Treg-γδ T cell axis determines sexual dimorphism in hepatocarcinogenesis

Domain: neuroscience
Published: 2026-02-11
Editorial outcome: major_revision

Abstract
Sexual dimorphism in the incidence and mortality of hepatocellular carcinoma (HCC) has been observed worldwide. Sex hormones play an essential role in determining male predominance in hepatocarcinogenesis. Here we reveal significant sexual dimorphism in regulatory T cells (Tregs). Compared with HCC in female mice/patients, tumours in male mice/patients are more enriched in Tregs. The male sex hormone androgen is shown to activate the NFκB pathway and reinforce the binding of NFκB to Ccl2 promoter loci in HCC cells, thereby promoting Ccl2 production, which facilitates the intratumoral recruitment of Tregs via the Ccl2-Ccr2 axis. Additionally, Treg infiltration shapes the HCC microenvironment through the suppression of the anti-tumorigenic activity of γδ T cells rather than CD8+ T cells. Moreover, intratumoral hypoxia induces the secretion of Treg-derived extracellular vesicle (EV)-S100a4, which serves as a transcriptional corepressor to impede γδ T cell activation in an epigenetic manner. Overall, these results demonstrate that sex-specific differences in Tregs from HCC are related to the ability of androgen-dependent Ccl2 secretion to enable immune evasion through the suppression of γδ T cell activity.

Introduction
Hepatocellular carcinoma (HCC) is among the most prevalent solid malignancies, with apparent sex disparities both in humans and rodents. According to the cancer statistics between 2018 and 2023, the incidence and mortality rates in men are twice those in women1. In an HCC mouse model, compared with females, males were more susceptible to developing tumour lesions2. Pathogenically, HCC typically develops rapidly and has a poor prognosis in male patients1. While recent studies have established a sex hormone-dominated theory for sexual dimorphism in HCC, the underlying mechanism remains to be elucidated. For example, androgen receptor antagonists did not demonstrate beneficial effects in patients with HCC in several clinical trials3. This evidence indicates that sex hormones alone cannot comprehensively account for the sexual dimorphism in HCC.

Accumulating evidence suggests that males and females exhibit substantial discrepancies in immune function and disease susceptibility4–6. Sex-related steroid axes are particularly important for determining differences in the incidence, severity and prognosis of diseases across sex7. Sexual bias in host immunity can affect the tumour microenvironment (TME) composition and systemic anticancer immunity4. Emerging evidence also demonstrates clearly reveals sexual dimorphism in the immune response in the context of anticancer immunotherapy8,9. Nevertheless, to date, the role of sex steroids in anticancer immunotherapy and the underlying mechanism remain largely underexplored.

Regulatory T (Treg) cells are a subset of T cells characterized by the expression of the transcription factor forkhead-like P3 (Foxp3). Classic regulatory T cells express surface markers such as CD4, CD25, and CTLA4, and secrete inhibitory cytokines such as interleukin-10 (IL-10), interleukin-35 (IL-35), and transforming growth factor β (TGF-β), thereby leading to immunosuppressive effects10. Sexual dimorphism in Tregs is determined by the tissue microenvironment in a sex-hormone-dependent manner in various tissues, which is acknowledged for its impact on multiple pathophysiological conditions, such as tissue injury, hypoxia, and inflammation11–13. Sex-specific differences in Tregs were also observed in tumour tissues. Differential gene expression was identified in pathways related to Treg function in colorectal cancer14. Oestrogen alleviates colorectal cancer progression by inhibiting Treg function and downregulating the expression of transforming growth factor β1 (TGF-β1)15. These results suggest that Tregs modulate differences in the tumorigenesis of colorectal cancer between males and females.

Recent studies have reported that Tregs negatively impact immune surveillance, thereby driving tumour tolerance. Increased infiltration levels of Tregs have been observed in HCC tissue, suggesting that these cells play an indispensable role in hepatocarcinogenesis16,17. Although Tregs govern TME properties in HCC, the effects of sex-biased differences in Tregs on tumorigenesis remain unclear. Therefore, definite mechanistic studies investigating sexual dimorphism in Tregs in HCC are warranted to develop sex-specific immunotherapies. In this study, expanded sex-specific Tregs were abundant in HCC tissue and were recruited via hepatoma-derived Ccl2 in an androgen-dependent manner. Subsequently, infiltrated Tregs create an immunosuppressive hepatic microenvironment by influencing γδ T cell antitumor activity.

Results
Sex-specific Treg infiltration in hepatocarcinogenesis
IMC was performed on human HCC and paracarcinoma tissues to investigate differences in the immune microenvironment between males and females (Fig. 1a). A 35-marker panel was constructed for immunophenotyping, including markers for hepatic nonparenchymal cells and innate and adaptive immune cells (Supplementary Data 1). A total of 6-cell clusters were exported for dedicated automatically-gated analyses across all subjects (Fig. 1b; Supplementary Fig. 1a). Notably, compared with females, males had proportionally higher proportions of Tregs, T cells, and B cells in HCC tissue (Fig. 1c). IMC, IHC and qPCR also revealed significantly higher expression levels of the Treg marker Foxp3 in male patients with HCC, which was correlated with a significantly lower overall survival rate (Fig. 1d–f; Supplementary Fig. 1b). In contrast, no significant differences were noted in the expression levels of CD3, CD4 and CD8 between males and females (Supplementary Fig. 1c, d). However, Treg infiltration was not correlated with HCC proliferation in human specimens (Supplementary Fig. 1e, f; Supplementary Data 2).

To validate these findings, a diethylnitrosamine (DEN)-induced mouse model was constructed to detect Treg infiltration using flow cytometry (Fig. 1g). As anticipated, DEN treatment aggravated hepatocarcinogenesis and resulted in increased tumour volume in male mice (Fig. 1h). Consistent with the results observed in humans, liver/body weight was elevated following DEN treatment in male mice with HCC (Supplementary Fig. 1g, h), accompanied by an increase in the level of Treg infiltration (Fig. 1i; Supplementary Fig. 1i). Interestingly, no significant differences were detected in normal hepatic Tregs or peripheral or splenic Tregs between males and females (Supplementary Fig. 1j–l). Taken together, these results suggest that sex-specific Treg infiltration may contribute to hepatocarcinogenesis and influence HCC prognosis.

The Ccl2–Ccr2 axis recruits Tregs to HCC
To identify factors triggering Treg infiltration during hepatocarcinogenesis, the expression levels of several cytokines and chemokines were evaluated in HCC, and the results revealed an increase in Ccl2 expression levels in male HCC and an increase in Ccr2 expression levels in Tregs (Fig. 2a, b; Supplementary Fig. 2a). In addition, a clear correlation was identified between the proportion of Tregs and the levels of Ccl2 or Ccr2 in both mouse and human HCC (Fig. 2c; Supplementary Fig. 2b). In contrast, the expression levels of other Treg-associated cytokines or chemokines (Tnfa, Ifng, IL6, Ccl19 and Ccl21) remained unchanged in male and female HCC tissues (Supplementary Fig. 3c). Ccl2-deficient (Ccl2−/−) mice were used to explore the effect of Ccl2 in a DEN-induced HCC model. There was an increase in Ccl2 expression with high levels of Treg infiltration in DEN-induced HCC in male mice. Depletion of Ccl2 reduced tumour burden and Treg infiltration in HCC (Fig. 2d–f). Furthermore, the results of immunofluorescence analysis indicated that the elevated Ccl2 expression originated from hepatoma cells (Supplementary Fig. 2d, e). Additionally, we genetically deleted Ccl2 in Hepa1-6 cells using CRISPR/Cas9, followed by orthotopic Hepa1-6 implantation. Immunofluorescence assays demonstrated that Ccl2 is mainly secreted by HCC cells and is triggered by androgen. In contrast, lower Ccl2 expression was detected in HCC in the absence of Ccl2 or androgen in both WT and Ccl2−/− mice (Supplementary Fig. 2f, g). Deservedly, deficiency of Ccl2 showed a lower tumour burden with reduced Treg infiltration in HCC (Supplementary Fig. 2h, i). Ccr2-deficient (Ccr2−/−) and Foxp3+ cell-specific Ccr2 knockout (Ccr2cKO) mice were used to analyse Treg recruitment in HCC (Fig. 2g). Although depletion of the Ccl2–Ccr2 axis did not alter Treg development in the spleen or blood (Supplementary Fig. 2j, k), male mice had a lower tumour burden with lower levels of Treg infiltration in HCC (Fig. 2h, i; Supplementary Fig. 2l, m). Taken together, these results suggest that hepatoma-derived Ccl2 is essential for male-specific recruitment of Tregs to HCC.

Androgen-dependent Ccl2 production contributes to Treg recruitment
A key question is why Tregs tend to infiltrate into male HCC tissue. Sex hormones are central to sexual dimorphism despite their role in HCC being controversial. Next, a mouse gonadectomy HCC model was used to explore the effects of sex hormones on Treg recruitment (Fig. 3a). The proportion of Tregs in the spleen and blood remained unaltered following gonadectomy (Supplementary Fig. 3a, b). Compared with those in the sham group, the levels of HCC-induced Treg infiltration and Ccl2 production were lower in castrated male mice. In contrast, castration did not alter Ccl2 expression in female HCC (Fig. 3b–d). Moreover, there was no difference in monocyte or macrophage infiltration in HCC between male and female mice or after castration (Supplementary Fig. 3c, d). Hepatic monocyte-, neutrophil-, Kupffer cell- and Treg-derived Ccl2 levels were comparable between the sham and castration groups (Supplementary Fig. 3e, f). Ccl2 was minimally expressed in healthy hepatocytes or immune cells (Supplementary Fig. 3g). These data suggested that androgen actively participates in HCC-derived Ccl2 production.

To identify the role of androgen in Ccl2 production, HCC cells were exposed to different concentrations of testosterone. As expected, testosterone treatment increased Ccl2 expression levels in a dose-dependent manner in HepG2 and Hepa1-6 cells (Fig. 3e), whereas estradiol treatment had no effect on HCC cell-derived Ccl2 expression (Supplementary Fig. 3h). Strong evidence supports that the NFκB pathway is important for Ccl2 production18,19. Indeed, the expression of NFKB1 was correlated with that of CCl2 in human HCC specimens (Fig. 3f). Constitutive activation of NFκB in HCC promotes Ccl2 expression and Treg infiltration, which leads to a high HCC burden in female mice (Supplementary Fig. 3i–k). A recent study revealed an AR-dependent pathway that acts through integrin α6/PAK1 to stimulate NFκB activity independent of PI3K signalling20. Indeed, testosterone treatment increased the levels of integrin α6 and PAK1 in vitro, which activated the NFκB pathway in a dose-dependent manner (Fig. 3g). AR-deficient HCC showed reduced HCC growth with lower Ccl2 expression and less Treg infiltration (Fig. 3h–j). Cut&Tag analysis using specific antibodies against NFκB in HepG2 cells revealed that the overall binding of NFκB at Ccl2-binding sites was significantly enhanced with or without testosterone administration, and these changes focused mainly on promoter regions (Fig. 3k, l). Furthermore, the potential binding sequences of NFκB in the Ccl2 promoter locus (−676 to 667, 5’-GGGAATTTAC-3’) were examined via qPCR, which revealed direct binding of NFκB to the Ccl2 promoter region (Fig. 3m, n). These results collectively indicated that androgen served as a positive regulator of Ccl2 expression in HCC cells by activating the AR/NFκB signalling pathway.

