BATF2 is a glutamine-responsive tumour suppressor required for type-I interferon-dependent anti-tumour immunity

Domain: chemistry
Published: 2025-12-29
Editorial outcome: major_revision

Abstract
Recent evidence highlights the significance of a new type of tumour suppressors, which are not frequently mutated but inhibited by metabolic cues in cancers. Here, we identify BATF2 as a tumour suppressor whose expression is epigenetically silenced by glutamine in Head and Neck Squamous Cell Carcinomas (HNSCC). BATF2 correlates with type-I interferon and Th1 signatures in human HNSCC, with correlation coefficients even stronger than those of the positive control, STING. The phosphorylation of BATF2 at serine 227 promotes the oligomerization of STING. BATF2 deficiency or high glutamine levels result in higher oxygen consumption rates and metabolic profiles unfavorable for type-I interferon production. An isocaloric glutamine-rich diet abolishes STING-mediated effector cell expansion in tumours, weakening STING agonist-induced tumour control. Cancer cell-specific BATF2 expression promotes an Id2-centered T-cell effector signature, reduces T-cell exhaustion, and triggers spontaneous HNSCC rejection in a type-I interferon-dependent fashion. Utilizing syngeneic subcutaneous, orthotopic, and 24-week-long cigarette smoke carcinogen-induced HNSCC models, we demonstrate that host Batf2 deficiency results in increased infiltration of CD206+ myeloid cells and reduced effector CD8+ T-cells, accelerating the initiation of cancers. Overall, we reveal a tumour suppressor BATF2 whose loss is mediated by unique metabolic cues in the TME and drives cancer immune escape.

Introduction
The clinical success of immune checkpoint inhibitors (ICI) proves that cancers can be separated from the normal tissue in which they arise by the immune system. The tumour mutational burden has been extensively characterized to test the hypothesis that the number of tumour-specific antigens underpins the immunogenicity of cancers. Recognizing the evidence substantiating this theory in several cancer types, some cancers with modest responses to ICI, however, harbor high mutational loads1. To understand the mechanism governing the immunogenicity of these cancers, we employed head and neck squamous cell carcinoma (HNSCC) as a model. Two distinct sets of etiological factors drive HNSCC initiation and both subsets contain high and similar levels of mutational burdens2. The human papillomavirus (HPV)-negative HNSCC is associated with cigarette smoke and heavy alcohol consumption. The HPV+ HNSCC often occurs in the Waldeyer’s ring, where lymphoid structures are enriched. Gleaning evidence from two large cohorts of patients with HNSCC, we found that the Stimulator of Interferon Genes (STING)–type-I interferon (IFN-I) pathway underpins the immunogenicity of HNSCC. Oncogenes, high-risk HPV and metabolic conditions can all dampen STING response3–5. Recent structural evidence shows that STING functions by assembling into large multimeric protein complexes6. Despite the clear delineation of the canonical STING-TANK Binding Kinase 1 (TBK1)-Interferon Regulatory Factor 3 (IRF3) signaling axis, key components in the STING complex contributing to cancer immune surveillance remain to be discovered.

Here, we show that Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2) functions as a tumour suppressor by sustaining the fitness of STING. BATF2 is indispensable for the activation of the STING pathway and not required for cytoplasmic dsRNA-mediated IFN-I activation. BATF2 translocates to endoplasmic reticulum (ER)-associated membranes upon STING stimulation and interacts with STING. We identify Serine 227 as a critical site for phosphorylation during BATF2 activation. High glutamine levels suppress the expression of BATF2 epigenetically and elevate oxidative phosphorylation to dampen IFN-I responses. BATF2 expression leads to IFN-I and γδ T-cell-dependent HNSCC rejection. A deficiency in BATF2 results in the expedited onset of cigarette smoke carcinogen-induced HNSCC with infiltration of increased M2-like macrophages and decreased Th1 effector cells. Using human HNSCC specimens, we show that high levels of BATF2 correlate with increased CD8+ T-cell infiltration and that glutamine metabolism is inversely correlated with BATF2-IFN-I signatures.

Results
BATF2 positively correlates with an IFN-I high and effector T-cell high signature in patients with HNSCC
HNSCC frequently dampens the production of IFN-I through distinct mechanisms. To identify central pathways that determine HNSCC immunogenicity, we analyzed a single-cell RNA-Seq dataset of human HNSCC tumour-infiltrating immune cells (PRJNA579178). After filtering out low-quality cells, we integrated 38,862 transcriptomes from HPV− HNSCC and 20,703 transcriptomes from HPV+ HNSCC. To stabilize immune cell lineage segregation, we included ~70 additional marker genes in addition to the top 3000 most differentially expressed genes to identify immune populations, as we recently reported7,8. We identified a total of 25 clusters and eight of them were significantly different from HPV− and HPV+ tumour microenvironment (TME). We developed a robust visualization tool for single-cell datasets, supervised capacity preserving mapping (supCPM), which is adept at preserving the global geometry and correlating biological distance with geographic distance8,9. We rendered the structure of cellular subsets in human HNSCC using supCPM (Fig. 1a). Clusters 10, 19 and 20 were characterized by myeloid cells expressing high levels of CXCL10, ISG15 and CD80. Notably, these clusters were also distinguished by the cells expressing high levels of BATF2 (Supplementary Fig. 1b). HPV+ contained fewer γδ T-cells (cluster 4), Batf3+ conventional dendritic cells type 1 (cDC1) (cluster 25), fewer mast cells (cluster 22) and fewer M1-like macrophages (clusters 10, 19 and 20) (Fig. 1a and Supplementary Fig. 1a). Because cells in clusters 10, 19 and 20 expressed high levels of IFN-I signatures, we next assessed the correlation between BATF2 and IFN-I target genes in the TME. We analyzed 520 HNSCC specimens in the TCGA database and found that the levels of BATF2 were significantly positively correlated with those of IFNB1 (ρ=0.28, ****p<0.0001) and IFN target genes, including MX1 (ρ=0.66, ****p<0.0001), ISG54 (ρ=0.66, ****p<0.0001), ISG15 (ρ=0.63, ****p<0.0001), PD-L1 (ρ=0.54, ****p<0.0001) and TNF (ρ=0.23, p<0.0001) (Fig. 1b and Supplementary Fig. 1c). Notably, the Spearman coefficients noted in these correlations were even stronger than those for STING in the same dataset4. We deconvolved the immune subsets in this dataset using a robust cellular deconvolution tool, FARDEEP10. We found that the expression levels of BATF2 were significantly positively correlated with the abundance of CD8+ T-cells (ρ=0.39, ****p<0.0001), CD4+ memory T-cells (ρ=0.53, ****p<0.0001), γδ T-cells (ρ=0.29, ****p<0.0001) and M1-like macrophages (ρ=0.38, ****p<0.0001). The expression levels of BATF2 were significantly negatively correlated with the abundance of regulatory T-cells (ρ=−0.27, ****p<0.0001) (Fig. 1c and Supplementary Fig. 1d).

To validate the key findings, we stained a human HNSCC tissue microarray (TMA) consisting of 677 cores from 258 patients with a median follow-up of 60.1 months. After removing suboptimal cores, we had cores from 200 patients whose demographic characteristics are summarized (Supplementary Data 1). BATF2 showed a broad range of staining intensity in tumour cells and stroma. HNSCC-specific and TME-specific BATF2 staining scores were both positively correlated with CD8 scores (Fig. 1d, e). Next, we analyzed five TCGA datasets, including HNSCC (n=522, *p=0.034), high-grade serous carcinoma (OV, n=303, *p=0.0181), sarcoma (SARC, n=260, *p=0.0208), skin cutaneous melanoma (SKCM, n=471, **p=0.00106), and stomach adenocarcinoma (STAD, n=415, *p=0.0428), and found that higher expression levels of BATF2 were positively correlated with patient outcomes (Supplementary Fig. 1e–i). To assess the role of BATF2 in suppressing tumour initiation, we next analyzed a group of patients with oral potentially malignant disorders (OPMD) specimens with confirmed malignant transformation (Supplementary Data 2). We performed BATF2 staining on the paired longitudinal specimens and found that BATF2 expression levels were lower in paired HNSCC than in OPMD (Fig. 1f, g). Next, to directly assess whether the expression levels of BATF2 in fresh HNSCC regulate their response to IFN agonists, we analyzed fresh human HNSCC from eight patients. We trisected the tissue into three pieces of comparable size. The first piece was injected with PBS ex vivo, the second piece was injected with 5.0 μg CDA, and the third piece was formalin fixed and integrated into a tissue microarray (Fig. 1h). We performed qPCR using tissue homogenates and normalized the levels of IFNB1 by PTPRC, the latter of which encodes CD45. We quantified BATF2 staining using ImageJ and generated BATF2 scores. Then, we performed a Spearman correlation analysis between BATF2 scores and normalized IFNB1 expression and identified a strong positive correlation with a ρ-value of 0.86 (**p<0.01) (Fig. 1i). The HNSCC TCGA data set does not have responder status for immunotherapy. Thus, we next examined four datasets that contained responder status to anti-PD111. We found that the responders among patients with STAD (****p<0.0001), SKCM (****p<0.0001), bladder squamous cell carcinoma (BLCA, *p=0.02), or non-small cell lung cancer (NSCLC, ****p<0.0001) showed significantly higher levels of BATF2 expression than non-responders (Supplementary Fig. 1j–m). Overall, these data show that BATF2 inhibits the initiation of HNSCC and promotes anti-tumour immunity in human specimens.

BATF2 controls cytoplasmic DNA-mediated IFN-I response in TME macrophages
Because of the strong correlation between BATF2 and IFN-I signatures in the TME of HNSCC, we generated littermate control wild type and Batf2−/− mice and validated the expression of Batf2 in bone-marrow-derived macrophages (BMDM) (Fig. 2a). We transfected the cells with a STING agonist, interferon-stimulatory DNA (ISD), or poly(I:C), which activates the cytoplasmic dsRNA-sensing mitochondrial antiviral signaling protein (MAVS) pathway. Then, we performed bulk RNA-Seq to compare the transcriptomes between wild type and Batf2−/− BMDM at baseline and stimulated conditions. We found that ISD and poly(I:C) induced potent IFN-I activation, with the upregulation of 13 Ifna subtypes, Ifnb1, Sting and Tnf. The absence of Batf2 lead to dampened IFN-I activation in response to ISD. However, Batf2−/− BMDM showed comparable IFN-I production when transfected with poly(I:C) (Fig. 2b). Notably, the expression levels of Arg1 and Mrc1 were suppressed when IFN-I was activated. The deletion of Batf2 resulted in elevated levels of Arg1 and Mrc1 at baseline and ISD-stimulated conditions. No apparent difference in the expression of Arg1 or Mrc1 was noted when cells were transfected with poly(I:C) (Fig. 2b). Then, we performed gene set enrichment analysis (GSEA) and identified the most significantly altered pathways between wild type and Batf2−/− BMDM when stimulated with ISD. The top five enriched pathways were cytokine receptor signaling, RNA splicing, unfolded protein binding, defense response and response to virus (Fig. 2c). To validate these findings, we performed qPCR to assess the mRNA abundance of Ifnb1, pan-Ifna, Cxcl10 in wild type and Batf2−/− BMDM treated with two STING agonists, ISD or 2′3′-Cyclic GMP-AMP (cGAMP). We found that the deletion of Batf2 significantly reduced the transcription of IFN-I signature genes (Fig. 2d, e). In contrast, when the wild type and Batf2−/− BMDM were transfected with MAVS agonists poly(I:C) or 5′ppp-dsRNA, both stimuli induced potent IFN-I pathway activation. However, there was no difference between the wild type and Batf2−/− BMDM (Fig. 2f, g).

To substantiate the findings, we performed ELISA to measure the protein concentration of Ifn-β in the supernatant. ISD transfection resulted in about 20 ng/ml Ifn-β release by the wild type BMDM. ISD-induced Batf2−/− BMDM produced less than 10 ng/ml of Ifn-β in the supernatant (Fig. 2h). Similarly, a defect in Batf2 almost abrogated cGAMP-mediated Ifn-β production (Fig. 2i). We also noted a significant yet less prominent reduction in Ifn-β production from the Batf2−/− BMDM when the cells were incubated with a non-nucleotide-based STING agonist, diABZI (Fig. 2j). In contrast, Batf2−/− BMDM were equally competent as the wildtype control cells in producing Ifn-β when transfected with poly(I:C) or 5′ppp-dsRNA (Fig. 2k, l). Similar results were observed when we measured the protein levels of Tnf-α (Supplementary Fig. 2a–e), showing the selectivity of Batf2 in regulating IFN-I pathways. Then, we sought to characterize the signaling events downstream of Batf2. Transfection with ISD resulted in the phosphorylation of Tbk1, Irf3, Sting and Stat1 as early as 4 hrs post-treatment in wild type BMDM. Batf2 deficiency resulted in substantial repression of these key signaling events, abrogating the phosphorylation of Irf3 (Fig. 2m). Similarly, Batf2−/− BMDM exhibited lower levels of phosphorylation of Tbk1, Irf3, Sting and Stat1 when transfected with cGAMP (Fig. 2n). In contrast, no difference in signaling was noted between wild type and Batf2−/− BMDM when they were challenged with MAVS agonists (Supplementary Fig. 2f, g). These findings suggest that BATF2 selectively engages STING to maintain the optimal levels of IFN-I production.

BATF2 promotes STING signaling in HNSCC cells
Because BATF2 is highly expressed by myeloid cells and epithelial cells, we performed a screen of a panel of HNC cell lines. FaDu and UMSCC22A cells express low levels of BATF2, while UMSCC108 and UMSCC49 express high endogenous levels of BATF2 (Supplementary Fig. 3a). First, we generated empty vector (EV) control and stable BATF2-expressing FaDu cells using retroviruses. We transfected EV control or BATF2-expressing FaDu cells with STING plasmid and found that BATF2 significantly promoted the transcription of IFN-I signature genes, including IFNB1, CXCL10, CXCL9 and ISG54 (Fig. 3a). This finding was also recapitulated in UMSCC22A cells (Fig. 3b). Although most HNSCC cells, including FaDu and UMSCC22A, do not show robust IFN-I activation when transfected with cGAMP4,5, we found that BATF2 could still enhance the IFN-I response (Supplementary Fig. 3b, c). During tumour initiation, when immune surveillance determines the outcomes of epithelial transformation, the proper activation of the innate immune pathways enables the maturation of antigen-presenting cells and subsequent cross-priming of T-cells. To evaluate whether BATF2 helps to launch the IFN-I pathway against epithelial malignant transformation, we expressed BATF2 in NOM9-TK normal oral keratinocytes, which directly triggered the phosphorylation of TBK1 and STING (Supplementary Fig. 3d). To substantiate the findings, we generated EV and BATF2-deficient UMSCC108 and UMSCC49 cells using CRISPR-Cas9 lentiviruses. In agreement, BATF2 deficiency in UMSCC108 and UMSCC49 cells significantly reduced STING-mediated IFN-I signature gene expression (Fig. 3c, d). Then, we examined signaling alteration by immunoblotting phosphorylation of TBK1 and STING in HNSCC cells. The expression of BATF2 substantially increased the activation markers of the STING pathway (Fig. 3e and Supplementary Fig. 3e). BATF2 was STING-inducible, and BATF2 depletion resulted in compromised STING activation in UMSCC108 and UMSCC49 cells (Fig. 3f).

BATF2 translocates to the endoplasmic reticulum (ER) to interact with STING
To characterize the staining pattern of BATF2 in the TME, we performed an immunohistochemical stain of human HNSCC specimens with an anti-BATF2 antibody. Among the BATF2+ HNSCC cases, BATF2 shows a perinuclear punctate staining pattern, which can be seen in proteins localized to ER or Golgi apparatus, in addition to nuclear staining (Fig. 4a). Thus, we performed a sucrose gradient to fractionate cytosol, nuclei and ER as reported12–14. We found that STING was enriched in the ER compartment and not identified in free cytosol, as expected. BATF2 was present in the cytosol and at a lower level in the ER in the resting state. When THP-1 cells were treated with cGAMP overnight, there was a substantial translocation of BATF2 into the ER (Fig. 4b). We also identified phosphorylated TBK1 and IRF3, two central STING signaling molecules enriched in the ER fraction when the cells were treated with cGAMP. Consistent with the transcription factor function, phosphorylated IRF3 was most highly enriched in the nuclei fraction. As controls, VDAC, a mitochondrial protein was largely absent in the ER and cytosol fractions. Lamin B1 (LMNB1) is a component of the nuclear lamina and was enriched in the nuclear fraction, as expected (Fig. 4b). To visualize the localization of BATF2, we performed a confocal imaging analysis of BATF2. We co-stained HNSCC cells with STING and MitoTracker. We noticed that BATF2 colocalized with STING but not with mitochondria (Fig. 4c). This prompted us to directly assess BATF2-STING interaction using biochemical methods. We pulled down STING under stringent buffer conditions and identified a strong interaction with BATF2 in HEK-293T cells (Fig. 4d), a model cell line free of oncogenic mutations that often interfere with the STING-IFN-I pathway. Similarly, we pulled down BATF2 and identified STING as a binding partner (Fig. 4e). Then, we performed co-immunoprecipitation in FaDu and RAW 264.7 cells and found that BATF2 interacted with STING strongly (Fig. 4f–h). To further investigate which domain of STING specifically interacts with BATF2, we deleted the N-terminus, the Ligand-Binding Domain, or the C-terminus of STING. All STING domain truncation mutants can be expressed. We immunoprecipitated BATF2 and found that only the LBD domain was required for the BATF2-STING interaction (Fig. 4i). LBD is also the domain critical for STING oligomerization15. Thus, it is likely that the BATF2-STING interaction stabilizes the STING oligomers in an LBD-dependent fashion.

To directly assess whether BATF2-mediated IFN-I activation is STING-dependent, we depleted STING in Batf2-expressing tumour cells (Supplementary Fig. 4a). Batf2 significantly increased the transcription levels of Ifnb1, Isg54 and Cxcl9 in response to transfection of 1.0 μg/mL ISD. Sting deficiency abrogated Batf2-driven IFN-I activation (Supplementary Fig. 4b–d). Similarly, Batf2 expression directly increased the transcription levels of Ifnb1, Pan-Ifna and Cxcl10 in wild-type BMDM, which was abrogated in Sting−/− BMDM (Supplementary Fig. 4e–g). These results substantiate the BATF2-STING-IFN-I signaling pathway.

