Fatty acid β-oxidation enhances immune regulatory function of double-negative T cells through pSTAT4–OX40 signaling pathway

Abstract

Background:

Double-negative T (DNT) cells (CD3⁺CD4⁻CD8⁻NK1.1⁻) demonstrate immunoregulatory functions in maintaining hepatic immune homeostasis. This study investigates how energy metabolism impacts DNT cell survival and immunoregulatory functions, exploring potential therapeutic applications for autoimmune hepatitis.

Methods:

We compared DNT cells with conventional CD4⁺ T cells through lipidomic analysis, fatty acid β-oxidation (FAO) assessment, and single-cell RNA sequencing. Cells were treated with fatty acids (oleic acid and palmitic acid) and the FAO inhibitor Etomoxir (Eto). We evaluated cell survival, proliferation, and function through flow cytometry and reverse transcription–quantitative polymerase chain reaction. Transcriptome sequencing identified key regulatory molecules. The therapeutic potential was assessed in a Concanavalin A (ConA)-induced autoimmune hepatitis mouse model receiving DNT and DNT-Eto cell treatments.

Results:

DNT cells showed higher fatty acid content, FAO levels, and related gene expression compared with CD4⁺ T cells. Fatty acid supplementation enhanced DNT cell proliferation and immunoregulatory function, whereas FAO inhibition significantly impaired cell survival and function. Transcriptome analysis identified OX40 as a key regulator of DNT cell survival and function, regulated by FAO-activated pSTAT4. In the ConA-induced murine model, therapeutic administration of DNT cells significantly ameliorated the severity of autoimmune hepatitis compared with the ConA-treated control group. Meanwhile, DNT-Eto–treated groups showed more severe liver injury and elevated liver enzymes compared with DNT-treated groups. In vivo analyses revealed that DNT cells exhibited superior survival, function, and CD4⁺ T cell inhibition compared with Eto-treated or OX40 KO-DNT cells.

Conclusions:

FAO regulates DNT cell survival and immunoregulatory function through the pSTAT4–OX40 pathway, enhancing their protective effect against autoimmune hepatitis.

INTRODUCTION

TCRαβ⁺ double-negative T (DNT) cells, characterized as CD3⁺TCRαβ⁺CD4⁻CD8⁻CD56⁻ (human)/NK1.1⁻ (mouse) T lymphocytes, represent a unique subset of T cells that possess immunoregulatory functions within the immune system.¹ Accumulating evidence has demonstrated the immunoregulatory function of DNT cells in controlling transplant rejection,² graft-versus-host disease,³ asthma,⁴ psoriasis, and type 1 diabetes.⁵,⁶ DNT cells exert cytotoxic effects through perforin–granzyme and Fas–FasL pathways, with IFN-γ further enhancing FasL-mediated cytotoxicity.⁷,⁸ Notably, the TNF receptor superfamily member OX40 has been implicated in DNT cell proliferation, survival, and regulatory function, while interleukin-10 (IL-10) acts as a negative regulator, inhibiting DNT cell expansion and activation.⁹,¹⁰ This intricate interplay of molecular mechanisms collectively governs the functional capacity of DNT cells, highlighting their potential as therapeutic targets in various immune-mediated disorders, including autoimmune hepatitis.

Energy metabolism plays a crucial role in regulating immune cell function.¹¹,¹² Different immune cell subsets exhibit distinct metabolic profiles to support their specialized functions. For instance, effector T cells primarily rely on glycolysis for rapid proliferation and cytokine production, while regulatory T cells (Tregs) predominantly utilize fatty acid β-oxidation and oxidative phosphorylation. Metabolic reprogramming can significantly influence T cell fate and function. Glycolysis has been shown to regulate T cell differentiation and facilitate rapid cytokine production, proliferation, and activation.¹³,¹⁴ Fatty acid metabolism is critical for inducing Th17 differentiation, as well as promoting Treg development and maintenance.¹⁵,¹⁶,¹⁷,¹⁸ However, the role of energy metabolism in regulating the survival and function of DNT cells remains poorly understood. Given the importance of metabolic pathways in shaping T cell responses, elucidating the metabolic requirements of DNT cells could provide valuable insights into their immunoregulatory capabilities.

In this study, we investigate the energy metabolism of DNT cells. Our findings reveal that DNT cells are characterized by robust fatty acid β-oxidation (FAO), distinguishing them from other immune cell populations. Furthermore, we elucidate the critical role of OX40 in modulating FAO within DNT cells, which influences their survival and function. By unraveling the relationship between fatty acid metabolism and the immunoregulatory capabilities of DNT cells, our research opens promising avenues for therapeutic strategies in autoimmune hepatitis and other autoimmune disorders.

