherry (Invitrogen, PA5-34974), anti-b-actin (Proteintech, 66009-1-Ig), and rabbit IgG isotype ctrl (Invitrogen, 31235). HRP-conjugated secondary antibodies included mouse-anti-rabbit IgG light chain (CST, 93702S/ Jackson ImmunoResearch, 211-032-171), goat-anti-mouse IgG (H+L) (Proteintech, SA00001-1), and goat-anti-rabbit IgG (H+L) (Proteintech, SA00001-2). Antibodies and isotype controls used in co-immunoprecipitation experiments were at 1\u0026nbsp;mg/mL, and antibodies for immunoblotting were used at 1:1000 dilution unless other described.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed with two-tailed unpaired Student\u0026rsquo;s t-test, Mann-Whitney test, Wilcoxon matched-pairs signed rank test, two-way ANOVA, and log-rank (Mantel-Cox) test, using GraphPad Prism software. Colocalization analyses were performed with ImageJ software (Fiji). *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, and ****P\u0026lt;0.0001 were considered statistically significant. ns, not significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSY. B., QL. W., XR. C., and J. P. performed experiments, T. Y. and LF. X. generated knockout\u003cem\u003e\u0026nbsp;\u003c/em\u003emice, SS. Z., YW. Z., SH. L., D. X., CX.T., L. W., SC. F, X. L., AM. J., D. L., JJ. H., YH. X., T. X., ZG. T., and RB. Z. provided materials, developed methods, or discussed experiments. SY. B., QL. W., HM. Z., and L. B. conceived the idea, designed the experiments, and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank NIH Tetramer Core Facility for providing CD1d-PBS57 tetramer. This work was supported by National Key R\u0026amp;D Program of China 2021YFC2300600, National Natural Science Foundation of China 91942310, 91954122, 81771671, 82071736, 82101912, Fundamental Research Funds for the Central Universities WK9110000149 and WK9100000001, the University Synergy Innovation Program of Anhui Province GXXT-2021-066, and the Natural Science Foundation of Hefei 2021024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGodfrey, D. I., MacDonald, H. R., Kronenberg, M., Smyth, M. J. \u0026amp; Van Kaer, L. NKT cells: what\u0026apos;s in a name? \u003cem\u003eNat Rev Immunol\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 231-237, doi:10.1038/nri1309 (2004).\u003c/li\u003e\n\u003cli\u003eBendelac, A., Savage, P. B. \u0026amp; Teyton, L. 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The Vam6 and Gtr1-Gtr2 pathway activates TORC1 in response to amino acids in fission yeast. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 1920-1928, doi:10.1242/jcs.094219 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vam6, mTORC1, AMPK, Rab7a-Vam6-AMPK complex, iNKT cells","lastPublishedDoi":"10.21203/rs.3.rs-1990874/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1990874/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eActivation of mTORC1 is essential for anti-tumor function of iNKT cells. The mechanisms underlying impaired mTORC1 activation in intratumoral iNKT cells remain unclear. Here, we showed that lactic acid from tumor cells increased Vam6 expression in iNKT cells which led to impaired mTORC1 activation and IFN-γ production. Mechanistically, Vam6 in iNKT cells was essential for Rab7a-Vam6-AMPK complex formation and thus for recruitment of AMPK to lysosome to activate AMPK, a negative regulator of mTORC1. Additionally, Vam6 relieved inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation at mitochondria-lysosome contact site. Given the key roles of lactic acid-increased Vam6 in promoting AMPK activation in intratumoral iNKT cells, reducing Vam6 expression significantly enhanced the mTORC1 activation in intratumoral iNKT cells as well as their anti-tumor efficacy. Together, we propose Vam6 as a target for iNKT cell-based immunotherapy.\u003c/p\u003e","manuscriptTitle":"Vam6 upregulated by lactic acid inhibits anti-tumor effects of intratumoral iNKT cells via modulating AMPK/mTOR pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-31 16:23:53","doi":"10.21203/rs.3.rs-1990874/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"21574a3d-2cfb-4316-954a-5c15132f52f9","owner":[],"postedDate":"August 31st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-21T01:45:34+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-31 16:23:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1990874","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1990874","identity":"rs-1990874","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}