Pentose phosphate pathway fuels cGAS-STING signalling to boost function of intratumoral conventional dendritic cells

Domain: chemistry
Published: 2026-03-23
Editorial outcome: minor_revision

Abstract
Tumors employ diverse mechanisms to impair conventional dendritic cells (cDCs) function within the tumor microenvironment, yet the underlying processes remain unclear. Here, we demonstrate that pentose phosphate pathway (PPP) reduction in late-stage intratumoral cDCs compromises their function. Both pharmacological and genetic inhibition of the PPP attenuate cDC-mediated antitumor responses. Conversely, PPP augmentation restores late-stage intratumoral cDCs antitumor capacity. PPP deficiency in cDCs enhances the immune checkpoint PD-L1 expression level, and combining cDC-specific PPP potentiation with immune checkpoint blockade synergistically enhances immunotherapy efficacy. Mechanistically, PPP activation fuels purine metabolism, thereby increasing ATP and GTP levels required for 2’3’-cGAMP synthesis, ultimately promoting cGAS-STING signaling and STING-dependent cDC antitumor responses. The PPP is associated with STING-dependent cDC activities in tumor tissues from female patients with breast cancer. Collectively, our findings establish PPP as an intrinsic metabolic checkpoint in STING-mediated cDC antitumor immunity, and suggest targeting PPP in cDCs as a promising cancer immunotherapy strategy.

Introduction
Immune checkpoint blockade (ICB) has revolutionized clinical cancer therapy1,2. However, only a subset of patients responds well to ICB3. Emerging evidence suggests that dysfunctional dendritic cells (DCs) in the tumor microenvironment (TME) limit ICB efficacy4,5. DCs, particularly conventional DCs (cDCs), play a critical role in antitumor immunity by presenting tumor antigens to prime and activate T cells4,6,7. Nevertheless, tumor immunoediting has evolved multiple strategies to suppress DC function within the TME7-9. Therefore, elucidating the mechanisms underlying DC functional modulation in the TME and developing strategies to enhance DC activity are essential for improving cancer immunotherapy.

Tumor immunoediting is a dynamic process that shapes immune cells function to dictate tumor evolution10-14. Naive and activated CD8+ T cells present in early tumors are replaced by exhausted and precursor-exhausted populations in late tumors. Late-stage tumors exhibit suppression of antitumor immunity genes, resulting in diminished immune cell infiltration and impaired tumor-immune communication. Notably, innate immune genes, particularly interferon-stimulated genes are downregulated in late tumors13. Latest finding has revealed that decreased cGMP levels in intratumoral DCs during the late stage compromise their interstitial motility and antitumor activity14. However, the underlying mechanism of tumor immunoediting mediating the DC phenotype remains elusive.

Metabolic reprogramming plays a central role in the development and maturation of DCs, providing essential energy for their functions9,15-18. Synergizing metabolic interventions with immunotherapy significantly enhances antigen-presentation capabilities and T cell activation by DCs, thereby improving the efficacy of current antitumor therapies7,19,20. Despite these advancements, the intrinsic metabolic disruptions within tumor-infiltrating DCs remain poorly understood, presenting a barrier to the development of innovative immunotherapy strategies. The pentose phosphate pathway (PPP) stands as a critical branch of glucose metabolism essential for producing ribose 5-phosphate (R5P) and nicotinamide adenine dinucleotide phosphate (NADPH). R5P acts as a foundational component for nucleotide synthesis, and NADPH plays a pivotal role in maintaining redox balance and supporting various biosynthetic processes21,22. Recent studies emphasize the PPP's impact on regulating T cell functions, underscoring its pivotal role in boosting CD8+ effector T cell activity23. Furthermore, the PPP enhances antioxidant capacity and mitigates exhaustion in CAR T cells24. Notably, Pyruvate Kinase M2 (PKM2) deficiency results in increased PPP flux, fostering the generation of TCF1+ progenitor CD8+ T cells25. Despite the growing evidence showcasing the PPP as an immune effector, how and whether PPP regulates DC function, especially in the TME is largely unknown.

In this work, we perform single-cell analyses on early- and late-stage tumor-infiltrating cDCs isolated from MC38 tumor-bearing mice. Our results reveal that late-stage intratumoral cDCs exhibited significant dysfunction and marked impaired PPP activity. Notably, enhancing PPP activity in late-stage intratumoral cDCs restores their functionality and augmentes ICB efficacy. Mechanistically, the PPP fuels cGAS-STING signaling and sustains STING-dependent cDC-mediated antitumor immunity. Importantly, the PPP supports STING-dependent cDC activities in tumor tissue samples from patients with breast cancers. Our findings elucidate how tumor immunoediting compromises cDC function in the TME and highlight the PPP as a critical metabolic regulator of STING-driven cDC antitumor responses. Our study suggests that targeting the PPP in cDCs could be a promising strategy for improving cancer immunotherapy.

Results
Tumor-infiltrating cDCs from late tumors show signs of dysfunction
The TME modulates DC function and influences ICB efficacy. To investigate tumor-infiltrating DC dynamics during tumor progression, we performed single-cell RNA sequencing (scRNA-seq) on DCs (live CD45+CD24-F480-Ly6C-CD11c+MHC-II+) isolated from MC38 tumor-bearing mice at early (8-10 days) and late (20-30 days) stages (Figure 1a, and Supplementary Figure 1a)13. For the identification of DC populations in the single-cell dataset, we referred to established DC-specific signatures and markers from published studies26-28. First, cell clusters expressing high levels of lineage markers (Cd3d, Cd19, Ncr1, Adgre1) were excluded. Tumor-infiltrating DCs were divided into four major clusters, including monocyte-derived DCs (mo-DCs), plasmacytoid DCs (pDCs), conventional type 1 DCs (cDC1s) and conventional type 2 DCs (cDC2s) (Figure 1b). Dirichlet multinomial regression analysis revealed temporal changes in DC composition, with pDCs and cDCs decreasing while mo-DCs became predominant in late-stage tumors (Figure 1c). Flow cytometry analysis revealed lower levels of typical DC maturation markers, including major histocompatibility complex class I (MHC-I), CD80 and CD86 in late-stage intratumoral cDCs (Figure 1, d and e). qPCR analysis confirmed significant downregulation of genes associated with DC maturation, including co-stimulatory factors, antigen processing and presentation molecules and adhesion molecules in late-stage intratumoral cDCs (Supplementary Figure 2, a-c). Furthermore, key DC functional genes were suppressed in late-stage intratumoral cDCs (Figure 1f, and Supplementary Figure 2d). CCR7 is necessary for the migration of tumor-infiltrating DCs into tumor-draining lymph nodes29,30, and CXC-chemokine ligand 9 (CXCL9) and CXCL10, predominantly secreted by tumor-infiltrating DCs, promote the recruitment of CD8+ T cells into the TME6. Our data further confirmed that Ccr7, Cxcl9 and Cxcl10 were all downregulated in late-stage intratumoral cDCs (Figure 1g). IRF4 and IRF8 are both necessary for the development of cDCs31,32. Late-stage intratumoral cDCs displayed lower expression levels of both Irf4 and Irf8 (Supplementary Figure 2e). Collectively, our results demonstrate that tumor-infiltrating cDCs from late tumors displayed dysfunctional phenotype.

Given the pivotal role of cDCs in T cell priming and antitumor immunity, we further distinguished cDC subpopulations from total DCs in our scRNA-seq data, which were defined based on canonical markers: Itgax, Flt3, and Zbtb46. Gene set enrichment analysis (GSEA) revealed impaired antigen processing and presentation capacity in late-stage intratumoral cDCs, characterized by negative regulation of both adaptive immune responses and T cell-mediated immunity (Supplementary Figure 2f and Figure 1h). To validate these findings in vitro, we isolated early- and late-stage intratumoral cDCs and co-cultured them with OT-I T cells in the presence of SIINFEKL peptides. Late-stage intratumoral cDCs demonstrated significantly reduced capacity to stimulate OT-I T cell proliferation (Figure 1, i and j) and effector cytokine production (Figure 1k) compared to their early-stage counterparts. These results collectively indicate that late-stage tumor-infiltrating cDCs exhibit markedly impaired T cell priming functionality.

As supported by latest studies, cDCs in the TME can be classified into three main subsets: cDC1, cDC2, and mature DCs (also referred to as mregDCs)33-36. Among these, mature DCs represent the dominant subset responsible for mediating antitumor immunity and are thought to originate from cDC1 and cDC2 following tumor antigen uptake33,34,36. Further analysis of our scRNA-seq data revealed a notable subset of mature DCs (defined by expression of Ccl22, Ccl17, and Ccr7) in late-stage tumors (Supplementary Figure 2, g and h). Flow cytometry analysis revealed reduced proportions of mature DCs and cDC1 in late-stage tumors (Supplementary Figure 2i). Furthermore, the absolute numbers of infiltrating mature DCs, cDC1, and cDC2 per gram of tumor tissue were significantly lower in late-stage compared to early-stage tumors (Supplementary Figure 2j). GSEA analysis indicated that mature DCs from late-stage tumors displayed impaired antigen presentation capacity (Supplementary Figure 2k). Next, we isolated mature DCs from early- and late-stage tumors and observed decreased expression of maturation markers and functional cytokines in the late-stage mature DCs (Supplementary Figure 2, l and m). To further assess the functional capacity of cDC subsets, equal numbers of early- and late-stage tumor-infiltrating mature DCs, cDC1, and cDC2 were isolated. Mature DCs and cDC1 were co-cultured with OT-I T cells, while cDC2 were co-cultured with OT-II T cells, all in the presence of SIINFEKL peptide. Compared to early-stage cells, late-stage mature DCs showed significantly impaired ability to stimulate OT-I T cell proliferation (Figure 1, l and m) and effector cytokine production (Figure 1n). Similarly, late-stage cDC1 and cDC2 also exhibited reduced T cell priming capacity (Figure 1, o-t). Together, these results demonstrate that all major cDC subsets in late-stage tumors are functionally compromised relative to their early-stage counterparts.

Late-stage tumor-infiltrating cDCs exhibit the PPP activity impairment
To explore what’s the dominant different events between late- and early-stage intratumoral cDCs, we next performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis using scRNA-seq data. The results displayed significant alterations in key biological processes within intratumoral cDCs during tumor progression. These changes affected pathways involved in antigen processing and presentation, T cell receptor signaling and critical inflammatory cascades, as well as the TNF, JAK-STAT and NF-κB signaling pathways, suggesting the notable differences between late- and early-stage intratumoral cDCs (Figure 2a). Given the pivotal role of metabolic rewiring in modulating intratumoral DC function and antitumor immunity, we focused our investigation on central carbon metabolism (Figure 2a). Central carbon metabolism-including glycolysis, the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle-contributes a fundamental metabolic framework essential for sustaining cellular function37. To validate these observations, we performed central carbon metabolomics on early- and late-stage intratumoral cDCs. Our analysis revealed obviously reduced levels of PPP-associated metabolites in late-stage intratumoral cDCs, whereas glycolytic metabolites remained no unified changed (Figure 2b, and Supplementary Figure 3a). The PPP, a critical branch of glucose metabolism, generates nicotinamide adenine dinucleotide phosphate (NADPH) and ribose 5-phosphate (R5P) to support cellular functions22,38. Consistent with the metabolic profiling, late-stage intratumoral cDCs exhibited markedly decreased NADPH and R5P production (Supplementary Figure 3a). Given the established role of R5P in nucleotide biosynthesis21,25, we further observed significantly reduced nucleotide abundance in late-stage intratumoral cDCs (Figure 2c). The initial step of the PPP is catalyzed by glucose-6-phosphate dehydrogenase (G6PD), which converts glucose-6-phosphate (G6P) to 6-phosphogluconolactone (6PGL). We found that 6PGL level was reduced in late-stage intratumoral cDCs, whereas glucose and G6P remained comparable between early- and late-stage cells (Supplementary Figure 3a). Moreover, protein expression of G6PDX (the murine ortholog of human G6PD) and mRNA levels of key PPP enzymes were significantly lower in late-stage intratumoral cDCs (Supplementary Figure 3, b and c). Together, these findings demonstrate that late-stage intratumoral cDCs exhibit impaired PPP activity. To further validate our observation, we performed KEGG pathway analysis on mature DCs using scRNA-seq data. The analysis identified the PPP as one of the most significantly altered pathways in late-stage compared to early-stage intratumoral mature DCs (Supplementary Figure 3d). We then isolated early- and late-stage intratumoral mature DCs and observed reduced levels of the key PPP metabolite NADPH in late-stage mature DCs (Supplementary Figure 3e). Additionally, qPCR analysis showed decreased mRNA expression of major PPP enzymes in late-stage mature DCs (Supplementary Figure 3f). Moreover, early-stage intratumoral mature DCs were treated in vitro with the PPP inhibitor G6PDi-1. Flow cytometry showed that PPP inhibition reduced the frequency of mature DCs (Supplementary Figure 3g) and downregulated expression of key maturation markers including MHC-I, CD80 and CD86 within this subset (Supplementary Figure 3h). Conversely, treatment of late-stage intratumoral mature DCs with the PPP agonist AG139 significantly increased both the proportion of mature DCs and the expression of maturation markers (Supplementary Figure 3, i and j). In summary, these results demonstrate that PPP activity is essential for maintaining both the abundance and function of mature DCs in the TME.

To validate our results in vivo, we treated early-stage intratumoral cDCs with PPP inhibitors G6PDi-1 and 6-Aminonicotinamide (6-ATN). PPP inhibition markedly downregulated DC maturation markers including MHC-I, CD80 and CD86 (Figure 2, d and e), as well as DC functional genes including Tnfa, Il12 and Ccr7 (Figure 2f) without affecting their viability (Supplementary Figure 3k). We then adoptively transferred PPP inhibitors-treated early-stage intratumoral cDCs into tumor-bearing mice. Compared to non-treated DCs, G6PDi-1- or 6-AN-treated DCs displayed reduced efficacy in suppressing tumor growth (Figure 2g). Additionally, G6PDi-1- or 6-AN-treated DCs exhibited decreased proportions of IFN-γ- and TNF-α-producing CD4+ and CD8+ T cells in the TME (Figure 2, h and i). Most significantly, when we augmented the PPP activity using AG1 in late-stage intratumoral cDCs, we observed upregulated expression of both maturation markers and functional genes in vitro (Figure 2, j-m) without affecting their viability (Supplementary Figure 3l), along with restored antitumor function in vivo (Figure 2, n-p). In summary, our results demonstrate that the PPP activity impairment contributes to late-stage intratumoral cDC dysfunction, and modulating PPP activity in cDCs effectively regulates their antitumor responses.

PPP deficiency in cDCs restricts antitumor function
The first step of PPP branch is G6PD catalyzing the conversion of G6P to 6PGL (Figure 3a). Additionally, G6PD is the sole rate-limiting enzyme in the PPP branch22. Therefore, we crossed G6pdx-floxed mice with Zbtb46-Cre mice to obtain G6pdxfl/flZbtb46-Cre (KO) mice (Supplementary Figure 4, a and b), representing PPP deficiency in cDCs. First, we observed that PPP deficiency did not affect DC viability (Supplementary Figure 4c). Furthermore, no significant differences were observed in the populations of DCs in the spleens (Supplementary Figure 4, d-f), as well as the frequencies of T cells in the thymus (Supplementary Figure 4g), lymph nodes and spleens (Supplementary Figure 4h) between 6-12-week-old gender-matched G6pdx+/+Zbtb46-Cre (WT) and KO mice, indicating PPP abolition was dispensable for DC and T cell development. To delve deeper into the impact of the PPP on DC function, we primed OT-I T cells using WT and KO bone marrow-derived DCs (BMDCs) (Supplementary Figure 5a) loaded with the SIINFEKL peptides. We observed that PPP-depleted DCs induced decreased proliferation (Figure 3, b and c) and effector cytokine expression (Figure 3d) of OT-I T cells compared with WT DCs. To explore the effect of PPP on DCs antitumor immunity in vivo, WT and KO mice were inoculated with MC38 tumor cells. Notably, our findings revealed that PPP deficiency within DCs expedited tumor growth (Figure 3e) and led to premature lethality (Figure 3f). Moreover, the frequencies of mature DCs and cDC1 were reduced in KO mice, whereas cDC2 proportions remained comparable between WT and KO mice (Supplementary Figure 5b). The absolute numbers of infiltrating mature DCs, cDC1, and cDC2 per gram of tumor tissue were significantly lower in KO mice (Supplementary Figure 5c). The absence of PPP activity led to decreased expression of key functional genes such as Tnfa, Il12 and Ccr7 in tumor-infiltrating cDCs (Figure 3g). Notably, PPP deletion correlated with diminished proportions of IFN-- and TNF--producing CD4+ and CD8+ T cells within the TME (Figure 3, h and i). We extended our studies in mice challenged with B16-F10 and LLC tumor cells. Also, PPP deficiency in DCs notably impairs antitumor immunity (Figure 3, j-n, and Supplementary Figure 5, d-h).

To explore the PPP effect on DC-mediated tumor immunotherapy, we employed the BMDC-based therapeutic tumor model8,17,18,40,41. We injected WT and KO BMDCs to the wild-type MC38-OVA tumor-bearing mice (Supplementary Figure 5i). Compared to WT DCs, KO DCs promoted tumor growth (Supplementary Figure 5j) and decreased T cell effector cytokine expression in the TME (Supplementary Figure 5, k and l). Overall, our data strongly suggest that PPP deficiency in cDCs restricts antitumor responses.

