CD36 promotes iron accumulation and dysfunction in CD8+ T cells via the p38-CEBPB-TfR1 axis in early-stage hepatocellular carcinoma

Yifei Qin; Fei Huo; Zhuan Feng; Jialu Hou; Yaxin Ding; Quancheng Wang; Yu Gui; Ziwei Yang; Jiali Yang; Gang Zhou; Ling Li; Jianli Jiang; Lingmin Kong; Shijie Wang; Gang Nan; Dingqiao Xu; Xiaohang Xie; Lijuan Wang; Qian He; Ruibin Yang; Peng Lin; Huijie Bian; Zhi-Nan Chen; Jiao Wu

doi:10.3350/cmh.2024.0948

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Original Article

CD36 promotes iron accumulation and dysfunction in CD8⁺ T cells via the p38-CEBPB-TfR1 axis in early-stage hepatocellular carcinoma

Yifei Qin^1,2,3,*, Fei Huo^2,3,*, Zhuan Feng^2,3,*, Jialu Hou^2,3,*, Yaxin Ding^2,3, Quancheng Wang^2,3,4, Yu Gui^2,3, Ziwei Yang^2,3, Jiali Yang^2,3, Gang Zhou^2,3, Ling Li^2,3, Jianli Jiang^2,3, Lingmin Kong^2,3, Shijie Wang^2,3, Gang Nan^2,3, Dingqiao Xu^2,3, Xiaohang Xie^2,3, Lijuan Wang^2,3, Qian He^2,3, Ruibin Yang^2,3, Peng Lin^2,3

, Huijie Bian^2,3

, Zhi-Nan Chen^2,3

, Jiao Wu^2,3

Clinical and Molecular Hepatology 2025;31(3):960-980.
Published online: March 4, 2025

DOI: https://doi.org/10.3350/cmh.2024.0948

¹Institutes of Biomedicine and Department of Cell Biology, Jinan University, Guangzhou, China

²Department of Cell Biology, National Translational Science Center for Molecular Medicine, Fourth Military Medical University, 169 Changle West Road, 710032, China

³State Key Laboratory of New Targets Discovery and Drug Development for Major Diseases, 169 Changle West Road, 710032, China

⁴Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, China

Corresponding author : Peng Lin Department of Cell Biology, National Translational Science Center for Molecular Medicine, Fourth Military Medical University, Changle western road No.169, 710032, China Tel: +86-29-84774547, E-mail: linpengxtal@126.com

Huijie Bian Department of Cell Biology, National Translational Science Center for Molecular Medicine, Fourth Military Medical University, Changle western road No.169, 710032, China Tel: +86-29-84774547, E-mail: hjbian@fmmu.edu.cn

Zhi-Nan Chen Department of Cell Biology, National Translational Science Center for Molecular Medicine, Fourth Military Medical University, Changle western road No.169, 710032, China Tel: +86-29-84774547, E-mail: znchen@fmmu.edu.cn

Jiao Wu Department of Cell Biology, National Translational Science Center for Molecular Medicine, Fourth Military Medical University, Changle western road No.169, 710032, China Tel: +86-29-84774547, E-mail: jiaowubio@hotmail.com

These authors contributed equally to this work.

Editor: Su Hyung Park, Korea Advanced Institute of Science and Technology (KAIST), Korea

• Received: November 3, 2024 • Revised: February 11, 2025 • Accepted: March 1, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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ABSTRACT
Study Highlights
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
SUPPLEMENTAL MATERIAL
Abbreviations
REFERENCES

ABSTRACT

Background/Aims
The identification of factors that lead to CD8⁺ T cell dysfunction within the tumor microenvironment (TME) holds great promise for the development of innovative immunotherapies. However, the mechanisms underlying the exhausted phenotype of CD8⁺ T cells infiltrating early-stage hepatocellular carcinoma (HCC) tumors remain unclear.
Methods
Single-cell RNA sequencing was performed using a murine HCC model. Flow cytometry and additional experimental approaches were employed to investigate the mechanisms of CD8⁺ T cell exhaustion.
Results
CD8⁺ T cells infiltrating early-stage HCC exhibited a functionally exhausted phenotype, which escalated with HCC progression. At early stages of HCC, the TME was characterized by significant iron accumulation. Moreover, tumor-infiltrating CD8⁺ T cells in murine HCC exhibited higher levels of intracellular ferrous iron compared to splenic CD8⁺ T. This excessive iron led to increased lipid peroxide levels and impaired the effector function of CD8⁺ T cells. Mechanistically, CD36 upregulated the iron uptake protein transferrin receptor 1 (TfR1) by mediating the activation of oxidized low-density lipoprotein (oxLDL)-p38-CEBPB axis. Depletion of CD36 in CD8⁺ T cells inhibited the upregulation of TfR1 and the increase of iron levels. Furthermore, constitutively activated nuclear factor erythroid 2-related factor 2 (NRF2) effectively suppressed lipid peroxidation, thereby preserving the effector functions of intratumoral CD8⁺ T cells and ultimately inhibiting tumor growth.
Conclusions
Our findings reveal a previously unidentified mechanism mediated by CD36 that regulates the progressive dysfunction of CD8⁺ T cells in early HCC TME and provide a potential novel therapeutic approach to restore T cell function.
Keywords: CD8⁺ T cell dysfunction; Lipid peroxidation; Iron accumulation; Hepatocellular carcinoma; CD36

Study Highlights

• CD36 promotes iron accumulation and lipid peroxidation in CD8⁺ T cells by activating oxLDL-p38-CEBPB-TfR1 axis, which is a previously unidentified mechanism in regulating CD8⁺ T cells dysfunction.

• Constitutively activated NRF2 effectively reduces lipid peroxidation, mitigates T cell exhaustion, and enhances the anti-tumor functions, providing a potential approach to restore T cell function.

• These findings provide valuable insights for enhancing antitumor immune responses by targeting the dysregulated interplay of iron and lipid metabolism regulated by CD36 in CD8⁺ T cells.

Graphical Abstract

INTRODUCTION

Previously, CD8⁺ T cell dysfunction was believed to arise late in tumor progression, driven by the immunosuppressive tumor microenvironment (TME) of established solid tumors [1]. However, even in the early stages of tumor development, tumor-specific CD8⁺ T cells exhibit impaired cytotoxicity [2]. Thus, identifying the factors that cause CD8⁺ T cell dysfunction in the early stages of tumor development is essential for enhancing their cytotoxic function and restoring their anti-tumor activity.

Emerging evidence revealed a close relationship between CD8⁺ T cell dysfunction and ferroptosis induced by metabolic by-products in the TME [3], Ferroptosis is a form of cell death characterized by the oxidation of phospholipid membranes via an iron-dependent mechanism [4]. Tumor-infiltrating T cells accumulate significantly more lipids compared to peripheral CD8⁺ T cells [5], and the lipid accumulation is largely dependent on CD36, a widely expressed transmembrane protein that mediates lipid uptake [6-8]. The combination of CD36 inhibitors and PD-1 antibody has been shown to rescue the anti-tumor immune responses of T cells [8]. Although CD36 expression is associated with increased intracellular iron, the underlying mechanism of iron accumulation remains to be investigated [8]. Further investigation uncovering the role of CD36 in iron metabolism of CD8⁺ T cells could offer new therapeutic strategies for cancer treatment.

In this study, we elucidate the mechanisms underlying iron accumulation in CD8⁺ T cells of early hepatocellular carcinoma (HCC) and its detrimental effects on CD8⁺ T cell function. Uptake of oxidized low-density lipoprotein (oxLDL) by CD36 activated the p38-CEBPB signaling axis in CD8⁺ T cells, which promoted transferrin receptor 1 (TfR1) expression and intracellular iron transport. Increased transportation of iron by tumor-infiltrating CD8⁺ T cells from ironoverload TME of early-stage HCC induced enhanced lipid peroxidation and dampened the anti-tumor functions of CD8⁺ T cells. We found that the antioxidant capability of nuclear factor erythroid 2-related factor 2 (NRF2) was partially ruined in CD8⁺ T cells in the environment enriched with iron and oxLDL. The constitutively active form of NRF2 greatly inhibited CD8⁺ T cells exhaustion and enhanced their antitumor effects. The intricate interplay between TfR1 and CD36 may uncover crucial pathways for developing anti-tumor strategies aimed at preserving CD8⁺ T cells function.

MATERIALS AND METHODS

Animal study

All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Translational Science Center for Molecular Medicine, Fourth Military Medical University (Approval No. 2023-NTSCMM-ID005). All mice were bred under specific pathogen-free (SPF) conditions. At the end of the study, blood samples were collected under deep anesthesia, and mice were euthanized by an intraperitoneal injection of pentobarbital.

The detailed methods are in the supplementary material.

RESULTS

CD8⁺ T cells infiltrating early-stage hepatocellular carcinoma exhibit functionally exhausted phenotype

The hydrodynamic tail-vein injection (HTVi) system has been used to establish a novel mouse model of liver cancer by injecting Sleeping Beauty (SB) plasmids and oncogenic plasmids. To more clearly demonstrate the progress and changes of murine HTVi HCC model, we conducted preliminary experiments to determine the tumor development status at each time point. At week 8, the liver showed small white tumor foci (maximum tumor diameter ≤2 mm). At week 9, the maximum tumors grew to ≤5 mm. At week 10, tumors continued to grow but remained smaller than 10 mm in diameter. However, from 11–13 weeks, numerous large nodules emerged on the liver (maximum tumor diameter ≥10 mm), while mice developed ascites and showed cachexia (Fig. 1A, Supplementary Fig. 1A–1E). H&E staining revealed the presence of necrosis within these large tumors during weeks 11 to 13 (Supplementary Fig. 1F). Therefore, we designate the 8–9 week period as earlystage HCC and the 11-13 weeks as late-stage HCC, characterized by significant increases in maximum tumor diameter and number, the emergence of larger nodules with necrotic areas, and the performance of mice (Fig. 1B). Immunohistochemical staining showed high expression of CD147 and glypian-3 (GPC3) within the tumor lesions of murine and human HCC, which are widely accepted as biomarkers for HCC (Supplementary Fig. 2A) [9,10]. Notably, early HCC lesions showed significant enrichment of cancer-associated fibroblasts (CAFs), endothelial cells, and T cells (Supplementary Fig. 2B, 2C). These immunostaining patterns aligned with observations in tumor tissues of earlystage HCC patients (Supplementary Fig. 2D). Multiplex immunofluorescence (mIHC) staining confirmed that CD8 expression within early-stage HCC tissue was markedly elevated compared to the adjacent non-tumor tissue or normal liver tissue (Fig. 1C, 1D and Supplementary Fig. 2E). Tumor-infiltrating CD8⁺ T cells from 2-week subcutaneously grown Hepa1-6 tumors had higher expression of exhaustion markers including PD-1, TIM-3 and TIGIT, and an increased frequency of PD-1⁺ TIM-3⁺ terminal exhausted T (Tex) cells compared with T cells from spleen (Fig. 1E and Supplementary Fig. 3A, 3B). Consistently, CD8⁺ T cells from 9-week tumors of HTVi HCC model showed increased levels of exhaustion markers and an increased frequency of PD-1⁺ TIM-3⁺ Tex cells (Fig. 1F and Supplementary Fig. 3C, D). These results indicate that the exhaustion of tumorinfiltrating CD8⁺ T cells show up even at the early stage of tumorigenesis.

