Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: major_revision

Abstract
Discovering more targets is of great importance for developing alternative interventions for tumor therapy. The roles of transmembrane protein 175 (TMEM175) in neurodegeneration diseases have been reported, however its functions in tumor immune surveillance are not known. We show that TMEM175 conditional knockout in macrophages inhibits the tumor growth and metastasis through promoting the anti-tumor immunity in the tumor microenvironment (TME), including elevated M1-like polarization, reduced M2-like polarization, and facilitated recruitment and activation of T cells and nature killer cells (NKs). The anti-tumor immunity is abrogated by caspase-1 inhibitor VX-765, anti-IL-1β, and anti-IL-18. Tmem175−/− bone marrow-derived macrophages (BMDMs) show enhanced tumor antigen cross-presentation that is further strengthened by IL-1β and IL-18. NLRP3 is robustly elicited in Tmem175−/− BMDMs by the tumor cell debris through lysosomal permeabilization and cathepsin B leakage. Finally, Tmem175−/− mice are more responsive to anti-PD-1. Our works imply TMEM175 to be a potential target for immunotherapy.

Introduction
Immune surveillance in TME is crucial for the clearance of tumor cells. Diving into the mechanisms of immune surveillance could help us find more targets to propagate the anti-tumor immunity. Immunotherapy of malignant tumors have developed a constellation of therapeutic methods providing diverse choice for clinicians1. For example, anti-PD-1 antibody is commonly applied in melanoma therapy, however, drug resistance has been reported repeatedly2. Developing novel immunotherapy strategies may help improving anti-tumor immunity through alternative mechanisms.

Macrophages are a subset of crucial immune cells in TME, whose M2-like polarization and other behaviors have been shown to promote the tumor progression3,4. M2-like tumor associated macrophages (TAMs) attenuate the anti-tumor immunity through diverse mechanisms, including secretion of anti-inflammatory cytokines5, up-regulation of immune-suppressive PD-L16, and down-regulation of immune-stimulatory major histocompatibility complex class II (MHC-II)7, resulting in the immune evasion. On the contrary, reprogramming M2-like TAMs into M1-like TAMs through some interventions, such as TLR ligands8, chloroquine9, and hydroxychloroquine10, restored the recruitment and activation of T cells. Notably, the behaviors of TAMs are much more complicated beyond polarization. For instance, TAMs have been demonstrated to degrade extracellular matrix and promote the tumor invasion via secreted cathepsins and cathepsins in lysosomes4,11,12.

Lysosomes are the organelles that profoundly mediate the behaviors of macrophages in many diseases13. Macrophages intake extracellular substances through phagocytosis and transport them into lysosomes, where the low pH provides an optimal condition for nearly 60 kinds of hydrolytic enzymes13,14. The acidic luminal pH in lysosomes is maintained by the vacuolar H+-ATPase (v-ATPase) and other ion channels on lysosomal membrane. The v-ATPase hydrolyzes ATP to pump protons into lysosomes, and at the same time, cation efflux or anion influx through other ion channels is needed to balance the transmembrane potential15,16. TMEM175 is a potassium channel on lysosomal membrane, whose knockout causes incapacity to maintain acidic luminal pH during starvation17,18. Additionally, later research also illustrates the H+ permeability of TMEM17519,20. Present results have revealed that the dysfunction of TMEM175 causes Parkinson disease18,21. However researches focusing on the potential role of TMEM175 in the tumor immunity is limited. The crosstalk between lysosomal proton and calcium mediates the polarization of macrophages and anti-tumor immunity9. We thus hypothesize that TMEM175 might influence the homeostasis of lysosomal ions and regulate the behaviors of TAMs.

Dysfunction of lysosomes can activate inflammasome signal and alter the anti-tumor immunity in TME22. Inflammasome signals are composed of several kinds of intracellular sensors including, NLR family pyrin domain containing 3 (NLRP3), NLR family pyrin domain containing 1 (NLRP1), NLR family apoptosis inhibitory protein (NAIP)/NLR family CARD domain containing 4 (NLRC4), absent in melanoma 2 (AIM2), and Pyrin that recognize various danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs)23. NLRP3 is the most extensively studied inflammasome sensor, partially because it can be activated by diverse stimuli, while the others can only be provoked by specific kinds of substances. Activation of NLRP3 requires signal 1 that initiates the expression of proteins in NLRP3 pathway by ligands of Toll-like receptors (TLRs) and signal 2 that engages assembly of NLRP3 inflammasomes by DAMPs and PAMPs resulting in the secretion of interleukin-1β (IL-1β) and interleukin-18 (IL-18)24. Rupture of lysosomes and leakage of lysosomal content have been shown to elicit NLRP3 signal during the pathology of atherosclerosis25. In TME, NLRP3 activation has been observed in not only tumor cells but also stromal cells26–29. Both pro- and anti-tumor effects of NLRP3 activation have been reported in massive researches26,27,30–32. Although many details about the relationship between NLRP3 inflammasomes and tumor progression remain fuzzy, some inhibitors of NLRP3 pathways have been developed and tested for their anti-tumor potential33–35. Interestingly, an NLRP3 activator, BMS-986299, was also tested in a clinical research for solid cancers (NCT03444753). More investigations about the role of NLRP3 in TME are needed for a better understanding of inflammasomes in tumors.

Here, we found that conditional knockout of TMEM175 in macrophages significantly attenuated the tumor growth of melanoma B16-F10 and lung cancer LL-2 subcutaneous tumors, as well as the lung metastasis of B16-F10. TMEM175 knockout increased the MHC-II+ M1-like macrophages and decreased the CD206+ M2-like macrophages, resulting in the fostered recruitment and activation of CD4+ T cells, CD8+ T cells, and NKs in TME. The effects of TMEM175 knockout were underpinned by the inflammasome activation, since VX-765, anti-IL-1β, anti-IL-18, and Nlrp3 silencing abrogated the anti-tumor effects. Due to the tempered lysosomal degradation, Tmem175−/− BMDMs demonstrated accentuated tumor antigen cross-presentation that was further strengthened by IL-1β and IL-18. We also found how Tmem175−/− BMDMs possessed robust inflammasome activation. The cell debris engulfed by BMDMs caused the lysosomal membrane permeabilization, resulting in the NLRP3 activation by the released cathepsin B (CTSB). During this process, the potentiated cytosolic calcium ions that might be owed to the attenuated lysosomal acidification could be the key factor facilitating the NLRP3 activation in Tmem175−/− BMDMs. Finally, the potential of clinical application of TMEM175 knockout was further verified by the result that Tmem175−/− mice showed better response to anti-PD-1 therapy.

In summary, conditional knockout of Tmem175 in macrophages demonstrates obvious anti-tumor immunity in mice. The underlying mechanisms are compromised by the cross-presentation and NLRP3 inflammasome, enhanced by the lysosomal dysfunction in Tmem175−/− BMDMs. Our findings reveal the role of TMEM175 in tumor immunity, highlighting its potential in immunotherapy. Development of interventions targeting TMEM175 is needed for alternative immunotherapy strategies in the future.

Results
TMEM175 is up-regulated in melanoma and related with M2-like TAMs
In order to investigate the role of TMEM175 in tumors, we compared the expression of TMEM175 in melanoma and paracancerous tissues. TMEM175 expression was higher in melanoma tissues than that in paracancerous tissues (Fig. 1a). In order to validate the relationship between immune-suppressive CD206+ TAMs and TMEM175 expression in different cell subsets, B16-F10 subcutaneous tumors from wild type C57BL/6J mice were analyzed by FCM. Percentages of CD206+ TAMs were positively correlated with the expression of TMEM175 in TAMs, instead of other CD45- cells (Fig. 1b, c). Thus, we decided to focus on the TMEM175 in macrophages.

Validation of TMEM175 conditional knockout mice
Conditional knockout of TMEM175 in macrophages was achieved through cre/loxp system in C57BL/6J mice (Supplementary Fig. 1a). Briefly, exon 5 and exon 6 were floxed (Supplementary Fig. 1a, b), then the Tmem175 flox/flox mice were crossbred with Lyz2-Cre+/+ mice to generate Tmem175 flox/flox Lyz2-Cre+/+ and Tmem175 flox/flox Lyz2-Cre+/− mice (referred as Tmem175−/− mice for brief hereafter) (Supplementary Fig. 1c). The age-matched Tmem175 flox/flox Lyz2-Cre−/− littermates were used as control, which are referred as WT mice for brief hereafter.

The abrogated TMEM175 expression in BMDMs from Tmem175−/− mice was confirmed by western blot (Fig. 1d, e). The down-regulated TMEM175 expression was also confirmed by the RNA-seq (Supplementary Data 1). Since TMEM175 is the potassium channel on the lysosomal membranes, whole-lysosomal voltage clamp was applied to confirm the attenuated potassium conductance in lysosomal membranes from Tmem175−/− macrophages. The significantly dampened cross-membrane potassium current confirmed that the function of TMEM175 channels was abrogated by the conditional knockout (Fig. 1f).

TMEM175 conditional knockout suppresses tumor growth through accentuating immune-suppressive microenvironment
To investigate the role of TMEM175 in tumor progression, we used melanoma cell line, B16-F10, to establish subcutaneous tumor models in WT and Tmem175−/− mice. We observed elevated tumor cell death indicated by the cleaved caspase-3 (Fig. 2a and Supplementary Fig. 2a), and significantly decreased tumor weights (Fig. 2b) in Tmem175−/− mice. The tumors were analyzed via flow cytometry to evaluate the recruitment and activation of diverse cell subsets in TME. M1-like MHC-II+ macrophages were augmented and M2-like CD206+ macrophages were mitigated in Tmem175−/− mice (Fig. 2c, d). Other myeloid cells, including granulocytic myeloid derived suppressor cells (G-MDSCs), monocytic myeloid derived suppressor cells (M-MDSCs), and dendritic cells (DCs), showed no significant difference between WT and Tmem175−/− mice (Supplementary Fig. 2b–d), indicating that macrophages were the mainly affected myeloid cell subsets here. We additionally evaluated the recruitment and activation of T cells and NKs in TME. Recruitment and activation of CD4+, CD8+ T cells, and NKs were significantly augmented in Tmem175−/− mice (Fig. 2e–h, Supplementary Fig. 2e, f). Expression of interferon-γ (IFN-γ) and granzyme B (GZMB) in CD4+ T cells, CD8+ T cells, and NKs was enhanced (Supplementary Fig. 2g–l).

We also explored whether the anti-tumor effect of TMEM175 conditional knockout was reproducible in different tumors. Tumor cell death of LL-2 was promoted (Fig. 2i and Supplementary Fig. 2m), and the tumor weights were decreased in Tmem175−/− mice (Fig. 2j). MHC-II+ M1-like macrophages were heightened, while CD206+ M2-like macrophages were not obviously changed (Fig. 2k, l). Similarly, the granulocytic myeloid derived suppressor cells, monocytic myeloid derived suppressor cells, and DCs were not the influenced myeloid cell subsets (Supplementary Fig. 2n–p). Recruitment of CD4+/CD8+ T cells in Tmem175−/− mice was potentiated; however, the percentages of NKs were not changed (Fig. 2m–p, Supplementary Fig. 2q, r). The activation of CD8+ T cells was potentiated in Tmem175−/− mice (Fig. 2o); however, the activation of CD4+ T cells and NKs was not significantly changed between the two genotypes (Fig. 2n, p). Expression of IFN-γ was significantly promoted in CD4+/CD8+ T cells and NKs from Tmem175−/− mice (Supplementary Fig. 2s-u). Additionally, the granzyme B expression in CD8+ T cells was accentuated in Tmem175−/− mice, but the granzyme B expression in CD4+ T cells and NKs was not obviously elevated (Supplementary Fig. 2v–x).

In order to further validate the importance of macrophages and CD8+ T cells in the anti-tumor immunity in Tmem175−/− mice, clodronate liposomes (Clo lipo) and anti-CD8a antibody were applied to eliminate the macrophages and CD8+ T cells, respectively. Clo lipo treatment diminished the TAMs and abrogated the inhibitory effects of TMEM175 in B16-F10 tumors (Supplementary Fig. 3a–c). The recruitment and activation of CD4+ T cells in Tmem175−/− mice were reduced by Clo lipo (Supplementary Fig. 3d, e). IFN-γ and granzyme B expression in CD4+ T cells in Tmem175−/− mice were also suppressed by Clo lipo (Supplementary Fig. 3f, g). As expected, the recruitment and activation of CD4+ T cells in Clo lipo treated WT mice were elevated to a similar level in Clo lipo treated Tmem175−/− mice (Supplementary Fig. 3d, e). More M2-like TAMs in WT mice and M1-like TAMs in Tmem175−/− mice were observed (Fig. 2c, d), thus Clo lipo tempered the anti-inflammatory effect of TAMs in WT mice and pro-inflammatory effect of TAMs in Tmem175−/− mice. Similar trends were also observed in CD8+ T cells, whose recruitment and activation were modulated to the equal level in WT and Tmem175−/− mice by Clo lipo (Supplementary Fig. 3h, i). IFN-γ expression and granzyme B expression in CD8+ T cells were inhibited in Tmem175−/− mice instead of WT mice (Supplementary Fig. 3j, k). Anti-CD8a treatment also eradicated the differences in tumor weights between WT and Tmem175−/− mice (Supplementary Fig. 3l, m). The clearance of CD8+ T cells was validated (Supplementary Fig. 3n).

Together, these results illustrated that TMEM175 conditional knockout hampered the tumor growth through increasing M1-like macrophages, decreasing M2-like macrophages, and facilitating T cell and NK-mediated anti-tumor immunity. This anti-tumor immunity was accordant in different tumors, although some differences existed.

TMEM175 conditional knockout suppresses B16-F10 lung metastasis through activating the anti-tumor immunity
Besides subcutaneous tumor growth, we were also curious about the influence of TMEM175 conditional knockout on lung metastasis. WT and Tmem175−/− mice were intravenously injected with B16-F10 cells to establish lung metastasis models. Tmem175−/− mice demonstrated obviously fewer metastatic nodules and lower lung weights (Fig. 3a–c). Then the subsets of lung macrophages, CD11c+ CD11b- CD64+ alveolar macrophages (AMs) and CD11b+ CD24- CD64+ interstitial macrophages (IMs), were analyzed by FCM. Previous works have shown that MHC-II was down-regulated in AMs in tumor-bearing mice36 and that CD206+ AMs were increased in bronchoalveolar lavage fluid from lung cancer patients37. We observed the elevated MHC-II and mitigated CD206 expression in AMs (Fig. 3d, e). Similarly, we found the augmented MHC-II high IMs (Fig. 3f), which might be superior in antigen-presentation38, as well as the reduced CD206 high IMs (Fig. 3g), which show immunosuppressive phenotypes39. Another classification of IM subsets based on CD11c and MHC-II was also performed. Tmem175−/− mice possessed significantly attenuated CD11c low MHC-II low IM1, CD11c low MHC-II high IM2, and expanded CD11c+ MHC-II high IM3 (Fig. 3h–k). IM1 and IM2 are relatively similar and show immunoregulatory roles, while IM3 resembles typically activated monocytes40. As expected, the recruitment, activation, and IFN-γ expression of CD4+ and CD8+ T cells were promoted in Tmem175−/− mice (Fig. 3l–q). Notably, CD8+ T cells recognizing p15E, a tumor antigen in B16-F10, were also expanded (Fig. 3r). However, neither cDC1 nor cDC2 recruitment in lungs was obviously changed (Supplementary Fig. 4a, b), which implied the pivotal roles of macrophages in eliciting the anti-tumor immunity.

Then we used single cell transcriptome to analyze the underlying mechanisms of the anti-tumor immunity (Fig. 3s). In accordance with the FCM results, T cells and NKs were expanded in Tmem175−/− mice (Fig. 3s, Supplementary Fig. 4c, and Supplementary Data 2). We focused on the transcriptome changes in AMs and IMs. Both AMs and IMs showed up-regulated antigen presentation pathway, which included several genes coding histocompatibility molecules such as H2-D1, H2-M3, and H2-K1 (Supplementary Fig. 5a, b and Supplementary Data 3 and 4). And this result was in accordance with our FCM results. The differently expressed genes were then analyzed by GO and KEGG enrichment (Supplementary Fig. 5c–f). In AMs, we found some up-regulated genes that have been reported to promote M1-like polarization, such as Tlr2, Irf7, Nlrp3, Malt1, Map3k1, and Nod2 (Supplementary Data 3). These genes were enriched in pro-inflammatory pathways such as NOD-like receptor signaling pathway (mmu04621), NF-kappa B signaling pathway (mmu04064), and MAPK signaling pathway (mmu04010) (Supplementary Fig. 5e and Supplementary Data 3). These genes were also enriched in GO terms that facilitate immunity and inflammation, such as immune response (GO:0006955), antigen processing and presentation of peptide antigen via MHC class I (GO:0002474), and inflammatory response (GO:0006954) (Supplementary Fig. 5c and Supplementary Data 3). In IMs, we also found some up-regulated genes that have been reported to promote M1-like polarization, such as Gbp2b, Irf7, Irf9, Gbp4, and Tlr9 (Supplementary Data 4). These genes were enriched in pro-inflammatory pathways such as NOD-like receptor signaling pathway (mmu04621), Toll-like receptor signaling pathway (mmu04620), and NF-kappa B signaling pathway (mmu04064) (Supplementary Fig. 5f and Supplementary Data 4). These genes were also enriched in GO terms that facilitate immunity and inflammation, such as immune system process (GO:0002376), antigen processing and presentation (GO:0002474), positive regulation of interleukin-1 beta production (GO:0032731), and positive regulation of NF-kappaB transcription factor activity (GO:0051092) (Supplementary Fig. 5d and Supplementary Data 4). Thus, TMEM175 knockout promoted the antigen presentation and inflammatory process, resulting in the M1-like polarization of macrophages, which could involve several different pathways, including inflammasome, TLRs, and NF-kappaB.

In summary, Tmem175−/− mice developed fewer lung metastatic nodules owing to the activated tumor immune microenvironment. Our data implied that the promoted inflammasome and cross-presentation could be the underlying mechanisms of the anti-tumor effect.

Inflammasome signal is required for the anti-tumor effects of tmem175 conditional knockout
We attempted to dissect the mechanisms of the anti-tumor immunity in Tmem175−/− mice. The single cell transcriptome analysis indicated that TLRs, NF-kappaB, and inflammasome signal were up-regulated in macrophages (Supplementary Fig. 5e, f). Indeed, TLRs and NF-kappaB are the signal 1 of NLRP3 inflammasome22. TMEM175 knockout has been reported to cause lysosomal dysfunction17,20,21. Dysfunction of lysosomes activated inflammasomes in macrophages25,41, and the activated inflammasome signal in macrophages showed anti-tumor potential31,42, which inspired us to test the role of inflammasome signal in the anti-tumor effect of TMEM175 conditional knockout mice. We validated the high expression level of caspase-1 p10 and IL-1β in B16-F10 from Tmem175−/− mice (Fig. 4a–c). VX-765 is an inhibitor of caspase-143 that is the common downstream protease of several inflammasome signals. Tumor-bearing Tmem175−/− mice and WT mice were treated with VX-765 or vehicle everyday. The anti-tumor effects of TMEM175 conditional knockout were abrogated by the VX-765 treatment, which heightened the tumor weights and suppressed the tumor cell death in Tmem175−/− mice (Fig. 4d–f and Supplementary Fig. 6a). These data indicated that inflammasome signal was likely to be the key pillar of the anti-tumor effects in Tmem175−/− mice. We then used flow cytometry to explore whether the VX-765-mediated promotion of tumor growth was achieved through the deprivation of anti-tumor immunity. VX-765 reduced the high percentages of MHC-II+ M1-like macrophages in Tmem175−/− mice to the same level as WT mice, leaving CD206+ M2-like macrophages scarcely changed (Fig. 4g, h). Consistent with the curtailed M1-like polarization, recruitment and activation of CD4+ T cells and CD8+ T cells in Tmem175−/− mice were diminished to the comparable level as WT mice (Fig. 4i–l). The blunted CD8+ T cells and NKs were also illustrated by immunofluorescence (Fig. 4m and Supplementary Fig. 6b–d). We also tested the influence of inflammasome stimulator, chitosan, on B16-F10 tumor-bearing mice. Chitosan is able to activate inflammasomes in macrophages, depending on phagocytosis44. Peritoneal administration of chitosan significantly curbed the tumor weights and induced the tumor cell death in both WT and Tmem175−/− mice (Fig. 4n–p and Supplementary Fig. 6e). This effect was more remarkable in Tmem175−/− mice than in WT mice (Fig. 4n–p and Supplementary Fig. 6e). In vitro cultured Tmem175−/− macrophages were more susceptible to phagocytosis-dependent inflammasome activator, because chitosan induced more IL-1β secretion in peritoneal macrophages (PMs) and BMDMs (Fig. 4q, r). The IL-1β treatment promoted the MHC-II expression in both WT and Tmem175−/− BMDMs (Supplementary Fig. 6f), indicating the role of inflammasome signal in the M1-like polarization of macrophages. Furthermore, more caspase-1 p10 was generated in Tmem175−/− BMDMs treated by aluminium hydroxide, another phagocytosis-depended inflammasome activator45 (Fig. 4s and Supplementary Fig. 6g).

Present works have reported the conflict roles of inflammasome in TME. The downstream cytokines IL-1β and IL-18 could promote or suppress the anti-tumor immunity in different works28,31,32,46–49. To further validate the influences of IL-1β and IL-18 on our tumor-bearing models, we injected IL-1β and IL-18 into wild type B16-F10 subcutaneous tumors. Intratumoral administration of IL-1β and IL-18 significantly decreased tumor weights (Supplementary Fig. 7a, b). Both IL-1β and IL-18 intensified the MHC-II and IFN-γ expression in TAMs, although they did not obviously alter CD206 expression (Supplementary Fig. 7c–e). IL-18 did not significantly promote the IFN-γ expression in CD4+ T cells and the granzyme B expression in CD8+ T cells, nevertheless IL-1β and IL-18 promoted the recruitment and activation of CD4+ T and CD8+ T cells in general (Supplementary Fig. 7f–m). IL-1β also increased the recruitment, activation, IFN-γ expression, and granzyme B expression in NKs (Supplementary Fig. 7n–q). Thus the tumor immune microenvironment in wild type mice receiving IL-1β and IL-18 intratumoral administration at least partially resembled that in Tmem175−/− mice.