METHODS

Animals

Male C57BL/6, CD45.1 congenic C57BL/6, and OX40-knockout mice of 8 weeks were primarily used in this study. The mice were obtained from Beijing Vital River Laboratory (China) and The Jackson Laboratory (USA). All animals were housed in specific pathogen-free (SPF) conditions at the Experimental Animal Center of Capital Medical University and the Experimental Animal Center of Beijing Chao-Yang Hospital. The mice were maintained in a temperature-controlled environment with a 12-hour light/dark cycle and given free access to food and water. All animal experiments were conducted in accordance with the guidelines for animal care and use and were approved by the Institutional Animal Care and Ethics Committee of Capital Medical University (IACUC approval number: AEEI-2023-282).

Conversion of DNT cells in vitro

The conversion of DNT cells from CD4⁺ T cells was performed as previously described.¹⁹ CD4⁺CD25⁻ T cells were isolated from the spleens and lymph nodes of C57BL/6 mice using magnetic separation. These purified CD4⁺CD25⁻ T cells were then co-cultured with mature dendritic cells (mDCs) derived from C57BL/6 mice in the presence of recombinant mouse IL-2 (50 ng/mL; Peprotech). After 7 days of co-culture, DNT cells were isolated using magnetic separation. The purity of DNT cells used in this manuscript consistently exceeded 95% (Supplemental Figure S1, http://links.lww.com/HC9/C73).

Cell treatment

DNT cells were given different stimuli to explore their mechanisms of action. The fatty acids palmitic acid (PA, Sigma-Aldrich) and oleic acid (OA, Sigma-Aldrich), the FAO inhibitor etomoxir (Eto, Sigma-Aldrich), and the STAT4 inhibitor (R)-lisofylline (MCE) were added to the culture medium. Cells were analyzed or used for subsequent experiments after 24 hours of stimulation.

Concanavalin A–induced immune-mediated liver injury model

The animal model of Concanavalin A (ConA)-induced immune-mediated autoimmune hepatitis was performed. CD45.1 congenic C57BL/6 mice were randomly divided into 4 groups (n=5 per group): PBS control group, ConA group, DNT treatment group, and DNT-Eto treatment group. Autoimmune hepatitis was induced by tail vein injection of ConA (15 mg/kg body weight). DNT cells (3×10⁶ cells/mouse) were adoptively transferred via tail vein injection immediately following ConA administration. Control group mice received an equal volume of PBS. Mice were sacrificed 48 hours after ConA administration, and samples were collected. Serum ALT and AST activities were measured using assay kits (Nanjing Jiancheng Company) according to the manufacturer’s instructions. Liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm thickness, and stained with hematoxylin and eosin (H&E). Areas of liver necrosis were assessed and quantified by light microscopy. The evaluator was blinded to the treatment groups.

Flow cytometric analysis of hepatic immune cells

Hepatic immune cells were isolated from fresh liver tissue following standard procedures of mechanical dissociation and enzymatic digestion. The tissue was filtered through a 70 μm cell strainer and centrifuged to collect cells. Mononuclear cells were isolated by density gradient centrifugation using lymphocyte separation medium (Percoll, GE Healthcare). Isolated cells were suspended and stained with fluorochrome-conjugated antibodies. For intracellular cytokine staining, peripheral blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A (Sigma-Aldrich) for 4 hours before surface and intracellular staining. Flow cytometric analysis was performed using a FACSAria II flow cytometer (BD), and data were analyzed using FlowJo v10 software (BD).

In vitro suppression assays

DNT cells were pretreated with free fatty acids or Eto before co-culture with CD4⁺ T cells. CD45.1⁺ CD4⁺ T cells (5×10⁴/well) were pre-stained with carboxyfluorescein succinimidyl ester (CFSE), and co-cultured with DNT cells (DNT:CD4⁺ T cells=4:1) in the presence of mDCs for 5–7 days. The suppressive effect of DNT cells on CD4⁺ T cells was evaluated by CFSE fluorescence reduction and Annexin V staining of CD45.1⁺ CD4⁺ T cells using flow cytometry.

Transcriptome sequencing analysis

Total RNA was extracted from CD4⁺ T cells and DNT cells (with or without 24-h Eto treatment) using the QIAGEN RNeasy Mini Kit (Qiagen). Complementary DNA (cDNA) libraries were synthesized and sequenced on an Illumina HiSeq 2500 platform (Illumina). Differential gene expression analysis was conducted using the edgeR package in R, with a significance threshold set at padj<0.05 and |log2(fold change)| ≥1. Differentially expressed genes (DEGs) were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis through DAVID Bioinformatics Resources (https://david.ncifcrf.gov). Raw sequencing data are available in NCBI’s Gene Expression Omnibus (accession number: GSE279790).

Single-cell RNA sequencing analysis

Single-cell sequencing analysis was conducted using our previously published data.²⁰,²¹ The data are available in the Gene Expression Omnibus (GEO) database under accession numbers GSE129030 and GSE246302.