PPP ablation attenuates STING signaling in cDCs
To delve into the foundational mechanism by which the PPP supports cDC function, we isolated intratumoral cDCs from MC38 tumor-bearing WT and KO mice for smart RNA sequencing. Notably, Principal Component Analysis (PCA) unveiled distinct transcriptomic differences between WT and KO intratumoral cDCs (Figure 4a). Intriguingly, our KEGG analysis highlighted an enrichment in the cytosolic DNA-sensing pathway, JAK-STAT signaling pathway and PI3K-AKT signaling pathway (Figure 4b). Previous research had underscored the pivotal role of the cGAS-STING signaling pathway-an essential player in cytosolic DNA sensing-in orchestrating crucial DC-mediated antitumor functions within the TME42-44. And STING signaling activation stimulate the downstream activation of the JAK-STAT pathway45. Our prior investigation also had proved glycolysis improved STING-dependent DC antitumor responses18. Yet, the precise metabolic regulations governing the STING signature within intratumoral DCs remain largely enigmatic. To investigate these observations, we isolated early- and late-stage intratumoral cDCs from MC38 tumor-bearing wild-type mice. Western blot analysis revealed significantly reduced phosphorylation of STING and STAT1 in late-stage intratumoral cDCs (Figure 4c), indicating impaired activation of both the cGAS-STING and JAK-STAT signaling pathways. In contrast, PI3K-AKT signaling remained unchanged, as evidenced by comparable AKT phosphorylation levels between early- and late-stage cDCs (Figure 4c). To further validate these findings, we isolated intratumoral cDCs from both WT and KO MC38 tumor-bearing mice. Our results confirmed that G6PDX deficiency obviously impaired STING and STAT1 phosphorylation, but had no effect on AKT phosphorylation (Figure 4d). Moreover, the expression level of type I interferons was notably decreased in KO DCs (Figure 4e). Furthermore, we cultured WT and KO BMDCs in vitro and stimulated them with the STING signaling agonist Interferon Stimulatory DNA (ISD, a synthetic double-stranded DNA that sensed by cGAS to activate the cGAS-STING pathway). Our results indeed confirmed that PPP deficiency impaired STING signaling (Supplementary Figure 6, a and b).

To confirm the intricate crosstalk between the PPP and STING signaling, we exposed BMDCs to the PPP inhibitors G6PDi-1 and 6-AN following stimulation of the STING signaling. Notably, our findings revealed a clear impairment in STING phosphorylation, as well as the expression of type I interferons upon the PPP inhibition (Supplementary Figure 6, c-h). Tumor-infiltrating DCs recognize tumor-derived DNA to engage STING signaling, thereby amplifying DC antitumor responses46. Consequently, we freshly isolated intratumoral cDCs from MC38 tumor-bearing mice and subjected them to treatment with the PPP inhibitors. Encouragingly, the results validated that PPP deficiency hindered the STING signaling activation (Supplementary Figure 6, i-l). Expanding on our investigations, ectopic expression of G6PDX in murine DC cell lines (DC2.4) to heighten the PPP flux resulted in elevated activation of the STING signaling (Supplementary Figure 6, m and n). Likewise, treatment with the PPP agonist AG1 facilitated STING phosphorylation (Supplementary Figure 6o) and type I interferons expression (Supplementary Figure 6p). In summary, our data indicate that the PPP plays a supportive role in STING signaling within cDCs. To further explore the interplay between the PPP and STING signaling, we collected STING signaling stimulated and non-stimulated cDCs for metabolomics. Interestingly, STING stimulation elicited an upregulation of metabolites associated with the PPP (Supplementary Figure 6q), suggesting a positive correlation between the PPP flux and STING signaling. Collectively, our results demonstrate that PPP deficiency in cDCs impairs STING signaling activation.

PPP supports STING-dependent cDC antitumor immunity
Given the supportive role of the PPP in STING signaling within cDCs, we proceeded to employ the BMDC-based therapeutic tumor model. Relative to WT DCs, STING deficient (Tmem173-/-) DCs resulted in accelerated tumor growth (Figure 4f), decreased frequencies of IFN-- and TNF--producing CD4+ and CD8+ T cells in the TME (Figure 4g and Supplementary Figure 6r). In contrast, when comparing STING deficient DCs to double-deficient knockout (DKO) DCs lacking both G6PDX and STING, no further alterations were discerned in terms of tumor growth or the proportions of IFN-- and TNF--producing CD4+ and CD8+ T cells (Figure 4, f and g, and Supplementary Figure 6r). To confirm the phenotype, we treated WT and STING-deficient DCs with AG1. In WT DCs, AG1 stimulation obviously decelerated tumor growth (Figure 4h). Conversely, in STING-deficient DCs, AG1 stimulation had no effect on tumor progression and antitumor immunity (Figure 4, h and I, and Supplementary Figure 6s). In conclusion, our results indicate that the PPP supports STING-dependent DC antitumor immunity.

To further test whether STING agonists retain their ability to activate DCs in PPP-deficient conditions, we treated BMDCs with the PPP inhibitor 6-AN, either alone or in combination with the STING agonist DMXAA (a direct small-molecule binder of murine STING). We found that 6-AN treatment suppressed STING signaling activation and downstream type I interferons expression in BMDCs, whereas co-treatment with 6-AN and DMXAA effectively restored STING signaling and downstream type I interferons expression (Supplementary Figure 6, t and u). In the BMDC-based tumor therapy model, DCs stimulated with 6-AN alone accelerated tumor growth (Figure 4j) and reduced the proportions of IFN-- and TNF--producing CD4+ and CD8+ T cells (Figure 4k and Supplementary Figure 6v) in the TME relative to untreated DCs. In contrast, DCs treated with both 6-AN and DMXAA exhibited delayed tumor growth and restored T cell cytokine production, achieving outcomes comparable to those of untreated DCs (Figure 4, j and k, and Supplementary Figure 6v). Furthermore, we performed similar BMDC-based tumor therapy experiment using WT and KO BMDCs. While PPP deficiency impaired STING signaling and downstream type I interferons expression, DMXAA stimulation in KO DCs successfully restored STING pathway activation and downstream type I interferons expression in vitro (Supplementary Figure 6, w and x), as well as DC-mediated antitumor function in vivo (Figure 4, l and m, and Supplementary Figure 6y). These results collectively demonstrate that PPP deficiency in cDCs compromises STING-dependent antitumor function, and that restoration of STING activity in PPP-deficient cDCs rescues their antitumor response.

Next, we isolated early- and late-stage tumor-infiltrating cDCs from MC38 tumor-bearing mice and treated late-stage cDCs with or without the DMXAA in vitro. These cells were then subcutaneously injected adjacent to the tumors to wild-type MC38 tumor-bearing mice. Compared with untreated late-stage intratumoral cDCs, those receiving DMXAA treatment exhibited delayed tumor growth and enhanced T cell cytokine production, achieving functional levels comparable to early-stage intratumoral cDCs (Figure 4, n and o, and Supplementary Figure 6z). These results demonstrate that restoring STING signaling in late-stage intratumoral cDCs rescues their antitumor immunity. In summary, the findings confirm that impaired STING activity contributes to the dysfunctional state of late-stage intratumoral cDCs.

PPP-driving purine metabolism boosts STING signaling
Subsequently, we delved deeper into the intricacies of how the PPP governed the regulation of STING signaling. PPP supplies NADPH and R5P (Supplementary Figure 7a). NADPH actively contributes to maintaining redox homeostasis. Measurement of both cellular and mitochondrial reactive oxygen species (ROS) revealed significantly higher levels in late-stage intratumoral cDCs compared to early-stage counterparts (Supplementary Figure 7, b and c). Therefore, we treated WT and KO BMDCs with a ROS scavenger, MITO-TEMPO. Remarkably, PPP deficiency in cDCs led to heightened ROS accumulation (Supplementary Figure 7d). Subsequent combined treatment with MITO-TEMPO successfully mitigated ROS levels in both WT and KO DCs (Supplementary Figure 7d). However, despite the reduction in ROS levels upon MITO-TEMPO administration, the phosphorylation of STING (Supplementary Figure 7e) and the expression of type I interferons (Supplementary Figure 7f) were not restored, even decreased in KO DCs. This suggested that the compromised STING signature resulting from the PPP deficiency was not attributable to ROS accumulation. R5P plays a pivotal role in nucleotide synthesis, encompassing purines and pyrimidines21,25. Next, we treated WT and KO BMDCs with R5P. We observed that PPP deficiency markedly suppressed STING signaling. Supplementation with R5P restored both STING activation and downstream type I interferons production (Supplementary Figure 7, g and h). These results confirm that impaired PPP activity curtails R5P production, thereby inhibiting STING signaling in DCs.

Furthermore, we treated DCs with the purine metabolism inhibitor 6-Mercaptopurine (6-MP). Remarkably, stimulation with 6-MP significantly hampered STING phosphorylation (Supplementary Figure 7i) and the expression of type I interferons (Supplementary Figure 7j). Conversely, inhibition of pyrimidine metabolism showed no discernible effect on STING signaling (Supplementary Figure 7, k and l). Building upon these findings, we then isolated intratumoral cDCs from MC38 tumor-bearing mice and subjected them to treatment with 6-MP. Encouragingly, the results validated that perturbation of purine synthesis impaired STING signaling (Supplementary Figure 7, m and n). In conclusion, our data demonstrate the pivotal role of PPP-derived purine metabolism in augmenting STING signaling. Since PPP deficiency reduces purine synthesis, we asked whether it induces DNA damage. Using etoposide as a positive control, we observed no significant difference in the DNA damage marker H2AX between WT and KO DCs (Supplementary Figure 7o). To further validate this finding, we compared early- and late-stage intratumoral cDCs and similarly detected no increase in DNA damage (Supplementary Figure 7p). In summary, our data indicate that PPP deficiency and the consequent purine depletion in cDCs do not lead to elevated DNA damage.

PPP deficiency impairs 2’ 3’-cGAMP production to restrict STING signaling
PPP deficiency undermines STING signaling. We delved deeper into elucidating the specific stage at which the PPP regulated STING signaling. By activating STING signaling using the STING direct agonists, cGAMP (the natural enzymatic product of cGAS that directly binds and activates STING) and DMXAA, we observed that PPP deficiency did not impact STING signaling upon stimulation with these agonists (Supplementary Figure 8, a-d). This suggests that the impairment of STING signaling mediated by the PPP deficiency occurs upstream of the STING molecule.

Cyclic GMP-AMP synthase (cGAS) senses double-stranded DNA, catalyzing ATP and GTP to generate 2’3’-cGAMP as a second messenger, which serves as the initiator for activating STING signaling47,48. Initially, we evaluated the production of 2’3’-cGAMP in both intratumoral cDCs and BMDCs. Interestingly, late-stage intratumoral cDCs exhibited significantly reduced levels of 2’3’-cGAMP compared to early-stage populations (Figure 5a). Furthermore, we observed that PPP deficiency markedly hampered 2’3’-cGAMP production in cDCs (Figure 5, b and d). Drawing from our aforementioned findings revealing PPP-driven purine metabolism supports STING signaling, we were spurred by existing studies demonstrating that deficiencies in purine synthesis diminished the nucleotide pool, including ATP and GTP49,50. This led us to postulate that PPP deficiency might restrict ATP and GTP production, thereby hindering 2’3’-cGAMP generation and impeding STING signaling. To validate this hypothesis, we examined the levels of ATP and GTP in both intratumoral cDCs and BMDCs. Strikingly, our data unveiled a notable reduction in ATP and GTP levels due to PPP ablation in cDCs (Figure 5, c and e). Our metabolomic analysis also confirmed significantly reduced ATP and GTP levels in late-stage intratumoral cDCs (Figure 2c). To determine whether the decline in ATP and GTP levels caused by PPP deficiency impairs DC function, we treated WT and KO BMDCs in vitro with varying concentrations of ATP and GTP. Supplementation with 100 µM ATP and GTP restored the expression of maturation markers in KO DCs to levels comparable to WT DCs, without affecting WT DCs (Supplementary Figure 8, e and f). Higher concentrations (300 and 500 µM) enhanced maturation in both genotypes (Supplementary Figure 8, e and f). Based on these results, 100 µM was selected as the working concentration for subsequent experiments. Correspondingly, qPCR analysis confirmed that 100 µM ATP and GTP supplementation also rescued the expression of functional cytokines in KO DCs, while leaving WT DCs unchanged (Supplementary Figure 8g). Furthermore, 100 µM ATP and GTP supplementation restored intracellular ATP and GTP levels in KO DCs to WT levels (Figure 5e) and reversed the decrease in 2’3’-cGAMP observed in KO DCs (Figure 5f). To further confirm this phenotype, we isolated late-stage tumor-infiltrating cDCs and treated them with ATP and GTP. ATP and GTP supplementation restored the expression of maturation markers in late-stage cDCs to levels seen in early-stage cDCs (Supplementary Figure 8h) and also rescued the expression of functional cytokines in late-stage cDCs (Supplementary Figure 8i). Notably, the restoration of ATP and GTP levels facilitated the recovery of STING phosphorylation (Figure 5g) and the expression of type I interferons (Figure 5h) in KO DCs. Interestingly, individual supplementation of ATP or GTP did not suffice in rescuing STING signaling in the KO DCs (Supplementary Figure 8, j and k). Subsequent validation through 6-MP treatment confirmed our observations, showcasing that inhibition of purine metabolism impaired 2’3’-cGAMP production following STING activation, unlike pyrimidine metabolism inhibition (Figure 5i). Crucially, the impairment in purine metabolism also led to restricted ATP and GTP levels within the DCs (Figure 5j). Intriguingly, the supplementation of ATP and GTP successfully restored 2’3’-cGAMP production and STING signaling upon 6-MP administration (Figure 5, j-n). Also, individual administration of either ATP or GTP failed to resuscitate STING signaling (Supplementary Figure 8, l and m). Recognizing that compromised 2’3’-cGAMP production serves as a linchpin in the inhibition of STING signaling by PPP deficiency, we directly provided KO DCs with 2’3’-cGAMP to normalize its levels to that of WT DCs (Figure 5o). Notably, the reinstatement of 2’3’-cGAMP showcased a recovery of STING signaling (Figure 5, p and q). Our collective findings indicate that PPP deficiency-induced restrictions in purine metabolism result in diminished ATP and GTP levels, thereby impeding 2’3’-cGAMP production and subsequently impairing STING signaling.

PPP improves the efficacy of DC-mediated tumor immunotherapy
Given the vital role of the PPP in sustaining STING signaling, which is crucial for DC-mediated antitumor function. We delved into exploring whether PPP could bolster DC-based immunotherapy. Employing the BMDC-based therapeutic tumor model, we administered ATP and GTP to KO DCs and noted a significant reduction in tumor size compared to untreated KO DCs (Figure 6a). Furthermore, this treatment led to the restoration of the number of tumor-infiltrating CD11c+ lymphocytes, CD4+ T, CD8+ T cells (Figure 6b), as well as enhanced frequencies of IFN-- and TNF--producing CD4+ and CD8+ T cells (Figure 6, c and d). Moreover, the activation of PPP by the agonist AG1 augmented the effectiveness of DC-mediated tumor therapy (Figure 6e). Notably, in KO DCs, treatment with AG1 did not result in any significant alterations in tumor size (Figure 6e), the populations of tumor-infiltrating CD11c+ lymphocytes, CD4+ T, CD8+ T cells (Figure 6f), or the frequencies of IFN-- and TNF--producing CD4+ and CD8+ T cells (Figure 6, g and h). PD-L1 plays a critical role in mediating tumor immune evasion following various cancer treatments51. In KO tumor-bearing mice, we observed elevated PD-L1 expression in intratumoral DCs compared to WT mice (Supplementary Figure 9, a-c). To investigate whether activating the PPP in DCs could enhance the effectiveness of immune checkpoint blockade, we inoculated wild-type mice with MC38 tumor cells and administered the anti-PD-L1 antibody treatment every 3 days for a total of three doses. Simultaneously, we subcutaneously injected AG1-treated or non-treated BMDCs adjacent to the tumors on days 3 and 6 post-tumor inoculation. Treatment with anti-PD-L1 antibody alone resulted in reduced tumor size (Figure 6i and Supplementary Figure 9d). Intriguingly, combining AG1 with anti-PD-L1 antibody treatment led to a notable reduction in tumor burden (Figure 6i and Supplementary Figure 9d), accompanied by increased populations of CD11c+ lymphocytes, CD4+ T, and CD8+ T cells (Figure 6j), along with higher frequencies of IFN-- and TNF--producing CD4+ and CD8+ T cells (Figure 6, k and l) in the TME. Taken together, our findings highlight that PPP activation has implications for enhancing the efficacy of DC-mediated tumor immunotherapy.

PPP supports STING signaling in DCs from human BC patient samples
Our study had shown that the PPP supported STING signaling in mice DCs. We had now extended our investigation to determine whether this phenomenon also held true in DCs derived from patients with cancer. Interestingly, we observed a positive correlation between G6PD expression and the level of DC infiltration, rather than tumor cells in Breast invasive carcinoma (BRCA) and Rectum adenocarcinoma (READ) (Supplementary Figure 10a). We further analyzed the correlation between cDC- and PPP-related gene signatures using TCGA datasets. The results revealed a positive correlation between these signatures in BRCA (Figure 7a). We next examined whether incorporating the PPP gene signature could improve the prognostic value of the cDC signature alone. Analysis showed that patients classified as cDChiPPPhi had the most favorable overall survival, while those in the cDClowPPPlow group exhibited the poorest outcomes in BRCA (Supplementary Figure 10b). Collectively, these findings position the PPP as a key metabolic determinant of cDC-mediated antitumor immunity, with combined cDC and PPP signatures offering enhanced prognostic stratification in BRCA.