Phosphorylation of BATF2 is essential for its function in supporting STING signaling
Because the activation of STING depends on its oligomerization into a higher-order molecular complex15 and BATF2 binds to the STING LBD domain essential for oligomerization, we sought to determine whether BATF2 was involved in the STING complex formation under native conditions. We stimulated wild type and Batf2−/− BMDM with ISD and performed native gel electrophoresis. We found that the STING oligomers started to form as early as 4 h post-transfection and became prominent at the 8-h time point. Batf2 depletion substantially repressed the oligomerization of STING (Fig. 4j). The oligomerization of STING promotes its phosphorylation and downstream signaling. Then, we investigated whether BATF2 was phosphorylated during STING activation. Using PhosphoSitePlus, we found that there were two predicted phosphorylation sites for Batf2 at serine 179 and serine 227 (Fig. 4k). Then, we set out to determine whether Batf2 is phosphorylated upon STING stimulation. Because there is no phosphorylation-specific antibody available for Batf2 yet, we ran a phos-tag gel to determine whether the protein was phosphorylated. THP-1 cells were incubated with cGAMP admixed with human albumin, and we found that BATF2 was indeed phosphorylated upon stimulation. IRF3 was also immunoblotted using phos-tag gel as a positive control (Fig. 4l). To determine which phosphorylation site is indispensable for Batf2’s function in regulating STING, we performed a reconstitution experiment where we transfected full-length and site-specific mutant Batf2 plasmids to Batf2−/− BMDM. We treated wild-type and Batf2−/− BMDM, in the absence or presence of reconstitution, with ISD. In agreement with Fig. 2m, Batf2 deficiency suppressed STING signaling. Reconstituting the Batf2−/− BMDM with wild-type Batf2 or Batf2-179A mutant, the latter of which contained a serine to alanine mutation and cannot be phosphorylated at 179S, restored STING signaling. In contrast, reconstitution with the Batf2-227A mutant failed to restore signaling, suggesting that the phosphorylation of the 227 serine residue of Batf2 is essential for its function (Fig. 4m). To confirm whether S227 is also essential for BATF2 activation in cancer cells, we expressed wild-type BATF2 and BATF2-227A mutant in the human UMSCC22A cell line. We found that the full-length BATF2 substantially enhanced cGAMP-mediated STING activation, which was abolished by mutating S227 to an alanine (Supplementary Fig. 4h).

Glutamine inhibits the STING-IFN-I pathway by suppressing the expression of BATF2
By examining the TCGA dataset, the loss of BATF2 at the genetic level is uncommon. We next sought to investigate the mechanism underpinning the suppression of BATF2 expression in the TME. First, we tested BATF2 expression in nutrient-defined media specifically deficient for one of the essential or non-essential amino acids (Supplementary Fig. 5a). We found that deficiency in glutamine, tyrosine, valine, isoleucine and methionine resulted in significantly elevated expression of BATF2 (Supplementary Fig. 5a). Recent evidence suggests that glutamine is a central anaplerotic nutrient sustaining the tricarboxylic acid cycle in cancers. Inhibition of glutamine metabolism inhibits the generation of myeloid-derived suppressor cells (MDSC) and suppresses tumour growth16. Conceptually in agreement, glutaminolysis is pivotal for alternative (M2-like) activation of macrophages17. In addition to immune cells, cancer cells show an even higher ability to uptake glutamine in the TME18. Because the lack of IFN-I signaling in the TME leads to MDSC expansion and M2-like differentiation, we next sought to determine whether glutamine inhibits STING-mediated IFN-I activation. We cultured BMDM in glutamine-free media, media with 0.5 g/L glutamine, i.e., at the concentration of regular complete media, and media with 5.0 g/L glutamine. Transfection of BMDM with cGAMP resulted in significantly increased transcription of Batf2 and IFN-I genes. Higher levels of glutamine significantly repressed cGAMP-induced expression of Batf2, Ifnb1 and pan-Ifna (Fig. 5a and Supplementary Fig. 5b, c). Similarly, we transfected BMDM with ISD and found that glutamine significantly suppressed the STING-induced expression of Batf2 and IFN-I signature genes (Fig. 5b and Supplementary Fig. 5d–g). Treatment of cGAMP-activated BMDM with a selective glutaminase inhibitor, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), significantly elevated the expression levels of Batf2, Ifnb1 and pan-Ifna in media containing either 0.5 g/L or 5.0 g/L glutamine (Fig. 5c, d and Supplementary Fig. 5h, i). To substantiate the findings, we measured the protein levels of Ifn-β (Fig. 5e, f), Tnf-α (Supplementary Fig. 5j, k) and Cxcl10 (Supplementary Fig. 5l) in the supernatant and recapitulated the findings using qPCR. We harvested the cells and performed immunoblotting of STING signaling markers. The addition of glutamine dampened the phosphorylation of Tbk1, Sting and Irf3 (Fig. 5g). Similarly, in THP-1 cells, we found that 0.5 g/L and 5 g/L glutamine suppressed the transcriptional expression of BATF2 and IFN-I target genes (Supplementary Fig. 6a). At the protein level, high concentrations of glutamine not only reduced BATF2 expression but also led to decreased phosphorylation of TBK1 and IRF3, ultimately resulting in impaired IFN-β production (Supplementary Fig. 6b, c). To confirm glutamine-mediated BATF2-IFN-I suppression also similarly occurs in cancer cells, given their high dependence on glutamine metabolism, we transfected UMSCC49 cells with 2.0 μg/mL ISD with or without glutamine in the culture media. In agreement, glutamine potently inhibited the expression of BATF2 and IFN-I target genes (Supplementary Fig. 6d). At protein levels, glutamine also inhibited the expression of BATF2, the phosphorylation of TBK1 and the phosphorylation of STING (Supplementary Fig. 6e). To determine whether glutamine-mediated IFN-I suppression depends on BATF2, we transfected Batf2−/− BMDM with ISD with or without glutamine. In contrast to wildtype BMDM, the deficiency in Batf2 abolished glutamine-driven suppression of Ifn-β production, suggesting that glutamine inhibits IFN-I in a Batf2-dependent fashion (Supplementary Fig. 6f).

To understand the mechanism underpinning glutamine-mediated suppression of BATF2, we performed a chromatin immunoprecipitation (ChIP) experiment to examine glutamine-mediated epigenetic regulation of Batf2 and IFN-I target genes in BMDM. We found that glutamine significantly increased the levels of a repressive marker, H3K27me3 on the promoters of Batf2, Ifnb1 and Cxcl9 (Fig. 5h and Supplementary Fig. 6g). This glutamine-mediated epigenetic regulation was specific because glutamine had no impact on the H3K4me3 marker on the promoters of Batf2 and Ifnb1 (Supplementary Fig. 6h). We showed that depletion of Batf2 increased the expression of marker genes for the alternatively activated macrophages, such as Arg1 and Mrc1 (Fig. 2b). STING-IFN-I signaling promotes the classical activation of macrophages, which primarily relies on aerobic glycolysis to meet the rapidly increased energy demand. In contrast, alternative activation in macrophages favors oxidative metabolism and maintains a robust glutaminolysis program. The addition of glutamine increased the basal and maximal oxygen consumption rates (OCR) at baseline and STING-induced conditions (Fig. 5i and Supplementary Fig. 7a, b). Although wild-type and Batf2−/− BMDM showed comparable basal and maximal OCR at baseline without immune activation, Batf2−/− BMDM demonstrated a significantly higher basal and maximal OCR than the wild-type cells under STING-induced conditions (Fig. 5j and Supplementary Fig. 7c, d), similar to the high glutamine culture conditions. To further evaluate the impact of glutamine upon STING activation in vivo, we employed an isocaloric diet model where we fed C57BL/6J mice with a control diet or a glutamine-rich diet. Mice in both groups showed comparable body weight throughout the experiments (Supplementary Fig. 7e). We implanted an aggressive HNSCC model, NOOC1, which shows ~91% similarity in mutational signatures to human tobacco-associated cancers and is refractory to ICB19, in mice on a control regular diet and an isocaloric, glutamine-enriched diet. NOOC1 grew even more aggressively in mice on the glutamine-rich diet at baseline (Fig. 5k). To assess whether glutamine-mediated tumour aggression depends on the suppression of Batf2, we implanted the same tumour model into Batf2−/− mice on either a regular diet or an isocaloric, glutamine-rich diet. We found that a glutamine-rich diet could no longer promote tumour growth in the absence of host expression of Batf2 (Supplementary Fig. 7f). For mice on a regular diet, intra-tumoural injection of STING agonists resulted in significant tumour volume reduction, as expected (Fig. 5k). However, in mice fed with a glutamine-rich diet, STING agonists failed to show any efficacy (Fig. 5k). We used a tumour volume of 500 mm³ as the cutoff point to assess mouse survival. We found that mice on a glutamine-enriched diet had significantly lower survival rates compared to those on a control diet, and CDA treatment did not improve their survival. In contrast, CDA treatment extended the survival of mice on a regular diet (Supplementary Fig. 7g). Then, we assessed the impact of inhibition of glutamine metabolism on tumour response to IFN-I agonists. In the same experiment shown in Fig. 5k, we also included two additional groups that received intra-tumoural injection of CDA and a glutaminase inhibitor, JHU083. One of these groups was on a regular diet, and the other was on an isocaloric glutamine-rich diet. Interestingly, although JHU083 did not further improve the anti-tumour efficacy of CDA in the regular diet group, it significantly improved tumour control in mice on a glutamine-rich diet (Fig. 5l, m).

To determine the mechanism of glutamine-rich diet-mediated desensitization of cancer to STING stimulation, we performed scRNA-Seq to examine changes in the TME (Fig. 5n). We utilized an optimized analytical pipeline where we included additional feature genes for lineage segregation and stabilization to identify ten main clusters7,8. In mice on a regular diet who were treated with STING agonists, we observed an increase in CXCR5+ B cells (cluster 7) and T-cells (cluster 6), along with a reduction in granulocytic MDSC infiltration (clusters 3 and 8) in the TME. In contrast, when mice were fed with an isocaloric glutamine-rich diet, intra-tumoural STING stimulation failed to increase T-cell infiltration (cluster 6) but increased the expansion of granulocytic MDSC (clusters 3 and 8). In the glutamine-rich diet group, we also observed an expansion of endothelial cells (cluster 9) and basophils (cluster 10) (Fig. 5o). To assess whether glutamine suppressed Batf2 in vivo as a mechanistic link of dampening STING-mediated anti-tumour immunity, we examined the expression levels of Batf2 and found that Batf2 was most highly expressed by tumour cells (cluster 5), Ifnb1+ Cxcl10+ myeloid cells (cluster 1), MHC-II+ myeloid cells (cluster 2) and dendritic cells (cluster 4) (Fig. 5p). Notably, glutamine significantly decreased the expression of Batf2 in vivo (Fig. 5q). In the myeloid cell cluster that features high expression levels of Ifnb1 and Cxcl10, a glutamine-rich diet significantly reduced the levels of Batf2 and Ifnb1 (Supplementary Fig. 7h, i). We and others have shown that STING agonists significantly expanded the infiltration of effector and stem-like T-cells5,20,21. Thus, we sub-clustered T-cells (cluster 6) into functional subsets to better assess the impact of a glutamine-rich diet on cancer STING response (Supplementary Fig. 7j, k). In agreement with the literature, STING agonists substantially expanded intra-tumoural CD8+ T-cells (subcluster 2), NK cells (subcluster 3) and Tcf7+ T-cells (subcluster 4) in mice on a regular diet. In contrast, STING agonists failed to expand these key anti-tumour immune subsets in mice on an isocaloric glutamine-rich diet (Supplementary Fig. 7j, k). As we saw a noted B-cell expansion in response to STING stimulation only among mice on a regular diet, we next examined the B-cell signatures and found that STING stimulation is more prominently promoting the upregulation of multiple functional B-cell subset markers in tumours grown in mice on a regular diet than those grown in mice on a glutamine-rich diet, including Cr2, Fcer2a, Igkc, Ighm, Ighd, Sell, Ms4a1, Sdc1, CD79a, CD19 and Cxcr5 (Supplemental Fig. 7l). In agreement, we quantified CD20 staining of tumours from different treatment groups and found that STING stimulation expanded B-cells in the tumours grown in mice on a regular diet but not in mice on a glutamine-rich diet (Supplemental Fig. 7m). To substantiate the physiological relevance of the glutamine-BATF2 pathway, we collected fresh HNSCC tissue from 15 patients (Supplementary Data 3) and performed bulk RNA-Seq. Then, we performed a correlation analysis between BATF2-IFN-I pathway genes and glutamine metabolism markers. We found that BATF2 was strongly positively correlated with Th1 genes, including CXCL9, CXCL10, ISG15, IFNB1, OASL and TNF. Notably, we also found that BATF2 was inversely correlated with glutamine metabolism pathway genes, such as SLC7A5, SLC1A5, SLC38A2 and GLS. In agreement, the Th1 cytokines, including IFNG, IFNB1 and TNF, all show an inverse correlation with glutamine metabolism (Fig. 5r). Collectively, these findings suggest that high glutamine levels silence the expression of BATF2 by increasing the trimethylation of lysine 27 on histone H3 protein, promoting a metabolic profile recalcitrant to STING stimulation.

HNSCC-specific BATF2 promotes IFN-I- and γδ T-cell-dependent tumour immunogenicity
The use of a cigarette smoke carcinogen, 4-nitroquinoline 1-oxide (4-NQO), could generate tumour cells with high similarity mutational profiles to human tobacco-associated cancers, such as HNSCC19,22. Thus, we treated C57BL/6J mice with 50 μg/mL 4-NQO-containing drinking water for 16 weeks. Then, we switched the mouse cages to regular water and monitored the development of HNSCC. A histology-verified tongue squamous cell carcinoma was processed to produce single-produce single-cell clones. Every single cell-derived line was expanded and screened in C57BL6/J mice for tumour establishment. Most clones were spontaneously rejected except for two 4-NQO-induced Oral Cancer (NOOC) lines, NOOC1 and NOOC2. Similar to NOOC1, which has a combined 91% similarity in mutational signatures to human tobacco-associated cancers, NOOC2 has a combined >95% similarity in mutational signatures to human tobacco-associated cancers, including 68.1% similarity to Single Base Substitution (SBS) 4 (tobacco smoking), 18.3% similarity to SBS5 (tobacco smoking), and 13.6% similarity to SBS14 (defective DNA mismatch repair) (Supplementary Fig. 8a). Common HNSCC missense mutations, such as Trp53 (R178P), Erbb2 (P1034L), Nsd1 (E1342*), Kmt2d (R5157L, R3551L, E3060K), Kdm5c (E1496*), Kdm6a (Q475H), Fgfr3 (G444S), Fat1 (Q901K), Fat1 (S1372I) and Fat1 (V2754M), were identified in NOOC2 (Supplementary Data 4). Unlike NOOC1, which is completely refractory to high doses of ICI19, NOOC2 partially responds to a combination of anti-Pd-1 and anti-Ctla4 (Supplementary Fig. 8b).

To directly assess the role of cancer cell-specific Batf2 expression in tumour immunogenicity, we first measured Batf2 expression levels in MOC2-E6/E7, NOOC1 and NOOC2 cells using qPCR and found that Batf2 expression was lowest in MOC2-E6/E7, which corresponded to the lowest transcription levels of Ifnb1 at baseline (Supplementary Fig. 9a). Then, we generated stable Batf2-expressing MOC2-E6/E7 cells (Supplementary Fig. 9b). We previously showed that MOC2-E6/E7 is refractory to anti-PD-L1 and partially responsive to non-formulated CDN5. Similar to human HNSCC cells shown in Fig. 3, Batf2 upregulated STING-induced transcription of IFN-I target genes and Ifn-β secretion into the supernatant (Fig. 6a, b). Batf2 expression also increased STING-induced phosphorylation of Tbk1, Irf3 and Sting in MOC2-E6/E7 cells (Fig. 6c). We did not find any differences in colony formation or proliferation between the empty vector (EV) control and Batf2-expressing cancer cells in vitro (Supplementary Fig. 9c, d). However, Batf2-expressing tumours were spontaneously rejected in wild-type C57BL6/J mice (Fig. 6d). We extracted mRNA from homogenized tumour tissues from both groups and found that the transcription levels of IFN signature genes, including Mx1, Cxcl9, Cxcl10 and Ifng were much higher in Batf2-expressing tumours (Supplementary Fig. 9e). To determine whether Batf2-mediated tumour rejection was dependent on IFN-I, we implanted EV and Batf2-expressing tumour cells in Ifnar1−/− mice and completed rescued the phenotype (Fig. 6e). To determine whether Batf2-mediated tumour rejection depends on Sting, we generated stable empty vector control and Sting-deficient Batf2-expressing MOC2-E6/E7 cells. We found that cancer cell-specific Batf2-mediated tumour rejection depended on cancer cell expression of Sting because knocking down Sting abrogated Batf2-mediated protection (Supplementary Fig. 9f, g). To discover the mechanism of cancer cell-specific Batf2 activation in tumour suppression, we characterized the temporal dynamics of the TME between control and Batf2-expressing tumours by analyzing TILs procured on days 7, 14 and 21 post-tumour implantation (Supplementary Fig. 10a). Batf2-expressing tumours demonstrated a more favorable TME at the earliest time point, as evidenced by the early expansion of CD45+CD3−CD11b+ myeloid cells that are positive phospho-Tbk1 and phospho-Irf3. Also noticeable at this early time point was the expansion of γδ T-cells. Then, at the later time points, Batf2-expressing tumours started to show significantly more CD8+ T-cells infiltration than control tumours, although this advantage appeared to become smaller as the tumour size continued to increase (Fig. 6f–i). These findings suggested that cancer cell-specific Batf2 expression reprograms innate and adaptive immune wiring in a stepwise fashion, with IFN-I activation in CD11b+ myeloid cells and γδ T-cells expansion as the earliest response feature, followed by expansion of CD8+ T-cells.

Then, we profiled the tumour-infiltrating immune cells at single-cell resolution. The significantly expanded clusters in the Batf2-expressing tumours included a subset of Cxcl10high myeloid cells, endothelial cells and γδ T-cells (Fig. 6j, k). To interrogate the differences in T-cell functions, we separated T-cells (clusters 13 and 16) and identified a list of genes that were differentially expressed between T-cells from EV and Batf2-expressing tumours. The T-cells in the control TME expressed high levels of exhaustion signatures such as Pdcd1, Havcr2 and Icos. In contrast, the T-cells in the TME of Batf2-expressing tumours expressed significantly higher levels of Id2 (Fig. 6l), which is indispensable for the generation of memory and effector T-cells23. Then, we performed a GSEA. We found that the pathways that were significantly upregulated in T-cells from Batf2-expressing tumours included effector functions, such as Ifn-γ response, allograft rejection, Ifn-α response, as well as cell proliferation, such as E2F targets, mitotic spindle, G2M checkpoint and Myc targets (Fig. 6m). Because we observed a significant expansion of γδ T-cells in BATF2-expressing human and murine HNSCC (Figs. 1c and 6h, j, k), we then investigated whether this population was essential for tumour immunogenicity. We implanted EV and Batf2-expressing tumours in Tcrd−/− mice and found that the lack of γδ T-cells abrogated Batf2-mediated tumour rejection, highlighting the importance of this subset in early tumour suppression (Supplementary Fig. 9h). Cxcl10 is a key target gene of Ifn-β, and Batf2 led to a significant expansion of Cxcl10+ myeloid cells. Thus, we evaluated whether the anti-tumoural function of BATF2 depends on Cxcl10. We administered 200 μg of anti-Cxcl10 neutralizing antibody on day 7 after Batf2-MOC2-E6/E7 tumour implantation, followed by 100 μg twice per week for a total of five dosages. We found that neutralizing Cxcl10 reversed the antitumour effect of Batf2 (Supplementary Fig. 9i). Then, we sought to determine whether Batf2-mediated protection depends on αβ effector T-cells. We administered 0.5 mg of anti-CD8 antibody intraperitoneally for three consecutive days starting three days before tumour implantation, followed by twice per week injections for a total of five doses. We found that depletion of CD8+ T cells abrogated Batf2-mediated protection (Supplementary Fig. 9j). To determine whether cancer cell-specific Batf2 expression has an abscopal effect, we implanted the EV control and Batf2-expressing tumours on the right flank. As expected, we observed a significant reduction in tumour volume when Batf2 was expressed. However, the EV control tumours grown in the left flank of mice that bear Batf2-expressing tumours in the right flank did not show a difference in volume compared to those EV tumours grown in the left flank of mice that bear EV tumours in the right flank (Supplementary Fig. 9k). These data suggest that the BATF2-expressing tumour activates a local immune response without triggering a notable abscopal effect.