Single-cell RNA sequencing reveals increased CD8⁺ T cells exhaustion in HCC progression

To elucidate the impact of the TME on the functional status of CD8⁺ T cells during the progression of HCC, we performed single-cell RNA sequencing (scRNA-seq) analysis on 3 samples of early-stage and 3 samples of late-stage tumors from the HTVi HCC model (Fig. 2A left, Supplementary Fig. 4A, 4B). T cells were significantly more enriched in early-stage tumors than in late-stage tumors, whereas late-stage tumors exhibited a higher proportion of HCC cells (Fig. 2A right and Supplementary Fig. 4C). The proportion of exhausted CD8⁺ T cells increased with disease progression (Fig. 2B, Supplementary Fig. 4D). Trajectory analysis performed using monocle3 uncovered a sequential developmental trajectory that began with CD8⁺ Tn cell cells and progressed over KIR⁺TXK⁺NK-like CD8⁺ T cells into CD8⁺ Tex cells (Fig. 2C). Density analysis shows that more KIR⁺TXK⁺NK-like CD8⁺ T cells were becoming exhausted as the tumor progressed (Fig. 2D). In addition, compared with early-stage tumors, CD8⁺ Tex cells in latestage tumors showed significant enrichment for PD-L1 expression and PD-1 checkpoint pathway (Fig. 2E, left). Like NK cells, effector memory KIR+ CD8⁺ T cells are cytotoxic and can produce IFNγ [11]. CD8⁺ Tex cells from late-stage tumors highly expressed multiple exhaustion markers and certain genes related with T cell exhaustion, including Pdcd1, Tox, Tox2, Cd38, Entpd1, Batf, and Rbpj (Fig. 2E, right). The exhaustion of CD8⁺ T cells is orchestrated by a sophisticated network of exhaustion-inducing transcription factors (TFs). Therefore, we evaluated the specifically expressed TFs and regulon activity in CD8⁺ Tex cells infiltrating early- and late-stage HCC tumors by SCENIC analysis. We found that early-stage CD8⁺ Tex cells showed higher activity levels of TFs associated with effector differentiation (Rel, Nfkb1, Jund), stemness (Foxo1 and Runx1) and survival (Runx2). Late-stage CD8⁺ Tex cells showed higher activity levels of TFs associated with exhaustion, including Fli1 and Irf2 (Fig. 2F). Unsupervised analysis further classified exhausted CD8⁺ T cells into 6 sub-clusters (Supplementary Fig. 5A, 5B). Clusters 0, 2, and 4 were enriched in early-stage tumors, while clusters 1, 3, and 5 were enriched in late-stage tumors (Supplementary Fig. 5C). To reveal the dynamics of transcriptional profiles of CD8⁺ Tex cells, we applied trajectory analysis. Partition-based graph abstraction (PAGA) revealed that cluster 2 emerged the earliest while cluster 4 appeared the latest (Supplementary Fig. 5D, left). Unsupervised monocle3 pseudotime analysis confirmed that the transition tendency originated from cluster 2 enriched in early-stage tumors (Supplementary Fig. 5D, right). Cluster 2 expressed high levels of the memoryrelated transcripts Tcf7 and Il7r, which are reported as essential markers for progenitor exhausted T cells (Supplementary Fig. 5B). These findings highlight the transition tendency of exhausted T cells during HTVi tumor progression. We further analyzed the scRNA-seq data of human HCC samples at early stage and the adjacent liver tissues (Supplementary Fig. 6A, 6B) [12]. We found 3 cluster subtypes, which were defined as CD4+, CD8⁺ and Treg cells (Supplementary Fig. 6C). Consistent with the results in murine HCC models, the infiltration of CD8⁺ T cells in tumor samples was found to be significantly increased compared with adjacent liver tissues (Supplementary Fig. 6D). Genes differentially expressed in tumor-infiltrating CD8⁺ T cells also showed enrichment of PD-L1 expression and PD-1 checkpoint pathway (Supplementary Fig. 6E, 6F). In addition, CD8⁺ T cells infiltrated in HCC tissue expressed higher levels of inhibitory molecules (PDCD1, HAVCR2, TIGIT, CTLA4) and exhibited a reduced capacity to secrete effector cytokines such as IFNγ (Supplementary Fig. 6G). In summary, these results reveal a correlation between HCC progression and the molecular signature associated with CD8⁺ T cells exhaustion, which contributes to HCC progression.

The accumulation of iron and lipid ROS in earlystage HCC tumors and tumor-infiltrating CD8⁺ T cells

To investigate how TME influences the metabolism and the effector functions of CD8⁺ T cells, we co-cultured CD8⁺ T cells with tumor mass obtained from the murine HTVi model. Notably, the presence of tumor mass induced the expression of immune exhaustion markers, including TIM-3, CTLA4, and TIGIT (Fig. 3A). As tumor progressed, the number of tumor-infiltrating CD8⁺ T cells decreased (Supplementary Fig. 6H). Genes upregulated in late-stage CD8⁺ Tex cells compared with early-stage CD8⁺ Tex cells showed enrichment of several processes related to ferroptosis, including unsaturated fatty acid metabolic process, iron ion binding and reactive oxygen species (ROS) metabolic process (Fig. 3B and Supplementary Fig. 6I). Consistently, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs) between CD8⁺ T cells in human HCC tumors and paracancerous tissues revealed enrichment of ferroptosis signaling pathway, suggesting a potential link between the functional loss of CD8⁺ T cells and ferroptosis in the progression of HCC (Supplementary Fig. 6F). Meanwhile, diaminobenzidine-enhanced Prussian blue iron staining revealed the accumulation of iron in murine and human HCC tumors at early stage (Fig. 3C). Moreover, tumor-infiltrating CD8⁺ T cells in early-stage murine HCC tumors had higher level of ferrous iron compared with splenic CD8⁺ T cells, indicating the import of iron from TME and the intracellular labile ferrous accumulation (Fig. 3D). In addition to elevated ferrous levels, the production of 4-hydroxynonenal (4HNE), a putative biomarker of lipid peroxidation, was enhanced in CD8⁺ T cells of early-stage murine and human HCC tumors compared with CD8⁺ T cells of liver tissues (Fig. 3E, 3F). These data confirm the accumulation of lipid peroxides and iron overload in tumor-infiltrating CD8⁺ T cells in HCC tumors, which occur at the early-stage of tumor development and are simultaneously enhanced during the progression of HCC.

Iron accumulation increases lipid peroxidation and impairs the effector function of murine and human CD8⁺ T cells

To determine the effects of iron overload in CD8⁺ T cells, we treated human and murine CD8⁺ T cells with ferric ammonium citrate (FAC). CD8⁺ T cells isolated from healthy donors’ peripheral blood mononuclear cells were activated and then exposed to FAC. Compared to control cells, FACtreated CD8⁺ T cells exhibited significantly higher intracellular labile Fe²⁺ level (Supplementary Fig. 7A) and increased lipid ROS (Supplementary Fig. 7B). Notably, lipid ROS accumulation induced by FAC was inhibited by ferrostatin-1, a lipid peroxide-trapping agent and ferroptosis inhibitor (Supplementary Fig. 7B). While the percentage of cell death in CD8⁺ T cells viability remained unchanged at 15 and 18 hours without FAC treatment, FAC-treated cells showed a significant increase of cell death (Supplementary Fig. 7C). Additionally, FAC-treated cells exhibited increased expression of immunosuppressive molecules PD-1 and TIM-3 (Supplementary Fig. 7D, 7E) and a significant decrease in the production of key cytotoxic cytokines, including TNFα, Perforin, and Granzyme B (Supplementary Fig. 7F–7H). Similar effects were observed in CD8⁺ T cells isolated from the spleens of mice (Fig. 4A–4D). Iron chelation with DFO effectively reduced intracellular iron levels of splenic CD8⁺ T cells induced by FAC, FeSO₄ or FeCl₃, as evidenced by decreased of lipid peroxidation. The addition of DFO also significantly mitigated the decrease IFNγ and the increase of T cell exhaustion marker PD-1 observed under iron overload conditions, highlighting the role of iron in driving CD8⁺ T cell dysfunction (Supplementary Fig. 8). Then we performed RNA-seq to detect if T cell metabolism and signaling pathways were influenced in CD8⁺ T cells with FAC stimulation. The differential expression analysis of genes between FAC-stimulated CD8⁺ T cells and control cells revealed that 703 genes were upregulated and 1,641 genes were downregulated (Supplementary Fig. 8A). FAC-stimulated CD8⁺ T cells showed decreased expression of memory control gene Cxcr4, stem-like gene Runx1 and Jun, a TF associated with productive T cell activation [13-15]. They also showed increased expression of terminal exhaustion genes, including PDCD1, CTLA4, Tox, and LAG3, TFs implicated in fostering T cell exhaustion, including Nr4a1 and Batf (Supplementary Fig. 8B) [16-18]. KEGG analysis showed that DEGs were enriched in pathways including cytokinecytokine receptor interaction, p38 MAPK signaling, and PD-L1 expression and PD-1 checkpoint pathway (Supplementary Fig. 8C). The Gene Ontology analysis revealed that they were involved in critical biological process terms associated with ferroptosis, such as response to oxidative stress, unsaturated fatty acid metabolic process and iron ion transport (Supplementary Fig. 8D). GSEA also confirmed the enrichment of PD-L1 expression and PD-1 checkpoint pathway in FAC-stimulated CD8⁺ T cells (Supplementary Fig. 8E). To explore the in vivo effects of iron overload, we fed mice with standard or high-iron diet (HID) for 16 weeks, starting at 4 weeks of age. Subcutaneous tumor model was then established, and tumor and spleen tissues were analyzed by flow cytometry 2 weeks later (Supplementary Fig. 10A). The results revealed that tumor-infiltrating CD8⁺ T cells from tumors in HID mice had increased intracellular labile Fe²⁺ levels and significantly elevated levels of TIGIT and PD-1 compared to CD8⁺ T cells from the standard diet group, with no significant change in TIM-3 expression (Fig. 4E, 4F). Similarly, CD8⁺ T cells in the spleens of HID mice showed increased iron and elevated expression of TIGIT and PD-1 relative to the standard diet group (Supplementary Fig. 10B–10D). In summary, these findings suggest that iron overload impairs CD8⁺ T cells effector function, potentially diminishing their ability to mount an effective anti-tumor immune response. Furthermore, we cultured CD8⁺ T cells under cystine-deprived and iron-enriched conditions to investigate the role of cysteine metabolism and iron accumulation in the context of CD36^-mediated T cell dysfunction. Our results showed that cystine deprivation and iron accumulation exerted a synergistic effect on the expression of T cell exhaustion markers, including PD-1 and TIM-3, accompanied by increased lipid peroxidation (Supplementary Fig. 11). These findings are consistent with the previous study which demonstrated that cystine deprivation triggers CD36^-mediated ferroptosis and T cell exhaustion [6].