To validate our discoveries, we freshly isolated DCs from breast cancer (BC) tissues. In line with our observations in mice, inhibiting the PPP through G6PDi-1 and 6-AN treatment impaired STING phosphorylation (Figure 7b) and type I interferons expression (Figure 7c). Furthermore, we treated human BC DCs separately with purine and pyrimidine metabolism inhibitors. Consistent with our murine findings, inhibition of purine but not pyrimidine metabolism suppressed STING signaling in human BC DCs (Figure 7, d-f, and Supplementary Figure 10, c and d). We noted that inhibition of PPP and purine metabolism both led to reduced ATP and GTP levels (Figure 7, g and l), along with impaired synthesis of 2’3’-cGAMP (Figure 7, h and m), ultimately resulting in diminished STING phosphorylation (Figure 7, i and n) and type I interferons expression (Figure 7, j, k and o). Notably, we found that supplementing ATP and GTP restored depleted pools in DCs exposed to G6PDi-1, 6-AN and 6-MP treatments, consequently reviving STING signaling (Figure 7, i, j, k, n and o). Moreover, direct supplementation of 2’3’-cGAMP was able to rescue STING signaling in the absence of PPP and purine metabolism (Figure 7, p-s). Collectively, our results demonstrate that the PPP maintains STING signaling in DCs from human BC patient samples.

Methods
Study approval
BC tissues samples were gathered from female patients with breast cancer at the Women's Hospital of Nanjing Medical University. Informed consent was obtained from all participants prior to sample collection, including consent to publish potentially identifying clinical information. The Clinical Research Ethics Committee of the Women’s Hospital of Nanjing Medical University granted the study protocol, ensuring adherence to ethical standards. Animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University, which sets a maximum allowable tumor size of 225 mm². Mice that exceeded this limit were humanely euthanized and used for subsequent in vitro analyses.

Human samples
BC tissues were obtained from 30 randomly selected female patients (See Supplementary Table 1) in Women's Hospital of Nanjing Medical University, with informed consent achieved from all patients. Tumor-infiltrating lymphocytes were isolated and concentrated via density-gradient centrifugation with Lymphoprep (78071, Stem cell). DCs were enriched via the Human Pan-DC Pre-Enrichment Kit (19521, Stem cell). The enriched DCs were treated with the indicated drugs and subsequently lysed for protein and RNA extraction for downstream analyses. This study was approved by the Medical Ethics Committee of Nanjing Women and Children's Healthcare Hospital (Approval No. 2024KY-137-01). All participants provided written informed consent prior to enrollment and did not receive financial compensation. All samples were collected and used exclusively for scientific research purposes in accordance with the approved ethical guidelines.

Mice
All mice strains were maintained with a C57BL/6 background. G6pdx-floxed mice (Cyagen) were crossed with Zbtb46-Cre mice (Shanghai Model Organisms) to generate G6pdx+/+Zbtb46-Cre mice and G6pdx fl/flZbtb46-Cre mice. Tmem173-/- mice (STING deficient) (Shanghai Model Organisms) crossed with G6pdxfl/flZbtb46-Cre mice to obtain G6pdxfl/flZbtb46-Cre Tmem173-/- mice. All mice used in our experiments were 6-12-week-old gender-matched mice. Both male and female mice were included to avoid sex bias. All mice were housed in a specific pathogen-free (SPF) facility at the Animal Core Facility of Nanjing Medical University under a standard 12-hour light/dark cycle, with ambient temperature maintained at 22 ± 1 °C and relative humidity at 50 ± 10%.

Cell lines and cell culture
DC2.4 (murine dendritic cells, SCC142) was purchased from Sigma-Aldrich. MC38 (murine colon cancer, ENH204-FP) was obtained from Kerafast. LLC (murine Lewis lung carcinoma, CRL-1642), B16-F10 (murine melanoma, CRL-6475), and HEK293T (human embryonic kidney cells, CRL-3216) were obtained from ATCC. LLC, MC38, and HEK293T cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. DC2.4 and B16-F10 cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin.

BMDC generation and culture
Bone marrow cells were extracted from tibiae and femurs of mice and subjected to treatment with red blood cell lysis buffer. The cells were then cultured for 7 days in RPMI 1640 medium containing 200 ng/mL FLT-3L (BE0098, Bio X Cell), 10% FBS and 1% penicillin/streptomycin to generate BMDCs. Live CD11c+MHC-II+ cells were isolated by flow cytometry for further experiments.

Cell isolation
Single-cell suspensions of the spleen were prepared for cell isolation. Splenic DCs (Spl-DC) were isolated utilizing the Mouse CD11c Positive Selection Kit (18780, Stem Cell). For tumor samples, tissues were enzymatically digested, followed by density-gradient centrifugation with Percoll (Cytiva) to enrich TILs. Tumor-infiltrating DCs (Tu-DC) were isolated via flow cytometry by gating on CD45+CD24-F4/80-Ly6C-CD11c+MHC-II+ cells.

Flow cytometry
Single-cell suspensions were prepared for flow cytometry analysis. For surface marker analysis, cells were incubated with specific fluorochrome-labeled antibodies in MACS buffer (PBS supplemented with 2% FBS and 1 mM EDTA) for 30 minutes following by flow cytometry analysis. For intracellular staining, cells were labeled with surface marker antibodies and then fixed with fixation/permeabilization buffer at 4°C for 1 hour. Subsequently, the cells were labeled with the relevant fluorochrome-conjugated antibodies in permeabilization buffer. To evaluate intracellular cytokine production, the cells underwent stimulation with phorbol 12-myristate 13-acetate (PMA), monensin and ionomycin for 4 hours, then harvested and incubated with appropriate fluorochrome-labeled antibodies for flow cytometry analysis.

Co-culture assay of OT-I T cells with DCs
OT-I T cells were isolated from the spleens of OT-I mice using the Mouse CD8 Positive Selection Kit (18953, Stem Cell). OT-II T cells were isolated from the spleens of OT-II mice using the Mouse CD4 T cell Isolation Kit (480033, Biolegend). For the detection of T cell expansion, 1x105 OT-I or OT-II T cells were labeled with CFSE (C34570, Thermo Fisher Scientific), then co-cultured with 3x104 DCs and supplemented with SIINFEKL peptides (A5503, Sigma) for 72 hours. The proliferation of T cells was analyzed via CFSE dilution using flow cytometry. To assess cell proliferation and effector cytokine expression of T cells, 1x105 OT-I or OT-II T cells were incubated with 3x104 DCs in the presence of SIINFEKL peptide for 72 hours, and the proportions of T cells producing Ki-67, GZMB, IFN- and TNF- were determined by flow cytometry.

Western blot
2x106 Cell pellets were gathered and then resuspended in the lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA, supplemented with phosphatase and protease inhibitor cocktails) at 4℃ for 20 minutes. The cell extracts were then centrifuged at 4°C for 10 minutes. After centrifugation, the supernatants resulting from the separation were collected for subsequent western blot analysis.

RNA extraction and qRT-PCR
Total RNA was extracted utilizing TRIzol Reagent (AG21102, Accurate Biology). Subsequently, cDNA was generated utilizing the one-step RT-gDNA digestion SuperMix for qPCR (11142ES60, YEASEN). The qRT-PCR was performed with specific primers as detailed in Supplementary Table 2, coupled with qPCR SYBR Green Master Mix (11202ES60, YEASEN).

RNA sequencing
For smart-RNA sequencing, tumor-infiltrating cDCs were isolated and purified via flow cytometry. Total RNA was enriched utilizing TRIzol Reagent and sequenced on the Illumina NovaSeq™ 6000 platform to acquire 150bp paired-end reads. Subsequent mapping to the Mus musculus reference genome (mm10) was achieved using HISAT2. GSEA software facilitated GSEA analysis, and KEGG pathway enrichment analysis was utilized by clusterProfiler. The RNA sequencing data have been uploaded to the Genome Sequence Archive of the National Genomics Data Center, available at https://ngdc.cncb.ac.cn/gsa/search?searchTerm=CRA020726 (accession numbers: CRA020726).

Signal-cell RNA sequencing
For single-cell RNA sequencing (scRNA-seq), tumor-infiltrating DCs were harvested from MC38 tumor-bearing mice. Transcriptomic profiling was conducted using the BD Rhapsody system. scRNA-seq libraries were constructed following the BD Rhapsody single-cell whole-transcriptome amplification workflow and sequenced utilizing DNBSEQ-T7 for 150 bp paired-end reads. Data analysis was proceeded in R (v4.4.0) via a Seurat (v5.1.0) pipeline. Cells displaying > 20% mitochondrial gene expression and > 5000 detected genes were filtered out. Clustering was done using the FindClusters() function with a resolution of 0.5. High lineage marker-expressing cell clusters (Cd3d, Cd19, Ncr1, Adgre1) were excluded. To investigate the potential function of cDCs, we further distinguished cDCs subpopulations (identified by Itgax, Flt3 and Zbtb46) from total DCs. Differential gene expression within specific clusters was explored with the FindMarkers() function, employing a two-sided Wilcoxon rank-sum test by default. The RNA sequencing data have been uploaded to the Genome Sequence Archive of the National Genomics Data Center, available at https://ngdc.cncb.ac.cn/gsa/search?searchTerm=CRA017022 (accession numbers: CRA017022).

Metabolite extraction and mass spectrometry
2x106 late- and early-stage intratumoral cDCs (live CD45+CD24-F4/80-Ly6C-CD11c+MHC-II+) were isolated by flow cytometry from MC38 inoculated wild-type mice. Cell pellet was collected by centrifuging at 100 × g and 4 ℃ for 5 minutes. Samples were mixed with 500 µL extraction buffer (methanol: water = 3:1, containing 0.5×internal standard). After placement on dry ice for pre-cooling, the samples underwent three freeze-thaw cycles in liquid nitrogen, followed by vortexing for 30 s to mix well. Subsequent sonication was performed in an ice-water bath for 15 minutes, and samples were then incubated at -40 °C for 1 hours. Following centrifugation at 13800 × g and 4 °C for 15 minutes, 200 µL of supernatant was collected into a 2.0 mL tube and dried under vacuum. The dried residue was then reconstituted in 160 µL of methanol: acetonitrile: water (1:1:2, v/v/v). The solution was filtered and placed into an autosampler vial for liquid chromatography-high-resolution mass spectrometry (LC-MS). For reversed-phase system, an Agilent 1290 UPLC system equipped with an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 × 150 mm) was used. For hydrophilic interaction liquid chromatography (HILIC) system, the same UPLC system was employed with an Atlantis Premier BEH Z-HILIC column (1.7 µm, 2.1 × 150 mm). An AB Sciex Triple Quad 6500+ mass spectrometer was used for metabolite detection. Raw LC-MS/MS data were processed using SCIEX Analyst Workstation Software (v1.7.3), and metabolite quantification was performed with the automated analysis software BIOTREE Bio Bud (v2.0.3)

DC stimulation and drug treatment
DC2.4, BMDCs and tumor-infiltrating DCs were quantified and then seeded into designated cell culture plates. To trigger STING signaling in DC2.4 and BMDCs, the cells were subjected to treatment with ISD, cGAMP or DMXAA for 4 hours. ISD and cGAMP were transfected utilizing PEI (40815ES03, YEASEN) transfection reagent. DCs were treated with MITO-TEMPO (20 μM), R5P (200 μM), AG1 (3 μM), G6PDi-1 (100 μM), 6-AN (50 μM), 6-MP (50 μM), 5-FU (10 μM), LEF (50 μM) or BRQ (1 μM) for 12 hours and then harvested for further experiments. The ISD used in this study was prepared with equimolar amounts of oligonucleotides: F: 5′-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACATACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3′; R: 5′-TGTAGATCATGTACAGATCAGTCATAGATCACTAGTAGATCTGTATGTAGATCATGTACAGATCAGTCATAGATCACTAGTAGATCTGTA-3′. The oligonucleotides were annealed in PBS at 70℃ for 30 minutes before cooling to room temperature.

Nucleotides supplement assay
BMDCs were pre-treated with ATP (100 μM) and GTP (100 μM) for 4 hours, followed by treatment with the corresponding drugs for 12 hours. Subsequently, the quantified cells were plated into specified cell culture plates and stimulated with ISD for 4 hours before being harvested for subsequent experiments. Tumor-infiltrating DCs were counted and then seeded into designated cell culture plates. After pre-treatment with ATP and GTP for 12 hours, the cells were collected for further investigations.

ATP, GTP and cGAMP measurement
To measure ATP levels, 3x105 cells were plated in the 12-well cell culture plate, and the intracellular ATP levels were detected by the Luminescent Cell Viability Assay (G7570, Promega) in accordance with the instructions of manufacturer. For GTP and cGAMP measurements, 1x106 cell pellets were lysed in Protein Extraction Reagent (78503, Thermo Fisher Scientific). Subsequently, the cell lysates were utilized for assessing intracellular GTP levels and cGAMP levels using the GTP ELISA Kit (66393A, MEIBIAO BIOLOGY) and the 2’3’-cGAMP ELISA Kit (501700, Cayman Chemical) respectively, according to the instructions of manufacturer.

Stable cell lines generation
Mouse G6pdx was cloned into the MSCV2.2 plasmids. HEK293T cells were transfected with MSCV2.2-G6PDX plasmid in conjunction with pge plasmid to generate lentivirus. After 48 hours, the supernatants containing the lentivirus were collected. DC2.4 cells were then exposed to the lentivirus-containing supernatants with polybrene (10 μg/mL) for 12 hours. Subsequently, the medium was refreshed for another 12 hours, repeating this cycle. The transduced G6PDX-overexpressing cells (GFP+) were isolated using flow cytometry for subsequent experiments.

Tumor model
Tumor size, calculated as length × width (mm²), served as an indicator of tumor growth. Tumor-induced lethality was defined as a tumor reaching 225 mm². For the subcutaneous tumor model, MC38 (5x105 cells per mouse), B16-F10 (1x106 cells per mouse) and LLC (1x106 cells per mouse) were injected subcutaneously into G6pdx+/+Zbtb46-Cre and G6pdxfl/flZbtb46-Cre mice. For the BMDC-based therapeutic tumor model, wild-type mice were injected subcutaneously of MC38-OVA (5x105 cells per mouse) tumor cells. On day 3 and day 6 post tumor cell inoculation, the mice were subcutaneously injected adjacent to the tumors with BMDCs (3x106 cells per mouse) that had been pre-treated with SIINFEKL peptides (A5503, Sigma) overnight. For another DC-based therapy tumor model, MC38 (5x105 cells per mouse) tumor-bearing wild-type mice (on day 6 post tumor cell inoculation) were subcutaneously injected adjacent to the tumors with 3x106 intratumoral cDCs. Early- and late-stage intratumoral cDCs were isolated from wild-type MC38 tumor-bearing mice (5×10⁵ cells per mouse) at day 10 and day 20 post-inoculation, respectively. Intratumoral cDCs (live CD45+CD24-F4/80-Ly6C-CD11c+MHC-II+) cells were isolated by flow cytometry. For in vitro treatment, early-stage intratumoral cDCs were treated with PPP inhibitors (G6PDi-1 or 6-AN) or untreated (Ctrl) for 12 hours, and late-stage intratumoral cDCs were treated with the PPP agonist AG1 or untreated (Ctrl) for 12 hours prior to subcutaneous injection. For the combined anti-PD-L1 therapeutic tumor model, wild-type mice received an injection of MC38-OVA (5x105 cells per mouse) tumor cells and treated with anti-PD-L1 antibodies or IgG2b isotype control (100 ug per mouse) every 3 days for a total of three doses. Additionally, AG1-treated or non-treated BMDCs (3x106 cells per mouse), pre-incubated with SIINFEKL peptides overnight, were subcutaneously injected adjacent to the tumors on days 3 and 6 post-tumor inoculation.

Quantification and statistical analysis
Statistical analysis was carried out utilizing GraphPad Prism 8.0 software, with results presented as mean ± SEM. Two-tailed unpaired Student’s t-tests were utilized to assess parameters between two distinct groups. One-way ANOVA was employed to analyze the comparisons involving more than two groups with a single variable. Two-way ANOVA analyses were conducted for comparing multiple experimental groups or continuous outcomes. Survival curves underwent assessment via the log-rank (Mantel-Cox) test. Gene expression levels from scRNA-seq data were examined using a two-sided Wilcoxon rank-sum test.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Liu investigates how tumor immunoediting affects DC function, focusing on the role of the pentose phosphate pathway (PPP). Using single-cell analysis and functional assays, the authors report that late-stage tumor–infiltrating DCs exhibit reduced PPP activity, which compromises their antitumor capacity. Mechanistically, PPP-driven purine metabolism is proposed to sustain cGAS–STING signaling and thereby promote DC-mediated immune responses. The study suggests PPP as an intrinsic metabolic checkpoint of DC function and a potential therapeutic target to enhance immune checkpoint blockade (ICB). This work addresses an important question at the intersection of tumor immunology and immunometabolism, and the link between PPP and STING signaling is intriguing. However, several aspects of the data presentation and mechanistic claims require clarification and further support.\nMajor Comments\n1. The study demonstrates that PPP deficiency leads to DC dysfunction, while pharmacological or genetic activation of the PPP only modestly restores DC function. This suggests that PPP is necessary but may not be sufficient for full DC activation. The authors should clarify this point and discuss whether direct STING agonist treatment might represent a more effective strategy than PPP augmentation.\n2. In Figure 4L, it remains unclear whether STING agonists retain their ability to activate DCs in PPP-deficient conditions or PPP knockout mice. Testing this point would clarify whether PPP is strictly upstream of STING signaling or acts in parallel.\n3. In Supplementary Figure 6, the use of 6-MP shows that purine metabolism is required for STING signaling. However, no data are provided on whether supplementing R5P can restore signaling. Without this, the claim that“PPP- driving purine metabolism boosts STING signaling”might be overstated. Rescue experiments or a toned-down interpretation is needed.\n4. In Figure 6A, the model suggests that reduced PPP activity limits ATP/GTP production and thereby compromises DC function. If so, supplementation with ATP/GTP in wild-type DCs at late tumor stages should alleviate dysfunction. The lack of such an effect requires explanation.\n5. Tumor growth curves across treatment groups appear modest and could be problematic, dose and timing optimization for better therapeutic effects? To better illustrate therapeutic effects, the authors should provide individual tumor growth trajectories in addition to group averages.\n6. While PPP activation is proposed to synergize with ICB, the therapeutic benefit appears modest. Stronger preclinical evidence or more nuanced discussion of clinical implications might be helpful to understand whole picture.\nMinor Comments\n1. Figure 2D, 2H, S2B, S2C, S2F, S2G, S8A-C: The data presentation is not appropriate. The authors normalized all values by dividing by 10³, which obscures the actual magnitude of differences. It would be more informative to present the raw MFI values directly and then assess the differences between groups accordingly. Please revise the figure and reanalyze the data.\n2. In Figure 4D, the actin loading control appears inconsistent across samples, which raises concerns about normalization. The authors should clarify or provide quantification to ensure reliability of the results.\n3. In Figure 6B, DC numbers are presented without accounting for differences in tumor size. Normalization to tumor weight (e.g., DCs per gram tumor) would allow more accurate comparisons.", "reviewer_2": "In the manuscript “Tumor immunoediting shapes pentose phosphate pathway to dictate DC function”, Liu et al. demonstrate that late-stage tumors exhibit reduced pentose phosphate pathway (PPP) activity, which correlates with a diminished ability to activate T cells within the tumor. Their findings suggest that the tumor microenvironment suppresses PPP activity, which is essential for fueling purine metabolism and providing ATP and GTP for 2’3’-cGAMP synthesis and cGAS activity. Reduced PPP activity ultimately leads to impaired cGAS signaling in dendritic cells and decreased expression of type I interferon genes.\nThe concept of the manuscript is well formulated, and the in vitro experiments using bone marrow–derived dendritic cells to show how PPP activity supports cGAS activation were interesting and well executed. However, the in vivo results that form the basis of the authors’ hypothesis and subsequent experiments lack critical controls and fail to account for the functional diversity of dendritic cells, which varies by origin (type 1 or type 2 DC) and maturation state (e.g., CCR7⁺ DCs). Although the authors performed single-cell RNA sequencing of dendritic cells and highlighted their heterogeneity, all in vivo experiments were conducted on total dendritic cells. This is problematic because CCR7⁺ mature dendritic cells are known to be the primary subset capable of activating T cells in tumors, and DC1 and DC2 populations differ in their ability to cross-present antigens.\nBelow are specific comments to improve and strengthen the manuscript:\nMajor Points\n• Figure 1: The metabolomics assay is not described in the Materials and Methods section. How was it performed on tumor-infiltrating DCs? Without this information, it is difficult to interpret the results—specifically, the number and type of cells analyzed.\n• How did the authors define the DC population in the single-cell dataset? Can they compare their data to publicly available dendritic cell signatures? How do they explain the absence of mature DCs in their single-cell analysis?\n• If different DC subsets were identified in the single-cell data, why were subsequent analyses performed on total DCs? Why were maturation markers and cytokines assessed in immature DCs rather than focusing on mature CCR7⁺ DCs?\n• Figure 1: Why did the authors validate the single-cell data using qPCR? Can they validate CCR7 expression at the protein level? Does the downregulation of maturation genes indicate fewer mature DCs in the tumor or reduced protein levels within mature DCs?\n• The GSEA was performed on total DCs. Could the authors identify mature DCs in the single-cell dataset and perform GSEA specifically on this population, which is responsible for T-cell activation?\n• For KEGG pathway analysis, it would be important to determine whether mature DCs exhibit lower PPP activity.\n• Figure 2: The authors treated tumors with PPP inhibitors and assessed maturation markers. Instead, could they quantify the proportion of mature CCR7⁺ DCs and measure the gMFI of maturation markers in this subset?\n• Figure 2: The description of how tumor DCs were recovered, stimulated, and re-injected is unclear. Were these total DCs? Were mature DCs excluded? Were DC1 and DC2 subsets considered? What was the viability of these cells after sorting and 12-hour in vitro stimulation prior to re-injection?\n• Related to the previous point: Did the treatment induce maturation of immature DCs in vitro? How were injected and non-injected tumor DCs distinguished for comparison? The experimental design is not well explained. Were control samples injected with untreated tumor DCs recovered and re-injected after 12 hours? Did the treatment itself alter maturation markers before injection?\n• Still in Figure 2: When augmenting PPP activity, the authors assessed maturation markers by qPCR in total DCs. Could they instead quantify mature DCs in the tumor and measure gMFI of maturation and activation markers? It is difficult to conclude that PPP activity impacts DC maturation and activation based solely on RNA levels in total DCs.\n• Figure 3: Could the authors quantify mature DCs in tumors from G6pdx⁺/⁺ Zbtb46-Cre (WT) and KO mice? Also, assess immature DC1 and DC2 infiltration to determine whether KO DCs have reduced tumor infiltration capacity.\n• Figure 4: The rationale for injecting G6pdx⁺/⁺ Zbtb46-Cre (WT) and KO BMDCs into tumors and monitoring tumor growth is unclear. If the tumor microenvironment impairs PPP activity in DCs, why would WT BMDCs be unaffected? Wouldn’t WT BMDCs also experience PPP suppression? Please clarify.\n• What is ISD, which was used to stimulate BMDCs?\n• Figure 4J: The rationale for comparing tumor growth after injection of WT versus STING-pathway KO BMDCs is unclear. How do the authors explain that injected BMDCs promote tumor growth compared to WT BMDCs? Please comment.\n• Figure 6: Does treatment with ATP and GTP induce maturation in KO DCs? Is the observed effect due to injecting mature BMDCs? Why is there no effect in WT BMDCs treated with ATP and GTP? The lack of tumor growth reduction in WT suggests that supplementation is insufficient to overcome PPP deficiency. How do the authors explain that KO BMDCs promote tumor growth and that supplementation restores tumor growth to WT levels?\n• Do KO BMDCs become more immunosuppressive in the tumor compared to WT BMDCs?", "reviewer_3": "Several studies in pre-clinical models have demonstrated an indespensible role for conventional dendritic cells (cDCs), particularly cDC1s, in promoting T cell responses to cancer and augmenting the efficacy of T cell‐based immunotherapies. While the link between cancer immunity and cDCs has been extensively studied, there is now growing interest in understanding the cel intrinsic determinants of cDC that govern cDC function.\nWork from Liu et al. reports cDC metabolism as a key component of their anti-tumour activity. The authors showed that the pentose phosphate pathway (PPP) is essential for cDC activation and their capacity to prime T cells. Although PPP activity is evident in intratumoural cDCs at early stages, it becomes suppressed during later stages of tumour progression, thereby contributing to immune evasion. Using conditional knockout mouse models and ex vivo/in vitro assays, the authors further showed that PPP deficiency compromises STING phosphorylation and downstream signalling. Pharmacological activation of the PPP restores antitumor immunity and enhances the efficacy of immune checkpoint blocakde. Finally, analysis of human breast cancer samples reveals that the expression of key PPP enzymes correlates with signatures of intratumoral cDC accumulation. While the mechanism proposed by the authors meets the novelty criteria expected for publication in Nature Communications and the experiments are generally well designed, the following points need to be addressed to improve the clarity and overall quality of the manuscript.\nMajor points:\n- It would be important to clarify whether the cDC1/cDC2 ratio is maintained between early- and late-stage tumours. If this is not the case, it becomes difficult to draw firm conclusions from the T cell activation assays, as the observed differences in ex vivo T cell priming could result either from functional impairment of cDC subsets or from numerical differences in cDC1 and cDC2 populations between early and late tumours. The authors should therefore normalise the T cell activation assays based on the cDC1/cDC2 ratio to ensure comparability between the groups.\n- In Figure 1, it would be important to determine whether the activation of both cDC subsets (cDC1 and cDC2) as well as their ability to prime T cells are equally affected by the suppression of PPP-pathway in late tumours. Identifying which subset is primarily impacted would enhance the mechanistic understanding of this study. To this end, the authors should repeat the T cell activation assays using separately purified cDC1 and cDC2 populations.\n- In Figures 3E and F, the appropriate control for G6pdxfl/flZbtb46Cre mice is G6pdxfl/fl; the authors should repeat the tumour growth profile using this control line for accurate comparison. Alternatively, the authors could provide tumour growth data of WT, Zbtb46Cre and G6pdxfl/fl within the same experiment to demonstrate that tumour progression is comparable across these lines and that they can be used interchangeably.\n- Given that PPP-deficient cDCs show impaired ability to prime antigen-specific T cells, it would be important to clarify whether the tumour growth retardation observed in mice with enhanced cDC PPP activity is CD8⁺ T cell-dependent. The authors could address this by depleting CD8⁺ T cells using specific antibodies in mice adoptively transferred with cDCs exhibiting enhanced PPP activity.\nMinor but essential points:\n- Figure legends should be revised for clarity and completeness, explicitly stating the number and type of replicates in each panel (e.g., whether each point in the graphs represents a biological or technical replicate).\n- The statement on lines 152–153 “Consistent with the metabolic profiling, late-stage intratumoral cDCs exhibited markedly decreased NADPH and R5P production (Figure 2B).\" is not adequately supported by data and should be revised or removed.\n- The authors should clearly define every gene, abbreviation, and mouse model used in the figures and main text. For example, specify which protein is absent in TmemKO mice. Figure legends should be self-explanatory and allow readers to interpret the data without referring to the methods section.\nSimilarly, in figure 5 and 6, please clarify in the panels the exact genotypes of the WT and KO mice.\n- The combination “A + G + ISD” requires clarification. The authors should specify in both the main text and figure legend what each acronym represents and briefly describe the mechanism of action (text and methods) of these STING agonists, particularly in contrast to the other cGAS/STING agonists used elsewhere in the manuscript.\n- It would be interesting to investigate whether a high PPP-related gene signature enhances the predictive value of the cDC1 gene signature (e.g. comparing patients with high PPP/high cDC1, low PPP/high cDC1, low PPP/low cDC1, high PPP/low cDC1 expression). The authors could analyse some of the TCGA datasets previously used to show that intratumoral cDC1 gene signatures positively correlate with patient survival, as reported by JP Böttcher etal., 2018 (DOI: 10.1016/j.cell.2018.01.004 )\n- The title should be changed and refined based on the data the authors have provided. There is no experimental evidence for tumour immunoediting – immunoediting refers historically to the experiments that have been conducted by R D Schreiber’s group, none of which are listed in this manuscript.", "reviewer_4": "In the current study, Ben Liu et al. demonstrate that defects in the pentose phosphate pathway (PPP) occur in late-stage tumor-infiltrated dendritic cells (DCs). Impairment of the PPP reduces the levels of purine nucleotides ATP and GTP, which are essential substrates for cGAS enzymatic activity. This, in turn, results in insufficient activation of the STING pathway and consequently dampens DC function, limiting their antigen presentation capacity and cytokine production. The study identifies a critical role for the PPP in dysfunctional tumor-derived DCs and highlights a positive feedback loop between the PPP and STING pathways, providing potential therapeutic targets and mechanistic insights for enhancing DC function in cancer therapy.\nThe following are our comments and suggestions that the authors may consider to further strengthen their study:\n1. Can the authors examine the levels of pyrimidines in early- versus late-stage tumor-derived DCs?\n2. The authors report that ROS accumulation is a consequence of PPP deficiency in DCs. Could they provide data on both mitochondrial and cellular ROS levels in early- and late-stage DCs?\n3. Since purine levels are reduced in late-stage DCs, does this lead to DNA damage in these cells?\n4. Could the authors elaborate on why PPP deficiency induces PD-L1 expression in DCs? Given that STING activation has previously been shown to promote PD-L1 expression and support DC activation, how do these findings align?\n5. Metabolic rewiring is a hallmark of DC activation as well as of dysfunctional DCs in the tumor microenvironment. Can the authors describe whether deficiency or inhibition of the PPP and STING pathways affects DC metabolism more broadly?\n6. Is PPP deficiency in late-stage DCs due to limited glucose availability, and is the expression or activity of G6PD altered?\n7. Is treatment with a STING agonist alone sufficient to restore the function of late-stage DCs?\n8. Could the authors clarify which DC subsets were included in the gating strategy for tumor-infiltrating DCs (Tu-DCs)? The current gating of CD45⁺CD24⁻F4/80⁻Ly6C⁻CD11c⁺MHC II⁺ cells may encompass both cDC1 and cDC2 populations. In addition, please specify the gating strategy used for Flt3L-derived BMDCs.\nOther comments:\n1. Please provide more detailed information on the experimental procedures. In particular, the ratios and cell numbers used are not clearly described. For example:\n  DC cell numbers used for Western blot analysis\n  The number of DCs transferred in adoptive transfer experiments (early vs. late DCs from tumors)\n  The ratios and cell numbers of DCs and T cells used in co-culture experiments\nThese details are essential for reproducibility and should be revised accordingly."}, "author_response": "We deeply appreciate the thorough analysis and constructive suggestions provided by the reviewers for further improving our manuscript. We have studied reviewer’s comments carefully and have made revision in blue in the revised manuscript. As described in more details below, we have performed the requested experiments. With these extensive revisions, we believe that we have now addressed all of the raised issues in a satisfactory manner and, consequently, substantially strengthened our paper. In the following, we provide point-by-point response to the reviewers’ comments.\nReviewer #1 (Remarks to the Author):\nThis manuscript by Liu investigates how tumor immunoediting affects DC function, focusing on the role of the pentose phosphate pathway (PPP). Using single-cell analysis and functional assays, the authors report that late-stage tumor–infiltrating DCs exhibit reduced PPP activity, which compromises their antitumor capacity. Mechanistically, PPP-driven purine metabolism is proposed to sustain cGAS-STING signaling and thereby promote DC-mediated immune responses. The study suggests PPP as an intrinsic metabolic checkpoint of DC function and a potential therapeutic target to enhance immune checkpoint blockade (ICB). This work addresses an important question at the intersection of tumor immunology and immunometabolism, and the link between PPP and STING signaling is intriguing. However, several aspects of the data presentation and mechanistic claims require clarification and further support.\nResponse: We sincerely thank the reviewer for their careful consideration of our manuscript and for providing these valuable comments, which have helped to improve our study. Please see below our point-by-point response to each comment, including revisions to the manuscript. Major revisions to the manuscript text have been highlighted in blue.\nMajor Comments\n1. The study demonstrates that PPP deficiency leads to DC dysfunction, while pharmacological or genetic activation of the PPP only modestly restores DC function. This suggests that PPP is necessary but may not be sufficient for full DC activation. The authors should clarify this point and discuss whether direct STING agonist treatment might represent a more effective strategy than PPP augmentation.