Metabolomics analysis

CD4⁺ T cells and DNT cells were isolated from C57BL/6. The cells (1×10⁶) were lysed, and the intracellular free fatty acids (FFAs) were extracted using a modified Bligh and Dyer method. The extracted FFAs were identified and quantified using a Q300 Metabolite Array Kit (Metabo-Profile).

Fatty acid β-oxidation detection

A Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies, USA) was used to assess the FAO level in CD4⁺ T cells and DNT cells. Cells (2 × 10⁵ per well) were cultured in Seahorse XF Base Medium supplemented with 2 mM L-glutamine, 10 mM glucose, and 1 mM sodium pyruvate. After incubation at 37 °C for 1 hour in a non-CO₂ incubator, vehicle (DMSO, 0.1%) or etomoxir (100 μM) was applied for 20 minutes, followed by palmitic acid (PA, 200 μM complexed with BSA) addition where indicated.

Plates were equilibrated in the Seahorse XF96 Analyzer for 30 minutes. The mitochondrial stress test was performed through sequential administration of 1 μM oligomycin, 1.5 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and a mixture of 0.5 μM rotenone and 0.5 μM antimycin A. Each compound plays a specific role in assessing mitochondrial function: oligomycin inhibits ATP synthase (complex V), allowing measurement of ATP-linked respiration and proton leak; FCCP uncouples oxygen consumption from ATP production, revealing the maximal respiratory capacity; rotenone and antimycin A inhibit complex I and complex III respectively, shutting down mitochondrial respiration to determine non-mitochondrial oxygen consumption. Oxygen consumption rate (OCR) was recorded at baseline and after each injection. OCR measurements data were used to calculate basal respiration (difference between baseline OCR and non-mitochondrial respiration), ATP production (difference between baseline OCR and oligomycin-induced OCR), maximal respiration (difference between FCCP-induced OCR and non-mitochondrial respiration), and spare respiratory capacity (difference between maximal and basal respiration) according to standard protocols. The etomoxir-sensitive OCR was calculated as the difference between vehicle-treated and etomoxir-treated cells, representing the specific contribution of FAO to cellular respiration.

In addition, basal fatty acid β-oxidation capacity of CD4⁺ T cells and DNT cells was measured using an OCR Plate Assay Kit (DOJINDO, China) according to the manufacturer’s instructions.

RNA extraction and real-time PCR analysis

Total RNA was extracted from DNT cells using the FreeZol Reagent Kit (Vazyme) according to the manufacturer’s protocol. Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Takara). Quantitative PCR was conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems) with Hieff qPCR SYBR Green Master Mix (Yeasen). Primer sequences are listed in Supplemental Table S1, http://links.lww.com/HC9/C73. Relative gene expression levels were calculated using the 2^−ΔΔCt method and normalized to GAPDH as an endogenous control. All samples were run in triplicate to ensure accuracy and reproducibility.

Protein extraction and western blot

DNT cells were stimulated with or without FAO inhibitor Eto for 24 hours. Protein of DNT cells was extracted with lysis buffer, and the protein concentration was accurately determined using the BCA assay kit. The extracted proteins were separated by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred onto polyvinylidene difluoride membranes. Primary antibodies of STAT4 (Cell Signaling Technology, 1:1000 dilution), p-STAT4 (Thermo Fisher Scientific, 1:1000 dilution), and β-actin (Cell Signaling Technology, 1:10000 dilution) were used. Results were visualized using the ECL method and quantified by ImageJ software.

Promoter-binding transcription factor profiling array assay

To identify potential transcription factors (TFs) binding to the OX40 core promoter region (−1500bp/+100bp) in DNT cells, TF activation profiling plate array kits (Signosis, USA) were used according to the manufacturer’s instructions.

Statistical analysis

Data analysis was performed using R software (R Foundation for Statistical Computing, Austria) and GraphPad Prism 7 (GraphPad Software, USA). Data are presented as mean ± SEM. The normality of data distribution was assessed using the Shapiro–Wilk test. Two-group comparisons were analyzed using a 2-tailed Student t test for normally distributed data or a Mann–Whitney U test for non-parametric data. Multiple group comparisons were analyzed using one-way ANOVA with Tukey post hoc test for normally distributed data, or Kruskal–Wallis test with Dunn post hoc test for non-normally distributed data. Statistical significance was defined as p<0.05.

RESULTS

DNT cells possess higher levels of FAO

Previous research established DNT cells as a distinct T cell subset derivable from activated CD4⁺ T cells. Given the unique characteristics of DNT cells, we compared their fatty acid metabolism levels with those of CD4⁺ T cells. Following cell collection, intracellular lipid droplet content was assessed, revealing significantly higher levels in DNT cells compared with CD4⁺ T cells (Figure 1A). Metabolomic analysis and profiling demonstrated fatty acid enrichment in DNT cells (Figures 1B, C).

FIGURE 1.