CD36 mediates the dysregulation of iron homeostasis via the p38-CEBPB-TfR1 axis in CD8⁺ T cells

To investigate the mechanisms contributing to the functional loss of CD8⁺ T cells in HCC TME enriched with iron, we stimulated CD8⁺ T cells with FAC and examined the expression of molecules related to iron homeostasis and lipid metabolism. After 12 hours of FAC exposure, CD8⁺ T cells exhibited significantly elevated expression of TfR1, while the expression of divalent metal transporter 1 (DMT1) was decreased (Supplementary Fig. 12A). Although the expres-sion of acyl-CoA synthetase long-chain family 4 (ACSL4) was increased under short-term stimulation with FAC, the effect was diminished under long-term stimulation. Meanwhile, the expression of CD36 was maintained at a high level (Supplementary Fig. 12A). The depletion of CD36 (CD36^-/-) significantly inhibited the increase of intracellular labile Fe²⁺ level and the accumulation of lipid peroxides triggered by FAC treatment (Supplementary Fig. 12B, Fig. 5A left and median). Compared with WT CD8⁺ T cells, culture of CD36^-/- CD8⁺ T cells with addition of FAC showed notably downregulated PD-1 and TIM-3 expression (Fig. 5A, right). In line with these results, FAC exposure induced elevated expression of TfR1, FTH1 and SLC40A1 in CD8⁺ T cells, and the depletion of CD36 significantly inhibited the upregulation (Fig. 5B). Overexpression of CD36 in CD36^-/- CD8⁺ T cells significantly elevated intracellular ferrous iron levels and lipid peroxidation, as well as the expression of PD-1 and TfR1 (Fig. 5C, 5D). These results confirmed the role of CD36 in mediating the dysregulation of iron homeostasis and lipid peroxidation in CD8⁺ T cells, probably by upregulating TfR1 expression. Since the expression of TfR1 can be regulated by the IRE/IRP regulatory system, we examined the effects of FAC stimulation on IRP1 and IRP2 expression under WT and CD36 knockout conditions. We found that IRP1 levels remained unchanged, while IRP2 expression was significantly increased in FAC-stimulated WT CD8⁺ T cells compared to controls, and was markedly reduced after CD36 knockout (Supplementary Fig. 12C). Therefore, the IRE/IRP system is likely involved, but its specific role requires further investigation. Among the DEGs between FAC-stimulated CD8⁺ T and control cells that were enriched in MAPK signaling pathway, MAPK11, one of the four p38 MAPKs, was upregulated (Supplementary Fig. 12D). It has been found that oxLDL uptake via CD36 in HCC CAFs leads to p38 phosphorylation and subsequent C/EBPβ activation [19]. Additionally, utilizing ENCODE chromatin immunoprecipitation sequencing (ChIP-seq) data, we identified TFRC (encoding TfR1) as a target gene for CEBPB (GSM1519638) and CEBPD (GSM1010777). Based on the above evidence, we focused on the p38-CEBPs axis to validate its regulatory effect on the expression of TfR1. We found that the simulation with FAC in WT CD8⁺ T cells significantly elevated the protein levels of p-p38, CEBPB and CEBPD, which were enhanced by oxLDL (Supplementary Fig. 12E, 12F). These effects were greatly mitigated by CD36 knockout or the CD36 inhibitor SSO (Supplementary Fig. 12E, 12F). Meanwhile, overexpression of CD36 led to a significant increase in p-p38, CEBPB and CEBPD in CD36^-/- CD8⁺ T cells (Supplementary Fig. 12E). To investigate the role of p38 in TfR1 regulation, we treated cells with a specific p38 inhibitor SB203580. Our data showed that inhibiting p38 significantly mitigated the elevation of p-p38, CEBPB, CEBPD and TfR1, as well as exhaustion markers induced by FAC and oxLDL in WT CD8⁺ T cells, indicating that p38 activation is essential for TfR1 upregulation and CD8⁺ T cells dysfunction (Supplementary Fig. 12G–12J). Immunofluorescence staining revealed that either FAC or oxLDL alone could result in a noticeable nuclear translocation of both CEBPB and CEBPD in WT CD8⁺ T cells, which was mitigated by CD36 knockout (Supplementary Fig. 13A, 13B). Furthermore, knockdown of CEBPB resulted in a significant decrease of TfR1 expression at both the protein and mRNA levels. However, while CEBPD knockdown had minimal impact, CEBPB knockdown significantly reduced TfR1 and intracellular ferrous iron levels (Supplementary Fig. 13C–13J). This suggests that CEBPB, rather than CEBPD, is the key TF mediating TfR1 upregulation. As shown in Fig. 5E left, transfection of the CEBPB plasmid into Jurkat T cells markedly increased the luciferase activity of the TFRC promoter region. The binding of CEBPB to the TFRC promoter was promoted by either oxLDL or FAC, and the combination of oxLDL and FAC caused enhanced luciferase activity. Further, suppressing CD36 by SSO reduced TFRC promoter activity (Fig. 5E, right). ChIP analysis revealed that CEBPB binds to the TfR1 promoter region, indicating direct transcriptional regulation (Fig. 5F). These findings suggest that CD36 plays a critical role in regulating TfR1 expression and iron metabolism through the p38-CEBPB axis in the presence of high-iron microenvironment. mIHC revealed high expression of CD36 on tumor-infiltrating CD8⁺ T cells in early-stage HCC tumors of the HTVi model (8 weeks post injection) (Supplementary Fig. 14A). Compared to WT mice, CD36^-/- mice exhibited slower subcutaneous tumor growth (Supplementary Fig. 14B, 14C), lower intracellular labile ferrous levels in tumor-infiltrating CD8⁺ T cells (Supplementary Fig. 14D), reduced accumulation of iron in tumors (Supplementary Fig. 14E), reduced serum levels of the lipid peroxidation product MDA (Supplementary Fig. 14F), and decreased expression of immunosuppressive molecules, including PD-1, TIM-3, and TIGIT (Supplementary Fig. 14G). These findings indicate that CD36 deficiency rescues the CD8⁺ T cells effector functions in part through reducing the iron accumulation.

In the early and late stage scRNA-seq data, GSEA showed that processes associated with long-chain fatty acid metabolism, unsaturated fatty acid metabolism, and positive regulation of lipid metabolism were also enriched in tumorinfiltrating CD36+ CD8⁺ T cells, while negative regulation of oxidative stress-induced cell death process was enriched in CD36^-/- CD8⁺ T cells (Supplementary Fig. 14H). Tumor-infiltrating CD36+ CD8⁺ T cells also had higher activation of signaling pathways involved in PD-L1 expression and PD-1 checkpoint pathway and ROS, revealed by GSEA (Supplementary Fig. 14I). Altogether, these results confirm that CD36 not only facilitates the uptake of oxLDL, but also mediates the induction of iron accumulation through the ox-LDL-p38-CEBPB-TfR1 axis in CD8⁺ T cells in the iron-enriched TME of early-stage HCC, contributing to lipid peroxidation and subsequent CD8⁺ T cells dysfunction.

Activated NRF2 suppresses lipid peroxidation in CD8⁺ T cells induced by iron and oxLDL and restores the effector functions

The accumulation of ferrous iron in cells can lead to lipid peroxidation and ferroptosis [20]. To investigate whether the ferroptosis-resistant pathways are activated in tumor-infiltrating CD8⁺ T cells with intracellular labile ferrous accumulation, we examined the DEGs between PDCD1 (PD-1)+ HAVCR2 (TIM-3)low and PDCD1+ HAVCR2^high tumor-infiltrating CD8⁺ T cells in scRNA-seq of human early HCC [12]. PDCD1+ HAVCR2^high CD8⁺ T cells expressed lower levels of NRF2 and higher levels of KEAP1 than PDCD1+ HAVCR2^low cells (Supplementary Fig. 15A). In scRNA-seq of murine HTVi HCC model, KIR⁺TXK⁺ NK^-like CD8⁺ T cells from early-stage tumors express high levels of Nfe2l2 (encoding NRF2), while CD8⁺ Tex cells from late-stage tumors had lower expression of NRF2 than that from early-stage tumors (Fig. 6A). Furthermore, Nfe2l2^low intratumoral CD8⁺ T cells exhibited enrichment of processes associated with higher levels of lipid peroxidation and ferroptosis compared with Nfe2l2^high CD8⁺ T cells, including mitochondrial respiratory chain, fatty acid metabolic process and lipid oxidation (Fig. 6B). Nfe2l2^low cells also displayed highly enriched gene set related to ROS production (Fig. 6C).

Given these observations, we focused on the KEAP1/ NRF2 signaling pathway, which is critical for the antioxidant stress response [21]. With FAC stimulation, the expression of NRF2 and its downstream effector FSP1 significantly increased. However, the expression of other NRF2 downstream targets associated with ferroptosis defense, including NQO1, HO-1 and GPX4 declined in response to 12 hours of FAC treatment (Fig. 6D). NRF2 activity is generally inhibited through its interaction with KEAP1, which facilitates NRF2 degradation. Under conditions of oxidative stress, this interaction is disrupted, allowing NRF2 to translocate to the nucleus and activate its target genes [22,23]. Immunofluorescence analysis indicated that FAC exposure led to slightly increased nuclear localization of NRF2 in CD8⁺ T cells (Supplementary Fig. 15B). The combined stimuli of FAC and NRF2 inhibitor ML385 suppressed IFNγ production (Supplementary Fig. 15C). This result indicates that NRF2 activation is essential for the maintenance of antitumor capabilities of CD8⁺ T cells. It has been reported that ratios of NRF2 over KEAP1 point to activation of NRF2 signaling pathway and increased antioxidant capability [24]. We next investigated the effects of FAC and oxLDL on NRF2/KEAP1 ratio by treating CD8⁺ T cells with increasing concentrations of oxLDL, with or without the presence of FAC. Although oxLDL at 40 μg/mL with the addition of FAC slightly increased the NRF2/KEAP1 ratio compared with FAC-treatment, higher doses (80 and 160 μg/mL) of oxLDL reduced the ratio, likely due to the upregulation of KEAP1 (Fig. 6E). Additionally, the expression of TfR1 was induced with increasing concentrations of oxLDL, which imports iron and thus further promotes the risk of ferroptosis. Under the combined effect of these factors, the antioxidant capability of NRF2 may not be sufficient to halt the lipid peroxidation caused by the combination of FAC and oxLDL in tumor-infiltrating CD8⁺ T cells (Fig. 6E). We then stimulated CD8⁺ T cells with Ki696, a selective inhibitor of KEAP1/ NRF2 interaction. Although NRF2 expression did not exhibit significant changes, Ki696 treatment under high iron conditions resulted in a pronounced increase in NRF2 nuclear translocation (Fig. 6F and Supplementary Fig. 15D). Given that the ferroptosis-defending capability of NRF2 in CD8⁺ T cells was insufficient to counteract the oxidative damage caused by the environment enriched with iron and oxLDL, we assumed that prevention of lipid peroxidation via NRF2 activation may rescue effector functions of T cells. To test this hypothesis, we infected CD8⁺ T cells with lentivirus overexpressing a constitutively active form of NRF2 (caNRF2), which lacks the first 89 amino acids necessary for binding KEAP1 (Supplementary Fig. 16A). CD8⁺ T cells overexpressing caNRF2 demonstrated robust nuclear localization following FAC stimulation (Fig. 7A and Supplementary Fig. 16B). Furthermore, in these cells, TfR1 expression was significantly reduced, independent of the CD36^-CEBPB signaling (Supplementary Fig. 16C). Notably, CD8⁺ T cells overexpressing caNRF2 exhibited lower levels of lipid peroxidation (Fig. 7B), reduced expression of PD-1, and increased levels of key effector cytokines such as IFNγ and TNFα (Fig. 7C). These findings indicate that transfection of active NRF2 effectively reduces lipid peroxidation and mitigates T cells exhaustion.