\nResponse: We thank the reviewer for these insightful comments and have included additional explanation and experiments.\nFirst, we agree with the reviewer's viewpoint that PPP is necessary but may not be sufficient for full DC activation. PPP deficiency creates a broad metabolic crisis including redox imbalance and nucleotide shortage. Therefore, PPP activity is necessary to establish a permissive metabolic foundation. Its restoration rescues this foundation, allowing DCs to respond to stimuli. However, the magnitude of the final immune response is also contingent on the availability and integration of other specific signals. Thus, while PPP is indispensable, maximal DC activation requires coordinated metabolic and signaling inputs.\nSecond, our findings at least established that PPP activity was essential for DC function and antitumor immunity (Figure 2 and 3). Notably, treatment with the PPP agonist AG1 restored the functionality of late-stage intratumoral cDCs (Figure 2, j-o). Furthermore, enhancing PPP activity via AG1 in wild-type DCs significantly boosted their function and antitumor efficacy (Figure 4, h and i, Supplemental Figure 6s, and Figure 6, e-l). Together, these results demonstrate that PPP is indispensable for DC function.\nThird, we also agree with the reviewer's insightful suggestion regarding the comparative efficacy of PPP and STING agonists in modulating DC function and antitumor immunity. To address this point, we conducted BMDC-based tumor therapy experiments in which wild-type BMDCs were treated with either the PPP agonist AG1 or the STING agonist DMXAA. Both treatments induced comparable levels of DC activation, as measured by expression of maturation markers including MHC-I, CD80 and CD86. Furthermore, BMDCs stimulated with either AG1 or DMXAA similarly delayed tumor growth and enhanced T cell cytokine production in the TME relative to untreated controls, with no significant differences observed between the two agonist treatments (Figure I). These results collectively indicate that both PPP augmentation and STING activation represent effective strategies for enhancing DC-based antitumor therapy at an appropriate activation intensity.\nFigure I: Both PPP augmentation and STING activation enhance DC-based antitumor immunity. Wild-type BMDCs were pulsed with OVA peptide for 12h and subsequently incubated with the PPP agonist AG1 (3 µM), the STING agonist DMXAA (7.5 µM), or untreated (Ctrl) for 4h in vitro. On day 3 and 6 post-inoculation, the treated BMDCs (3 × 10⁶ cells per mouse) were injected subcutaneously adjacent to the established MC38-OVA tumors. Flow cytometry analysis (left) and graph presentation (right) of MHC-I (a), CD80 (b) and CD86 (c) of BMDCs (n = 4). d: Tumor growth of wild-type mice (n = 10). Flow cytometry analysis of the proportions of IFN-- (e) and TNF--producing (f) tumor-infiltrating CD4+ and CD8+ T cells from (d) (n = 6). Data were presented as mean ± SEM. (a-c, e and f) were analyzed through one-way ANOVA analyses with Tukey’s multiple comparison test. (d) was analyzed through two-way ANOVA analyses with Tukey’s multiple comparison test. In a-c (right), n indicates the number of biological replicates. In d-f, n indicates the number of mice.\nMore importantly, this comparative result provides a strategic insight: PPP augmentation may hold particular therapeutic value in tumor contexts where metabolic dysfunction could render DC unresponsive to signaling agonists alone. Thus, our work not only identifies PPP as a critical regulator but also suggests a metabolic priming strategy to potentially broaden the efficacy of existing immunotherapies.\n2. In Figure 4L, it remains unclear whether STING agonists retain their ability to activate DCs in PPP-deficient conditions or PPP knockout mice. Testing this point would clarify whether PPP is strictly upstream of STING signaling or acts in parallel.\nResponse: We appreciate the reviewer’s helpful comments and have included additional experiments.\nFirst, we treated BMDCs with the PPP inhibitor 6-AN, either alone or in combination with the STING agonist DMXAA. We found that 6-AN treatment suppressed STING signaling activation and downstream type I interferons expression in BMDCs, whereas co-treatment with 6-AN and DMXAA effectively restored STING signaling and downstream type I interferons expression (Supplemental Figure 6, t and u). In the BMDC-based tumor therapy model, BMDCs stimulated with 6-AN alone accelerated tumor growth (Figure 4j) and reduced the proportions of IFN-- and TNF--producing CD4+ and CD8+ T cells (Figure 4k and supplemental Figure 6v) in the TME relative to untreated BMDCs. In contrast, BMDCs treated with both 6-AN and DMXAA exhibited delayed tumor growth and restored T cell cytokine production, achieving outcomes comparable to those of untreated BMDCs (Figure 4, j and k, and supplemental Figure 6v).\nSecond, we further performed similar BMDC-based tumor therapy experiments using wild-type and G6pdx-KO (PPP deficient) BMDCs. While PPP deficiency impaired STING signaling and downstream type I interferons expression, DMXAA stimulation in G6pdx-KO DCs successfully restored STING pathway activation and downstream type I interferons expression in vitro (Supplemental Figure 6, w and x), as well as DC-mediated antitumor function in vivo (Figure 4, l and m, and Supplemental Figure 6y).\nThird, we treated wild-type (WT) and STING deficient (Tmem173-/-) BMDCs with the PPP agonist AG1. AG1 enhanced NADPH production-the primary metabolic output of the PPP-in both WT and STING deficient BMDCs (Figure II), confirming that PPP activation does not act downstream of STING signaling in DCs.\nFigure II: AG1 enhances NADPH production in both WT and STING-deficient BMDCs. Wild-type (WT) and STING deficient (Tmem173-/-) BMDCs were treated with AG1 (3 µM), or untreated (Ctrl) for 4h in vitro. Graph presentation of NADPH level (left) and NADP+/NADPH ratio (right) (n = 4). Data were presented as mean ± SEM. Data were analyzed through one-way ANOVA analyses with Tukey’s multiple comparison test. n indicates the number of biological replicates.\nOverall, these results demonstrate that direct STING agonist treatment indeed rescues the functional impairment of DCs induced by PPP inhibition, and PPP act as upstream of STING signaling in DCs.\n3. In Supplementary Figure 6, the use of 6-MP shows that purine metabolism is required for STING signaling. However, no data are provided on whether supplementing R5P can restore signaling. Without this, the claim that “PPP-driving purine metabolism boosts STING signaling” might be overstated. Rescue experiments or a toned-down interpretation is needed.\nResponse: We sincerely appreciate the reviewer’s helpful comments and comprehensive consideration of our manuscript. To address these comments, we treated wild-type (WT) and G6pdx-KO (PPP deficient) BMDCs with R5P. We observed that PPP deficiency markedly suppressed STING signaling. Supplementation with R5P restored both STING activation and downstream type I interferons production (Supplemental Figure 7, g and h). These results confirm that PPP impairment disrupts purine synthesis by limiting R5P availability, thereby inhibiting STING signaling in DCs.\n4. In Figure 6A, the model suggests that reduced PPP activity limits ATP/GTP production and thereby compromises DC function. If so, supplementation with ATP/GTP in wild-type DCs at late tumor stages should alleviate dysfunction. The lack of such an effect requires explanation.\nResponse: We sincerely thank the reviewer for your valuable comments. In accordance with the suggestion, we isolated late-stage tumor-infiltrating DCs from MC38 tumor-bearing wild-type mice and treated them in vitro with ATP and GTP for 12h. Supplementation with ATP and GTP restored the expression of key maturation markers including MHC-I, CD80, and CD86 in late-stage intratumoral DCs to levels comparable to early-stage intratumoral DCs (Supplemental Figure 8h). Correspondingly, qPCR analysis confirmed that ATP and GTP supplementation also rescued the expression of functional cytokines in late-stage intratumoral DCs (Supplemental Figure 8i). Taken together, these findings demonstrate that PPP dysfunction in late-stage intratumoral DCs disrupts purine metabolism, resulting in diminished ATP and GTP levels that contribute to functional impairment. Importantly, supplementation with ATP and GTP effectively rescues the functional capacity of late-stage intratumoral DCs.\n5. Tumor growth curves across treatment groups appear modest and could be problematic, dose and timing optimization for better therapeutic effects? To better illustrate therapeutic effects, the authors should provide individual tumor growth trajectories in addition to group averages.\nResponse: We thank the reviewer for your valuable comments. In accordance with the suggestion to enhance tumor therapeutic efficacy, we had modified the treatment protocol by initiating PD-L1 administration at an earlier timepoint and incorporating an increased dose of AG1. The updated experimental scheme, now included in the revised manuscript as follows: “For the combined anti-PD-L1 and AG1 therapeutic tumor model, wild-type mice were received an injection of MC38-OVA (5x105 cells per mouse) tumor cells and treated with anti-PD-L1 antibodies (100 μg per mouse) at days 6, 9 and 12 post-tumor inoculation for a total of three doses. Additionally, AG1-treated (4 μM) or non-treated BMDCs (3x106 cells per mouse), pre-incubated with OVA overnight, were subcutaneously injected adjacent to the tumors on days 3 and 6 post-tumor inoculation.”\nWe observed that anti-PD-L1 antibody treatment or AG1 stimulation alone resulted in reduced tumor size (Figure 6i and Supplemental Figure 9d). Notably, combining anti-PD-L1 antibody with AG1 led to a significant reduction in tumor size (Figure 6i and Supplemental Figure 9d), accompanied by increased populations of CD11c+ lymphocytes, CD4+ T, and CD8+ T cells (Figure 6j), along with higher frequencies of IFN-- and TNF--producing CD4+ and CD8+ T cells (Figure 6, k and l) in the TME. To better illustrate the therapeutic efficacy, we had included individual tumor growth curves in Supplemental Figure 9d. These data clearly show that the combination of anti-PD-L1 antibody and AG1 results in the smallest tumor volumes among all treatment groups.\n6. While PPP activation is proposed to synergize with ICB, the therapeutic benefit appears modest. Stronger preclinical evidence or more nuanced discussion of clinical implications might be helpful to understand whole picture.\nResponse: We sincerely thank the reviewer for this constructive feedback, which has significantly strengthened our study.\nFirst, in response to the observation that the efficacy of the ICB and AG1 combination was modest, we had optimized the treatment protocol by initiating anti-PD-L1 administration earlier and increasing the AG1 dosage. This revised strategy achieved the strongest antitumor effect among all treatment groups (Figure 6, i-l, and Supplemental Figure 9d). These results provide stronger preclinical evidence that PPP activation can effectively synergize with ICB.\nSecond, we fully agree that a more in-depth discussion of the clinical implications of PPP activation for tumor therapy would strengthen our study. We had therefore added the following discussion to the revised manuscript:\n“Enhancing the PPP in immune cells represents an emerging strategy to improve the efficacy of ICB in cancer therapy. Recent studies highlight its potential through improved T cell functionality and persistence in the TME. For instance, PPP activation has been shown to promote the generation and maintenance of TCF1 progenitor-like exhausted CD8 T cells, thereby enhancing tumor control when combined with PD-1 blockade. Another study demonstrates that the PPP metabolite Xu5P sustains the expansion of effector SLC35E2 CD8 progenitor-like exhausted T cells, and Xu5P supplementation synergizes with PD-1 blockade in tumor therapy. In our study, we found that PPP activation in DCs improved DC-mediated antitumor immunity. Moreover, PPP activation in DCs enhanced the therapeutic efficacy of anti-PD-L1 therapy. In conclusion, both previous reports and our current work suggest that activating the PPP in T cells or DCs constitutes a promising strategy for improving immunotherapy outcomes. Thus, selectively activating the PPP in T cells or DCs, in combination with ICB, represents a promising tumor therapy strategy worthy of investigation.”\nIn summary, the incorporation of above additional experiments and expanded discussion has strengthened the overall quality of our manuscript.\nMinor Comments\n1. Figure 2D, 2H, S2B, S2C, S2F, S2G, S8A-C: The data presentation is not appropriate. The authors normalized all values by dividing by 10³, which obscures the actual magnitude of differences. It would be more informative to present the raw MFI values directly and then assess the differences between groups accordingly. Please revise the figure and reanalyze the data.\nResponse: We thank for the reviewer’s helpful comments and for raising this point regarding the data presentation in our figures. Regarding your comments on Figures 2D, 2H, S2B, S2C, S2F, S2G, and S8A-C, we sincerely apologize for the confusion caused by our axis labeling. We would like to clarify that the raw MFI values were not normalized or divided by 10³. To make the axis labels more compact, we used the notation “10³” to represent thousands (e.g., labeling '1000' as '10³'). We realize now that this presentation was ambiguous and misleading, for which we take full responsibility.\nTo address these comments, we had revised all the mentioned figures in our revised manuscript. The axis labels had been updated to display the full, raw MFI values without the \"10³\" abbreviation. And we had confirmed that all statistical analyses and conclusions remain unchanged, as they were always based on the raw data. We sincerely appreciate your attention to detail, which has been instrumental in improving the clarity of our manuscript.\n2. In Figure 4D, the actin loading control appears inconsistent across samples, which raises concerns about normalization. The authors should clarify or provide quantification to ensure reliability of the results.\nResponse: We appreciate the reviewer’s careful observation and helpful suggestion for our manuscript. To address the comments, we had now quantified the Western blot signals for p-STING, p-STAT1, p-AKT, and actin using ImageJ in Figure 4d. The results confirmed that PPP deficiency significantly suppressed STING signaling and downstream JAK-STAT activation, without notably affecting the PI3K-AKT pathway (Figure 4d). And we had incorporated the quantitative data, expressed as phosphorylation-to-actin ratios, into the revised manuscript to enhance clarity and strengthen the support for our conclusions.\n3. In Figure 6B, DC numbers are presented without accounting for differences in tumor size. Normalization to tumor weight (e.g., DCs per gram tumor) would allow more accurate comparisons.\nResponse: We sincerely apologize for the lack of clarity in our original manuscript and thank the reviewer for highlighting this important point. We would like to clarify that the immune cell counts presented in Figure 6b, 6f, and 6j were already normalized per g of tumor tissue. To prevent any potential misunderstanding, we have revised the corresponding figure legends to explicitly state: \"Flow cytometry analysis of the numbers of tumor-infiltrating CD11c , CD4 T, and CD8 T cells per g of tumor tissue.\" This revision ensures transparency in data normalization and enables accurate cross-group comparisons independent of tumor size variations.\nReviewer #2 (Remarks to the Author):\nIn the manuscript “Tumor immunoediting shapes pentose phosphate pathway to dictate DC function”, Liu et al. demonstrate that late-stage tumors exhibit reduced pentose phosphate pathway (PPP) activity, which correlates with a diminished ability to activate T cells within the tumor. Their findings suggest that the tumor microenvironment suppresses PPP activity, which is essential for fueling purine metabolism and providing ATP and GTP for 2’3’-cGAMP synthesis and cGAS activity. Reduced PPP activity ultimately leads to impaired cGAS signaling in dendritic cells and decreased expression of type I interferon genes.\nThe concept of the manuscript is well formulated, and the in vitro experiments using bone marrow–derived dendritic cells to show how PPP activity supports cGAS activation were interesting and well executed. However, the in vivo results that form the basis of the authors’ hypothesis and subsequent experiments lack critical controls and fail to account for the functional diversity of dendritic cells, which varies by origin (type 1 or type 2 DC) and maturation state (e.g., CCR7 DCs). Although the authors performed single-cell RNA sequencing of dendritic cells and highlighted their heterogeneity, all in vivo experiments were conducted on total dendritic cells. This is problematic because CCR7 mature dendritic cells are known to be the primary subset capable of activating T cells in tumors, and DC1 and DC2 populations differ in their ability to cross-present antigens.\nResponse: We thank the reviewer for their time and constructive comments on our manuscript. We fully acknowledge the importance of DC functional diversity. In response, we have performed additional experiments and analyses focusing on cDC1, cDC2, and CCR7 DC subsets to address the concerns raised. We believe these additions substantially strengthen the study. Below is our point-by-point response. Corresponding revisions in the manuscript text are highlighted in blue.\nBelow are specific comments to improve and strengthen the manuscript:\nMajor Points\n1. Figure 1: The metabolomics assay is not described in the Materials and Methods section. How was it performed on tumor-infiltrating DCs? Without this information, it is difficult to interpret the results-specifically, the number and type of cells analyzed.\nResponse: We thank for the valuable comments. To address the reviewer’s question, we had added the detailed methods of metabolomics in the Materials and Methods section in the revised manuscript as follows: “Metabolite extraction and mass spectrometry-based metabolomics analysis: 2x106 late- and early-stage intratumoral cDCs (live CD45+CD24-F4/80-Ly6C-CD11c+MHC-II+) were isolated by flow cytometry from MC38 inoculated wild-type mice. Cell pellet was collected by centrifuging at 1000 rpm and 4 for 5 min. Samples were mixed with 500 µL extraction buffer (methanol: water = 3:1, containing 0.5×internal standard). After placement on dry ice for pre-cooling, the samples underwent three freeze-thaw cycles in liquid nitrogen, followed by vortex for 30 s to mix well. Subsequent sonication was performed in an ice-water bath for 15 min, and samples were then incubated at -40 °C for 1h. Following centrifugation at 12000 rpm and 4 °C for 15 min, 200 µL of supernatant was collected into a 2.0 mL tube and dried under vacuum. The dried residue was then reconstituted in 160 µL of methanol: acetonitrile: water (1:1:2, v/v/v). The solution was filtered and placed into an autosampler vial for liquid chromatography-high-resolution mass spectrometry (LC-MS). For reversed-phase (RP) system, an Agilent 1290 UPLC system equipped with an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 × 150 mm) was used. For hydrophilic interaction liquid chromatography (HILIC) system, the same UPLC system was employed with an Atlantis Premier BEH Z-HILIC column (1.7 µm, 2.1 × 150 mm). An AB Sciex Triple Quad 6500+ mass spectrometer was used for metabolite detection. Raw LC–MS/MS data were processed using SCIEX Analyst Workstation Software (v1.7.3), and metabolite quantification was performed with the automated analysis software BIOTREE Bio Bud (v2.0.3)”.\n2. How did the authors define the DC population in the single-cell dataset? Can they compare their data to publicly available dendritic cell signatures? How do they explain the absence of mature DCs in their single-cell analysis?