To ascertain the influences of IL-1β and IL-18 on anti-tumor effects by TMEM175 knockout, antibodies targeting IL-1β and IL-18 were applied. Intraperitoneal anti-IL-1β administration increased the tumor weights in Tmem175−/− mice (Fig. 5a, b). MHC-II expression in Tmem175−/− TAMs was decreased, while CD206 expression was not altered (Fig. 5c, d). Recruitment, activation, IFN-γ expression and granzyme B expression in CD4+ T cells, CD8+ T cells, and NKs in Tmem175−/− mice were mitigated by anti-IL-1β (Fig. 5e–l and Supplementary Fig. 8a–d). Similarly, anti-IL-18 abrogated the anti-tumor effects in Tmem175−/− mice (Fig. 5m, n). Percentages of M1-like TAMs in Tmem175−/− mice declined to the similar level to WT mice, while M2-like TAMs were not obviously changed (Fig. 5o, p). Recruitment, activation, IFN-γ expression and granzyme B expression in CD4+ T cells, CD8+ T cells, and NKs in Tmem175−/− mice were suppressed by anti-IL-18 (Fig. 5q–x and Supplementary Fig. 8e–h). CD8+ T/NK cell recruitment, CD4+ T/CD8+ T/NK cell activation, and granzyme B expression in CD8+ T cells were inhibited by anti-IL-18 in WT mice (Fig. 5r, u, v, x, Supplementary Fig. 8e, f), and NK cell recruitment was inhibited by anti-IL-1β in WT mice (Supplementary Fig. 8a) indicating less importance of inflammasome in the anti-tumor immunity in WT mice than that in Tmem175−/− mice. Thus increased IL-1β and IL-18 production is the key step of the anti-tumor immunity in Tmem175−/− mice.

Collectively, the anti-tumor effects of TMEM175 knockout mice were abrogated by VX-765, anti-IL-1β, and anti-IL-18 through restraining anti-tumor immunity, highlighting the crucial role of inflammasome signal in this process.

TMEM175 knockout augments the cross-presentation of tumor antigens
Since the tumor antigen-specific CD8+ T cells were increased in Tmem175−/− mice (Fig. 3r), we sought to compare the different cross-presentation capacities between WT and Tmem175−/− BMDMs that activate CD8+ T cells. Referring to MacNabb et al. 7 days were sufficient for priming tumor antigen specific CD8+ T cells50. B16-F10-OVA cells were implanted in wild type C57BL/6J mice and grew for 10 days to prime CD8+ T cells (Fig. 6a). Then the splenic lymphocytes from tumor bearing mice were harvested, CFSE-labeled, and cultured with WT or Tmem175−/− BMDMs with/without B16-F10-OVA cell debris for 48h before FCM and ELISA analysis (Fig. 6a). Tmem175−/− BMDMs promoted the expansion of CD8+ T cells indicated by CFSE low CD8+ T cells (Fig. 6b, c) and increased the percentages of OVA specific CD8+ T cells (Fig. 6d). Additionally, activated CD8+ CD69+ T cells, IFN-γ secretion, and TNF secretion were all elevated in Tmem175−/− group (Fig. 6e–g). IL-1β and IL-18 supplements facilitated the proliferation or activation of CD8+ T cells in different aspects (Fig. 6b–g). Thus, knockout of TMEM175 promoted the cross-presentation signal, which could be enhanced by IL-1β and IL-18.

There have been many researches revealing that limited lysosomal degradation of antigens favors antigen cross-presentation and presentation of APCs51–53. We therefore compared the intracellular tumor antigen levels between WT and Tmem175−/− BMDMs (Fig. 6h). In vitro cultured BMDMs were treated with B16-F10 cell debris or FITC-dextran for desired period of time and then analyzed by FCM. Higher levels of tumor antigen fluorescence were observed in Tmem175−/− BMDMs (Fig. 6i). However, the fluorescent intensities of FITC-dextran were comparable between WT and Tmem175−/− BMDMs (Fig. 6j). Thus, the delayed antigen degradation in Tmem175−/− BMDMs could explain the enhanced cross-presentation.

Then RNA-seq was applied to illustrate the signal changes during the cross-presentation. In the absence of B16-F10-OVA cell debris, Tmem175−/− group showed higher expression of several genes than WT group including Tlr13, Tlr8, Malt1, Irf4, Irf5, Trim29, Pcbp2, Cd9, and Sema4a that have been reported to promote T cell proliferation and activation (Supplementary Data 5). And these genes were enriched in innate immune response (GO:0045087), immune response (GO:0006955), adaptive immune response (GO:0002250), and inflammatory response (GO:0006954) (Fig. 6k and Supplementary Data 5). Although no tumor antigen exists, Tmem175−/− BMDMs showed pro-inflammatory phenotypes, thus promoting non-specific T cell proliferation and activation, which could explain the Tmem175−/− promoted CFSE low CD8+ and CD69+ CD8+ T cells without antigen stimulation in Fig. 6c, e. In B16-F10-OVA debris treated Tmem175−/− group, cell cycle (GO:0007049), cell division (GO:0051301), and mitotic spindle organization (GO:0007052) were up-regulated comparing with WT group treated with tumor antigen (Fig. 6l and Supplementary Data 6), which might be attributed to the more robust antigen specific T cell proliferation induced by Tmem175−/− BMDMs (Fig. 6c, d). We also attempted to investigate the effects of IL-1β and IL-18 on cross-presentation. Totally 7 common up-regulated genes (Cd164, Il1b, Gbp8, Iigp1, Csf2rb, Gbp4, and Gbp5) and 3 common down-regulated genes (Bcl6, Fubp1, and Atf4) between WT and Tmem175−/− group were altered by IL-1β (Supplementary Fig. 9a). However no common gene between WT and Tmem175−/− group was altered by IL-18 (Supplementary Fig. 9a). Despite this, several common altered KEGG pathway and GO terms were found to be altered by IL-1β and IL-18 (Supplementary Figs. 9b–e and 10a–d). The KEGG pathways and GO terms up-regulated by IL-1β in both WT and Tmem175−/− group included IL-17 signaling pathway (Path:04657), NOD-like receptor signaling pathway (Path:04621), cellular response to type II interferon (GO:0071346) and interleukin-1 beta production (GO:0032611) (Supplementary Figs. 9b, c and 10a, b and Supplementary Data 7 and 8). Additionally, positive regulation of canonical NF-kappaB signal transduction (GO:0043123) was up-regulated by IL-1β in WT group and regulation of canonical NF-kappaB signal transduction (GO:0043122) was up-regulated by IL-1β in Tmem175−/− group (Supplementary Data 7 and 8). Thus the common down-stream events of IL-1β in WT and Tmem175−/− group might include NF-kappaB signal, down-stream IFN-γ secretion, and possible positive feedback of IL-1β secretion. The common up-regulated KEGG pathways by IL-18 included IL-17 signaling pathway (Path:04657) and Toll-like receptor signaling pathway (Path:04625) (Supplementary Fig. 9d, e and Supplementary Data 9 and 10), indicating these two pathways to be the common down-stream events of IL-18 in WT and Tmem175−/− groups. In order to validate the down-stream events of IL-1β and IL-18, we used siRNA to silence Myd88, a known down-stream protein of IL-1R, and Csf2rb, a common DEG up-regulated by IL-1β (Supplementary Fig. 9f, g). Silencing Myd88 suppressed the effects of IL-1β and IL-18. For IL-1β, better inhibitory effects were observed in siMyd88 than that in siCsf2rb, indicating that Myd88 is the main down-stream event (Supplementary Fig. 9f–i).

To confirm the in vivo effects of the enhanced cross-presentation in Tmem175−/− BMDMs, two batches of CD8+ spleen T cells from the culture system were adopted to Cd8a−/− B16-F10-OVA tumor bearing mice (Fig. 6m). T cells cultured with Tmem175−/− BMDMs showed more obvious anti-tumor effects (Fig. 6n).

In summary, probably owing to the delayed lysosomal antigen degradation, Tmem175−/− BMDMs possess higher cross-presentation capacity, which was further enhanced by the down-stream cytokines of inflammasomes, IL-1β and IL-18.

B16-F10 cell debris activates NLRP3 inflammasomes in TMEM175 knockout BMDMs more extensively
Our previous experiments have proven that inflammasome signal was involved in the anti-tumor effects of TMEM175 conditional knockout. We then attempted to pursue the inflammasome activator in TME. We suspected that two groups of factors in TME were the potential inflammasome stimulators. One is composed of the soluble cytokines and metabolites derived from tumor cells, and the other is the dead tumor cells. Thus the conditioned medium from in vitro cultured B16-F10 was collected and filtered to remove insoluble materials, and the B16-F10 cells were harvested and repeatedly blown by syringes followed by two freeze-thaw cycles to generate cell debris. In order to test the activation of inflammasome pathway by B16-F10 supernatant and B16-F10 cell debris, LPS was applied to elicit signal 1 of inflammasomes in primary BMDMs, then the B16-F10 supernatant or B16-F10 cell debris was incubated overnight with BMDMs, from which the secreted IL-1β was detected by ELISA. The B16-F10 cell debris, instead of B16-F10 conditioned medium, provoked obvious IL-1β production more extensively in Tmem175−/− BMDMs (Fig. 7a). B16-F10 cell debris induced the secretion of IL-1β in time-dependent manner, during which the differences between WT and Tmem175−/− groups became distinct around 4h (Fig. 7b). Decreasing FBS in the culture medium could further facilitate the IL-1β production (Supplementary Fig. 11a). We also detected the released IL-18 in cell supernatant of BMDMs after B16-F10 cell debris treatment (Fig. 7c and Supplementary Fig. 11b). Rising extracellular potassium to 40mM almost completely inhibited the B16-F10 cell debris-mediated IL-1β production, which resembled the feature of NLRP3 inflammasomes54 implying NLRP3 to be the activated inflammasome sensor here (Fig. 7d). The enhanced recruitment and polymerization of ASC by NLRP3 in Tmem175−/− BMDMs was confirmed by co-immunoprecipitation, in which the Nigericin was used as a positive control (Fig. 7e and Supplementary Fig. 11c). The weak ASC signal in blank group could be owed to the engulfment of dead cells inevitable during the primary cell culture (Fig. 7e and Supplementary Fig. 11c). LPS alone could elicit uncanonical inflammasome signal that causes pyroptosis resulting in downstream NLRP3/ASC/caspase-1 activation55 (Fig. 7e and Supplementary Fig. 11c).

The next question is how the inflammasomes were activated. Untreated WT and Tmem175−/− BMDMs were analyzed by RNA-seq to explore whether any pro-inflammasome genes were altered in resting state. One up-regulated gene and 25 down-regulated genes were found (Supplementary Data 1), none of which was related with inflammasome strongly. And these genes were not significantly enriched in inflammasome related GO term or KEGG pathway (Supplementary Fig. 11d and Supplementary Data 1). These results indicated that the Tmem175−/− BMDMs did not show pro-inflammasome phenotype in resting state and that post transcription mechanisms might exist during the inflammasome activation. Since the chitosan and aluminium hydroxide elicited inflammasome through lysosomal destabilization44,45, we observed the morphological changes of the lysosomes through electron microscope. B16-F10 cell debris was engulfed into lysosomes leading to lysosomal membrane permeabilization featured with the discontinuous high density lysosomal membranes (Fig. 7f). Leakage of lysosomal contents, including cathepsin B, has been proven to stimulate NLRP3 inflammasome during the progression of atherosclerosis and multiple myeloma25,41. The lysosomal membrane permeabilization was confirmed by the results that round, and light spots of CTSB, CTSD, and CTSL became fuzzy and dim after B16-F10 cell debris treatment (Fig. 7g and Supplementary Fig. 11e–g). The activation of NLRP3 and production of IL-1β were dependent on the interaction between cathepsin B and NLRP3, which was abrogated by CA-074, a specific cathepsin B inhibitor (Fig. 7d, h, Supplementary Fig. 11h, i).

To further validate the role of NLRP3 in the anti-tumor effects of TMEM175 knockout, we silenced NLRP3 in BMDMs through siRNA and adopted these cells into WT B16-F10 tumor bearing mice. As expected, silencing NLRP3 expression in Tmem175−/− BMDMs abrogated the anti-tumor effects (Fig. 7i and Supplementary Fig. 11j, k).

Previous work has demonstrated that lysosomal rupture activates NLRP3 signal through not only cathepsin B but also reactive oxygen species (ROS)41. We tested the level of ROS in B16-F10 cell debris treated BMDMs. B16-F10 cell debris significantly increased intracellular ROS, and the highest ROS level was found in Tmem175−/− BMDMs (Supplementary Fig. 12a). Inhibiting ROS through acetylcysteine (N-Acetyl-L-cysteine, NAC) mitigated the activation of NLRP3 inflammasome and IL-1β secretion (Supplementary Fig. 12b–d).

In summary, B16-F10 cell debris was engulfed by the BMDMs, resulting in the lysosomal membrane permeabilization and NLRP3 activation, and stronger inflammasome activation was observed in Tmem175−/− BMDMs. These results explained the strong inflammasome signal in Tmem175−/− mice.

Calcium ions are involved in the promoted inflammasome activation in TMEM175 knockout BMDMs
We tried to interrogate how TMEM175 knockout promoted the NLRP3 inflammasome activation. Previous research reported that knockout of TMEM175 suppressed the acidification of lysosomes during starvation17. Through pH sensitive lysosomal probe, we found that after B16-F10 cell debris treatment, lysosomal pH in Tmem175−/− BMDMs was higher than that in WT BMDMs (Supplementary Fig. 13a) indicating a hampered lysosomal acidification in Tmem175−/− BMDMs by cell debris treatment. Elevated lysosomal pH has been reported to induce the efflux of lysosomal calcium9,56. Lysosomal calcium concentration determined by rhod-dextran illustrated less lysosomal calcium in Tmem175−/− BMDMs after B16-F10 cell debris treatment (Supplementary Fig. 13b). Additionally, cytosolic calcium determined by Fluo-4-AM indicated higher cytosolic calcium level in Tmem175−/− BMDMs treated by B16-F10 cell debris (Supplementary Fig. 13c). Inhibiting the lysosomal acidification in WT BMDMs by BafA1 promoted the efflux of lysosomal calcium and NLRP3 activation (Supplementary Figs. 12b–d and 13b, c). Thus, we speculated that the hampered lysosomal acidification could explain the enhanced lysosomal calcium efflux and the elevated cytoplasm calcium in Tmem175−/− BMDMs treated with cell debris.

Cytosolic calcium promoted the NLRP3 activation through calcium-sensing receptor or mitochondrial damage57,58, but it was fuzzy whether the direct interaction between calcium and NLRP3 existed. Chelating cytosolic calcium via BAPTA-AM before B16-F10 cell debris treatment significantly impeded the IL-1β production and ASC polymerization (Supplementary Fig. 13d–f), which suggested the higher cytosolic calcium to be the reason of the more intensified inflammasome activation in Tmem175−/− BMDMs. The cytosolic calcium was required for the direct interaction between NLRP3 and ASC, because both BAPTA-AM administration before B16-F10 cell debris treatment and EDTA-Na2 administration in cell lysate during CO-IP hampered the interaction between NLRP3 and ASC (Supplementary Fig. 13e, f). Additionally, calcium ions might also participate in the interaction between NLRP3 and CTSB (Supplementary Fig. 13g, h). On the contrary, inhibiting CTSB did not obviously alter the cytosolic calcium level (Supplementary Fig. 13c), indicating the lysosomal calcium efflux to be the up-stream event of CTSB activation.

We additionally investigated the role of CTSB, cytosolic calcium ions, and ROS in the anti-tumor immunity through inhibitors. The anti-tumor effects of TMEM175 knockout were blunted by the treatment of CA-074 and BAPTA-AM (Supplementary Fig. 14a, b). M1-like polarization of TAMs in Tmem175−/− mice was reversed by CA-074 and BAPTA-AM, and more obvious effects were observed in BAPTA-AM treatment (Supplementary Fig. 14c, d). Similarly, recruitment and activation of CD4+ T cells were curbed by CA-074 and BAPTA-AM in Tmem175−/− mice, while IFN-γ expression and granzyme B expression were only repressed by BAPTA-AM (Supplementary Fig. 14e–h). Recruitment, activation, IFN-γ expression and granzyme B expression of CD8+ T cells were restrained by CA-074 and BAPTA-AM in Tmem175−/− mice (Supplementary Fig. 14i–l). Thus, cytosolic calcium elevation seemed to be a more crucial factor than CTSB activation for the pro-inflammatory phenotypes in TMEM175 knockout. Comparing with the distinctly inhibitory effects of BAPTA-AM, the ROS scavenger, NAC, showed partial inhibitory effects on the anti-tumor immunity. NAC administration through drinking water59,60 increased the tumor weights, decreased the M1-like TAMs, and elevated the M2-like TAMs in Tmem175−/− mice (Supplementary Fig. 14m–p). These results are in accordance with previous reports60,61. NAC partially inhibited the recruitment and activation of CD4+ T cells, but it did not obviously alter the IFN-γ and granzyme B expression in CD4+ T cells in Tmem175−/− mice (Supplementary Fig. 14q–t). Recruitment, activation, IFN-γ expression, and granzyme B expression of CD8+ T cells were suppressed by NAC in Tmem175−/− mice (Supplementary Fig. 14u–x). These effects were not obvious in NAC-treated WT mice, which could be owed to the low level of ROS activation in WT mice. Although NAC inhibited the inflammasome activation in WT BMDMs in vitro, the inhibitory effects of NAC on immune microenvironment in vivo could only be clearly observed in mice with high basal ROS activation level. Our in vivo research demonstrated that the cytosolic calcium seems to be the key factor, although CTSB and ROS are also involved in the NLRP3 activation.

We also tested whether calcium was involved in the PYD-PYD interaction of ASC and NLRP3 through molecular dynamics simulation. The PYD-PYD interaction complex was used for the molecular dynamics simulation in the existence of ten calcium ions for 100ns. Eight of the ten calcium ions demonstrated obvious interaction with the amino-acid residues through hydrogen bonds (Supplementary Fig. 15a). The PYD-PYD interaction complex reached a stable state after 20ns (Supplementary Fig. 15b). The radius of gyration (Rg), solvent accessible surface area (SASA), and distance between two PYD domains of the PYD-PYD interaction complex decreased during the 100ns simulation, indicating that the PYD domains of ASC and NLRP3 get closer to each other with the help of calcium ions (Supplementary Fig. 15c-e). The detailed interactions between each calcium ion and amino-acid residues were shown (Supplementary Fig. 15f). Thus calcium ions facilitated the PYD-PYD interaction of ASC and NLRP3 through direct interactions.

Together, the augmented NLRP3 activation in Tmem175−/− BMDMs could be owed to the elevated cytosolic calcium, which might result from the suppressed lysosomal acidification during cell debris treatment. Although other factors, such as ROS and cathepsins, may also participate in this process, the interaction between lysosomal protons and calcium ions could at least partially explain the elevated NLRP3 activation.

TMEM175 conditional knockout mice show better therapeutic responses to anti-PD-1 therapy
Knowing that TMEM175 knockout ameliorated the immune-suppressive microenvironment, we attempted to interrogate whether it could be combined with other immunotherapy methods. Since T cells were vigorously recruited into TME in Tmem175−/− mice, we chose an anti-PD-1 antibody. Tumor-bearing Tmem175−/− and WT mice were treated with anti-PD-1 or isotype antibodies every 3 days. Tmem175−/− mice showed promoted tumor cell death and decreased tumor weights in response to anti-PD-1 antibody, while WT mice were seldom relieved by the anti-PD-1 therapy (Fig. 8a, b and Supplementary Fig. 16a). This result could be explained by the sparse infiltration of T cells in the tumors of WT mice. MHC-II+ M1-like macrophages were increased by the anti-PD-1 therapy both in Tmem175−/− and WT mice (Fig. 8c), which coincided with previous report62. On the contrary, CD206+ M2-like macrophages were not obviously altered by the anti-PD-1 therapy (Fig. 8c). The recruitment of CD4+ T cells, CD8+ T cells, and NKs was significantly elevated by the anti-PD-1 therapy in Tmem175−/− mice, while NKs in WT mice were scarcely altered by the anti-PD-1 (Fig. 8d-h, Supplementary Fig. 16b, c). Anti-PD-1 antibody promoted the activation of CD4+/CD8+ T cells both in Tmem175−/− mice and WT mice (Fig. 8f, g), however no matter whether anti-PD-1 antibody was administrated, the recruitment and activation of CD4+/CD8+ T cells in Tmem175−/− mice were higher than that in WT mice (Fig. 8f, g). Additionally, recruitment and activation of NKs were selectively expanded in Tmem175−/− mice (Fig. 8h).

Overall, conditional knockout of TMEM175 in macrophages improved the effects of anti-PD-1 therapy through promoting the recruitment and activation of T cells and NKs, implying a promising application of the interventions targeting TMEM175 in immunotherapy. The figure summarizing the immune mechanisms involved in the Tmem175−/− BMDMs has been presented (Supplementary Fig. 17a). The FCM gating strategies in our work have been presented (Supplementary Fig. 18a, b and Supplementary Fig. 19a).

Methods
Mouse breeding and generation of TMEM175 conditional knockout mice
All animal studies in this work were approved by the Animal Ethics Committee of Sichuan University (Chengdu, Sichuan, China). Our experiments are in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Mice were housed in specific pathogen-free (SPF) conditions with 22–23°C, 45–55% relative humidity and a 12–12h light/dark cycle with free food and water supplies. In this work, experimental and control animals were co-housed. All animals used were euthanized with carbon dioxide in accordance with approved animal protocols.

Tmem175 flox/wt mice were created by Nanjing Biomedical Research Institute of Nanjing University. A pair of sgRNAs was designed and validated to target wild type Tmem175. The sequences of sgRNA were 5′-end: GGAGTTTTAACTGTTCAGG and 3′-end: CATTATAGCCCTGCA TACTTGG. Referring to the sgRNA, a donor vector carrying exon 5 and exon 6 encircled by two loxP sequences was constructed. The donor vector was injected into mouse oosperm, then the E5-E6 of the wild type Tmem175 gene was replaced by the loxp-E5-E6-loxp through homologous recombination with the help of Cas9 system to generate Tmem175 flox/wt mice. The targeting strategy and loxp-E5-E6-loxp sequence have been shown in Supplementary Fig. 1. Tmem175 flox/wt mice mated to generate Tmem175 flox/flox mice, which mated with Lyz2-Cre+/+ mice (Jackson Lab, stock number: 004781) and then self-crossed to generate Tmem175 flox/flox Lyz2-Cre+/+ and Tmem175 flox/flox Lyz2-Cre+/− mice. Both Tmem175 flox/flox Lyz2-Cre+/+ and Tmem175 flox/flox Lyz2-Cre+/− mice were regarded as TMEM175 conditional knockout mice (Tmem175−/− mice). The Tmem175 flox/flox Lyz2-Cre−/− littermates were used as control (WT mice). Every WT or Tmem175−/− mouse was identified by PCR before being included in our experiments. To eliminate inevitable mutations during the self-fertilization, the 15th generation of Tmem175 flox/flox Lyz2-Cre+/+ mice mated with wild type Tmem175 wt/wt Lyz2-Cre−/− C57BL/6J mice, and the offspring self-crossed to generate a novel line of Tmem175−/− mice. Both previous and novel lines of WT and Tmem175−/− mice were used in our experiments.