Overexpression of activated NRF2 in intratumoral CD8⁺ T cells inhibits tumor growth

Tumor-bearing CD8-cre mice were treated with rAAVCMV-DIO-flag-caNRF2-WPRE-hGH to enable specific overexpression of caNRF2 in tumor-infiltrating CD8⁺ T cells (Supplementary Fig. 16D). Compared with control virus, caNRF2 overexpressing in CD8⁺ T cells resulted in a deceleration in tumor growth, marked decreased lipid peroxidation and increased number of tumor-infiltrating CD8⁺ T cells (Fig. 7D–7F). Additionally, both serum MDA levels and serum iron levels were reduced in mice treated with caNRF2 AAV (Supplementary Fig. 16E, 16F).

After infecting OT-1 CD8⁺ T cells with caNRF2-lentivirus, we adoptively transferred these cells into C57BL/6 mice bearing Hepa1-6-OVA tumors (Supplementary Fig. 17A). CD8⁺ T cells overexpressing caNRF2 exhibited significantly enhanced antitumor activity compared to those transferred with a control virus, resulting in marked tumor growth inhibition (Supplementary Fig. 17B) and decreased serum iron levels (Supplementary Fig. 17C). Collectively, these data indicate that both strategies to activate NRF2 in CD8⁺ T cells could effectively rescue the anti-tumor function of tumor-infiltrating CD8⁺ T cells, eventually resulting in elimination of tumor cells.

Targeting CD36 significantly enhances the efficacy of PD-1 therapy

After subcutaneous implantation of Hepa1-6 cells in WT and CD36^-/- mice, intraperitoneal injections of IgG or PD-1 antibody were administered, respectively. Compared with the WT mice, CD36^-/- mice or PD-1 antibodies alone, the combination of CD36 knockout and PD-1 antibodies significantly delayed the growth of subcutaneous tumors (Supplementary Fig. 18A, 18B), reduced the levels of lipid peroxides and iron in the serum (Supplementary Fig. 18C, 18D). Particularly, lipid peroxidation and iron accumulation were reduced in tumor-infiltrating CD8⁺ T cells (Supplementary Fig. 18E, 18F), with decreased expression of inhibitory receptors TIM-3, PD-1, TIGIT, and CTLA4 and increased expression of TNFα (Supplementary Fig. 18G– 18K), indicating enhanced antitumor function of CD8⁺ T cells. These results demonstrate that the blockade of CD36 significantly enhances the efficacy of PD-1 therapy. This finding also suggests a potential novel strategy for cancer immunotherapy, potentially offering improved outcomes for patients with tumors.

High expression of CD36 in CD8⁺ T cells is associated with worse prognosis in HCC patients

We utilized HCC tissue microarrays to conduct mIHC to detect the expression of TfR1, CD36, and CD8. The tissue microarray consists of 57 cases of T₁N₀M₀ and 29 cases of T₂N₀M₀ human HCC tissues (5 cases without stage information). We then assessed CD36 and TfR1 in tumor-infiltrating CD8⁺ T cells. We found that in HCC tissues, the proportion of CD8 and CD36 double-positive cells and the proportion of CD8 and TfR1 double-positive cells among CD8⁺ T cells were both significantly higher than those in adjacent non-cancerous tissues (Fig. 8A, 8B). The expression levels of TfR1 in tumor-infiltrating CD36+CD8⁺ T cells were significantly higher than that in CD36^-CD8⁺ T cells, validating the correlation between CD36 and TfR1 within patient-derived tumor environments (Fig. 8C, left). Given that T₁N₀M₀ generally represents early-stage HCC according to the American Joint Committee on Cancer (AJCC) tumor/ node/metastasis (TNM) classification system, we specifically analyzed the relationship between CD36 or TfR1 expression in T₁N₀M₀ HCC tissues and found the same result (Fig. 8C, right). After assessing CD36 and TfR1 in tumor-infiltrating CD8⁺ T cells in HCC tissue microarrays, we investigated the correlation between CD36 and TfR1 levels and overall survival. We categorized the CD8⁺ T cells into different groups according to the positivity percentages of CD36 or TfR1 and analyzed the prognosis of patients. Compared to the CD36^high group, the prognosis of the CD- 36low group was significantly better (Fig. 8D). Additionally, CD36 and TfR1 expression levels didn’t show strong correlations with TNM staging (Supplementary Fig. 19). These findings suggest that the expression of CD36 in CD8⁺ T cells infiltrating HCC tumors may serve as a prognostic indicator in predicting patient outcomes and highlight potential therapeutic targets for improving prognosis in HCC.

DISCUSSION

Most HCC patients are diagnosed at advanced stages, resulting in poor prognosis and limited treatment options. Furthermore, the immunosuppressive TME of HCC and the tolerogenic characteristic of the liver may work together to prevent the development of anti-tumor immunity against HCC [25]. T cell dysfunction in the TME is characterized by several key features, including the upregulation of inhibitory receptors (e.g., PD-1, CTLA-4, TIM-3, LAG-3), the presence of immunosuppressive cells (e.g., Tregs, MDSCs, TAMs), and metabolic alterations that impair T cell function [26-28]. This emphasizes the critical need to elucidate factors contributing to immune suppression in the early-stage HCC TME, which may serve as targets for therapeutic intervention. To address this need, we utilized an HTVi murine HCC model to examine the molecular pathways involved in the development of the early-stage TME in HCC. Once the HCC tumors are generated, it allows further analyses of the TME by techniques such as scRNA-seq and spatial transcriptome [29]. In mouse models representing normal liver, early-stage, and advanced HCC, we observed that while CD8⁺ T cells were abundantly present in earlystage liver tumors, most of them exhibited functionally exhausted phenotype. As the tumor progressed, these cells transitioned to a highly exhausted state, characterized by the upregulation of T cell exhaustion-related features.

These findings suggest that the immune status of CD8⁺ T cells in HCC varies significantly in stages, and highlights the importance of focusing on immunotherapeutic strategies targeting early-stage HCC. The use of plasmid-based tail vein HCC models provides an effective tool for studying tumor formation. However, the definitions of early and late stage tumor progression in these models have not been clearly established in the past. Most tumors in these models form within 6-8 weeks post-injection, but due to individual variability among mice, it is difficult to ensure that tumor growth adheres to a precise timeline. In this study, we defined the early and late stages based on tumor progression and the state of mice observed at different time points post-injection, presenting the general condition of the liver in the majority of mice at an indicated time point.

As a scavenger receptor, CD36 uptakes lipids, including arachidonic acid and oxLDL from the TME, and consequently triggers T cells ferroptosis [8]. Here our study suggests a new role of CD36 in regulating CD8⁺ T cells dysfunction. We found that CD36 depletion decreased intracellular iron levels in CD8⁺ T cells under excessive iron conditions. Mechanically, CD36 mediates the upregulation of the major iron uptake protein TfR1 by activating ox-LDL-p38-CEBPB axis. Increased transportation of iron by tumor-infiltrating CD8⁺ T cells from iron-enriched TME of early-stage HCC leads to greater lipid peroxidation and impaired anti-tumor functions of CD8⁺ T cells, and this effect escalates with tumor progression (Supplementary Fig. 17D). Therefore, blocking CD36 might be potentially beneficial in restoring anti-tumor immunity and improving the effectiveness of immune checkpoint blockade therapies. Meanwhile, it is important to note that the potential effects on normal T cells must be carefully considered to avoid compromising immune function. Strategies that selectively target CD36 in the TME while sparing normal T cells are crucial. Additionally, combining approaches targeting the CD36^-iron axis with existing immunotherapies, such as checkpoint inhibitors, could provide a synergistic effect in overcoming T cell dysfunction.

In light of the essential role that excess iron and TfR1 play in ferroptosis of CD8⁺ T cells, it is possible that blockade of ferroptosis through the depletion of iron and TfR1 preferentially contributes to the restoration of CD8⁺ T cells effector functions. Several studies have suggested that cancer cells exhibit ‘iron addiction’ traits. Cancer cells contain high iron levels, which improve malignant properties, and iron deprivation is a new strategy in cancer therapy. The iron chelator Triapine that trial, can effectively reduce the tumorigenicity of cancer cells and slow down their migration speed which had entered the clinical II [30]. It remains to be clarified whether iron deprivation therapy could rescue the effector function of tumor-infiltrating CD8⁺ T cells.

NRF2 is a central regulator of the cellular antioxidant defense system [31]. KEAP1-mediated ubiquitin-proteasomal degradation regulates NRF2 activity [32]. Moreover, KEAP1-independent mechanisms, such as the β-TrCP/Cul1 system, also play a significant role in regulating NRF2 stabilization and activation [33]. The above mechanisms may explain the observed increase in NRF2 levels despite high KEAP1 expression under the stimulation of FAC and ox-LDL. Under oxidative stress or other stress conditions, KEAP1 undergoes conformational changes that weaken its binding to NRF2. This allows NRF2 to escape degradation and translocate to the nucleus [34]. However, the upregulation of KEAP1 may still, to some extent, limit the antioxidant capacity of NRF2. We thus introduced active NRF2 into CD8⁺ T cells. The results were promising: caNRF2 effectively reduced lipid peroxidation, mitigated T cell exhaustion, and enhanced the anti-tumor functions. Additionally, NRF2 activation led to a decrease of TfR1 expression in FAC-stimulated CD8⁺ T cells independent of CD36. These findings also highlight the need to further explore the broader implications of active NRF2 in competing ferroptotic-cell death in organ damage.