\nResponse: We appreciate the reviewer's insightful comments and valuable suggestions.\nFor the identification of DC populations in the single-cell dataset, we referred to established DC-specific signatures and markers from previous published studies (Brown et al., 2019; Gerhard et al., 2021; Zhang et al., 2020). First, cell clusters expressing high levels of lineage markers (Cd3d, Cd19, Ncr1, Adgre1) were excluded. Subsequently, cDCs were defined based on canonical markers: Itgax, Flt3, and Zbtb46 (Figure III).\nFigure III: The identification of DC populations in the single-cell dataset. (a and b) Dot plots show the expression levels of marker genes across different clusters. The expression levels of canonical marker genes used to exclude other cells (a), and the expression levels of DC lineage marker genes in each cluster from a (b).\nThe protocol was employed as follows and had been added in the Method section of the revised manuscript: “Cells displaying > 20% mitochondrial gene expression and > 5000 detected genes were filtered out. Clustering was done using the FindClusters() function with a resolution of 0.5. High lineage marker-expressing cell clusters (Cd3d, Cd19, Ncr1, Adgre1) were excluded. To investigate the potential function of cDCs, we further distinguished cDCs subpopulations (identified by Itgax, Flt3 and Zbtb46) from total DCs. Differential gene expression within specific clusters was explored with the FindMarkers() function, employing a two-sided Wilcoxon rank-sum test by default.”\nWe apologize for the lack of clarity in our original manuscript, which led to the impression that mature DCs were absent. In fact, mature DCs are present in our dataset, as explicitly shown in the re-analyzed figures (Supplemental Figure 2, g and h). We fully agree with the reviewer's comments that CCR7 mature DCs (also known as mregDC) are known to be the primary subset capable of activating T cells in tumors, which were also proved by multitudinous previous studies (Lee et al., 2024; Maier et al., 2020; Ng et al., 2025; Zhang et al., 2020; Zhang et al., 2019). Based on publicly established markers of mature DCs (identified by expression of Ccl22, Ccl17, and Ccr7) (Ng et al., 2025; Zhang et al., 2020), we reanalyzed our single-cell RNA sequencing data to delineate this mature DC subpopulation. Our results further indicated that these existed a significant mature DC subpopulation both in early- and late-stage intratumoral DCs (Supplemental Figure 2, g and h), and the population of mature DCs from late tumors were downregulated compared to that in early tumors (Supplemental Figure 2i).\n3. If different DC subsets were identified in the single-cell data, why were subsequent analyses performed on total DCs? Why were maturation markers and cytokines assessed in immature DCs rather than focusing on mature CCR7 DCs?\nResponse: We appreciate the reviewer's insightful comments, which led to a valuable improvement of our study.\nWe sincerely thank the reviewer for raising this important point regarding the limitation of focusing solely on the total cDC population in our study. Our initial analyses on bulk cDCs, while identifying a global functional decline, lacked the resolution to attribute defects to specific functional subsets. In direct response to this comment, we have now performed a comprehensive re-analysis of our scRNA-seq data and conducted new experiments specifically on the sorted mature DC subset. As supported by latest studies, cDCs in the tumor microenvironment (TME) can be classified into three main subsets: cDC1, cDC2, and mature DCs (also referred to as mregDCs) (Lee et al., 2024; Maier et al., 2020; Ng et al., 2025; Zhang et al., 2019). Among these, mature DCs represent the dominant subset responsible for mediating antitumor immunity and are thought to originate from cDC1 and cDC2 following tumor antigen uptake (Maier et al., 2020; Ng et al., 2025; Zhang et al., 2019). Further analysis of our scRNA-seq data revealed a notable subset of mature DCs (defined by expression of Ccl22, Ccl17, and Ccr7) in late-stage tumors (Supplemental Figure 2, g and h). Flow cytometry analysis indicated the proportion of mature DCs was decreased in late-stage tumors (Supplemental Figure 2i). KEGG analysis confirmed that mature DCs from late-stage tumors displayed impaired antigen presentation capacity (Supplemental Figure 2k). Furthermore, we isolated mature DCs from early- and late-stage tumors and observed decreased expression of maturation markers and functional cytokines in the late-stage mature DCs (Supplemental Figure 2, l and m). Moreover, we isolated equal numbers of early- and late-stage mature DCs and co-cultured them with OT-I T cells in the presence of SIINFEKL peptide. Late-stage intratumoral mature DC demonstrated significantly reduced capacity to stimulate OT-I T cell proliferation (Figure 1, l and m) and effector cytokine production (Figure 1n) compared to their early-stage counterparts. In summary, these findings indicate that mature DCs in late-stage tumors exhibit functional impairment compared to those in early-stage tumors, which is consistent with our total cDCs phenotype.\nThese new findings do not contradict our initial observations on bulk cDCs; rather, they identify the mature DC subset as an important contributor to the overall functional impairment in our study. We believe this revision significantly strengthens the precision and impact of our study.\n4. Figure 1: Why did the authors validate the single-cell data using qPCR? Can they validate CCR7 expression at the protein level? Does the downregulation of maturation genes indicate fewer mature DCs in the tumor or reduced protein levels within mature DCs?\nResponse: Thanks for the reviewer’s valuable comments. In response to the reviewer’s question, we assessed CCR7 protein expression by flow cytometry in early- and late-stage tumors. The results showed that CCR7 protein levels were lower in late-stage tumors (Supplemental Figure 2i). Further analysis revealed a notable reduction in the numbers of CCR7 DCs per g of tumor tissue in late-stage tumors (Supplemental Figure 2j). Together, these findings indicate that both CCR7 expression and the abundance of CCR7 DCs are diminished in late-stage tumors.\n5. The GSEA was performed on total DCs. Could the authors identify mature DCs in the single-cell dataset and perform GSEA specifically on this population, which is responsible for T-cell activation?\nResponse: Thanks for the reviewer’s helpful suggestions. In response to the reviewer's comments, we reanalyzed our single-cell RNA sequencing data using established markers for mature DC subset (defined by expression of Ccl22, Ccl17, and Ccr7) (Maier et al., 2020; Ng et al., 2025; Zhang et al., 2019), enabling clear identification of this subset within the total DC population (Supplemental Figure 2, g and h). Subsequent GSEA analysis revealed impaired antigen processing and presentation capacity in late-stage intratumoral mature DCs (Supplemental Figure 2k), a finding consistent with the functional defects observed in the total cDCs population.\n6. For KEGG pathway analysis, it would be important to determine whether mature DCs exhibit lower PPP activity.\nResponse: We thank the reviewer for their valuable comments. To address the question, we performed KEGG pathway analysis. The analysis identified the PPP as one of the most significantly altered pathways in late-stage compared to early-stage mature DCs (Supplemental Figure 3d). We then isolated early- and late-stage intratumoral mature DCs and observed reduced levels of the key PPP metabolite NADPH in late-stage mature DCs (Supplemental Figure 3e). Additionally, qPCR analysis showed decreased mRNA expression of major PPP enzymes in late-stage mature DCs (Supplemental Figure 3f). Together, these findings demonstrate that late-stage intratumoral mature DCs exhibit impaired PPP activity.\n7. Figure 2: The authors treated tumors with PPP inhibitors and assessed maturation markers. Instead, could they quantify the proportion of mature CCR7 DCs and measure the gMFI of maturation markers in this subset?\nResponse: We greatly appreciate the valuable suggestions provided by the reviewer and have performed additional experiments to address this concern. We isolated early-stage intratumoral DCs from MC38 tumor-bearing wild-type mice and treated them with the PPP inhibitor G6PDi-1 for 12 in vitro. Flow cytometric analysis revealed that PPP inhibition reduced the proportion of mature CCR7 DCs (Supplemental Figure 3g), consistent with our single-cell RNA sequencing data. Furthermore, PPP downregulation decreased expression of key maturation markers including MHC-I, CD80, and CD86 within the mature DC subset (Supplemental Figure 3h). These results demonstrate that PPP activity is essential for maintaining both the abundance and functional maturation of CCR7 DCs in the TME.\n8. Figure 2: The description of how tumor DCs were recovered, stimulated, and re-injected is unclear. Were these total DCs? Were mature DCs excluded? Were DC1 and DC2 subsets considered? What was the viability of these cells after sorting and 12-hour in vitro stimulation prior to re-injection? Did the treatment induce maturation of immature DCs in vitro? How were injected and non-injected tumor DCs distinguished for comparison? The experimental design is not well explained. Were control samples injected with untreated tumor DCs recovered and re-injected after 12 hours? Did the treatment itself alter maturation markers before injection?\nResponse: We thank the reviewer for these detailed and critical questions, which have helped us notably improve the clarity of our experimental design.\nFor the DC population used, subset consideration, and exclusion: The transferred cells were total intratumoral cDCs, defined as live CD45 CD24- F4/80 Ly6C CD11c MHC-II cells. No subsets (including mature DCs, cDC1, or cDC2) were excluded. This population naturally contains a mixture of these subsets (Supplemental Figure 1). We chose this approach to model the therapeutic transfer of a total tumor-derived DC population. Furthermore, we had added detailed experimental design protocols of our DC-based therapy tumor model in the Method section of our revised manuscript “For another DC-based therapy tumor model, MC38 (5x105 cells per mouse) tumor-bearing wild-type mice (on day 6 post tumor cell inoculation) were subcutaneously injected adjacent to the tumors with 3x106 intratumoral cDCs. Early- and late-stage intratumoral cDCs were isolated from wild-type MC38 tumor-bearing mice (5×10⁵ cells per mouse) at day 10 and day 20 post-inoculation, respectively. Intratumoral cDCs (live CD45+CD24-F4/80-Ly6C-CD11c+MHC-II+) were isolated by flow cytometry. Early-stage intratumoral cDCs were treated with PPP inhibitors (G6PDi-1 or 6-AN) or untreated (Ctrl) for 12h in vitro, and late-stage intratumoral cDCs were treated with the PPP agonist AG1 or untreated (Ctrl) for 12h in vitro prior to subcutaneous injection.”\nFor the cell viability post-sorting and treatment: After sorting and the 12h in vitro treatment with PPP modulators (G6PDi-1, 6-AN, AG1) or control, cell viability was uncompromised (Supplemental Figure 3, k and l).\nFor the effect of in vitro treatment on DC maturation: The results demonstrated that the 12h treatment itself directly altered the maturation state of DCs prior to injection. PPP inhibition via G6PDi-1 or 6-AN downregulated DC maturation markers and functional gene expression (Figure 2, d-f), whereas PPP activation via AG1 enhanced these parameters (Figure 2, j-l).\nFor the distinguishing injected compared to non-injected DCs: In this model, different groups of mice received DCs subjected to different in vitro treatments. The comparison is made between these independent experimental groups.\nFor the control group: The critical control group consisted of mice injected with DCs that underwent the identical process of sorting and 12h in vitro culture but in the absence of PPP modulators (the “untreated Ctrl” group in our description).\nWe have incorporated all these details into the revised Methods section and Figure legends. This incorporation has notably helped improve the quality of our manuscript.\n9. Still in Figure 2: When augmenting PPP activity, the authors assessed maturation markers by qPCR in total DCs. Could they instead quantify mature DCs in the tumor and measure gMFI of maturation and activation markers? It is difficult to conclude that PPP activity impacts DC maturation and activation based solely on RNA levels in total DCs.\nResponse: We are deeply grateful for the reviewer's insightful suggestions. In response to this valuable feedback, we have conducted additional experiments to address this important concern. Late-stage tumor-infiltrating cDCs were isolated from MC38 tumor-bearing mice and treated in vitro with the PPP agonist AG1 or untreated for 12h. Flow cytometry analysis revealed that PPP activation increased the proportion of mature DCs (Supplemental Figure 3i). Further analysis on mature DCs showed elevated expression of maturation and activation markers including MHC-I, CD80, and CD86 following AG1 treatment (Supplemental Figure 3j). These results collectively demonstrate that PPP activation promotes the maturation and activation of intratumoral mature DCs.\n10. Figure 3: Could the authors quantify mature DCs in tumors from G6pdx / ⁺⁺ Zbtb46-Cre (WT) and KO mice? Also, assess immature DC1 and DC2 infiltration to determine whether KO DCs have reduced tumor infiltration capacity.\nResponse: We sincerely appreciate the reviewer's valuable comments. To address the questions raised, we inoculated G6pdx / Zbtb46-Cre (WT) and ⁺⁺ G6pdxfl/flZbtb46-Cre (KO) mice with MC38 tumor cells. Our results indicated that the proportions of CCR7 DCs (mature DCs) and cDC1s were decreased in KO mice compared to WT mice, whereas the percentage of cDC2s remained unchanged (Supplemental Figure 5b). Furthermore, the absolute numbers of CCR7 DCs, cDC1s and cDC2s per g of tumor tissue were all reduced in KO mice (Supplemental Figure 5c). These findings demonstrate that PPP deficiency in cDCs impairs the tumor infiltration capacity of mature and cDC1 subsets.\n11. Figure 4: The rationale for injecting G6pdx / Zbtb46-Cre (WT) and KO BMDCs into tumors and monitoring tumor growth is unclear. If the TME impairs ⁺⁺ PPP activity in DCs, why would WT BMDCs be unaffected? Wouldn’t WT BMDCs also experience PPP suppression? Please clarify.\nResponse: We sincerely appreciate the reviewer’s insightful comments, which raise an important theoretical consideration regarding the suppressive nature of the TME. We agree that if the TME broadly impairs PPP activity in all infiltrating DCs, the functional difference between wild-type (WT) and PPP-deficient (KO) cells might be attenuated.\nThe primary objective of our adoptive DC transfer model is precisely to isolate and evaluate the cell-intrinsic functional consequence of PPP deficiency, independent of the complex systemic metabolic suppression imposed by the established TME. To achieve this, we pulsed and activated BMDCs with a defined antigen (SIINFEKL peptide) and a potent stimulant (ISD) in vitro before transfer. This critical step generates a cohort of DCs that have initiated their antigen-presentation and maturation programs under controlled, optimal conditions prior to encountering the TME. Our in vitro data already established that PPP deficiency intrinsically impairs DC antigen-presenting capacity (Figure 3, b-d).\nIn this experimental framework, the transferred WT BMDCs serve as a baseline for “the maximal antitumor function achievable by DCs upon proper in vitro activation.” Upon peritumoral injection, both WT and KO DCs are subjected to the same immunosuppressive TME. The key question our experiment addresses is: Does an intrinsic, genetic deficiency in the PPP impair the ability of KO DCs to sustain this pre-activated functional state and effectively prime tumor-specific CD8 T cells in vivo, compared to PPP competent (WT) DCs? Our in vivo data provide a clear answer: Despite identical in vitro activation and facing the same TME, KO DCs exhibited a significantly reduced capacity to control tumor growth compared to the WT DC group (Figure 4, f and g, and Supplemental Figure 5, i-l). These results demonstrate that PPP is an indispensable, cell-intrinsic determinant of DC function. Therefore, our model does not assume that WT DCs are unaffected by the TME. Instead, it reveals that intrinsic PPP activity is a critical metabolic node governing DC efficacy, and its loss undermines DC function.\n12. What is ISD, which was used to stimulate BMDCs?\nResponse: We apologize for any confusion. Interferon-stimulatory DNA (ISD) is a well-established agonist of the cGAS-STING signaling pathway. It can be recognized by cGAS, leading to this pathway activation, and has been widely utilized across numerous studies to stimulate cGAS-STING signaling (Cho et al., 2024; Fang et al., 2023; Li et al., 2024; Mukai et al., 2016; Xu et al., 2025). Furthermore, we have now included a detailed description of the ISD sequence and its synthesis method in the Methods section of the revised manuscript: “The ISD used in this study was prepared with equimolar amounts of oligonucleotides: F: 5′-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACATACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3′; R: 5′-TGTAGATCATGTACAGATCAGTCATAGATCACTAGTAGATCTGTATGTAGATCATGTACAGATCAGTCATAGATCACTAGTAGATCTGTA-3′. The oligonucleotides were annealed in PBS at 70 for 30 minutes before cooling to room temperature.” Additionally, we have also added the following description of ISD in the Results section of the revised manuscript:\n“Furthermore, we cultured WT and KO BMDCs in vitro and stimulated them with the STING signaling agonist Interferon Stimulatory DNA (ISD, a synthetic double-stranded DNA that sensed by cGAS to activate the cGAS-STING pathway). Our results indeed confirmed that PPP deficiency impaired STING signaling (Supplemental Figure 6, a and b).”\n13. Figure 4J: The rationale for comparing tumor growth after injection of WT versus STING-pathway KO BMDCs is unclear. How do the authors explain that injected BMDCs promote tumor growth compared to WT BMDCs? Please comment.\nResponse: We thank the reviewer for this comment and apologize for any lack of clarity.\nFirst, Figure 4f (former manuscript Figure 4j) is a DC adoptive transfer model to evaluate DC function. In this model, BMDCs were pulsed with SIINFEKL peptide and transferred into MC38-OVA tumor-bearing wild-type mice. Impaired function of the transferred DCs would be expected to result in accelerated tumor growth. As shown in Figure 4f, mice receiving STING-KO DCs exhibited faster tumor growth compared to those receiving wild-type DCs, suggesting that STING deficiency impairs DC function. While this phenotype is also supported by our previous work (Hu et al., 2021; Hu et al., 2023), we acknowledge that the direct evidence linking STING deficiency in DCs to their functional impairment was not explicitly provided in the manuscript, which likely caused confusion. To address this directly, we added an in vitro assay. WT and STING-KO BMDCs were pulsed with SIINFEKL peptide and co-cultured separately with OT-I T cells. Compared to WT BMDCs, STING-KO BMDCs showed a significantly reduced capacity to stimulate OT-I T cell proliferation and effector cytokine production. These results confirm STING deficiency intrinsically impairs DC function (Figure IV).\nFigure IV: STING deficiency intrinsically impairs DC function. 1x105 OT-I T cells were cultured with 3x104 wild-type (WT) and Tmem173-/- (STING deficient) BMDCs in the presence of SIINFEKL peptides (n = 5). (a) Flow cytometry analysis (left) and graph presentation (right) of CFSE. (b and c) Flow cytometry analysis of Ki-67+ (b), IFN-+, TNF-+ and GZMB+ (c) T cells. Data were presented as mean ± SEM. Data were analyzed through two-tailed unpaired Student’s t-tests. n indicates the number of biological replicates.\nSecond, the purpose of Figure 4f was not to prove that STING is critical for DC function-a well-established concept in the literature-but rather to determine whether the antitumor function impairment caused by PPP deficiency is STING-dependent. To this end, we transferred WT, PPP-KO, STING-KO, and double-knockout (DKO; PPP-KO & STING-KO) DCs into MC38-OVA tumor-bearing mice. As expected, PPP-KO DCs accelerated tumor growth compared to WT DCs. Crucially, tumor growth in mice receiving STING-KO DCs was not further accelerated by the additional loss of PPP (DKO vs STING-KO). This indicates that PPP deficiency does not exacerbate the functional impairment observed upon STING deletion, demonstrating that PPP mediates DC antitumor function in a STING-dependent manner.\n14. Figure 6: Does treatment with ATP and GTP induce maturation in KO DCs? Is the observed effect due to injecting mature BMDCs? Why is there no effect in WT BMDCs treated with ATP and GTP? The lack of tumor growth reduction in WT suggests that supplementation is insufficient to overcome PPP deficiency. How do the authors explain that KO BMDCs promote tumor growth and that supplementation restores tumor growth to WT levels?\nResponse: We sincerely thank the reviewer for their valuable comments and apologize for any confusion.\nIn Figure 6a, the concentration of ATP and GTP (100 µM) was selected based on our experimental data. This dose of ATP and GTP was sufficient to restore the function of PPP deficient (KO) DCs without affecting wild-type (WT) DCs. We acknowledge that these supporting data were not included in the original manuscript, which led to the misunderstanding. Therefore, we have now added them in the revised manuscript.\nSpecifically, WT and KO BMDCs were cultured and treated with different concentrations of ATP and GTP for 12h in vitro. First, we confirmed that PPP deficiency reduced the expression of maturation markers including MHC-I, CD80, and CD86 in DCs (Supplemental Figure 8, e and f). Supplementation with 100 µM ATP and GTP restored the expression of these maturation markers in KO DCs to levels comparable to WT DCs, without altering expression in WT DCs (Supplemental Figure 8, e and f). Higher concentrations (300 and 500 µM) enhanced maturation in both WT and KO DCs (Supplemental Figure 8, e and f). To ensure the rationality of our experiments, 100 µM was selected as the working concentration for subsequent studies. Correspondingly, qPCR analysis confirmed that 100 µM ATP and GTP supplementation also rescued the expression of functional cytokines in KO DCs without affecting WT DCs (Supplemental Figure 8g). Therefore, in Figure 6a, we observed that supplementing WT DCs with 100 µM ATP and GTP did not affect tumor growth compared to untreated WT DCs. More importantly, supplementing KO DCs with ATP and GTP significantly delayed tumor growth compared to untreated KO DCs, restoring it to the level observed with WT DCs.\nIn summary, our results demonstrate that PPP deficiency in DCs impairs purine metabolism, leading to reduced ATP and GTP levels. Supplementation with ATP and GTP rescues the maturation, functional cytokine expression, and antitumor immunity of PPP-deficient DCs.\n15. Do KO BMDCs become more immunosuppressive in the tumor compared to WT BMDCs?\nResponse: We thank the reviewer for their helpful suggestion and have performed additional experiments to address this point. We cultured WT and PPP deficient BMDCs, and then labeled them with CFSE (carboxyfluorescein succinimidyl ester) in vitro. The labeled BMDCs were then injected subcutaneously adjacent to established tumors. After 24h, we checked the PD-L1 expression on CFSE-labelled DCs. Our results indicated that KO DCs displayed higher expression level of PD-L1, indicating an acquisition of an immunosuppressive molecular profile. Furthermore, we isolated CFSE-labeled WT and KO BMDCs from the tumors in equal numbers and co-cultured them separately with OT-I T cells in the presence of the SIINFEKL peptide. Compared to WT BMDCs, the KO BMDCs exhibited a significantly reduced capacity to stimulate OT-I T cell proliferation and effector cytokine production (Figure V).\nIn summary, our results demonstrate KO BMDCs become more immunosuppressive in the tumor compared to WT BMDCs.\nFigure V: PPP deficient DCs acquire a suppressive phenotype in the tumors. (a) Flow cytometry analysis of CFSE+ BMDCs. (b) Flow cytometry analysis (left) and graph presentation (right) of expression of PD-L1 on DC (n = 5). (c) Flow cytometry analysis (left) and graph presentation (right) of CFSE+ T cells. (d-e) Flow cytometry analysis of Ki-67+ (d, n = 5), IFN-+, TNF-+ and GZMB+ (e,n = 5) T cells. Data were presented as mean ± SEM. Data were analyzed through two-tailed unpaired Student’s t-tests. In b-e, n indicates the number of biological replicates.\nReviewer #3 (Remarks to the Author):\nSeveral studies in pre-clinical models have demonstrated an indispensable role for conventional dendritic cells (cDCs), particularly cDC1s, in promoting T cell responses to cancer and augmenting the efficacy of T cell based immunotherapies. While the link between cancer immunity and cDCs has been extensively studied, there is now growing interest in understanding the cell intrinsic determinants of cDC that govern cDC function.\nWork from Liu et al. reports cDC metabolism as a key component of their anti-tumour activity. The authors showed that the pentose phosphate pathway (PPP) is essential for cDC activation and their capacity to prime T cells. Although PPP activity is evident in intratumoural cDCs at early stages, it becomes suppressed during later stages of tumour progression, thereby contributing to immune evasion. Using conditional knockout mouse models and ex vivo/in vitro assays, the authors further showed that PPP deficiency compromises STING phosphorylation and downstream signaling. Pharmacological activation of the PPP restores antitumor immunity and enhances the efficacy of immune checkpoint blocakde. Finally, analysis of human breast cancer samples reveals that the expression of key PPP enzymes correlates with signatures of intratumoral cDC accumulation. While the mechanism proposed by the authors meets the novelty criteria expected for publication in Nature Communications and the experiments are generally well designed, the following points need to be addressed to improve the clarity and overall quality of the manuscript.\nResponse: We sincerely thank the reviewer for their careful consideration of our manuscript and for providing these valuable comments, which have helped to improve our study. Please see below our point-by-point response to each comment, including revisions to the manuscript. Major revisions to the manuscript text have been highlighted in blue.\nMajor points:\n1. It would be important to clarify whether the cDC1 or cDC2 ratio is maintained between early- and late-stage tumours. If this is not the case, it becomes difficult to draw firm conclusions from the T cell activation assays, as the observed differences in ex vivo T cell priming could result either from functional impairment of cDC subsets or from numerical differences in cDC1 and cDC2 populations between early and late tumours. The authors should therefore normalize the T cell activation assays based on the cDC1 and cDC2 ratio to ensure comparability between the groups.\nResponse: We sincerely thank the reviewer for this valuable suggestion. To address these comments, we performed the following additional experiments.\nFirst, we quantified cDC1 and cDC2 proportions in early- and late-stage tumors by flow cytometry. The proportion of cDC1 was significantly lower in late-stage than in early-stage tumors, whereas the proportion of cDC2 was comparable between the two stages (Supplemental Figure 2i).\nSecond, to assess the functional capacity of these subsets, we isolated equal numbers of cDC1 from early- and late-stage tumors and co-cultured them separately with OT-I T cells in the presence of SIINFEKL peptide. Late-stage cDC1 showed markedly reduced ability to stimulate OT-I T cell proliferation (Figure 1, o and p) and effector cytokine production (Figure 1q) compared to early-stage cDC1. Furthermore, we performed similar experiments with cDC2 using OT-II T cells and observed a similar functional decline. Late-stage cDC2 were significantly less capable of promoting OT-II T cell proliferation and cytokine production than early-stage cDC2 (Figure 1, r-t).\nTogether, these results demonstrate that even when controlling for subset frequency by using equal cell numbers, both cDC1 and cDC2 in late-stage tumors exhibit intrinsic functional impairment. This confirms that the diminished T cell priming capacity observed in bulk assays is due to cell-autonomous dysfunction, not merely changes in DC subset composition.\n2. In Figure 1, it would be important to determine whether the activation of both cDC subsets (cDC1 and cDC2) as well as their ability to prime T cells are equally affected by the suppression of PPP-pathway in late tumours. Identifying which subset is primarily impacted would enhance the mechanistic understanding of this study. To this end, the authors should repeat the T cell activation assays using separately purified cDC1 and cDC2 populations.\nResponse: We sincerely appreciate the reviewer’s valuable suggestion. To address these comments, we isolated equal numbers of cDC1 or cDC2 from early- and late-stage tumors, respectively. We then co-cultured cDC1 with OT-I T cells, and cDC2 with OT-II T cells in the presence of SIINFEKL peptide. Our results showed that late-stage intratumoral cDC1 exhibited a markedly reduced capacity to stimulate OT-I T cell proliferation (Figure 1, o and p) and effector cytokine production (Figure 1q) compared to early-stage cDC1. Similarly, late-stage cDC2 were significantly less effective at promoting OT-II T cell proliferation and cytokine production than early-stage cDC2 (Figure 1, r-t).\nTogether, these findings demonstrate that both cDC1 and cDC2 from late-stage tumors exhibit substantially impaired T cell priming capacity. Importantly, the degree of functional impairment was comparable between the two subsets. This indicates that the suppression of the PPP in the late tumor microenvironment does not preferentially affect one conventional DC subset over another; rather, it broadly compromises the functional fitness of the entire intratumoral cDC compartment.\n3. In Figures 3E and F, the appropriate control for G6pdxfl/flZbtb46-Cre mice is G6pdxfl/fl; the authors should repeat the tumour growth profile using this control line for accurate comparison. Alternatively, the authors could provide tumour growth data of WT, Zbtb46-Cre and G6pdxfl/fl within the same experiment to demonstrate that tumour progression is comparable across these lines and that they can be used interchangeably.\nResponse: We greatly appreciate the valuable suggestions provided by the reviewer, and have performed additional experiments to address this concern.\nWe subcutaneously inoculated G6pdxfl/fl and G6pdxfl/flZbtb46-Cre mice with MC38 tumor cells. Consistent with our observations in Figure 3e, the results confirmed G6PDX deficiency in cDCs accelerates tumor growth (Figure VI).\nFigure VI: G6PDXdeficiency in cDCs accelerates tumor growth. Tumor growth of G6pdxfl/fland G6pdxfl/flZbtb46-Cremice (n = 9). Data were presented as mean ± SEM and were analyzed through two-way ANOVA analyses. n indicates the number of mice.\n4. Given that PPP-deficient cDCs show impaired ability to prime antigen-specific T cells, it would be important to clarify whether the tumour growth retardation observed in mice with enhanced cDC PPP activity is CD8 T cell-dependent. The authors could address this by depleting CD8 T cells using specific antibodies in mice adoptively transferred with cDCs exhibiting enhanced PPP activity.\nResponse:We thank the reviewer for this important suggestion. To directly test whether the antitumor effect of enhanced PPP activity in cDCs depends on CD8 T cells, we performed an in vivo depletion experiment. Wild-type mice receiving adoptive transfer of AG1-augmented cDCs were treated with anti-CD8 depleting antibody. Flow cytometry confirmed efficient depletion of CD8 T cells in vivo. Compared to untreated DCs, AG1-augmented DCs significantly inhibited tumor growth. As anticipated, CD8 T cell depletion completely abrogated the tumor growth inhibition mediated by AG1-treated DCs. Furthermore, CD8 T cell depletion eliminating the therapeutic difference between AG1-treated and untreated DC groups (Figure VII). Together, these results demonstrate that the antitumor effect mediated by enhanced PPP activity in cDCs is strictly CD8 T cell-dependent.\nFigure VII: The antitumor effect mediated by enhanced PPP activity in cDCs is strictly CD8 T cell-dependent. Wild-type mice were inoculated with MC38-OVA tumor cells. Anti-CD8 (200 μg per mouse) or isotype control IgG was administered intraperitoneally one day prior to tumor challenge and then every three days for a total of four doses. BMDCs were pulsed with SIINFEKL peptide for 12h, incubated with AG1 (3 µM) or untreated (Ctrl) for 4h, and subsequently injected subcutaneously adjacent to established tumors on days 3 and 6 post-tumor inoculation. a: Flow cytometry analysis of CD8. b: Tumor growth of wild-type mice (n = 9). Data were presented as mean ± SEM. (b) was analyzed through two-way ANOVA analyses with Tukey’s multiple comparison test.In b, n indicates the number of mice.\nMinor but essential points:\n1. Figure legends should be revised for clarity and completeness, explicitly stating the number and type of replicates in each panel (e.g., whether each point in the graphs represents a biological or technical replicate).\nResponse: We sincerely thank the reviewer for this important suggestion. We have thoroughly revised the legends for all figures in the revised manuscript to ensure clarity and completeness.\n2. The statement on lines 152-153 “Consistent with the metabolic profiling, late-stage intratumoral cDCs exhibited markedly decreased NADPH and R5P production (Figure 2B).\" is not adequately supported by data and should be revised or removed.\nResponse: We sincerely appreciate the reviewer's comprehensive evaluation of our manuscript and so sorry for the mistake. To address the reviewer’s suggestion, we had included statistical analysis of the metabolic profiling data in Supplemental Figure 3a. The results confirmed that levels of key PPP metabolites were significantly reduced in late-stage intratumoral cDCs. Accordingly, we had revised the corresponding statement in the revised manuscript to: \"Consistent with the metabolic profiling, late-stage intratumoral cDCs exhibited markedly decreased NADPH and R5P production (Supplemental Figure 3a).\"\n3. The authors should clearly define every gene, abbreviation, and mouse model used in the figures and main text. For example, specify which protein is absent in TmemKO mice. Figure legends should be self-explanatory and allow readers to interpret the data without referring to the methods section. Similarly, in figure 5 and 6, please clarify in the panels the exact genotypes of the WT and KO mice.\nResponse: We thank the reviewer for this critical point regarding manuscript clarity. We have thoroughly revised the manuscript to address this as follows:\nFor the Definitions and abbreviations: All non-standard gene symbols, protein names, and abbreviations have been clearly defined upon first use in both the main text and the figure legends.\nFor the mouse model clarification: The specific genetic alteration for each knockout mouse model is now explicitly stated in the Methods section and reiterated in the relevant figure legends. For example, we have specified that \"Tmem173-/-\" refers to mice with a knockout of the STING gene.\nFor the Figure legends: All figure legends have been revised in revised manuscript. We have stated the exact genotypes of the mice used in each experiment directly in the legends for Figures 5 and 6.\nThese comprehensive edits ensure that readers can accurately interpret all data presented without needing to cross-reference the Methods section, significantly improving the quality of our manuscript.\n4. The combination “A + G + ISD” requires clarification. The authors should specify in both the main text and figure legend what each acronym represents and briefly describes the mechanism of action (text and methods) of these STING agonists, particularly in contrast to the other cGAS/STING agonists used elsewhere in the manuscript.\nResponse: We sincerely thank the reviewer for highlighting this lack of clarity.\nFirst, we have specified in both the main text and figure legends what each acronym represents. For example, we have clarified the \"A+G+ISD\" combination throughout the manuscript, now explicitly defining it as \"ATP+ GTP+ISD\" in the relevant text and Figure legends.\nSecond, we have briefly described the mechanism of action of these STING agonists used in our study and added in our revised manuscript: ISD (a synthetic double-stranded DNA that sensed by cGAS to activates the cGAS-STING pathway); DMXAA (a direct small-molecule binder of murine STING); cGAMP (the natural enzymatic product of cGAS that directly binds and activates STING).\n5. It would be interesting to investigate whether a high PPP-related gene signature enhances the predictive value of the cDC1 gene signature (e.g. comparing patients with high PPP/high cDC1, low PPP/high cDC1, low PPP/low cDC1, high PPP/low cDC1 expression). The authors could analysis some of the TCGA datasets previously used to show that intratumoral cDC1 gene signatures positively correlate with patient survival, as reported by JP Böttcher et al., 2018 (DOI: 10.1016/j.cell.2018.01.004 )\nResponse: We thank the reviewer for the valuable comments. In response to the reviewer's suggestion (Böttcher et al., 2018), we first analyzed the correlation between cDC1- and PPP-related gene signatures using TCGA datasets. The results revealed a positive correlation between these signatures in breast cancer (BRCA) (Figure 7a). We next examined whether incorporating the PPP gene signature could improve the prognostic value of the cDC signature alone. Analysis showed that patients classified as cDChiPPPhi had the most favorable overall survival, while those in the cDClowPPPlow group exhibited the poorest outcomes in BRCA (Supplemental Figure 10b). Collectively, these findings position the PPP as a key metabolic determinant of cDC-mediated antitumor immunity, with combined cDC and PPP signatures offering enhanced prognostic stratification in BRCA.\n6. The title should be changed and refined based on the data the authors have provided. There is no experimental evidence for tumour immunoediting- immunoediting refers historically to the experiments that have been conducted by R D Schreiber’s group, none of which are listed in this manuscript.\nResponse: We appreciate the reviewer’s comprehensive consideration of our manuscript. We agree that our study does not provide direct experimental evidence for the broader process of tumor immunoediting. Our work instead focuses on how alterations in the PPP within intratumoral cDCs impact their antitumor functionality. In response to the reviewer's suggestion, we have revised the manuscript title to: \"Pentose phosphate pathway fuels cGAS-STING signaling to boost intratumoral cDC function.\" We believe this title more accurately reflects the central findings and scope of our study.\nReviewer #4 (Remarks to the Author):\nIn the current study, Ben Liu et al. demonstrate that defects in the pentose phosphate pathway (PPP) occur in late-stage tumor-infiltrated dendritic cells (DCs). Impairment of the PPP reduces the levels of purine nucleotides ATP and GTP, which are essential substrates for cGAS enzymatic activity. This, in turn, results in insufficient activation of the STING pathway and consequently dampens DC function, limiting their antigen presentation capacity and cytokine production. The study identifies a critical role for the PPP in dysfunctional tumor-derived DCs and highlights a positive feedback loop between the PPP and STING pathways, providing potential therapeutic targets and mechanistic insights for enhancing DC function in cancer therapy.