DNT cells exert higher levels of FAO. DNT cells were converted from CD4⁺ T cells in vitro. Both converted DNT cells and conventional CD4⁺ T cells were harvested from wild-type C57BL/6 mice. (A) Flow cytometry detection of intracellular lipid droplet content of DNT cells and CD4⁺ T cells. (B, C) Metabolomics comparison of fatty acid content in DNT and CD4⁺ T cells. (D, E) Flow cytometry detection of intracellular lipid droplet content of DNT subgroups with high and low proliferation levels (Ki67⁺/Ki67⁻) and high and low expression of functional molecule GzmB (GzmB⁺/GzmB⁻). (F, G) GO enrichment and FAO-related gene expression differences of Gzmb^high DNT cells and Gzmb^low DNT cells. DNT cells were divided into Gzmb^high and Gzmb^low groups based on Gzmb expression level by single-cell sequencing analysis. (H) Basal OCR of DNT cells and CD4⁺ T cells detected by the OCR Plate Assay Kit. (I, J) Seahorse analysis of DNT cells and CD4⁺ T cells. Arrows indicate sequential injection of metabolic modulators: oligomycin (1 μM) at the first arrow to inhibit ATP synthase, FCCP (1.5 μM) at the second arrow to uncouple oxygen consumption from ATP production, and a mixture of rotenone and antimycin A (0.5 μM each) at the third arrow to inhibit mitochondrial electron transport chain. Vehicle (DMSO) or Eto (100 μM) was applied 20 minutes before the experiment, followed by PA (200 μM complexed with BSA) addition, where indicated. ***p<0.001. Abbreviations: BSA, bovine serum albumin; DNT, double-negative T; Eto, etomoxir; FAO, fatty acid β-oxidation; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; GO, Gene Ontology; GzmB, Granzyme B; OCR, oxygen consumption rate; PA, palmitic acid; Rot/AA, rotenone and antimycin A.

Flow cytometry analysis revealed that the proliferative DNT cell subset marked by Ki67⁺ had significantly higher intracellular lipid droplet content compared with the Ki67⁻ subset. Similarly, the subset expressing the classic cytotoxic molecule Granzyme B (GzmB⁺) also exhibited higher lipid droplet content than the GzmB⁻ subset, suggesting a link between DNT cell proliferation, cytotoxicity, and fatty acid metabolism (Figures 1D, E). Differential analysis of the single-cell sequencing results confirmed FAO-pathway upregulation in Gzmb^high DNT cells, which displayed higher levels of FAO gene expression compared with Gzmb^low DNT cells (Figures 1F, G).

Direct measurement of oxygen consumption rates using a specific assay kit disclosed that DNT cells exhibited markedly higher FAO levels than CD4⁺ T cells (Figure 1H). Dynamic monitoring of both DNT and CD4⁺ T cells via Seahorse analysis demonstrated substantially higher FAO levels in DNT cells regardless of Etomoxir treatment or PA supplementation (Figures 1I, J). Since our DNT cells are mainly derived from CD4⁺ T cells, we further analyzed the FAO level of natural DNT cells. We performed single-cell RNA sequencing analysis comparing natural DNT cells directly isolated from healthy mice with conventional CD4⁺ T cells.²¹ Similarly, natural DNT cells exhibited significantly higher expression of FAO-related genes compared with CD4⁺ T cells. The differential expression of key FAO genes such as Cpt1a, Cpt2, Acadl, and Hadha was particularly notable (Supplemental Figure S2, http://links.lww.com/HC9/C73).

Regulating the FAO level of DNT cells affects their survival and function

To determine whether elevated FAO levels influence DNT cell survival and function, we supplemented DNT cells with PA and OA and assessed changes in DNT cell survival and the expression of functional molecules. Annexin V staining via flow cytometry indicated that apoptosis in DNT cells was significantly reduced following the replenishment of free fatty acids (Figure 2A). Assessment of the functional molecule GzmB revealed a notable increase in its expression upon fatty acid supplementation (Figure 2B). PCR analysis demonstrated significant upregulation of anti-apoptotic genes Bcl-2 and Bcl-xL, and the functional molecules Gzmb and Prf1 after fatty acid supplementation (Figure 2C). In vitro suppression assay of CD45.1⁺ CD4⁺ T cells showed enhanced immunoregulatory function of fatty acid-pretreated DNT cells, evidenced by increased suppression of CD4⁺ T cell proliferation and elevated apoptosis (Figures 2D, E).

FIGURE 2.