Androgen orchestrates Tregs to impair γδ T cell antitumor immunity
Recent studies have shown that γδ T cells possess unique advantages for recognizing and responding to HCC, offering promising avenues for HCC immunotherapy21,22. The antitumorigenic function of γδ T cells is substantially impaired in HCC, which is partially mediated by Tregs23. Although the infiltration level of γδ T cells was relatively low in healthy liver tissue, it was significantly greater in female HCC than in male HCC (Fig. 4a). Furthermore, compared with mice in the sham group, male HCC-bearing mice subjected to gonadectomy exhibited lower tumour volume and greater γδ T cell infiltration (Fig. 4b, c). Consistently, higher proportions of total IFNγ+ and GzmB+ γδ T cells were detected in HCC after castration. In contrast, castration did not affect the percentage or cytotoxicity of CD8+ T cells (Fig. 4d, e).

To explore the interactions among sex hormones, Tregs and γδ T cells, primary Tregs and γδ T cells were isolated from the spleen and cocultured with Hepa1-6 cells (Fig. 4f). Interestingly, neither sex hormones (estradiol and testosterone) nor Tregs alone influenced HCC cell growth (Supplementary Fig. 4a–d). As expected, coculture Hepa1-6 cells with γδ T cells alone resulted in increased cytotoxicity without affecting cell proliferation, whereas testosterone treatment did not suppress γδ T cell cytotoxicity (Fig. 4g; Supplementary Fig. 4e). In agreement with the in vivo findings, coculture with γδ T cells and Tregs impaired the antitumour immunity of γδ T cells but not that of CD8+ T cells in a dose-dependent manner (Fig. 4h, i), whereas coculturing of Tregs with γδ T cells or CD8+ T cells did not affect HCC cell proliferation (Supplementary Fig. 4f, g).

S100a4 expression is upregulated in Tregs under hypoxic conditions
To investigate the mechanisms through which Tregs regulate the antitumour immunity of γδ T cells in the TME, the scRNA-seq data of human HCC samples from 16 males and 15 females were analysed. A total of 11 cell clusters and 5 T-cell subsets were identified and manually gated for analysis (Fig. 5a; Supplementary Fig. 5a–c). These findings revealed that male patients with HCC had a proportionally greater proportion of Tregs and a lower proportion of γδ T cells (Fig. 5b; Supplementary Fig. 5d). Notably, both CD8+ T cells and γδ T cells highly expressed genes characteristic of cytotoxic markers, namely GZMK, GZMB, IFNG, and PRF1 (Fig. 5c). In addition, immune checkpoint inhibitory markers (LAG3, HAVCR2, PDCD1, INPP5F, TIGIT, and CTLA4) were enriched exclusively in CD8+ T cells rather than in γδ T cells, suggesting that γδ T cells are promising candidates for HCC immunotherapy (Fig. 5d). Moreover, the expression levels of cytotoxic markers were significantly greater in female HCC-derived γδ T cells (Fig. 5e).

Unexpectedly, a population of S100A4+ T cells and Tregs was detected in the immune milieu of HCC (Fig. 5f; Supplementary Fig. 5e). Interestingly, increased levels of S100A4 (Fig. 5g–i; Supplementary Fig. 5f; Supplementary Data 3), which were positively correlated with FOXP3 expression (Fig. 5j) and negatively correlated with the overall survival rate (Fig. 5k), were detected in Tregs from human male HCC tissue. By contrast, the levels of other Treg-associated cytokines (IL-10, TGF-β, and IL-35) and γδ T cell-derived S100a4 remained unchanged in male and female HCC samples (Supplementary Fig. 5f–h). Immunofluorescence staining revealed an increase in S100a4 expression levels in Tregs acquired from male mice with HCC (Fig. 5l). Recent studies have suggested that S100a4 contributes to HCC proliferation, invasion and metastasis, which are correlated with the clinicopathological features and prognosis of HCC24–26. More importantly, S100a4 was identified as a hypoxia-inducible gene whose transcription is governed by Hif1α binding27. Indeed, compared with adjacent tissue, HCC is more hypoxic in both males and females. Enhanced Hif1α expression was detected in male HCC-derived Tregs compared with female HCC-derived Tregs because of increased intratumoral necrosis (Supplementary Fig. 5i). Moreover, treatment with PX-478, an inhibitor of Hif1α, restrained S100a4 expression, leading to an equal tumour burden (Supplementary Fig. 5j, k). Next, primary Tregs were isolated from the spleen and cultured under hypoxic conditions. These results revealed that hypoxia induced S100a4 production in Tregs (Fig. 5m, n).

Finally, the results of the Cut&Tag analysis indicated that Hif1α binds to the motif located at +76 to +195 of the S100a4 promoter locus to increase S100a4 expression (Fig. 5o). These data collectively indicated that HCC-mediated hypoxia triggers S100a4 transcription in Tregs through interactions with Hif1α.

EV-S100a4 suppresses the programme of γδ T cell activation
Subsequently, the colocalization of S100a4 with Tregs and γδ T cells was investigated using immunohistochemical analysis. The results indicated that S100a4 was highly expressed in Tregs and γδ T cells (Fig. 6a). Treatment with the S100a4 protein directly did not significantly affect the cytolytic activity of γδ T cells or CD8+ T cells against target cells, suggesting an alternative mechanism for S100a4 transport (Supplementary Fig. 6a, b). Recently, S100a4 has been shown to regulate oncogenic properties via EV-mediated intercellular communication26,28. Therefore, a fluorescently labelled S100a4-overexpressing lentiviral vector (LV) was constructed to overexpress S100a4 in Hepa1-6 cells, Tregs and γδ T cells (Supplementary Fig. 6c). The Treg-mediated transfer of S100a4 to γδ T cells was subsequently investigated using a coculture model (Fig. 6b). Tregs were preinfected with S100a4-overexpressing LV prior to coculture with γδ T cells. The results of flow cytometry analysis revealed that the fluorescent signal steadily increased in γδ T cells and decreased in Tregs in a time-dependent manner following coculture (Fig. 6c). S100a4, originally identified only in Tregs, was detected in γδ T cells (Fig. 6d; Supplementary Fig. 6d). These observations prompted us to test whether Treg-derived S100a4 is contained in EVs. EVs were isolated from coculture supernatants via ultracentrifugation, and typical EV markers and S100a4 levels were validated. Notably, both western blotting, transmission electron microscopy (TEM) and imaging flow cytometry revealed significant S100a4 expression in EVs after coculture (Fig. 6e; Supplementary Fig. 6e). These results indicated that Treg-derived EV-S100a4 can be transported into γδ T cells.

Altering S100a4 expression using LVs in vitro did not influence the cytotoxicity of CD8+ T cells (Fig. 6f). Moreover, lentiviral transduction of S100a4 into Hepa1-6 cells did not significantly impact HCC growth (Supplementary Fig. 6f). In contrast, γδ T cells (from human PBMCs or mouse spleens) overexpressing S100a4 exhibited decreased cytotoxicity, lower cytokine production, and shorter survival times, suggesting a universal mechanism involving S100a4 in different species of γδ T cells (Fig. 6g–i; Supplementary Fig. 6g–i). Consistently, S100a4 overexpression repressed TCR signalling activation of γδ T cells, as evidenced by the decreased phosphorylation of Zap70, Akt, and NFκB (Fig. 6j). As expected, the enhanced EVs-S100a4 expression and impaired TCR signalling activation were also detected in male DEN-induced HCC (Supplementary Fig. 6j, k). To determine changes in TCR clonotype-induced γδ T cell inactivation, the TCRδ repertoire was tracked using TCR-seq in human HCC samples (Supplementary Data 4, 5). The use of TCRs by γδ T cells was comparable across different sexes in patients with HCC. Most γδ T cells are Vγ9/10 and Vδ1 in both males and females. VDJ frequency, TCR diversity, TCR overlap and CDR3 amino acid bias were also similar between males and females, indicating that TCR usage does not play a pivotal role in γδ T cell antitumor activity (Supplementary Fig. 7).

Emerging evidence suggests that S100a4 acts as a cotranscription factor to modulate the activity of several transcription factors (including NFκB and STAT3)26,29. ATAC-seq and RNA-seq were subsequently performed to explore the potential underlying mechanisms. A total of 2,916 differentially accessible regions in chromatin and 24,642 DEGs involved in transcription were identified by ATAC-seq or RNA-seq (Supplementary Data 6, 7). The overexpression of S100a4 in γδ T cells resulted in significantly different chromatin accessibility and transcriptional profiles (Supplementary Fig. 8a, e). Most notably, decreased chromatin accessibility was detected in S100a4-overexpressing γδ T cells (Fig. 6k, l). More than 85% of these changes were in promoter regions, whereas fewer than 15% were in other regions (Supplementary Fig. 8b). Among these changes, a decrease in chromatin accessibility was detected for genes associated with cytotoxic cytokines (Ifng, prf1, Tnf), as well as signal transduction molecules in the TCR signalling pathway (Zap70, Lck, Tab3, Grb2, Cbll1, Vav1, Tagap, Akt, Nfkbia) (Fig. 6m, n; Supplementary Fig. 8c, d). RNA-seq analysis also revealed similar changes in transcription levels, which are in agreement with chromatin accessibility (Supplementary Fig. 8f, g). Differentially expressed genes (DEGs) were also enriched in the “TCR signalling pathway” gene sets, which indicated that S100a4 inhibits the accessibility of genes involved in TCR signalling (Fig. 6o; Supplementary Fig. 8h). In contrast, genes associated with exhaustion (Havcr2, Jun, Lag3, Pdcd1, Tigit, and Tox) did not significantly change in transcription level (Supplementary Fig. 8i). These findings demonstrated that S100a4 might serve as a transcription corepressor to induce epigenetic reprogramming at gene loci associated with TCR signalling in γδ T cells.

Treg/S100a4/γδ T cell axis controls sex differences in hepatocarcinogenesis
We subsequently observed an increase in intratumor γδ T cell expansion in Ccr2cKO and Treg-deficient (Foxp3DTR knock-in) mice (Supplementary Fig. 9a, b). Moreover, the deletion of γδ T cells increased the tumour burden in female mice and decreased sex disparity (Supplementary Fig. 9c). To determine the effects of eliminating S100a4 in Tregs on the antitumour capacity of γδ T cells and mitigating sex differences in hepatocarcinogenesis between males and females, orthotopic HCC implantation was performed in Foxp3+ cell-specific S100a4 knockout (S100a4cKO) mice (Supplementary Fig. 9d, e). Although S100a4 knockout did not influence the maturation, development, or liver homing of Tregs under steady-state conditions (Supplementary Fig. 9f), the deletion of S100a4 in Tregs markedly decreased the HCC S100a4 level and tumour burden in male mice (Fig. 7a, b). Additionally, S100a4 deficiency in Tregs did not affect Treg infiltration (Fig. 7c, d) or Ccl2 expression levels in HCC (Fig. 7e, f). The abundance of γδ T cells, as well as the expression levels of GzmB and IFN-γ, were greater in S100a4-deficient male HCC tissues (Fig. 7g–i). These results demonstrated that the deletion of S100a4 enhanced the efficacy of γδ T cells against HCC and that androgen-induced Treg infiltration and S100a4 production contribute primarily to γδ T cell immune surveillance and sexual dimorphism in hepatocarcinogenesis.

Methods
Humansamples
Treatment-naïve patients pathologically diagnosed with HCC were included in this study. Fifty-seven human HCC and paracarcinoma tissues were collected from the Department of Pathology, The 971 Hospital of People’s Liberation Army Navy, between 2020 and 2024. The resected specimens of all patients were histologically confirmed by an experienced pathologist. The basic demographics of this study were summarized in Supplementary Data 8. All the samples were collected with informed consent of the patients, and all the experiments were approved by the ethics committee of Xiamen University (XDYX202411K71).