There is substantial evidence supporting the use of NRF2 activators to protect cells from oxidative damage. DMF, by activating the NRF2-dependent anti-oxidant response pathway, stimulates the anti-inflammatory and cytoprotective responses [35]. Another NRF2 activator, apigenin could protect hepatocytes against oxidative condition in HFD-induced metabolic dysfunction-associated steatotic liver disease [36]. These findings suggest that NRF2 can be harnessed to enhance cellular resilience against oxidative insults, which is particularly relevant in conditions such as neurodegenerative diseases and ischemia-reperfusion injury. However, NRF2 activation can be a double-edged sword in cancer. While transient activation of NRF2 is beneficial for countering oxidative stress and preventing tumor initiation in normal cells, prolonged activation can promote tumor progression and chemoresistance. This duality raises concerns about the potential for tumor cells to exploit NRF2 activation for excessive proliferation, especially in the absence of precise targeting mechanisms (such as mutations in KEAP1) [37]. Several studies have provided evidence that preventing the permanent activity of NRF2 by its inhibitors renders cancer cells susceptible to apoptosis and enhances the efficacy of chemotherapeutics. Therefore, inhibitors of NRF2, rather than activators, have garnered more attention in cancer treatment. Although our research confirms that NRF2 activation can protect CD8⁺ T cells from dysfunction, the use of NRF2 activators in cancer treatment may also pose the risk of enhancing the survival of cancer cells. In this regard, we propose two potential solutions. On one hand, the potential of incorporating constitutively active NRF2 into CAR-T cells could be explored. This approach aims to develop CAR-T cells that are resistant to ferroptosis, thereby enhancing their anti-tumor efficacy. On the other hand, future research should investigate the development of targeted NRF2 activators that can specifically enhance NRF2 activity in T cells without promoting tumor cell proliferation. Additionally, targeting CD36 with small molecule inhibitors, function-blocking antibodies or genetic ablation has been shown to reduce tumor burden and metastasis in various cancer models [38,39]. This suggests that CD36 inhibition could enhance the efficacy of existing cancer therapies by disrupting tumor metabolism and immunosuppression. Importantly, targeting CD36 in the TME can also alleviate immunosuppression, creating a more favorable environment for immune-mediated tumor clearance. Combining CD36 inhibition with NRF2 activation could offer a synergistic approach to enhance immune cancer therapy by bolstering the antioxidant capacity of immune cells like CD8⁺ T cells and enhancing their functionality. In summary, while the clinical application of NRF2 activators remains a promising yet challenging area, we believe that targeted approaches and innovative cell engineering strategies can overcome current limitations.

FOOTNOTES

Authors’ contribution

Y.Q., F.H., Z.F. and J.H. contributed equally to this work. Y.Q. was responsible for data acquisition, analysis, investigation, and writing original draft; F.H., Z.F., J.H., G.N., D.X. and X.X. were responsible for data acquisition and analysis and provided the methods; Y.D., Q.W., Y.G., Z.Y., J.Y. and G.Z. were responsible for provision of study materials, reagents, materials, patients, laboratory samples, animals and instrumentation. L.L., J.J., L.K. and S.W. were responsible for the application of statistical, mathematical, computational, or other formal techniques to analyze data. L.W., Q.H. and R.Y. were responsible for research activity planning and execution. P.L., H.B., Z.N.C. and J.W. were responsible for the supervision, conceptualization and design of the study.

Acknowledgements

This study was supported by the National Natural Science Foundation of China 82270078 and 8202205, Top Team in Strategy of Sanqin Talent Special Support Program of Shaanxi Province, Youth Innovation Team of Shaanxi Province, the Key Research and Development Program of Shaanxi (2023-YBSF-176), State Key Laboratory of Cancer Biology (CBSKL2022ZZ31), Shaanxi Natural Science Basic Research Program (2023-JC-ZD-45), Independent Project of State Key Laboratory of Cancer Biology (CBSKL2022ZZ36).

Conflicts of Interest

The authors have no conflicts to disclose.

SUPPLEMENTAL MATERIAL

Supplementary material is available at Clinical and Molecular Hepatology website (http://www.e-cmh.org).

SUPPLEMENTARY MATERIALS AND METHODS

cmh-2024-0948-Supplementary-Materials-and-Methods.pdf

Supplementary Figure 1.

Detailed description of the HCC development process based on the HTVi system. (A) Liver images of the HTVi mice model at different weeks. Statistical data including maximum tumor diameter (B), tumor number (C), body weight (D) and liverto- body weight ratio (E) at various time points of the process. (F) H&E images of the HTVi mice model at different weeks. Scale bar: 2,000 μm.

cmh-2024-0948-Supplementary-Figure-1.pdf

Supplementary Figure 2.

Enrichment of CAFs, endothelial cells, and T cells in early HCC. (A) IHC staining of CD147 and GPC3 in Hepa1-6 tumors, HTVi tumors, human HCC tumors and the paired adjacent tissues or normal liver tissues. Scale bar: 100 μm. (B–D) Immunohistochemical staining of FAP, α-SMA, CD31, and CD3 in serial tissue sections from representative tumor and control/adjacent tissues. (C) n=3 mice per condition. (D) n=3 patients per condition. Scale bar: 100 μm. (E) mIHC staining and quantification of CD8 (red) and CD4 (green) in human HCC tumors and adjacent liver tissues. Scale bar: 50 μm.

cmh-2024-0948-Supplementary-Figure-2.pdf

Supplementary Figure 3.

Tumor-infiltrating CD8⁺ T cells in murine HCC exhibit exhaustion-related signature. (A) The expression of TIGIT and CTLA4 in splenic CD8⁺ T cells and tumor-infiltrating CD8⁺ T cells from 1-week and 2-week Hepa1-6 tumors was measured by flow cytometry. n=5 mice per condition. (B) The percentage of terminally exhausted CD8⁺ T cell subset (PD-1⁺ TIM-3⁺) in tumor-infiltrating CD8⁺ T cells from 1-week and 2-week Hepa1-6 tumors. n=5 mice per condition. (C) The expression of TIGIT and CTLA4 in splenic CD8⁺ T cells and tumor-infiltrating CD8⁺ T cells from 8-week and 9-week HTVi tumors was measured by flow cytometry. n=3 mice per condition. (D) The percentage of terminally exhausted CD8⁺ T cells subset (PD-1⁺ TIM-3⁺) in tumor-infiltrating CD8⁺ T cells from 8-week and 9-week HTVi tumors. n=3 mice per condition.

cmh-2024-0948-Supplementary-Figure-3.pdf

Supplementary Figure 4.

Single-cell RNA sequencing of murine HTVi HCC tumors at early and late stages. (A) Dot plot showing the expression of top three most highly expressed genes in each cell type. The color and size indicate the effect size. (B) UMAP visualization of subclusters of HTVi HCC model tumor tissue formation at early or late stage. (C) Box plots comparing the number of T cells and HCC cells between early and late HCC stages. Statistical analysis was performed using unpaired two-tailed t-tests. (D) Dot plot depicting expression levels of selected immune-related genes in each T cell subcluster. The color and size indicate the effect size.

cmh-2024-0948-Supplementary-Figure-4.pdf

Supplementary Figure 5.

Reclustering of CD8⁺ Tex cells and trajectory analysis. (A) UMAP showing the subpopulation of CD8⁺ Tex cells from early and late stage HCC, cells were colored by clusters. (B) Left, dot plot for expression of top5 DEGs in exhausted CD8⁺ T cell sub-clusters. Median and right visualization of Tcf7 and Havcr2 transcript levels within individually identified CD8⁺ Tex cell populations. (C) Boxplot illustrating the proportions of sub-clusters in early and late stage HTVi tumors. (D) Left, PAGA algorithm for analyzing transition tendency among different sub-clusters. Right, monocle3 pseudotime analysis showing the transition tendency among different sub-clusters.

cmh-2024-0948-Supplementary-Figure-5.pdf

Supplementary Figure 6.

Single-cell RNA sequencing of human HCC samples at BCLC A stage. (A) UMAP plots displaying the 24 clusters of cells in scRNA-seq of human HCC samples at early stages and the adjacent liver tissues. (B) UMAP plots showing the proportion of tumor and adjacent. (C) Cell-type annotation of each subpopulation from human HCC samples at early stages and the adjacent liver tissues. (D) Ratio of 4 major cell types showing in bar plots of human HCC tumor and adjacent. (E) Ratio of up and down genes from 4 major cell types showing in bar plots of human HCC tumor and adjacent. (F) Enriched KEGG pathway based on CD8⁺ T cells from HCC tumors and adjacent tissues. (G) Volcano plot of differentially expressed genes between CD8⁺ T cells from HCC tumors and adjacent tissues. (H) mIHC staining of CD8 (green) in HTVi HCC tumors at different time points. Scale bar: 50 μm. (I) Gene Set Enrichment Analysis of late-stage CD8⁺ Tex cells compared with early-stage CD8⁺ Tex cells in HTVi HCC tumors.

cmh-2024-0948-Supplementary-Figure-6.pdf

Supplementary Figure 7.

Enhanced accumulation of iron and lipid ROS in human and murine CD8⁺ T cells impairs the effector function. (A) Intracellular Fe²⁺ levels in human CD8⁺ T cells treated with FAC (5 mM) for 15 hours assessed by flow cytometry using FerroOrange probe. (B) CD8⁺ T cells treated with FAC (5 mM, 15 hours) and ferrostatin-1 (2 μM, 15 hours) (HY-100579 MCE) were analyzed for lipid peroxidation using a lipid peroxidation assay kit. Decreased PE/FITC ratio means the increase of lipid peroxidation. (C) Cell death of human CD8⁺ T cells measured after FAC treatment for 15 hours and 18 hours. Percentage of PD-1⁺ (D), TIM-3⁺ (E), TNFα⁺ (F), Perforin⁺ (G), Granzyme B⁺ (H) cells of the human CD8⁺ T cells measured after FAC treatment.

cmh-2024-0948-Supplementary-Figure-7.pdf

Supplementary Figure 8.

Iron chelation with DFO reduces intracellular iron levels, lipid peroxidation and T cell exhaustion markers of splenic CD8⁺ T cells induced by FAC (2.5 mM, 12 hours), FeSO₄ (100 μM, 12 hours) or FeCl₃ (200 μM, 12 hours). Flow cytometry analysis of the impact of three different forms of iron on CD8⁺ T cell iron levels (A, E, I), lipid ROS levels (B, F, J), IFNγ (C, G, K) and PD-1 (D, H, L) in the presence of DFO.

cmh-2024-0948-Supplementary-Figure-8.pdf

Supplementary Figure 9.

RNA sequencing of CD8⁺ T cells with FAC stimulation. (A) Number of DEGs between control and FAC-stimulated CD8⁺ T cells. (B) Volcano plot of DEGs between control and FAC-stimulated CD8⁺ T cells. KEGG (C) and Gene Ontology (GO) (D) results of DEGs. (E) Gene Set Enrichment Analysis of FAC-stimulated CD8⁺ T cells compared with control cells.

cmh-2024-0948-Supplementary-Figure-9.pdf

Supplementary Figure 10.