Survival and function of DNT cells are related to FAO. (A) Flow cytometry of Annexin V detection of DNT cells with the supplement of free fatty acid OA and PA. (B) Flow cytometry of GzmB expression of DNT cells with the supplement of OA and PA. (C) Relative gene expression of survival-related genes (Bcl-2 and Bcl-xl) and function-related genes (Gzmb and Prf1) of DNT cells with the supplement of OA and PA. (D) In vitro suppression rate of DNT cells toward CD4⁺ T cells with the supplement of OA and PA. (E) Annexin V detection of CD4⁺ T cells from in vitro suppression assays with the supplement of OA and PA. (F) Flow cytometry of Annexin V detection, Ki67 and GzmB expression level of DNT cells with the supplement of FAO inhibitor Eto. (G) In vitro suppression rate of DNT cells toward CD4⁺ T cells with the supplement of Eto. (H) Annexin V detection of CD4⁺ T cells from in vitro suppression assays with the supplement of Eto. *p<0.05 and **p<0.01. Abbreviations: BSA, bovine serum albumin (solvent for OA and PA); DNT, double-negative T; Eto, etomoxir; FAO, fatty acid β-oxidation; GzmB, Granzyme B; OA, oleic acid; PA, palmitic acid.

Furthermore, we treated DNT cells with the FAO inhibitor Etomoxir (Eto) and examined the subsequent changes in DNT cell survival and function. Treatment with Eto significantly increased DNT cell apoptosis while decreasing proliferation marker Ki67 and functional molecule Granzyme B (GzmB) (Figure 2F). In addition, after Eto treatment, the suppression effect of DNT cells on CD4⁺T cells was significantly inhibited (Figures 2G, H). Based on the above results, we speculate that in DNT cells, the FAO level plays a crucial role in maintaining their survival and function and regulating FAO can affect their immunoregulatory function.

FAO regulates DNT cells through the key molecule OX40

To further investigate the regulatory mechanisms underlying the FAO levels in DNT cells, we collected DNT cells with and without Eto treatment and performed transcriptome sequencing (Figure 3A). As shown in Figure 3B, 364 upregulated genes and 908 downregulated genes were differentially expressed after Eto treatment. Functional enrichment analysis of the KEGG and GO pathways revealed significant enrichment in pathways related to cell proliferation, survival, and cytotoxicity (Figures 3C, D), with pathway-associated genes markedly downregulated in Eto-treated DNT cells (Figures 3E, F). Among genes involved in both survival and cytotoxicity, we identified 5 common regulators that participate in the regulation of both processes (Figure 3G). PCR at the mRNA level revealed a most obvious reduction in the expression of Tnfrsf4 (OX40), and flow cytometry at the protein level also confirmed the significant reduction of OX40 in Eto-treated DNT cells (Figures 3H, I).

FIGURE 3.

DNT cells regulate FAO through OX40. (A) Transcriptome sequencing of DNT cells with and without FAO inhibitor Eto treatment. (B) Differential gene expression of DNT cells with and without Eto treatment. (C, D) Functional enrichment analysis of DNT cells with and without Eto treatment. (E, F) Expression levels of genes related to survival and cytotoxicity of DNT cells with and without Eto treatment. (G) The intersection of differentially expressed survival and cytotoxicity-related genes. (H) Relative gene expression of 5 common genes in the intersection. (I) Flow cytometry of the OX40 expression level of DNT cells with the supplement of Eto. (J) Relative gene expression of survival-related genes (Bcl-2 and Bcl-xl) and function-related genes (Gzmb, Klrk1, and Prf1) of WT DNT cells and OX40 KO-DNT cells. (K, L) Flow cytometry of Annexin V detection and Edu level of WT DNT cells and OX40 KO-DNT cells. *p<0.05. Abbreviations: DNT, double-negative T; Eto, Etomoxir; FAO, fatty acid β-oxidation; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; KO, knockout; WT, wild type.

To confirm the key role of OX40 in regulating DNT cells, DNT cells were harvested from the spleens and lymph nodes of wild-type (WT) mice and OX40-knockout (KO) mice to assess the differences in survival and function between the 2 groups. Results showed a decrease in the expression of survival-related genes Bcl-2 and Bcl-xl, cytotoxicity-related genes Gzmb and Prf1, and activation-related gene Klrk1 in OX40 KO-DNT cells (Figure 3J). Flow cytometry confirmed increased apoptosis and reduced proliferation in OX40 KO-DNT cells (Figures 3K, L), suggesting that FAO inhibition may suppress DNT cell immunoregulatory function through OX40 downregulation.

STAT signaling upregulates OX40 expression to promote the survival and function of DNT cells

We then focused on the main mechanism through which FAO changes in DNT cells affect OX40 expression. Bioinformatic prediction identified multiple STAT family-binding sites, particularly STAT4-binding sites, in the OX40 promoter region (−1500 bp/+100 bp region) (Figure 4A). A competitive promoter-binding transcription factor profiling array was performed with or without the binding of the OX40 promoter. STAT4 demonstrated the most significant change in chemiluminescence signal among all TFs tested (Figure 4B).

FIGURE 4.