Mice and in vivo models
Ccl2 and Ccr2 gene knockout mice (Ccl2−/− and Ccr2−/−) were purchased from GemPharmatech Co., Ltd (Nanjing, China). Foxp3DTR knock-in mice were purchased from The Jackson Laboratory (Bar Harbour, USA). S100a4cKO and Ccr2cKOmice were generated by crossing S100a4f/f and Ccr2f/f mice (Gempharmatech Co., Ltd) with Foxp3YFP-Cre mice (Jackson Laboratory, Bar Harbour, USA), respectively. All mice were housed and maintained under specific-pathogen-free (SPF) conditions. All animal experiments, housing, and feeding conditions were approved by the Institutional Ethical Commission for Animal Research at Xiamen University (XMULAC20240007).
For chemical induction of hepatocarcinogenesis, mice at postnatal day 14 were injected intraperitoneally with 25 mg/kg diethylnitrosamine (DEN, Sigma-Aldrich) in 0.9% saline and then maintained for 10 months after initial injection. Tumours were measured twice weekly after treatment unless mice died or were euthanized. Mice were euthanized via CO2 asphyxiation when tumour size exceeded 2 cm3 or recommended by an independent animal health monitor for morbidity or moribund behaviour. For Tregs ablation, 6-week-old Foxp3DTR mice were intraperitoneally injected twice a week with 500ng dose of diphtheria toxin (DT, ListLabs, California, USA) for 2 weeks. Hepa1-6 cells (5×106 per mice) were suspended in 100 μl of PBS (Hyclone) and Matrigel (BD Biosciences) mix (1:1) and implanted into the liver of C57BL/6J mice to establish orthotopic HCC implantation models. Mice were sacrificed 4 weeks after HCC cell injection for further experiments. For experiments with the Hif1α inhibitor PX-478 (Shanghai Yuanye Bio-Technology Co., Ltd), mice were treated by intraperitoneal injection at a dose of 30mg/kg body weight twice a week after orthotopic implantation. For the mouse gonadectomy model, 6-week-old male or female mice were castrated or sham-operated6,45. In brief, mice were anesthetized with isoflurane for an abdominal midline incision. Subsequently, the testes or the uterus adnexa were pulled out, and then the scrotum or the ovary was excised. Mice were sacrificed 6 weeks after gonadectomy for further experiments.

Cell culture and transfection
The Hepa1-6, HepG2 and 293T cell lines (Shanghai cell bank, Chinese Academy of Sciences) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Solarbio Life Sciences, Beijing, China) supplemented with 10% foetal bovine serum (FBS, Yeasen, Shanghai, China) and 1% penicillin/streptomycin (P/S) (VivaCell Biotechnology GmbH, Denzlingen, Germany). Splenic CD8+ T cells and Tregs (CD4+YFP+) from Foxp3YFP-Cre mice were sorted and cultured in X-Vivo 15 medium (Lonza, Basel, Switzerland) supplemented 100 ng/ml anti-mouse CD3 (BioLegend, San Diego, CA, USA), 1 μg/ml anti-mouse CD28 (BioLegend), and 10 ng/ml IL2 (Novoprotein, Shanghai, China). γδ T cells (TCRγδ+) were cultured in the medium supplemented with 1 μg/ml anti-mouse CD3 (BioLegend, San Diego, CA, USA), 1 μg/ml anti-mouse CD28 (BioLegend), 10 ng/ml IL2 (Novoprotein, Shanghai, China), and 5 μM zoledronic acid. For the establishment of hypoxic cell growth conditions, an AnaeroPack™ MicroAero (Mitsubishi Gas Chemical, Tokyo, Japan) was placed inside a sealed air-tight container. Cells were incubated in the hypoxic container at 37°C for 48h. Cell viability was measured using the Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China). For cytotoxic assay, Propidium Iodide (PI, Yuanye Bio-Technology, Shanghai, China) and Zombie NIR™ Fixable Viability Kit (BioLegend) were used to stain the dead cells.

Construction of lentiviral vector (LV) and stable cell lines
Human S100A4 cDNA (primer forward: GCTAGCGAATTCGCCACCATGGCGTGCCCTCTGGAG; reverse: TCTGCCCTCAGCGGCCGCTATTTCTTCCTGGGCTG) or mouse S100a4 cDNA (primer forward: GCTAGCCTCAACGGTTACC; reverse: GCGGCCGCTAGTTTCAAAACCT) was cloned into pCDH-MSCV lentiviral vector (LV) followed by transfected into 293T cells to generate pCDH-MSCV-S100a4-GFP LV, respectively. For the overexpression of S100a4, γδ T cells were infected with pCDH-MSCV-S100a4-GFP LV with 10 MOI for 72h. Full-length IKK2ca was cloned into pCDH-MSCV LV and infected with Hepa1-6 for constitutive activation of NFκB. All procedures involving LVs were conducted in a Biosafety Level 2 (BSL2) laboratory.
Guide RNAs (sgRNAs) were designed with the online tool (SYNTHEGO, https://design.synthego.com/#/) and synthesized from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of the sgRNAs were as follows: sgRNA‑Ccl2-1, AGTGGGGCGTTAACTG CATC; sgRNA‑Ccl2-2, TGAGTAGCAGCAGGTGAGTG; sgRNA‑AR-1, ACAGACAAGCTCAAGGATGG; sgRNA‑AR-2, TCAAGGATGGAGGTGCAGTT. Subsequently, sgRNAs were inserted into the LentiCRISPRv2 plasmid to generate recombinant plasmids, followed by transfected with Hepa1-6 to generate the Cas9‑sgRNA cell lines. Furthermore, puromycin was used to select stably transfected cells.

Imaging mass cytometry (IMC) and data processing
Hyperion Mass Imaging System (Fluidigm Corporation, CA, USA) was used according to the tuning protocol provided by the manufacturer. Briefly, the regions of interest (ROIs) in the human HCC and paracarcinoma tissue were collected based on consecutive tissue sections stained with hematoxylin and eosin. ROIs of 1000×1000µm were laser ablated at 200 Hz, and raw data were acquired using the Fluidigm MCDTM viewer (v1.0.560.2, Fluidigm Corporation). In order to identify an antibody signal, a minimum signal threshold of one or two dual counts was set. Then, each image was checked by visually inspecting all markers' staining patterns to avoid background noise.
For cell segmentation of cells, DNA-Iridium staining was used as a nuclear and cell membrane marker to define primary and secondary objects. Scikit-image (v0.16.2, Python package) was subsequently used to define the cell masks and quantify the mean expression of 35 markers (Supplementary Data 9). Furthermore, the normalized expressions of the segmented single cells were processed by a standard pipeline to remove the potential batch effect between ROIs. To identify cell types and subpopulations, we initially distinguished 6 cell types based on the expression of canonical marker genes. Specifically, CD4+ or CD8+ T cells were identified by positive expression of CD4 or CD8, while Tregs were recognized by high expression of CD4 and Foxp3. Myeloid cells were characterized by CD16 and CD14. Hepatic stellate cells (HSC) were marked by α-SMA and Collagen Ⅰ, and B cells were differentiated by CD20 expression. Hepatic parenchymal cells were identified by low expression of all markers. All clustering analyses were implemented using the R package FlowSOM (v1.18.0) for a self-organizing map. Subsequently, the R package FlowSOM (v0.99.1) was applied to the mean expression values of each group to ensure the final clustering data.

Western blotting
Cells were collected and lysed in RIPA buffer (Solarbio Life Sciences) supplemented with a protease inhibitor cocktail (Bimake, Shanghai, China), followed by centrifugation at 10,000×g for 10 min at 4°C. The total protein concentration was measured using a Bio-Rad Kit (Hercules, CA, USA). Forty micrograms of protein were subjected to SDS–PAGE gel and transferred to PVDF membranes (LABLEAD, Beijing, China). Membranes were probed with primary antibodies and incubated with HRP-conjugated secondary antibodies. Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA) was used to visualize the protein bands. The antibodies used were as follows: Integrin α6 (Huabio, SR45-00l, 1:1000), PAK1 (Baijia, IPB5302, 1:1000), Androgen receptor (Huabio, PO00-29, 1:1000), Phospho-NFκB p65 (Ser468, Proteintech, 6N1, 1:1000), NFκB p65 (Proteintech, 4C7, 1:1000), Hif1α (Servicebio, GB111339, 1:1000), S100a4 (Huabio, SD200-08, 1:1000), HSP90 (Servicebio, GB15284, 1:1000), ZAP70 (Zenbio, R26132, 1:1000), phospho-ZAP70 (Tyr319, Zenbio, 310211, 1:1000), Akt (HUABIO, ET1609-51, 1:1000), phospho-Akt (Ser473, Sino Biological, 110852-R0069, 1:1000), and Tubulin (Huabio, 1-B11, 1:5000).

Immune cell isolation and flow cytometry
Isolation of intrahepatic immune cells was conducted as described in our previous study46. The single-cell suspension was then stained with fluorescently conjugated antibodies at 4°C for 20 min to avoid light. For cytokine detection, the cells were fixed with Fixation/Permeabilization Solution (eBioscience, Shanghai, China) for 30 min at 4°C, followed by washing with Permeabilization Buffer (eBioscience). The stained cells were examined using a Cytoflex S (Beckman) and Cytoflex LX (Beckman) and analysed with FlowJo software (version 10.6.2, BD, Ashland, USA). The antibodies PE-CD3 (17A2, 1:500), APC-CD3 (17A2, 1:500), PerCP-CD45 (30-F11, 1:500), APC-Ccr2 (SA203G11, 1:500), PE-Foxp3 (150D, 1:500), FITC-CD4 (GK1.5, 1:500), FITC-F4/80 (BM8, 1:500), PE-CD11b (M1/70, 1:500), PE-Ccl2 (2H5, 1:500), FITC-TCRγδ (UC7-13D5, 1:500), APC-IFN-γ (XMG1.2, 1:500), PE-Perforin (S16009A, 1:500), and Pacific Blue-GzmB (QA16A02, 1:500) from BioLegend.

Immunohistochemistry
The fresh liver samples were immediately excised, fixed in 4% paraformaldehyde, and then embedded in paraffin after ethanol gradient dehydration. For brightfield staining, after neutralizing endogenous peroxidases, the sections were incubated with primary antibodies at 4°C for 24h, followed by incubation with secondary antibodies at room temperature for 30 min, then images were acquired using a Leica DM2700 microscope after DAB staining. For immunofluorescence staining, the sections were blocked with normal donkey serum. To obtain multiple immunofluorescence, the following procedure was repeated for multiple immunostaining: (1) incubation with citric acid buffer at 100°C for 30 min to repair antigen; (2) blocking with blocking solution after incubation with 3% hydrogen peroxide for 15 min avoid light; (3) incubation with the primary antibody (mouse anti-TCRγδ, rabbit anti-Foxp3 and rabbit anti-S100a4 in tune) overnight at 4°C; (4) incubation with the HRP-conjugated secondary antibodies (which appropriately respond to the species of the primary antibody) at room temperature for 50 min; and (5) incubation with fluorescein-conjugated tyramide signal amplification (TSA, Abclonal, Wuhan, China) (cy3-Tyramide, FITC-Tyramide, or iF647-Tyramide) at room temperature for 10 min. Finally, the sections were stained with DAPI. The images of the processed sections were scanned using a Leica TCS SP8 DLSSystem. Primary antibodies were used as following: mouse anti-CD3 (MXB Biotechnologies, MX036, 1:100), rabbit anti-CD4 (MXB Biotechnologies, SP35, 1:100), mouse anti-CD8 (MXB Biotechnologies, MX117, 1:100), rabbit anti-Foxp3 (Servicebio, GB112325, 1:200), rabbit anti-HNF4a (Servicebio, GB115549, 1:200), rabbit anti-Foxp3 (Huabio, JF0898, 1:200), rabbit anti-S100a4 (Huabio, SD200-08, 1:200), mouse anti-TCRγδ (Biolegend, UC7-13D5, 1:200), rat anti-Ly6c (Abcam, RM1151, 1:200), rabbit anti-AFP (Servicebio, GB15287, 1:200), rabbit anti-Ccl2 (Servicebio, GB11199, 1:200), rabbit anti-albumin (Huabio, JF32-10, 1:200), anti-rabbit IgG (Abcam, 1:500) and anti-mouse IgG (Abcam, 1:500). The images of the processed sections were scanned using a panoramic (3DHISTECH) digital slide scanner. All images were manually coded and randomized for a subsequent, independent blind analysis, conducted by two researchers.