The PCR primers (Tsingke biotechnology) used in the identification of our mice include: Tmem175 F: 5′-CCCATAACTCCTATTAGCA TATCAAC-3′, Tmem175 R: 5′- GGCTGGGCTAGGTATATTCAATC-3′, (WT=195bp, flox=285bp); Lyz2-Cre A: 5′-CCCAGAAATGCCA GATTACG-3′, Lyz2-Cre B: 5′-TTACAGTCGGCCAGGCTGAC-3’, Lyz2-Cre AB: 5′- CTTGGGCTGCCAGAATTTCTC-3′, (WT=350bp, Mut=~700bp).

Cd8a−/− mice were purchased from Jackson Lab (stock number: 002665), and the genotypes were identified according to the protocol provided by Jackson Lab. The PCR primers (Tsingke biotechnology) used in the identification of Cd8a−/− mice include: oIMR1098: 5′- GACCTGGTATGTGAAGTGTTGG-3′; oIMR1099: 5′-ACATCACC GAGTTGCTGATG-3′; oIMR6828: 5′-CATAGCGTTGGCTACCCGTG-3′ (WT=265bp, Mut=343bp).

Both male and female mice were included in our experiments. Sex-matched WT and Tmem175−/− mice at 6–8 weeks old were included in the in vivo and in vitro experiments. The oligonucleotide sequences have been summarized in Supplementary Data 11.

Cell culture
Mouse melanoma cell line (B16-F10) and Lewis lung cancer cell line (LL-2) were purchased from ATCC. B16-F10, B16-F10-OVA, and LL-2 cells were cultured in DMEM medium (Gibco, C11009955CP) supplied with 10% FBS (Gibco, 10091148) and 100U/mL penicillin+100µg/mL streptomycin (Beyotime, C0222). G418 (0.4mg/mL) was applied in the B16-F10-OVA culture. Mouse BMDMs were extracted from tibias and femurs. The bone marrow was treated by red blood cell lysis, filtered by a 70µm cell strainer (Falcon, CLS352350), and cultured in RPMI-1640 medium (Gibco, C11875500BT) supplied with 10% FBS (Gibco, 10091148), 100U/mL penicillin+100µg/mL streptomycin (Beyotime, C0222), and 20ng/mL M-CSF (Novoprotein, CB34) for 3 days. Then the culture medium was discarded, and the cells were cultured in fresh medium for an additional 2–3 days. Afterward, the suspension cells were removed, and the adherent cells were applied in our experiments.

For mouse peritoneal macrophages, humanely euthanized mice were intraperitoneally injected with 5mL cold saline. The ascites was extracted after gentle massage on abdomen. The peritoneal cells were centrifuged and cultured in RPMI-1640 medium (Gibco, C11875500BT) supplied with 10% FBS (Gibco, 10091148) and 100U/mL penicillin + 100µg/mL streptomycin (Beyotime, C0222) for 2h to allow the adherence of macrophages and remove the suspension cells. All the cells were maintained at 37°C and 5% CO2.

Electrophysiology
Whole-organelle voltage-clamp recording was applied according to previously reported methods17,83. Macrophages were seeded onto coverslips for 48-well plates. Lysosomes were enlarged by 5µM vacuolin-1 (Sigma, 673000) treatment for 2–3h. The enlarged lysosomes were dissected out with a glass pipette. Then another fire-polished glass pipette was applied to form the gigaseal formation with the lysosomes. When the pipette tips touch the lysosomes, slight suction through a plastic pipette connected to the pipette holder might help with the gigaseal formation. When the current decreased to less than 10pA at 5mV, or the seal resistance increased to around 8–20 GΩ, a successful gigaseal was formed. Then ZAP pulses (0.1–50ms, −200 to −1000mV voltage pulses) were used to rupture the part of the membrane in the glass pipette tips and to establish whole-lysosomal mode. Successful establishment of whole-lysosomal configuration was indicated by the decrease in seal resistance to 20–100 MΩ. Whole-organelle voltage-clamp recording of current from −100mV to 100mV was performed with Axon patch-clamp 700B (Molecular Devices). The bath solution contained 145mM K-methanesulfonate, 5mM KCl, and 10mM HEPES (pH 7.2); alternatively, 150mM NMDG-methanesulfonate was used to replace the potassium. The pipette solution contained 145mM K-methanesulfonate, 5mM KCl, and 10mM MES (pH 5.5).

Animal subcutaneous tumor models
WT and Tmem175−/− sex-matched mice at 6–8 weeks old were used in our experiments. B16-F10 and LL-2 subcutaneous tumor models were used in our research. Mice were subcutaneously injected with 1×106 tumor cells. For VX-765 treatment, VX-765 (Selleck, S2228) was administrated at the 8th day after inoculation. Vehicle (containing 5% DMSO, 40% PEG 400, 5% Tween 80, and 50% saline) or VX-765 solution (0.4mg dissolved in 200µL) was administrated every day through intraperitoneal injection. For clodronate liposomes treatment, mice were intravenously injected with the clodronate liposomes (YEASEN Biotechnology, 40337ES08) or control liposomes (YEASEN Biotechnology, 40338ES08) at a dose of 1mg/20g body weight at day −1, 6, and 13. For anti-CD8a (Bio X Cell, BE0061), anti-IL-1β (Bio X Cell, BE0246), and anti-IL-18 (Bio X Cell, BE0237) treatment, mice were intraperitoneally injected with 100µg/100µL antibody or isotype control (rat IgG2b isotype (Bio X Cell, BE0090) for anti-CD8a, Armenian hamster IgG isotype (Bio X Cell, BE0091) for anti-IL-1β, and rat IgG2a isotype (Bio X Cell, BE0089) for anti-IL-18) diluted with saline at day −1, 6, and 13. For CA-074 and BAPTA-AM treatment, mice were intratumorally injected with CA-074 (MCE, HY-103350) or BAPTA-AM (MCE, HY-100545) or vehicle (containing 10% DMSO, 40% PEG 400, and 50% saline) at a dose of 200µg/20g body weight at days 7, 10, and 13. For anti-PD-1 therapy, 200µg anti-PD-1 antibody (Bio X Cell, BE0146) or rat IgG2a isotype (Bio X Cell, BE0089) dissolved in 200µL saline was administered every 3 days through intraperitoneal injection, and the 1st treatment was administrated at the 3rd day after tumor cell inoculation. For chitosan treatment, vehicle (containing 2% acetic acid and 98% saline) or chitosan (Sigma, 448869) solution (2mg dissolved in 200µL) was administered every 3 days through intraperitoneal injection, and the 1st treatment was administrated at the 3rd day after tumor cell inoculation. For NAC treatment, 1g/L NAC (MCE, HY-B0215) was administrated in drinking water since day 759,60.

For the administration of IL-1β and IL-18 in wild type mice, intratumoral injections of 100ng IL-1β (Novoprotein, C042) or 100ng IL-18 (Novoprotein, CK06) dissolved in 50µL saline were implemented everyday from day 8 to day 17.

For the adoption of in vitro cultured T cells, spleen lymphocytes from B16-F10-OVA tumor bearing mice were harvested and cultured with WT or Tmem175−/− BMDMs in the existence of B16-F10-OVA for 48h. Then the CD8+ T cells were isolated by immunomagnetic negative selection (STEMCELL TECHNOLOGIES, 19853) and injected intravenously into Cd8a−/− mice (1×106 cells per mouse) 1 day before B16-F10-OVA implantation and seven days after B16-F10-OVA implantation. Cd8a−/− mice were subcutaneously injected with 1×106 B16-F10-OVA tumor cells.

For the adoption of BMDMs, in vitro cultured WT or Tmem175−/− BMDMs were transfected with siRNA (GenePharma) targeting Nlrp3 (sense: 5′-CCAGGAGAGAACCUCUUAUTT-3′ anti-sense: 5′-AUAA GAGGUUCUCUCCUGGTT-3′) or control siRNA via Lipofectamine RNAiMAX (Thermo Fisher, 13778030). One day after transfection, the BMDMs were harvested and injected intravenously into WT B16-F10 tumor bearing mice (1×106 cells per mouse) at 1 day before B16-F10 implantation and 7 days after B16-F10 implantation. The oligonucleotide sequences have been summarized in Supplementary Data 11.

Tumor sizes were monitored by vernier calipers every 2 days. The largest tumor should not exceed 2cm at the largest diameter according to the ethics committee; otherwise, the mouse should be euthanized. At the end of our experiments, the mice were euthanized with carbon dioxide. The tumors were removed and weighed.

B16-F10 lung metastatic model
B16-F10 cells (2×105) were intravenously injected into WT and Tmem175−/− sex-matched mice at 6–8 weeks old. Referring to the experience in our laboratory, B16-F10 lung metastatic nodules do not grow excessively on day 21, which minimizes animal suffering. Mice were euthanized with carbon dioxide on day 21. The lungs were weighed, and the metastatic nodules were counted.

Single-cell RNA sequencing analysis
Lung tissues of the B16-F10 lung metastatic WT and Tmem175−/− mouse models were used in the single cell transcriptome analysis. Lungs were minced and digested to create single cell suspension. Then the ratio of live cells was confirmed to be around 90%. Single-cell RNA sequencing (scRNA-seq) libraries were constructed via the 10x Chromium Single Cell 5′ platform (10X Genomics), referring to the manufacturer’s protocol. Briefly, single-cell suspensions were added onto a Chromium microfluidic chip in order to generate single-cell Gel Beads in Emulsion. Then the scRNA-seq libraries were prepared through the Chromium Single Cell 5′ Reagent Kit v2 (10X Genomics). Library quality was assessed by the Agilent 2100 Bioanalyzer system (Agilent Technologies), and the sequencing was performed by the Illumina NovaSeq X Plus platform (Illumina) with a sequencing depth of higher than 50,000 reads per cell.

The acquired sequencing data were aligned to the mouse reference genome (GRCm39) and processed through CellRanger (version 9.0.0, https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger). The gene expression matrix obtained from the CellRanger pipeline was filtered and normalized via the Seurat R package (version 5.2, https://cran.r-project.org/web/packages/Seurat/index.html)84. Cells were included if they met the following criteria: (i) >200 genes and <4500 genes; (ii) <5% of mitochondrial gene expression in the UMI counts. After removing low-quality cells, the gene expression matrices were normalized to the total UMI counts per cell and transformed to a natural log scale. All datasets from the same group were integrated through the “FindIntegrationAnchors” and “IntegrateData” functions in Seurat. We identified “anchors” among all the individual datasets in the same group with multiple canonical correlation analyses (CCA) that have been implemented by the “FindIntegrationAnchors” function. These “anchors” were applied in the “IntegrateData” function to create a batch-corrected expression matrix of all the cells, which allowed the cells from different datasets to be integrated and analyzed.

The integrated data were scaled to a unit variance and zero mean, and the dimensionality was reduced by principal component analysis (PCA). A shared nearest neighbor (SNN) graph was constructed based on the distances in PCA space (top 1:50 principal components) with the “FindNeighbors” function, in which the k-nearest neighbors (KNN) of each cell were determined. Then this KNN graph was used to construct the SNN graph by calculating the neighborhood overlaps (Jaccard index) between each cell and its KNN. The Louvain algorithm was used to iteratively group the proximal cells together with the “FindClusters” function, with a resolution of 1. UMAP analysis was performed through RunUMAP (dims=1:47) functions. Cell type annotation was performed via Azimuth based on Azimuth Mouse Lung CellRef (https://app.lungmap.net/app/azimuth-mouse-lung-cellref-seed)85 in company with marker-based annotations.

The “FindMarkers” function in Seurat was applied to find the markers for alveolar macrophages and interstitial macrophages. Genes that were significantly differentially expressed between the Tmem175−/− and WT groups (|log2FC|≥0.5, P adj ≤0.01) were included in the functional enrichment analysis, which has been implemented by KOBAS. The P values of differently expressed genes were calculated by Wilcoxon rank-sum test and adjusted by Bonferroni correction. The enrichment analysis of comprehensive functions, GO terms, and KEGG pathways. The P values of GO and KEGG analysis were calculated by Fisher’s exact test and adjusted by Benjamini–Hochberg procedure.

Flow cytometry
For the analysis of TME, tumor tissues from euthanized mice were cut up and digested in DMEM medium supplied with 0.5mg/mL collagenase I (Gibco, 17018029), 1mg/mL collagenase VI (Gibco, 17104019), and 1U/mL DNase I (KeyGEN BioTECH, KGA1506-1) for 30min (for B16-F10) or 1h (for LL-2). For lung metastatic models, the lungs were cut up and digested in DMEM medium supplied with 1mg/mL collagenase I (Gibco, 17018029), 0.5mg/mL collagenase VI (Gibco, 17104019), and 1U/mL DNase I (KeyGEN BioTECH, KGA1506-1) for 1h. The suspensions were filtered through 70µm strainers (Falcon, CLS352350) and treated by red blood cell lysis. For lung metastatic models, cells were stained by H-2Kb MuLV p15E Tetramer (MBL, TS-M507) on ice for 1h. Then cells were stained by antibodies and LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher, L34975) for 30min on ice. Alternatively, dead cells might also be stained with DAPI (Beyotime, C1005) for 5min. For intracellular staining, the cells were fixed and permeabilized by Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, 554714) after surface antigen staining. Then the cells were stained by the desired antibodies for 1h on ice. The cells were analyzed by ACEA NovoCyte (ACEA Biosciences).

For the analysis of intracellular antigen degradation, WT and Tmem175−/− BMDMs (1×106) were treated with B16-F10 cell debris (2.5×105/mL) for desired time (12, 24, and 48h). At the same time, FITC-dextran (MCE, HY-128868E) (0.2mg/mL) was incubated with WT and Tmem175−/− BMDMs (1×106) for 48h. At the end of the treatment, the cells were harvested and stained by desired antibodies and LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher, L34975). Then the cells were fixed and permeabilized by Cytofix/Cytoperm Fixation/Permeabilization Kit (BD bioscience, 554714) for intracellular p15E staining. The cells were analyzed by ACEA NovoCyte (ACEA Biosciences).

The following antibodies were used: TMEM175 rabbit pAb (Proteintech, 19925-1-AP), FeLV p15e Antibody (Santa Cruz Biotechnology, sc-65622), FITC anti-mouse CD45 (BD Biosciences, 553080), PerCP-Cy5.5 anti-mouse CD45 (BioLegend, 103132), BV421 anti-mouse CD45 (BioLegend, 103133), BV510 anti-mouse CD45 (BioLegend, 103137), BV650 anti-mouse CD45 (BioLegend, 103151), BV785 anti-mouse CD45 (BioLegend, 103149), PE anti-mouse CD206 (BioLegend, 141706), APC anti-mouse CD206 (BioLegend, 141708), FITC anti-mouse CD206 (BioLegend, 141704), APC anti-mouse F4/80 (BioLegend, 123116), PE anti-mouse F4/80 (BioLegend, 123110), FITC anti-mouse F4/80 (BioLegend, 123108), BV421 anti-rabbit IgG (BD Biosciences, 565014), BV650 anti-mouse CD11b (BioLegend, 101239), FITC anti-mouse CD11b (BioLegend, 101206), PE-Cy7 anti-mouse CD11b (BioLegend, 101216), BV510 anti-mouse CD11b (BioLegend, 101245), BV421 anti-mouse CD11b (BioLegend, 101235), APC anti-mouse CD11b (BioLegend, 101212), PerCP-Cy5.5 anti-mouse CD11b (BioLegend, 101228), BV711 anti-mouse-I-A/I-E (BioLegend, 107643), FITC anti-mouse-I-A/I-E (BioLegend, 107606), BV650 anti-mouse-I-A/I-E (BioLegend, 107641), PE-Cy7 anti-mouse-I-A/I-E (BioLegend, 107630), PE-Cy7 anti-mouse-CD11c (BD Biosciences, 558079), FITC anti-mouse-CD11c (BioLegend, 117306), BV510 anti-mouse CD11c (BioLegend, 117337), APC anti-mouse-Ly6G (BD Biosciences, 560599), PE anti-mouse-Ly6G (BioLegend, 127608), BV421 anti-mouse-Ly6C (BioLegend, 128031), APC anti-mouse-Ly6C (BioLegend, 128016), FITC anti-mouse-CD8a (BD Biosciences, 553031), BV421 anti-mouse CD8a (BioLegend, 100737), BV510 anti-mouse CD8a (BioLegend, 100751), PE anti-mouse CD8a (BD Biosciences, 553033), PE anti-mouse-IFN-γ (BioLegend, 505808), BV421 anti-mouse-IFN-γ (BioLegend, 505829), PE-Cy7 anti-mouse-IFN-γ (BioLegend, 505826), PerCP-Cy5.5 anti-mouse CD3 (BioLegend, 100218), PE anti-mouse CD3 (BD Biosciences, 555275), BV421 anti-mouse CD3 (BioLegend, 100227), PE-Cy7 anti-mouse CD3 (BioLegend, 100220), PE anti-mouse granzyme B (BioLegend, 372208), PE-Cy7 anti-mouse granzyme B (BioLegend, 372214), AF647 anti-mouse granzyme B (BioLegend, 515406), BV421 anti-mouse CD69 (BioLegend, 104527), APC anti-mouse CD69 (BioLegend, 104514), PE anti-mouse CD69 (BD Biosciences, 553237), PE-Cy7 anti-mouse CD69 (BioLegend, 104512), FITC anti-mouse CD69 (BD Biosciences, 553236), PerCP-Cy5.5 anti-mouse CD69 (BD Biosciences, 561931), BV510 anti-mouse CD4 (BioLegend, 100553), FITC anti-mouse CD4 (BioLegend, 100406), PE anti-mouse CD4 (BioLegend, 100512), APC anti-mouse CD4 (BioLegend, 100516), PerCP-Cy5.5 anti-mouse CD4 (BD Biosciences, 550954), BV421 anti-mouse CD4 (BioLegend, 100543), BV650 anti-mouse NK1.1 (BioLegend, 108735), APC anti-mouse NK1.1 (BioLegend, 108710), BV421 anti-mouse NK1.1 (BioLegend, 108731), FITC anti-mouse CD64 (BioLegend, 139316), APC anti-mouse CD24 (BioLegend, 121435), BV711 anti-mouse CD103 (BioLegend, 138506), APC goat anti-mouse IgG (BioLegend, 405308).

The gating strategies of our FCM have been shown in Supplementary Figs. 18 and 19. The antibodies have been summarized in Supplementary Data 11.

Immunohistochemistry and immunofluorescence
Tissue chip of paired melanoma and paracancerous tissues was purchased from WEIAOBIO (Catalog: ZL-MEL963). All uses of human material have been approved. The IRB approval (approval number: LLS M-15-01) has been provided by the ethics committee of Shanghai Zhuoli Biotechnology Co., Ltd, whose general distributor is the WEIAOBIO. Tumor tissues from humanely euthanized mice were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned at 3µm, hydrated, and underwent antigen retrieval.

For immunohistochemistry, the sections were pre-treated with 3% H2O2 to remove endogenous peroxidase activity. Then the cell slides were blocked with goat serum and incubated with desired antibody and detect antibody successively. The cell slides were incubated with DAB substrate, counterstained by hematoxylin, dehydrated, and mounted. The following antibodies were used: TMEM175 antibody (Proteintech, 19925-1-AP), cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, 9661L), caspase-1 p10 antibody (SANTA CRUZ, sc-56036), IL-1β (3A6) Mouse antibody (Cell Signaling Technology, 12242S), HRP anti-rabbit IgG antibody (ServiceBio, GB23303), and HRP anti-mouse IgG antibody (ServiceBio, G1214). For immunofluorescence, the slides were incubated with NK1.1 mouse mAb (Thermo Fisher, 14-5941-82) and CD8 rat mAb (Abcam, ab25478) overnight. Then the slides were incubated with AF488 anti-rat IgG (Thermo Fisher, A-11006) and Cy3 anti-mouse IgG (Thermo Fisher, M30010). Finally, the slides were incubated with DAPI (Beyotime, C1005) and mounted. The TMEM175 positive area, caspase-1 p10 positive area, IL-1β positive area, cleaved caspase-3 positive area, CD8-AF488 positive area, and NK1.1-Cy3 positive area were measured by ImageJ software. The antibodies have been summarized in Supplementary Data 11.

Cross-presentation assay
B16-F10-OVA cells were implanted in wild type C57BL/6J mice and grew for 10 days to prime CD8+ T cells. Then the mice were humanely euthanized to collect the spleens, from which the lymphocytes were separated by Mouse lymphocyte separation medium (DAKEWE, DKW33-R0100). The lymphocytes (1×106) were labeled by CFSE kit (Thermo Fisher) and then cultured with BMDMs (1×106) with IL-2 (20U/mL) (Novoprotein, CK24) and B16-F10-OVA cell debris (2.5×105/mL) for 48h with or without IL-1β (500pg/mL) and IL-18 (500pg/mL). After 48h, the cells were harvested and stained by H-2Kb OVA Tetramer (MBL, TS-5001-1C), antibodies, and LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher, L34975). The cells were analyzed by ACEA NovoCyte (ACEA Biosciences). The culture medium was collected for ELISA assay.

For the siRNA silencing, in vitro cultured WT or Tmem175−/− BMDMs were transfected with siRNA (GenePharma) targeting Myd88 (sense: 5′-GAAGCGACUGAUUCCUAUUTT-3′ anti-sense: 5′-AAUAG GAAUCAGUCGCUUCTT-3′) or Csf2rb (sense: 5′-GCCUGCAUUGG GAAACUCATT-3′ anti-sense: 5′-UGAGUUUCCCAAUGCAGGCTT-3′) via Lipofectamine RNAiMAX (Thermo Fisher, 13778030). The oligonucleotide sequences have been summarized in Supplementary Data 11.