OX40 expression is regulated by STAT signaling. (A) Bioinformatic prediction of STAT4-binding sites in the promoter region of OX40 (−1500 bp/+100 bp region). (B) Relative chemiluminescence signal of the STAT family with or without the binding of the OX40 promoter. (C) Flow cytometry of phosphorylated STAT4 expression level of DNT cells with the supplement of FAO inhibitor Eto. (D) Western blot of total and phosphorylated STAT4 expression level of DNT cells with the supplement of Eto, and statistical analysis of the relative p-STAT4/total STAT4 ratio. (E) Flow cytometry of OX40 expression level of DNT cells with the supplement of STAT4 inhibitor, LSF. (F) Flow cytometry of Annexin V detection and Bcl-2 expression level of DNT cells with the supplement of LSF. (G) Flow cytometry of the Ki67 expression level of DNT cells with the supplement of LSF. (H) Flow cytometry of cytotoxicity-related molecules PRF1 and GzmB in DNT cells with the supplement of LSF. (I) Flow cytometry of activation-related molecules NKG2D and NKG2A in DNT cells with the supplement of LSF. *p<0.05. Abbreviations: DNT, double-negative T; Eto, Etomoxir; FAO, fatty acid β-oxidation; GzmB, Granzyme B; LSF, Lisofylline.

Flow cytometry staining of phosphorylated STAT4 within the nuclei revealed a significant downregulation of p-STAT4 in Eto-treated DNT cells (Figure 4C), suggesting the role of p-STAT4 in regulating FAO. To further confirm this finding, Western blot analysis was performed and demonstrated a marked decrease in the expression level of p-STAT4 in Eto-treated DNT cells compared with the control group, while total STAT4 expression remained unchanged (Figure 4D). These consistent results from both flow cytometry and western blot analyses provide strong evidence for the regulatory role of p-STAT4 in FAO.

Furthermore, treatment with the STAT4 inhibitor (R)-Lisofylline (LSF) significantly decreased OX40 expression in DNT cells (Figure 4E). LSF treatment (40 or 100 μM) increased apoptosis while decreasing Bcl-2 and Ki67 expression in DNT cells (Figures 4F, G). Similarly, LSF treatment significantly decreased expression of cytotoxicity-related molecules PRF1 and GzmB, and cell activation-related molecules NKG2D, while increasing the inhibitory molecule NKG2A expression (Figures 4H, I). These results suggest that FAO changes of DNT cells affect the expression of OX40 through the p-STAT4 pathway, thereby regulating the survival and function of DNT cells.

FAO inhibition impairs the therapeutic effect of DNT cells on ConA-induced autoimmune hepatitis

As an immunoregulatory T cell, DNT cells play a significant role in maintaining immune homeostasis and combating autoimmune diseases. To evaluate whether FAO regulation affects the therapeutic effect of DNT cells in vivo, we established an acute autoimmune hepatitis model in mice by tail vein injection of ConA. Mice received adoptive transfer of control or Eto-pretreated DNT cells as treatment. After 48 hours, mice were sacrificed, and liver tissues were harvested. The TUNEL staining results of liver tissues showed a significant increase in the apoptotic cells in the ConA model group. In contrast, the apoptotic cells were markedly decreased in the DNT treatment group, while the reduction of apoptotic cells in the DNT-Eto group was not as significant as that in the DNT group (Figure 5A). Liver necrosis was evident in the ConA group, while DNT cell treatment significantly reduced necrotic areas, but this protective effect was diminished in the Eto-pretreated DNT group (Figures 5B, C). Measurement of serum ALT and AST showed a significant decrease in the DNT treatment group compared with the ConA group, while the DNT-Eto treatment group showed reduced therapeutic efficacy compared with the DNT group (Figure 5D).

FIGURE 5.

FAO contributes to the therapeutic effect of DNT cells on ConA-induced autoimmune liver models. (A) TUNEL staining schematic diagrams of control, ConA, DNT treatment, and DNT-Eto treatment groups, and a statistical chart of relative TUNEL fluorescence. (B) H&E staining schematic diagrams of control, ConA, DNT treatment, and DNT-Eto treatment groups. (C) Statistical chart of liver necrosis area of control, ConA, DNT treatment, and DNT-Eto treatment group. (D) Serum ALT and AST levels of control, ConA, DNT treatment, and DNT-Eto treatment group. (E) Flow cytometry analysis of intrahepatic CD4⁺ T, CD8⁺ T, and NKT cells in different treatment groups. (F) Flow cytometry of Annexin V detection of DNT cells in the DNT treatment and DNT-Eto treatment groups. (G) Flow cytometry of Ki67 levels of DNT cells in the DNT treatment and DNT-Eto treatment groups. (H) Flow cytometry of Bcl-2, GzmB, and NKG2D levels of DNT cells in the DNT treatment and DNT-Eto treatment groups. (I) Flow cytometry of OX40 expression levels of DNT cells in the DNT treatment and DNT-Eto treatment groups. (J) Flow cytometry of relative p-STAT4 expression levels of DNT cells in the DNT treatment and DNT-Eto treatment groups. *p<0.05. Abbreviations: ConA, concanavalin A; DNT, double-negative T; Eto, etomoxir; FAO, fatty acid β-oxidation; GzmB, Granzyme B; H&E, hematoxylin and eosin; NKT, natural killer T.