T cell receptor repertoire sequencing (TCR-seq) and analysis
Human HCC samples (male 8, female 8) were used to detect TCRδ-chain repertoire. Total RNA from tissue was isolated using TRIzol (ABclonal, Wuhan, China). Subsequently, 200 ng RNA was reverse transcribed into cDNA using a Transcriptor First Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai, China) on a C1000 TouchTM Thermal Cycler (Bio-Rad). For TCRδ library construction, two-round nested amplicon arm-PCR was performed. The amplicons libraries were constructed using VAHTS Universal Pro DNA Library Prep Kit (Vazyme, Nanjing, China) followed by paired-end sequenced (2×150 bp) on an Illumina platform according to standard protocols.
The MiXCR software was used to process the raw data. Relative data analysis was performed as previously described38.

CUT&Tag
CUT&Tag assay was performed using NovoNGS® CUT&Tag 4.0 High-Sensitivity Kit (Novoprotein, N259-YH01-01B) according to the manufacturer’s instructions. Approximately 1×105 cells were treated with 10 μl of binding ConA beads at room temperature for 10 min. Primary antibodies (NFκB, ET1603-12, Huabio; Hif1α, Huabio) were incubated at room temperature for 2h, followed by the addition of secondary antibodies at room temperature for 1h. Subsequently, the mixture was treated with 1 μl ChiTag Transposome at room temperature for 1h. For DNA fragmentation, 65 μl Tagmentation Buffer was added at 37°C for 1h. DNA was then collected for subsequent PCR analysis and sequencing. Primer sequences as follows: S100a4 forward, GGGCGCTGTGGGGATAAATA, reverse, TTTGCAGAGTCCAAGTGGGG; Ccl2 forward, CTGGGTTCTGTGCTCGAGG, reverse, AAATGCCAGTGTCATACTCACA. The libraries were amplified and subjected to sequencing using the Illumina NovaSeq 6000 platform. The raw data underwent quality filtering using Cutadapt to obtain clean data. To visualize the genomic occupancy of the peaks, Integrative Genomics Viewer and UCSC Genome Browser were applied. HOMER software was used to analyse the motif.

Extracellular vesicle (EV) isolation and immunogold electron microscopy
γδ T cells were indirectly cocultured with Tregs that were preinfected with S100a4 LV for 48h. The coculture medium was removed. Tregs were washed once with PBS and then cultured in cell culture medium containing no serum for 48h. The supernatant was collected and ultracentrifuged at 100,000×g for 2h at 4°C to isolate EVs (JXN-30, Beckman Coulter). EVs were resuspended in 100 μl PBS. A 7 μl drop of the sample solution containing EVs was adsorbed to a glow-discharged carbon-coated copper grid, blocked with 1% BSA for 10 min at room temperature, and incubated with an antibody against s100a4 (HUABIO, SD200-08) diluted 1:20 in 1% BSA for 1h at 4°C. The samples were washed 3 times with deionized H2O for 3 min and incubated with a 10 nm gold-conjugated anti-rabbit antibody (Solarbio) diluted 1:100 in 1% BSA for 3h at room temperature. After washing 3 times with deionized H2O for 10 min, samples were fixed with 20 μl of freshly prepared 2.5% glutaraldehyde for 15 min at room temperature. Then, samples were washed 3 times with deionized H2O and stained with uranyl acetate and lead citrate for 5 min. Samples were imaged at room temperature on a Hitachi HT-7800 transmission electron microscope.

RNA/DNA extraction and quantitative PCR (qPCR)
Total RNA was extracted from frozen liver samples using TRIzol (ABclonal, Wuhan, China) following the manufacturer’s protocol. A total of 1 μg RNA was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Yeasen Biotechnology). To detect mRNA expression, qPCR was performed using SYBR Green PCR Mix (Yeasen Biotechnology) and conducted with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Relative gene expression levels of mRNA were normalized against Gapdh and calculated using the 2−ΔΔCt method. Cells' genomic DNA was extracted using TIANamp Genomic DNA Kit (TIANGEN, Shanghai, China) according to the standard protocols. The relative genetic level of S100a4-exon2 was normalized against S100a4-exon3. Primer sequences are shown in Supplementary Data 10.

Bulk RNA-seq and ATAC-seq
The RNA-seq libraries were generated using TruePrep RNA Library Prep Kit for Illumina (Vazyme, Nanjing, China) and sequenced on an Illumina NovaSeq 6000 instrument (Illumina, San Diego, USA) using the PE150 strategy. The R package DESeq2 was used to compare the differentially expressed genes (DEGs). Genes with a q value < 0.05 and log2|FoldChange| > 0.05 were deemed differentially expressed. Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses were performed using the R package ClusterProfiler.
For ATAC-seq libraries preparation, a commercial Hyperactive ATAC-Seq Library Prep Kit for Illumina (Vazyme, Nanjing, China) was used according to the manufacturer’s instructions, followed by pair-end sequences with a Novaseq 6000 system (Illumina). For data analysis, adaptors were trimmed using Cutadapt (v2.10). The resulting clean reads were aligned to the mouse reference genome (mm10) using Bowtie2 (v2.2.1). Peaks were called using MACS2 (v2.1.1)64 with the parameter ‘macs2 callpeak -g mm –broad –bdg -f BAMPE’. To generate bigwig files, deepTools (v3.4.3)65 and the function bamCoverage (v3.1.2) were employed, using BAM files. Read coverage in the bigwig files was normalized as Counts Per Million mapped reads (CPM). The union peak list was determined with the Diffbind package. Peaks were annotated by ChIPseeker. ATAC-seq results were visualized in the bigwig format using IGV (v2.4.14).

scRNA-seq data acquisition and processing
scRNA-seq data (16 male and 15 female HCC samples) were obtained from the GSA-Human database. The scRNA-seq data were analysed using the Seurat 5.0 R package. Cells with fewer than 250 genes, more than 4000 genes, or over 20% mitochondrial gene content were excluded. Data integration was performed with the Harmony R package. Principal component analysis (PCA) was employed for dimensionality reduction, and UMAP was used for visualization of the cellular landscape.
Eleven cell types were identified based on the expression of canonical marker genes. T cells were characterized by high expression of CD3D and CD3E, B cells by MS4A1, CD79A, and CD79B, macrophages by CSF1R and CD68, and monocytes by S100A8, S100A9, and LST1. Endothelial cells expressed PTPRB, PECAM1, and VWF, epithelial cells expressed KRT5 and KRT14, and mast cells expressed KIT, TPSAB1, TPSB2, and CPA3. Plasma cells were marked by IGHG3 and MZB1, plasmacytoid dendritic cells by IL3RA, IRF8, and CLEC4C, hepatocytes by AFP, ALB, APOA1, HNF4A, TAT, and TTR, and smooth muscle cells by MYL9 and ACTA2. T cells were further classified into CD4+ T cells (CD4, CCR6, LTB), CD8+ T cells (CD8A, CD8B), natural killer T cells (NKT; SLC4A10, IL4I1, NCR3), gamma delta T cells (γδ T; TRGC1, TRDC), and regulatory T cells (Tregs; FOXP3, RTKN2, CTLA4). DEGs between groups within each T cell subpopulation were identified using the “FindMarkers” function with the Wilcoxon rank-sum test. Genes with a q value < 0.05, log2|FoldChange| > 0.5, and a minimum percentage > 0.1 were considered differentially expressed.