High-iron diet impairs the effector function of splenic and tumor-infiltrating CD8⁺ T cells. (A) Schematic diagram of high-iron diet feeding and tumor implantation. Mean fluorescence intensity (MFI) of Fe²⁺ (B) and percentages of PD-1⁺ (C), and TIGIT⁺ (D) cells in splenic CD8⁺ T cells infiltrating Hepa1-6 tumors, measured after a high-iron diet. n=3 mice per condition.

cmh-2024-0948-Supplementary-Figure-10.pdf

Supplementary Figure 11.

Cystine deprivation and iron accumulation exerted a synergistic effect on CD8⁺ T cell dysfunction. CD8⁺ T cells were treated with cystine-deprived (72 hours) and iron-enriched (FAC, 2.5 mM 12 hours) conditions and the levels of Fe²⁺ (A), lipid ROS (B), PD-1 (C), TIM-3 (D), TNFα (E) and Granzyme B (F) were measured.

cmh-2024-0948-Supplementary-Figure-11.pdf

Supplementary Figure 12.

CD36 contributes to the activation of p38-CEBPB-TfR1 signaling. (A) Protein levels of TfR1, divalent metal transporter 1 (DMT1), CD36 and acyl-CoA synthetase long-chain family 4 (ACSL4) in CD8⁺ T cells following treatment with FAC for 4 and 12 hours. (B) Efficiency of CD36 depletion in splenic CD8⁺ T cells of CD36^-/- mice, measured by qRT-PCR and western blotting. n=3 mice. (C) Protein levels of IRP1 and IRP2 in CD8⁺ T cells or CD36^-/- CD8⁺ T cells following treatment with FAC for 4 and 12 hours. (D) Heatmap showing relative expression of genes enriched in MAPK pathway. (E) phopho-p38, CEBPB and CEBPD protein expression levels in WT CD8⁺ T cells, CD36^-/- CD8⁺ T cells and CD36^-/- CD8⁺ T cells overexpressing CD36 with or without FAC stimulation (5 mM) for 12 hours. (F) WT CD8⁺ T cells were treated with FAC, the combination of oxLDL and FAC with or without the presence of CD36 inhibitor SSO (HY- 112847A MCE, 10 μM). The levels of p-p38, CEBPB, CEBPD and TfR1 were analyzed by western blotting. (G) Protein levels of p-p38, CEBPB, CEBPD, TfR1 and FTH1 in CD8⁺ T cells or CD36^-/- CD8⁺ T cells following treatment with oxLDL (80 μg/mL), FAC (2.5 mM, 12 hours), the combination of oxLDL and FAC, or the combination of oxLDL, FAC and SB (SB203580, inhibitor of p38, 1 μM, 24 hours). n=3 samples per condition. Levels of Fe²⁺ (H), PD-1, TIM-3, TIGIT (I) and IFNγ (J) were measured by flow cytometry.

cmh-2024-0948-Supplementary-Figure-12.pdf

Supplementary Figure 13.

CEBPB mediates TfR1 upregulation. Immunofluorescence staining of CEBPB (A) and CEBPD (B) in WT CD8⁺ T and CD36^-/- CD8⁺ T cells treated with either vehicle control, oxLDL (50 μg/mL), FAC (5 mM), or the combination of oxLDL and FAC. Scale bar: 5 μm. (C) WT CD8⁺ T cells were transfected with siCEBPB and the expression levels of TfR1 were analyzed by western blotting. (D) The mRNA levels of CEBPB and TfR1 in WT CD8⁺ T cells transfected with siCEBPB. (E) WT CD8⁺ T cells were transfected with siCEBPB treated with or without FAC and the expression levels of TfR1 were analyzed by western blotting. (F) Levels of Fe²⁺ were analyzed by flow cytometry. (G) WT CD8⁺ T cells were transfected with siCEBPD and the expression levels of TfR1 were analyzed by western blotting. (H) The mRNA levels of CEBPD and TfR1 in WT CD8⁺ T cells transfected with siCEBPD. (I) WT CD8⁺ T cells were transfected with siCEBPD treated with or without FAC and the expression levels of TfR1 were analyzed by western blotting. (J) Levels of Fe²⁺ were analyzed by flow cytometry.

cmh-2024-0948-Supplementary-Figure-13.pdf

Supplementary Figure 14.

CD36 deficiency rescues the CD8⁺ T cells effector functions through reducing the iron accumulation. (A) mIHC staining of CD8 and CD36 in early-stage HCC tissues and normal liver tissues. Scale bar: 50 μm. (B) Schematic diagram of tumor implantation in WT and CD36^-/- mice. (C) WT and CD36^-/- mice were implanted with Hepa1-6 cells as indicated, and tumor growth curves were shown. n=3 mice per condition. (D) Intracellular Fe²⁺ levels in tumor-infiltrating CD8⁺ T cells from WT and CD36^-/- mice. n=3 mice per condition. (E) Diaminobenzidine-enhanced Prussian blue iron staining and quantification in tumors of WT and CD36^-/- mice. Areas with positive Prussian staining were quantified. n=3 mice per condition. Scale bar: 100 μm. (F) Serum MDA levels in WT and CD36^-/- mice. n=3 mice per condition. (G) Percentages of PD-1⁺, TIM-3⁺, and TIGIT⁺ cells in tumor-infiltrating CD8⁺ T cells from WT and CD36^-/- mice were detected by flow cytometry. n=3 mice per condition. (H) Volcano plot showing GO terms enriched in CD36⁺ CD8⁺ T cells compared with CD36^- CD8⁺ T cells in the single-cell sequencing data of murine HTVi HCC. (I) GSVA showing enriched pathways in CD36⁺ CD8⁺ T cells compared with CD36^- CD8⁺ T cells.

cmh-2024-0948-Supplementary-Figure-14.pdf

Supplementary Figure 15.

The role of KEAP1 and NRF2 in CD8⁺ T cells under high iron conditions. (A) The levels of NRF2 and KEAP1 in PDCD1⁺ HAVCR2^low and PDCD1⁺ HAVCR2^high tumor-infiltrating CD8⁺ T cells in scRNA-seq of human early HCC. (B) WT CD8⁺ T cells were treated with FAC for 12 hours, and the translocation of NRF2 was detected by immunofluorescence. Scale bar: 5 μm. (C) Percentages of IFNγ-positive WT CD8⁺ T cells incubated with ML385 (10 μM, 15 hours) (HY-100523 MCE), FAC (5 mM, 12 hours) and ferrostatin- 1 (2 μM, 15 hours) as indicated. n=3 samples per condition. (D) NRF2 and KEAP1 expression in WT CD8⁺ T cells treated with Ki696 (1 μM, 24 hours), FAC (5 mM, 12 hours), or the combination of Ki696 and FAC detected by western blotting.

cmh-2024-0948-Supplementary-Figure-15.pdf

Supplementary Figure 16.

Activated NRF2 attenuates lipid peroxidation and restores the effector functions of CD8⁺ T cells. (A) Schematic representation of caNRF2. (B) Efficiency of caNRF2 overexpression in WT CD8⁺ T cells. (C) The expression of TfR1 and CEBPB in the indicated CD8⁺ T cells treated with FAC. (D) Schematic diagram of the experimental design of adoptive T cell transfer. CD8-cre mice were implanted with Hepa1-6 cells, and rAAV-DIO-flag-caNRF2 and rAAV-DIO-flag-GAPDH were intratumorally injected 7 days later. (E) Serum MDA levels in mice treated with AAVs. n=5 samples per condition. (F) Serum iron levels in mice treated with AAVs. n=3 samples per condition.

cmh-2024-0948-Supplementary-Figure-16.pdf

Supplementary Figure 17.

Adoptive transfer of CD8⁺ T cells overexpressing activated NRF2 inhibits tumor growth. (A) Schematic diagram of the experimental design of adoptive T cell transfer. (B) C57BL/6 mice were implanted with Hepa1-6-OVA cells and in vitro activated OT-1 CD8⁺ T cells transduced with lentivirus overexpressing caNRF2 were adoptively transferred 7 days later. Tumor growth was measured every 2 days. (C) Serum iron levels in tumor-bearing mice with adoptive T cell transfer. n=5 mice per condition. (D) Schematic diagram showing that CD36 induces accumulation of iron and lipid ROS through activating the p38-CEBPB-TfR1 signaling axis, causing dysfunction in CD8⁺ T cells in the early HCC tumor microenvironment.

cmh-2024-0948-Supplementary-Figure-17.pdf

Supplementary Figure 18.

Targeting CD36 significantly enhances the efficacy of PD-1 therapy. Hepa1-6 cells were subcutaneously implanted into the flanks of C57BL/6 mice. The mice were then treated with anti-IgG or anti-PD-1. Tumor images (A) and tumor weights (B) at the endpoint of the experiment. n=6 for each group. Serum MDA levels (C) and serum iron levels (D). n=3 samples per condition. Intracellular lipid ROS (E) and Fe²⁺ (F) levels in tumor-infiltrating CD8⁺ T cells. n=3 samples per condition. Percentages of PD-1⁺ (G), TIM-3⁺ (H), TIGIT⁺ (I), CTLA4⁺ (J) and TNFα⁺ (K) cells in tumor-infiltrating CD8⁺ T cells. n=3 samples per condition.

cmh-2024-0948-Supplementary-Figure-18.pdf

Supplementary Figure 19.

Comparison of the proportions of CD36⁺ CD8⁺ cells (A) and TfR1⁺ CD8⁺ cells (B) among CD8⁺ T cells between T₁N₀M₀ and T₂N₀M₀ stages in HCC patients. T₁N₀M₀, n=57. T₂N₀M₀, n=29.

cmh-2024-0948-Supplementary-Figure-19.pdf

Supplementary Figure 20.

Gating strategy to analyze human peripheral blood or mouse isolated CD8⁺ T cells for levels of Fe²⁺, lipid ROS and indicated proteins.

cmh-2024-0948-Supplementary-Figure-20.pdf

Figure 1.

CD8⁺ T cells infiltrating early-stage HCC exhibit exhaustion-related signature. (A) HCC murine models, including subcutaneous tumor model and hydrodynamic tail-vein injection (HTVi) HCC model. (B) Left, representative bioluminescence image of HTVi mice 7 weeks post-tail vein injection. Right, liver images of HTVi mice at indicated time points. (C) Multiplex immunofluorescence (mIHC) staining of CD8 (red) and CD4 (green) in 1-week Hepa1-6 tumors. The percentages of CD4⁺ T cells and CD8⁺ T cells among total cells were quantified. n=4 images from 4 mice per condition. Scale bar: 50 μm. (D) mIHC staining and quantification of CD8 (red) and CD4 (green) in 7-week HTVi tumors. n=11 images from 3 mice per condition. Scale bar: 50 μm. (E) The expression of PD-1 and TIM-3 in splenic CD8⁺ T cells and tumor-infiltrating CD8⁺ T cells from 1-week and 2-week Hepa1-6 tumors. n=5 mice per condition. (F) The expression of PD-1 and TIM-3 in splenic CD8⁺ T cells and tumor-infiltrating CD8⁺ T cells from 8-week and 9-week HTVi tumors. n=3 mice per condition.