In vitro stimulation of inflammasomes
B16-F10 tumor cell debris was the main inflammasome stimulator in our experiment. The concentration of cell debris was 2.5×105/mL. The harvested cells were repeatedly blown by syringes followed by two freeze-thaw cycles to generate cell debris. BMDMs (1×106) were activated by 500ng/mL LPS (Sigma, L2880) for 4h, then the medium was discarded, and the BMDMs were treated with cell debris for a desired period of time. During the treatment of cell debris, VX-765, CA-074, or KCl were added, whose final concentrations were 10µg/mL, 100µM, and 40mM respectively. For positive control, 10µM Nigericin (MCE, HY-127019) was added after LPS stimulation. For calcium chelation, cells were incubated with 20µM BAPTA-AM (MCE, HY-100545) for 30min after the LPS treatment. For ROS inhibition, cells were incubated with 10mM NAC (MCE, HY-B0215) during the cell debris treatment. For Bafilomycin A1 treatment, cells were incubated with 1µM Bafilomycin A1 (MCE, HY-100558) during the cell debris treatment. For the treatment of chitosan, BMDMs or peritoneal macrophages were activated by 500ng/mL LPS (Sigma, L2880) for 4h and then treated with 40µg/mL chitosan (Sigma, 448869) for 5h. For the treatment of aluminium hydroxide (Invivogen, vac-alu-50), BMDMs were activated by 500ng/mL LPS (Sigma, L2880) for 4h and then treated with 300µg/mL aluminium hydroxide for a desired period of time. For the treatment of B16-F10 supernatant, BMDMs were activated by 500ng/mL LPS (Sigma, L2880) for 4h and then treated with 25% volume of filtered B16-F10 supernatant overnight.

ELISA
Cell culture supernatants were analyzed by Mouse IL-1beta Uncoated ELISA Kit (Invitrogen, 88-7013A-88), Mouse TNF Uncoated ELISA Kit (Invitrogen, 88-7324-88), and Mouse IFN gamma “Femto-HS” High Sensitivity Uncoated ELISA Kit (Invitrogen, 88-8314-88) according to the manufacturer’s instructions.

Western blot
For the western blot analysis of cell lysis, BMDMs were lysed by RIPA (Beyotime, P0013B) and boiled with 1× SDS buffer (Beyotime, P0015L) for 5min at 95°C. Protein lysates were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, IPVH00010). The membranes were probed by desired antibodies.

For the western blot analysis of cell supernatant, total proteins in the cell supernatant were precipitated by an equal volume of 10% trichloroacetic acid (TCA). The protein precipitants were washed by cold acetone three times. Then precipitants were subjected to air-drying for 5min. The precipitants were dissolved by alkaline 1 × SDS buffer (Beyotime, P0015L) plus 200mM DTT (Thermo Fisher Scientific, R0861) and then boiled for 5min at 95°C. The grayscale values of TMEM175, β-actin, caspase-1 p10, IL-18 p24, ASC polymer, and cathepsin B were measured by ImageJ software. If the immunoblotting included β-actin, the results were presented as the ratio of the target protein/β-actin; otherwise, the grayscale values of the target protein were presented directly.

The following antibodies were used: TMEM175 mouse mAb (Proteintech, 68172-1-Ig), caspase-1 rabbit mAb (Abcam, 3345-1), IL-18 mouse mAb (LSBio, LS-C179849), β-actin mouse mAb (Santa Cruz Biotechnology, sc-47778), anti-rabbit IgG (HRP) (Thermo Fisher Scientific, 31460), anti-mouse IgG (HRP) (Thermo Fisher Scientific, 31430), and anti-rabbit IgG-Fc (HRP) (SinoBiological, SSA003). The antibodies have been summarized in Supplementary Data 11.

Co-immunoprecipitation
After the desired treatment, BMDMs were lysed by RIPA. Protein lysates were incubated with 1µg/mL rabbit IgG isotype (Cell Signaling Technology, 3900S) and 20µL Protein A+G Agarose (Beyotime, P2012) at 4°C for 30min to avoid non-specific combination. The lysates were centrifuged to remove the agarose beads and incubated with 1µg/mL NLRP3 rabbit mAb (Cell Signaling Technology, 15101S) at 4°C overnight, during which 5mM EDTA-Na2 was added to the desired group. For negative control, the lysates were incubated overnight with 1µg/mL isotype rabbit IgG. Afterward, the lysates were incubated with 20µL Protein A+G Agarose (Beyotime, P2012) at 4°C for 3h. The beads were washed 3 times with RIPA. Finally, the beads were resuspended in 1× SDS buffer and boiled for 5min at 95°C.

The following antibodies were used: Cathepsin B rabbit mAb (Cell Signaling Technology, 31718S), ASC mouse mAb (Santa Cruz Biotechnology, sc-514414), NLRP3 rabbit mAb (Cell Signaling Technology, 15101S), Rabbit IgG isotype (Cell Signaling Technology, 3900S), anti-rabbit IgG (HRP) (Thermo Fisher Scientific, 31460), anti-mouse IgG (HRP) (Thermo Fisher Scientific, 31430), and anti-rabbit IgG-Fc (HRP) (SinoBiological, SSA003). The antibodies have been summarized in Supplementary Data 11.

Transcriptome analysis
Tmem175−/− and WT BMDMs were cultured, harvested, and lysed in TRIzol (Invitrogen, 15596018CN). Novogene Bioinformatics Institute, Beijing, China, performed the RNA extraction and sequencing. The raw data were processed by adapter trimming, and reads were filtered by trimgalore version 0.5.0 to ensure data quality. The reads were mapped to Mus_musculus.GRCm38.dna.toplevel.fa and annotated with Mus_musculus.GRCm38.98.gtf.

To identify differentially expressed genes between Tmem175−/− and WT groups, fold change (FC) was calculated. The cut-off number of log2FC was set to be 1. The P values were calculated by likelihood ratio test, and adjusted by Benjamini–Hochberg procedure. The differentially expressed genes were further annotated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes database. The P values of GO and KEGG analysis were calculated by Fisher’s exact test and adjusted by Benjamini–Hochberg procedure.

The RNA-seq of the co-culture system was performed by DNBSEQ-T7 (MGITech). Clean reads were obtained by removing the adaptor sequences, reads with >5% ambiguous bases, and low-quality reads containing more than 20% of bases with qualities of <13. The clean reads were aligned to mouse genome (version: GRCh38 NCBI) using hisat2. The DESeq algorithm was used to filter the differentially expressed genes (FoldChange >1.1 or <0.9, FDR <0.05). The P values were calculated by Negative binomial distribution test, and adjusted by Benjamini–Hochberg procedure. For GO enrichment analysis, GO annotations were downloaded from NCBI, UniProt, and the Gene Ontology. Significant GO categories were identified by Fisher’s exact test, and the p-value was corrected by FDR through Benjamini–Hochberg procedure. Significant KEGG pathways were selected by Fisher’s exact test, and the significance thresholds were defined by p-value and FDR through Benjamini–Hochberg procedure.

Electron microscopy
BMDMs were harvested, fixed with 3% glutaraldehyde, and re-fixed with 1% osmium tetroxide. The pellets were dehydrated with a series of acetone and embedded in Ep812. The ultrathin sections were cut by a diamond knife and stained by uranyl acetate and lead citrate. Images were taken by JEM-1400-FLASH Transmission Electron Microscope.

Lysosomal pH determination
The determination of lysosomal pH was performed using LysoSensor Yellow/Blue DND-160 (Invitrogen, L7545) according to manufacturers’ instructions. BMDMs were seeded in non-transparent 96-well plates. BMDMs were incubated with 5µM LysoSensor Yellow/Blue DND-160 for 5min after cell debris stimulation. Then the cells were washed twice by phenol red-free 1640 (Gibco, 11835030). The plates were read by Microplate Reader (PerkinElmer). Two fluorescence intensities, 329nm excitation/440nm emission and 384nm excitation/540nm emission, were collected. The ratio of 440nm/540nm was calculated for every well. We created a standard curve for pH calculation with a series of solutions with known pH at 3, 4, 5, 6, and 7. The LysoSensor Yellow/Blue DND-160 fluorescents were collected, and the 440nm/540nm ratio of each solution was calculated and used to create the standard curve. The values of lysosomal pH were calculated by referring to the standard curve.

Lysosomal calcium determination
The determination of lysosomal calcium determination was performed using AF488-dextran (Invitrogen, D34682) and rhod-dextran (AAT Bioquest, 20451) according to previous report86. Briefly, BMDMs were seeded in non-transparent 96-well plates and incubated with 0.1mg/mL rhod-dextran and 0.2mg/mL AF488-dextran overnight. The cells were then washed twice and stimulated with LPS and B16-F10 tumor cell debris as mentioned above in phenol red-free 1640 (Gibco, 11835030). The plates were read by a Microplate Reader (PerkinElmer). At the same time, standard wells with known calcium concentration were used as control. Two fluorescence intensities, 543nm excitation/560nm emission and 488nm excitation/530nm emission, were collected. The lysosomal calcium concentrations were calculated using the following equations: [Ca2+] = Kd × (R - Rmin) / (Rmax - R) × (Sf2 / Sb2). R was the ratio of 560nm/530nm for a single well at a certain time point; Rmin was the lowest 560nm/530nm ratio; Rmax was the highest 560nm/530nm ratio of a saturating calcium concentration. Sf2 and Sb2 showed fluorescence of 530nm in the absence and presence of calcium, respectively. Kd is the dissociation constant determined by the standard curve with pH at 5, which equals to the concentration of calcium at which 560nm fluorescence of rhod-dextran reached 50% brightness. In the desired wells, 1µM Bafilomycin A1 (MCE, HY-100558) was added during the cell debris treatment.

Cytoplasmic calcium determination
The determination of cytoplasmic calcium was performed using Fluo-4-AM (Invitrogen, F14201). Briefly, BMDMs were seeded in non-transparent 96-well plates. BMDMs were incubated with 2µM Fluo-4-AM for 30min after cell debris stimulation. Then the cells were washed twice in phenol red-free 1640 (Gibco, 11835030). The plates were read by a Microplate Reader (PerkinElmer). Fluorescence intensity 480nm excitation/525nm emission was collected. The fluorescence of Fluo-4 was positively related to the concentration of cytoplasmic calcium; thus, the final results were presented as the ratio of 525nm fluorescence in each group to the average 525nm fluorescence in the blank group. In the desired wells, 1µM Bafilomycin A1 (MCE, HY-100558) or 100µM CA-074 (MCE, HY-103350) was added during the cell debris treatment.

Confocal microscope
BMDMs were seeded in glass slips and treated by LPS and B16-F10 tumor cell debris mentioned above. The cells were fixed and permeabilized by Cytofix/Cytoperm Fixation/Permeabilization Kit (BD bioscience, 554714). Then the cells were incubated with the desired antibodies overnight and the desired fluorescent secondary antibodies at room temperature. For cathepsin B rabbit mAb (Cell Signaling Technology, 31718S), the fluorescent secondary antibodies were Cy3 goat anti-rabbit IgG (Abcam, ab98416). For cathepsin D mAb (HUABIO, ET1608-49) and cathepsin L mAb (HUABIO, HA722063), the fluorescent secondary antibodies were AF488 goat anti-rabbit IgG (Thermo Fisher Scientific, A-11034). Nuclei were stained with DAPI (Beyotime, C1005). Imaging was performed using laser scanning confocal microscope (Olympus), and the images were analyzed using CellSens Dimension Software (Olympus).

ROS detection
The determination of ROS was performed using ROS probe, H2DCFDA (Invitrogen, D399). Briefly, BMDMs were seeded in non-transparent 96-well plates. BMDMs were incubated with 1µM H2DCFDA for 30min after cell debris stimulation. Then the cells were washed twice in phenol red-free 1640 (Gibco, 11835030). The plates were read by a Microplate Reader (PerkinElmer). Fluorescence intensity 488nm excitation/520nm emission was collected. The fluorescence intensity was positively related to the concentration of ROS.

Molecular dynamics simulation
The model of PYD domain from ASC was downloaded from protein data bank (PDB: 2N1F). The model of PYD domain from NLRP3 was built by Swiss-model87. The GMQE was 0.88, and the QMEANS was 0.81±0.05, indicating a reliable structure generation. The molecular docking was performed using HDock88, in which the docking score was 176.33 kcal/mol. The molecular docking result was used as the initiating conformation in the molecular dynamics simulation.

Gromacs 2019.689 was applied, and Amber14SB was selected as protein force field. The water model was established by TIP3P. Ten calcium ions were randomly added, and the sodium ion/chloride system was founded. The particle-mesh Ewald algorithm was used for electrostatic calculations, in which the system was minimized via steepest descent algorithm for 50,000 steps. The cut-off distance of both Coulomb force and the van der Waals radii was 1.4nm. Canonical ensemble (NVT) and constant-temperature, constant-pressure ensemble (NPT) were adopted. Molecular dynamics simulation was performed for 100ns at normal temperature and pressure. The results were displayed by PyMOL 2.5.190 and LigPlot 2.191.