Statistical analysis
Statistical analyses were performed using GraphPad Prism Version 9 (GraphPad) and expressed as the geometric mean ± standard error of mean (SEM). The unpaired Student’s t test or Fisher’s exact test between two groups, one-way or two-way ANOVA with Bonferroni’s multiple comparison test among three or more groups, were used as indicated in the figure legends. Log rank (Mantel–Cox) test was used to compare the overall survival rate. In addition, the Pearson correlation analysis was performed to determine the possible correlation. All statistical tests were conducted in a two-sided manner with a significance level set at P < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript the authors have investigated the basis for the well-established male bias in hepatocellular carcinoma (HCC). They report that HCCs in males contain more Treg cells than HCCs in females. Examining DEN-induced HCC in mice they also find more Tregs in male tumors than in female tumors. They attribute these differences to increased production of the Treg chemoattractant CCL2 by male HCC cells compared to female HCC cells. They also show that CCL2 expression in HCC cells, but not in other cell types, is androgen inducible. Overall, the manuscript describes intriguing and potentially interesting findings. Unfortunately, however, the current experimental evidence in support of differential CCL2 expression by HCC cells as the basis for the sex differences in HCC as well as the role of T cells in HCC immune surveillance are not totally convincing and many important controls are missing, rendering their work incomplete.\n- In all experiments the authors need to determine Treg absolute number per g or mg of tissue both in tumor and tumor adjacent areas.\n- Which HCC cell line was used in Fig. 2? Also need to show tumor growth in males vs. females.\n- Need to compare CCL2 expression by HCC cells to that in immune cells, using CCL2+ and CCL2- versions of the same HCC cell line in both male and female hosts that are either WT or CCL2KO. The antibody staining could be misleading without such controls.\n- Previous work demonstrated higher IL-6 expression by HCC associated macrophages in male mice. Please explain the basis for the discrepancy between your study and the earlier study.\n- Moreover, the authors need to examine the effect of global CCL2 and IL-6 knockouts on DEN-induced HCC in male and female mice. As mentioned above, the staining for CCl2 in Figs. 2d and 3d lack specificity controls, which ideally would be a liver from CCL2KO mice.\n- The activation of NF-kB by testosterone is unprecedented. The authors need to provide a clear mechanistic explanation for the pathway leading from AR to NF-kB. It should be noted that the authors found no effect of testosterone on NF-kB recruitment to the CCL2 control region.\n- Moreover, if the AR to NF-kB pathway is the main driver of the male bias in HCC, then females with constitutive NF-kB activation in hepatocytes should also exhibit high HCC burden that should not go up after androgen administration.\n- If the authors wish to claim that the major mechanism by which Treg promote HCC development is impairment of T cell function they need to compare the effect of TCR or TCR KO to that CD8KO, which was already shown to greatly enhance and accelerate HCC development. Besides, it is important to compare the absolute numbers of T cells that are CD8+ or CD8- and - CD8+ T cells per g of HCC tissue under the different experimental conditions.", "reviewer_2": "The manuscript by Liang and colleagues seeks to elucidate the sex-based differences in the incidence and mortality of hepatocellular carcinoma (HCC). The study demonstrates that males exhibit increased tumor infiltration of regulatory T (Treg) cells, driven by testosterone-induced upregulation of CCL2, which promotes the recruitment of CCR2+ Treg cells. These Treg cells, in turn, secrete extracellular vesicles enriched in S100A4, which impair the cytotoxic activity of γδ T cells. The manuscript is well-organized, presents compelling, novel and interesting findings of broad relevance to the field, and supports its conclusions with carefully designed experiments, including validation in human samples.\nNotwithstanding, there are some important issues to be addressed, namely:\n1) Throughout the manuscript, the authors report impacts on percentage of cells, however, the absolute numbers of cells/ mg of tumor are highly relevant, especially when quantifying migration of cells – do the changes in percentages reflect an alteration in Treg and γδ T cell absolute numbers?\n2) The authors perform the first set of experiments in a chemically-induced HCC model but then move to an orthotopic model. Are the mechanistic findings (CCL2/SV-s100a7-γδ T cells) validated in the DEN-induced model?\n3) The authors show that Tregs are more prevalent in the tumors of male mice, but not in adjacent tissue. However, it is unclear if the basal infiltration of Treg cells in healthy liver (non-tumor-bearing) differs between males and females?\n4) What drives CCR2 upregulation in Treg cells in male mice? Is it testosterone-mediated?\n5) Other populations also express CCR2 - such as monocytes, macrophages and γδ T cells themselves. How do the levels of CCR2 expression compare between the different populations? Do monocytes and macrophages also exhibit different infiltration in males and females? And upon castration? Are γδ T cells impacted by monocyte depletion?\n6) Related to the previous point – why do the authors think the differences in Treg infiltration in CCR2 KO models are restricted to the tumor? Are there increased levels of testosterone in tumors compared to tumor-adjacent liver, spleen or blood?\n7) How are the levels of s100a4 expression in Treg from CCR2cKO vs fl/fl mice? Is the impact of CCL2/CCR2 only on cell infiltration or are Treg cells qualitatively different? Does CCR2 deficiency impact the release of extracellular vesicles? Along the same lines, authors show that supernatants of Treg cells and γδ T cells cocultures contain EV loaded with S100a4. How authors know that these EV come from Treg cells? Is the secretion of EV-containing S100a4 specifically increased in the presence of CCL2 or is it the amount of S100a4 what varies?\n8) How do the authors explain the lack of suppressive activity of Tregs over CD8 T cells in in vitro killing assays? This is very surprising and difficult to interpret.\n9) Since the mechanism seems to be TCR dependent, as the pathways identified are downstream signaling of the TCR, what are the subtypes of γδ T cell (Vγ TCR usage) expanded in males vs females?\n10) The authors suggest that S100a4 expression is induced by hypoxia, however the in vivo relevance of hypoxia-driven upregulation of this molecule is unclear. Any evidence that the tumor is more hypoxic than the adjacent tissue? Along the same lines –any evidence that male mice/patients have higher hypoxic levels in HCC than females?\n11) Are the male vs female differences abrogated in TCRδKO mice lacking γδ T cells?\nMinor comments:\n1) How is the fold change of γδ T cells in male/female in IHC from HCC patients (Fig1c)?\n2) Results from Extended fig 3f/h are more impactful than fig 2e – thus the authors should maybe move the conditional KO data to main figure?\n3) Figure legends are very informative and complete. However, figure panels could be more self-explanatory by adding some more information such as plot titles.\n4) Scheme in fig 4f is difficult to understand.", "reviewer_3": "In this study, the authors aim to explain the male bias observed in hepatocellular carcinoma (HCC) incidence. Mechanistically, they demonstrate that regulatory T cells (Tregs) are enriched in male HCCs, where they promote immune suppression and facilitate tumour growth. The recruitment of Tregs to the tumour microenvironment (TME) is driven by testosterone-regulated Ccl2, produced exclusively by male cancer cells. Ablation of CCR2 in Tregs corrected the male bias in HCC incidence and was associated with the expansion of cytotoxic γδ T cells, but not CD8⁺ T cells. The authors further show that Tregs suppress γδ T cells through secretion of extracellular vesicle (Ev)-associated S100a4, which reduces γδ T cell cytotoxicity. Notably, deletion of S100a4 in Tregs normalized the sex bias in HCC incidence.\nThis is an elegant study that combines analysis of human HCC samples with sophisticated mouse models to uncover immune mechanisms underpinning sex differences in HCC. However, there are a few minor concerns that should be addressed:\nComments:\n1. The authors present the percentage of Tregs in mouse HCC models. Please also provide absolute numbers of Tregs in male versus female DEN-induced HCC.\n2. Are Tregs infiltrating male and female HCC phenotypically and transcriptionally similar? Please provide evidence.\n3. The authors should show intra-tumoral γδ T cells in Ccr2⁻/⁻ or Treg-specific Ccr2 conditional knockout mice.\n4. Is CCR2 expression (by FACS) different in male and female HCC Tregs? Please include this data.\n5. To definitively prove sex hormones regulate Ccl2, consider using an androgen receptor (AR) knockout HCC cell line and assess Treg infiltration in both male and female tumour-bearing mice.\n6. While γδ T cells expand in HCCs from castrated male mice (coinciding with Treg loss), the in vitro co-culture experiments are insufficient to rule out a direct role of testosterone as a negative regulator of γδ T cells. To clarify this, Tregs should be selectively depleted in vivo without hormonal manipulation, and γδ T cell expansion assessed.\n7. Castration at 6 weeks, does it reduce circulating testosterone levels. Provide reference or data.\n8. To rule out transcriptional induction of S100a4 in HCC infiltrating gdT cells, please provide qPCR/transcriptome data.\n9. Could hypoxia mediate Treg-induced sex bias? Since S100a4-deficient Tregs infiltrate and expand within tumours but appear functionally impaired, this warrants further investigation. Can hypoxia inhibitors revert sex-bias?\n10. Is hypoxia differentially regulated in male versus female HCC? If so, can Hif1a expression in Tregs explain the sex bias? Please show Hif1a levels in male and female HCC Tregs.\n11. Does S100a4 deletion affect immune homeostasis in other tissues?\n12. Please revise the manuscript for English language clarity. Despite the sound science, there are multiple incomplete or unclear sentences that limit readability as well as typos.", "reviewer_4": "The authors must be congratulated on a remarkable study which significantly extends our understanding of sexual dimorphism in hepatocellular carcinoma (HCC). The authors demonstrate that male predominance in HCC may be driven by an androgen-dependent Treg-γδ T cell axis. Specifically, they suggest that androgen signaling acts upon HCC cells to promote CCL2 production, which recruits CCR2+ regulatory T cells to the tumor microenvironment. These infiltrating Tregs are proposed to suppress γδ T cell anti-tumor immunity through secretion of S100a4. Notably, host deletion of CCR2 or conditional deletion of S100a4 in Foxp3+ Tregs abolishes the sexual dimorphism in HCC growth. The models employed are largely appropriate (with the exception noted below regarding Foxp3-YFP-Cre mice) and the scale of the study and depth to which the authors have mechanistically dissected the in vivo system are commendable.\nMajor Comments:\nValidation of androgen-CCL2-Treg axis: While the finding that androgens drive CCL2 expression to recruit CCR2+ Tregs to HCC is remarkable, it requires further validation. The authors should genetically delete CCL2 and AR in HCC cell lines using CRISPR or other approaches, then assess sexual dimorphism of tumor growth and Treg recruitment.\nFoxp3-YFP-Cre specificity: Foxp3-YFP-Cre mice are known to exhibit leaky expression (e.g., https://pubmed.ncbi.nlm.nih.gov/25533288/). The authors should confirm the specificity of S100a4 deletion to the Treg lineage by FACS-sorting B cells, conventional T cells, and Tregs, followed by genomic DNA qPCR analysis.\nγδ T cell specificity: It is surprising that increased Treg recruitment results in a phenotype entirely dependent on suppression of a single anti-tumor effector population—the γδ T cell. Tregs are known to exert pleiotropic suppressive functions, and the impact of this work would not be diminished if increased Treg recruitment acted through suppression of multiple lymphocyte subsets. Does depletion of γδ T cells reverse the observed sexual dimorphism of HCC growth?