Figure 2.

Single-cell RNA sequencing reveals CD8⁺ T cells exhaustion in the progression of HTVi HCC model. (A) Left, UMAP plot of cells from 3 samples of early-stage and 3 samples of late-stage tumors of the HTVi HCC model. Right, average proportion of each cell type between early and late HCC stages. (B) Left, UMAP plot showing the identified cell types of T cells subtypes from early and late HCC stages, annotated and colored by cluster. Right, percentages of clusters illustrating the percentage distribution of T cell subsets between early and late HCC stages. (C) Differentiation trajectory of CD8⁺ T cells by monocle3 analysis. Left, location of Tn, terminal exhausted T (Tex) and KIR⁺TXK⁺NK-like CD8⁺T cells in the differentiation trajectory of CD8⁺ T cells from single-cell RNA sequencing datasets; Tn, naive T cells; Tex, exhausted T cells. Right, pseudotime analysis for profiling trajectory of differentiating CD8⁺ T cells. (D) Density analysis of CD8⁺ T cells in the differentiation trajectory of early-stage and late-stage tumors. (E) Left, top 10 enriched pathways of differentiated genes between early- and late-stage HCC CD8⁺ Tex. Right, dot plot showing the expression of classic exhaustion markers in indicated subtypes. (F) Heatmap showing normalized activity of transcription factor regulons predicted by SCENIC in CD8⁺ T cells from early-stage and late-stage HCC tumor.

Figure 3.

Elevated iron and lipid reactive oxygen species accumulation in early-stage HCC and tumor-infiltrating CD8⁺ T cells. (A) TIM-3, TIGIT, and CTLA4 proportion of splenic CD8⁺ T cells cultured with HTVi HCC tumor mass. n=3 samples per condition. (B) Volcano plot showing Gene Ontology (GO) terms enriched in late CD8⁺ terminal exhausted T (Tex) compared with early CD8⁺ Tex. (C) Diaminobenzidine- enhanced Prussian blue iron staining and quantification of 1-week hepa1-6 tumors, early-stage HTVi tumors, human HCC tissues and the respective control tissues. Hepa1-6 tumors and, HTVi tumors n=3 tissue samples from different individuals per condition. Human HCC n=18. Scale bar: 100 μm. (D) Left, intracellular Fe²⁺ levels in CD8⁺ T cells from 1-week Hepa1-6 tumors and spleens. n=5 mice. Right, intracellular Fe²⁺ levels in CD8⁺ T cells from 8-week HTVi tumors and spleens. n=3 mice. (E) mIHC staining and quantification of CD8 and 4-hydroxynonenal (4HNE) of early HTVi tumors and normal liver tissues. n=11 images from 3 mice per condition. Scale bar: 50 μm. (F) mIHC staining and quantification of CD8 and 4HNE of human HCC tumors and paired adjacent tissues. n=11 images from 5 patients per condition. Scale bar: 50 μm. GSEA, Gene Set Enrichment Analysis.

Figure 4.

Enhanced accumulation of iron and lipid reactive oxygen species in murine CD8⁺ T cells impairs the effector function. (A) Intracellular Fe²⁺ levels in murine splenic CD8⁺ T cells treated with ferric ammonium citrate (FAC). (B) Murine splenic CD8⁺ T cells treated with FAC and ferrostatin-1 (Fer-1) were analyzed for lipid peroxidation. (C) Cell death of murine splenic CD8⁺ T cells measured after FAC treatment. Percentage of IFNγ⁺ (D, left), TNFα⁺ (D, median) and PD-1⁺ (D, right) cells of the murine CD8⁺ T cells measured after FAC treatment. (E) Intracellular Fe²⁺ levels in tumor-infiltrating CD8⁺ T cells of Hepa1-6 tumor-bearing mice fed with normal diet or high-iron diet. (F) Percentage of PD-1⁺ and TIGIT⁺ cells in tumor-infiltrating CD8⁺ T cells. HID, high-iron diet; ND, normal diet.

Figure 5.

CD36 modulates iron metabolism through the p38-CEBPB-TfR1 axis. (A) Left, intracellular Fe²⁺ levels in WT CD8⁺ T and CD36^-/- CD8⁺ T cells incubated with or without FAC for 12 hours. n=3 samples per condition. Median, WT CD8⁺ T and CD36^-/- CD8⁺ T cells treated with FAC were analyzed for lipid peroxidation. n=3 samples per condition. Right, WT CD8⁺ T and CD36^-/- CD8⁺ T cells treated with FAC were analyzed for positive proportions of PD-1 and TIM-3. n=3 samples per condition. (B) TfR1, CD36, FTH1 and SLC40A1 expression from WT CD8⁺ T and CD36^-/- CD8⁺ T cells incubated with FAC for 4 hours or 12 hours. (C) Left, intracellular Fe²⁺ levels in CD36^-/- CD8⁺ T cells and overexpression of CD36 in CD36^-/- CD8⁺ T cells incubated with or without FAC for 12 hours. n=3 samples per condition. Median, CD36^-/- CD8⁺ T cells and overexpression of CD36 in CD36^-/- CD8⁺ T cells treated with FAC were analyzed for lipid peroxidation. n=3 samples per condition. Right, CD36^-/- CD8⁺ T cells and overexpression of CD36 in CD36^-/- CD8⁺ T cells treated with FAC were analyzed for positive proportions of PD-1 and TIM-3. n=3 samples per condition. (D) CD36, TFR1, and FTH1 protein expression levels in WT CD8⁺ T cells, CD36^-/- CD8⁺ T cells and overexpression of CD36 in CD36^-/- CD8⁺ T cells incubated with FAC for 12 hours. (E) Left, TFRC promoter luciferase activity in Jurkat T cells transfected with CEBPB plasmid. n=3 samples per condition. Right, TFRC promoter luciferase activity in Jurkat T cells treated with oxLDL, FAC or the combination of oxLDL and FAC, with or without the presence of SSO. n=3 samples per condition. (F) Chromatin immunoprecipitation was utilized to detect the binding of CEBPB and TFRC at promoter regions of CEBPB in Jurkat T cells (n=3).

Figure 6.

The antioxidant capability of NRF2 is not sufficient to halt the lipid peroxidation caused by the combination of FAC and oxLDL. (A) Nfe2l2 gene expression in indicated populations in single-cell RNA sequencing data of murine hydrodynamic tail-vein injection hepatocellular carcinoma. (B) Volcano plot showing Gene Ontology (GO) terms enriched in Nfe2l2^high CD8⁺ T cells compared with Nfe2l2^low CD8⁺ T cells. (C) Kyoto Encyclopedia of Genes and Genomes pathway enriched in Nfe2l2^high CD8⁺ T cells compared with Nfe2l2^low CD8⁺ T cells. (D) The expression levels of KEAP1, NRF2, NQO1, HO-1, SLC7A11, GPX4, and FSP1 in WT CD8⁺ T cells. (E) The expression levels of CD36, KEAP1, NRF2, and TfR1 and the ratio of NRF2 to KEAP1 expression. (F) Immunofluorescence staining showing the translocation of NRF2 in CD8⁺ T cells treated with or without FAC or Ki696. Scale bar: 5 μm.

Figure 7.

Activated nuclear factor erythroid 2-related factor 2 (NRF2) attenuates lipid peroxidation and restores the effector functions of CD8⁺ T cells. (A) Immunofluorescence staining showing NRF2 localization in mock CD8⁺ T cells and caNRF2-overexpressing CD8⁺ T cells treated with FAC. Scale bar: 5 μm. Mock CD8⁺ T cells and caNRF2-overexpressing CD8⁺ T cells were treated with FAC and analyzed for the levels of lipid peroxidation (B), PD-1, IFNγ and TNFα (C). n=3 samples per condition. (D) CD8-cre mice were implanted with Hepa1-6 cells and rAAV-DIO-flag-caNRF2 and rAAV-DIO-flag-GAPDH were intratumorally injected 7 days later. n=5 mice per condition. (E) Lipid peroxidation in splenic and tumor-infiltrating CD8⁺ T cells. n=5 mice per condition. (F) Left, the percentage of CD8⁺ T cells in Hepa1-6 tumors. Right, multiplex immunofluorescence staining of CD8 and NRF2 in Hepa1-6 tumors. n=5 mice per condition. Scale bar: 50 μm.

Figure 8.

Correlations between CD36 and transferrin receptor 1 (TfR1) expression in CD8⁺ T cells and patient outcomes. (A) Representative images for multiplexed immunofluorescence staining of CD8⁺ T cells in human HCC tissue microarrays (CD8, green; CD36, red; TfR1, white). DAPI was used to highlight all nuclei. Scale bars=500 μm. (B) Comparison of the proportion of CD8 and CD36 doublepositive cells and the proportion of CD8 and TfR1 double-positive cells among CD8⁺ T cells between tumor and adjacent tissues from HCC patients. CD8, CD36 and TfR1 positivity thresholds were determined using the Inform software. Tumor n=91, adjacent n=89. (C) The expression levels of TfR1 in tumor-infiltrating CD36⁺CD8⁺ T cells and CD36-CD8⁺ T cells. (D) Kaplan–Meier overall survival curves for HCC patients.

Abbreviations

AAV

adenoassociated virus

ACSL4

acyl-CoA synthetase long-chain family 4

AJCC

American Joint Committee on Cancer

CAF

cancer-associated fibroblast

caNRF2

constitutively active form of NRF2

ChIP-seq

chromatin immunoprecipitation sequencing

DEG

differentially expressed gene

DFO

deferoxamine mesylate

FAC

ferric ammonium citrate

GPC3

glypian-3

GSEA

Gene Set Enrichment Analysis

HCC

hepatocellular carcinoma

HID

high-iron diet

HTVi

hydrodynamic tail-vein injection

ISG

interferon-stimulated genes

KEGG

Kyoto Encyclopedia of Genes and Genomes

KIR

Killer cell immunoglobulin-like receptor

MDA

malondialdehyde

mIHC

multiplex immunofluorescence

MOI

multiplicity of infection

normal diet

natural killer

NRF2

nuclear factor erythroid 2-related factor 2

oxLDL

oxidized low-density lipoprotein

PAGA

partition-based graph abstraction

PBMCs

peripheral blood mononuclear cells

ROS

reactive oxygen species

Sleeping Beauty

scRNAseq

singlecell RNA sequencing

Tex

terminal exhausted T

transcription factor

Tfh

helper T cells

TfR1

transferrin receptor 1

TME

tumor microenvironment

naïve T cells

TNM

tumor/node/metastasis

Treg

regulatory T cells

4HNE

4-hydroxynonenal

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CD36 promotes iron accumulation and dysfunction in CD8⁺ T cells via the p38-CEBPB-TfR1 axis in early-stage hepatocellular carcinoma