Flow cytometric analysis of intrahepatic immune cells revealed a significant reduction in T cell proportions in the DNT treatment group, whereas the decrease was not pronounced in the DNT-Eto treatment group (Figure 5E). Detection of the transferred-DNT cells showed increased apoptosis and decreased proliferation in Eto-treated DNT cells in vivo (Figures 5F, G). In addition, expression of Bcl-2, GzmB, and NKG2D was significantly lower in the DNT-Eto group compared with the DNT group (Figure 5H). Assessment indicated a significant downregulation of the key FAO regulator OX40 in the DNT-Eto group, with a corresponding decrease in the expression of the TF p-STAT4 (Figures 5I, J).

OX40 knockout impairs the therapeutic effect of DNT cells on ConA-induced autoimmune hepatitis

To evaluate whether FAO regulation affects the therapeutic effect of DNT cells through the key molecule OX40 in vivo, we established the ConA model with WT DNT and OX40 KO-DNT treatment.

The TUNEL staining results of liver tissues demonstrated a significant increase in the apoptotic cells in the OX40 KO-DNT treatment group, compared with the DNT treatment group (Figure 6A). Liver necrosis was evident in the ConA group, while DNT cell treatment significantly reduced necrotic areas, and this protective effect was diminished in the OX40 KO-DNT treatment group (Figures 6B, C). Measurement of serum ALT and AST levels also revealed a significant decrease in the DNT treatment group compared with the ConA group, with the OX40 KO-DNT treatment group showing reduced therapeutic efficacy compared with the DNT group (Figure 6D).

FIGURE 6.

OX40 knockout impairs the therapeutic effect of DNT cells on ConA-induced autoimmune liver models. (A) TUNEL staining schematic diagrams of control, ConA, DNT treatment, and OX40 KO-DNT treatment group and statistical chart of relative TUNEL fluorescence. (B) H&E staining schematic diagrams of control, ConA, DNT treatment, and OX40 KO-DNT treatment group. (C) Statistical chart of liver necrosis area of control, ConA, DNT treatment, and OX40 KO-DNT treatment group. (D) Serum ALT and AST levels of control, ConA, DNT treatment, and OX40 KO-DNT treatment group. (E) Flow cytometry analysis of intrahepatic CD4⁺ T, CD8⁺ T, and NKT cells in different treatment groups. (F) Flow cytometry of Annexin V detection of DNT cells in DNT treatment and OX40 KO-DNT treatment group. (G) Flow cytometry of Ki67 levels of DNT cells in the DNT treatment and OX40 KO-DNT treatment groups. (H) Flow cytometry of Bcl-2, GzmB, and NKG2D levels of DNT cells in DNT treatment and OX40 KO-DNT treatment groups. (I) The graphic abstract of this study. *p<0.05. Abbreviations: ConA, concanavalin A; DNT, double-negative T; Eto, etomoxir; GzmB, Granzyme B; H&E, hematoxylin and eosin; KO, knockout; NKT, natural killer T.

Flow cytometric analysis of intrahepatic immune cells showed a significant reduction in T cell proportions in the DNT treatment group, whereas the decrease was not pronounced in the OX40 KO-DNT treatment group (Figure 6E). Detection of the transferred-DNT cells indicated increased apoptosis and decreased proliferation in OX40 KO-DNT cells in vivo (Figures 6F, G). In addition, the expression of Bcl-2, GzmB, and NKG2D was significantly lower in the OX40 KO-DNT group compared with the DNT group (Figures 6H, I). These results suggest that OX40 knockout impairs the therapeutic effect of DNT cells in the ConA-induced autoimmune hepatitis model, further emphasizing the importance of OX40 in maintaining the therapeutic efficacy of DNT cells in liver diseases.

DISCUSSION

β-oxidation of fatty acids, which breaks down fatty acids and generates energy, is an important metabolic pathway for the survival and function of immune cells.²² The key enzymes CPT1a and CPT2, located on the outer and inner mitochondrial membranes, respectively, facilitate the initial step of long-chain fatty acid transport into mitochondria.²³ Previous studies have demonstrated the importance of CPT1a/CPT2 in regulating various T cell subsets. CPT1a expression, enhanced by IL-15, promotes FAO and supports the development and survival of CD8⁺ memory T cells.²⁴ While in regulatory T cells, CPT1a influences induced Treg (iTreg) differentiation, although some conflicting evidence exists.²³,²⁵ However, in effector T cells, CPT1a expression is generally lower, reflecting their preference for glycolytic metabolism.²⁴ Our findings demonstrate that DNT cells are characterized by an abundance of lipid droplets and exhibit robust fatty acid metabolism, and DNT cells with high GzmB expression and stronger immunoregulatory function show more vigorous FAO levels, suggesting that FAO is the primary energy metabolism pathway supporting DNT cell function.