\nClinical relevance: Given that androgen receptor antagonists have shown limited efficacy in human HCC treatment, have the authors considered modeling this in their system? The timing of castration relative to tumor growth differs from therapeutic administration of anti-androgen therapy in progressive HCC patients. Would the authors consider testing whether anti-androgens are effective in their murine model, either before or after HCC implantation? Alternatively, a discussion of why their mouse model differs from the human clinical setting would be valuable.\nThe involvement of S100A4-containing extracellular vesicles in epigenetically reprogramming γδ T cells by acting as transcriptional corepressors is an intriguing proposition, supported by in vitro observations. However, the authors should be sufficiently circumspect about the level of evidence provided, and that the involvement in the observed dimorphic phenotype in vivo is not fully validated (this would quite reasonably be beyond the scope of the present extensive work).\nMinor Comments:\nThe manuscript contains grammatical and typographical errors throughout and would benefit from careful proofreading. Additionally, the text is verbose in places and could be more concise to improve readability.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear Reviewers,\nWe are so grateful for reviewers' insightful suggestions. We have conscientiously\naddressed the comments raised by the reviewers and have provided our detailed\nresponses below.\nReviewer #1:\nPoint 1: In all experiments the authors need to determine Treg absolute number per g\nor mg of tissue both in tumor and tumor adjacent areas.\nResponse 1: Thanks for your suggestion. In order to validate these results, the absolute\nnumbers of Treg and γδ T cell were measured by flow cytometry in this study. Notably,\nhigher level of Tregs and lower level of γδ T cells were found in male HCC compared\nwith female HCC (Fig.R1a,b).\nFigure R1 (a,b) Hepatic immune cells were verified by flow cytometry. The absolute\nnumbers of Tregs (a) and γδ T cells (b) in DEN-induced HCC (right) (n = 8).\nPoint 2: Which HCC cell line was used in Fig. 2? Also need to show tumor growth in\nmales vs. females.\nResponse 2: DEN-induced HCC (Fig.2a-f) and orthotopic HCC implantation (Fig.2g-\ni) models were used in Figure 2. Hepa1-6 cells was used to orthotopic HCC\nimplantation. We have added relevant content and tumor growth after orthotopic\nHepa1-6 implantation in males and females (Extended Data Fig.2d,e).\nPoint 3: Need to compare CCL2 expression by HCC cells to that in immune cells, using\nCCL2+ and CCL2- versions of the same HCC cell line in both male and female hosts\nthat are either WT or CCL2KO. The antibody staining could be misleading without\nsuch controls.\nResponse 3: Thanks for your suggestion. We genetically deleted Ccl2 in Hepa1-6 cells\nusing CRISPR/Cas9 followed by orthotopic Hepa1-6 implantation.\nImmunofluorescence assays demonstrated that Ccl2 is mainly secreted by HCC cells\nand is triggered by androgen. In contrast, lower Ccl2 expression was detected in HCC\nin the absence of Ccl2 or androgen in both WT and Ccl2-/- mice (Extended Data\nFig.2f,g). Deservedly, deficiency of Ccl2 showed a lower tumor burden with reduced\nTreg infiltration in HCC (Extended Data Fig.2h,i).\nPoint 4: Previous work demonstrated higher IL-6 expression by HCC associated\nmacrophages in male mice. Please explain the basis for the discrepancy between your\nstudy and the earlier study.\nResponse 4: A previous study indicates the gender disparity in liver cancer due to\nKupffer cells (KCs)-derived IL-6 production. It is indicated that estrogen inhibited the\nsecretion of IL-6 from KCs exposed to necrotic hepatocytes in DEN-induced mice1.\nThis estrogen/IL-6/STAT3 axis may contribute to gender disparity in liver cancer, and\nestrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk.\nIndeed, inflammatory cytokines (including IL-6) are essential for tumour occurrence\nand progression. Our previous study demonstrated that a large amount of IL-6 is\nsecreted from KCs after liver injury2. An earlier study elucidated the protective effect\nof estrogen and the inflammatory microenvironment in hepatocarcinogenesis. The\ncurrent study focused on the effects of androgen on Treg recruitment and γδ T cell\nfunction and elucidated the mechanism through which androgen controls the immune\nmicroenvironment. We added relevant content in Discussion section (Page 14, Line 15-\n25).\nPoint 5: Moreover, the authors need to examine the effect of global CCL2 and IL-6\nknockouts on DEN-induced HCC in male and female mice. As mentioned above, the\nstaining for CCl2 in Figs. 2d and 3d lack specificity controls, which ideally would be a\nliver from CCL2KO mice.\nResponse 5: Thanks for your suggestion. Ccl2-deficient (Ccl2-/-) mice were used to\nexplore the effect of Ccl2 in a DEN-induced HCC model. There was an increase in Ccl2\nexpression with high levels of Treg infiltration in DEN-induced HCC in male mice.\nDepletion of Ccl2 reduced tumour burden and Treg infiltration in HCC, leading to\nsimilar hepatocarcinogenesis between male and female (Fig.2d-f). Furthermore,\nMichael Karin etc. demonstrated that ablation of IL-6 abolished the gender differences\nin hepatocarcinogenesis in mice1.\nPoint 6: The activation of NF-kB by testosterone is unprecedented. The authors need\nto provide a clear mechanistic explanation for the pathway leading from AR to NF-kB.\nIt should be noted that the authors found no effect of testosterone on NF-kB recruitment\nto the CCL2 control region.\nResponse 6: Previous studies indicated that close regulatory link between AR and NF-\nκB both in human cells and in mouse models of prostate cancer3-5. Recent studies\nidentified an AR-dependent pathway acting through integrin α6/PAK1 that stimulates\nNF-κB activity independently of PI3K signaling6,7. I Indeed, testosterone treatment\nincreased the levels of integrin α6 and PAK1 in vitro, which activated the NFκB\npathway in a dose-dependent manner (Fig.3g).\nPoint 7: Moreover, if the AR to NF-kB pathway is the main driver of the male bias in\nHCC, then females with constitutive NF-kB activation in hepatocytes should also\nexhibit high HCC burden that should not go up after androgen administration.\nResponse 7: In order to interpret this issue, IKK2ca (required for activation of the\nclassical NFκB pathway) was cloned into pCDH-MSCV lentiviral vector (LV) followed\nby transfected into 293T cells to generate pCDH-MSCV- IKK2ca -GFP LV. After LV\ninfection for 48 hours and selection for 2 days, Hepa1-6 cells were cultured in regular\nmedium for orthotopic HCC implantation. As expected, constitutive activation of NFκB\nin HCC promotes Ccl2 expression and Treg infiltration, which leads to a high HCC\nburden in female mice (Extended Data Fig.3i-k).\nPoint 8: If the authors wish to claim that the major mechanism by which Treg promote\nHCC development is impairment of γδ T cell function they need to compare the effect\nof TCR or TCR KO to that CD8KO, which was already shown to greatly enhance and\naccelerate HCC development. Besides, it is important to compare the absolute numbers\nof γδ T cells that are CD8+ or CD8- and γδ- CD8+ T cells per g of HCC tissue under\nthe different experimental conditions.\nResponse 8: Thanks for your suggestion. We subsequently observed an increase in\nintratumor γδ T cell expansion in Ccr2cKO and Treg-deficient mice (Extended Data\nFig.9a,b). Furthermore, Tcrd-/- mice were used to compare sexual dimorphism in\nhepatocarcinogenesis. γδ T cell deficiency definitely accelerated HCC development.\nInterestingly, the deletion of γδ T cells increased the tumour burden in female mice and\ndecreased sex disparity (Extended Data Fig.9c). These results also demonstrate that\nTreg-γδ T cell axis contributes to sexual dimorphism in hepatocarcinogenesis. Besides,\nthe absolute numbers of HCC-derived γδ+ T cells and CD8+ T cells were measured by\nflow cytometry in this study.\nReviewer #2:\nMajor Comments:\nPoint 1: Throughout the manuscript, the authors report impacts on percentage of cells,\nhowever, the absolute numbers of cells/ mg of tumor are highly relevant, especially\nwhen quantifying migration of cells – do the changes in percentages reflect an alteration\nin Treg and γδ T cell absolute numbers?\nResponse 1: Thanks for your suggestion. In order to validate these results, the absolute\nnumbers of Treg and γδ T cell were measured by flow cytometry in this study. Notably,\nhigher level of Tregs and lower level of γδ T cells were found in male HCC compared\nwith female HCC (Fig.R1a,b).\nFigure R1 (a,b) Hepatic immune cells were verified by flow cytometry. The absolute\nnumbers of Tregs (a) and γδ T cells (b) in DEN-induced HCC (right) (n = 8).\nPoint 2: The authors perform the first set of experiments in a chemically-induced HCC\nmodel but then move to an orthotopic model. Are the mechanistic findings (CCL2/SV-\ns100a7-γδ T cells) validated in the DEN-induced model?\nResponse 2: Thanks for your suggestion. The mechanistic findings have also been\nvalidated in the DEN-induced model. As expected, enhanced Ccl2 expression and EVs-\nS100a4 secretion was found in male HCC. Impaired TCR signaling activation also was\nfound in γδ T cells from male HCC, as evidenced by the decreased phosphorylation of\nZap70 and Akt (Fig.2d-f; Extended Data Fig.6j,k).\nPoint 3: The authors show that Tregs are more prevalent in the tumors of male mice,\nbut not in adjacent tissue. However, it is unclear if the basal infiltration of Treg cells in\nhealthy liver (non-tumor-bearing) differs between males and females?\nResponse 3: We have measured the absolute number of Treg in mice healthy liver. No\nsignificant differences were detected in normal hepatic Tregs or peripheral or splenic\nTregs between males and females (Extended Data Fig.1j-l).\nPoint 4: What drives CCR2 upregulation in Treg cells in male mice? Is it testosterone-\nmediated?\nResponse 4: In order to investigate what factor drives Ccr2 upregulation in Treg cells,\nCcl2 or testosterone was used to treat Treg cells in vitro. Notably, neither Ccl2 nor\ntestosterone influences Ccr2 expression in Tregs after treatments (Fig.R2a,b). Therefore,\nwe speculated that HCC-related microenvironment disparity led to differential\nexpression of Ccr2 in Tregs.\nFigure R2 (a,b) Splenic Tregs were treated with Ccl2 (100 ng/ml) (a) or testosterone\n(20 nM) (b) for 72 hours. Representative flow cytometry plots (left) and the statistical\nquantification (right) of Ccr2+ Tregs after treatment (n = 5).\nPoint 5: Other populations also express CCR2 - such as monocytes, macrophages and\nγδ T cells themselves. How do the levels of CCR2 expression compare between the\ndifferent populations? Do monocytes and macrophages also exhibit different infiltration\nin males and females? And upon castration? Are γδ T cells impacted by monocyte\ndepletion?\nResponse 5:Indeed, other cell subsets (monocytes and macrophages) also express Ccr2.\nThe level of Ccr2 expression can be detected using cytometry. There was no difference\nin monocyte or macrophage infiltration in HCC between male and female mice or after\ncastration (Extended Data Fig.3c,d). We didn’t evaluate the impact of monocyte\ndepletion on γδ T cell function due to specific knockout mice. However, Sacchi etc.\ndemonstrated that myeloid-derived suppressor cells (MDSC) inhibited IFN-γ\nproduction and antitumor cytotoxic activity of Vδ2 T cells, implying γδ T cells may be\nimpacted by monocyte depletion8.\nPoint 6: Related to the previous point – why do the authors think the differences in\nTreg infiltration in CCR2 KO models are restricted to the tumor? Are there increased\nlevels of testosterone in tumors compared to tumor-adjacent liver, spleen or blood?\nResponse 6: In this study, HCC-derived Ccl2 secretion (triggered by androgen)\nattributes to Treg recruitment in HCC. In contrast, Ccl2 was minimally expressed in\nhealthy hepatocytes or immune cells (Extended Data Fig.3g). Therefore, the difference\nin Treg infiltration is restricted to HCC due to intratumoural Ccl2 disparity between\nmale and female (Extended Data Fig.2d,e). It is hard to compare testosterone level in\ndifferent tissue. Our results also demonstrate no obvious Treg distribution in tumor-\nadjacent liver, spleen or blood (Fig.1i; Extended Data Fig.1j-l).\nPoint 7: How are the levels of s100a4 expression in Treg from CCR2cKO vs fl/fl mice?\nIs the impact of CCL2/CCR2 only on cell infiltration or are Treg cells qualitatively\ndifferent? Does CCR2 deficiency impact the release of extracellular vesicles? Along\nthe same lines, authors show that supernatants of Treg cells and γδ T cells cocultures\ncontain EV loaded with S100a4. How authors know that these EV come from Treg cells?\nIs the secretion of EV-containing S100a4 specifically increased in the presence of CCL2\nor is it the amount of S100a4 what varies?