Clin Mol Hepatol. 2025;31(3):960-980. Published online March 4, 2025

DOI: https://doi.org/10.3350/cmh.2024.0948

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CD36 promotes iron accumulation and dysfunction in CD8⁺ T cells via the p38-CEBPB-TfR1 axis in early-stage hepatocellular carcinoma

Clin Mol Hepatol. 2025;31(3):960-980. Published online March 4, 2025

DOI: https://doi.org/10.3350/cmh.2024.0948

Figure

Development of risk scores for prognosis prediction among patients with early-stage hepatocellular carcinoma

CD36 promotes iron accumulation and dysfunction in CD8⁺ T cells via the p38-CEBPB-TfR1 axis in early-stage hepatocellular carcinoma

Figure 1. CD8+ T cells infiltrating early-stage HCC exhibit exhaustion-related signature. (A) HCC murine models, including subcutaneous tumor model and hydrodynamic tail-vein injection (HTVi) HCC model. (B) Left, representative bioluminescence image of HTVi mice 7 weeks post-tail vein injection. Right, liver images of HTVi mice at indicated time points. (C) Multiplex immunofluorescence (mIHC) staining of CD8 (red) and CD4 (green) in 1-week Hepa1-6 tumors. The percentages of CD4+ T cells and CD8+ T cells among total cells were quantified. n=4 images from 4 mice per condition. Scale bar: 50 μm. (D) mIHC staining and quantification of CD8 (red) and CD4 (green) in 7-week HTVi tumors. n=11 images from 3 mice per condition. Scale bar: 50 μm. (E) The expression of PD-1 and TIM-3 in splenic CD8+ T cells and tumor-infiltrating CD8+ T cells from 1-week and 2-week Hepa1-6 tumors. n=5 mice per condition. (F) The expression of PD-1 and TIM-3 in splenic CD8+ T cells and tumor-infiltrating CD8+ T cells from 8-week and 9-week HTVi tumors. n=3 mice per condition.

Figure 2. Single-cell RNA sequencing reveals CD8+ T cells exhaustion in the progression of HTVi HCC model. (A) Left, UMAP plot of cells from 3 samples of early-stage and 3 samples of late-stage tumors of the HTVi HCC model. Right, average proportion of each cell type between early and late HCC stages. (B) Left, UMAP plot showing the identified cell types of T cells subtypes from early and late HCC stages, annotated and colored by cluster. Right, percentages of clusters illustrating the percentage distribution of T cell subsets between early and late HCC stages. (C) Differentiation trajectory of CD8+ T cells by monocle3 analysis. Left, location of Tn, terminal exhausted T (Tex) and KIR+TXK+NK-like CD8+T cells in the differentiation trajectory of CD8+ T cells from single-cell RNA sequencing datasets; Tn, naive T cells; Tex, exhausted T cells. Right, pseudotime analysis for profiling trajectory of differentiating CD8+ T cells. (D) Density analysis of CD8+ T cells in the differentiation trajectory of early-stage and late-stage tumors. (E) Left, top 10 enriched pathways of differentiated genes between early- and late-stage HCC CD8+ Tex. Right, dot plot showing the expression of classic exhaustion markers in indicated subtypes. (F) Heatmap showing normalized activity of transcription factor regulons predicted by SCENIC in CD8+ T cells from early-stage and late-stage HCC tumor.

Figure 3. Elevated iron and lipid reactive oxygen species accumulation in early-stage HCC and tumor-infiltrating CD8+ T cells. (A) TIM-3, TIGIT, and CTLA4 proportion of splenic CD8+ T cells cultured with HTVi HCC tumor mass. n=3 samples per condition. (B) Volcano plot showing Gene Ontology (GO) terms enriched in late CD8+ terminal exhausted T (Tex) compared with early CD8+ Tex. (C) Diaminobenzidine- enhanced Prussian blue iron staining and quantification of 1-week hepa1-6 tumors, early-stage HTVi tumors, human HCC tissues and the respective control tissues. Hepa1-6 tumors and, HTVi tumors n=3 tissue samples from different individuals per condition. Human HCC n=18. Scale bar: 100 μm. (D) Left, intracellular Fe2+ levels in CD8+ T cells from 1-week Hepa1-6 tumors and spleens. n=5 mice. Right, intracellular Fe2+ levels in CD8+ T cells from 8-week HTVi tumors and spleens. n=3 mice. (E) mIHC staining and quantification of CD8 and 4-hydroxynonenal (4HNE) of early HTVi tumors and normal liver tissues. n=11 images from 3 mice per condition. Scale bar: 50 μm. (F) mIHC staining and quantification of CD8 and 4HNE of human HCC tumors and paired adjacent tissues. n=11 images from 5 patients per condition. Scale bar: 50 μm. GSEA, Gene Set Enrichment Analysis.

Figure 4. Enhanced accumulation of iron and lipid reactive oxygen species in murine CD8+ T cells impairs the effector function. (A) Intracellular Fe2+ levels in murine splenic CD8+ T cells treated with ferric ammonium citrate (FAC). (B) Murine splenic CD8+ T cells treated with FAC and ferrostatin-1 (Fer-1) were analyzed for lipid peroxidation. (C) Cell death of murine splenic CD8+ T cells measured after FAC treatment. Percentage of IFNγ+ (D, left), TNFα+ (D, median) and PD-1+ (D, right) cells of the murine CD8+ T cells measured after FAC treatment. (E) Intracellular Fe2+ levels in tumor-infiltrating CD8+ T cells of Hepa1-6 tumor-bearing mice fed with normal diet or high-iron diet. (F) Percentage of PD-1+ and TIGIT+ cells in tumor-infiltrating CD8+ T cells. HID, high-iron diet; ND, normal diet.

Figure 5. CD36 modulates iron metabolism through the p38-CEBPB-TfR1 axis. (A) Left, intracellular Fe2+ levels in WT CD8+ T and CD36-/- CD8+ T cells incubated with or without FAC for 12 hours. n=3 samples per condition. Median, WT CD8+ T and CD36-/- CD8+ T cells treated with FAC were analyzed for lipid peroxidation. n=3 samples per condition. Right, WT CD8+ T and CD36-/- CD8+ T cells treated with FAC were analyzed for positive proportions of PD-1 and TIM-3. n=3 samples per condition. (B) TfR1, CD36, FTH1 and SLC40A1 expression from WT CD8+ T and CD36-/- CD8+ T cells incubated with FAC for 4 hours or 12 hours. (C) Left, intracellular Fe2+ levels in CD36-/- CD8+ T cells and overexpression of CD36 in CD36-/- CD8+ T cells incubated with or without FAC for 12 hours. n=3 samples per condition. Median, CD36-/- CD8+ T cells and overexpression of CD36 in CD36-/- CD8+ T cells treated with FAC were analyzed for lipid peroxidation. n=3 samples per condition. Right, CD36-/- CD8+ T cells and overexpression of CD36 in CD36-/- CD8+ T cells treated with FAC were analyzed for positive proportions of PD-1 and TIM-3. n=3 samples per condition. (D) CD36, TFR1, and FTH1 protein expression levels in WT CD8+ T cells, CD36-/- CD8+ T cells and overexpression of CD36 in CD36-/- CD8+ T cells incubated with FAC for 12 hours. (E) Left, TFRC promoter luciferase activity in Jurkat T cells transfected with CEBPB plasmid. n=3 samples per condition. Right, TFRC promoter luciferase activity in Jurkat T cells treated with oxLDL, FAC or the combination of oxLDL and FAC, with or without the presence of SSO. n=3 samples per condition. (F) Chromatin immunoprecipitation was utilized to detect the binding of CEBPB and TFRC at promoter regions of CEBPB in Jurkat T cells (n=3).

Figure 6. The antioxidant capability of NRF2 is not sufficient to halt the lipid peroxidation caused by the combination of FAC and oxLDL. (A) Nfe2l2 gene expression in indicated populations in single-cell RNA sequencing data of murine hydrodynamic tail-vein injection hepatocellular carcinoma. (B) Volcano plot showing Gene Ontology (GO) terms enriched in Nfe2l2high CD8+ T cells compared with Nfe2l2low CD8+ T cells. (C) Kyoto Encyclopedia of Genes and Genomes pathway enriched in Nfe2l2high CD8+ T cells compared with Nfe2l2low CD8+ T cells. (D) The expression levels of KEAP1, NRF2, NQO1, HO-1, SLC7A11, GPX4, and FSP1 in WT CD8+ T cells. (E) The expression levels of CD36, KEAP1, NRF2, and TfR1 and the ratio of NRF2 to KEAP1 expression. (F) Immunofluorescence staining showing the translocation of NRF2 in CD8+ T cells treated with or without FAC or Ki696. Scale bar: 5 μm.

Figure 7. Activated nuclear factor erythroid 2-related factor 2 (NRF2) attenuates lipid peroxidation and restores the effector functions of CD8+ T cells. (A) Immunofluorescence staining showing NRF2 localization in mock CD8+ T cells and caNRF2-overexpressing CD8+ T cells treated with FAC. Scale bar: 5 μm. Mock CD8+ T cells and caNRF2-overexpressing CD8+ T cells were treated with FAC and analyzed for the levels of lipid peroxidation (B), PD-1, IFNγ and TNFα (C). n=3 samples per condition. (D) CD8-cre mice were implanted with Hepa1-6 cells and rAAV-DIO-flag-caNRF2 and rAAV-DIO-flag-GAPDH were intratumorally injected 7 days later. n=5 mice per condition. (E) Lipid peroxidation in splenic and tumor-infiltrating CD8+ T cells. n=5 mice per condition. (F) Left, the percentage of CD8+ T cells in Hepa1-6 tumors. Right, multiplex immunofluorescence staining of CD8 and NRF2 in Hepa1-6 tumors. n=5 mice per condition. Scale bar: 50 μm.

Figure 8. Correlations between CD36 and transferrin receptor 1 (TfR1) expression in CD8+ T cells and patient outcomes. (A) Representative images for multiplexed immunofluorescence staining of CD8+ T cells in human HCC tissue microarrays (CD8, green; CD36, red; TfR1, white). DAPI was used to highlight all nuclei. Scale bars=500 μm. (B) Comparison of the proportion of CD8 and CD36 doublepositive cells and the proportion of CD8 and TfR1 double-positive cells among CD8+ T cells between tumor and adjacent tissues from HCC patients. CD8, CD36 and TfR1 positivity thresholds were determined using the Inform software. Tumor n=91, adjacent n=89. (C) The expression levels of TfR1 in tumor-infiltrating CD36+CD8+ T cells and CD36-CD8+ T cells. (D) Kaplan–Meier overall survival curves for HCC patients.

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CD36 promotes iron accumulation and dysfunction in CD8⁺ T cells via the p38-CEBPB-TfR1 axis in early-stage hepatocellular carcinoma

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CD36 promotes iron accumulation and dysfunction in CD8+ T cells via the p38-CEBPB-TfR1 axis in early-stage hepatocellular carcinoma

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CD36 promotes iron accumulation and dysfunction in CD8⁺ T cells via the p38-CEBPB-TfR1 axis in early-stage hepatocellular carcinoma