Statistical analysis
Results from two or three independent experiments are presented as average values ± SEM. The number of biological replicates is shown in the figure legends. All statistical analyses were performed by linear regression, two-sided unpaired t-test (for 2 groups), one-way ANOVA (for 3 groups), or two-way ANOVA (for ≥2 variables or ≥4 groups) using GraphPad Prism 8 (GraphPad Prism Software Inc., La Jolla). The statistical methods to calculate the statistical significances were indicated in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this MS by Zhang et al., the authors investigated the roles of lysosomal TMEM175 channels in tumor growth through an interactive regulation of tumor microenvironment. Conditional knockout of Tmem175 in mouse macrophages resulted in reduced tumor weight and metastasis. This effect was reversed by VX-765, a broad-spectrum inflammasome inhibitor, suggesting that inflammasome signaling is essential for the anti-tumor effects mediated by TMEM175 deficiency. It was claimed that in Tmem175 KO cells, lysosomes were hypo-acidified, resulting in increased lysosomal cathepsin B (CTSB) leakage and Ca2+ efflux. The subsequent cytosolic Ca2+ elevation was proposed to regulate cell debris-activated inflammation, as both CTSB inhibitors and membrane-permeable Ca2+ chelator BAPTA-AM completely eliminated the differences between WT and KO cells. Overall, there were some interesting observations in the study, but many experimental details and controls were missing. Hence, the evidence presented in this article is insufficient to support the conclusions drawn in many sections.\nMajor Concerns:\n1. In Figure 1, the weak band intensity observed in the western blot for the Tmem175-/- group was attributed to the presence of remaining exons 1-4. However, one would expect a size-shifted band in KO samples. It was not clear whether the antibody used was specific, as KO efficiency appeared to be only slightly more than 50%, judging from the biochemical validation.\n2. In Figures 6 and 7, both CA-074 and BAPTA-AM abolished the differences in cell debris-activated inflammation between WT and KO cells. What is the relationship between cytosolic CTSB accumulation and Ca2+ elevation? Additional controls are necessary to delineate the relationship between these two events. For instance, what is the effect on CTSB activity following BAPTA-AM treatment, and what is the effect of CA-074 treatment on cytosolic Ca2+ elevation?\n3. In Figure 7, it was suggested that elevated lysosomal pH is associated with the efflux of lysosomal Ca2+. Can pre-treatment with Baf-A1, a V-ATPase inhibitor, mimic all the effects of TMEM175 KO?\n4. As mentioned in Discussion, the effect of TMEM175 KO on lysosomal pH is controversial. What is the basal lysosomal pH of BMDMs without LPS treatment? It was also not clear about the effects of LPS and cell debris treatments on lysosomal pH and Ca2+ in WT and KO cells.\n5. In Figures 7b and 7c, the authors showed decreases in lysosomal pH and calcium concentrations. However, most Ca2+ dyes are known to be pH-sensitive. How can you separate the effects of your treatment on Ca2+ versus pH?\n6. For the electrophysiological recordings in Figure 1, it was difficult to evaluate the results without providing detailed recording solutions and protocols. How did you know whether the currents were not caused by leakage?\n7. In Figure 6g, there was no significant difference for the leakage of cathepsin B between WT and Tmem175 KO groups. Statistical analysis was missing. What was the rationale that only CTSB, but not other cathepsins, e.g., CTSD or CTSL, was investigated? Previous studies suggested that CTSL might be more relevant for lysosome membrane permeability.\n8. The levels of reactive oxygen species (ROS) were high in Tmem175-/- BMDMs. Do ROS contribute to the observed phenotypes?\nMinor Concerns:\n1. What does the time point \"0\" represent in Figure 7a? The Y-axis indicates lysosomal pH, but it is unclear on how lysosomal pH was calibrated in the study.\n2. In Figure 9, are you sure about the direction of K+ flow across lysosomal membranes? Cytosolic K+ concentration is known to be as high as 150 mM.\n3. In Figure 1e, why did the scale of the ratio start from 0.2?\n4. There was lack of statistical analysis in Figure 7c.", "reviewer_2": "In this study by Zhang et al., the authors elucidate a critical role for TMEM175 within myeloid cells in orchestrating the anti-tumour immune response. The myeloid-specific Tmem175 knockout in murine models exhibited notable anti-tumour efficacy, accompanied by a reconfiguration of the tumour microenvironment. Furthermore, TMEM175 ablation impaired lysosomal acidification and potentiated cross-presentation of tumour antigens. These findings, highlighting the therapeutic promise of targeting TMEM175 in myeloid cells, are both innovative and promising. Nevertheless, the manuscript would benefit from refined data presentation and an augmented mechanistic investigation, as outlined below.\nMajor Concerns:\nClinical Relevance: The study proposes TMEM175 as an intriguing target for tumour immunotherapy. However, it remains unclear whether TMEM175 is differentially expressed between normal and cancerous tissues. Additionally, does TMEM175 deficiency influence the polarisation of tissue-resident macrophages?\nIn Figures 2 and 3, TMEM175 deficiency demonstrates anti-tumour effects by impeding tumour growth and metastasis, with concurrent alterations in tumour-associated macrophage (TAM) composition and T-cell infiltration. These findings, however, are predominantly observational. A mechanistic elucidation is necessary to substantiate whether, and through what pathways, TMEM175 deficiency attenuates the immunosuppressive tumour microenvironment (TME). Does TMEM175 directly modulate TAM polarisation? What are the underlying molecular mechanisms? Moreover, the functional contributions of distinct immune cell populations to the observed anti-tumour effects remain ambiguous. Depletion of TAMs and T-cells would substantiate these findings.\nAn enigmatic observation lies in the insensitivity of B16-F10 tumours to inflammasome inhibition (via VX-765) juxtaposed with their heightened sensitivity to inflammasome stimulation (through chitosan), as illustrated in Figure 4.\nThe antigen cross-presentation data presented in Figure 5 are perplexing. Tmem175-/- bone marrow-derived macrophages (BMDMs) markedly induce proliferation of OVA-specific CD8+ T cells, even in the absence of B16-F10 OVA cell debris. The addition of B16-F10 OVA debris results in only a minimal further increase in T cell proliferation. This suggests that the observed T cell proliferation and activation may occur via mechanisms beyond antigen cross-presentation.\nMinor Points:\n1. In Figure 1d, there remains a residual band, albeit weaker than wild-type, in BMDMs from Tmem175-/- mice. The authors attribute this to residual expression from exons 1-4; however, the band appears at the same molecular weight as full-length TMEM175. Additional validation is warranted to confirm effective knockout of Tmem175 in myeloid cells.\n2. Vehicle controls are conspicuously absent in Figure 4j.", "reviewer_3": "Overall comments:\nIn this manuscript, Zhang, et al investigate the role of TMEM175 in macrophages and tumors reporting macrophage-specific TMEM175 deficiency inhibited tumor growth through promoting M1-like polarization and the recruitment of T cells and NKs. In addition, they found TMEM175 KO BMDMs showed enhanced tumor antigen cross-presentation. To further explore the mechanism, authors determined the importance of inflammasome activation in macrophages and found TMEM175 deficiency inhibited lysosomal acidification and promoted lysosomal calcium efflux, leading to increased ASC-NLRP3 interaction. As a possible therapeutic strategy, macrophage-specific TMEM175 KO mice had better therapeutic response to anti-PDL1 therapy.\nTheir work is interesting and provides new insights into the function of macrophage TMEM175 in regulation of the tumor microenvironment. Their finding that macrophage-specific KO reduced tumor growth and improved the therapeutic effect of anti-PDL1 enhance the translational potential of their study. However, the mechanism by which TMEM175 promotes the anti-tumor immunity is too diffuse and complicated and not well-supported at several places. The mechanistic aspect can be strengthened with some additional experiments and significant reorganizations as I detail below:\nMajor points:\nThe authors made a significant effort in exploring the mechanisms by which TMEM175 deficiency promotes the anti-tumor immunity. However, their concept includes multiple parallel pathways (inflammasome activation by cathepsin, ROS, and calcium efflux; cross-presentation of tumor antigens; and autophagy dysfunction) which leads to an unfocused assessment resulting in confusion of the reader. In addition, beyond usage of VX-765 to test the importance of inflammasome activation during tumor progression, other pathways are not fully characterized in their tumor models. It’s not clear how much each pathway contributes to the anti-tumor immunity in TMEM175 KO macrophages, or this effect is just a simple mixture of phenotypes from different pathways.\n1) The authors indicate that increased IL-1β and IL-18 secreted from TMEM175 KO macrophages drive the recruitment and activation of T cells and NK cells in TME. To support their conclusion, the anti-tumor immunity in vivo should be evaluated using neutralizing antibodies or ways to perturb this cytokine signaling.\n2) The authors raise three hypotheses about how TMEM175 deficiency leads to increased inflammasome activation: increased cathepsin B leakage, higher ROS levels, and elevated cytosolic calcium. Although all three mechanisms may result from TMEM175-induced lysosomal dysfunction, the effects of inhibitors (CA-074, BAPTA-AM, etc) for these pathways on blocking inflammasome activation should be evaluated to identify whether there’s a dominant pathway responsible for the pro-inflammatory phenotype in TMEM175 KO macrophages. Moreover, the dominant inhibitor should be tested in vivo for the effect of pathways on anti-tumor immunity and tumor growth. VX-765 is only a general inhibitor of caspase-1. For the precise machinery of anti-tumor immunity, siRNA-mediated gene silencing in macrophages should be conducted to verify the pharmacological inhibition data, and the anti-tumor effect of macrophage conditional depletion of the predominant pathway should be evaluated in vivo. In this way, the manuscript becomes more focused on a predominant pathways as the key mechanism underlying their phenotype. By evaluating all types of pathways in superficial ways, it leaves more to be desired and confuses the reader.\n3) Authors also demonstrated TMEM175 deficiency augmented the cross-presentation of tumor antigens. The in vitro work is nice but without evaluation of the effect of this pathway on anti-tumor immunity and tumor growth weakens their conclusion. In addition, authors mentioned the elevated cross-presentation signal in TMEM175 KO macrophages can be enhanced by IL-1β and IL-18 but didn’t provide any explanation. Does it mean those cytokines promote phagocytosis, or the antigen-presentation relies on inflammasome activation? The utilization of siRNA may help answer these questions.\n4) The data about autophagy dysfunction and inflammasome activation is very superficial and lacks the evaluation of IL-1β secretion, so can be removed from the revised manuscript and only mentioned in the discussion. Again, by doing superficial work on multiple pathways as a mechanism is not focused.\n5) The study on PYD-PYD interaction (Fig. 7f-k) seems redundant and not directly linked to their main findings.\nMinor points:\n1) Similarly, the Discussion Section is too long and includes lots of review about TMEM175 and lysosomal dysfunction. Based on their findings, authors should focus on how TMEM175 expression is regulated in macrophages during tumor progression and what area therapeutic strategies can be developed.\n2) The description of generating conditional TMEM175 mice can be moved to the Method Section or Supplemental figure legends and not main figures.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear editor,\nThank you for your letter and the reviewers comments concerning our manuscript\nDeficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor\nimmunity via inflammasome and cross-presentation pathway (NCOMMS-24-\n59454A) submitted to Nature communications. The comments are very helpful for us\nto improve our manuscript. Newly revised portions are highlighted in yellow. The\npoint-by-point response for the reviewers’ comments are listed as follows.\nReviewer #1\nMajor Concerns:\n1. In Figure 1, the weak band intensity observed in the western blot for the\nTmem175-/- group was attributed to the presence of remaining exons 1-4. However,\none would expect a size-shifted band in KO samples. It was not clear whether the\nantibody used was specific, as KO efficiency appeared to be only slightly more\nthan 50%, judging from the biochemical validation.\nResponse: Thank you for your comment. The antibody used in our previous experiment\nwas a polyclone antibody (Proteintech, 19925-1-AP). To improve the accuracy, we\nreplicated the western blot using a monoclonal antibody (Proteintech, 68172-1-Ig).\nMore accurate result demonstrated higher KO efficiency, and we found the weak size-\nshifted band (40-55 kD) you mentioned. We speculate that the remaining weak 55-\n70KD band in Tmem175-/- group might be owed to the inevitable inclusion of cells that\ndid not express Lyz2 genes during the primary cell culture.\nTMEM175 expression was obviously decreased in Tmem175-/- BMDMs. Lysates of\nTmem175-/- and WT BMDMs were analyzed by western blot using anti-TMEM175\nantibody.\n2. In Figures 6 and 7, both CA-074 and BAPTA-AM abolished the differences in\ncell debris-activated inflammation between WT and KO cells. What is the\nrelationship between cytosolic CTSB accumulation and Ca2+ elevation?\nAdditional controls are necessary to delineate the relationship between these two\nevents. For instance, what is the effect on CTSB activity following BAPTA-AM\ntreatment, and what is the effect of CA-074 treatment on cytosolic Ca2+ elevation?\nResponse: Thank you for your question. We used CO-IP to analyze the influence of\nBAPTA-AM on CTSB and Fluo-4-AM to analyze the influence of CA-074 on cytosolic\ncalcium ions. Our present results demonstrated that the cytosolic calcium ions elevation\nseemed to be the upstream event of CTSB-NLRP3 interaction. Calcium chelating by\nBAPTA-AM inhibited the CTSB-NLRP3 interaction, while inhibiting CTSB by CA-\n074 did not obviously altered the cytosolic calcium.\nFig. 8f Calcium chelation through BAPTA-AM inhibited the interaction between\ncathepsin B and NLRP3. Cells were treated by B16-F10 cell debris for 4 hours.\nFig. 8c Dynamic changes of cytoplasmic calcium were determined using Fluo-4-AM.\nThe higher level of cytoplasmic calcium in Tmem175-/- BMDMs than WT BMDMs was\nobserved by B16-F10 cell debris treatment. BAPTA-AM did not obviously alter the\ncytoplasmic calcium levels.\n3. In Figure 7, it was suggested that elevated lysosomal pH is associated with the\nefflux of lysosomal Ca2+. Can pre-treatment with Baf-A1, a V-ATPase inhibitor,\nmimic all the effects of TMEM175 KO?\nResponse: Thanks for your constructive suggestion. During the B16-F10 cell debris\ntreatment Baf-A1 was applied, which promoted the ASC polymerization and IL-1\nsecretion in both WT and Tmem175-/- BMDMs. Notably, the inflammasome activatio β n\nin Baf-A1 treated WT group was comparable with that in Baf-A1 untreated Tmem175-\n/- group, thus Baf-A1 treatment mimicked the effects of TMEM175 KO.\nFig. S12(b to d) Tmem175-/- and WT BMDMs were stimulated with 500 ng/ml LPS for\n4 hours to activate signal 1 of NLRP3 inflammasomes, then treated with B16-F10 cell\ndebris (2.5 105/ml) to activate NLRP3 inflammasomes. For ROS inhibition, cells were\n×\nincubated with 10 mM NAC during the cell debris treatment. For Bafilomycin A1\ntreatment, cells were incubated with 1 M Bafilomycin A1 during the cell debris\nμ\ntreatment.\n4. As mentioned in Discussion, the effect of TMEM175 KO on lysosomal pH is\ncontroversial. What is the basal lysosomal pH of BMDMs without LPS treatment?\nIt was also not clear about the effects of LPS and cell debris treatments on\nlysosomal pH and Ca2+ in WT and KO cells.\nResponse: Thank you for your question. We repeated our lysosomal pH, lysosomal\ncalcium, and cytosolic calcium measurement and added the results of WT and KO cells\nwithout LPS treatment. The basal lysosomal pH was higher in KO BMDMs than that\nin WT BMDMs, but we did not observed a significant difference between the basal\nlysosomal calcium ions in WT and KO BMDMs. We speculate that the lysosome\nmembrane permeabilization after B16-F10 cell debris treatment was needed for the\nlysosomal calcium efflux. Likewise, we also added the ROS production level of WT\nand KO cells without LPS treatment.\nFig. 8(a to c) For dynamic observation of lysosomal pH, lysosomal calcium, and\ncytosolic calcium, the cells were pre-treated with desired fluorescent probes and the\nfluorescence was collected by fluorescent reader.\nFig. S12a Tmem175-/- and WT BMDMs were stained with ROS probe H2DCFDA and\ntreated with LPS and B16-F10 cell debris. The fluorescence reflexed the amount of\nROS. Tmem175-/- BMDMs generated higher level of ROS than WT BMDMs.\n5. In Figures 7b and 7c, the authors showed decreases in lysosomal pH and calcium\nconcentrations. However, most Ca2+ dyes are known to be pH-sensitive. How can\nyou separate the effects of your treatment on Ca2+ versus pH?\nResponse: Thank you for your question. The lysosomal Ca2+ concentration was\ndetermined referring to the protocol published by Anthony J Morgan et al. (Imaging\napproaches to measuring lysosomal calcium, PMID: 25665446). It’s true that the\ncalcium dye, rhod-dextran, is pH-sensitive, however the key dissociation constant (K )\nd\nused to calculate the lysosomal Ca2+ concentration was measured at the acidic pH. In\naddition, they applied pH-insensitive AF488-dextran to rule out other interference\nfactors.\nAccording to the lysosomal pH measurement, we know that the lysosomal pH was\naround 5 after the LPS and B16-F10 cell debris treatment. Thus we prepared a series of\ncalcium ion solution in gradient dilution at pH 5 to calculate the dissociation constant\n(K ) of rhod-dextran at pH 5. Then, free rhod-dextran has little fluorescent excitation,\nd\nwhile Ca2+-rhod-dextran has strong fluorescent excitation. Thus according to the\nformula , when equals to\n2+\n[𝐶𝑎 ]×[𝑟ℎ𝑜𝑑−𝑑𝑒𝑥𝑡𝑟𝑎𝑛] 2+\n𝐾𝑑 = [𝐶𝑎 2\n(\n+\nh\n−\na\n𝑟\n𝑙\nℎ\n𝑓\n𝑜 𝑑\n−\n𝑓\n𝑑𝑒\n𝑡ℎ\n𝑥\n𝑒\n𝑡𝑟 𝑎\n𝑚\n𝑛]\naximum C\n[\n𝑎\n𝑟2ℎ+𝑜\n-r\n𝑑\nho\n−\n𝑑\n𝑑\n𝑒\n𝑥\n𝑡\n𝑟\n𝑎\n𝑛\n𝑛 ]\nfluorescence\n[\n)\n𝐶\n, \n𝑎 −\n𝑟ℎ𝑜𝑑 −𝑑𝑒𝑥𝑡𝑟𝑎𝑛] 𝐾𝑑 =\n.\n2+\n[𝐶𝑎 The ] formula to calculate lysosomal Ca2+ concentration is supposed to be\n2+\n[𝐶𝑎 ] =\n. However Anthony J Morgan et al. modified it to be\n2+\n[𝐶𝑎 −𝑟ℎ𝑜𝑑−𝑑𝑒𝑥𝑡𝑟𝑎𝑛]\n𝐾𝑑 × [𝑟ℎ𝑜𝑑−𝑑𝑒𝑥𝑡𝑟𝑎𝑛] . R was the ratio of rhod-dextran/AF488-dextran for a\n2+ 𝑆𝑓2 (𝑅−𝑅𝑚𝑖𝑛)\n[𝐶𝑎 ] = 𝐾𝑑 ×𝑆𝑏2 ×(𝑅𝑚𝑎𝑥−𝑅)\nsingle well in a certain time point. R was the lowest 560 nm/530 nm ratio; R was\nmin max\nthe highest 560 nm/530 nm ratio of a saturating calcium concentration. We speculate\nthat the rhod-dextran and AF488-dextran are engulfed into cells at a certain ratio. Thus\nrhod-dextran/AF488-dextran is used to rule out the differences caused by the different\nengulfment capacities. refers to , because only Ca2+-\n2+\n(𝑅−𝑅𝑚𝑖𝑛) [𝐶𝑎 −𝑟ℎ𝑜𝑑−𝑑𝑒𝑥𝑡𝑟𝑎𝑛]\n(𝑅𝑚𝑎𝑥−𝑅) [𝑟ℎ𝑜𝑑−𝑑𝑒𝑥𝑡𝑟𝑎𝑛]\nrhod-dextran has strong fluorescent excitation. S and S were the fluorescence of\nf2 b2\nAF488-dextran in the absence and presence of calcium respectively. The addition of\nis supposed to rule out the influence of calcium on the AF488-dextran fluorescence.\n𝑆𝑓2\n𝑆𝑏2\n6. For the electrophysiological recordings in Figure 1, it was difficult to evaluate\nthe results without providing detailed recording solutions and protocols. How did\nyou know whether the currents were not caused by leakage?\nResponse: Thank you for your question. We provided additional details for our whole-\norganelle voltage-clamp recording in our method section. The lysosomal\nelectrophysiological recordings were performed referring to the protocol published by\nCheng-Chang Chen et al. (Patch-clamp technique to characterize ion channels in\nenlarged individual endolysosomes, PMID: 28726848). In commonly studied whole-\ncell electrophysiology, the leakage currents are usually caused by incomplete gigaseal\nformation or loss of seal during the whole-cell mode. For incomplete gigaseal formation,\nit was very easy to form complete gigaseal (resistance around 8-20 GΩ) by the fire-\npolished glass pipettes in our whole-lysosomal mode. For loss of seal, the situation\nseems to be different between whole-cell and whole-lysosomal electrophysiology. In\nwhole-cell electrophysiology, the pipette tips could be partially separated from the cell\nmembrane to cause leakage current and the incomplete seal could be maintained for a\nperiod of time, but we did not observe such situation in our whole-lysosomal\nelectrophysiology. When the pipette tips were separated from the lysosomal membranes\nthrough slightly positive pressure or moving the pipette tips, the current dramatically\nincreased and the lysosomes sometimes ruptured. We speculate that lysosomal\nmembranes are more fragile than cell membrane, therefore incomplete seal could not\nexist stably either during the gigaseal formation nor during the whole-lysosomal mode.\nThus our results were not likely to be the leakage currents.\n7. In Figure 6g, there was no significant difference for the leakage of cathepsin B\nbetween WT and Tmem175 KO groups. Statistical analysis was missing. What was\nthe rationale that only CTSB, but not other cathepsins, e.g., CTSD or CTSL, was\ninvestigated? Previous studies suggested that CTSL might be more relevant for\nlysosome membrane permeability.\nResponse: Thank you for your question. It is true that CTSL is more relevant for\nlysosome membrane permeability, however CTSB is more wildly studied for the\ninflammasome activation (PMID: 18604214, PMID: 18604209, PMID: 31611559).\nThus we focused on CTSB as an indicator of lysosomal membrane permeabilization\nand activator of NLRP3 signal. Nevertheless, we added the immunofluorescence results\nof CTSD and CTSL for more sufficient results. We also added the statistical analysis.\nIt is true that we did not observe the difference for the leakage of cathepsins between\nWT and Tmem175-/- groups through immunofluorescence. We suspect that the\ndifferences were made in the down-stream event of lysosomal membrane\npermeabilization, which promoted the activation of NLRP3 through lysosomal calcium\nefflux.\nFig. 7g Tmem175-/- and WT BMDMs treated with LPS and B16-F10 cell debris were\nfixed and permeabilized. Then cells were stained by cathepsin B rabbit mAb, cathepsin\nD rabbit mAb, or cathepsin L rabbit mAb. The spots of cathepsin B, cathepsin D, and\ncathepsin L diffused in cytoplasm by cell debris treatment. Scale bars indicate 10 .\nμm\nFig. S11(d to f) Bright fluorescent areas of CTSB, CTSD, and CTSL in Fig. 7g were\ncalculated (n = 3).\n8. The levels of reactive oxygen species (ROS) were high in Tmem175-/- BMDMs.\nDo ROS contribute to the observed phenotypes?\nResponse: Thank you for your question. We used acetylcysteine (NAC) to inhibit ROS\nin our inflammasome stimulation experiments. NAC inhibited the ASC polymerization\nand IL-1 secretion. Thus ROS contributed the observed phenotypes.\nβ\nFig. S12(b to d) Tmem175-/- and WT BMDMs were stimulated with 500 ng/ml LPS for\n4 hours to activate signal 1 of NLRP3 inflammasomes, then treated with B16-F10 cell\ndebris (2.5 105/ml) to activate NLRP3 inflammasomes. For ROS inhibition, cells were\n×\nincubated with 10 mM NAC during the cell debris treatment. For Bafilomycin A1\ntreatment, cells were incubated with 1 M Bafilomycin A1 during the cell debris\nμ\ntreatment.\nMinor Concerns:\n1. What does the time point \"0\" represent in Figure 7a? The Y-axis indicates\nlysosomal pH, but it is unclear on how lysosomal pH was calibrated in the study.\nResponse: Thank you for your question. The time point “0” represent the beginning of\nB16-F10 cell debris treatment. We have added the clarification in our revised figure\nlegend. For the calibration of pH, we prepared a series of solution with known pH at 3,\n4, 5, 6, and 7. Then the solution and LysoSensor™ Yellow/Blue DND-160 were added\ninto 96-well plates. Two fluorescence intensity, 329 nm excitation/440 nm emission and\n384 nm excitation/540 nm emission, were collected and the ratio of 440 nm/540 nm\nwere used for creating a standard curve. We also collected the 440 nm/540 nm from\ncells in different groups. The values of lysosomal pH from cells in different groups were\ncalculated referring to the standard curve. We modified our method description in our\nrevised manuscript.\n2. In Figure 9, are you sure about the direction of K+ flow across lysosomal\nmembranes? Cytosolic K+ concentration is known to be as high as 150 mM.\nResponse: Thank you for your question. The K+ flow is not necessarily to be outward,\nwe checked the related published works and changed the illustration in our revised\nmanuscript.\n[Figure Redacted]\n(Figure 6 from Patch-clamp technique to characterize ion channels in enlarged\nindividual endolysosomes, PMID: 28726848)\nFig. 10 Mechanisms of the elevated anti-tumor immunity in TMEM175 knockout mice.\nTumor cell debris engulfed by BMDMs caused lysosomal membrane permeabilization\nand cathepsin B leakage, which activated NLRP3 inflammasome. TMEM175 knockout\npromoted lysosomal calcium efflux into cytoplasm thus promoting NLRP3\ninflammasome activation and IL-1 /IL-18 secretion. IL-1 and IL-18 facilitated the\nβ β\nrecruitment and activation of T cells and NKs. The augmented antigen cross-\npresentation in Tmem175-/- BMDMs enhanced CD8+ T cell expansion and activation,\nwhich was further promoted by IL-1 and IL-18.\nβ\n3. In Figure 1e, why did the scale of the ratio start from 0.2?\nResponse: Thank you for your comment. We have changed the scale and we are sorry\nfor the mistake.\nFig. 1(d and e) TMEM175 expression was obviously decreased in Tmem175-/- BMDMs.\nLysates of Tmem175-/- and WT BMDMs were analyzed by western blot using anti-\nTMEM175 antibody. Ratios of the grayscale values of TMEM175 and -actin were\nβ\ncalculated.\n4. There was lack of statistical analysis in Figure 7c.\nResponse: Thank you for your comment. We have added the statistical analysis in not\nonly this figure but also other similar figures including the determination of lysosomal\npH and lysosomal calcium concentrations.\nFig. 8(a to c) For dynamic observation of lysosomal pH, lysosomal calcium, and\ncytosolic calcium, the cells were pre-treated with desired fluorescent probes and the\nfluorescence was collected by fluorescent reader.\nReviewer #2\nMajor Concerns:\n1. Clinical Relevance: The study proposes TMEM175 as an intriguing target for\ntumour immunotherapy. However, it remains unclear whether TMEM175 is\ndifferentially expressed between normal and cancerous tissues. Additionally, does\nTMEM175 deficiency influence the polarisation of tissue-resident macrophages?\nResponse: Thank you for your question. We detected the TMEM175 expression in\nhuman melanoma and paracancerous tissues through IHC. The results showed that\nTMEM175 was expressed more robustly in melanoma tissues. Tissue-resident\nmacrophages are a group of macrophages distinct from the recruited monocyte-derived\nmacrophages. According to Tomi Lazarov et al. (PMID: 37344646), part of the alveolar\nmacrophages are tissue-resident macrophages. We found that Tmem175-/- alveolar\nmacrophages expressed higher MHC-II and lower CD206 than WT cells in B16-F10\nlung metastatic models. We additionally tested the polarization of Kupffer cells, which\nare resident macrophages in the livers. Tmem175-/- Kupffer cells showed higher MHC-\nII expression. It seemed that although the polarization pattern might differ, TMEM175\ndeficiency promoted M1-like polarization in tissue-resident macrophages.\nFig. 1a Tissue chip of paired melanoma and paracancerous tissues was stained by anti-\nTMEM175 to detect the expression of TMEM175 through IHC (n = 6 pairs).\nTMEM175 was expressed more robustly in melanoma tissues than paracancerous\ntissues. Scale bars indicate 500 for left panel and 50 for right panel.\nμm μm\nTMEM175 positive area was calculated.\nFig. 3(d and e) MHC-IIhigh AMs (gated from CD45+ CD11c+ CD11b- CD64+ cells) were\nincreased in Tmem175-/- mice. CD206high AMs (gated from CD45+ CD11c+ CD11b-\nCD64+ cells) were decreased in Tmem175-/- mice.\nExpressions of MHC-II and CD206 in kupffer cells (gated from CD45+ F4/80+ CD11b-\nLy6G+ cells) were tested by FCM.\n2. In Figures 2 and 3, TMEM175 deficiency demonstrates anti-tumour effects by\nimpeding tumour growth and metastasis, with concurrent alterations in tumour-\nassociated macrophage (TAM) composition and T-cell infiltration. These findings,\nhowever, are predominantly observational. A mechanistic elucidation is necessary\nto substantiate whether, and through what pathways, TMEM175 deficiency\nattenuates the immunosuppressive tumour microenvironment (TME). Does\nTMEM175 directly modulate TAM polarisation? What are the underlying\nmolecular mechanisms? Moreover, the functional contributions of distinct\nimmune cell populations to the observed anti-tumour effects remain ambiguous.\nDepletion of TAMs and T-cells would substantiate these findings.\nResponse: Thank you for your comment. In order to investigate the mechanisms of\nmacrophage polarization in Tmem175-/- mice, we applied single-cell sequencing to\nanalysis the tumor microenvironment in B16-F10 lung metastatic models. Both AMs\nand IMs showed up-regulated antigen presentation pathway, which included several\ngenes coding histocompatibility molecules such as H2-D1, H2-M3, and H2-K1. And\nthis result was in accordance with our FCM results. In AMs, we found some up-\nregulated genes that have been reported to promote M1-like polarization, such as Tlr2,\nIrf7, Nlrp3, Malt1, Map3k1, and Nod2. These genes were enriched in pro-inflammatory\npathways such as NOD-like receptor signaling pathway (mmu04621), NF-kappa B\nsignaling pathway (mmu04064), and MAPK signaling pathway (mmu04010). These\ngenes were also enriched in GO terms that facilitate immune process and inflammatory\nprocess, such as immune response (GO:0006955), antigen processing and presentation\nof peptide antigen via MHC class I (GO:0002474), and inflammatory response\n(GO:0006954). In IMs, we also found some up-regulated genes that have been reported\nto promote M1-like polarization, such as Gbp2b, Irf7, Irf9, Gbp4, and Tlr9. These genes\nwere enriched in pro-inflammatory pathways such as NOD-like receptor signaling\npathway (mmu04621), Toll-like receptor signaling pathway (mmu04620), and NF-\nkappa B signaling pathway (mmu04064). These genes were also enriched in GO terms\nthat facilitate immune process and inflammatory process, such as immune system\nprocess (GO:0002376), antigen processing and presentation (GO:0002474), positive\nregulation of interleukin-1 beta production (GO:0032731), positive regulation of NF-\nkappaB transcription factor activity (GO:0051092). In summary, TMEM175 knockout\npromoted the antigen presentation and inflammatory process resulting in the M1-like\npolarization of TAMs, which could involve several different pathways including\ninflammasome, NF-kappa B, and TLRs.\nAdditionally, we confirmed that IL-1 modulated the M1-like polarization of\nβ\nmacrophages in vitro. Thus the mechanism of inflammasome-mediated M1-like\npolarization in Tmem175-/- mice was investigated by single cell transcriptome analysis\nand in vitro experiment.\nWe used clodronate liposomes to delete macrophages and anti-CD8 antibody to delete\nCD8+ T cells to substantiate their roles in the anti-tumor immunity in Tmem175-/- mice.\nClodronate liposomes and anti-CD8 antibody abrogated the differences of tumor\nweights between WT and Tmem175-/- mice. Furthermore, clodronate liposomes\ninhibited the T cell immunity in Tmem175-/- mice. Thus macrophage deletion smoothed\nthe differences of immune microenvironment between WT and Tmem175-/- mice.\nFigure S5. related to Figure 3. TMEM175 Conditional Knockout Suppressed B16-F10\nLung Metastasis through Activating Anti-tumor Immunity.\nThe lungs of B16-F10 metastatic models were analyzed by single cell sequencing.\nDifferentally expressed genes (Tmem175-/- vs WT) in AMs and IMs were identified and\nanalyzed by GO and KEGG.\n(a and b) Volcano plots of differentially expressed genes in AMs (a) and IMs (b).\n(c and d) Up-regulated GO terms in AMs (c) and IMs (d).\n(e and f) Up-regulated KEGG pathways in AMs (e) and IMs (f).\nFig. 4s Primary Tmem175-/- and WT BMDMs were treated with IL-1 (500 pg/ml) for\n48 hours. Then the MHC-II expression in CD45+ CD11b+ F4/80+ cells β were determined\nthrough FCM (n = 3).\nFigure S3. related to Figure 2. The anti-tumor immunity in Tmem175-/- mice relied on\nmacrophages and CD8+ T cells.\n(a to k) Tmem175-/- and WT mice (n = 5 mice) were treated with 1 mg/20 g body weight\nclodronate liposomes or control liposomes intravenously one day before the\nsubcutaneous implantation with B16-F10. Then same doses of liposomes\n6\n1×10\nwere injected intravenously on day 6 and 13. Mice were humanely sacrificed on day 14\nto weigh the tumors and analyze the TME through FCM.\n(b) Tumor weights in Tmem175-/- mice were elevated by clodronate liposomes.\n(c) CD11b+ F4/80+ TAMs (gated from CD45+ cells) in B16-F10 tumors were\nsignificantly diminished by clodronate liposomes.\n(d to g) Recruitment, activation, IFN- expression and granzyme B expression in CD4+\nT cells (gated from CD3+ cells) in T γ mem175-/- mice were suppressed by clodronate\nliposomes.\n(h to k) Recruitment, activation, IFN- expression and granzyme B expression in CD8+\nT cells (gated from CD3+ cells) in T γ mem175-/- mice were suppressed by clodronate\nliposomes.\n(l to n) Tmem175-/- and WT mice (n = 5 mice) were treated with 100 /100 anti-\nμg μl\nCD8a or rat IgG2b isotype intraperitoneally one day before the subcutaneous\nimplantation with B16-F10. Then same doses of liposomes were injected\n6\n1×10\nintraperitoneally on day 6 and 13. Mice were humanely sacrificed on day 14 to weigh\nthe tumors and analyze the TME through FCM.\n(m) Tumor weights in Tmem175-/- mice were elevated by anti-CD8a.\n(n) CD8+ T cells (gated from CD3+ cells) in B16-F10 tumors were significantly\ndiminished by anti-CD8a.\nRepresentative results from two independent experiments are presented as mean ± SEM.\nStatistical significances in (b-k and m-n) were determined by two-way ANOVA.\n*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 NS, not significant.\n3. An enigmatic observation lies in the insensitivity of B16-F10 tumours to\ninflammasome inhibition (via VX-765) juxtaposed with their heightened\nsensitivity to inflammasome stimulation (through chitosan), as illustrated in\nFigure 4.\nResponse: Thank you for your comment. We speculate that inflammasome signal was\nseldom activated in B16-F10 tumors from WT mice. It is reasonable that the untreated\nstate with little inflammasome signal showed insensitivity to inflammasome inhibition\nand sensitivity to inflammasome stimulation. To confirm our hypothesis, we used IHC\nto investigate the markers of inflammasome signal in B16-F10 tumors from WT and\nTmem175-/- mice. We found that WT B16-F10 tumors showed significantly lower\ncaspase-1 p10 and IL-1 expression level. Thus it could explain why WT B16-F10\nβ\ntumors were not sensitive to VX-765, because the signal was too low at untreated state.\nFig. 4(a to c) B16-F10 tumors from Fig. 2 were fixed, sliced, and stained by anti-\ncaspase-1 p10 (a) or anti- IL-1 (b) to detect the inflammasome activation through IHC.\nEnhanced inflammasome activ β ation was observed in tissues from Tmem175-/- mice (c).\nScale bars indicate 50 μm.\n4. The antigen cross-presentation data presented in Figure 5 are perplexing.\nTmem175-/- bone marrow-derived macrophages (BMDMs) markedly induce\nproliferation of OVA-specific CD8+ T cells, even in the absence of B16-F10 OVA\ncell debris. The addition of B16-F10 OVA debris results in only a minimal further\nincrease in T cell proliferation. This suggests that the observed T cell proliferation\nand activation may occur via mechanisms beyond antigen cross-presentation.\nResponse: Thank you for your comment. We used RNA-seq to investigate the\nunderlying mechanisms. We found that in the absence of B16-F10-OVA cell debris,\nTmem175-/- group up-regulated molecules (such as Tlr13, Tlr8, Malt1, Irf4, Irf5, Trim29,\nPcbp2, Cd9, Sema4a) that have been reported to promote T cell proliferation and\nactivation. And these genes were enriched in innate immune response (GO:0045087),\nimmune response (GO:0006955 adaptive immune response (GO:0002250) , and\ninflammatory response (GO:0006954). Besides antigen specific T cell proliferation and\nactivation, the non-specific T cell proliferation and activation through theses\ninflammation signal might exist in Tmem175-/- group. We speculate that Tmem175-/-\nBMDMs demonstrated pro-inflammatory phenotypes thus promoting non-specific T\ncell proliferation and activation. In fact, the inflammatory signals have been reported\nto promote T cell proliferation (PMID: 31405895, PMID: 35309296, and PMID:\n34772757). In B16-F10-OVA debris treated Tmem175-/- group, cell cycle\n(GO:0007049), cell division (GO:0051301), and mitotic spindle organization\n(GO:0007052) were up-regulated, which might be attributed to the more robust antigen\nspecific T cell proliferation induced by Tmem175-/- BMDMs.\nFig. 6(k and l) Up-regulated GO terms by TMEM175 knockout without (k) or with (l)\nB16-F10-OVA cell debris treatment.\nMinor Points:\n1. In Figure 1d, there remains a residual band, albeit weaker than wild-type, in\nBMDMs from Tmem175-/- mice. The authors attribute this to residual expression\nfrom exons 1-4; however, the band appears at the same molecular weight as full-\nlength TMEM175. Additional validation is warranted to confirm effective\nknockout of Tmem175 in myeloid cells .\nResponse: Thank you for your comment. The antibody used in our previous experiment\nwas a polyclone antibody (Proteintech, 19925-1-AP). To improve the accuracy, we\nreplicated the western blot using a monoclonal antibody (Proteintech, 68172-1-Ig).\nMore accurate result demonstrated higher KO efficiency, and we found the weak size-\nshifted band (40-55 kD). We speculate that the remaining weak 55-70KD band in\nTmem175-/- group might be owed to the inevitable inclusion of cells that did not express\nLyz2 genes during the primary cell culture.\nTMEM175 expression was obviously decreased in Tmem175-/- BMDMs. Lysates of\nTmem175-/- and WT BMDMs were analyzed by western blot using anti-TMEM175\nantibody.\n2. Vehicle controls are conspicuously absent in Figure 4j.\nResponse: Thank you for your comment. The vehicle controls have been added in our\nrevised manuscript.\nFig. 4(q and r) Primary Tmem175-/- and WT BMDMs or PMs were stimulated with 500\nng/ml LPS for 4 hours to activate signal 1 of NLRP3 inflammasomes, and then treated\nwith 40 g/ml chitosan for 5 hours. Tmem175-/- BMDMs and PMs secreted more IL-\nμ\n1 in response to chitosan (n = 5).\nβ\nReviewer #3\nMajor Concerns:\n1. The authors indicate that increased IL-1β and IL-18 secreted from TMEM175\nKO macrophages drive the recruitment and activation of T cells and NK cells in\nTME. To support their conclusion, the anti-tumor immunity in vivo should be\nevaluated using neutralizing antibodies or ways to perturb this cytokine signaling.\nResponse: Thank you for your comment. We applied anti-IL-1 and anti-IL-18\nβ\nantibody i.p. to inhibit IL-1 and IL-18. The results demonstrated that anti-IL-1 and\nβ β\nanti-IL-18 inhibited M1-like polarization, and T & NK cells recruitment and activation\nin Tmem175-/- mice, while the effects were less obvious in WT mice. The low tumor\nweights in Tmem175-/- mice were elevated by anti-IL-1 and anti-IL-18.\nβ\nThus the\nincreased IL-1β and IL-18 production is the key step of the anti-tumor immunity in\nTmem175-/- mice.\nFig. 5 The anti-tumor immunity in Tmem175-/- mice could be inhibited by anti-IL-1\nβ\nand anti-IL-18.\n(a to l) Tmem175-/- and WT mice (n = 5 mice) were treated with 100 /100 anti-\nμg μl\nIL-1 or Armenian hamster IgG isotype intraperitoneally one day before the\nβ\nsubcutaneous implantation with B16-F10. Then same doses of IgG were\n6\n1×10\ninjected intraperitoneally on day 6 and 13. Mice were humanely sacrificed on day 14 to\nweigh the tumors and analyze the TME through FCM.\n(b) The anti-tumor effects of TMEM175 knockout was suppressed by anti-IL-1 .\n(c) Expression of MHC-II in TAMs (gated from CD45+ CD11b+ F4/80+ cell β s) was\nsignificantly attenuated by anti-IL-1 in Tmem175-/- mice.\n(d) Expression of CD206 in TAMs ( β gated from CD45+ CD11b+ F4/80+ cells) was not\nobviously altered by anti-IL-1 in Tmem175-/- mice.\nβ\n(e to h) Recruitment, activation, IFN- expression, and granzyme B expression in\nCD4+ T cells (gated from CD3+ cells) w γ ere significantly diminished by anti-IL-1 in\nTmem175-/- mice. β\n(i to l) Recruitment, activation, IFN- expression, and granzyme B expression in CD8+\nT cells (gated from CD3+ cells) w γ ere significantly diminished by anti-IL-1 in\nTmem175-/- mice. β\n(m to x) Tmem175-/- and WT mice (n = 5 mice) were treated with 100 /100 anti-\nμg μl\nIL-18 or rat IgG2a isotype intraperitoneally one day before the subcutaneous\nimplantation with B16-F10. Then same doses of IgG were injected\n6\n1×10\nintraperitoneally on day 6 and 13. Mice were humanely sacrificed on day 14 to weigh\nthe tumors and analyze the TME through FCM.\n(n) The anti-tumor effects of TMEM175 knockout was suppressed by anti-IL-18.\n(o) Expression of MHC-II in TAMs (gated from CD45+ CD11b+ F4/80+ cells) was\nsignificantly attenuated by anti-IL-18 in Tmem175-/- mice.\n(p) Expression of CD206 in TAMs (gated from CD45+ CD11b+ F4/80+ cells) was not\nobviously altered by anti-IL-18 in Tmem175-/- mice.\n(q to t) Recruitment, activation, IFN- expression, and granzyme B expression in CD4+\nT cells (gated from CD3+ cells) γ were significantly diminished by anti-IL-18 in\nTmem175-/- mice. Activated CD4+ T cells (gated from CD3+ cells) were significantly\ndiminished by anti-IL-18 in WT mice.\n(i to l) Recruitment, activation, IFN- expression, and granzyme B expression in CD8+\nT cells (gated from CD3+ cells) γ were significantly diminished by anti-IL-18 in\nTmem175-/- mice. Recruitment, activation, and granzyme B expression in CD8+ T cells\n(gated from CD3+ cells) were significantly diminished by anti-IL-18 in WT mice.\nRepresentative results from two independent experiments are presented as mean ± SEM.\nStatistical significances in (b-l and n-x) were determined by two-way ANOVA.\n*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS, not significant.\nFigure S8. related to Figure 5. The anti-tumor immunity in Tmem175-/- mice could be\ninhibited by anti-IL-1 and anti-IL-18.\nβ\nTmem175-/- and WT mice (n = 5 mice) were treated with 100 /100 anti-IL-\nμg μl\n1 /Armenian hamster IgG isotype or anti-IL-18/IgG2a isotype intraperitoneally one\nβ\nday before the subcutaneous implantation with B16-F10. Then same doses of\n6\n1×10\nIgG were injected intraperitoneally on day 6 and 13. Mice were humanely sacrificed on\nday 14 to weigh the tumors and analyze the TME through FCM.\n(a to d) Recruitment, activation, IFN- expression, and granzyme B expression in NKs\n(gated from CD3- cells) were signif γ icantly diminished by anti-IL-1 in Tmem175-/-\nmice. NKs recruitment (gated from CD3+ cells) was significantly dim β inished by anti-\nIL-1 in WT mice.\nβ\n(e to h) Recruitment, activation, IFN- expression, and granzyme B expression in NKs\n(gated from CD3- cells) were significa γ ntly diminished by anti-IL-18 in Tmem175-/- mice.\nNKs recruitment and activation (gated from CD3+ cells) were significantly diminished\nby anti-IL-18 in WT mice.\nRepresentative results from two independent experiments are presented as mean ± SEM.\nStatistical significances in (a-h) were determined by two-way ANOVA. *P<0.05,\n**P<0.01, ***P<0.001, NS, not significant.\n2. The authors raise three hypotheses about how TMEM175 deficiency leads to\nincreased inflammasome activation: increased cathepsin B leakage, higher ROS\nlevels, and elevated cytosolic calcium. Although all three mechanisms may result\nfrom TMEM175-induced lysosomal dysfunction, the effects of inhibitors (CA-074,\nBAPTA-AM, etc) for these pathways on blocking inflammasome activation should\nbe evaluated to identify whether there’s a dominant pathway responsible for the\npro-inflammatory phenotype in TMEM175 KO macrophages. Moreover, the\ndominant inhibitor should be tested in vivo for the effect of pathways on anti-\ntumor immunity and tumor growth. VX-765 is only a general inhibitor of\ncaspase-1. For the precise machinery of anti-tumor immunity, siRNA-mediated\ngene silencing in macrophages should be conducted to verify the pharmacological\ninhibition data, and the anti-tumor effect of macrophage conditional depletion of\nthe predominant pathway should be evaluated in vivo. In this way, the manuscript\nbecomes more focused on a predominant pathways as the key mechanism\nunderlying their phenotype. By evaluating all types of pathways in superficial\nways, it leaves more to be desired and confuses the reader.\nResponse: Thank you for your comment. Intratumoral injection of CA-074 and\nBAPTA-AM inhibited the anti-tumor immunity in Tmem175-/- mice. Both CA-074 and\nBAPTA-AM significantly inhibited the anti-tumor immunity. However, the inhibitory\neffects were more obvious in BAPTA-AM group than that in CA-074 group. Thus the\nelevated cytosolic calcium seemed to be the dominant pathway responsible for the pro-\ninflammatory phenotype in Tmem175-/- macrophages.\nFig. 8(g to r) Tmem175-/- and WT mice (n = 5 mice) were subcutaneously implanted\nwith B16-F10. On day 7, 10, and 13, CA-074 (200 /20 g body weight) or\n6\n1×10 μg\nBAPTA-AM (200 /20 g body weight) or vehicle were intratumorally injected into\nμg\nB16-F10 tumors. Mice were humanely sacrificed on day 14 to weigh the tumors and\nanalyze the TME through FCM.\n(h) CA-074 and BAPTA-AM suppressed the anti-tumor effects of TMEM175 knockout.\n(i) CA-074 and BAPTA-AM decreased MHC-II+ TAMs (gated from CD45+ CD11b+\nF4/80+ cells) in Tmem175-/- mice. BAPTA-AM decreased MHC-II+ TAMs (gated from\nCD45+ CD11b+ F4/80+ cells) in WT mice.\n(j) CA-074 and BAPTA-AM increased CD206+ TAMs (gated from CD45+ CD11b+\nF4/80+ cells) in Tmem175-/- mice.\n(k and l) CA-074 and BAPTA-AM suppressed the recruitment and activation of CD4+\nT cells (gated from CD3+ cells) in Tmem175-/- mice. BAPTA-AM suppressed the\nrecruitment of CD4+ T cells (gated from CD3+ cells) in WT mice.\n(m and n) BAPTA-AM was more efficient than CA-074 in inhibiting IFN- and\ngranzyme B expression in CD4+ T cells (gated from CD3+ cells) in Tmem175-/- m γ ice.\n(o and r) CA-074 and BAPTA-AM suppressed the recruitment, activation, IFN-\nexpression, and granzyme B expression of CD8+ T cells (gated from CD3+ cells) i γ n\nTmem175-/- mice.\nRepresentative results from two independent experiments are presented as mean ± SEM.\nStatistical significances in (a-d and h-r) were determined by two-way ANOVA.\n*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS, not significant.\nAdditionally, ROS inhibitor, acetylcysteine (NAC), inhibited the ASC\npolymerization and IL-1β secretion. Thus ROS contributed the observed phenotypes.\nFig. S12(b to d) Tmem175-/- and WT BMDMs were stimulated with 500 ng/ml LPS for\n4 hours to activate signal 1 of NLRP3 inflammasomes, then treated with B16-F10 cell\ndebris (2.5 105/ml) to activate NLRP3 inflammasomes. For ROS inhibition, cells were\n×\nincubated with 10 mM NAC during the cell debris treatment. For Bafilomycin A1\ntreatment, cells were incubated with 1 M Bafilomycin A1 during the cell debris\nμ\ntreatment.\nTo confirm the role of NLRP3 in Tmem175-/- mice, we transfected siRNA to WT or\nTmem175-/- BMDMs and adopted the cells to WT B16-F10 tumor bearing mice.\nSilencing Nlrp3 in Tmem175-/- BMDMs abrogated the inhibitory effects in tumor\ngrowth. Thus NLRP3 is crucial for the present mechanism.\nFig. 7i In vitro cultured WT and Tmem175-/- BMDMs were transfected with siRNA\ntargeting Nlrp3 or control siRNA. Then the BMDMs were adopted to wild type mice\none day before and day 7th after B16-F10 implantation. Tumor bearing mice were\nhumanely sacrificed on day 14th after B16-F10 implantation to weigh the tumors.\n3. Authors also demonstrated TMEM175 deficiency augmented the cross-presentation\nof tumor antigens. The in vitro work is nice but without evaluation of the effect of\nthis pathway on anti-tumor immunity and tumor growth weakens their conclusion.\nIn addition, authors mentioned the elevated cross-presentation signal in TMEM175\nKO macrophages can be enhanced by IL-1β and IL-18 but didn’t provide any\nexplanation. Does it mean those cytokines promote phagocytosis, or the\nantigen-presentation relies on inflammasome activation? The utilization of siRNA\nmay help answer these questions.\nResponse: Thank you for your comment. We designed an adoptive experiment to\nelaborate the in vivo effects of the enhanced cross-presentation in Tmem175-/- BMDMs.\nCD8+ spleen T cells from B16-F10-OVA tumor bearing mice were co-cultured with WT\nor Tmem175-/- BMDMs with B16-F10-OVA cell debris for 48 hours. The CD8+ T cells\nwere injected i.v. into Cd8a-/- mice. Then these mice were challenged with B16-F10-\nOVA cells subcutaneously. Within the period of tumor growth, one additional T cell\nadoption was applied. The results proved the anti-tumor immunity and tumor growth in\nvivo.\nFig. 6(n and o) CD8+ T cells in the co-culture system were harvested and adopted to\nCd8a-/- mice one day before and day 7th after B16-F10-OVA implantation. Tumor\nbearing mice were humanely sacrificed on day 14th after B16-F10-OVA implantation to\nweigh the tumors.\nConcerning the down-stream events of IL-1 and IL-18 is a good question. It has\nβ\nbeen reported that cross-presentation could be facilitated by IL-1 (PMID: 33963060\nβ\nand PMID: 38053332) and IL-18 (PMID: 36516765). T cell expansion and activation\ncould be induced by IL-1 (PMID: 24092469, PMID: 31405895, and PMID:\nβ\n34108686) and IL-18 (PMID: 34108686, PMID: 32581358, and PMID:\n36933554),\nwhich coincided with our results. Generally, the receptors of IL-1 and IL-18 elicited\nβ\nMyD88/NF-kappaB signal whose down-stream events are production of pro-\ninflammatory molecules (PMID: 32770571). In order to investigate the underlying\nmechanisms, we used RNA-seq to compare the difference of transcriptome between\nWT and Tmem175-/- group. We attempted to find the common differently expressed\ngenes (DEGs) or pathways between WT and Tmem175-/- groups regulated by IL-1 /IL-\n18 (WT+B16-F10-OVA+IL-1 /IL-18 VS WT+B16-F10-OVA) and (Tmem175-/-+ β B16-\nF10-OVA+IL-1 /IL-18 VS Tm β em175-/-+B16-F10-OVA).\nβ\nFor IL-1 , we noticed the common up-regulated GO terms including cellular\nβ\nresponse to type II interferon (GO:0071346), which could explain the promoted IFN-\nγ\nsecretion in our results, and interleukin-1 beta production (GO:0032611), which might\nindicate a positive feedback of IL-1 secretion. Consistent with previously review\nβ\n(PMID: 32770571) that the down-stream signal of IL-1 and IL-18 is NF-kappaB,\nβ\npositive regulation of canonical NF-kappaB signal transduction (GO:0043123) was\nenriched in WT+B16-F10-OVA+IL-1 VS WT+B16-F10-OVA comparison while\nβ\nregulation of canonical NF-kappaB signal transduction (GO:0043122) was enriched in\nTmem175-/-+B16-F10-OVA+IL-1 VS Tmem175-/-+B16-F10-OVA comparison. The\nβ\nNF-kappaB signal is necessary for the survival and maturation of T cells (PMID:\n24352326). We found 7 up-regulated DEGs (Cd164, Il1b, Gbp8, Iigp1, Csf2rb, Gbp4,\nand Gbp5) and 3 down-regulated DEGs (Bcl6, Fubp1, and Atf4) to be the common\nDEGs between WT and Tmem175-/- groups regulated by IL-1 . Gbp4, Gbp5, and Gbp8\nβ\nwere related with cellular response to type II interferon (GO:0071346). Bcl6 and Atf4\nwere related with positive regulation of apoptotic process (GO:0043065) (not\nsignificant in WT). Thus after IL-1 treatment, the NF-kappaB signal, positive\nβ\nfeedback of IL-1 secretion, down-stream IFN- secretion and possible down-\nβ γ\nregulated apoptosis were responsible for the enhanced cross-presentation.\nFor IL-18, we noticed that response to type I interferon (GO:0034340) and non-\ncanonical NF-kappaB signal transduction (GO:0038061) were the up-regulated GO\nterms in WT+B16-F10-OVA+ IL-18 VS WT+B16-F10-OVA comparison, while\nregulation of inflammatory response (GO:0050727) and immune system process\n(GO:0002376) were the up-regulated GO terms in Tmem175-/-+B16-F10-OVA+ IL-18\nVS Tmem175-/-+B16-F10-OVA comparison. Although we did not found the common\nGO terms, these terms were pro-inflammatory and pro-immunity in general. In KEGG\nanalysis, the common up-regulated pathways were IL-17 signaling pathway\n(Path:04657) and Toll-like receptor signaling pathway (Path:04620). The cross-talk\nbetween IL-18 and IL-17 signal has been summarized (PMID: 24332029 and PMID:\n36211331). IL-17 is a pro-inflammatory cytokine in general. Interestingly, TLR signal\nis commonly regarded as an up-stream event of IL-18 production (PMID: 38081945),\nbut our results indicated a potential feedback loop between TLRs and IL-18.\nIn summary, the promoted antigen cross-presentation by Tmem175-/- BMDMs was\nvalidated in vivo. IL-1 stimulated several common genes between WT and Tmem175-\n/- group that were enric β hed in several different pathways (NF-kappaB, type II interferon,\npositive feedback of IL-1 , and down-regulated apoptosis) resulting in the promoted T\nβ\ncell proliferation and activation during the antigen cross-presentation. IL-18 did not\nstimulated common gene between WT and Tmem175-/- group, however the DEGs\nelicited by IL-18 enriched in IL-17 and TLR signals resulting in pro-inflammatory\nphenotypes. Further investigations of the down-stream events of IL-1 and IL-18 in\nβ\nthe cross-presentation mode are meaningful.\nFig. 6m Common up-regulated and down-regulated genes between WT and Tmem175-\n/- BMDMs altered by IL-1 and IL-18.\nβ\nFigure S9. related to Figure 6. TMEM175 knockout augmented the cross-presentation\nof tumor antigens.\n(a to d) Differently expressed genes modulated by IL-1 and IL-18 were analyzed by\nβ\nKEGG enrichment.\n(a) Up-regulated KEGG pathways by IL-1 treatment in WT group.\n(b) Up-regulated KEGG pathways by IL-1 β treatment in Tmem175-/- group.\nβ\n(c) Up-regulated KEGG pathways by IL-18 treatment in WT group.\n(d) Up-regulated KEGG pathways by IL-18 treatment in Tmem175-/- group.\nFigure S10. related to Figure 6. TMEM175 knockout augmented the cross-presentation\nof tumor antigens.\n(a to d) Differently expressed genes modulated by IL-1 and IL-18 were analyzed by\nβ\nGO enrichment.\n(a) Up-regulated GO terms by IL-1 treatment in WT group.\n(b) Up-regulated GO terms by IL-1 β treatment in Tmem175-/- group.\nβ\n(c) Up-regulated GO terms by IL-18 treatment in WT group.\n(d) Up-regulated GO terms by IL-18 treatment in Tmem175-/- group.\n4. The data about autophagy dysfunction and inflammasome activation is very\nsuperficial and lacks the evaluation of IL-1β secretion, so can be removed from the\nrevised manuscript and only mentioned in the discussion . Again, by doing\nsuperficial work on multiple pathways as a mechanism is not focused.\nResponse: Thank you for your comment. These data have been removed. We will\nlaunch additional in-depth work and publish this data in the future.\n5. The study on PYD-PYD interaction (Fig. 7f-k) seems redundant and not directly\nlinked to their main findings.\nResponse: Thank you for your comment. These data are just a supplementary evidence\nfor the role of calcium ions in ASC-NLRP3 interaction. Since additional in vivo data\nshould be exhibited in this figure and the dynamics simulation data are not that\nimportant, these data are now exhibited in the supplementary figure. 13.\nMinor points:\n1. Similarly, the Discussion Section is too long and includes lots of review about\nTMEM175 and lysosomal dysfunction. Based on their findings, authors should\nfocus on how TMEM175 expression is regulated in macrophages during tumor\nprogression and what area therapeutic strategies can be developed.\nResponse: Thank you for your comment. We have removed the redundant review in our\nDiscussion Section. We focused on the role of TMEM175 in the tumor immune\nsurveillance.\n2. The description of generating conditional TMEM175 mice can be moved to the\nMethod Section or Supplemental figure legends and not main figures\nResponse: Thank you for your comment. We have moved the description of generating\nconditional TMEM175 mice to Supplemental figure and Method Section.\nAll the point-by-point responses are presented above. We are willing to make further\nrevision to improve the manuscript. We look forward to your decision.\nYours sincerely,\nXiawei Wei, Ph. D\nFull Professor and Director of Lab of Cancer Drug Target and Aging Research\nState Key Laboratory of Biotherapy,\nWest China Hospital, Sichuan University\nEmail: xiaweiwei@scu.edu.cn;\nAddress：No. 17, Block 3, Southern Renmin Road, Chengdu, Sichuan 610041,\nPeople’s Republic of China"}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revised manuscript, the authors have performed several new experiments to address the reviewers' concerns, and the revised manuscript is improved. The roles of TMEM175 in anti-tumor immunity via inflammasome activation are convincingly established. However, the proposed mechanisms that link the channel properties of lysosomal TMEM175 with Ca²⁺ dys-homeostasis and oxidative stress are still not clear. In the new working model in Figure 10, the authors now claim that there is no lysosomal K⁺ defect associated with TMEM175 deficiency. Instead, they attribute the abnormalities in Ca²⁺ and ROS levels to a lysosomal pH defect (hypo-acidification). It should be noted that the basal lysosomal pH was reported to be normal in early TMEM175 studies (PMID 26317472) but decreased (hyper-acidification) in more recent studies (e.g., PMID: 35750034, 35333573). However, the new data in the revision showed that the basal lysosomal pH was elevated (see Figure 8), which contradicts all previous studies. Additionally, lysosomal pH in macrophages was reported to be more acidic upon M-CSF treatment (PMID: 17314396). These discrepancies must be addressed if the authors aim to provide mechanistic explanations for the role of a lysosomal K⁺/H⁺ channel in anti-tumor immunity. It is also important to note that Ca²⁺ defects were proposed to cause ROS elevation, which in turn promotes NLRP3 inflammasome activation and IL-1β/IL-18 secretion. Yet, lysosomal Ca²⁺ levels were shown to be normal in TMEM175 knockout (KO) cells (Figure 8b). These findings suggest that the Ca²⁺/ROS/inflammasome effects may be secondary to lysosomal dysfunction associated with TMEM175 deficiency. Additionally, TMEM175 overexpression was reported to cause lysosomal hypo-acidification (e.g., PMID: 35750034, 35333573). This raises the question: would TMEM175 overexpression also exert effects in anti-tumor immunity? Some of the specific comments were not satisfactorily addressed. For instance, in the response to major concern 1, this reviewer was unable to find the mentioned 40-55 kD band in the attached Figure 1. Regarding the response to major concern 6, membrane leak can be easily investigated using Na⁺ or NMDG⁺ containing recording solutions, as neither of these ions is permeable to TMEM175 (PMID 26317472).Overall, while the revised manuscript addresses some of the reviewers' concerns, several critical issues regarding the mechanisms linking TMEM175 to Ca²⁺ dys-homeostasis, oxidative stress, and lysosomal pH remain unresolved and require further clarification.", "reviewer_2": "The authors have done an excellent job revising the manuscript, and my previous comments have been adequately addressed.", "reviewer_3": "Overall comments:\nThe authors have addressed my comments including adding more data and discussion to improve the manuscript. However, a few issues remain to address in the revision. These are listed below:\n1) Authors did a great job to evaluate the effect of CA-074 and BAPTA-AM on the inhibition of anti-tumor immunity in Tmem175-/- mice. However, without an in vivo experiment using NAC, the contribution of ROS to the anti-tumor immunity is still unclear.\n2) Authors did a great amount of work on the mechanism by which cross-presentation signals in TMEM175 KO macrophages was enhanced by IL-1β and IL-18. However, the explanation is still unclear and too complicated. If authors thought NF-kappaB pathway is the main mediator of IL-1β/IL18-enhanced cross-presentation signal in macrophages, a treatment of Myd88 siRNA or related inhibitors will help determine their hypothesis. Similarly, the roles of DEGs they found could also be verified in macrophages with siRNA.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear editor,\nThank you for your letter and the reviewers comments concerning our manuscript\nDeficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor\nimmunity via inflammasome and cross-presentation pathway (NCOMMS-24-59454B)\nsubmitted to Nature communications. The comments are very helpful for us to\nimprove our manuscript. Newly revised portions are highlighted in yellow. The point-\nby-point response for the reviewers’ comments are listed as follows.\nReviewer #1\n1. In this revised manuscript, the authors have performed several new experiments\nto address the reviewers' concerns, and the revised manuscript is improved. The\nroles of TMEM175 in anti-tumor immunity via inflammasome activation are\nconvincingly established. However, the proposed mechanisms that link the channel\nproperties of lysosomal TMEM175 with Ca²⁺ dys-homeostasis and oxidative stress\nare still not clear. In the new working model in Figure 10, the authors now claim\nthat there is no lysosomal K⁺ defect associated with TMEM175 deficiency. Instead,\nthey attribute the abnormalities in Ca²⁺ and ROS levels to a lysosomal pH defect\n(hypo-acidification). It should be noted that the basal lysosomal pH was reported\nto be normal in early TMEM175 studies (PMID 26317472) but decreased (hyper-\nacidification) in more recent studies (e.g., PMID: 35750034, 35333573). However,\nthe new data in the revision showed that the basal lysosomal pH was elevated (see\nFigure 8), which contradicts all previous studies. Additionally, lysosomal pH in\nmacrophages was reported to be more acidic upon M-CSF treatment (PMID:\n17314396). These discrepancies must be addressed if the authors aim to provide\nmechanistic explanations for the role of a lysosomal K⁺/H⁺ channel in anti-tumor\nimmunity.\nResponse: Thank you for your kind and constructive comments. We appreciate your\nprofessional insights into the current literature on TMEM175. In our response, we first\naddress the seemingly contradictory findings reported in previous studies and then\nexplain our results. To address your comments thoroughly, we have repeated the\nrelevant experiments using improved methodologies and have updated the manuscript\nwith additional discussion.\nIn the earlier articles (TMEM175 Is an Organelle K(+) Channel Regulating\nLysosomal Function. Chunlei Cang et al. Cell. 2015, PMID: 26317472 and\nTMEM175 deficiency impairs lysosomal and mitochondrial function and increases\nα-synuclein aggregation. Sarah Jinn et al. Proc Natl Acad Sci USA. 2017, PMID:\n28193887), lysosomal acidification was reported to be impaired in TMEM175\nknockout cells under starvation conditions. However, under nutrient-rich (fed)\nconditions, lysosomal pH was found to be slightly lower in TMEM175 KO cells\ncompared to wild-type (WT) cells. Additionally, in another article (A growth-factor-\nactivated lysosomal K+ channel regulates Parkinson's pathology. Jinhong Wie et al.\nNature. 2021 PMID: 33505021), lysosomal pH was observed to be higher in\nTMEM175 KO cells during starvation, while differences were minimal under fed\nconditions.\nIn the more recent studies (Parkinson's disease-risk protein TMEM175 is a\nproton-activated proton channel in lysosomes. Meiqin Hu et al. Cell. 2022 PMID:\n35750034 and pH regulates potassium conductance and drives a constitutive proton\ncurrent in human TMEM175. Wang Zheng et al. Sci Adv. 2022 PMID: 35333573),\nit appears that the experiments were conducted without subjecting cells to starvation.\nThe lysosomal pH was measured in fed cells with/without Baf-A1. A possible\nexplanation, therefore, is that the effects of TMEM175 knockout on lysosomal pH\ndepends on the state of the cells. The phenotypes observed under fed conditions are\nconsistent across studies, and the phenotypes observed under starvation are not\ncontradictory.\nIn our experiment, measurements were taken using a plate reader, which\nmaintained an optimal temperature of 37°C for the cells. However, this instrument does\nnot provide a sterile environment, and extended measurement periods could introduce\nadditional variables. A two-hour timeframe was insufficient to observe the full effects\nof the treatment, as BMDMs and culture media were typically harvested 4–10 hours\nafter cell debris treatment in our ELISA and Co-IP experiments. Therefore, we aimed\nto accelerate the inflammasome activation. According to the present articles,\nstarvation\nexacerbates lysosomal acidification defects and impairs lysosomal enzyme function (A\ngrowth-factor-activated lysosomal K+ channel regulates Parkinson's pathology.\nJinhong Wie et al. Nature. 2021 PMID: 33505021). This inspired us to investigate\nwhether FBS starvation could promote the inflammasome activation. In preliminary\nexperiments, we observed that reducing FBS concentrations enhanced the\ninflammasome activation. Specifically, at 1% FBS, IL-1β secretion was more robust.\nThus for our dynamic measurements of lysosomal pH, lysosomal calcium, cytosolic\ncalcium, and ROS, cells were treated in medium containing 1% FBS to observe changes\nwithin a 2-hour window. These conditions resemble the “starved” state, and our results\nwere consistent with previous studies. We apologize for not clearly stating these details\nin the original manuscript. To avoid further confusion, we repeated the experiments\nunder 10% FBS conditions and measured the lysosomal pH, lysosomal calcium,\ncytosolic calcium, and ROS at the end of the treatment. Under these revised conditions,\nwe found that lysosomal pH in untreated Tmem175-/- BMDMs was slightly lower than\nin WT BMDMs, whereas lysosomal pH in cell debris-treated Tmem175-/- BMDMs was\nhigher than in WT BMDMs. We propose that cell debris treatment creates a stress\ncondition that stimulates lysosomal activation, thereby revealing impaired lysosomal\nacidification in Tmem175-deficient cells. These results parallels the response seen\nunder starvation. We also added the related information in our discussion section.\nAdditionally, we thank the reviewer for pointing out that M-CSF promotes\nlysosomal acidification. Both Tmem175-/- and WT BMDMs were treated in medium\ncontaining M-CSF, thus this factor is unlikely to influence the comparative conclusions\nof our experiments.\nIn our pre-experiment, we found that decreasing FBS promoted the inflammasome\nactivation. Thus the dynamic changes of lysosomal pH, lysosomal calcium, cytosolic\ncalcium, and ROS were measured in medium containing 1% FBS to observe changes\nwithin a 2-hour window\nFig.8 Calcium ions were involved in the promoted inflammasome activation in\nTMEM175 knockout BMDMs. (a) Lysosomal pH were determined using\nLysoSensor™ Yellow/Blue DND-160. The impeded lysosomal acidification in\nTmem175-/- BMDMs was observed after B16-F10 cell debris treatment.\n2. It is also important to note that Ca²⁺ defects were proposed to cause ROS\nelevation, which in turn promotes NLRP3 inflammasome activation and IL-1β/IL-\n18 secretion. Yet, lysosomal Ca²⁺ levels were shown to be normal in TMEM175\nknockout (KO) cells (Figure 8b). These findings suggest that the\nCa²⁺/ROS/inflammasome effects may be secondary to lysosomal dysfunction\nassociated with TMEM175 deficiency.\nResponse: Thank you for your comment. It is indeed true that Ca²⁺ defects can reduce\nROS levels through mitochondrial-related mechanisms (Calcium and ROS: A mutual\ninterplay. Agnes Görlach et al. Redox Biol. 2015 PMID: 26296072). However, as\nnoted by Agnes Görlach et al., the relationship between Ca²⁺ and ROS is condition-\ndependent. When the respiratory chain is inhibited or the Ca²⁺ overload occurs, a\npositive correlation between Ca²⁺ and ROS can be observed. Additionally, we found a\nstudy that demonstrated Ca²⁺-induced mitochondrial damage, which led to ROS\ngeneration and NLRP3 inflammasome activation (Critical role for calcium\nmobilization in activation of the NLRP3 inflammasome. Tomohiko Murakami et al.\nProc Natl Acad Sci USA. 2012 PMID: 22733741). Therefore, the interplay between\nCa²⁺ and ROS may vary depending on the specific cellular conditions.\nIn contrast to previous studies suggesting that Ca²⁺ activates the NLRP3\ninflammasome indirectly through ROS or other mechanisms (Critical role for calcium\nmobilization in activation of the NLRP3 inflammasome. Tomohiko Murakami et al.\nProc Natl Acad Sci USA. 2012 PMID: 22733741 and The calcium-sensing receptor\nregulates the NLRP3 inflammasome through Ca2+ and cAMP. Geun-Shik Lee et al.\nNature. 2012 PMID: 23143333), the primary hypothesis of our study is that Ca²⁺\ndirectly participates in the ASC/NLRP3 interaction. We agree with your comment that\nROS may play a secondary role in this process. Although ROS could potentially be\ninvolved in our mechanism, its contribution may be less significant than that of Ca²⁺.\nThis is supported by our findings showing that in vivo administration of NAC (a ROS\ninhibitor) partially suppressed T cell immunity, whereas BAPTA-AM (a Ca²⁺ chelator)\ncompletely suppressed T cell immunity.\nThank you again for sharing your insights on Ca²⁺ and ROS. This is an intriguing\narea of research that could be explored more deeply in future studies. However in our\ncurrent work it seems that Ca²⁺ regulated NLRP3 activation directly, because EDTA-\nNa2 treatment in cell lysate inhibited the ASC polymerization.\nFigure S12. B16-F10 cell debris activated NLRP3 inflammasomes more\nextensively in TMEM175 knockout BMDMs through lysosomal dysfunction. (k to\nn) Recruitment and activation of CD4+ T cells (gated from CD3+ cells) were partially\ninhibited by NAC in Tmem175-/- mice, however the IFN-γ and granzyme B expression\nin CD4+ T cells (gated from CD3+ cells) were not significantly altered in Tmem175-/-\nmice. (o to r) Recruitment, activation, IFN-γ expression, and granzyme B expression\nin CD8+ T cells (gated from CD3+ cells) were significantly diminished by NAC in\nTmem175-/- mice.\nFig.8 Calcium ions were involved in the promoted inflammasome activation in\nTMEM175 knockout BMDMs. (k and l) CA-074 and BAPTA-AM suppressed the\nrecruitment and activation of CD4+ T cells (gated from CD3+ cells) in Tmem175-/- mice.\nBAPTA-AM suppressed the recruitment of CD4+ T cells (gated from CD3+ cells) in\nWT mice. (m and n) BAPTA-AM was more efficient than CA-074 in inhibiting IFN-γ\nand granzyme B expression in CD4+ T cells (gated from CD3+ cells) in Tmem175-/-\nmice. (o to r) CA-074 and BAPTA-AM suppressed the recruitment, activation, IFN-γ\nexpression, and granzyme B expression of CD8+ T cells (gated from CD3+ cells) in\nTmem175-/- mice.\nFig.8 Calcium ions were involved in the promoted inflammasome activation in\nTMEM175 knockout BMDMs. (d) Calcium chelation through BAPTA-AM inhibited\nthe IL-1β secretion (n = 3). Cells were treated by B16-F10 cell debris for 8 hours. (e)\nCalcium chelation through BAPTA-AM or EDTA-Na2 blocked the interaction between\nASC and NLRP3 (n = 3). Cells were treated by B16-F10 cell debris for 4 hours.\nFig.10 Mechanisms of the elevated anti-tumor immunity in TMEM175 knockout\nmice.\nTumor cell debris engulfed by BMDMs caused lysosomal membrane permeabilization\nand cathepsin B leakage, which activated NLRP3 inflammasome. TMEM175 knockout\npromoted lysosomal calcium efflux into cytoplasm thus promoting NLRP3\ninflammasome activation and IL-1β/IL-18 secretion. IL-1β and IL-18 facilitated the\nrecruitment and activation of T cells and NKs. The augmented antigen cross-\npresentation in Tmem175-/- BMDMs enhanced CD8+ T cell expansion and activation,\nwhich was further promoted by IL-1β and IL-18.\n3. Additionally, TMEM175 overexpression was reported to cause lysosomal hypo-\nacidification (e.g., PMID: 35750034, 35333573). This raises the question: would\nTMEM175 overexpression also exert effects in anti-tumor immunity?\nResponse: Thank you for your insightful inspiration about the TMEM175 and anti-\ntumor immunity. In the previous works the role of TMEM175 over-expression was\nanalyzed in fed condition rather than during starvation or cell debris treatment. Notably,\nTMEM175 over-expression resulted in its localization to the plasma membranes, in\naddition to lysosomal membranes (Parkinson's disease-risk protein TMEM175 is a\nproton-activated proton channel in lysosomes. Meiqin Hu et al. Cell. 2022 PMID:\n35750034 and pH regulates potassium conductance and drives a constitutive proton\ncurrent in human TMEM175. Wang Zheng et al. Sci Adv. 2022 PMID: 35333573).\nThus it is difficult to determine whether TMEM175 over-expression could promote the\nanti-tumor immunity under these conditions. However, we believe the key factor for\npromoting the anti-tumor immunity is lysosomal dysfunction, which occurs during\nstarvation or cell debris treatment in TMEM175 knockout cells. Meiqin Hu et al.\nreported that TMEM175 deficiency caused lysosomal over-acidification, impaired\nproteolytic activity, and facilitated a-synuclein aggregation. Both their findings and\nours demonstrated that TMEM175 knockout induces lysosomal dysfunction, which is\ncritical for the downstream immune responses. TMEM175 overexpression is unlikely\nto exert effects in anti-tumor immunity, because it is unlikely to cause lysosomal\ndysfunction during cell debris treatment. Additionally, our results found that TMEM175\nexpression was higher in melanoma tissues than that in paracancerous tissues.\nFurthermore, higher TMEM175 expression in TAMs is associated with\nimmunosuppressive CD206+ TAMs.\nFig.1 TMEM175 was elevated in melanoma tissues and related with M2-like TAMs\nindicating that TMEM175 in myeloid cells should be deleted.\n(a) Tissue chip of paired melanoma and paracancerous tissues was stained by anti-\nTMEM175 to detect the expression of TMEM175 through IHC (n = 6 pairs).\nTMEM175 was expressed more robustly in melanoma tissues than paracancerous\ntissues. Scale bars indicate 500 μm for left panel and 50 μm for right panel.\nTMEM175 positive area was calculated.\n(b and c) Wild type mice were subcutaneously injected with 1×106 B16-F10 tumor\ncells (n = 18 mice). Mice were humanely sacrificed at day 18 to analyze the TME\nthrough FCM.\n4. Some of the specific comments were not satisfactorily addressed. For instance,\nin the response to major concern 1, this reviewer was unable to find the mentioned\n40-55 kD band in the attached Figure 1.\nResponse: Thank you for your comment. The 40-55 kD band was too weak. In order to\nshow it clearly, we lengthened the exposure time of our WB membrane.\nFig.1 TMEM175 was elevated in melanoma tissues and related with M2-like TAMs\nindicating that TMEM175 in myeloid cells should be deleted. (d) TMEM175\nexpression was obviously decreased in Tmem175-/- BMDMs. Lysates of Tmem175-/-\nand WT BMDMs were analyzed by western blot using anti-TMEM175 antibody (n =\n3).\n5. Regarding the response to major concern 6, membrane leak can be easily\ninvestigated using Na⁺ or NMDG⁺ containing recording solutions, as neither of\nthese ions is permeable to TMEM175 (PMID 26317472).\nResponse: Thank you for your comment. According to previously reported (TMEM175\nIs an Organelle K(+) Channel Regulating Lysosomal Function. Chunlei Cang et al.\nCell. 2015, PMID: 26317472), we included the results obtained with the NMDG bath\nsolution. Our findings are consistent with those presented in Figure 7B-E of Chunlei\nCang's study, where NMDG in the bath solution inhibited the positive currents.\nFig.1 TMEM175 was elevated in melanoma tissues and related with M2-like TAMs\nindicating that TMEM175 in myeloid cells should be deleted. (f) Lysosomal\npotassium conductance in Tmem175-/- BMDMs was significantly decreased.\nLysosomes from Tmem175-/- and WT BMDMs were analyzed by lysosomal voltage\nclamp. The current was recorded when the bath solution contained 150 mM K+ or 150\nmM NMDG. Representative result of voltage clamp was displayed, and the average\ncurrent intensities in +100 mV and 150 mM K+ were compared (n = 5).\n6. Overall, while the revised manuscript addresses some of the reviewers' concerns,\nseveral critical issues regarding the mechanisms linking TMEM175 to Ca²⁺ dys-\nhomeostasis, oxidative stress, and lysosomal pH remain unresolved and require\nfurther clarification.\nResponse: Thank you for your comment. Our previously presented data demonstrated\nthat ROS acted as a NLRP3 activator, although its role may be secondary to that of\ncalcium ions. We further investigated the Ca²⁺ dys-homeostasis through BafA1. BafA1\ntreatment decreased the lysosomal calcium concentration and increased the cytosolic\ncalcium, indicating the enhanced calcium efflux from lysosomes, which was in\naccordance with previous report (Chloroquine modulates antitumor immune response\nby resetting tumor-associated macrophages toward M1 phenotype. Degao Chen et al.\nNat Commun. 2018, PMID: 29491374). In conclusion, we appreciate your\nconstructive suggestions, which have significantly contributed to improving our\nmanuscript.\nFig.8 Calcium ions were involved in the promoted inflammasome activation in\nTMEM175 knockout BMDMs. (b) Lysosomal calcium were determined using rhod-\ndextran and AF488-dextran. The lower level of lysosomal calcium in Tmem175-/-\nBMDMs than WT BMDMs was observed after B16-F10 cell debris treatment.\nNeutralizing lysosomal pH with Bafilomycin A1 further decreased the lysosomal\ncalcium. (c) Cytoplasmic calcium were determined using Fluo-4-AM. The higher level\nof cytoplasmic calcium in Tmem175-/- BMDMs than WT BMDMs was observed by\nB16-F10 cell debris treatment. CA-074 did not obviously alter the cytoplasmic calcium\nlevels. Neutralizing lysosomal pH with Bafilomycin A1 further increased the\ncytoplasmic calcium.\nReviewer #2\nThe authors have done an excellent job revising the manuscript, and my previous\ncomments have been adequately addressed.\nResponse: Thank you for your comment.\nReviewer #3\n1. Authors did a great job to evaluate the effect of CA-074 and BAPTA-AM on the\ninhibition of anti-tumor immunity in Tmem175-/- mice. However, without an in\nvivo experiment using NAC, the contribution of ROS to the anti-tumor immunity\nis still unclear.\nResponse: Thank you for your comment. We added the in vivo experiment using NAC\naccording to previously reported (Antioxidants accelerate lung cancer progression in\nmice. Volkan I Sayin et al. Sci Transl Med. 2014, PMID: 24477002 and BACH1\nStabilization by Antioxidants Stimulates Lung Cancer Metastasis. Clotilde Wiel et al.\nCell. 2019, PMID: 31257027). And the related description were added in our\nmanuscript.\nFigure S12. B16-F10 cell debris activated NLRP3 inflammasomes more\nextensively in TMEM175 knockout BMDMs through lysosomal dysfunction. (g to\nr) In order to investigate the influence of ROS on the anti-tumor immunity, Tmem175-\n/- and WT mice (n = 5 mice) were subcutaneous implantation with 1×106 B16-F10\nand treated with 1g/L NAC through drinking water after day 7. Mice were humanely\nsacrificed on day 14 to weigh the tumors and analyze the TME through FCM. (h) The\nanti-tumor effects of TMEM175 knockout was suppressed by NAC. (i) Expression of\nMHC-II in TAMs (gated from CD45+ CD11b+ F4/80+ cells) was significantly\nattenuated by NAC in Tmem175-/- mice. (j) Expression of CD206 in TAMs (gated from\nCD45+ CD11b+ F4/80+ cells) was elevated by NAC in Tmem175-/- mice. (k to n)\nRecruitment and activation of CD4+ T cells (gated from CD3+ cells) were partially\ninhibited by NAC in Tmem175-/- mice, however the IFN-γ and granzyme B expression\nin CD4+ T cells (gated from CD3+ cells) were not significantly altered in Tmem175-/-\nmice. (o to r) Recruitment, activation, IFN-γ expression, and granzyme B expression\nin CD8+ T cells (gated from CD3+ cells) were significantly diminished by NAC in\nTmem175-/- mice.\n2. Authors did a great amount of work on the mechanism by which cross-\npresentation signals in TMEM175 KO macrophages was enhanced by IL-1β and\nIL-18. However, the explanation is still unclear and too complicated. If authors\nthought NF-kappaB pathway is the main mediator of IL-1β/IL18-enhanced cross-\npresentation signal in macrophages, a treatment of Myd88 siRNA or related\ninhibitors will help determine their hypothesis. Similarly, the roles of DEGs they\nfound could also be verified in macrophages with siRNA.\nResponse: Thank you for your comment. We used siRNA to inhibit Myd88 (a well-\nestablished downstream signaling molecule of IL-1R) and Csf2rb (a shared DEG\nregulated by IL-1β in both Tmem175-/- and WT group). Silencing Myd88 abrogated the\neffects of IL-1β and IL-18. For IL-1β , better inhibitory effects were observed in\nsiMyd88, indicating that Myd88 is the main down-stream event. We did not silence\nCsf2rb in IL-18 group, because it was not identified as a DEG in response to IL-18.\nThank you again for your constructive suggestions, which have greatly improved the\nquality of our manuscript.\nFigure S9. related to Figure 6. TMEM175 knockout augmented the cross-\npresentation of tumor antigens. (e and f) In order to validate the down-stream events\nof IL-1β and IL-18, we used siRNA to inhibit Myd88 (a known down-stream protein\nof IL-1R) and Csf2rb (a common DEG up-regulated by IL-1β ). (e) Spleenic\nlymphocytes from B16-F10-OVA tumor bearing mice were co-cultured with BMDMs\ntransfected with desired siRNA. (f) Silencing MyD88 inhibited the promoted CD8+ T\ncell proliferation by IL-1β and IL-18. Silencing CSF2RB partially inhibited the\npromoted CD8+ T cell proliferation by IL-1β.\nReviewer #4\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.\nResponse: Thank you for your comments.\nAll the point-by-point responses are presented above. We are willing to make further\nrevision to improve the manuscript. We look forward to your decision.\nYours sincerely,\nXiawei Wei, Ph. D\nFull Professor and Director of Lab of Cancer Drug Target and Aging Research\nState Key Laboratory of Biotherapy,\nWest China Hospital, Sichuan University\nEmail: xiaweiwei@scu.edu.cn;\nAddress：No. 17, Block 3, Southern Renmin Road, Chengdu, Sichuan 610041,\nPeople’s Republic of China"}, {"round": 3, "reviewer_comments": {"reviewer_1": "In the re-revised manuscript, it appears that some data were deleted or replaced without explanation. For example, the new data in Figure 8 show lysosomal hyper-acidification (no LPS and cell debris treatment) in the KO cells. These results are opposite to what the authors showed in the previous submission, i.e., lysosomal hypo-acidification in the KO cells. Why were several figure panels regarding lysosomal acidification deleted in the current version? As mentioned repeatedly in the reviewers’ comments and now also in the Discussion of the manuscript, the effect of TMEM175 KO on lysosomal pH is controversial. After the two original studies in 2022 (PMID: 35750034, Cell 2022, and PMID: 35333573, Science Advances 2022) showed that TMEM175 is a lysosomal H+ channel and that TMEM175 KO cells exhibit lysosomal hyper-acidification, several follow-up studies have confirmed the hyper-acidification phenotype in TMEM175 KO cells, e.g., PMID: 37390818, Mol. Cell 2023; PMID: 40865534, Neuron 2025; PMID: 40473625, Nature Communications 2025. Note that it is possible that LPS and cell debris treatment might affect lysosomal acidification differently in WT versus KO cells. However, that was not what the data from the earlier versions of the manuscript showed. Also, in the new Figure 8a, LPS induces lysosomal hyper-acidification in WT cells, yet the opposite results were shown previously. Note that the proposed Ca2+ link can make sense only when KO cells have a lysosomal hypo-acidification phenotype. In addition, it appears that the Western blots in Figure 1d were also replaced, yet it was mentioned in the rebuttal letter that the 40-55 kD band could only be seen by increasing exposure time. In Figure 1f, I thought new experiments were performed with a K+ versus NMDG+ comparison. However, although the data from WT cells are new, the data from the KO cells and the statistical analysis are the same as before.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear editor,\nThank you for your letter and the reviewers comments concerning our manuscript\nDeficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor\nimmunity via inflammasome and cross-presentation pathway (NCOMMS-24-59454C)\nsubmitted to Nature communications. Newly revised portions are highlighted in\nyellow. The point-by-point response for the reviewers’ comments are listed as follows.\nReviewer #1\n1. In the re-revised manuscript, it appears that some data were deleted or replaced\nwithout explanation. For example, the new data in Figure 8 show lysosomal hyper-\nacidification (no LPS and cell debris treatment) in the KO cells. These results are\nopposite to what the authors showed in the previous submission, i.e., lysosomal\nhypo-acidification in the KO cells. Why were several figure panels regarding\nlysosomal acidification deleted in the current version?\nResponse: Thank you for your question. We apologize for the insufficient explanation\nprovided in the revised manuscript. In our previous submission, lysosomal pH,\nlysosomal calcium, and cytosolic calcium levels were measured using a fluorescent\nplate reader to monitor dynamic changes over a 2-hour period (i.e., dynamic\nmeasurement). However, we found that a more accurate approach would be to collect\ndata at the end of the LPS and cell debris treatments (i.e., end-point measurement) for\ntwo key reasons.\nFirst, according to the instruction manual of the lysosomal pH probe LysoSensor™\nYellow/Blue DND-160 (Thermo), it is recommended to acquire the data within 5 min.\nTherefore, we chose to measure the lysosomal pH once, immediately following the\ndesired treatment, rather than monitoring dynamic changes over a 2-hour period.\nSecond, although the plate reader maintained an optimal temperature of 37°C for\nthe cells, it does not provide a sterile environment with 5% CO . Moreover, the 2-hour\n2\ntime frame was insufficient to capture the full effects of the LPS and cell debris\ntreatment, as BMDMs and culture media were harvested 4–10 hours after treatment in\nour ELISA and Co-IP experiments. An extended dynamic measurement period would\nnot be accurate, as fluorescence readings were conducted outside a sterile environment\nwith 5% CO . Therefore, in the current version of the manuscript, we cultured the cells\n2\nunder standard conditions (sterile, 37°C, and 5% CO ) and collected data on lysosomal\n2\npH, lysosomal calcium, and cytosolic calcium once, immediately after treatment.\nThe discrepancy between the dynamic measurement (in our previous version) and\nthe end-point measurement (in the present version) arises from differences in the cell\ntreatment protocols. In the end-point measurement, BMDMs were cultured in complete\nmedium and treated with LPS and cell debris. In contrast, during the dynamic\nmeasurement, BMDMs were cultured in medium containing 1% FBS in an attempt to\naccelerate inflammasome activation by reducing FBS, as extended measurement\nperiods in the plate reader could introduce additional variables. In our preliminary\nexperiments, reducing FBS concentrations was found to expedite inflammasome\nactivation. However, these conditions mimicked a \"starvation\" state and did not\naccurately reflect the effect of TMEM175 knockout on lysosomal pH under normal\nculture conditions. Consequently, we preferred to replace the dynamic measurement\nwith the end-point measurement in our current manuscript.\nIn our pre-experiment, we found that decreasing FBS promoted the inflammasome\nactivation. Thus the dynamic changes of lysosomal pH, lysosomal calcium, and\ncytosolic calcium were measured in medium containing 1% FBS to observe changes\nwithin a 2-hour window in our previous manuscript.\n2. As mentioned repeatedly in the reviewers’ comments and now also in the\nDiscussion of the manuscript, the effect of TMEM175 KO on lysosomal pH is\ncontroversial. After the two original studies in 2022 (PMID: 35750034, Cell 2022,\nand PMID: 35333573, Science Advances 2022) showed that TMEM175 is a\nlysosomal H+ channel and that TMEM175 KO cells exhibit lysosomal hyper-\nacidification, several follow-up studies have confirmed the hyper-acidification\nphenotype in TMEM175 KO cells, e.g., PMID: 37390818, Mol. Cell 2023; PMID:\n40865534, Neuron 2025; PMID: 40473625, Nature Communications 2025.\nResponse: Thank you for providing additional articles on the influence of TMEM175\nknockout (KO) on lysosomal pH. We have carefully reviewed these references. It\nappears that in these studies (PMID: 35750034, Cell 2022; PMID: 35333573, Science\nAdvances 2022; PMID: 37390818, Mol. Cell 2023; PMID: 40865534, Neuron 2025;\nPMID: 40473625, Nature Communications 2025), WT and TMEM175 KO cells were\ncultured in complete medium and subsequently stained with lysosomal pH probes. In\ncontrast, in our previous dynamic measurement, cells were measured in a \"starvation\"\nstate, as explained earlier, which resembles conditions in earlier studies (PMID:\n26317472, Cell 2015; PMID: 28193887, PNAS 2017; PMID: 33505021, Nature 2021).\nIn our end-point measurement, cells were cultured in complete medium, yielding results\nconsistent with the articles you provided. Thus, the \"contradictory\" results between\ndifferent studies may stem from differences in cell treatment. Under resting conditions,\nTMEM175 KO results in a hyper-acidification phenotype. However, when cells are\nchallenged with starvation or cell debris, TMEM175 KO induces an unstable lysosomal\npH and hypo-acidification.\n3. Note that it is possible that LPS and cell debris treatment might affect lysosomal\nacidification differently in WT versus KO cells. However, that was not what the\ndata from the earlier versions of the manuscript showed. Also, in the new Figure\n8a, LPS induces lysosomal hyper-acidification in WT cells, yet the opposite results\nwere shown previously. Note that the proposed Ca2+ link can make sense only\nwhen KO cells have a lysosomal hypo-acidification phenotype.\nResponse: Thank you for your comments. We acknowledge that TMEM175 knockout\nmay influence lysosomal acidification differently depending on whether cells are\ncultured in complete medium or challenged with starvation or cell debris. In the present\nFig. 8a, LPS induced lysosomal hypo-acidification in WT cells, which aligns with our\nprevious data. As explained earlier, we believe that our current end-point measurement\nprovides a more accurate method for determining lysosomal pH, lysosomal calcium,\nand cytosolic calcium, and thus we have replaced the figures accordingly. We suspect\nthat the \"Ca²⁺ link\" may not be the optimal model for describing cells treated with LPS\nalone, as LPS treatment did not result in lysosomal membrane permeabilization (Fig.\n7g). We hypothesize that lysosomal hypo-acidification, which occurs during lysosomal\nmembrane permeabilization induced by cell debris, promotes lysosomal calcium efflux,\nthereby contributing to NLRP3 activation. As previously suggested, inhibiting\nacidification by BafA1 in WT BMDMs mimicked the phenotypes of TMEM175 KO\nBMDMs, which supported the \"Ca²⁺ link\" during the lysosomal membrane\npermeabilization.\nFig. 8a Lysosomal pH were determined using LysoSensor™ Yellow/Blue DND-160.\nThe impeded lysosomal acidification in Tmem175-/- BMDMs was observed after B16-\nF10 cell debris treatment.\nFig. 7g Tmem175-/- and WT BMDMs treated with LPS and B16-F10 cell debris were\nfixed and permeabilized. Then cells were stained by cathepsin B rabbit mAb, cathepsin\nD rabbit mAb, or cathepsin L rabbit mAb (n = 3). The spots of cathepsin B, cathepsin\nD, and cathepsin L diffused in cytoplasm by cell debris treatment. Scale bars indicate\n10 μm.\n4. In addition, it appears that the Western blots in Figure 1d were also replaced,\nyet it was mentioned in the rebuttal letter that the 40-55 kD band could only be\nseen by increasing exposure time.\nResponse: Thank you for your comments. We have replaced this figure in response to\nyour previous suggestion to show the weak 40-55 kD bands. To address this, we\nrepeated the western blot assay and increased the exposure time to enhance detection.\n5. In Figure 1f, I thought new experiments were performed with a K+ versus\nNMDG+ comparison. However, although the data from WT cells are new, the data\nfrom the KO cells and the statistical analysis are the same as before.\nResponse: Thank you for your comments. We have updated all of our voltage clamp\ndata and included the K⁺ versus NMDG comparison as requested.\nFig.1f Lysosomal potassium conductance in Tmem175-/- BMDMs was significantly\ndecreased. Lysosomes from Tmem175-/- and WT BMDMs were analyzed by lysosomal\nvoltage clamp. The current was recorded when the bath solution contained 150 mM K+\nor 150 mM NMDG. Representative result of voltage clamp was displayed, and the\naverage current intensities in +100 mV with 150 mM K+ or 150 mM NMDG were\ncompared (n = 5).\nReviewer #3 & #4\n1. The new in vivo experiment with NAC confirmed the contribution of ROS to\nthe anti-tumor immunity in Tmem175-/- mice. However, it seems that NAC\nadministration caused no phenotypes in control mice. Considering authors showed\nNAC was able to suppress inflammasome activation and IL-1β secretion in control\nmacrophages (Supplemental Fig. 12C,D), they should give an explanation in the\nmanuscript.\nResponse: Thank you for your comments. NAC partially inhibited the anti-tumor\nimmunity in Tmem175-/- mice, suggesting that the elevated ROS levels in Tmem175-/-\nmacrophages contributed to the anti-tumor immunity. This inhibitory effects were not\nas pronounced in WT mice, which could be owed to the lower ROS activation in WT\nmice. Although NAC inhibited the inflammasome activation in WT BMDMs in vitro,\nits inhibitory effects on the immune microenvironment in vivo were more evident in\nmice with higher basal ROS activation. When the ROS in macrophages was low,\ninhibiting ROS did not further suppress downstream events. The related explanation\nhas been added in our manuscript (RESULTS section line 548-552).\n2. The reduction the promoted CD8+ T cell proliferation by siRNA treatment\nseems convincing. A measurement of knock down efficiency in macrophages by\nsiRNA would further elevate the solidity of this data.\nResponse: Thank you for your comments. We confirmed the knockdown efficiency by\nFCM, and the data have been added in Supplementary Fig. 9g and h.\nFigure S9. (g and h) The decreased MyD88 and CSF2RB expression by siRNA was\nconfirmed by FCM.\nAll the point-by-point responses are presented above. We are willing to make further\nrevision to improve the manuscript. We look forward to your decision.\nYours sincerely,\nXiawei Wei, Ph. D\nFull Professor and Director of Lab of Cancer Drug Target and Aging Research\nState Key Laboratory of Biotherapy,\nWest China Hospital, Sichuan University\nEmail: xiaweiwei@scu.edu.cn;\nAddress：No. 17, Block 3, Southern Renmin Road, Chengdu, Sichuan 610041,\nPeople’s Republic of China"}, {"round": 4, "reviewer_comments": {"reviewer_1": "If some of the key results were obtained from blind experiments, it is necessary to indicate that in the methods and figure legends.", "reviewer_3": "The authors have address all this reviewer's comments. No further comments.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear editor,\nThank you for your letter and the reviewers comments concerning our manuscript\nDeficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor\nimmunity via inflammasome and cross-presentation pathway (NCOMMS-24-59454C)\nsubmitted to Nature communications. Newly revised portions are highlighted in\nyellow. The point-by-point response for the reviewers’ comments are listed as follows.\nReviewer #1\nIf some of the key results were obtained from blind experiments, it is necessary to\nindicate that in the methods and figure legends.\nResponse: Thank you for your comment. Data of this work are not obtained from blind\nexperiments.\nReviewer #3\nThe authors have address all this reviewer's comments. No further comments.\nResponse: Thank you for your comment.\nReviewer #4\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: Thank you for your comment.\nAll the point-by-point responses are presented above. We are willing to make further\nrevision to improve the manuscript. We look forward to your decision.\nYours sincerely,\nXiawei Wei, Ph. D\nFull Professor and Director of Lab of Cancer Drug Target and Aging Research\nState Key Laboratory of Biotherapy,\nWest China Hospital, Sichuan University\nEmail: xiaweiwei@scu.edu.cn;\nAddress：No. 17, Block 3, Southern Renmin Road, Chengdu, Sichuan 610041,\nPeople’s Republic of China"}]

Decision letter