FAO affects immune cell function through multiple mechanisms. CD8⁺ memory T cells depend on FAO-derived free fatty acids from lysosomal hydrolysis to support the metabolic programming necessary for development.²⁶ By promoting mitochondrial biosynthesis and CPT1a expression, FAO enhances the mitochondrial spare respiration capacity of CD8⁺memory T cells, thereby promoting their survival.²⁴ FAO can also alter deacetylase SIRT1 activity through the NAD⁺/NADH ratio, thereby affecting the stability of Treg cells.²⁷ Specifically, our study reveals the relationship between FAO and DNT cell immunoregulatory function, particularly highlighting the pivotal role of the OX40 molecule. OX40, a member of the TNF receptor superfamily, is known for its critical involvement in T cell activation, proliferation, survival, and differentiation of memory T cells.²⁸ Previous reports have elucidated the multifaceted role of OX40 in immune cell regulation, including augmenting T cell survival by upregulating anti-apoptotic molecules Bcl-xl and Bcl-2,²⁹ extending the proliferative cycle of T cells through the activation of the Akt axis,³⁰ mediating T cell differentiation into Th1 and Th2 cells,³¹,³² and promoting transition of T cells into Tregs.³³ Our previous studies have confirmed that OX40 is highly expressed on DNT cells and plays an important role in maintaining the survival of DNT cells, promoting DNT proliferation, and inhibiting apoptosis.³⁴ Our experimental data confirm that inhibiting FAO can suppress OX40 levels in DNT cells, which leads us to propose that FAO regulates DNT cell function through the OX40 pathway.

Our findings also reveal that the STAT signaling pathway influences DNT cell survival and function through modulation of OX40, highlighting the critical role of STAT signaling cascades in immune cell regulation. Previous studies have demonstrated the importance of STAT signaling in controlling immune cell differentiation and function.³⁵ For instance, STAT1 and STAT3 participate in T follicular helper cell differentiation and function,³⁶ while STAT1 and STAT4 are key determinants in Th1 cell polarization, with STAT4 being essential for IL-12 signaling and STAT1 critical for IFN-γ signaling.³⁷,³⁸ Inversely, STAT6 inhibits Th1 polarization but promotes the development of Th2 cells, with hallmark IL-4, IL-13, and IL-5.³⁸,³⁹ STAT3 intimately associates with the development of Th17 cells, cooperating with the retinoic acid receptor–related orphan receptor γ (RORγt) to induce IL-17 expression in the clearance of extracellular bacteria and antifungal responses.⁴⁰,⁴¹ STAT5 promotes Treg differentiation by regulating Foxp3 expression, maintaining immune tolerance.³⁹,⁴² Furthermore, metabolic alterations within cells can intricately link to STAT signaling pathways, influencing how immune cells respond to pathogens and inflammation. Cellular metabolic states affect STAT protein activity through mitochondrial dynamics,⁴³ and metabolic shifts influence STAT3-mediated host–pathogen interactions via DENV-associated proteins.⁴⁴

The parallel dysregulation of STAT and OX40–OX40L pathways exhibits similar patterns in atopic dermatitis, where targeting either pathway shows therapeutic potential,⁴⁵,⁴⁶ suggesting a functional synergy or correlation between STAT signaling and OX40 expression. Consistent with this notion, our results demonstrate that STAT inhibition downregulates OX40 expression and DNT cell function, indicating that the interplay between STAT signaling and OX40 may represent a novel mechanism by which FAO enhances the immune regulatory function of DNT cells.

Immune cell energy metabolism regulation offers promising therapeutic potential in various diseases. In the tumor microenvironment, fatty acid metabolism has been proven to complement glycolysis in the selective expansion of regulatory T cells, highlighting the potential of manipulating lipid metabolism to enhance immunoregulatory function.⁴⁷ And targeting ACC1, the rate-limiting enzyme of fatty acid synthesis, shows capacity to treat TH17 cell-mediated autoimmune disease by inhibiting pro-inflammatory TH17 cell development and promoting anti-inflammatory Treg cell induction.¹⁵ As important immune regulatory cells that maintain immune homeostasis, DNT cell therapy has also been proven effective in various diseases, including hematological and solid tumor models,⁴⁸,⁴⁹ graft-versus-host disease⁵⁰ and autoimmune disease.⁵

In conclusion, our study demonstrates that DNT cells upregulate fatty acid metabolism, particularly FAO, and enhance their survival and function through the key molecule OX40. This metabolic shift significantly enhances the immunoregulatory capabilities of DNT cells. By leveraging this newfound knowledge of DNT cell metabolism, we propose a novel strategy for immune therapy in autoimmune diseases, including autoimmune hepatitis, offering a promising avenue to enhance the efficacy of DNT cell-based therapies.

Supplementary Material

hc9-9-e0783-s001.docx ^{(1.1MB, docx)}