\nResponse 7: Thanks for your questions. In order to explore the impact of Ccr2\ndeficiency on S100a4 expression and extracellular vesicles release, Tregs were isolated\nfrom Ccr2cKO and Ccr2fl/fl mice. qPCR analysis revealed that S100a4 expression\nshowed no difference in Tregs from Ccr2cKO and Ccr2fl/fl mice (Fig.R3a). These data\nindicated that Ccl2/Ccr2 axis only influences Treg infiltration rather than Treg-derived\nS100a4 production. Moreover, Ccr2 deficiency didn’t impact the release of EVs in Treg\n(Fig.R3b). The Treg-mediated transfer of S100a4 to γδ T cells was subsequently\ninvestigated using a coculture model (Fig.6b). Tregs were preinfected with S100a4-\noverexpressing LV prior to coculture with γδ T cells. The results of flow cytometry\nanalysis revealed that the fluorescent signal steadily increased in γδ T cells and\ndecreased in Tregs in a time-dependent manner following coculture (Fig.6c). S100a4,\noriginally identified only in Tregs, was detected in γδ T cells (Fig.6d; Extended Data\nFig.6d). These results demonstrate that Treg-derived EV-S100a4 can be transported into\nγδ T cells. We also demonstrated that hypoxia is a critical factor for the secretion of EV-\ncontaining S100a4.\nFigure R3 Splenic Tregs were isolated from Ccr2cKO and Ccr2fl/fl mice. a The relative\nmRNA expression of S100a4 in Tregs measured by qPCR (n = 5). b The quantification\nof EVs from medium (n = 5).\nPoint 8: How do the authors explain the lack of suppressive activity of Tregs over CD8\nT cells in in vitro killing assays? This is very surprising and difficult to interpret.\nResponse 8: This is a good question! CD8+ T cells exert powerful cytotoxicity against\ntumor cell. However, CD8+ T cells will fall into exhaustion in short time (in 4 hours).\nscRNA-seq data also support this conclusion that CD8+ T cells are prone to exhaustion\nthan γδ T cells. In vitro killing assays, Hepa1-6 cells and γδ T cells or CD8+ T cells\nwere co-cultured with Tregs for 12 hours. Therefore, the lack of suppressive activity of\nTregs over CD8+ T cells was due to exhaustedstatus within CD8+ T cells. In contrast,\nγδ T cells display a longer-lasting cytotoxicity against tumor cell without exhaustion,\npositioning them as a promising strategy for the treatment of liver cancer.\nPoint 9: Since the mechanism seems to be TCR dependent, as the pathways identified\nare downstream signaling of the TCR, what are the subtypes of γδ T cell (Vγ TCR usage)\nexpanded in males vs females?\nResponse 9: We have detected the Vγ and Vδ TCR usage by TCR-seq in human HCC\nsamples (Extended Data Table 4,5). The TCR usages of γδ T cell were comparable in\nHCC from different gender. Most γδ T cells are Vγ9/10 and Vδ1 in both males and\nfemales, indicating that TCR usage does not play a pivotal role in γδ T cell antitumour\nactivity (Extended Data Fig.7).\nPoint 10: The authors suggest that S100a4 expression is induced by hypoxia, however\nthe in vivo relevance of hypoxia-driven upregulation of this molecule is unclear. Any\nevidence that the tumor is more hypoxic than the adjacent tissue? Along the same lines\n–any evidence that male mice/patients have higher hypoxic levels in HCC than females?\nResponse 10: Previous studies identify a hypoxic microenvironment in tumor9,10.\nIndeed, compared with adjacent tissue, HCC is more hypoxic in both males and females.\nEnhanced Hif1α expression was detected in male HCC-derived Tregs compared with\nfemale HCC-derived Tregs because of increased intratumoral necrosis (Extended Data\nFig.5i). Moreover, treatment with PX-478, an inhibitor of Hif1α, restrained S100a4\nexpression, leading to an equal tumour burden (Extended Data Fig.5j,k). These results\nsuggest that hypoxia is key factor to drive S100a4 expression in Treg.\nPoint 11: Are the male vs female differences abrogated in TCRδKO mice lacking γδ T\ncells?\nResponse 11: Furthermore, Tcrd-/- mice were used to compare sexual dimorphism in\nhepatocarcinogenesis. γδ T cell deficiency definitely accelerated HCC development.\nInterestingly, deletion of γδ T cell enhanced tumor burden in female mice and decreased\ngender disparity, demonstrating that Treg-γδ T cell axis determines sexual dimorphism\nin hepatocarcinogenesis (Extended Data Fig.9c). Thank you very much!\nMinor comments:\nPoint 1: How is the fold change of γδ T cells in male/female in IHC from HCC patients\n(Fig1c)?\nResponse 1: Sorry, we didn’t detect γδ T cells in IMC assay. The expression of γδ T\ncells was detected by immunofluorescence staining (Fig.4a).\nPoint 2: Results from Extended fig 3f/h are more impactful than fig 2e – thus the\nauthors should maybe move the conditional KO data to main figure?\nResponse 2: Thank you for your suggestion! We moved these results to main figure\n(Fig.2g-i).\nPoint 3: Figure legends are very informative and complete. However, figure panels\ncould be more self-explanatory by adding some more information such as plot titles.\nResponse 3: We added plot titles and annotations to make figure intelligibility.\nPoint 4: Scheme in fig 4f is difficult to understand.\nResponse 4: We revised this scheme to make it understand easily (Fig.4f).\nReviewer #3:\nPoint 1: The authors present the percentage of Tregs in mouse HCC models. Please\nalso provide absolute numbers of Tregs in male versus female DEN-induced HCC.\nResponse 1: Thanks for your suggestion. In order to validate these results, the absolute\nnumbers of Treg and γδ T cell were measured by flow cytometry in this study. Notably,\nhigher level of Tregs and lower level of γδ T cells were found in male HCC compared\nwith female HCC (Fig.R1a,b).\nFigure R1 (a,b) Hepatic immune cells were verified by flow cytometry. The absolute\nnumbers of Tregs (a) and γδ T cells (b) in DEN-induced HCC (right) (n = 8).\nPoint 2: Are Tregs infiltrating male and female HCC phenotypically and\ntranscriptionally similar? Please provide evidence.\nResponse 2: FOXP3 transcriptional level was measured by qPCR. A higher level of\nFOXP3 mRNA was detected in human male HCC compared with female HCC\n(Extended Data Fig.1b).\nPoint 3: The authors should show intra-tumoral γδ T cells in Ccr2⁻/⁻ or Treg-specific\nCcr2 conditional knockout mice.\nResponse 3: The absolute numbers of γδ T cell was measured by flow cytometry. As\nexpected, HCC samples had a higher γδ T cell infiltration in Ccr2cKO mice (Extended\nData Fig.9a).\nPoint 4: Is CCR2 expression (by FACS) different in male and female HCC Tregs?\nPlease include this data.\nResponse 4: There is an increase in Ccr2 expression levels in Tregs from male HCC.\nIn order to investigate what factor drives Ccr2 upregulation in Treg cells, Ccl2 or\ntestosterone was used to treat Treg cells in vitro. Notably, neither Ccl2 nor testosterone\ninfluences Ccr2 expression after treatment (Fig.R2a,b). Therefore, we speculated that\nHCC-related microenvironment disparity led to differential expression of Ccr2 in Treg\ncells.\nFigure R2 (a,b) Splenic Tregs were treated with Ccl2 (100 ng/ml) (a) or testosterone\n(20 nM) (b) for 72 hours. Representative flow cytometry plots (left) and the statistical\nquantification (right) of Ccr2+ Tregs after treatment (n = 5).\nPoint 5: To definitively prove sex hormones regulate Ccl2, consider using an androgen\nreceptor (AR) knockout HCC cell line and assess Treg infiltration in both male and\nfemale tumour-bearing mice.\nResponse 5: Thanks for your suggestion. We genetically deleted AR in Hepa1-6 using\nCRISPR/Cas9 followed by orthotopic Hepa1-6 implantation. Indeed, AR-deficient\nHCC showed reduced HCC growth with lower Ccl2 expression and less Treg\ninfiltration (Fig.3h-j).\nPoint 6: While γδ T cells expand in HCCs from castrated male mice (coinciding with\nTreg loss), the in vitro co-culture experiments are insufficient to rule out a direct role\nof testosterone as a negative regulator of γδ T cells. To clarify this, Tregs should be\nselectively depleted in vivo without hormonal manipulation, and γδ T cell expansion\nassessed.\nResponse 6: Thanks for your suggestion. To explore the role of testosterone as a\nnegative regulator of γδ T cells in vivo, Foxp3DTR mice were used to selectively deplete\nTreg before orthotopic HCC implantation. We subsequently observed an increase in\nintratumor γδ T cell expansion in Treg-deficient mice (Extended Data Fig.9b).\nPoint 7: Castration at 6 weeks, does it reduce circulating testosterone levels.\nProvide reference or data.\nResponse 7: Mouse gonadectomy standard procedure to study androgen or estrogen\nfunction. This model has been proved in prior studies11,12.\nPoint 8: To rule out transcriptional induction of S100a4 in HCC infiltrating gdT cells,\nplease provide qPCR/transcriptome data.\nResponse 8: In order to rule out transcriptional induction of S100a4 in HCC infiltrating\nγδ T cells, γδ T cells were isolated from orthotopic HCC. qPCR analysis revealed that\nS100a4 expression showed no difference in γδ T cells between male and female mice\n(Extended Data Fig.5h). These data reveal the upregulation of S100a4 in HCC owing\nto Treg infiltration rather than γδ T cell-derived S100a4 production.\nPoint 9: Could hypoxia mediate Treg-induced sex bias? Since S100a4-deficient Tregs\ninfiltrate and expand within tumours but appear functionally impaired, this warrants\nfurther investigation. Can hypoxia inhibitors revert sex-bias?\nResponse 9: This is a good question! To identify whether hypoxia mediate Treg-\ninduced sex bias, PX-478, an inhibitor of Hif1α, was injected intraperitoneally twice\nper week following orthotopic Hepa1-6 implantation. Indeed, treatment with PX-478,\nan inhibitor of Hif1α, restrained S100a4 expression, leading to an equal tumour burden\n(Extended Data Fig.5j,k). These results suggest that hypoxia is key factor to drive\nS100a4 expression in Treg.\nPoint 10: Is hypoxia differentially regulated in male versus female HCC? If so, can\nHif1a expression in Tregs explain the sex bias? Please show Hif1a levels in male and\nfemale HCC Tregs.\nResponse 10: Indeed, compared with adjacent tissue, HCC is more hypoxic in both\nmales and females. Enhanced Hif1α expression was detected in male HCC-derived\nTregs compared with female HCC-derived Tregs because of increased intratumoral\nnecrosis (Extended Data Fig.5i).\nPoint 11: Does S100a4 deletion affect immune homeostasis in other tissues?\nResponse 11: Foxp3+ cell-specific S100a4 knockout (S100a4cKO)mice show normal\nthymus, spleen and lymph node. There is normal development of the T-cell lineage.\nPatterns of CD4 and CD8 T cells are apparently normal. S100a4 knockout didn’t\ninfluence the maturation, development, or liver homing of Tregs under steady-state\nconditions (Extended Data Fig.9f).\nPoint 12: Please revise the manuscript for English language clarity. Despite the sound\nscience, there are multiple incomplete or unclear sentences that limit readability as well\nas typos.\nResponse 12: The revised manuscript has been re-edited by Springer Nature English\nLanguage Editing Services (Verification code 28CA-2EC9-2181-3123-598F) to make\nit readability (Fig. R5). Thank you for your suggestion and patience！\nFigure R5 The editing certificate of Springer Nature English Language Editing\nServices.\nReviewer #4:\nMajor Comments:\nPoint 1: Validation of androgen-CCL2-Treg axis: While the finding that androgens\ndrive CCL2 expression to recruit CCR2+ Tregs to HCC is remarkable, it requires\nfurther validation. The authors should genetically delete CCL2 and AR in HCC cell\nlines using CRISPR or other approaches, then assess sexual dimorphism of tumor\ngrowth and Treg recruitment.\nResponse 1: Thanks for your suggestion. We genetically deleted AR or Ccl2 in Hepa1-\n6 using CRISPR/Cas9 followed by orthotopic Hepa1-6 implantation. Indeed,\ndeficiency of AR or Ccl2 showed a lower tumor burden with reduced Treg infiltration\nin HCC (Fig.3h-j; Extended Data Fig.2h,i).\nPoint 2: Foxp3-YFP-Cre specificity: Foxp3-YFP-Cre mice are known to exhibit leaky\nexpression (e.g., https://pubmed.ncbi.nlm.nih.gov/25533288/). The authors should\nconfirm the specificity of S100a4 deletion to the Treg lineage by FACS-sorting B cells,\nconventional T cells, and Tregs, followed by genomic DNA qPCR analysis.\nResponse 2: Indeed, Foxp3 also expresses in other cell population (including B cell).\nThus, B cells, CD8 T cells, CD4 T cells and Treg cells were isolated from spleen in\nS100a4cKO and S100a4f/f mice. qPCR analysis demonstrated that S100a4 wasn’t found\nsignificant difference in B cells, CD4 T cells, CD8 T cellsexcept Tregs (Extended Data\nFig.9e).\nPoint 3: γδ T cell specificity: It is surprising that increased Treg recruitment results in\na phenotype entirely dependent on suppression of a single anti-tumor effector\npopulation—the γδ T cell. Tregs are known to exert pleiotropic suppressive functions,\nand the impact of this work would not be diminished if increased Treg recruitment acted\nthrough suppression of multiple lymphocyte subsets. Does depletion of γδ T cells\nreverse the observed sexual dimorphism of HCC growth?