To further support our findings, we knocked down Batf2 in NOOC2 cells, which have higher endogenous Batf2 expression levels. Depletion of Batf2 significantly reduced the production of Ifnb1 (Supplementary Fig. 9l). In agreement, sh-Batf2 NOOC2 tumours grew significantly faster than EV control tumours (Supplementary Fig. 9m). We demonstrated the role of the S227 phosphorylation site in Batf2 activation. To substantiate the physiological relevance of this finding, we established tumour models that stably express empty vector, wild-type Batf2 and 227A mutant Batf2. In agreement with our other data, expression of Batf2 inhibited tumour growth. However, Batf2-mediated protection was abrogated by mutating serine 227 to alanine and disabling phosphorylation at this amino acid (Supplementary Fig. 9n).

BATF2 expression in the host deters the initiation of HNSCC
To understand the impact of host BATF2 signaling on the immunogenicity of HNSCC, we first implanted MOC2-E6/E7 cells into littermate control wild-type and Batf2−/− mice. We observed significantly accelerated MOC2-E6/E7 tumour growth in the Batf2−/− host (Fig. 7a). Flow cytometric analysis of the TME showed that fewer p-Irf3+, CD4+, CD8+ and CD8+ Ifn-γ+ T cells were present in tumours growing in Batf2−/− mice (Fig. 7b and Supplementary Fig. 10b). Similarly, we observed fewer γδ T-cells in tumours grown in the Batf2−/− hosts (Supplementary Fig. 11a). Using another model, we implanted NOOC2 into littermate control wild-type and Batf2−/− mice and consistently found that the tumour burden was significantly higher in the Batf2−/− mice (Fig. 7c). Consistent with previous results, host deficiency in Batf2 resulted in fewer p-Irf3+, CD4+ and CD8+ T-cells infiltration in the TME (Fig. 7d). To further substantiate the findings, we employed the third model, 4MOSC1, which only grows orthotopically in the tongue. Batf2−/− hosts developed the largest tongue tumours (Fig. 7e).

Implantable tumour models can provide insight into the role of cancer cell-immune cell interaction in the TME, but bypass an important phase in cancer establishment – malignant transformation. HNSCC is preceded by precursor lesions known as epithelial dysplasia. Thus, we treated C57BL/6J mice with 50 μg/mL 4-NQO-containing drinking water for 16 weeks. Then, we switched the mouse cages to regular water and monitored the development of HNSCC. A histology-verified tongue squamous cell carcinoma was processed to produce single-cell clones. Every single cell-derived line was expanded and screened in C57BL6/J mice for tumour establishment. Most clones were spontaneously rejected except for two 4-NQO-induced Oral Cancer (NOOC) lines, NOOC1 and NOOC2. Similar to NOOC1, which has a combined 91% similarity in mutational signatures to human tobacco-associated cancers, NOOC2 has a combined >95% similarity in mutational signatures to human tobacco-associated cancers, including 68.1% similarity to Single Base Substitution (SBS) 4 (tobacco smoking), 18.3% similarity to SBS5 (tobacco smoking), and 13.6% similarity to SBS14 (defective DNA mismatch repair) (Supplementary Fig. 8a). Common HNSCC missense mutations, such as Trp53 (R178P), Erbb2 (P1034L), Nsd1 (E1342*), Kmt2d (R5157L, R3551L, E3060K), Kdm5c (E1496*), Kdm6a (Q475H), Fgfr3 (G444S), Fat1 (Q901K), Fat1 (S1372I) and Fat1 (V2754M), were identified in NOOC2 (Supplementary Data 4). Unlike NOOC1, which is completely refractory to high doses of ICI19, NOOC2 partially responds to a combination of anti-Pd-1 and anti-Ctla4 (Supplementary Fig. 8b).

To directly assess the role of cancer cell-specific Batf2 expression in tumour immunogenicity, we first measured Batf2 expression levels in MOC2-E6/E7, NOOC1 and NOOC2 cells using qPCR and found that Batf2 expression was lowest in MOC2-E6/E7, which corresponded to the lowest transcription levels of Ifnb1 at baseline (Supplementary Fig. 9a). Then, we generated stable Batf2-expressing MOC2-E6/E7 cells (Supplementary Fig. 9b). We previously showed that MOC2-E6/E7 is refractory to anti-PD-L1 and partially responsive to non-formulated CDN5. Similar to human HNSCC cells shown in Fig. 3, Batf2 upregulated STING-induced transcription of IFN-I target genes and Ifn-β secretion into the supernatant (Fig. 6a, b). Batf2 expression also increased STING-induced phosphorylation of Tbk1, Irf3 and Sting in MOC2-E6/E7 cells (Fig. 6c). We did not find any differences in colony formation or proliferation between the empty vector (EV) control and Batf2-expressing cancer cells in vitro (Supplementary Fig. 9c, d). However, Batf2-expressing tumours were spontaneously rejected in wild-type C57BL6/J mice (Fig. 6d). We extracted mRNA from homogenized tumour tissues from both groups and found that the transcription levels of IFN signature genes, including Mx1, Cxcl9, Cxcl10 and Ifng were much higher in Batf2-expressing tumours (Supplementary Fig. 9e). To determine whether Batf2-mediated tumour rejection was dependent on IFN-I, we implanted EV and Batf2-expressing tumour cells in Ifnar1−/− mice and completed rescued the phenotype (Fig. 6e). To determine whether Batf2-mediated tumour rejection depends on Sting, we generated stable empty vector control and Sting-deficient Batf2-expressing MOC2-E6/E7 cells. We found that cancer cell-specific Batf2-mediated tumour rejection depended on cancer cell expression of Sting because knocking down Sting abrogated Batf2-mediated protection (Supplementary Fig. 9f, g). To discover the mechanism of cancer cell-specific Batf2 activation in tumour suppression, we characterized the temporal dynamics of the TME between control and Batf2-expressing tumours by analyzing TILs procured on days 7, 14 and 21 post-tumour implantation (Supplementary Fig. 10a). Batf2-expressing tumours demonstrated a more favorable TME at the earliest time point, as evidenced by the early expansion of CD45+CD3−CD11b+ myeloid cells that are positive phospho-Tbk1 and phospho-Irf3. Also noticeable at this early time point was the expansion of γδ T-cells. Then, at the later time points, Batf2-expressing tumours started to show significantly more CD8+ T-cells infiltration than control tumours, although this advantage appeared to become smaller as the tumour size continued to increase (Fig. 6f–i). These findings suggested that cancer cell-specific Batf2 expression reprograms innate and adaptive immune wiring in a stepwise fashion, with IFN-I activation in CD11b+ myeloid cells and γδ T-cells expansion as the earliest response feature, followed by expansion of CD8+ T-cells.

Then, we profiled the tumour-infiltrating immune cells at single-cell resolution. The significantly expanded clusters in the Batf2-expressing tumours included a subset of Cxcl10high myeloid cells, endothelial cells and γδ T-cells (Fig. 6j, k). To interrogate the differences in T-cell functions, we separated T-cells (clusters 13 and 16) and identified a list of genes that were differentially expressed between T-cells from EV and Batf2-expressing tumours. The T-cells in the control TME expressed high levels of exhaustion signatures such as Pdcd1, Havcr2 and Icos. In contrast, the T-cells in the TME of Batf2-expressing tumours expressed significantly higher levels of Id2 (Fig. 6l), which is indispensable for the generation of memory and effector T-cells23. Then, we performed a GSEA. We found that the pathways that were significantly upregulated in T-cells from Batf2-expressing tumours included effector functions, such as Ifn-γ response, allograft rejection, Ifn-α response, as well as cell proliferation, such as E2F targets, mitotic spindle, G2M checkpoint and Myc targets (Fig. 6m). Because we observed a significant expansion of γδ T-cells in BATF2-expressing human and murine HNSCC (Figs. 1c and 6h, j, k), we then investigated whether this population was essential for tumour immunogenicity. We implanted EV and Batf2-expressing tumours in Tcrd−/− mice and found that the lack of γδ T-cells abrogated Batf2-mediated tumour rejection, highlighting the importance of this subset in early tumour suppression (Supplementary Fig. 9h). Cxcl10 is a key target gene of Ifn-β, and Batf2 led to a significant expansion of Cxcl10+ myeloid cells. Thus, we evaluated whether the anti-tumoural function of BATF2 depends on Cxcl10. We administered 200 μg of anti-Cxcl10 neutralizing antibody on day 7 after Batf2-MOC2-E6/E7 tumour implantation, followed by 100 μg twice per week for a total of five dosages. We found that neutralizing Cxcl10 reversed the antitumour effect of Batf2 (Supplementary Fig. 9i). Then, we sought to determine whether Batf2-mediated protection depends on αβ effector T-cells. We administered 0.5 mg of anti-CD8 antibody intraperitoneally for three consecutive days starting three days before tumour implantation, followed by twice per week injections for a total of five doses. We found that depletion of CD8+ T cells abrogated Batf2-mediated protection (Supplementary Fig. 9j). To determine whether cancer cell-specific Batf2 expression has an abscopal effect, we implanted the EV control and Batf2-expressing tumours on the right flank. As expected, we observed a significant reduction in tumour volume when Batf2 was expressed. However, the EV control tumours grown in the left flank of mice that bear Batf2-expressing tumours in the right flank did not show a difference in volume compared to those EV tumours grown in the left flank of mice that bear EV tumours in the right flank (Supplementary Fig. 9k). These data suggest that the BATF2-expressing tumour activates a local immune response without triggering a notable abscopal effect.

To further support our findings, we knocked down Batf2 in NOOC2 cells, which have higher endogenous Batf2 expression levels. Depletion of Batf2 significantly reduced the production of Ifnb1 (Supplementary Fig. 9l). In agreement, sh-Batf2 NOOC2 tumours grew significantly faster than EV control tumours (Supplementary Fig. 9m). We demonstrated the role of the S227 phosphorylation site in Batf2 activation. To substantiate the physiological relevance of this finding, we established tumour models that stably express empty vector, wild-type Batf2 and 227A mutant Batf2. In agreement with our other data, expression of Batf2 inhibited tumour growth. However, Batf2-mediated protection was abrogated by mutating serine 227 to alanine and disabling phosphorylation at this amino acid (Supplementary Fig. 9n).

BATF2 expression in the host deters the initiation of HNSCC
To understand the impact of host BATF2 signaling on the immunogenicity of HNSCC, we first implanted MOC2-E6/E7 cells into littermate control wild-type and Batf2−/− mice. We observed significantly accelerated MOC2-E6/E7 tumour growth in the Batf2−/− host (Fig. 7a). Flow cytometric analysis of the TME showed that fewer p-Irf3+, CD4+, CD8+ and CD8+ Ifn-γ+ T cells were present in tumours growing in Batf2−/− mice (Fig. 7b and Supplementary Fig. 10b). Similarly, we observed fewer γδ T-cells in tumours grown in the Batf2−/− hosts (Supplementary Fig. 11a). Using another model, we implanted NOOC2 into littermate control wild-type and Batf2−/− mice and consistently found that the tumour burden was significantly higher in the Batf2−/− mice (Fig. 7c). Consistent with previous results, host deficiency in Batf2 resulted in fewer p-Irf3+, CD4+ and CD8+ T-cells infiltration in the TME (Fig. 7d). To further substantiate the findings, we employed the third model, 4MOSC1, which only grows orthotopically in the tongue. Batf2−/− hosts developed the largest tongue tumours (Fig. 7e).

Given the critical role of IFN-I in immune surveillance, we next sought to determine whether BATF2 suppresses tumour initiation using a 22-week-long carcinogen exposure model for HNSCC. The 4-NQO-induced HNSCC follows the pathological course of epithelial dysplasia transformation. We fed wildtype and Batf2−/− mice with drinking water containing 50 μg/mL 4-NQO for 16 weeks and switched to regular water afterward. We found that Batf2−/− mice showed shorter oral lesion-free survival (Fig. 7f). We started monitoring oral lesions weekly 10 weeks post-treatment start date. The oral lesions started to appear 14 weeks post-treatment initiation. We classified tumours based on their clinical measurements and found that the Batf2−/− mice showed a higher disease burden (Fig. 7g and Supplementary Fig. 10b). Notably, the deficiency in Batf2 lead to broad-field cancerization. The 4-NQO model in wild-type mice almost exclusively induces lesions in the tongue, in agreement with the literature24. We found that Batf2−/− mice were much more prone to developing tumours in the buccal mucosa and palate, in addition to the oral tongue (Supplementary Fig. 11c). The 4-NQO model bears a high histologic resemblance to human HNSCC, with architectural disturbance, the transition between dysplastic epithelium and invasive tumour islands, keratin pearl formation, cytologic atypia, increased mitotic count, stromal complexity and variably dense inflammatory cell infiltrate (Supplementary Fig. 11d). To determine whether a Batf2 defect remodeled the infiltrating immune cell landscape, we performed spatial protein marker analyses. We first examined the percentage of different immune subsets and found that the HNSCC in the Batf2−/− hosts showed significantly increased levels of CD206+ macrophages, PD-L1+ myeloid cells, Ly6g+ MDSC, decreased levels of Tbet+ CD4+ and significantly decreased expression of nuclear T-bet in CD8+ T-cells (Fig. 7h, i and Supplementary Fig. 10e, f). To map the intercellular relationships, we performed the nearest neighbor analysis and found that the distances between the following pairs of markers were significantly reduced in 4-NQO-induced HNSCC in Batf2−/− mice: from CD206+ to CD206+ cells, from PD-L1+ to CD206+ cells, from CD4+ to CD206+ cells, and from Tbet+ CD4+ to CD206+ cells (Supplementary Fig. 11g). These findings suggest that BATF2 promotes the expansion of effector population in the TME of HNSCC.

Methods
Cell culture and treatments
The HNSCC cell lines FaDu, UMSCC22A, UMSCC22B, UMSCC108, PCI13 and UMSCC49 were maintained in DMEM (10-013-CV, Corning) supplemented with 10% FBS (Gibco, Life Technologies) and 100 U/mL penicillin-streptomycin (15-140-122, Gibco). THP-1 cells were cultured in RPMI 1640 (MT10040CV, Corning) supplemented with 10% heat-inactivated FBS (Gibco, Life Technologies and 100 U/mL penicillin-streptomycin (15-140-122, Gibco). HEK-293T and RAW 264.7 were cultured in the same medium as HNSCC cell lines. MOC2-E6/E7, NOOC1 and NOOC2 cell lines were cultured in the 60% IMDM (SH30228.01, HyClone) with 30% F12 nutrient mix (11764-054, Gibco), 5% FBS, 5 μg/mL insulin, 40 ng/mL hydrocortisone, 5 ng/mL EGF, 100 U/mL penicillin and 100 mg/mL streptomycin. 4MOSC1 cells were cultured as previously reported22. NOM9-TK cells were cultured in oral keratinocyte medium (Sciencell, Catalog No. #2611). The defined amino acid deficiency media were formulated as previously reported42. BMDM were isolated from both male and female littermate wild-type C57BL/6J and Batf2−/− mice femurs and differentiated as described earlier3. The experimental BMDM were plated in DMEM containing 20% heat-inactivated FBS and 100 U/mL penicillin/streptomycin for the indicated treatment. In the experiments evaluating the impact of glutamine on the IFN-I pathway, BMDM were seeded in a medium containing 90% glutamine-free RPMI 1640 (21870076, Gibco), 10% dialyzed FBS (SH30079.01, HyClone) and indicated levels of L-glutamine (25030081, Gibco). Cells were cultured in a 37°C humidified incubator with 5% CO2. Treatments included 2′3′-cGAMP (tlrl-nacga23, Invivogen), ISD (tlrl-isdn, Invivogen), poly(I:C) (tlrl-pic, Invivogen), 5′ppp-dsRNA (tlrl-3prna, Invivogen), diABZI (tlrl-diabzi-2, Invivogen) and BPTES (SML0601, Sigma) as described in paper. NOOC1, NOOC2 and UMSCC cell lines were authenticated by STR profiling. FaDu, HEK-293T cells and THP-1 cells were from ATCC. Other cell lines were not authenticated. All cell lines tested negative for mycoplasma contamination.

Animals
We obtained six to eight-week-old C57BL/6J (strain 000664) mice from the Jackson Laboratory. The Ifnar1−/− strain backcrossed to the C57BL6/J background for over nine generations was reported3. The Rag1−/− (strain 002216) and Sting−/− (Strain 017537) mice were acquired from the Jackson Laboratory. The sperms of Batf2−/− mice were kindly provided by Dr. Shizuo Akira. The cryorecovery was performed by Taconic. The mice were genotyped by the following primers: Batf2s extra 5′-ACATCGGTCTCAGAGAGTTTGGAGTGG-3′, Batf2 WT 5′-TAGGCC CCTTCTCTAGAAGAAGGATGG-3′, Batf2 KO 5′-CTAAAGCGCATGCTC CAGACTGCCTTG-3′. Mice were kept under specific pathogen-free conditions in a 12 h:12 h light:dark cycle with a room temperature of 72°F and an air relative humidity of 57%. For the 4-NQO-induced HNSCC mouse model, wildtype and Batf2−/− mice were fed with drinking water containing 50 μg/mL 4-NQO for 16 weeks before regular water feeding for an additional six weeks. We started to monitor oral lesions ten weeks post-starting date. For the glutamine diet experiments, 6–12-week-old C57BL/6J mice were provided with a diet that contained 15 kcal% fat, 200 × g glutamine and 181 × g carbohydrates/3947 kcal (A16070103, Research Diets) or a control diet that contained 15 kcal% fat and 381 × g carbohydrates/3947 kcal (A11112201, Research Diets) on the same day when tumour cells were implanted. Anti-mouse CD8α (BE0061, BioXCell, Clone: 2.43), anti-mouse Cxcl10 (BE0040, BioXCell, Clone: 1F11), anti-Pd-1 (BP0033-2, BioXCell, Clone: J43) and anti-Ctla-4 antibody (BP0131, Bio X Cell, Clone: 9H10) were administered by intraperitoneal injection. Free c-di-AMP (tlrl-nacda-5, Invivogen) was administered via intra-tumoural injections or in combination with JHU-083 (HY-122218, MCE) by PO at 1.82 mg/kg when the average tumour size reached 150 mm3. Mice were humanely euthanized when the tumour volume reached 2000 mm³ or the ulceration exceeded 50% of the tumour surface area.

Human specimen analysis
Human specimens were from the Oral Pathology and Head and Neck Surgery services. HNSCC specimen analysis was approved by IRB protocols HUM00113038 and HUM00042189 at the University of Michigan and 2024-0364 at the University of Texas M.D. Anderson Cancer Center. Informed consent was obtained for specimens utilized for bulk RNA-Seq. All specimens were de-identified, and the aggregated patient’s demographic information is summarized in Supplementary Data 1–3. The HNSCC tissues used in this experiment have been exhausted and are unavailable. In the TMA analysis shown in Fig. 1e, the investigators were blinded to the CD8+ T-cell counts within the TME.

Gene expression and knockout
We generated BATF2-expressing retroviruses and transduced human HNSCC cells (FaDu and UMSCC22A) and murine HNSCC cells MOC2-E6/7 with retroviruses to produce stable cell lines. We generated the BATF2-179A and BATF2-227A mutants, which contain serine-to-alanine mutations and cannot be phosphorylated at 179S and 227S by using a Site-Directed Mutagenesis Kit (E0554S, NEB). We generated BATF2-targeted CRISPR-Cas9 lentiviruses using a single-guide RNA (sgRNA): 5′-CACCGTGCACCTCTGTGGGGGCAAT-3′. We transduced UMSCC108 and UMSCC49 with the BATF2-targeted CRISPR/Cas9 lentiviruses and generated stable cell lines. We generated Batf2-targeted and Sting-targeted lentiviruses using shRNA (shBatf2: 5′-GCACGAATCCTTGGA GAAACA-3′ and shSting: 5′-CAACATTCGATTCCGAGATAT-3′) and transduced them with MOC2-E6/7 or NOOC2 cells. We transduced the same cell line with empty vector lentivirus to generate the control cells. STING domain truncation mutants were generated by PCR amplification using sequence-specific primers. The PCR product and the pcDNA3.1-Flag backbone were digested with NotI and EcoRI restriction endonucleases, gel-purified and subsequently ligated with T4 DNA ligase. The ligation mixture was transformed into Stbl3 competent cells, and colonies were selected on LB agar containing ampicillin (100 μg/mL). After plasmid extraction, the constructs were validated by Sanger sequencing and further confirmed by Western blot analysis to ensure optimal expression.

Immunoblot and immunoprecipitation
Cells were washed with 1× PBS twice and then lysed in 1× RIPA buffer (9806, Cell Signaling Technology) supplemented with an EDTA-free protease inhibitor cocktail (11836170001, Roche) and phosphatase inhibitor cocktail (78420, Thermo Fisher Scientific). The protein concentration was measured by a BCA kit (23225, Thermo Fisher Scientific) and boiled with 4× sample buffer (NP0007, Invitrogen) for 5 min at 95°C. Prepared lysates were loaded on NuPAGE 4 to 12% (NP0323BOX, Thermo Fisher Scientific) or 16% (XP00162BOX, Thermo Fisher Scientific) Bis-Tris Gel. Followed by blocking in 10% milk, membrane was incubated in 5% milk with the primary antibodies: BATF2 Antibody (ab204510, Abcam, dilution 1:400), Phospho-TBK1(Ser172) Antibody (5483S, Cell Signaling Technology, dilution 1:1000), TBK1 Antibody (3504S, Cell Signaling Technology, dilution 1:1000), Phospho-STING (Ser365) Antibody (72971S, Cell Signaling Technology, dilution 1:1000), STING Antibody (13647S, Cell Signaling Technology, dilution 1:1000), phospho-IRF-3 (Ser396) Antibody (4947S, Cell Signaling Technology, dilution 1:1000), IRF-3 Antibody (4302S, Cell Signaling Technology, dilution 1:1000), phospho-STAT1 (Tyr701) Antibody (9167S, Cell Signaling Technology, dilution 1:1000), STAT1 Antibody (14994S, Cell Signaling Technology, dilution 1:1000), VDAC Antibody (4661S, Cell Signaling Technology, dilution 1:1000), Lamin B1 Antibody (ab16048, Abcam, dilution 1:1000), α-Tubulin Antibody (3873S, Cell Signaling Technology, dilution 1:1000) and anti-β-actin antibody (ab49900, Abcam, dilution 1:1000). The secondary antibodies anti-rabbit IgG-HRP antibody (ab97051, Abcam, dilution 1:20000) and anti-mouse IgG-HRP antibody (ab97023, Abcam, dilution 1:20000) were incubated according to the manufacturer’s recommendations. To determine phosphorylated proteins, samples in 1× Sample Buffer (191-13272, Fujifilm) were loaded into SuperSep™ Phos-tag™ gel (199-18011, Fujifilm) and were blotted for BATF2 and IRF3.
To immunoblot native proteins, the cells were lysed in 1× NativePAGE™ Sample Buffer and 1% Digitonin (BN2008, Thermo Fisher Scientific) and sonicated 15 s for 3 times. The BCA protein assays were performed to quantify lysate protein concentrations. The samples were loaded into NativePAGE 4–16%, Bis-Tris, 1.0 mm, Mini Protein Gels (BN1002BOX, Thermo Fisher Scientific) and were run in the NativePAGE Running Buffer (BN2007, Thermo Fisher Scientific). Then, the proteins were transferred to a PVDF membrane in the NuPAGE Transfer Buffer and blotted by a STING Antibody (13647S, Cell Signaling Technology, dilution 1:1000).
HEK-293T and FaDu cells were seeded in 10 cm dishes and were transfected with 1 μg/mL FLAG-STING and 1 μg/mL BATF2-HA plasmids for 24 h to assess the interaction between BATF2 and STING. Experimental cells were lysed on ice by NP40 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.1% NP-40) supplemented with protease and phosphatase inhibitors. Anti-FLAG M2 Affinity gel (A2220, Millipore Sigma) and anti-HA Agarose (26181, Thermo Fisher Scientific) were used to pulldown FLAG-STING and BATF2-HA proteins. The immunoprecipitated proteins were blotted for anti-FLAG M2 Antibody (14793, Cell Signaling Technology, dilution 1:1000) and anti-HA-Tag Antibody (3724, Cell Signaling Technology, dilution 1:1000).

Immunofluorescent confocal microscopy
Cells grown in 4-well chamber culture slides were stained with 100 nM MitoTracker (M7510, Invitrogen) Orange for 15 min at 37°C, followed by fixation in 4% paraformaldehyde/PBS for 15 min at room temperature. Fixed cells were permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Cells were incubated with primary antibodies anti-HA-488 (2350S Cell Signaling Technology, dilution 1:800) and anti-STING (13647S, Cell Signaling Technology, dilution 1:50) for 1 h. After washing, cells were incubated with AF647 labeled secondary antibodies for 1 h followed by staining with Hoechst 33342 (H3570, Life Technologies, dilution 1:5000). Coverslips were mounted in ProLong antifade mountants and imaged with a Nikon A1 SI inverted confocal microscope.

Subcellular fractionation
The purification protocol of the nuclei, cytosol and ER membranes by serial centrifugation was adapted from a reported protocol43. Briefly, THP-1 STING WT cells were lysed by ~70 iterations of up and down passes of the pestle in a hypotonic buffer. Nuclei were obtained by centrifugation of the lysed cells at 600 × g for 5 min at 4°C. The nuclear pellet (P600) was resuspended and incubated in the nuclei lysis buffer for 30 min on ice and centrifuged at 10,000 × g for 30 min, and the resulting supernatant was collected as the nuclear fraction. Post-nuclear supernatant (S600) was centrifuged at 5000 × g for 10 min at 4°C to remove mitochondrial fraction (P5000) and then centrifuged at 20,000 × g for 30 min at 4°C to remove lysosomal and plasma membrane fraction (P20000). The supernatant was centrifuged at 100,000 × g for 1 h at 4°C to obtain the ER fraction (P100000) and the cytosolic fraction (S100000).

Quantification of gene expression levels
Total RNA was extracted by RNeasy Plus Mini Kit (74136, Qiagen). The RNA concentration was determined using a Nanodrop Spectrophotometer from Thermo Fisher Scientific. Subsequently, the RNA was reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit along with RNase inhibitor (4368814 and N8080119, respectively, Applied Biosystems). The primer sequences have been previously reported3,4. Additional primers are as follows: human BATF2 forward 5′-3′ CAGCTGAAGAAGCAGAAGAACCG, reverse 5′-3′ TTTTCC AGAGACTCGTGCTGC; mouse Batf2 forward 5′-3′, CACCGCGATCG CATGCAACTCTGTGGGAGTAG, reverse 5′-3′ CTAGAAGTGGACT TTTGCAGAAG; Mouse Oas1 forward 5′-3′, GCCTGGTCACG CACTGGTA, reverse 5′-3′ AAGCCCTGGGCTGTGTTG; mouse Mx1 forward 5′-3′, GGGGAGGAAATAGAGAAAATGAT, reverse 5′-3′ GTTTACAA AGGGCTTGCTTGCT; human CXCL9 forward 5′-3′, GTGGTGTTCTTTT CCTCTTGGG, reverse 5′-3′ ACAGCGACCCTTTCTCACTAC; human CXCL10 forward 5′-3′, CTCCAGTCTCAGCACCATGA, reverse 5′-3′ GCTCCCCTCTGGTTTTAAGG; mouse Ifng forward 5′-3′ TCAAGTGG CATAGATGTGGAAGAA, reverse 5′-3′ TGGCTCTGCAGGATTTTCATG; human ISG54 forward 5′-3′, ACGGTATGCTTGGAACGATTG, reverse 5′-3′ AACCCAGAGTGTGGCTGATG.

ELISA and Luminex
Cells were seeded into six-well or 96-well plates and treated with agonists as indicated. The amount of Ifn-β and Tnf-α in the supernatant was measured by a mouse Ifn-β kit (luex-mifnbv2, Invivogen) and a Tnf-α ELISA kit (BMS6073, Invitrogen) according to the manufacturer’s instructions. A mouse Luminex Discovery Assay kit (LXSAMSM, R&D Systems) was also used to quantify cytokines.

Tissue histology, immunohistochemistry and multispectral imaging
Tongue specimens from mice in the 4-NQO model were fixed in 10% formalin and then serially sectioned before paraffin embedding. The slides were deparaffinized and stained in Mayers Hematoxylin. After washing four times with tap water, the slides were washed with 1× PBS and distilled water. Then, they were stained with Alcoholic-Eosin and dehydrated through ethanol followed by xylene wash.
Human HNSCC specimens were included in tissue microarrays (TMAs). TMAs were stained with anti-BATF2 antibody at 1:200 dilution (ab204510, Abcam). Paired longitudinal specimens from patients who progressed from OPMD to oral cancer were stained for BATF2 using the same protocol. Anti-CD20 antibody (ab64088, Abcam, dilution 1:200) and anti-Tcrγ/δ antibody (#14-5711-82, Thermo Fisher Scientific, dilution 1:100) were used for syngeneic murine tumour specimens. The specimens were incubated with a secondary antibody (PK-4001, Vectastain, dilution 1:100). Immunohistochemical (IHC) staining intensity was quantitated in tumour tissue by selecting a region of interest using Aperio ImageScope or ImageJ, followed by quantification.
For immunofluorescence multispectral imaging, the slides were stained with antibodies against PD-L1 (AF1019, R&D, dilution 1:40), CD206 (24595S, Cell Signaling Technology, dilution 1:400), CD4 (25229S, Cell Signaling Technology, dilution 1:200), T-bet (97135S, Cell Signaling Technology, dilution 1:400), Ly-6G (87048S Cell Signaling Technology, dilution 1:100) and CD8 (98941S, Cell Signaling Technology, dilution 1:200). The slides were then stained with horseradish peroxidase HRP-conjugated secondary antibody and HRP-reactive OPAL fluorescent reagents. The signal was washed off between each staining. Whole-slide scanning was completed using the Vectra Polaris Automated Quantitative Pathology Imaging System by Akoya Biosciences. Regions of Interest (ROIs) were selected by an oral pathologist using the Akoya Phenochart software. ROIs were then scanned at 40× resolution.
The subsequent analysis involved employing machine learning algorithms developed within the inForm software package. Assurance of consistent staining intensities across the tissue specimen was established. Tissue regions were classified into distinct categories, namely “stroma,” or “other” encompassing elements like empty space. Cellular segmentation encompassed nuclear, cytoplasmic and membranous domains. We utilized the inForm software package to perform a texture analysis and cell segmentation for the classification of cell phenotypes. Then, we employed the R-language to process the single-cell dataset. We first collapsed the metric across multiple ROIs by the sample ID and then conducted a two-sample Wilcoxon test for each marker to compare its mean expression level between groups. We further conducted Akoya’s nearest neighbor analysis to reveal the mean cell distance between two phenotypes of interest. For each phenotype pair, we conducted a two-sample Wilcoxon test to compare the mean cell distance between groups. The test result is significant if the p-value is smaller than 0.05.

Chromatin immunoprecipitation (ChIP)
ChIP was performed using the SimpleChIP® Plus Sonication Chromatin IP Kit (56383, Cell Signaling Technology). BMDM were fixed with 1% formaldehyde and then lysed in cell lysis buffer and nuclear lysis buffer. The samples were sonicated at 65% amplitude for 10 min before being subjected to pull-down using an anti-H3K27me3 antibody (07-449, Millipore, dilution 1:50) and an anti-H3K4me3 antibody (9751S, Cell Signaling Technology, dilution 1:50). The immunoprecipitated and input DNA was quantified using qPCR. The primers are as follows: mouse Batf2 promoter forward 5′-ATCACAGCTGCCTTCTTTCC, reverse 5′-TGCATGTCCCTTGGATTGTG; mouse Ifnb1 promoter forward 5′-TCCCTCAAGGTTCTCTTCCTG, reverse 5′-AGCAAAGCCAC CAAGAAAGC; mouse Cxcl9 promoter forward 5′-TTGAAAGGGTG GTTGTTGGC, reverse 5′-3′ ATGCCCAATTTGCCTTCTGG.

Oxygen consumption rate assay
The BMDM were seeded in the seahorse XFp mini plate overnight and then transfected with 3.0 μg/mL ISD for 6 h. The XFp sensor cartridges were filled with 200 μL pre-warmed XF Calibrant the day before the assay. The cells were treated with 1.5 μM oligomycin, 1.0 μM FCCP and 0.5 μM Rot/AA. The oxygen consumption rate was quantified using a Seahorse XFp Analyzer.

Flow cytometry
Tumour infiltrating lymphocytes were purified by Ficoll and seeded in complete RPMI with 2-mercaptoethanol (55 μM) overnight. Phorbol myristate acetate (20 ng/ml), ionomycin (1.0 μg/mL) and 1× monensin were added into each well for 4 h. Cells were collected and stained for viability with Zombie Aqua (423101, BioLegend). Next, cells were incubated with Fc blocker (anti-CD16/32, clone 93, BioLegend, dilution 1:100) for 10 min on ice. The cells were then stained using the following antibodies: anti-CD45 (clone 30-F11, BioLegend, dilution 1:100), anti-CD8 (clone 56-6.7, BioLegend, dilution 1:50), anti-TCRb (clone H57-597, BioLegend, dilution 1:40), anti-CD4 (clone RM4-5, BioLegend, dilution 1:40), anti-IFNγ (clone XMG1.2, BioLegend, dilution 1:40), anti-p-IRF3 (53539S, Cell Signaling Technology, dilution 1:50), anti-p-TBK1 (14590S, Cell Signaling Technology, dilution 1:50), anti-mouse TCRγ/δ (clone GL3, BioLegend, dilution 1:40), anti-CD3 (clone 17A2, BioLegend, dilution 1:25). Data acquisition and compensation was conducted on flow cytometer and analyzed by FlowJo.

Next-generation sequencing and data analyses
Total RNA from non-treated or treated WT and Batf2−/− BMDM was processed to bulk sequencing. Samples with an RNA Integrity Number of 8 or greater were subjected to Poly(A) mRNA purification. After the cDNA libraries were validated, the samples were subjected to paired-end 150 bp sequencing on the Illumina NovaSeq S4 platform. Bcl2fastq2 Conversion Software by Illumina was used to de-multiplex Fastq files.
After tumour-infiltrating immune cells were enriched, dead cells in the cell suspension were removed using the EasySep Dead Cell Removal (Annexin V) Kit (17899, STEMCELL Technologies). Following cell preparation, the suspension underwent 10× Genomics 3′-single-cell processing and RNA-Seq. The quality of the final libraries was verified using the TapeStation 4200 (Agilent). The final libraries were quantified by Qubit (Thermo Fisher). Pooled libraries were subjected to paired-end sequencing on the Illumina NovaSeq platform. Bcl2fastq was used for de-multiplexing Fastq files. Our selection of high-quality transcriptomes was based on the following criteria: (i) qualified cells should exhibit unique feature counts ranging from 200 to 6000; (ii) mitochondrial reads were limited to fewer than 20%; and (iii) transcriptomes should demonstrate a unique molecular identifier count of over 500. To mitigate technical artifacts, we controlled the library size effects using the sctransform tool and applied the mutual nearest neighbor algorithm to eliminate potential batch effects during integration. In selecting genes for further analysis, we prioritized those exhibiting the highest cell-to-cell variation, totaling 3008 genes. Additional feature genes were chosen to improve the segregation of the immune subsets as reported44.
For whole-exome sequencing of NOOC2, genomic DNA was extracted by the Genomic DNA Mini Kit (K182001, Invitrogen). The sequencing dataset is deposited to the NCBI SRA with a BioProject ID PRJNA1147735. We only focused on high-quality reads that passed the Illumina RTA filter criteria. The reads were then aligned to the NCBI human reference genome (GRCh37) using BWA45. We used Picard to remove the duplicated reads. Putative single-nucleotide variants (SNVs) and small insertions/deletions (Indels) are identified by running four different variant callers, including Strelka, MuSE, VarDict and Mutect. Then, we filtered out potential false positives and mapping artifacts. To remove germline polymorphisms, we compared the nominated somatic mutations to dbSNP, 1000 Genomes Project, the National Heart, Lung, and Blood Institute’s Exome Sequencing Project, and the Genome Aggregation Database (gnomAD). All mutations were annotated by ANNOVAR using the NCBI RefSeq database46. The mutational signatures of SNVs were analyzed using SigProfilerSingleSample47.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Gong and colleagues provide a well written, well developed and rigorously performed study identifying BATF2 as a tumor suppressor gene acting via feed-forward activation of the STING/IFN-I/Tgd pathway, with BATF2 transcription/expression negatively modulated by local (dietary) glutamine concentrations. Increased intrinsic expression of BATF2 in tumors leads to enhanced anti-tumor T cell fitness and improved tumor growth control in vivo across multiple mouse (transplantable and inducible) HNSCC tumor models, while deficiency of BATF2 in tumors results in a pro-tumor TIME characterized by enriched M2 TAM and reduced Type-1 T cell content. Furthermore, BATF2-KO mice exhibited greater intrinsic susceptibility to tumor initiation/growth/progression in multiple model systems. Overall, the presented data support the ability of BATF2 to sustain optimal fitness of the STING signaling pathway within the TME and suggest that future therapeutic targeting to reinforce BATF2 expression and/or to reduce glutamine metabolism may improve (immune)therapeutic outcomes in the HNSCC (cancer) setting.\nThe report is interesting and informative and my only minor concerns reflect:\n1.) For the data presented in Fig. 5k and 5l where I would request that the authors redraft the Fig. 5l tumor growth curves with the same y-axis range (i.e., 1500 mm2) as was used for Fig. 5k. Also, the fact that the glutamine diet negated the significance of response to STING agonist may be true, but this does not appear to provide a survival advantage based on the perceived trajectories for tumor growth in Figs. 5k/l. Can the authors provide Kaplan-Meier plots and the relevant statistical analyses to support or refute the impact of the glutamine diet?\n2.) Per Fig. 5m/n, the major difference in the TIME impacted by the glutamine diet appears to reflect presence of CXCR5+ B cells under constitutive glutamine concentrations in the TME. The authors should provide data for comparative levels of CXCL13 expression (including which cells are producing via scRNAseq) in the TME under both conditions. Also, since B cells are a canonical component of mature tertiary lymphoid structures, are TLS prevalent in control tumors but devoid in mice fed the glutamine diet? Please discuss and provide supportive data is available.\n3.) The discussion is rather short overall and more discussion for practical means by which BATF2 might be pharmacologically upregulated in the TME for therapeutic gain is recommended. Can the authors speak to known promoters of BATF2 or epigenetic agents that might be targeted with known in clinic drugs for testing in prospective translational studies (alone or in combination with immunotherapeutic agents)? Such discussion would add to the merit of the report.", "reviewer_2": "Gong et al. identified BATF2 as a novel regulator of the STING-type I interferon axis. They show that BATF2 directly interacts with STING, thereby promoting downstream signaling and target gene expression. By integrating molecular and functional in vitro studies with in vivo experiments in transplantable tumor models, the authors conclude that BATF2 is a tumor suppressor in HNSCC and that loss of BATF2 expression is a mechanism of immune evasion, as it impairs STING signaling in tumor cells.\nComment: The BATF2-STING axis is a novel and very interesting finding. However, additional (control) experiments are needed to clearly demonstrate that BATF2 is a tumor suppressor and that downregulation of BATF2 in tumor cells is of relevance in immune evasion of HNSCC.\nComments on the role of BATF2 as novel tumor suppressor:\nSo far, there is no evidence for a tumor-suppressor function of BATF2 in HNSCC, as the manuscript does not compare the in situ expression of BATF2 between cancer cells and normal squamous cells. What is shown in Fig. 1f? Are BATF2 levels in cancer cells, or are the BATF2 levels for the entire tissue core presented? Since BATF2 can also be induced by interferon-gamma, the correlation between BATF2 and CD8 could be driven by IFN-γ release from CD8 T cells. In this case, HNSCC tumor cells may not lose BATF2 expression but rather acquire it due to signals from the microenvironment.\nMoreover, several single-cell RNA sequencing datasets for HNSCC are available (including those from different disease stages, e.g., PMID: 36828832), where BATF2 expression in cancer cells could be analyzed and plotted.\nThe authors analyzed BATF2 expression in tissue cores from 200 patients with a median follow-up of 60 months. Is there any association between BATF2 expression and survival or disease progression? If BATF2 is a strong tumor suppressor, patients with high BATF2 expression should have a significantly better outcome.\nFinally, introducing BATF2 as a general tumor suppressor is an overstatement, as the authors focused their studies solely on HNSCC.\nComments on the BATF2-STING axis in human squamous cell carcinoma cell lines.\nTo demonstrate that BATF2 impacts STING signaling in carcinoma cells, the authors had to overexpress both BATF2 and, notably, STING, since synthetic STING agonists do not induce “… robust IFN-I activation” (author statement, lines 205-207, page 10). Even in cell lines with high endogenous STING levels (Fig. 3F, cell lines UMSCC108, UMSCC49), STING must be overexpressed to achieve pathway activation. This raises the question: how relevant is the BATF2-STING axis in squamous cell carcinoma if STING cannot be activated upon transfection with synthetic agonists? This suggests that activation of endogenous STING is defective even in the presence of high BATF2 levels.\nComments on silencing of BATF2 by glutamine:\nThe authors conducted several in vitro studies to demonstrate that glutamine, by enhancing oxidative phosphorylation, leads to the epigenetic silencing of BATF2 expression. However, the effect of glutamine on BATF2 expression is shown only at the RNA level; important protein data are missing. All experiments related to glutamine were performed in murine macrophages. If the authors wish to claim that this mechanism allows tumor cells to downregulate BATF2, it is crucial to demonstrate that this regulation occurs in carcinoma cells and affects BATF2 protein levels. Furthermore, the effects of a glutamine diet on tumor growth following intratumoral injection of STING agonists appear to be relatively weak.\nComments on in vivo studies:\nIn vivo experiments were conducted using different murine squamous cell carcinoma cell lines.there is no information about endogenous BATF2 protein expression in those cells.\nFigure 6f demonstrates reduced growth of Batf2-overexpressing tumors. To what extent this effect is related to STING activation remains unclear. Experiments should be performed using BATF2-overexpressing STING knockout cells.\nAdditionally, survival curves should be included.\nIn Figure 7: Is the enhanced growth of transplanted tumor cells in BATF2-/- mice due to the lack of STING activation?\nFinally, the authors completely ignore relevant published data on BATF2 in cancer, even those studies which already showed that BATF2 has anti-tumor activity.", "reviewer_3": "This manuscript offers a novel perspective on BATF2 as a glutamine-responsive tumor suppressor essential for promoting STING signaling and IFN-I production. The study is comprehensive, leveraging both molecular and preclinical models to delineate BATF2's immunomodulatory roles. However, some areas of the manuscript could benefit from additional clarification, experimental reinforcement, and broader context to enhance its impact and rigor.\nMajor Revisions\n1. Mechanistic Insight on Glutamine and Epigenetic Regulation:\nWhile the role of glutamine in promoting H3K27me3 modifications at the BATF2 promoter is well demonstrated, the study does not explore whether this mechanism also affects other key immune genes regulated by STING. A broader analysis of glutamine-induced epigenetic changes at loci relevant to the immune response (e.g., CXCL10 or IFN-I target genes) would strengthen the conclusion that glutamine broadly suppresses antitumor immunity through epigenetic remodeling.\n2. Role of BATF2 in STING Oligomerization:\nThe manuscript demonstrates that BATF2 is essential for STING oligomerization (Fig. 4h). However, the specific role of BATF2 in stabilizing these oligomers remains unclear. Testing whether BATF2 interacts directly with key regions of STING (e.g., using mutational analysis or co-immunoprecipitation of truncated STING constructs) would clarify the molecular basis of this interaction.\n3. Function of CXCL10+ Myeloid Cells in Tumor Rejection:\nThe authors highlight the expansion of CXCL10+ myeloid cells downstream of BATF2 but do not directly assess their functional relevance. Including depletion experiments (e.g., using neutralizing antibodies or conditional knockouts) to test whether CXCL10+ myeloid cells are necessary for BATF2-mediated tumor rejection would substantiate this claim.\n4. Kinetics of BATF2 Expression and IFN-I Responses:\nThe timing of BATF2 expression relative to IFN-I signaling and T-cell recruitment is critical to understanding its role in tumor immunity. Longitudinal studies tracking BATF2 expression, STING activation, and immune infiltration in tumors (e.g., via time-course RNA-Seq or flow cytometry) would provide valuable insights.\n5. Relevance of p227 Phosphorylation Across Models:\nThe phosphorylation of BATF2 at serine 227 (p227) is presented as critical for its function, but this claim is primarily demonstrated in vitro (Fig. 4k). Testing whether p227 phosphorylation is similarly required in vivo (e.g., using BATF2 p227A knock-in mice) would solidify this mechanistic insight.\n6. STING Agonist Resistance and Glutamine Levels:\nThe manuscript concludes that high glutamine levels blunt STING agonist efficacy (Fig. 5). It would be helpful to explore whether glutamine-targeting interventions (e.g., glutaminase inhibitors) can rescue STING agonist responses in glutamine-rich conditions, thus supporting potential combination therapies.\n7. BATF2 in Human Clinical Samples:\nWhile BATF2 expression is correlated with immune markers in HNSCC patient datasets, there is little exploration of its prognostic or therapeutic relevance. Analyzing BATF2 expression in patients stratified by clinical outcomes (e.g., response to immunotherapy) would provide translational significance to the findings.\nMinor Revisions\nTerminology Consistency: Standardize terms like “IFN-I signature genes” and “IFN-I target genes” for clarity.\nText Clarity: Simplify the introduction by focusing on BATF2’s novelty in immune modulation while reducing background repetition.", "reviewer_4": "Gong et al describe a role for BATF2 as a glutamine-dependent tumor suppressor gene in HNSCC. BATF2 is phosphorylated at serine 227 to interact with STING, and is required for eliciting STING-dependent type I IFN response, thus affecting anti-tumor immunity.\nThe main findings are:\n1) BATF2 expression is positively correlated with the gene expression of type I IFN genes and with immune cell infiltration, both in published cohorts and in a separate tissue microarray analysis of n=200 samples.\n2) BATF2 KO in BMDM impairs type I IFN responses upon STING but not upon MAVS activation.\n3) Exogenous BATF2 expression in HNSCC cell lines that do not express BATF2 upregulates type I IFN response upon STING activation, whereas BATF2 knockout in BATF2-expressing HNSCC cell lines impairs type I IFN response.\n4) BATF2 localizes in the ER and is phosphorylated at serine 227 to interact with STING.\n5) BATF2 expression is silenced in glutamine-rich media, presumably by the regulation of histone marks.\n6) Glutamine-rich diet impairs the efficacy of STING agonists in a mouse model of s.c. HNSCC and reduces immune cell infiltration.\n7) BATF2 overexpression leads to tumor rejection in immunocompetent mice, which is dependent on the presence of gd T cells, and IFNAR1.\n8) BATF2 knockout increases tumor growth and suppresses anti-tumor immune responses in several different HNSCC mouse models.\nThe manuscript establishes a role for BATF2 as a tumor suppressor gene in HNSCC and combines data from human tumor samples and several mouse models. Robust genetic approaches and state-of-the-art read-outs are used to support the findings. The results are well-described, and the figures and figure legends are nicely presented.\nThe role of BATF2 as a tumor suppressor gene is well established by previous studies, including in epithelial cancers: PMID 26135942, 32650804, 37995570, 34778372 and more. Some of these studies show, similarly to the current one, that BATF2 expression on tumor cells shapes the TME immune infiltrate. The role of BATF2 in driving inflammatory responses, e.g. in macrophages, has also been previously observed: PMID 25957166 and others.\nThe current study presents novel points on the importance of BATF2 for type I IFN responses following STING, but not MAVS activation, in particular on the phosphorylation of BATF at serine 227 as a prerequisite for interaction with STING. A further novel point is the metabolic regulation of BATF2 expression through glutamine. However, I think that, in particular, the last point needs to be investigated in greater detail.\nMajor points:\n1. The manuscript is somewhat difficult to follow, as the authors frequently switch between the role of BATF2 in BMDM and in tumor cells. In the end, it doesn't really become clear to me whether BATF2 expression on one of these two populations is more important for the observed in vivo effects. Also, not all mechanisms are shown in both cell types. The interaction of STING and phosphorylation of BATF2 are shown in tumor cell lines, while the regulation of BATF2 through glutamine is shown in BMDM.\n2. I find the beginning of the paper confusing. The authors begin the manuscript with the question why HPV+ HNSCC do not respond to ICI blockade as well as expected and compare single-cell transcriptomes from HPV+ and HPV- patients to discover aberrations in the immune cell population composition. However, there is no correlation with clinical responses to ICI, which makes me wonder why this comparison was made in the first place. I personally think that leading into the paper by stating that high BATF2 expression was found in clusters 10, 19, 20 which also had high type I IFN gene expression would make a clearer point, and that splitting the samples in HPV+ and HPV- doesn't have an added value for the manuscript.\n3. The interaction between BATF2 and STING is shown for tumor cells, but not for macrophages. Do the authors have evidence that the same mechanism is active in macrophages? Vice versa, does glutamine concentration in the media affect the BATF2 expression in tumor cells, or only in macrophages?\n4. How much of the effects of high-glutamine media on type I IFN secretion is mediated through BATF2? The experiments in Fig. 5e-f should be repeated with BATF2-/- cells. Along the same lines, is the effect of glutamine-rich diet on tumor development in Fig. 5k-q mediated solely through BATF2? Experiments should include groups with BATF2 KO tumors and/or hosts as controls.\n5. What is the mechanism by which glutamine epigenetically regulates BATF2? Is this mediated through glutamine itself, or through some of its downstream metabolites, e.g. alpha-ketoglutarate. How are the metabolic profiles with increased oxidative phosphorylation (Figure 5i-j) related to the remaining mechanism? Would other metabolic conditions that increase OCR also affect BATF2 expression, or is this specific to glutamine-rich media?\n7. Are there data from human tumor samples supporting the notion that glutamine metabolism regulates BATF2-expression?\n8. Figure 6: are only gd T cells responsible for the rejection of BATF2-expressing tumors? I would be interested to see experiments with depletion of alpha-beta T cells, especially as these subsets are also affected by BATF2 expression in other figures. Vice versa, are gd T cells affected in the tumor models presented in Fig. 7?\n9. It would be interesting to know if BATF2 expression by tumor cells drives only local or also systemic immune responses. For example, the authors could use the model in FIgure 6f and inject WT tumor cells in one flank and BATF2-expressing or WT cells in the other flank, and monitor whether the presence of a BATF2-expressing tumor activates a systemic immune response leading to a better control of the WT tumor in the other flank, compared to animals bearing WT tumors on both sides.\n10. Would BATF2-expressing tumors grow more aggressively if mice are kept on a glutamine-rich diet (Figure 6)?\n11. Is there a possibility to target BATF2 or any downstream gene pharmacologically?\nMinor points:\n1. I would recommend to include in the figure legends how often experiments were performed and whether pooled or representative data are shown.\n2. Why are cells sometimes stimulated with ISD/cGAMP, and other times with STING?\n3. The discussion is quite short and could be extended to interpret the findings in the light of other relevant studies in the field."}, "author_response": "We are submitting our revised manuscript NCOMMS-24-80950-T, entitled “BATF2 is a glutamine-responsive tumour suppressor essential for type-I interferon-dependent anti-tumour immunity”, for your consideration. We appreciate the enthusiastic feedback and the opportunity to improve this study further. We are grateful for the effort, rigor, and insight of the editor and all four reviewers. We have kept the critiques raised in mind, carefully performed new experiments, and extensively revised our manuscript with new data listed below, which substantially improved the depth and breadth of this manuscript. Below is the list of new data we generated in response to reviewers’ comments.\nNew Fig. 1f-g BATF2 expression levels are compared between human OPMD and paired HNSCC specimens.\nNew Fig. 1h-i The association between BATF2 expression and IFN-I activation is analyzed using fresh human oral squamous cell carcinoma specimens.\nNew Fig. 4h Co-Immunoprecipitation of the BATF2-STING complex is performed in RAW 264.7 cells.\nNew Fig. 4i BATF2 specifically interacts with the LBD domain of STING.\nNew Fig. 5k-m Glutamine desensitizes tumours to STING agonists. Inhibition of glutamine metabolism with JHU083 sensitized tumours to CDA in mice on a glutamine-rich diet.\nNew Fig. 5r BATF2-IFN-I target gene expression levels are inversely correlated with glutamine metabolism genes in human oral squamous cell carcinomas.\nNew Fig. 6f-i Tumour-specific expression of Batf2 reprograms the TME in a stepwise fashion.\nNew Fig. 7b and 7d Host deficiency in Batf2 dampens anti-tumour immunity.\nNew Supplementary Fig. 1e-i BATF2 expression levels are positively correlated with overall survival in patients with HNSCC, OV, SAR, SKCM, or STAD.\nNew Supplementary Fig. 1j-m BATF2 expression levels are higher in responders to ICB.\nNew Supplementary Fig. 3d BATF2 activates the STING pathway in NOM9-TK normal oral keratinocytes.\nNew Supplementary Fig. 4a The Sting knockdown efficiency in stable cell lines is confirmed.\nNew Supplementary Fig. 4b-d Batf2-mediated IFN-I pathway activation depends on Sting in cancer cells.\nNew Supplementary Fig. 4e-g Batf2-mediated IFN-I pathway activation depends on Sting in BMDMs.\nNew Supplementary Fig. 4h S227 phosphorylation is required for STING activation in UMSCC22A cells.\nNew Supplementary Fig. 5a The expression levels of BATF2 are specifically regulated by defined amino acids in the culture media.\nNew Supplementary Fig. 6a-c Glutamine suppresses the BATF2-IFN-I pathway in THP1 cells.\nNew Supplementary Fig. 6d-e Glutamine suppresses the BATF2-IFN-I pathway in HNSCC cells.\nNew Supplementary Fig. 6f Glutamine-mediated inhibition of STING-induced Ifn-b production depends on Batf2 in BMDMs.\nNew Supplementary Fig. 7f Glutamine-rich diet-mediated tumour aggression depends on Batf2.\nNew Supplementary Fig. 7g Glutamine-rich diet reduced the survival of tumour-bearing mice treated with CDA.\nNew Supplementary Fig. 7l A glutamine-rich diet dampens STING-induced B-cell marker upregulation in the scRNA-Seq analysis of the TME.\nNew Supplementary Fig. 7m A glutamine-rich diet dampens STING-induced expansion of CD20+ B-cells.\nNew Supplementary Fig. 9a Endogenous expression levels of Batf2 and Ifnb1 in murine HNSCC cell lines are assessed using qPCR.\nNew Supplementary Fig. 9f-g Batf2-mediated immunity against MOC2-E6/E7 tumours depends on cancer-specific expression of Sting.\nNew Supplementary Fig. 9i Batf2-mediated anti-tumour immunity depends on Cxcl10.\nNew Supplementary Fig. 9j Batf2-mediated anti-tumour immunity depends on CD8+ T-cells.\nNew Supplementary Fig. 9k Cancer cell-specific expression of Batf2 does not show abscopal effects.\nNew Supplementary Fig. 9l Stable empty vector control and Batf2-deficient NOOC2 cells are validated. Batf2 deficiency leads to lower levels of Ifnb1at baseline.\nNew Supplementary Fig. 9m Cancer cell-specific deficiency of Batf2 leads more aggressive tumour growth.\nNew Supplementary Fig. 9n The phosphorylation of Batf2 S227 is indispensable for the tumour-inhibitory function of Batf2 in vivo.\nNew Supplementary Fig. 10a Tumours growing in Batf2-/- mice harbor fewer γδ T-cells quantified by IHC staining using an antibody specific for the δ chain of the murine T-cell receptor.\nWe feel that the reviewers’ comments have guided us to substantially improve the quality and expand the breadth of our study. Main changes are highlighted in yellow in the marked version of the manuscript. Please also find our point-by-point responses below.\nPOINT-BY-POINT RESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author): with expertise in cancer immunology, STING\n“Gong and colleagues provide a well written, well developed and rigorously performed study identifying BATF2 as a tumor suppressor gene acting via feed-forward activation of the STING/IFN-I/Tgd pathway, with BATF2 transcription/expression negatively modulated by local (dietary) glutamine concentrations. Increased intrinsic expression of BATF2 in tumors leads to enhanced anti-tumor T cell fitness and improved tumor growth control in vivo across multiple mouse (transplantable and inducible) HNSCC tumor models, while deficiency of BATF2 in tumors results in a pro-tumor TIME characterized by enriched M2 TAM and reduced Type-1 T cell content. Furthermore, BATF2-KO mice exhibited greater intrinsic susceptibility to tumor initiation/growth/progression in multiple model systems. Overall, the presented data support the ability of BATF2 to sustain optimal fitness of the STING signaling pathway within the TME and suggest that future therapeutic targeting to reinforce BATF2 expression and/or to reduce glutamine metabolism may improve (immune)therapeutic outcomes in the HNSCC (cancer) setting.”\nResponse: We appreciate the insightful comments from Reviewer #1 and have performed additional experiments to enhance the rigor of this study. Please refer to our point-by-point response below.\n1. “The report is interesting and informative and my only minor concerns reflect: For the data presented in Fig. 5k and 5l where I would request that the authors redraft the Fig. 5l tumor growth curves with the same y-axis range (i.e., 1500 mm2) as was used for Fig. 5k. Also, the fact that the glutamine diet negated the significance of response to STING agonist may be true, but this does not appear to provide a survival advantage based on the perceived trajectories for tumor growth in Figs. 5k/l. Can the authors provide Kaplan-Meier plots and the relevant statistical analyses to support or refute the impact of the glutamine diet?”\nResponse 1: Thank you for this thoughtful suggestion. To directly compare the impact of an isocaloric, glutamine-rich diet on tumor growth using this model, we repeated the experiment so that all mice were treated and monitored in the same cohort and included the new data in New Fig. 5k. Consistent with our prior submission, we found that intra-tumoral injection of CDA was sufficient to reduce tumor burden when mice were on a regular diet. A glutamine-rich diet dampened host response to STING stimulation, as shown by the diminished tumor control by CDA. Without any STING agonists, tumors grew more aggressively in mice fed with an isocaloric, glutamine-rich diet than those on a regular diet (New Fig. 5k, Line 374-378). We compared the Kaplan-Meier curves and found that a glutamine-enriched diet was associated with inferior overall survival at baseline. STING stimulation improved overall survival for mice on a regular diet. However, STING stimulation failed to offer any meaningful protection in mice on an isocaloric, glutamine-rich diet (New Supplementary Fig. 7g, Line 385-388). In addition, to directly evaluate the effect of glutamine metabolism in tumor response to STING agonists, we treated mice with a prodrug of 6-diazo-5-oxo-L-norleucine, JHU083, which specifically inhibits glutaminase. JHU083 did not further improve the tumor response to STING agonists when mice were fed a regular diet. However, inhibition of glutamine metabolism in mice on a glutamine-rich diet sensitized cancers that were refractory to STING agonists to CDA (New Fig. 5l-m, Line 388-393).\n2. “Per Fig. 5m/n, the major difference in the TIME impacted by the glutamine diet appears to reflect presence of CXCR5+ B cells under constitutive glutamine concentrations in the TME. The authors should provide data for comparative levels of CXCL13 expression (including which cells are producing via scRNAseq) in the TME under both conditions. Also, since B cells are a canonical component of mature tertiary lymphoid structures, are TLS prevalent in control tumors but devoid in mice fed the glutamine diet? Please discuss and provide supportive data is available.”\nResponse 2: Thank you for this insightful question. We first performed additional histologic and immunohistochemical staining of CD20, trying to identify TLS. In agreement with previous studies (reviewed in PMID: 37809103), which showed that most syngeneic cell lines implanted s.c. do not develop TLS naturally due to the rapid growth, we did not observe apparent mature TLS in this study. To determine whether the increase in B-cell infiltration post STING stimulation shown in the single-cell analysis can be validated, we quantified CD20 staining of tumors from different treatment groups. We found that STING stimulation expanded B-cells in the tumors grown in mice on a regular diet but failed to increase B-cell infiltration in tumors grown in mice on a glutamine-rich diet (New Supplementary Fig. 7l-m, Line 417-424). Then, we examined the single-cell data set to evaluate B-cell signatures and Cxcl13. We quantified the fold change in gene expression for B-cell and plasma-cell markers, including CD19, Cxcr5, Ms4a1 (CD20), CD79a, Sdc1 (CD138), Igkc, Ighm, Ighd, Fcer2a (CD23), Cr2 (CD21/CD35), and Sell (CD62L). Besides the lineage markers, we included some functional markers because marginal zone B-cells are defined as CD93-IghdlowIghmhighCD23lowCD21high and follicular B-cells are defined as CD93-IghdhighIghmlowCD62L+CD23highCD21mid. We found that STING stimulation is more prominently promoting the upregulation of multiple functional B-cell subset markers in tumors grown in mice on a regular diet than those grown in mice on a glutamine-rich diet (New Supplementary Fig. 7l-m, Line 417-424). This single-cell data set did not identify Cxcl13 in any clusters. Cxcl13 is mainly produced by follicular dendritic cells and follicular helper T-cells, which are usually more prominent in mature TLS. Syngeneic models typically do not produce TLS. Cxcl13 is the main chemokine to guide B cells into TLS. However, B-cells also respond to other chemokine gradients, such as Cxcl10, to infiltrate into the TME. We did observe an expansion of Cxcl10high myeloid cell subset in response to STING stimulation in mice on a regular diet (Fig. 5n-o). Overall, the STING activation promotes the infiltration of B-cell subsets with transitional features of marginal zone and follicular B-cells into the TME. Glutamine dampens STING-mediated B-cell infiltration.\n3. “The discussion is rather short overall and more discussion for practical means by which BATF2 might be pharmacologically upregulated in the TME for therapeutic gain is recommended. Can the authors speak to known promoters of BATF2 or epigenetic agents that might be targeted with known in clinic drugs for testing in prospective translational studies (alone or in combination with immunotherapeutic agents)? Such discussion would add to the merit of the report.”\nResponse 3: Thank you for this insightful suggestion. To assess the potential for reversing BATF2 inhibition through pharmacologic approaches, in the same experiment shown in Fig. 5k, we also included two additional groups that received intra-tumoral injection of CDA and a glutaminase inhibitor, JHU083. One of these groups was on a regular diet, and the other was on an isocaloric glutamine-rich diet. Interestingly, although JHU083 did not further improve the anti-tumor efficacy of CDA in the regular diet group, it significantly improved tumor control in mice on a glutamine-rich diet (New Fig. 5l-m, Line 387-393). EZH2 is a key enzyme that methylates H3K27. Recent studies show that inhibition of EZH2 potentiates IFN-I, which supports this mechanistic link. Thus, in addition to improving the pharmacokinetic properties of STING agonists, the development of strategies to overcome BATF2-dependence, such as inhibition of EZH2 or glutamine metabolism, is promising to sensitize cancers to STING-mediated immune priming approaches. We have expanded the discussion section and included more details (Line 585-596, Line 607-621, Line 622-649).\nReviewer #2 (Remarks to the Author): with expertise in cancer immunology, IFNs\n“Gong et al. identified BATF2 as a novel regulator of the STING-type I interferon axis. They show that BATF2 directly interacts with STING, thereby promoting downstream signaling and target gene expression. By integrating molecular and functional in vitro studies with in vivo experiments in transplantable tumor models, the authors conclude that BATF2 is a tumor suppressor in HNSCC and that loss of BATF2 expression is a mechanism of immune evasion, as it impairs STING signaling in tumor cells.”\n1. “Comment: The BATF2-STING axis is a novel and very interesting finding. However, additional (control) experiments are needed to clearly demonstrate that BATF2 is a tumor suppressor and that downregulation of BATF2 in tumor cells is of relevance in immune evasion of HNSCC.”\nResponse 1: Thank you for your thoughtful suggestion. We measured the transcription levels of Batf2 in syngeneic murine HNSCC cell lines, including MOC2-E6/E7, NOOC1, and NOOC2. We found that MOC2-E6/E7 exhibited the lowest endogenous level of Batf2 expression, while NOOC2 showed high endogenous levels of Batf2 expression (New Supplementary Fig. 9a, Line 456-459). In addition to the Batf2-expressing MOC2-E6/E7 model we included in the original submission (Fig. 6), we generated new stable Batf2-deficient NOOC2 cells with its empty vector control (New Supplementary Fig. 9l, Line 523-525). In agreement with our previous findings, Batf2-deficient tumors grew significantly more aggressively (New Supplementary Fig. 9l-m, Line 523-526).\n2. “Comments on the role of BATF2 as novel tumor suppressor: So far, there is no evidence for a tumor-suppressor function of BATF2 in HNSCC, as the manuscript does not compare the in situ expression of BATF2 between cancer cells and normal squamous cells. What is shown in Fig. 1f? Are BATF2 levels in cancer cells, or are the BATF2 levels for the entire tissue core presented? Since BATF2 can also be induced by interferon-gamma, the correlation between BATF2 and CD8 could be driven by IFN-γ release from CD8 T cells. In this case, HNSCC tumor cells may not lose BATF2 expression but rather acquire it due to signals from the microenvironment. Moreover, several single-cell RNA sequencing datasets for HNSCC are available (including those from different disease stages, e.g., PMID: 36828832), where BATF2 expression in cancer cells could be analyzed and plotted. The authors analyzed BATF2 expression in tissue cores from 200 patients with a median follow-up of 60 months. Is there any association between BATF2 expression and survival or disease progression? If BATF2 is a strong tumor suppressor, patients with high BATF2 expression should have a significantly better outcome. Finally, introducing BATF2 as a general tumor suppressor is an overstatement, as the authors focused their studies solely on HNSCC.”\nResponse 2: Thank you for raising this thoughtful point. We appreciate the importance of strengthening human specimen studies and included new analyses using three additional independent cohorts of clinical specimens. A common caveat of analyzing human OPMD specimens is the often lack of longitudinal monitoring, including the referenced study (PMID: 36828832), where leukoplakia of unknown malignant transformation potential is included. To substantiate the role of BATF2 in suppressing tumor initiation, we first identified patients who had OPMD progressing into HNSCC despite complete surgical removal of the OPMD. We performed BATF2 staining on the paired longitudinal specimens and found that BATF2 expression levels were lower in paired HNSCC than in OPMD (New Fig. 1f-g, Line 161-163). Next, to directly assess whether the expression levels of BATF2 in fresh human oral squamous cell carcinoma specimens regulate their response to IFN agonists, we procured fresh human oral squamous cell carcinomas from eight patients. We trisected the tissue into three pieces of comparable size. The first piece was injected with PBS ex vivo, the second piece was injected with 5.0 μg CDA, and the third piece was formalin fixed and integrated into a tissue microarray (New Fig. 1h-i, Line 163-172). We performed qPCR using tissue homogenates and normalized the levels of IFNB1 by PTPRC, the latter of which encodes CD45. We quantified BATF2 staining using Aperio Imagescope and generated BATF2 scores. Then, we performed a Spearman correlation analysis between BATF2 scores and normalized IFNB1 expression and identified a strong positive correlation with a r-value of 0.86 (**p<0.01) (New Fig. 1h-i, Line 163-172).\nOur previous Fig.1f (current Fig. 1e) showed cancer-specific BATF2 expression in the upper panel and TME-specific BATF2 expression in the lower panel. This TMA was based on a clinical cohort collected through the former University of Michigan (UMich) Head and Neck SPORE, which ended in 2012. The follow-up was stopped after the grant ended, which made the follow-up data complex and not ideal. Over a decade has passed, and the statisticians for the UMich SPORE also left. However, we appreciate the value of assessing the prognostic role of BATF2, given its tumor-inhibitory role. Thus, we resorted to five TCGA data sets that include HNSCC (n=522, *p=0.034), high-grade serous carcinoma (OV, n=303, *p=0.0181), sarcoma (SARC, n=260, *p=0.0208), skin cutaneous melanoma (SKCM, n=471, **p=0.00106), and stomach adenocarcinoma (STAD, n=415, *p=0.0428). We found that higher expression levels of BATF2 were positively correlated with patient outcomes (New Supplementary Fig. 1e-i, Line 154-158). The HNSCC TCGA data set does not have responder status for immunotherapy. Thus, we next examined four data sets that contained responder status to anti-PD1. We found that the responders among patients with STAD (****p<0.0001), SKCM (****p<0.0001), bladder squamous cell carcinoma (BLCA, *p=0.02), or non-small cell lung cancer (NSCLC, ****p<0.0001) showed significantly higher levels of BATF2 expression than non-responders (New Supplementary Fig. 1j-m, Line 173-177). Overall, these new exciting data show that BATF2 inhibits the initiation of HNSCC and promotes anti-tumor immunity in human specimens.\n3. “Comments on the BATF2-STING axis in human squamous cell carcinoma cell lines. To demonstrate that BATF2 impacts STING signaling in carcinoma cells, the authors had to overexpress both BATF2 and, notably, STING, since synthetic STING agonists do not induce “… robust IFN-I activation” (author statement, lines 205-207, page 10). Even in cell lines with high endogenous STING levels (Fig. 3F, cell lines UMSCC108, UMSCC49), STING must be overexpressed to achieve pathway activation. This raises the question: how relevant is the BATF2-STING axis in squamous cell carcinoma if STING cannot be activated upon transfection with synthetic agonists? This suggests that activation of endogenous STING is defective even in the presence of high BATF2 levels.”\nResponse 3: We appreciate this thoughtful comment. We and others have shown that cancer cells have evolved multiple mechanisms to suppress the STING-TBK1-IFN pathway, such as promoting the turnover of STING and sequestering STING away from interacting with TBK1. This pathway is central for the immune sensing of HNSCC, which is likely why the tumors need multiple mechanisms to dampen this pathway. The regulation of this pathway is highly relevant for the following reasons.\n(a) During tumor initiation, when immune surveillance determines the outcomes of epithelial transformation, the proper activation of the innate immune pathways enables the maturation of antigen-presenting cells and subsequent cross-priming of T-cells. Known oncogenes for HNSCC, such as SOX2 and HPV16 E7, potently shut down IFN-I production. To evaluate whether BATF2 helps to launch the IFN-I pathway before epithelial malignant transformation, we expressed BATF2 in normal oral epithelial NOM9-TK cells, which directly triggered the phosphorylation of TBK1 and STING, indicative of pathway activation (New Supplementary Fig. 3d, Line 234-240).\n(b) In murine HNSCC cells, high Batf2 expression was also associated with elevated transcription levels of Ifnb1 (New Supplementary Fig. 9a and l, Line 456-459 and 523-525). In our previous submission, we showed that Batf2-expressing tumors are much more immunogenic (Fig. 6). To substantiate the physiological relevance of the Batf2-Sting pathway, we generated additional stable cell lines, including vector control Batf2-expressing and Batf2-expressing, Sting-deficient tumor cells. We found that cancer cell-specific Batf2-mediated tumor rejection completely depended on cancer cell expression of Sting because knocking down Sting abrogated Batf2-mediated protection (New Supplementary Fig. 9f-g, Line 473-477).\n(c) Now we also show that HNSCC cells only showed a modest increase in the phosphorylation of TBK1 and STING with cGAMP transfection. However, the STING response was largely restored when we expressed BATF2 (New Supplementary Fig. 4h, Line 308-311).\n(d) Although squamous cell carcinoma cells show a modest response to STING agonists, the magnitude of such differential responses can be amplified through myeloid cells, eventually controlling the infiltration of effector immune cells. To further characterize how cancer cell-specific expression of Batf2 impacts the effectors as a function of time, we implanted empty vector control and Batf2-expressing MOC2-E6/E7 cells, derived from a tumor model that has low endogenous levels of Batf2 expression (New Supplementary Fig. 9a, line 456-459), and analyzed tumor-infiltrating immune cells on Days 7, 14, and 21 post implantation. We found that Batf2-expressing tumors rapidly recruit CD11b+ myeloid cells that showed higher levels of phospho-Irf3 (New Fig. 6g, Line 477-488) and gd T-cells (New Fig. 6h, Line 477-488). At later time points, including Days 14 and 21, cancer cell-specific Batf2 expression resulted in an increased infiltration of CD8+ T-cells (New Fig. 6i, Line 477-488), in agreement with our analysis presented in the original submission (Fig. 6j-m).\n4. “Comments on silencing of BATF2 by glutamine: The authors conducted several in vitro studies to demonstrate that glutamine, by enhancing oxidative phosphorylation, leads to the epigenetic silencing of BATF2 expression. However, the effect of glutamine on BATF2 expression is shown only at the RNA level; important protein data are missing. All experiments related to glutamine were performed in murine macrophages. If the authors wish to claim that this mechanism allows tumor cells to downregulate BATF2, it is crucial to demonstrate that this regulation occurs in carcinoma cells and affects BATF2 protein levels. Furthermore, the effects of a glutamine diet on tumor growth following intratumoral injection of STING agonists appear to be relatively weak.”\nResponse 4: We appreciate this constructive comment. Because BATF2 is primarily expressed in epithelial cells and myeloid cells, we validated the impact of glutamine on BATF2 in both cell types. We additionally tested the effect of glutamine on the expression levels of BATF2 in THP1 cells. In agreement with mouse studies, we found that glutamine potently suppressed the mRNA expression levels of BATF2 and IFN-I target genes when THP1 cells were treated with cGAMP (New Supplementary Fig. 6a, Line 339-341). Then, we confirmed that glutamine potently inhibited the expression of BATF2 at protein levels and the production of IFN-b (New Supplementary Fig. 6b-c, Line 341-343). Similarly, we found that glutamine suppressed the expression of BATF2 at mRNA and protein levels in HNSCC cells (Supplementary Fig. 6d-e, Line 343-349).\nTo strengthen the in vivo data related to glutamine metabolism, we first repeated the glutamine diet experiment by including all groups in the same experiment for direct multi-comparisons. Consistent with the data presented in the original submission, CDA reduced tumor burden when mice were on a regular diet but failed to show any protection in mice on an isocaloric diet (New Fig. 5k, Line 374-377). We compared surrogate animal survival and found that CDA extended host survival only in the regular diet group New Supplementary Fig. 7g, Line 384-387). Glutamine-rich diet alone enhanced tumor growth at the baseline (Fig. 5k and New Supplementary Fig. 7g, Line 374-381 and 384-387). Then, we assessed the impact of inhibition of glutamine metabolism on tumor response to IFN-I agonists. In the same experiment shown in Fig. 5k, we also included two additional groups that received intra-tumoral injection of CDA and a glutaminase inhibitor, JHU083. One of these groups was on a regular diet, and the other was on an isocaloric glutamine-rich diet. Interestingly, although JHU083 did not further improve the anti-tumor efficacy of CDA in the regular diet group, it significantly improved tumor control in mice on a glutamine-rich diet (New Fig. 5l-m, Line 387-393). Overall, this series of new experiments substantiated the physiological relevance of the novel glutamine-BATF2-IFN-I pathway. Glutamine inhibits BATF2 at mRNA and protein levels, leading to dampened IFN-I production. A glutamine-rich diet desensitized tumors to STING stimulation and promoted tumor growth. Direct inhibition of glutamine metabolism more effectively reduced tumor burden in mice on a glutamine-rich diet.\n5. “Comments on in vivo studies: In vivo experiments were conducted using different murine squamous cell carcinoma cell lines.there is no information about endogenous BATF2 protein expression in those cells. Figure 6f demonstrates reduced growth of Batf2-overexpressing tumors. To what extent this effect is related to STING activation remains unclear. Experiments should be performed using BATF2-overexpressing STING knockout cells. Additionally, survival curves should be included.”\nResponse 5: Thank you for the constructive comment. In our revised manuscript, we measured the endogenous levels of Batf2 in the mouse HNSCC cell lines MOC2-E6/E7, NOOC1, and NOOC2. MOC2-E6/E7 exhibited the lowest endogenous level of Batf2 expression, while NOOC2 showed high endogenous levels of Batf2 expression (New Supplementary Fig. 9a, Line 456-459). In addition to the Batf2-expressing MOC2-E6/E7 model we included in the original submission (Fig. 6), we generated new stable Batf2-deficient NOOC2 cells with its empty vector control (New Supplementary Fig. 9l, Line 523-525). In agreement with our previous findings, Batf2-deficient tumors grew significantly more aggressively (New Supplementary Fig. 9l-m, Line 523-526). Following this helpful suggestion, we also generated new vector control Batf2-expressing cell line and Sting-deficient, Batf2-expressing MOC2-E6/E7 cells. We found that cancer cell-specific Batf2-mediated tumor rejection completely depended on cancer cell expression of Sting because knocking down Sting abrogated Batf2-mediated protection (New Supplementary Fig. 9f-g, Line 471-477). We also included Kaplan-Meier survival analysis (New Supplementary Fig. 9g, Line 475-477). Please also refer to our response #3.\n6. “In Figure 7: Is the enhanced growth of transplanted tumor cells in BATF2-/- mice due to the lack of STING activation?”\nResponse 6: Thank you for this thoughtful question. To examine the Sting pathway activation in Batf2-/- mice in vivo, we assessed the levels of Irf3 phosphorylation by flow cytometry and found that the proportion of p-Irf3+ cells in TILs from Batf2-/- mice was significantly lower than that in the control group using two different HNSCC models (New Fig. 7b and d, Line 533-542).\n7. “Finally, the authors completely ignore relevant published data on BATF2 in cancer, even those studies which already showed that BATF2 has anti-tumor activity.”\nResponse 7: We appreciate the importance of published data. Thus, we revised the discussion to include these studies (Line 632-634). We also respectfully submit that our study has established the following novelties that effectively separate this study. (a) We thoroughly characterized a new molecular pathway that connects BATF2 with STING-specific IFN-I potentiation. We further strengthened the data to show that the S227 phosphorylation site is required for BATF2’s immune-activating function (please refer to details in our response to Reviewer 4 Comment #2). (b) We established a clear molecular mechanism of the BATF2-STING complex. Our new data identified the LBD domain of STING, which is essential for its oligomerization, as the critical binding site of BATF2 (please refer to details in our response to Reviewer #3 Comment #2). (c) We identified glutamine as a pivotal trans-regulatory element that suppresses the expression of BATF2. We characterized the impact of BATF2 on the metabolic activities of myeloid cells and established the role of glutamine in tumor response to IFN-I agonists. (d) Now we have shown that inhibition of glutamine metabolism did not further improve the anti-tumor efficacy of CDA in the regular diet group. However, inhibitors of glutamine metabolism significantly improved tumor control in mice on a glutamine-rich diet. (e) According to the 2025 Annual Report to the Nation on the Status of Cancer, oral cavity and pharynx cancer is one of the only three cancer types that showed a significant increase in incidence among both male and female patients. We created a rigorous new oral squamous cell carcinoma model NOOC2, which shows over 95% similarity in mutational signatures to human tobacco-associated cancers. None of the published studies was focused on the molecular biology of HNSCC. (f) We separately characterized the role of BATF2 in both epithelial and myeloid cell compartments, which are the principal cell types that express BATF2. None of the published studies examined the BATF2-IFN-I pathway in vivo. In addition, our new data show that BATF2-mediated tumor rejection depends on IFN-I signaling, Cxcl10, and cytotoxic T-cells. (g) We included complementary human specimen cohorts to substantiate the tumor-inhibitory function of BATF2. Our new data show that BATF2 levels were decreased as oral potentially malignant precursor lesions transformed and that BATF2 is associated with a favorable anti-tumor immune response profile.\nReviewer #3 (Remarks to the Author): with expertise in head and neck cancer, cancer immunology\n“This manuscript offers a novel perspective on BATF2 as a glutamine-responsive tumor suppressor essential for promoting STING signaling and IFN-I production. The study is comprehensive, leveraging both molecular and preclinical models to delineate BATF2's immunomodulatory roles. However, some areas of the manuscript could benefit from additional clarification, experimental reinforcement, and broader context to enhance its impact and rigor.”\n1. “Mechanistic Insight on Glutamine and Epigenetic Regulation: While the role of glutamine in promoting H3K27me3 modifications at the BATF2 promoter is well demonstrated, the study does not explore whether this mechanism also affects other key immune genes regulated by STING. A broader analysis of glutamine-induced epigenetic changes at loci relevant to the immune response (e.g., CXCL10 or IFN-I target genes) would strengthen the conclusion that glutamine broadly suppresses antitumor immunity through epigenetic remodeling.”\nResponse 1: Thank you for sharing this insight. In our original submission, we did include additional STING target genes, including Ifnb1 and Cxcl9 (Supplementary Fig. 6g, Line 354-358).\n2. “Role of BATF2 in STING Oligomerization: The manuscript demonstrates that BATF2 is essential for STING oligomerization (Fig. 4h). However, the specific role of BATF2 in stabilizing these oligomers remains unclear. Testing whether BATF2 interacts directly with key regions of STING (e.g., using mutational analysis or co-immunoprecipitation of truncated STING constructs) would clarify the molecular basis of this interaction.”\nResponse 2: We appreciate this insightful comment. Following this recommendation, we generated new truncation mutants for STING. We deleted the N-terminus, the Ligand-Binding Domain, or the C-terminus of STING. All STING domain truncation mutants can be expressed. We immunoprecipitated HA-BATF2 and found that only the LBD domain was required for the BATF2-STING interaction (New Fig. 4i, Line 271-276). LBD is also the domain critical for STING oligomerization. Thus, it is likely that the BATF2-STING interaction stabilizes the STING oligomers in an LBD-dependent fashion.\n3. “Function of CXCL10+ Myeloid Cells in Tumor Rejection: The authors highlight the expansion of CXCL10+ myeloid cells downstream of BATF2 but do not directly assess their functional relevance. Including depletion experiments (e.g., using neutralizing antibodies or conditional knockouts) to test whether CXCL10+ myeloid cells are necessary for BATF2-mediated tumor rejection would substantiate this claim.”\nResponse 3: Thank you for this thoughtful suggestion. We followed the advice and performed neutralization experiments. First, we administered 200 μg anti-Cxcl10 neutralizing antibody on day 7 after tumor implantation and continued 100 μg anti-Cxcl10 twice per week for a total of five doses. We found that Cxcl10 depletion abrogated Batf2-mediated protection (New Supplementary Fig. 9i, Line 505-510). We observed that T-cells from Batf2-expressing tumors showed significantly elevated effector function pathways, such as IFN-γ response, allograft rejection, IFN-α response, as well as cell proliferation, such as E2F targets, mitotic spindle, G2M checkpoint, and Myc targets (Fig. 6m). Thus, we then sought to determine whether Batf2-mediated protection depended on CD8+ T-cells. Our new data showed that anti-CD8-mediated T-cell depletion eliminated the protective effects of cancer cell-specific Batf2 expression (New Supplementary Fig. 9j, Line 510-515).\n4. “Kinetics of BATF2 Expression and IFN-I Responses: The timing of BATF2 expression relative to IFN-I signaling and T-cell recruitment is critical to understanding its role in tumor immunity. Longitudinal studies tracking BATF2 expression, STING activation, and immune infiltration in tumors (e.g., via time-course RNA-Seq or flow cytometry) would provide valuable insights.”\nResponse 4: Thank you for raising this insightful point. We have now collected tumor specimens on days 7, 14, and 21 post-implantation of empty vector and Batf2-expressing tumors. Batf2-expressing tumors demonstrated a more favorable TME at the earliest time point, as evidenced by the early expansion of CD45+Tcr-CD11b+ myeloid cells that are positive phospho-Tbk1 and phospho-Irf3. Also noticeable at this early time point was the expansion of γδ T-cells. Then, at the later time points, Batf2-expressing tumors started to show significantly more CD8+ T-cells infiltration than control tumors, although this advantage appeared to become smaller as the tumor size continued to increase (New Fig. 6f-i, Line 477-488). These new data suggest that cancer cell-specific Batf2 expression reprograms innate and adaptive immune wiring in a stepwise fashion, with IFN-I activation in CD11b+ myeloid cells and γδ T-cells expansion as the earliest response feature, followed by expansion of CD8+ T-cells. In addition to the number difference in effectors, the quality of these T-cells is also different. As shown in our previous submission, the T-cells from Batf2-expressing tumors exhibited an Id2 effector signature with upregulated functional pathways, such as IFN-γ and proliferation responses (Fig. 6j-m).\n5. “Relevance of p227 Phosphorylation Across Models: The phosphorylation of BATF2 at serine 227 (p227) is presented as critical for its function, but this claim is primarily demonstrated in vitro (Fig. 4k). Testing whether p227 phosphorylation is similarly required in vivo (e.g., using BATF2 p227A knock-in mice) would solidify this mechanistic insight.”\nResponse 5: Thank you for your thoughtful suggestion. Because our original and new data highlight the protective role of BATF2 in suppressing tumor initiation, here we substantiate the physiological relevance of serine 227 phosphorylation in vivo by generating new tumor models that stably express empty vector, wild-type Batf2, and 227A mutant Batf2. In agreement with our other data, expression of Batf2 inhibited tumor growth. However, Batf2-mediated protection was abrogated by mutating serine 227 to alanine and disabling phosphorylation at this amino acid (New Supplementary Fig. 9n, Line 526-531). Hence, here we show that the phosphorylation of S227 is critical for the tumor inhibitor role of Batf2 in vivo.\n6. “STING Agonist Resistance and Glutamine Levels: The manuscript concludes that high glutamine levels blunt STING agonist efficacy (Fig. 5). It would be helpful to explore whether glutamine-targeting interventions (e.g., glutaminase inhibitors) can rescue STING agonist responses in glutamine-rich conditions, thus supporting potential combination therapies.”\nResponse 6: Taking this helpful advice to heart, we performed new in vivo experiments to substantiate the physiological relevance of the glutamine-BATF2-IFN-I pathway. In our original submission, we showed that a selective glutaminase inhibitor, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), significantly elevated the expression levels of Batf2, Ifnb1, and pan-Ifna in BMDM under STING-stimulated conditions (Fig. 5c-d and Supplementary Fig. 5g-h). To strengthen the in vivo data related to glutamine metabolism, we first repeated the glutamine diet experiment by including all groups in the same experiment for direct comparisons. Consistent with the data presented in the original submission, CDA reduced tumor burden when mice were on a regular diet but failed to show any protection in mice on an isocaloric diet (New Fig. 5k, Line 374-377). We compared surrogate animal survival and found that CDA extended host survival only in the regular diet group (New Supplementary Fig. 7g, Line 384-387). Glutamine-rich diet alone enhanced tumor growth at the baseline (New Fig. 5k and New Supplementary Fig. 7g, Line 374-387). Then, we assessed the impact of inhibition of glutamine metabolism on tumor response to IFN-I agonists. In the same experiment shown in Fig. 5k, we also have two additional groups that received intra-tumoral injection of CDA and a glutaminase inhibitor, JHU083. One of these groups was on a regular diet, and the other was on an isocaloric glutamine-rich diet. Interestingly, although JHU083 did not further improve the anti-tumor efficacy of CDA in the regular diet group, it significantly improved CDA-mediated tumor control in mice on a glutamine-rich diet (New Fig. 5l-m, Line 387-393). Overall, this series of new experiments substantiated the physiological relevance of the novel glutamine-BATF2-IFN-I pathway. Glutamine inhibits BATF2 at mRNA and protein levels, leading to dampened IFN-I production. A glutamine-rich diet desensitized tumors to STING stimulation and promoted tumor growth. Direct inhibition of glutamine metabolism more effectively reduced tumor burden in mice on a glutamine-rich diet.\n7. “BATF2 in Human Clinical Samples: While BATF2 expression is correlated with immune markers in HNSCC patient datasets, there is little exploration of its prognostic or therapeutic relevance. Analyzing BATF2 expression in patients stratified by clinical outcomes (e.g., response to immunotherapy) would provide translational significance to the findings.”\nResponse 7: Thank you for sharing this important insight. To strengthen human specimen studies, we included new analyses using three additional independent cohorts of clinical specimens. A common caveat of analyzing human oral potentially malignant disorders (OPMD) specimens is the often lack of longitudinal monitoring, where leukoplakia of unknown malignant transformation potential is included. To substantiate the role of BATF2 in suppressing tumor initiation, we first identified patients who had OPMD progressing into HNSCC despite complete surgical removal of the OPMD. We performed BATF2 staining on the paired longitudinal specimens and found that BATF2 expression levels were lower in paired HNSCC than in OPMD (New Fig. 1f-g, Line 161-163).\nNext, to directly assess whether BATF2 in fresh human oral squamous cell carcinoma specimens regulates their response to IFN agonists, we procured fresh human oral squamous cell carcinomas from eight patients. We trisected the tissue into three pieces of comparable size. The first piece was injected with PBS ex vivo, the second piece was injected with 5.0 μg CDA, and the third piece was formalin fixed and integrated into a tissue microarray (Fig. 1h, Line 163-168). We performed qPCR using tissue homogenates and normalized the levels of IFNB1 by PTPRC, the latter of which encodes CD45. We quantified BATF2 staining using Aperio Imagescope and generated BATF2 scores. Then, we performed a Spearman correlation analysis between BATF2 scores and normalized IFNB1 expression and identified a strong positive correlation between BATF2 and STING response with a r-value of 0.86 (**p<0.01) (New Fig. 1i, Line 168-172).\nTo comprehensively assess the impact of BATF2 on overall survival, we resorted to five TCGA data sets that include HNSCC (n=522, *p=0.034), high-grade serous carcinoma (OV, n=303, *p=0.0181), sarcoma (SARC, n=260, *p=0.0208), skin cutaneous melanoma (SKCM, n=471, **p=0.00106), and stomach adenocarcinoma (STAD, n=415, *p=0.0428). We found that higher expression levels of BATF2 were positively correlated with patient outcomes (Supplementary Fig. 1e-i, Line 154-158). The HNSCC TCGA data set does not have responder status for immunotherapy. Thus, we next examined four data sets that contained responder status to anti-PD1. We found that the responders among patients with STAD (****p<0.0001), SKCM (****p<0.0001), bladder squamous cell carcinoma (BLCA, *p=0.02), or non-small cell lung cancer (NSCLC, ****p<0.0001) showed significantly higher levels of BATF2 expression than non-responders (New Supplementary Fig. 1j-m, Line 173-177). Overall, this analysis shows that BATF2 promotes anti-tumor immune responses in tumor types besides HNSCC.\nMinor Revisions\nTerminology Consistency: Standardize terms like “IFN-I signature genes” and “IFN-I target genes” for clarity.\nResponse: We changed all “IFN-I signature genes” to “IFN-I target genes”.\nText Clarity: Simplify the introduction by focusing on BATF2’s novelty in immune modulation while reducing background repetition.\nReviewer #4 (Remarks to the Author): with expertise in cancer immunology, metabolism\n1. “The manuscript establishes a role for BATF2 as a tumor suppressor gene in HNSCC and combines data from human tumor samples and several mouse models. Robust genetic approaches and state-of-the-art read-outs are used to support the findings. The results are well-described, and the figures and figure legends are nicely presented. The role of BATF2 as a tumor suppressor gene is well established by previous studies, including in epithelial cancers: PMID 26135942, 32650804, 37995570, 34778372 and more. Some of these studies show, similarly to the current one, that BATF2 expression on tumor cells shapes the TME immune infiltrate. The role of BATF2 in driving inflammatory responses, e.g. in macrophages, has also been previously observed: PMID 25957166 and others. The current study presents novel points on the importance of BATF2 for type I IFN responses following STING, but not MAVS activation, in particular on the phosphorylation of BATF at serine 227 as a prerequisite for interaction with STING. A further novel point is the metabolic regulation of BATF2 expression through glutamine. However, I think that, in particular, the last point needs to be investigated in greater detail.”\nResponse 1: Thank you for your thoughtful comments and for recognizing the novel aspects of this study. We have revised our discussion to recognize the previous literature more broadly. We also respectfully submit that our study has established the following novelties that effectively separate this study. (a) We thoroughly characterized a new molecular pathway that connects BATF2 with STING-specific IFN-I potentiation. We further strengthened the data to show that the S227 phosphorylation site is required for BATF2’s immune-activating function (please refer to details in our response to Reviewer 4 Comment #2). (b) We established a clear molecular mechanism of the BATF2-STING complex. Our new data identified the LBD domain of STING, which is essential for its oligomerization, as the critical binding site of BATF2 (please refer to details in our response to Reviewer #3 Comment #2). (c) We identified glutamine as a pivotal trans-regulatory element that suppresses the expression of BATF2. We characterized the impact of BATF2 on the metabolic activities of myeloid cells and established the role of glutamine in tumor response to IFN-I agonists. (d) Now we have shown that inhibition of glutamine metabolism did not further improve the anti-tumor efficacy of CDA in the regular diet group. However, inhibitors of glutamine metabolism significantly improved CDA-mediated tumor control in mice on a glutamine-rich diet. (e) According to the 2025 Annual Report to the Nation on the Status of Cancer, oral cavity and pharynx cancer is one of the only three cancer types that showed a significant increase in incidence among both male and female patients. We created a rigorous new oral squamous cell carcinoma model NOOC2, which shows over 95% similarity in mutational signatures to human tobacco-associated cancers. None of the published studies was focused on the molecular biology of HNSCC. (f) We separately characterized the role of BATF2 in both epithelial and myeloid cell compartments, which are the principal cell types that express BATF2. None of the published studies examined the BATF2-IFN-I pathway in vivo. In addition, our new data show that BATF2-mediated tumor rejection depends on IFN-I signaling, Cxcl10, and cytotoxic T-cells. (g) We included complementary human specimen cohorts to substantiate the tumor-inhibitory function of BATF2. Our new data show that BATF2 levels were decreased as oral potentially malignant precursor lesions transformed and that BATF2 is associated with a favorable anti-tumor immune response profile.\nTo substantiate the physiological relevance of serine 227 phosphorylation in vivo, we generated new tumor models that stably express empty vector, wild-type Batf2, and 227A mutant Batf2. In agreement with our other data, expression of Batf2 inhibited tumor growth. However, Batf2-mediated protection was completely abrogated by mutating serine 227 to alanine and disabling phosphorylation at this amino acid (New Supplementary Fig. 9n, Line 526-531). Hence, here we show that the phosphorylation of S227 is critical for the tumor inhibitor role of Batf2 in vivo.\nWe also performed additional experiments to strengthen the glutamine-BATF2-IFN-I signaling axis. Because BATF2 is primarily expressed in epithelial cells and myeloid cells, we validated the impact of glutamine on BATF2 in both cell types. In our original submission, we showed the inhibitory effect of glutamine on the BATF2 protein levels (Fig. 5g). During the revision, we additionally tested the effect of glutamine on the expression levels of BATF2 in THP1 cells. In agreement with mouse studies, we found that glutamine potently suppressed the mRNA expression levels of BATF2 and IFN-I target genes when THP1 cells were treated with cGAMP (New Supplementary Fig. 6a, Line 339-341). Then, we confirmed that glutamine potently inhibited the expression of BATF2 at protein levels and the production of IFN-b (Supplementary Fig. 6b-c, Line 341-343). Similarly, we found that glutamine suppressed the expression of BATF2 at mRNA and protein levels in HNSCC cells (New Supplementary Fig. 6d-e, Line 343-349).\nTo strengthen the in vivo data related to glutamine metabolism, we first repeated the glutamine diet experiment by including all groups in the same experiment for direct multi-comparisons. Consistent with the data presented in the original submission, CDA reduced tumor burden when mice were on a regular diet but failed to show any protection in mice on an isocaloric diet (New Fig. 5k, Line 381-387). We compared surrogate animal survival and found that CDA extended host survival only in the regular diet group (New Supplementary Fig. 7g, Line 381-387). Glutamine-rich diet alone enhanced tumor growth at the baseline (New Fig. 5k and New Supplementary Fig. 7g, Line 374-387). In addition, we implanted the same tumor model that showed more aggressive growth in wild-type mice on a glutamine-rich diet into Batf2-/- mice on either a regular diet or an isocaloric, glutamine-rich diet. We found that a glutamine-rich diet could no longer promote tumor growth in the absence of host expression of Batf2 (Supplementary Fig. 7f, Line 377-381), suggesting that glutamine-mediated tumor aggression depends on its suppression of Batf2. Then, we assessed the impact of inhibition of glutamine metabolism on tumor response to IFN-I agonists. In the same experiment shown in Fig. 5k, we also have two additional groups that received intra-tumoral injection of CDA and a glutaminase inhibitor, JHU083. One of these groups was on a regular diet, and the other was on an isocaloric glutamine-rich diet. Interestingly, although JHU083 did not further improve the anti-tumor efficacy of free CDA in the regular diet group, it significantly improved tumor control in mice on a glutamine-rich diet (New Fig. 5l-m, Line 387-393). Overall, this series of new experiments substantiated the physiological relevance of the novel glutamine-BATF2-IFN-I pathway. Glutamine inhibits BATF2 at mRNA and protein levels, leading to dampened IFN-I production. A glutamine-rich diet desensitized tumors to STING stimulation and promoted tumor growth. Direct inhibition of glutamine metabolism more effectively reduced tumor burden in mice on a glutamine-rich diet.\n“Major points:”\n2. “The manuscript is somewhat difficult to follow, as the authors frequently switch between the role of BATF2 in BMDM and in tumor cells. In the end, it doesn't really become clear to me whether BATF2 expression on one of these two populations is more important for the observed in vivo effects. Also, not all mechanisms are shown in both cell types. The interaction of STING and phosphorylation of BATF2 are shown in tumor cell lines, while the regulation of BATF2 through glutamine is shown in BMDM.”\nResponse 2: We appreciate the thoughtful comment. The reason we focused on two cell compartments is that BATF2 is most highly expressed in epithelial/carcinoma cells and myeloid cells. We established a novel glutamine-BATF2-STING pathway that determines HNSCC immunogenicity. We agree that thoroughly testing the key mechanisms in both compartments improves the rigor. First, we extended the testing for the interaction between BATF2 and STING from HEK-293T cells and the HNSCC cells to macrophages. We immunoprecipitated BATF2 and blotted STING to confirm such interaction (New Fig. 4h, Line 269-271). Then, we further refined the mechanism of the BATF2-STING binding by mapping the domain requirements for this interaction. We generated new STING mutants with the deletion of the N-terminus, the Ligand-Binding Domain, or the C-terminus. All STING domain truncation mutants can be expressed. We immunoprecipitated HA-BATF2 and found that only the LBD domain was required for the BATF2-STING interaction (New Fig. 4i, Line 271-276). LBD is also the domain critical for STING oligomerization. Thus, it is likely that the BATF2-STING interaction stabilizes the STING oligomers in an LBD-dependent fashion. Next, we confirmed the critical role of S227 phosphorylation in HNSCC cells. In our original submission, we showed that the BATF2 S227A mutant failed to reconstitute STING activation in Batf2-/- BMDM. During the revision, we performed new experiments to show that the expression of full-length BATF2 restored cGAMP-induced IFN-I response in an HNSCC cell line that is otherwise non-responsive to cGAMP transfection. Notably, the BATF2 S227A mutant abrogated the role of BATF2-mediated STING activation (New Supplementary Fig. 4h, Line 308-311). Then, we additionally tested the effect of glutamine on the expression levels of BATF2 in THP1 cells. In agreement with mouse studies, we found that glutamine potently suppressed the mRNA expression levels of BATF2 and IFN-I target genes when THP1 cells were treated with cGAMP (Supplementary Fig. 6a, Line 339-341). Then, we confirmed that glutamine potently inhibited the expression of BATF2 at protein levels and the production of IFN-b (New Supplementary Fig. 6b-c, Line 341-343). Similarly, we found that glutamine suppressed the expression of BATF2 at mRNA and protein levels in HNSCC cells (New Supplementary Fig. 6d-e, Line 343-349) (Please also refer to our response to Reviewer 4 Comment 1).\n3. I find the beginning of the paper confusing. The authors begin the manuscript with the question why HPV+ HNSCC do not respond to ICI blockade as well as expected and compare single-cell transcriptomes from HPV+ and HPV- patients to discover aberrations in the immune cell population composition. However, there is no correlation with clinical responses to ICI, which makes me wonder why this comparison was made in the first place. I personally think that leading into the paper by stating that high BATF2 expression was found in clusters 10, 19, 20 which also had high type I IFN gene expression would make a clearer point, and that splitting the samples in HPV+ and HPV- doesn't have an added value for the manuscript.\nResponse 3: We appreciate the suggestion and de-emphasized the HPV-related immune escape mechanism to focus on the link between BATF2 and IFN-I target genes in the Introduction section.\n4. “The interaction between BATF2 and STING is shown for tumor cells, but not for macrophages. Do the authors have evidence that the same mechanism is active in macrophages? Vice versa, does glutamine concentration in the media affect the BATF2 expression in tumor cells, or only in macrophages?”\nResponse 4: Thank you for this thoughtful suggestion. In our revised manuscript, we performed new experiments and validated the mechanism in both tumor cells and BMDM (New Supplementary Fig. 6a-e, Line 339-349). Please also see our response to Reviewer 4 Comment 1.\n5. “How much of the effects of high-glutamine media on type I IFN secretion is mediated through BATF2? The experiments in Fig. 5e-f should be repeated with BATF2-/- cells. Along the same lines, is the effect of glutamine-rich diet on tumor development in Fig. 5k-q mediated solely through BATF2? Experiments should include groups with BATF2 KO tumors and/or hosts as controls.”\nResponse 5: Thank you for raising this thoughtful point. We additionally evaluated whether glutamine-mediated STING suppression depended on Batf2. We cultured Batf2-/- BMDM in media containing 0 g/L, 0.5 g/L, or 5.0 g/L glutamine for 24 hours, followed by transfection with 3.0 µg/mL ISD for 16 hours. Notably, we did not observe a reduction in the protein levels of Ifn-b in the supernatant when glutamine was added to the culture compared with glutamine-free control (New Supplementary Fig. 6f, Line 349-353), suggesting that Batf2 is indispensable for glutamine-mediated STING inhibition. In addition, we implanted the same tumor model that showed more aggressive growth in wild-type mice on a glutamine-rich diet into Batf2-/- mice on either a regular diet or an isocaloric, glutamine-rich diet. We found that a glutamine-rich diet could no longer promote tumor growth in the absence of host expression of Batf2 (Supplementary Fig. 7f, Line 377-381).\n6. “What is the mechanism by which glutamine epigenetically regulates BATF2? Is this mediated through glutamine itself, or through some of its downstream metabolites, e.g. alpha-ketoglutarate. How are the metabolic profiles with increased oxidative phosphorylation (Figure 5i-j) related to the remaining mechanism? Would other metabolic conditions that increase OCR also affect BATF2 expression, or is this specific to glutamine-rich media?”\nResponse 6: Thank you for those thoughtful questions. Previous studies have shown that IL4 treatment, which promotes the M2-like alternative activation of macrophages, reduces the deposition of H3K27me3 on the promoters of Arg1, Ym1, Retnla, and Mrc1 genes. This finding is conceptually in agreement with our data, which show that glutamine potentiates an OCR profile conducive to M2-like alternative activation and promotes the deposition of repressive H3K27m3 on the promoters of Batf2, Ifnb1, and Cxcl9. EZH2 is a key enzyme that methylates H3K27. Recent studies show that inhibition of EZH2 potentiates type-I interferons, which supports this mechanistic link. We revised our discussion to include these future directions (Line 613-621). To test whether amino acids-potentiated suppression of BATF2 is specific to glutamine, we tested a panel of essential and non-essential amino acids. We cultured UMSCC108 cells in complete media and media specifically deficient only in one amino acid and tested the levels of BATF2. In agreement with the data presented in the original submission, glutamine deficiency resulted in higher levels of BATF2. Interestingly, we also found that tyrosine, valine, isoleucine, and methionine also suppress BATF2. None of the other non-essential and essential amino acids regulates BATF2, suggesting pathway specificity (New Supplementary Fig. 5a, Line 313-318). A recent study showed that methionine restriction increases the STING pathway activation, conceptually in agreement with our finding that methionine suppresses BATF2 (Line 629-631).\n7. “Are there data from human tumor samples supporting the notion that glutamine metabolism regulates BATF2-expression?”\nResponse 7: Thank you for this insightful suggestion. To address this question, we collected fresh oral squamous cell carcinoma tissue from 15 patients and performed bulk RNA-Seq. Then, we performed a correlation analysis between BATF2 and glutamine metabolism markers. We also included IFN-I target genes as controls. We found that BATF2 is strongly positively correlated with Th1 genes, including CXCL9, CXCL10, ISG15, IFNB1, OASL, and TNF. Notably, we also found that BATF2 is inversely correlated with glutamine metabolism pathway genes, such as SLC7A5, SLC1A5, SLC38A2 and GLS. In agreement, the Th1 cytokines, including IFNG, IFNB1, and TNF, all show an inverse correlation with glutamine metabolism (New Fig. 5r, Line 424-432). These new human data support the glutamine-BATF2-IFN pathway we characterized in depth in this study.\n8. “Figure 6: are only gd T cells responsible for the rejection of BATF2-expressing tumors? I would be interested to see experiments with depletion of alpha-beta T cells, especially as these subsets are also affected by BATF2 expression in other figures. Vice versa, are gd T cells affected in the tumor models presented in Fig. 7?”\nResponse 8: Thank you for this insightful suggestion. We have now collected tumor specimens on days 7, 14, and 21 post-implantation of empty vector and Batf2-expressing tumors to determine the dynamics of the infiltration of γδ T-cells and ab T-cells. Batf2-expressing tumors demonstrated a more favorable TME at the earliest time point, as evidenced by the early expansion of CD45+Tcr-CD11b+ myeloid cells that are positive phospho-Tbk1 and phospho-Irf3. Also noticeable at this early time point was the expansion of γδ T-cells. Then, at the later time points, Batf2-expressing tumors started to show significantly more CD8+ T-cells infiltration than control tumors, although this advantage appeared to become smaller as the tumor size continued to increase (New Fig. 6f-i, Line 477-485). These new data suggest that cancer cell-specific Batf2 expression reprograms innate and adaptive immune wiring in a stepwise fashion, with IFN-I activation in CD11b+ myeloid cells and γδ T-cells expansion as the earliest response feature, followed by expansion of CD8+ T-cells. In our original submission, we observed that pooled γδ T-cells and ab T-cells from Batf2-expressing tumors showed significantly elevated effector function pathways, such as IFN-γ response, allograft rejection, IFN-α response, as well as cell proliferation, such as E2F targets, mitotic spindle, G2M checkpoint, and Myc targets (Fig. 6m). Thus, we then sought to determine whether Batf2-mediated protection depended on CD8+ T-cells. Our new data showed that anti-CD8-mediated T-cell depletion eliminated the protective effects of cancer cell-specific Batf2 expression (New Supplementary Fig. 9j, Line 510-515). Next, we evaluated whether host Batf2 expression has an impact on γδ T-cell infiltration into the TME. We stained tumors grown in wild-type and Batf2-/- mice with a δ-TCR-specific antibody and found that there were fewer γδ T-cells in tumors grown in Batf2-/- mice (New Supplementary Fig. 10a, Line 537-538).\n9. “It would be interesting to know if BATF2 expression by tumor cells drives only local or also systemic immune responses. For example, the authors could use the model in FIgure 6f and inject WT tumor cells in one flank and BATF2-expressing or WT cells in the other flank, and monitor whether the presence of a BATF2-expressing tumor activates a systemic immune response leading to a better control of the WT tumor in the other flank, compared to animals bearing WT tumors on both sides.”\nResponse 9: We appreciate this thoughtful advice to examine the abscopal effects. We implanted the empty vector (EV) control and Batf2-expressing tumors on the right flank. As expected, we observed a significant reduction in tumor volume when Batf2 was expressed. However, the EV control tumors grown in the left flank of mice that bear Batf2-expressing tumors in the right flank did not show a difference in volume compared to those EV tumors grown in the left flank of mice that bear EV tumors in the right flank (New Supplementary Fig. 9k, Line 515-522). These data suggest that the BATF2-expressing tumors establish a favorable local TME without triggering a notable abscopal effect.\n10. “Would BATF2-expressing tumors grow more aggressively if mice are kept on a glutamine-rich diet (Figure 6)?”\nResponse 10: Thank you for this question. We first repeated the diet experiment so that all treatment groups could be monitored simultaneously. In the non-treated empty vector tumor groups, isocaloric glutamine-rich diet alone was sufficient to result in higher tumor burdens (New Fig. 5m, Line 374-377). Because the stable overexpression of Batf2 cannot be silenced by a glutamine-rich diet, we did not observe a difference in the tumor burden of Batf2-expressing tumors between regular and a glutamine-rich diet. To assess whether glutamine-mediated tumor aggression depends on the suppression of Batf2, we implanted the same tumor model that showed more aggressive growth in wild-type mice on a glutamine-rich diet into Batf2-/- mice on either a regular diet or an isocaloric, glutamine-rich diet. We found that a glutamine-rich diet could no longer promote tumor growth in the absence of host expression of Batf2 (Supplementary Fig. 7f, Line 377-381) (please also refer to our response to Reviewer 4 Comment 5).\n11. “Is there a possibility to target BATF2 or any downstream gene pharmacologically?”\nResponse 11: Thank you for this thoughtful advice. To target the glutamine-BATF2 pathway pharmacologically, we assessed the impact of inhibition of glutamine metabolism on tumor response to IFN-I agonists. In the same experiment shown in Fig. 5k, we also have two additional groups that received intra-tumoral injection of CDA and a glutaminase inhibitor, JHU083. One of these groups was on a regular diet, and the other was on an isocaloric glutamine-rich diet. Interestingly, although JHU083 did not further improve the anti-tumor efficacy of CDA in the regular diet group, it significantly improved tumor control in mice on a glutamine-rich diet (New Fig. 5l-m, Line 387-393). Please also refer to our response to Reviewer 4 Comment 1.\n12. “Minor points:\nI would recommend to include in the figure legends how often experiments were performed and whether pooled or representative data are shown.”\nResponse 12: Thank you for the advice. We have revised the figure legends to include these details.\n13. “Why are cells sometimes stimulated with ISD/cGAMP, and other times with STING?”\nResponse 13: Thank you for your careful review. In most cases, we transfected cGAMP or ISD in BMDM to activate STING and overexpress STING in HNSCC cells to activate the pathway. We and others have shown that cancer cells have evolved multiple mechanisms to suppress the STING-TBK1-IFN pathway, such as promoting the turnover of STING and sequestering STING away from interacting with TBK1. This pathway is central for the immune sensing of HNSCC, which is likely why the tumors need multiple mechanisms to dampen this pathway. The regulation of this pathway is highly relevant for the following reasons. (a) During tumor initiation, when immune surveillance determines the outcomes of epithelial transformation, the proper activation of the innate immune pathways enables the maturation of antigen-presenting cells and subsequent cross-priming of T-cells. Known oncogenes for HNSCC, such as SOX2 and HPV16 E7, potently shut down IFN-I production. To evaluate whether BATF2 helps to launch the IFN-I pathway before epithelial malignant transformation, we expressed BATF2 in normal oral epithelial NOM9-TK cells, which directly triggered the phosphorylation of TBK1 and STING, indicative of pathway activation (New Supplementary Fig. 3d, Line 234-240). (b) In murine HNSCC cells, high Batf2 expression was also associated with elevated transcription levels of IFNb1 (New Supplementary Fig. 9a and 9l, Line 456-459 and 523-525). In our previous submission, we showed that Batf2-expressing tumors are much more immunogenic (Fig. 6). To substantiate the physiological relevance of the Batf2-Sting pathway, we generated additional stable cell lines, including vector-controlled Batf2-expressing and Batf2-expressing, Sting-deficient tumor cells. We found that cancer cell-specific Batf2-mediated tumor rejection depended on cancer cell expression of Sting because knocking down Sting abrogated Batf2-mediated protection (New Supplementary Fig. 9f-g, Line 473-477). (c) Now we also show that HNSCC cells only showed a modest increase in the phosphorylation of TBK1 and STING with cGAMP transfection. However, the STING response was largely restored when we expressed BATF2 (New Supplementary Fig. 4h, Line 308-311).\n14. “The discussion is quite short and could be extended to interpret the findings in the light of other relevant studies in the field.”\nResponse 14: We have revised the discussion section.\nPOINT-BY-POINT RESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author):\n“The authors have suitably addressed my previous minor concerns, and their report is significantly improved.”\nResponse:\nWe appreciate the reviewer’s enthusiastic feedback and the insightful comments that helped us to improve the quality of this manuscript.\nReviewer #2 (Remarks to the Author):\n“The authors adequately addressed the major points I raised.\nMinor: Some figure panels are quite dense, for example, Fig. 2D-L. The colour code does not need to be repeated in every graph; the same applies to Fig. 3A–D.”\nResponse:\nWe thank the reviewer for the enthusiastic feedback regarding the figure presentation. Following the reviewer’s advice, we have simplified the figures by reducing panel density and avoiding unnecessary repetition of the colour code. Specifically, the colour legend is now shown only once for each group of related panels (e.g., Fig. 2D–L and Fig. 3A–D), which improves clarity and readability.\nReviewer #4 (Remarks to the Author):\n“The authors present a substantially revised manuscript and have answered my questions. I don't have further comments.”\nResponse:\nWe appreciate the reviewer’s insightful comments that helped us to improve the breadth and depth of this study."}]

Decision letter