\nThe following are our comments and suggestions that the authors may consider to further strengthen their study:\nResponse: We sincerely thank reviewer for their time and effort in reviewing our manuscript. We are particularly grateful for the insightful comments and constructive suggestions aimed at strengthening our study. We have carefully considered all points raised and have performed additional experiments. Below is our point-by-point response. Corresponding revisions in the manuscript text are highlighted in blue.\n1. Can the authors examine the levels of pyrimidines in early- versus late-stage tumor-derived DCs?\nResponse: We thank the reviewer for the valuable suggestions. To address this question, we measured pyrimidine levels in early- and late-stage tumor-infiltrating cDCs using mass spectrometry. The results showed decreased levels of cytosine and uracil in late-stage intratumoral cDCs (Figure VIII). We also assessed ribose 5-phosphate (R5P), a precursor metabolite for pyrimidine synthesis in the PPP. R5P levels were significantly lower in late-stage cDCs compared to early-stage cells (Supplemental Figure 3a). Moreover, supplementation with R5P restored STING signaling in G6PDX-deficient DCs (Supplemental Figure 7, g and h). In summary, these findings indicate that impaired PPP activity in late-stage intratumoral cDCs leads to reduced accumulation of pyrimidines.\nFigure VIII: PPP activity in late-stage intratumoral cDCs leads to reduced accumulation of pyrimidines. Graph presentation of cytosine (a) and uracil (b) in late-stage intratumoral cDCs (n = 4). Data were presented as mean ± SEM. Data were analyzed through two-tailed unpaired Student’s t-tests. In a and b, n indicates the number of mice.\n2. The authors report that ROS accumulation is a consequence of PPP deficiency in DCs. Could they provide data on both mitochondrial and cellular ROS levels in early- and late-stage DCs?\nResponse: We appreciate the reviewer's insightful comments. Our study demonstrated that impaired PPP activity in late-stage tumor-infiltrating cDCs compromised their functionality. As PPP supplies NADPH and R5P (Supplemental Figure 7a). NADPH actively contributes to maintaining redox homeostasis. Our data also showed that PPP deficiency in DCs led to heightened ROS accumulation in vitro (Supplemental Figure 7d). In response to the reviewer's suggestion, we further assessed whether ROS accumulation also occurs in late-stage intratumoral cDCs in vivo. Measurement of both cellular and mitochondrial ROS revealed significantly higher levels in late-stage intratumoral cDCs compared to early-stage counterparts (Supplemental Figure 7, b and c). Collectively, these findings demonstrate that PPP impairment in late-stage intratumoral cDCs induces ROS accumulation.\n3. Since purine levels are reduced in late-stage DCs, does this lead to DNA damage in these cells?\nResponse: We appreciate the reviewer’s helpful comments and have included additional experiments. To address the question, we isolated early- and late-stage tumor-infiltrating cDCs from MC38 tumor-bearing wild-type mice. As a positive control for DNA damage induction, BMDCs were treated with 100 μM etoposide. Western blot analysis of γH2AX-a well-established marker of DNA damage-revealed no significant difference between early- and late-stage intratumoral cDCs (Supplemental Figure 7p). To further validate this observation, we compared wild-type and G6PDX deficient BMDCs and similarly observed no increase in DNA damage upon PPP disruption (Supplemental Figure 7o). In summary, our data indicate PPP deficiency and the associated purine depletion in DCs do not lead to elevated DNA damage.\n4. Could the authors elaborate on why PPP deficiency induces PD-L1 expression in DCs? Given that STING activation has previously been shown to promote PD-L1 expression and support DC activation, how do these findings align?\nResponse: We are deeply grateful for the reviewer's insightful comments. This is an excellent and critical point which is deserved to be discussed in the manuscript.\nTo address the comments, we have included detailed discussion in the DISCUSSION section in our revised manuscript as follow: “PD-L1 expression on DCs is critical for their survival, functionality, and response to ICB (Oh et al., 2020; Peng et al., 2020; Xiao et al., 2024). Our findings indicated that PPP deficiency upregulates PD-L1 expression in intratumoral cDCs. This observation appears to contrast with a recent report demonstrating that STING activation robustly increases PD-L1 expression in monocytes via the STING-IRF3-type I interferon axis(Song et al., 2025). Such a discrepancy suggests that the relationship between STING signaling and PD-L1 expression may be cell-type-specific. PPP deficiency impairs NADPH production, leading to ROS accumulation. Elevated ROS levels are known to activate signaling pathways that induce PD-L1 expression in non-small cell lung cancer (Liu et al., 2023). We observed that PPP deficiency in cDCs induced ROS accumulation, which may contribute to increased PD-L1 levels. While our study establishes a link between PPP deficiency and PD-L1 upregulation in intratumoral cDCs, the precise molecular mechanisms warrant further investigation.”\n5. Metabolic rewiring is a hallmark of DC activation as well as of dysfunctional DCs in the tumor microenvironment. Can the authors describe whether deficiency or inhibition of the PPP or STING pathways affects DC metabolism more broadly?\nResponse: We thank the reviewer for your valuable comments and have performed additional experiments to address this question. Wild-type BMDCs were cultured in vitro and treated with or without the PPP inhibitor 6-AN (50 µM), followed by metabolomic profiling. KEGG pathway analysis revealed that PPP inhibition broadly perturbs cellular metabolism, affecting pathways including amino acid metabolism (e.g., alanine, aspartate, and glutamate metabolism; arginine biosynthesis; glycine, serine, and threonine metabolism), fatty acid metabolism (e.g., glycerophospholipid metabolism; biosynthesis of unsaturated fatty acids), and central carbon metabolism (e.g., TCA cycle; one-carbon pool by folate). Collectively, these results indicate PPP inhibition triggers extensive metabolic reprogramming beyond the pathway itself.\nFigure IX: PPP inhibition affects DC metabolism. KEGG pathways enrichment analysis of non-targeted metabolomics data of BMDCs treated with PPP inhibitor 6-AN (50 μM) or untreated (Ctrl). P values were calculated by Fisher’s exact test.\n6. Is PPP deficiency in late-stage DCs due to limited glucose availability, and is the expression or activity of G6PD altered?\nResponse: We appreciate the reviewer’s valuable comments and have included additional experiments.\nFirst, we measured intracellular glucose levels in early- and late-stage intratumoral cDCs from MC38 tumor-bearing wild-type mice using mass spectrometry. The results revealed no significant difference in glucose levels between the two stages (Supplemental Figure 3a). Similarly, glucose-6-phosphate (G6P) levels remained comparable between early- and late-stage cDCs (Supplemental Figure 3a).\nGiven the observed impairment in PPP activity in late-stage cDCs, we next examined the expression of key PPP enzymes. qPCR analysis showed that transcript levels of G6pdx and Pgd were both downregulated in late-stage cDCs (Supplemental Figure 3c). This finding was corroborated at the protein level by western blot, which confirmed reduced G6PDX expression (Supplemental Figure 3b). Since the first and rate-limiting step of the PPP involves the conversion of G6P to 6-phosphogluconolactone (6PGL) by G6PD, accompanied by R5P production, we further assessed downstream metabolites. Consistent with reduced G6PDX protein level, both 6PGL and R5P levels were lower in late-stage cDCs (Supplemental Figure 3a).\nIn summary, these results indicate that late-stage intratumoral cDCs exhibit diminished G6PDX expression and overall PPP metabolic flux.\n7. Is treatment with a STING agonist alone sufficient to restore the function of late-stage DCs?\nResponse: We sincerely appreciate the reviewer’s valuable suggestion and have included additional experiments. We isolated early- and late-stage tumor-infiltrating cDCs from MC38 tumor-bearing mice and treated late-stage cDCs with or without the STING agonist DMXAA in vitro for 12h. These cells were then subcutaneously injected adjacent to the tumors to wild-type MC38 tumor-bearing mice on day 3 and 6 post-tumor inoculation. Compared with untreated late-stage intratumoral cDCs, those receiving STING agonist treatment exhibited delayed tumor growth and enhanced T cell cytokine production, achieving functional levels comparable to early-stage intratumoral cDCs (Figure 4, n and o, and Supplemental Figure 6z). These results demonstrate that restoring STING signaling in late-stage intratumoral cDCs rescues their antitumor immunity. Furthermore, the findings confirm that impaired STING activity contributes to the dysfunctional state of late-stage intratumoral cDCs.\n8. Could the authors clarify which DC subsets were included in the gating strategy for tumor-infiltrating DCs (Tu-DCs)? The current gating of CD45 CD24- F4/80 Ly6C CD11c MHCII cells may encompass both cDC1 and cDC2 populations. In addition, please specify the gating strategy used for Flt3L-derived BMDCs.\nResponse: We sincerely thank the reviewer for your valuable feedback. In response to the comments, we have now included the gating strategy for tumor-infiltrating DCs in the revised manuscript (Supplementary Figure 1). Our gating strategy indicated that we isolated both intratumoral cDC1 and cDC2 subsets for our analyses and experiments. Additionally, we have added the corresponding gating strategy for Flt3L-induced BMDCs in the revised manuscript (Supplementary Figure 5a). Incorporating these revisions in response to the reviewer’s comments has significantly strengthened the manuscript.\nOther comments:\n1. Please provide more detailed information on the experimental procedures. In particular, the ratios and cell numbers used are not clearly described. For example: DC cell numbers used for Western blot analysis; The number of DCs transferred in adoptive transfer experiments (early vs. late DCs from tumors); The ratios and cell numbers of DCs and T cells used in co-culture experiments. These details are essential for reproducibility and should be revised accordingly.\nResponse: We sincerely thank the reviewer for this critical feedback, which is essential for ensuring the reproducibility of our work. We have thoroughly revised the Materials and Methods section, and Figure legends to provide comprehensive and precise experimental details as requested.\nReferences\nBöttcher, J. P., Bonavita, E., Chakravarty, P., Blees, H., Cabeza-Cabrerizo, M., Sammicheli, S., Rogers, N. C., Sahai, E., Zelenay, S., and Reis e Sousa, C. (2018). NK Cells Stimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control. Cell 172, 1022-1037.e1014.\nCho, M.-G., Kumar, R. J., Lin, C.-C., Boyer, J. A., Shahir, J. A., Fagan-Solis, K., Simpson, D. A., Fan, C., Foster, C. E., Goddard, A. M., et al. (2024). MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis. Nature 625, 585-592.\nFang, L., Hao, Y., Yu, H., Gu, X., Peng, Q., Zhuo, H., Li, Y., Liu, Z., Wang, J., Chen, Y., et al. (2023). Methionine restriction promotes cGAS activation and chromatin untethering through demethylation to enhance antitumor immunity. Cancer Cell 41, 1118-1133.e1112.\nHu, Z., Teng, X. L., Zhang, T., Yu, X., Ding, R., Yi, J., Deng, L., Wang, Z., and Zou, Q. (2021). SENP3 senses oxidative stress to facilitate STING-dependent dendritic cell antitumor function. Mol Cell 81, 940-952 e945.\nHu, Z., Yu, X., Ding, R., Liu, B., Gu, C., Pan, X.-W., Han, Q., Zhang, Y., Wan, J., Cui, X.-G., et al. (2023). Glycolysis drives STING signaling to facilitate dendritic cell antitumor function. Journal of Clinical Investigation 133.\nLee, C. Y. C., Kennedy, B. C., Richoz, N., Dean, I., Tuong, Z. K., Gaspal, F., Li, Z., Willis, C., Hasegawa, T., Whiteside, S. K., et al. (2024). Tumour-retained activated CCR7+ dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity. Nature Communications 15.\nLi, H., Liu, C., Li, R., Zhou, L., Ran, Y., Yang, Q., Huang, H., Lu, H., Song, H., Yang, B., et al. (2024). AARS1 and AARS2 sense l-lactate to regulate cGAS as global lysine lactyltransferases. Nature 634, 1229-1237.\nLiu, W.-J., Wang, L., Zhou, F.-M., Liu, S.-W., Wang, W., Zhao, E.-J., Yao, Q.-J., Li, W., Zhao, Y.-Q., Shi, Z., et al. (2023). Elevated NOX4 promotes tumorigenesis and acquired EGFR-TKIs resistance via enhancing IL-8/PD-L1 signaling in NSCLC. Drug Resistance Updates 70.\nMaier, B., Leader, A. M., Chen, S. T., Tung, N., Chang, C., LeBerichel, J., Chudnovskiy, A., Maskey, S., Walker, L., Finnigan, J. P., et al. (2020). A conserved dendritic-cell regulatory program limits antitumour immunity. Nature 580, 257-262.\nMukai, K., Konno, H., Akiba, T., Uemura, T., Waguri, S., Kobayashi, T., Barber, G. N., Arai, H., and Taguchi, T. (2016). Activation of STING requires palmitoylation at the Golgi. Nature Communications 7.\nNg, I.-I., Zhang, Z., Xiao, K., Ye, M., Tian, T., Zhu, Y., He, Y., Chu, L., and Tang, H. (2025). Targeting WEE1 in tumor-associated dendritic cells potentiates antitumor immunity via the cGAS/STING pathway. Cell Reports 44.\nOh, S. A., Wu, D.-C., Cheung, J., Navarro, A., Xiong, H., Cubas, R., Totpal, K., Chiu, H., Wu, Y., Comps-Agrar, L., et al. (2020). PD-L1 expression by dendritic cells is a key regulator of T-cell immunity in cancer. Nature Cancer 1, 681-691.\nPeng, Q., Qiu, X., Zhang, Z., Zhang, S., Zhang, Y., Liang, Y., Guo, J., Peng, H., Chen, M., Fu, Y.-X., and Tang, H. (2020). PD-L1 on dendritic cells attenuates T cell activation and regulates response to immune checkpoint blockade. Nature Communications 11.\nSong, H., Chen, L., Pan, X., Shen, Y., Ye, M., Wang, G., Cui, C., Zhou, Q., Tseng, Y., Gong, Z., et al. (2025). Targeting tumor monocyte-intrinsic PD-L1 by rewiring STING signaling and enhancing STING agonist therapy. Cancer Cell 43, 503-518.e510.\nXiao, K., Zhang, S., Peng, Q., Du, Y., Yao, X., Ng, I.-I., and Tang, H. (2024). PD-L1 protects tumor-associated dendritic cells from ferroptosis during immunogenic chemotherapy. Cell Reports 43.\nXu, Y., Wang, Q., Wang, J., Qian, C., Wang, Y., Lu, S., Song, L., He, Z., Liu, W., and Wan, W. (2025). The cGAS-STING pathway activates transcription factor TFEB to stimulate lysosome biogenesis and pathogen clearance. Immunity 58, 309-325.e306.\nZhang, L., Li, Z., Skrzypczynska, K. M., Fang, Q., Zhang, W., O’Brien, S. A., He, Y., Wang, L., Zhang, Q., Kim, A., et al. (2020). Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer. Cell 181, 442-459.e429.\nZhang, Q., He, Y., Luo, N., Patel, S. J., Han, Y., Gao, R., Modak, M., Carotta, S., Haslinger, C., Kind, D., et al. (2019). Landscape and Dynamics of Single Immune Cells in Hepatocellular Carcinoma. Cell 179, 829-845.e820.\nWe have carefully revised the manuscript (changes highlighted in blue) in accordance with the requirements. In the following, we provide point-by-point responses.\nReviewer #1:\nNo additional comment.\nResponse: We thank the reviewer for her/his careful evaluation of our manuscript.\nReviewer #2:\nThe authors have done considerable work to improve this manuscript that can now be accepted for publication.\nResponse: We appreciate the reviewer’s careful evaluation of our manuscript.\nReviewer #3:\nThe authors have addressed all points raised in a thorough and satisfactory manner. The revisions and additional clarifications have strengthened the manuscript, and the responses are clear, well reasoned, and supported by appropriate data and analyses. The manuscript is now significantly improved and meets the standards expected for publication. I therefore consider the manuscript suitable for publication in Nature Communications.\nResponse: We sincerely thank the reviewer for her/his valuable evaluation of our manuscript\nReviewer #4:\nIn the revised manuscript, the authors clearly delineate defects in the pentose phosphate pathway (PPP) in late-stage tumor-infiltrating dendritic cells, which lead to insufficient activation of the cGAS–STING pathway and impaired DC activation. The study provides compelling mechanistic insights into how dendritic cells become dysfunctional within the tumor microenvironment from a metabolic perspective. With the inclusion of new data in the rebuttal materials, the major concerns have been addressed and substantial mechanistic depth has been added. The manuscript is now suitable for publication in Nature Communications.\nResponse: We appreciate the reviewer’s careful evaluation of our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "No additional comment", "reviewer_2": "The authors have done considerable work to improve this manuscript that can now be accepted for publication.", "reviewer_3": "The authors have addressed all points raised in a thorough and satisfactory manner. The revisions and additional clarifications have strengthened the manuscript, and the responses are clear, well reasoned, and supported by appropriate data and analyses. The manuscript is now significantly improved and meets the standards expected for publication.\nI therefore consider the manuscript suitable for publication in Nature Communications.", "reviewer_4": "In the revised manuscript, the authors clearly delineate defects in the pentose phosphate pathway (PPP) in late-stage tumor-infiltrating dendritic cells, which lead to insufficient activation of the cGAS–STING pathway and impaired DC activation. The study provides compelling mechanistic insights into how dendritic cells become dysfunctional within the tumor microenvironment from a metabolic perspective. With the inclusion of new data in the rebuttal materials, the major concerns have been addressed and substantial mechanistic depth has been added. The manuscript is now suitable for publication in Nature Communications."}, "author_response": "We have carefully revised the manuscript (changes highlighted in blue) in accordance with the requirements. In the following, we provide point-by-point responses.\nReviewer #1:\nNo additional comment.\nResponse: We thank the reviewer for her/his careful evaluation of our manuscript.\nReviewer #2:\nThe authors have done considerable work to improve this manuscript that can now be accepted for publication.\nResponse: We appreciate the reviewer’s careful evaluation of our manuscript.\nReviewer #3:\nThe authors have addressed all points raised in a thorough and satisfactory manner. The revisions and additional clarifications have strengthened the manuscript, and the responses are clear, well reasoned, and supported by appropriate data and analyses. The manuscript is now significantly improved and meets the standards expected for publication. I therefore consider the manuscript suitable for publication in Nature Communications.\nResponse: We sincerely thank the reviewer for her/his valuable evaluation of our manuscript\nReviewer #4:\nIn the revised manuscript, the authors clearly delineate defects in the pentose phosphate pathway (PPP) in late-stage tumor-infiltrating dendritic cells, which lead to insufficient activation of the cGAS–STING pathway and impaired DC activation. The study provides compelling mechanistic insights into how dendritic cells become dysfunctional within the tumor microenvironment from a metabolic perspective. With the inclusion of new data in the rebuttal materials, the major concerns have been addressed and substantial mechanistic depth has been added. The manuscript is now suitable for publication in Nature Communications.\nResponse: We appreciate the reviewer’s careful evaluation of our manuscript."}]

Decision letter