\nResponse 3: Furthermore, Tcrd-/- mice were used to compare sexual dimorphism in\nhepatocarcinogenesis. γδ T cell deficiency definitely accelerated HCC development.\nInterestingly, deletion of γδ T cell enhanced tumor burden in female mice and decreased\ngender disparity, demonstrating that Treg-γδ T cell axis determines sexual dimorphism\nin hepatocarcinogenesis (Extended Data Fig.9c). Thank you very much!\nPoint 4: Clinical relevance: Given that androgen receptor antagonists have shown\nlimited efficacy in human HCC treatment, have the authors considered modeling this\nin their system? The timing of castration relative to tumor growth differs from\ntherapeutic administration of anti-androgen therapy in progressive HCC patients.\nWould the authors consider testing whether anti-androgens are effective in their murine\nmodel, either before or after HCC implantation? Alternatively, a discussion of why their\nmouse model differs from the human clinical setting would be valuable.\nResponse 4: This a good question! Although recent studies have established a sex\nhormone-dominated theory in which estrogens prevent and androgens promote HCC\ndevelopment, the differences in the tumour immune microenvironment (TME)\nunderlying sexual dimorphism in hepatocarcinogenesis remain elusive. Clinical results\nwhen antiandrogens are used to treat patients with HCC remain controversial. In our\nopinion, there are two reasons for this. First, androgen affects various functions in HCC,\nincluding cell proliferation, viral infection and the immune microenvironment13.\nTherefore, multiple factors should be considered when deciding whether to use\nandrogen ablation therapy to treat HCC. Second, androgen or androgen receptors\naccelerate oncogenesis, especially during the initial stage of HCC. Targeting androgen\nor androgen receptors before HCC implantation clearly suppresses androgen-induced\nHCC cell growth14. Furthermore, blocking androgen or androgen receptors in the early\nclinical stage mayresult in some positive improvement in response to HCC treatment,\nwhile patients with progressive disease fail to show any improvement in response to\nthe use of antiandrogens15,16. These findings reveal that cell growth is out of androgen\ncontrol in late-phase HCC. We have added relevant content in Discussion section (Page\n17, Line 9-23).\nPoint 5: The involvement of S100A4-containing extracellular vesicles in epigenetically\nreprogramming γδ T cells by acting as transcriptional corepressors is an intriguing\nproposition, supported by in vitro observations. However, the authors should be\nsufficiently circumspect about the level of evidence provided, and that the involvement\nin the observed dimorphic phenotype in vivo is not fully validated (this would quite\nreasonably be beyond the scope of the present extensive work).\nResponse 5:In vivo, enhanced EVs-S100a4 secretion was found in male DEN-induced\nHCC. Impaired TCR signaling activation also was found in γδ T cells from male HCC,\nas evidenced by the decreased phosphorylation of Zap70 and Akt (Extended Data\nFig.6j,k). These data indicate that EV-S100a4 suppresses the program of γδ T activation\nboth in vivo and in vitro.\nMinor comments:\nPoint 1: The manuscript contains grammatical and typographical errors throughout and\nwould benefit from careful proofreading. Additionally, the text is verbose in places and\ncould be more concise to improve readability.\nResponse 1: We have thoroughly and carefully examined our entire manuscript to\nensure that it caters to the requirements of the journal. The revised manuscript has been\nre-edited by Springer Nature English Language Editing Services (Verification code\n28CA-2EC9-2181-3123-598F) to make it readability (Fig. R5). Thank you for your\nsuggestion and patience！\nFigure R5 The editing certificate of Springer Nature English Language Editing\nServices.\nReviewer #5:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: Thank you very much！\nReferences\nIL-6 production. Science317, 121–124 (2007). https://doi.org/10.1126/science.1140485\nPolarization During Liver Reconstitution. Adv Sci (Weinh) 11, e2403849 (2024).\nhttps://doi.org/10.1002/advs.202403849\nand pro-inflammatory signaling remodels androgen receptor and NF-kappaB cistrome to\nreprogram the prostate cancer cell transcriptome. Nucleic Acids Res 45, 619–630 (2017).\nhttps://doi.org/10.1093/nar/gkw855\nCancer-Experimental and Computational Analyses. Cancers (Basel) 14 (2022).\nhttps://doi.org/10.3390/cancers14246164\nInduce NF-kappaB Activity via Androgen-Dependent and -Independent Pathways in Prostate\nCarcinoma Cells. Int J Mol Sci24 (2023). https://doi.org/10.3390/ijms24076245\nto promote prostate tumor cell survival via NF-kappaB and Bcl-xL Independently of PI3K\nsignaling. Cancer Res71, 2739–2749 (2011). https://doi.org/10.1158/0008-5472.CAN-10-2745\ndrive NF-kappaB-dependent resistance to apoptosis in 3D mammary acini. J Cell Sci120, 3700–\n3712 (2007). https://doi.org/10.1242/jcs.03484\nProduction and Antitumor Cytotoxic Activity of Vdelta2 T Cells. Front Immunol9, 1271 (2018).\nhttps://doi.org/10.3389/fimmu.2018.01271\nopportunities. Cancer Metastasis Rev42, 741–764 (2023). https://doi.org/10.1007/s10555-022-\n10071-1\nHepatology79, 926–940 (2024). https://doi.org/10.1097/HEP.0000000000000237\nLiver Dis41, 150–155 (2009). https://doi.org/10.1016/j.dld.2008.02.007\n200 (2024). https://doi.org/10.1038/s41586-024-07291-6\nCancer Lett555, 216037 (2023). https://doi.org/10.1016/j.canlet.2022.216037\nhepatocellular carcinoma. Gastroenterology 135, 947–955, 955 e941–945 (2008).\nhttps://doi.org/10.1053/j.gastro.2008.05.046\nacetate treatment in primary hepatocellular carcinoma is related to fall in free 5 alpha-\ndihydrotestosterone. Eur J Cancer Clin Oncol 23, 1659–1664 (1987).\nhttps://doi.org/10.1016/0277-5379(87)90446-9\nAdvanced Hepatocellular Carcinoma. Oncologist 25, e1825–e1836 (2020).\nhttps://doi.org/10.1634/theoncologist.2020-0521"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have adequately addressed my concerns and provided interesting additional data to support their claims.", "reviewer_3": "The authors have satisfactorily addressed all of this reviewer’s concerns. They have incorporated substantial new and revised data, resulting in a clear improvement in the manuscript’s quality.", "reviewer_4": "The authors have gone to significant lengths to respond to my original critiques. The majority of my concerns have now been addressed. However, the following points were not satisfactorily addressed, and further clarification would be helpful:\n1. Previous comment 2: FOXP3YFP-Cre is a notoriously leaky allele, and I had suggested that B cells, CD8 T cells, CD4 T cells, and Treg cells are facs sorted from such animals. The specificity of genetic deletion of the S100a4 locus be tested using quantitative PCR of genomic DNA. What is shown in Extended Figure 9 appears to be quantitative PCR of gene expression rather than S100a4 locus excision, and the primers used have not been specified, as far as I can tell. Additionally, the FACS sorting strategy has not been shown. Depending on the exon-exon boundary at which mRNA-QPCR is being measured, conditional deletion of alleles may have no effect on mRNA expression. What is needed is a specific Quantitative PCR reaction on genomic DNA where primers specifically designed to detect the floxed and excised alleles are used, and the quantity of the two products are compared with each other\n2. It is surprising that increased Treg recruitment results in a phenotype entirely dependent on suppression of a single anti-tumor effector population—the γδ T cell. Reasons for this surprising observation should be speculated upon in the discussion.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response letter\nDear Reviewers,\nWe are so grateful for reviewers' insightful suggestions. We have conscientiously\naddressed the comments raised by the reviewers and have provided our detailed\nresponses below.\nReviewer #4:\nPoint 1: Previous comment 2: FOXP3YFP-Cre is a notoriously leaky allele, and I had\nsuggested that B cells, CD8 T cells, CD4 T cells, and Treg cells are facs sorted from\nsuch animals. The specificity of genetic deletion of the S100a4 locus be tested using\nquantitative PCR of genomic DNA. What is shown in Extended Figure 9 appears to be\nquantitative PCR of gene expression rather than S100a4 locus excision, and the primers\nused have not been specified, as far as I can tell. Additionally, the FACS sorting strategy\nhas not been shown. Depending on the exon-exon boundary at which mRNA-QPCR is\nbeing measured, conditional deletion of alleles may have no effect on mRNA\nexpression. What is needed is a specific Quantitative PCR reaction on genomic DNA\nwhere primers specifically designed to detect the floxed and excised alleles are used,\nand the quantity of the two products are compared with each other.\nResponse 1: Thanks for your suggestion. We have checked the conditional knockout\nstrategy of S100a4 gene. The S100a4 gene has 2 transcripts. According to the structure\nof S100a4 gene, exon2 of S100a4-201 (ENSMUST00000001046.7) transcript was\nrecommended as the knockout region. Knock out the region will result in disruption of\nprotein function. Thus, CRISPR/Cas9 technology was used to modify exon2 of S100a4-\n201.The flox mice will be knocked out after mating with Foxp3YFP-Cre mice expressing\nCre recombinase, resulting in the loss of function of the S100a4 gene in Foxp3 positive\ncells.\nSplenic B cells (CD3-, B220+), CD8 T cells (CD3+, CD8+), CD4 T cells (CD3+,\nCD4+), and Tregs (CD3+, CD4+, CD25+) were FACS sorted from S100a4cKO mice.\nGenomic DNA was extracted using TIANamp Genomic DNA Kit (TIANGEN,\nShanghai, China) according to the standard protocols. The two paired primers (S100a4-\nexon2 forward, CCTGGATGTAATTGTGTCCACC, reverse, AGCTCCCTGGTCA\nGTAGCTC; S100a4-exon3 forward, CTCCTCAGATGAAGTGTTGGG, reverse,\nTGTGCGAAGAAGCCAGAGTA) were used to detect the specificity of genetic\nS100a4 (exon2) deletion by quantitative PCR. Relative level of exon2 was normalised\nagainst exon3. qPCR analysis demonstrated that the specificity of genetic S100a4\n(exon2) deletion in Tregs (Extended Data Fig.9e).\nFigure R1 (a) Gating strategy for sorting splenic B cells (CD3-, B220+), CD8 T cells\n(CD3+, CD8+), CD4 T cells (CD3+, CD4+), and Tregs (CD3+, CD4+, CD25+) from\nS100a4cKO mice. (b) qPCR assay of genetic S100a4 (exon2) levels in B cells, CD8+ T\ncells, CD4+ T cells and Tregs from S100a4f/for S100a4cKO mice.\nPoint 2: It is surprising that increased Treg recruitment results in a phenotype entirely\ndependent on suppression of a single anti-tumor effector population—the γδ T cell.\nReasons for this surprising observation should be speculated upon in the discussion.\nResponse 2: This is a good question! Several studies show the immunosuppressive\neffect of Tregs on CD8+ T cellsproliferation and cytotoxicity in HCC1-3. Indeed, CD8+\nT cells exert powerful cytotoxicity against tumor cell. However, CD8+ T cells will fall\ninto exhaustion in short time. scRNA-seq data also support this conclusion that CD8+\nT cells are prone to exhaustion than γδ T cells. Therefore, the lack of suppressive\nactivity of Tregs over CD8+ T cells in vitro killing assays was due to exhaustedstatus\nwithin CD8+ T cells. In contrast, γδ T cells exhibit increased cytotoxicity and do not\nexpress immune checkpoint markers. In this study, androgen-trigger Treg recruitment\nimpaired γδ T cell antitumor immunity, and deletion of γδ T cells increased the tumour\nburden in female mice and decreased sex disparity. These results indicate that γδ T cells\ndisplay a longer-lasting cytotoxicity against tumor cell without exhaustion,\npositioning them as a promising strategy for the treatment of liver cancer. We have added relevant\ncontent in Discussion section (Page 16, Line 1-6).\nReferences\nPromoting Regulatory T-Cells Infiltration and Immunosuppression. Adv Sci (Weinh) 11, e2310304\n(2024). https://doi.org/10.1002/advs.202310304\ninfection and related hepatocellular carcinoma. Immunology 171, 445–463 (2024).\nhttps://doi.org/10.1111/imm.13738\nsynergizes with 4-1BB agonist for liver cancer treatment. Hepatology 78, 1402–1417 (2023).\nhttps://doi.org/10.1097/HEP.0000000000000320"}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors have now addressed my remaining questions regarding the specificity of the Foxp3-Cre allele used in their experiments. With these clarifications, I now find the manuscript to be acceptable for publication. I would congratulate the authors on a remarkable piece of work", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter