LncBADR promotes T cell-mediated autoimmunity by binding Mccc1 and Pcca to regulate BCAAs degradation

Abstract

T cell dysfunction is a pivotal driving factor in autoimmune diseases, yet its underlying regulatory mechanisms remain incompletely understood. The role of long non-coding RNAs (lncRNAs) in immune regulation has gradually been recognized, although their functional mechanisms in T cells remain elusive. This study focuses on lncBADR (LncRNA Branched-chain Amino acids Degradation Regulator), elucidating its mechanism by which it regulates branched-chain amino acids (BCAAs) metabolism to influence T cell effector functions. Mice with specific knockout of lncBADR (T cell^{lncBADR−/−}) exhibited markedly ameliorated experimental autoimmune encephalomyelitis (EAE) symptoms. Mechanistic investigations revealed that lncBADR inhibits BCAAs degradation by binding to the enzymes Mccc1 and Pcca, leading to the accumulation of BCAAs within T-cells. This, in turn, activates the mTOR-Stat1 signaling pathway, promoting IFN-γ secretion and exacerbating EAE pathology. In contrast, knockout of lncBADR restored BCAAs degradation, significantly reducing IFN-γ secretion in T cells and suppressing their pathogenic functions. Further studies demonstrated that high-BCAAs feeding partially reversed the protective effects of lncBADR knockout, indicating that lncBADR plays a crucial role in autoimmune inflammation by regulating BCAAs metabolism. This study offers new insights into targeting lncBADR or modulating BCAAs metabolism as potential therapeutic strategies for autoimmune diseases.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12974-025-03538-9.

Background

Autoimmune diseases represent a group of severe pathological conditions characterized by the immune system erroneously attacking the host’s healthy cells. Despite ongoing research, the understanding of their cellular and molecular mechanisms remains limited, and effective therapeutic strategies are still lacking [1]. Multiple sclerosis (MS) is one of the quintessential autoimmune diseases, a chronic demyelinating disorder that affects the central nervous system (CNS) [2]. Dysfunction of T cells plays a pivotal role in the pathogenesis of MS [3, 4]. However, the precise molecular mechanisms underlying T cell dysfunction remain incompletely elucidated. Thus, unveiling the potential mechanisms through which T cells contribute to MS will not only enhance our understanding of autoimmune disease pathogenesis but may also offer novel therapeutic targets for these disorders.

T cells primarily develop into two subsets: CD4⁺ T cells and CD8⁺ T cells [5]. During the pathogenesis of MS and EAE, naïve CD4⁺ T and CD8⁺ T cells undergo activation, clonal expansion, and subsequent differentiation, thereby executing their effector functions, which include the elimination of infected cells, the secretion of cytokines, and the regulation of immune responses [4, 6]. The effector subsets of CD4⁺ T cells are defined by the characteristic patterns of cytokine production and the expression of key transcription factors. CD4⁺ T cell Subsets that are capable of inducing EAE include T helper 1 (Th1) and T helper 17 (Th17) cells [4]. The hallmark cytokine of Th1 cells is IFN-γ, which promotes the infiltration of immune cells through the blood-brain barrier, exacerbating the immune-inflammatory response in the CNS [4]. Th17 cells, characterized by the cytokine IL-17, induce inflammation in a less potent manner but synergize with other cytokines to maintain the activation of inflammatory cells, thereby exerting a robust inflammatory effect [7]. Regulatory T (Treg) cells, which express high levels of CD25 and the transcription factor FoxP3, are key regulators in suppressing excessive inflammation and maintaining immune tolerance. Studies have demonstrated that Treg counteract Th17-mediated inflammatory responses, and the dynamic balance between these two subsets is closely associated with the pathogenesis and progression of various autoimmune diseases [8, 9]. CD8⁺ T cells, also known as cytotoxic T lymphocytes, drive immune responses by secreting effector molecules such as IFN-γ, Granzyme B, and TNF-α. Notably, in the CNS of MS patients, the number of CD8⁺ T cells surpasses that of CD4⁺ T cells, suggests that CD8⁺ T cells may play an active pathogenic role in MS [10–12].

LncRNAs are a class of non-coding RNA molecules that can influence cellular metabolism and signal transduction through interactions with proteins, playing an important regulatory role in immune cells [13–15]. Dysregulation of lncRNA expression is closely linked to a variety of neurodegenerative diseases and can affect critical biological processes such as immune homeostasis, immune tolerance, immune cell development, differentiation, and proliferation, as well as the balance of pro-inflammatory and anti-inflammatory cytokines [16–18]. In the pathogenesis of EAE, T cells transition from a naïve to an effector state, rapidly regulating multiple signaling pathways to execute organized functions. During this process, does lncRNA participate in this regulation? To answer this question, we consulted relevant databases and compared the differentially expressed lncRNAs between naïve T cells and EAE T cells. After screening, we identified lncRNA-NONMMUT031096, which exhibited significant expression changes and had not been thoroughly studied [19], and selected it as the focus of our research, subsequently naming it lncBADR. In this study, we found that lncBADR exacerbates T cell-mediated pathological responses by promoting IFN-γ secretion. To elucidate the underlying mechanism, we employed RNA pull-down assays to identify proteins interacting with lncBADR, which revealed Mccc1 and Pcca as binding partners. These enzymes serve as critical regulators in the branched-chain amino acids (BCAAs) metabolic pathway [20, 21]. Further investigation demonstrated that lncBADR interacts with Mccc1 and Pcca, resulting in intracellular BCAAs accumulation, which subsequently activates the mTOR-Stat1 signaling pathway. This cascade of events influences T cell metabolic reprogramming and enhances IFN-γ secretion. Consequently, our findings not only advance the understanding of the role of lncRNA in immune cell function, but also propose a novel therapeutic strategy for autoimmune diseases by targeting lncBADR to regulate BCAAs metabolism.

Materials and methods

Animals

Female mice, aged 8 to 12 weeks, with a C57Bl/6 background, were used in all experiments. Wild-type mice were procured from Beijing HFK Bioscience Co., Ltd. CD4-Cre mice and lncBADR^fl/fl mice were obtained from Cyagen Biosciences. To achieve the knockout of lncBADR in T cells, we opted to use CD4-Cre mice instead of Lck-Cre mice. This choice was driven by the fact that generating conditional knockout (CKO) mice through mating Cre tool mice with flox/flox mice entails a lengthy experimental process. Moreover, our laboratory lacks Lck-Cre mice but possesses CD4-Cre mice. Additionally, during T cell development, there exists a CD4 + CD8 + double-positive (DP) stage, which theoretically enables CD4-Cre mice to efficiently delete lncBADR within T cells. We crossed CD4-Cre mice with lncBADR^fl/fl mice to generate CD4 Cre-lncBADR^fl/fl mice, and successfully confirmed the T cell-specific knockout of lncBADR, which we subsequently designated as T cell^{lncBADR−/−} mice. All mice were maintained under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle (7:00 AM to 7:00 PM) and were provided ad libitum access to standard rodent chow and sterile water. Environmental conditions were controlled, with the room temperature Maintained between 21 °C and 23 °C and humidity at 65%. All experimental procedures complied with Guidelines for the Care and Use of Laboratory Animals issued by the China National Institute of Health. The study protocol was approved by the Institutional Animal Care and Use Committee of Harbin Medical University (IRB reference number: SYXK2022-014; 81430035).

Induction of EAE

Mice were subcutaneously injected with 200 µg of MOG_35–55 peptide (Wuhan Bioyeargene), emulsified in incomplete Freund’s adjuvant containing heat-inactivated Mycobacterium tuberculosis (5 mg/mL; Sigma-Aldrich). Additionally, 300 ng of pertussis toxin (List Biological Laboratories) was administered via tail vein injection on the day of immunization and again 48 h later. The clinical progression of EAE was monitored daily using the following scoring system: 0, no disease; 1, full tail paralysis; 2, paralysis of one hind limb while the other remains functional; 3, complete hind limb paralysis; 4, quadriplegia; and 5, death. Mice displaying intermediate symptoms were scored in increments of 0.5.

In vivo high BCAA-fed mouse model

In the high BCAA-fed experiments, mice were provided with water containing 15 mg/mL valine, 15 mg/mL leucine, and 15 mg/mL isoleucine (all from Sigma-Aldrich) for the duration of the experiment. Control mice (normal BCAA group) were given standard sterile ddH2O.

Histopathology

Frozen sections, 10 μm thickness, were prepared from the enlarged lumbar segment of the spinal cord. Hematoxylin and eosin (H&E) staining (Salorbio) was performed according to the manufacturer’s instructions to assess inflammatory cell infiltration. Myelin Basic Protein (MBP) staining was conducted using FluoroMyelin^TM Green (Thermo Fisher Scientific kit) to evaluate demyelination, following the manufacturer’s protocol. The number of infiltrating inflammatory cells and the percentage of MBP loss were quantified using ImageJ software.

Primary cell isolation

The thymus, lymph nodes, and spleen were dissected from mice, and homogenized using 70 μm cell strainers to obtain single-cell suspensions. After perfusion with phosphate-buffered saline (PBS), the spinal cord and brain were collected and homogenized with a tissue grinder (Wheaton). Mononuclear cells were isolated via density gradient centrifugation using 30% Percoll (Cytiva).

Purification and culture of CD3⁺ T cells

Lymph nodes were collected from mice and processed into single-cell suspensions. T cells were isolated using the MojoSort™ Mouse CD3 T Cell Isolation Kit (BioLegend). The purity of each isolated immune cell population was routinely confirmed to exceed 96% by flow cytometry. For in vitro culture experiments, T cells were seeded at a density of 3 × 10⁵ cells per well in U-bottom 96-well plates, with a total volume of 200 µL per well. The plates were pre-coated with anti-CD3 antibody (2.5 µg/mL, BioLegend) and anti-CD28 antibody (2.5 µg/mL, BioLegend), and IL-2 (30 U/mL, PeproTech) was added to the culture medium. The culture medium consisted of RPMI 1640 (Procell) or custom BCAA-free RPMI medium (Procell), Supplemented with 10% fetal bovine serum (AusGeneX), 1% penicillin/streptomycin, 1% L-glutamine, 1% MEM non-essential amino acid solution, 0.1% 2-mercaptoethanol, and 0.1% sodium pyruvate (all from Thermo Fisher Scientific). T cells were cultured at 37 °C in a 5% CO₂ incubator for 48 h. In the BCAA supplementation experiment, custom BCAA-free RPMI medium (Procell) was used, and depending on the experimental needs, valine, leucine, and isoleucine (all purchased from Sigma-Aldrich) were added to the BCAA-free medium at concentrations ranging from 0 to 320 µM to stimulate T cells.

Purification of macrophages

Spleens were harvested from mice and processed into single-cell suspensions. Macrophages were subsequently isolated using the EasySep™ Mouse F4/80 Positive Selection Kit (STEMCELL Technologies), with isolated cell purity routinely exceeding 90% as verified by flow cytometric analysis.

Transfection of T cells

Mice lymph node purified CD3⁺ T cells were suspended in T buffer (2 × 10⁶ in 100 µL) of the Neon consumables (Thermo Fisher Scientific) with 50 nM siRNA targeting Mccc1 or Pcca or negative control (RiboBio). Electroporation was carried out using the Neon Transfection System (1825 V, 10 ms, 2 pulses). Transfected cells were transferred to 24-well plates and stimulated with anti-CD3 (2.5 µg/mL, BioLegend) + anti-CD28 (2.5 µg/mL, BioLegend) + IL-2 (30 U/mL, PeproTech) in RPMI 1640 medium (Procell) Supplemented with 10% fetal bovine serum (AusGeneX), 1% penicillin/streptomycin, 1% L-glutamine, 1% MEM non-essential amino acids, 0.1% β-mercaptoethanol, and 0.1% sodium pyruvate (all from Thermo Fisher Scientific), with a total volume of 1 mL/well, followed by 48-hour incubation at 37 °C with 5% CO₂.

Surface immunophenotyping

Cells were prepared and seeded into a 96-well U-bottom plate. After a single wash with PBS, Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific) was added, and the cells were incubated on ice for 30 min to exclude apoptotic or dead cells. Following another wash with PBS, the cells were incubated with Surface antibody on ice for 30 min. The cells were then washed, fixed, and analyzed by flow cytometry. All incubation steps were performed in the dark to minimize light exposure of the samples throughout the procedure. The antibodies used for flow cytometry staining are listed in supplementary Table 1.

Intracellular cytokine staining

To evaluate intracellular cytokine staining, cells were stimulated with the Cell Stimulation Cocktail (Thermo Fisher Scientific) at 37 °C in 5% CO₂ for 4 to 5 h to induce cytokine secretion. After standard staining with Fixable Viability Dye eFluor™ 780 and surface antibodies, the cells were fixed and permeabilized using the Fixation/Permeabilization Kit (BD Biosciences). Intracellular antibodies were then added, and the cells were incubated on ice for 30 min. Finally, the cells were washed and analyzed by flow cytometry. All incubation steps were performed in the dark to minimize light exposure to the samples throughout the procedure.

Intracellular transcription factor and nuclear protein staining

To evaluate intracellular transcription factor and nuclear protein staining, cells were first stained with Fixable Viability Dye eFluor™ 780 and surface antibodies according to standard protocols. The cells were then fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (Invitrogen). Following this, transcription factor and nuclear protein antibodies were added, and the samples were incubated on ice for 30 min. Finally, the cells were washed and analyzed by flow cytometry. All incubation steps were conducted under light-protected conditions to minimize sample exposure to light throughout the procedure.

Real-time PCR

Purified CD3⁺ T, CD4⁺ T, CD8⁺ T cells (5 × 10⁶ cells per subset) and macrophages (1 × 10⁶ cells) were used to extract total RNA with the RNAiso PLUS kit (Takara) following the manufacturer’s instructions. RNA concentration and quality were assessed using a NanoDrop (Thermo Fisher Scientific). RNA was then reverse-transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Real-time PCR was performed using the 2X TransStart^® Top Green qPCR SuperMix (TransGen Biotech) on an Applied Biosystems 7500 Fast Real-Time PCR system. Gene expression levels were normalized to the internal control gene GAPDH. Primer sequences were as follows: LncBADR forward primer, 5′-CCAGAAGCCAGCAGTGATCTT-3′, reverse primer, 5′-CCCTGCTGCCCAAATAACCA-3′; GAPDH forward primer, 5′-GGACCTCATGGCCTACATGG-3′, reverse primer, 5′-TAGGGCCTCTCTTGCTCAGT-3′; Mccc1 forward primer, 5′-GGGTGATACGAACAGCCAAAA-3′, reverse primer, 5′-CTCGTCTGCCATATCTACATGC-3′; Pcca forward primer, 5′-TTTGGCTAAAGCCGTGAAGTAT-3′, reverse primer, 5′-GGATGTTCGACCTGTAGTCTTGT-3′; Actin forward primer, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′, reverse primer, 5′-TAAAACGCAGCTCAGTAACAGTCC-3′.

Nuclear and cytoplasmic fractionation assay

T cells were isolated using the MojoSort™ Mouse CD3 T Cell Isolation Kit (BioLegend). Nuclear and cytoplasmic RNA fractions were separated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions, and subsequently subjected to reverse transcription and real-time PCR. lncRNA-Malat1 was used as the nuclear control, and GAPDH as the cytoplasmic control. The primer sequences were as follows: lncBADR forward primer, 5′-CCAGAAGCCAGCAGTGATCTT-3′; reverse primer, 5′-CCCTGCTGCCCAAATAACCA-3′; Malat1 forward primer, 5′-GAGTTGTAGGCTTCTGTGTA-3′; reverse primer, 5′-AGGCTTGTGGTAGGTCAT-3′; GAPDH forward primer, 5′-GGACCTCATGGCCTACATGG-3′; reverse primer, 5′-TAGGGCCTCTCTTGCTCAGT-3′.

Pull-down assay

A full-length lncBADR gene fragment was synthesized to construct a Linearized plasmid template for the preparation of biotin-labeled RNA probes. T cells were lysed in lysis buffer containing 1 mM PMSF, 100 U RNase inhibitor, and 1× protease inhibitor cocktail to ectract total cellular protein. Biotin-labeled sense and antisense RNAs (10 pmol each) were denatured at 90 °C for 2 min, followed by a 5-minute incubation on ice. RNA structure buffer was then added, and the RNAs were refolded at room temperature for 20 min. The sense and antisense RNA probes were each incubated with 1 mg of total protein in RNA binding buffer with gentle rotation at 25 °C for 2 h. Streptavidin magnetic beads (20 µL, Invitrogen) were Subsequently added to each reaction and incubated at 25 °C for an additional 2 h. The beads were collected and washed five times with RNA binding buffer. Bound proteins were eluted with 30 µL of 1× SDS loading buffer by heating at 95 °C for 10 min. Ten microliters of the eluted sample were separated by 8%−12% SDS-PAGE and visualized by high-resolution silver staining. Differential bands were excised and analyzed by mass spectrometry.

RNA sequencing

Total RNA was extracted from T cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. cDNA library construction was performed using the TruSeq^® RNA Sample Preparation Kit (Illumina Inc.) following the provided instructions. Key steps included poly-A RNA enrichment, fragmentation of purified total RNA, first-strand cDNA synthesis, second-strand cDNA synthesis, end repair, 3’ adenylation, adapter ligation, and library enrichment, which together completed the preparation of the sequencing library. The purified libraries were quantified using a Qubit^® 2.0 Fluorometer (Life Technologies, USA) and validated with an Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the molar concentration. Library clustering was performed using a cBot system after diluting the Library to 10 pM, followed by sequencing on the Illumina NovaSeq 6000 platform (Illumina, USA). Library construction and sequencing were carried out at Shanghai Biotechnology Corporation.

Western blot

T cells cultured in vitro were lysed using cell lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitors (Thermo Fisher). Protein samples were standardized and denatured at 100 °C for 7 min, followed by separation via 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were subsequently transferred onto PVDF membranes (Millipore) using a wet transfer method. The membranes were blocked with 5% non-fat milk at room temperature for 1–2 h. Primary antibodies were diluted in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated overnight at 4 °C. The primary antibodies used included: p-mTOR (Ser2448) (1:1000, CST), p-p70 S6K (Thr389) (1:500, CST), p-Stat1 (Tyr701) (1:1000, CST), p-Stat1 (Ser727) (1:1000, CST), and β-Actin (1:1000, CST). Subsequently, the appropriate secondary antibody was added and incubated at room temperature for 1 h. Protein bands were visualized using ECL detection reagents and quantified with ImageJ software.

Statistical analysis

All values are expressed as the mean ± standard error of the mean (SEM). P-values were determined using appropriate statistical tests, including Student’s t-test, one-way ANOVA, two-way ANOVA, or two-tailed paired t test, with all analyses performed in GraphPad Prism 9. Statistical significance was defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Flow cytometry data were analyzed using FlowJo V10.

Results

Deletion of LncBADR in T cells ameliorates autoimmune neuroinflammation

Previous studies have demonstrated that lncRNAs possess robust regulatory capabilities; however, our understanding of these molecules remains limited. Experimental autoimmune encephalomyelitis (EAE) is a well-established T cell-mediated autoimmune disease [22–24], during which the expression of numerous lncRNAs in T cells undergoes significant alterations [19, 25]. By consulting relevant databases, we compared the differentially expressed lncRNAs between naïve T cells and EAE T cells. From this analysis, we identified lncRNA-NONMMUT031096 as showing significant expression changes while remaining largely unexplored [19]. Consequently, we selected this lncRNA for further investigation and named it lncBADR. To assess the role of lncBADR in T cell-mediated immune responses in vivo, we utilized EAE, a well-established T cell-mediated autoimmune disease model, to mimic MS [26, 27]. Mice were immunized with MOG₃₅₋₅₅ peptide emulsified in complete Freund’s adjuvant (CFA) to induce EAE, as shown in Fig. 1A. Initial comparison revealed significantly elevated lncBADR expression in T cells from EAE mice versus controls (Fig. 1B), thereby validating our RNA sequencing data. To investigate the functional role of lncBADR in T cells, we utilized CD4-Cre mice instead of Lck-Cre mice to generate conditional knockout mice with T cell-specific deletion of lncBADR, referred to as T cell^{lncBADR−/−} (Fig. 1C). This decision was based on the relatively long breeding cycle required for generating conditional knockout mice and the unavailability of Lck-Cre mice in our laboratory. Additionally, the presence of the CD4 + CD8 + double-positive (DP) stage during T cell development supports the use of CD4-Cre mice for achieving lncBADR deletion in T cells. In this study, lncBADR^fl/fl mice were used as the control group throughout. RT-qPCR analysis confirmed the deletion of lncBADR in T cells (Fig. 1D). Additionally, we validated the absence of lncBADR in both CD4⁺ T and CD8⁺ T cells (Supplementary Fig. 1 A).

Fig. 1.

Effect of T cell-specific deletion of lncBADR on EAE. A Induction strategy for EAE. B Relative expression of lncBADR in lymph node CD3⁺ T cells 10 days post EAE induction (n = 5). C Experimental strategy for T cell-specific deletion of lncBADR. D RT-PCR validation of lncBADR knockout in T cells (n = 3 or 4). E Clinical score of EAE (n = 8 or 5). F Onset of disease in EAE (n = 5 or 8). G Disease severity calculated as the area under the curve (AUC) of clinical scores (n = 5 or 8). H and I HE staining of spinal cord sections to assess inflammatory cell infiltration (black arrows indicate areas of infiltration, n = 4 or 6). J and K MBP staining of the spinal cord sections to assess myelin loss (white arrows indicate areas of MBP loss, n = 3). L and M Total cell counts in the lymph nodes and spleen of control and T cell^{lncBADR−/−} mice. N Cell numbers of T cells in the lymph nodes and spleen of control and T cell^{lncBADR−/−} mice (day 9 post-EAE induction, n = 5 or 6). O Cell numbers of CD4⁺ T cells, CD8⁺ T cells, B cells, granulocytes, monocytes and NK cells in the lymph nodes and spleen of control and T cell^{lncBADR−/−} mice (day 9 post-EAE induction, n = 5 or 6). Statistical analysis was performed using two-way ANOVA or t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

We next evaluated the impact of T cell-specific lncBADR deletion on the development of immune organs. No significant differences were observed in the size or total cell count of the thymus, lymph nodes, or spleen between the control and T cell^{lncBADR−/−} mice (Supplementary Fig. 1B and C). To assess the influence of lncBADR on T cell development, we compared the proportions and numbers of various T cell subsets in the thymus of both groups, which also showed no differences (Supplementary Fig. 1D-F). Similarly, the proportion and absolute counts of CD4⁺ T cells, CD8⁺ T cells, B cells, monocytes, granulocytes, and NK cells in the lymph nodes and spleen were comparable between the two groups (Supplementary Fig. 1G, H). These results indicate that lncBADR deletion in T cells does not affect the development of immune organs or immune cells, nor does it alter thymic T cell selection or their homeostatic distribution in peripheral lymphoid organs.

Subsequently, we evaluated the impact of lncBADR on EAE pathogenesis. Compared to control mice, T cell^{lncBADR−/−} mice exhibited reduced Susceptibility to EAE induction. Specifically, control mice began showing symptoms around day 10, while T cell^{lncBADR−/−} mice showed disease onset around day 12. Clinical scores progressively increased and peaked on day 17 in control mice. Furthermore, T cell^{lncBADR−/−} mice exhibited a faster disease recovery between days 17 and 30, with significantly lower disease scores compared to control mice (Fig. 1E and F). Area under the curve (AUC) analysis of the clinical score confirmed that the severity of the disease was milder in T cell^{lncBADR−/−} mice compared to control mice (Fig. 1G). Histopathological analysis revealed that, at peak disease, T cell^{lncBADR−/−} mice had fewer inflammatory cell infiltrates and a smaller area of myelin loss in the CNS (Fig. 1H-K). Furthermore, early in EAE progression, both control and T cell^{lncBADR−/−} mice showed significant increases in total cell counts in the lymph nodes and spleen compared to naïve mice, indicating the activation of peripheral immune responses. However, no differences were observed in the total cell counts of the lymph nodes or spleen between the two groups (Fig. 1L and M). Since lncBADR deletion is specific to T cells, we compared T cell counts and found no significant differences (Fig. 1N). Additionally, the absolute cell numbers of CD4⁺ T cells, CD8⁺ T cells, B cells, monocytes, granulocytes, and NK cells in the lymph nodes and spleen were also similar between the two groups (Fig. 1O), these findings indicate that lncBADR deletion does not influence the expansion of immune cells. The above results suggest that the deletion of lncBADR in T cells significantly alleviates the symptoms of neuroinflammation, without causing changes in the number of immune cells.

Reduced IFN-γ production in T cells in the absence of LncBADR in vivo

Pathogenic T cells initiate disease after proliferating to a certain threshold of cell numbers. The previous results indicated that the deletion of lncBADR did not significantly affect the numbers of T cells, CD4⁺ T cells, or CD8⁺ T cells. Therefore, the alleviation of EAE symptoms is unlikely to be attributed to changes in the quantity of these cells. To further investigate the underlying mechanisms contributing to the reduction in EAE severity, we examined the secretion of inflammatory cytokines by these cells. On day 9 post-immunization, we harvested lymph nodes and spleens from the mice. Compared to the control group, T cell^{lncBADR−/−} mice exhibited a significant reduction in IFN-γ and Granzyme B secretion by T cells in the lymph nodes, while IL-17 levels showed no significant change (Fig. 2A-F). A similar trend was observed in the spleen (Supplementary Fig. 2A-F). We then further compared cytokine production in CD4⁺ T and CD8⁺ T cells between the two groups. The results demonstrated that CD4⁺ T cells in the lymph nodes of T cell^{lncBADR−/−} mice produced significantly less IFN-γ in both proportion and quantity compared to controls, while the expression of IL-17 and Granzyme B was comparable to the control group (Fig. 2G-L). Similarly, CD8⁺ T cells in the lymph nodes of T cell^{lncBADR−/−} mice secreted less IFN-γ and Granzyme B, with IL-17 levels unchanged (Fig. 2M-R). A similar pattern was observed in the spleen (Supplementary Fig. 2G-R). In summary, the absence of lncBADR significantly alters the expression of IFN-γ in both CD4⁺ T and CD8⁺ T cells. Additionally, lncBADR deficiency leads to a reduction in Granzyme B secretion by CD8⁺ T cells, although this change is less pronounced than the decrease in IFN-γ production. Notably, IL-17 production remains unaffected by the deletion of lncBADR. Based on these findings, We hypothesize that the absence of lncBADR in T cells alleviates the severity of neuroinflammation primarily by inhibiting the production of IFN-γ.

Fig. 2.

Impact of lncBADR deletion on T cell effector function during EAE in vivo. A to F Analysis of IFN-γ⁺, IL-17⁺, and Granzyme B⁺ cells gated on T cells in the lymph nodes 9 days after EAE induction. A Relative percentage and (B) cell number of IFN-γ⁺ T cells (n = 4 or 5). C Relative percentage and (D) cell number of IL-17⁺ T cells (n = 4 or 5). E Relative percentage and (F) cell number of Granzyme B⁺ T cells (n = 4). G to L Analysis of IFN-γ⁺, IL-17⁺ and Granzyme B⁺ cells gated on CD4⁺ T cells in the lymph nodes 9 days after EAE induction. G Relative percentage and (H) cell number of IFN-γ⁺ CD4⁺ T cells (n = 4 or 5). I Relative percentage and (J) cell number of IL-17⁺ CD4⁺ T cells (n = 4 or 5). K Relative percentage and (L) cell number of Granzyme B⁺ CD4⁺ T cells (n = 4). M to R Analysis of IFN-γ⁺, IL-17⁺ and Granzyme B⁺ cells gated on CD8⁺ T cells in the lymph nodes 9 days after EAE induction. M Relative percentage and (N) cell number of IFN-γ⁺ CD8⁺ T cells (n = 4 or 5). O Relative percentage and (P) cell number of IL-17⁺ CD8⁺ T cells (n = 4 or 5). Q Relative percentage and (R) cell number of Granzyme B⁺ CD8⁺ T cells (n = 4). Statistical analysis was performed using t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

Deletion of LncBADR inhibits IFN-γ production in T cells in vitro

In vivo data suggest that the absence of lncBADR in T cells leads to a reduction in EAE-associated neuroinflammation, primarily due to the suppression of IFN-γ secretion by T cells, including both CD4⁺ T and CD8⁺ T cells. To further investigate the immunoregulatory role of lncBADR in T cells, we conducted in vitro experiments. T cells were purified from the draining lymph nodes of control and T cell^{lncBADR−/−} mice. These cells were subsequently stimulated with anti-CD3, anti-CD28 antibodies, and interleukin-2 (IL-2). Following 48 h of culture, we comprehensively assessed T cell activation, proliferation, and effector function. The results, as shown in the figure, indicate that lncBADR deletion has minimal impact on T cell activation, with only a slight reduction in the expression of CD69 and CD44, and a significant decrease in CD25 expression (Fig. 3A-C). No change in T cell proliferative capacity was observed (Fig. 3D-F). However, lncBADR knockout markedly reduced IFN-γ production by T cells, while having no effect on IL-17 secretion (Fig. 3G-I). Additionally, lncBADR deletion inhibited the secretion of Granzyme B by T cells (Fig. 3J). Next, we analyzed the activation, proliferation, and cytokine production of CD4⁺ T cells. As shown in the figure, compared to the control group, lncBADR deletion had a minimal impact on CD4⁺ T cell activation, resulting in a reduction in CD25 expression, but without affecting the expression of CD69 or CD44 (Fig. 3K-M). LncBADR had no effect on the proliferation of CD4⁺ T cells (Fig. 3N-P), nor did it alter their capacity to secrete IL-17 and Granzyme B (Fig. 3S and T). However, lncBADR deletion significantly inhibited the secretion of IFN-γ by CD4⁺ T cells (Fig. 3Q and R). We further examined the effects of lncBADR on CD8⁺ T cells. Compared to the control group, CD8⁺ T cells from T cell^{lncBADR−/−} mice displayed reduced expression of CD69, CD25, and CD44 (Fig. 3U-W). We also compared the proliferative capacity of CD8⁺ T cells from control and T cell^{lncBADR−/−} mice, and found no differences in proliferation (Fig. 3X-Z). Next, we assessed the impact of lncBADR on the effector functions of CD8⁺ T cells. Compared to the control group, CD8⁺ T cells from T cell^{lncBADR−/−} mice exhibited significantly reduced IFN-γ production, with no change in IL-17 secretion, and a marked reduction in Granzyme B secretion (Fig. 3AA-AD). Stat1, a key transcription factor for IFN-γ, is phosphorylated and translocates to the nucleus, where it binds to the promoter regions of interferon-stimulated genes (ISGs), thereby promoting the expression of IFN-γ [28, 29]. Given the substantial effect of lncBADR deletion on IFN-γ production in T cells, we further examined the phosphorylation of Stat1 in T cells from both groups. Compared to the control group, CD4⁺ T and CD8⁺ T cells from T cell^{lncBADR−/−} mice exhibited lower mean fluorescence intensities for p-Stat1 (Tyr701) and p-Stat1 (Ser727) (Supplementary Fig. 3A-G), consistent with the observed trends in IFN-γ expression (Fig. 3H, R, and AB). These findings suggest that, under in vitro non-specific T cell activation conditions, lncBADR deletion significantly affects T cell activation and cytokine production, particularly IFN-γ secretion, which aligns with the results obtained from in vivo studies.

Fig. 3.

Effects of lncBADR deletion on T cells in vitro. T cells were purified from the draining lymph nodes of control and T cell^{lncBADR−/−} mice, and then stimulated with anti-CD3 (2.5 µg/ml) + anti-CD28 (2.5 µg/ml) + IL-2 (30 U/ml) for 48 h. A to C The percentages of CD69 (A, n = 5), CD25 (B, n = 4), and CD44 (C, n = 5) in T cells. D Representative histograms showing CFSE dilution in all T cells. E Proliferation index calculated from CFSE dilution curves (n = 4). F The percentage of Ki67⁺ cells in T cells (n = 5). G to I Dot plots and percentages of IFN-γ⁺ T cells and IL-17⁺ T cells (n = 4). J The percentage of Granzyme B⁺ cells in T cells (n = 4 or 6). K to M The percentages of CD69 (K, n = 5), CD25 (L, n = 4), and CD44 (M, n = 5) gated on CD4⁺ T cells. N Representative histograms showing CFSE dilution in all CD4⁺ T cells. O Proliferation index calculated from CFSE dilution curves (n = 4). P The percentage of Ki67⁺ cells in CD4⁺ T cells (n = 5). Q to S Dot plots and percentages of IFN-γ⁺ CD4⁺ T cells and IL-17⁺ CD4⁺ T cells (n = 4). T The percentage of Granzyme B⁺ cells in CD4⁺ T cells (n = 4 or 6). U to W The percentages of CD69 (U, n = 3 or 4), CD25 (V, n = 4), and CD44 (W, n = 3 or 4) gated on CD8⁺ T cells. X Representative histograms showing CFSE dilution in all CD8⁺ T cells. Y Proliferation index calculated from CFSE dilution curves (n = 4). Z The percentage of Ki67⁺ cells in CD8⁺ T cells (n = 4). AA to AC Dot plots and percentages of IFN-γ⁺ CD8⁺ T cells and IL-17⁺ CD8⁺ T cells (n = 4). AD The percentage of Granzyme B⁺ cells in CD8⁺ T cells (n = 4 or 6). Statistical analyses were performed using t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

LncBADR deletion in T cells impacts IFN-γ production by regulating BCAAs degradation in vitro

To further investigate the molecular mechanism by which lncBADR promotes T cell IFN-γ secretion, we performed subcellular localization of lncBADR using nuclear-cytoplasmic separation assays. The results revealed that lncBADR is primarily localized in the cytoplasm (Fig. 4A), leading us to hypothesize that lncBADR may regulate intracellular signaling pathways by interacting with proteins. Using RNA pull-down assays, we identified proteins binding to lncBADR, and mass spectrometry analysis showed that lncBADR interacts with Mccc1 and Pcca (Fig. 4B). Both Mccc1 and Pcca are key enzymes involved in the degradation of branched-chain amino acids (BCAAs) [20, 21]. To determine whether lncBADR affects BCAAs degradation, we cultured T cells in vitro and measured the concentration of BCAAs in T cells from control and T cell^{lncBADR−/−} mice. The results demonstrated a reduction in BCAAs concentration in T cells from T cell^{lncBADR−/−} mice (Fig. 4C). Notably, lncBADR deletion did not alter mRNA expression of BCAA catabolic enzymes Mccc1 and Pcca (Supplementary Figs. 4A-D), supporting its protein-binding mechanism rather than transcriptional regulation of BCAAs metabolism. BCAAs are essential amino acids that must be obtained through the diet [30]. To determine whether lncBADR specifically regulates T cell IFN-γ secretion by modulating BCAAs degradation, we cultured T cells in BCAA-free medium to block their uptake. The results showed that BCAA-free medium reversed the lncBADR-mediated secretion of IFN-γ, but did not affect IL-17 production (Fig. 4D and E). This phenomenon was also observed in both CD4⁺ T and CD8⁺ T cells (Fig. 4F to I). To further investigate the effect of BCAAs on T cell IFN-γ production, we treated T cells with different concentrations of BCAAs. The results indicated that higher BCAA concentrations led to increased IFN-γ production by T cells (Fig. 4J), suggesting that BCAAs can promote IFN-γ production in vitro to varying degrees.

Fig. 4.

LncBADR binds to Mccc1 and Pcca, inhibiting BCAAs degradation. A Nuclear-cytoplasmic fractionation assay. The control Malat1 is localized in the nucleus, and GAPDH is localized in the cytoplasm (n = 4). B Pull-down assay to identify proteins interacting with lncBADR. C T cells were purified from the draining lymph nodes of control and T cell^{lncBADR−/−} mice and stimulated for 48 h with anti-CD3 (2.5 µg/ml) + anti-CD28 (2.5 µg/ml) + IL-2 (30 U/ml). The BCAAs concentrations in T cells (n = 6). D to I T cells were cultured in complete lymphocyte medium with or without BCAAs, and stimulated for 48 h with anti-CD3 + anti-CD28 + IL-2. Flow cytometry analysis of IFN-γ and IL-17 expression. D and E The percentages of IFN-γ⁺ and IL-17⁺ cells in T cells (n = 4 or 5). F and G The percentages of IFN-γ⁺ and IL-17⁺ cells gated on CD4⁺ T cells (n = 4 or 5). H and I The percentages of IFN-γ⁺ and IL-17⁺ cells gated on CD8⁺ T cells (n = 4 or 5). J T cells were cultured in complete lymphocyte media supplemented with varying concentrations of BCAAs and stimulated with anti-CD3 + anti-CD28 + IL-2 for 48 h. The expression levels of IFN-γ were measured by flow cytometry (n = 3). K to N Purified mice T cells were transfected with either siNC or siMccc1 and stimulated with anti-CD3 + anti-CD28 + IL-2 for 48 h. K RT-PCR validation of Mccc1 knockdown in T cells (n = 5). L The BCAAs concentrations in T cells (n = 8). M and N The percentages of IFN-r (M, n = 7) and IL-17 (N, n = 7) gated on CD3⁺ T cells. O to R Purified mice T cells were transfected with either siNC or siPcca and stimulated with anti-CD3 + anti-CD28 + IL-2 for 48 h. O RT-PCR validation of Pcca knockdown in T cells (n = 5). P The BCAAs concentrations in T cells (n = 8). Q and R The percentages of IFN-r (Q, n = 8) and IL-17 (R, n = 8) gated on CD3⁺ T cells. Statistical analysis was performed using either the t-test, two-way ANOVA or two-tailed paired t test. *P < 0.05, **P < 0.01, ****P < 0.0001, ns: not significant

To further elucidate the roles of Mccc1 and Pcca in BCAAs metabolism and T cell cytokine production, naïve CD3⁺ T cells purified from mice were resuspended in electroporation buffer and transfected with siMccc1, siPcca, or control siRNA using the Neon™ Transfection System. Results demonstrate that siMccc1 effectively reduced Mccc1 mRNA levels (Fig. 4K), concomitant with significantly elevated intracellular BCAAs (Fig. 4L) and enhanced IFN-γ production (Fig. 4M), while exerting no effect on IL-17 expression (Fig. 4N). Correspondingly, siPcca transfection robustly decreased Pcca mRNA (Fig. 4O), increased BCAA accumulation (Fig. 4P), promoted IFN-γ secretion (Fig. 4Q), yet similarly did not alter IL-17 production (Fig. 4R). These data indicate that suppressing Mccc1 or Pcca expression inhibits BCAAs degradation, leading to intracellular BCAAs accumulation and consequent potentiation of IFN-γ secretion. This phenotype—opposite to the effect of lncBADR deletion—supports the model wherein lncBADR binding to Mccc1 and Pcca impairs their enzymatic function, thereby disrupting BCAAs metabolism and ultimately modulating T cell inflammatory responses.

Several studies have reported that the intake of BCAAs is linked to T cell metabolic reprogramming [31], and BCAAs effectively activate the mTORC1 signaling pathway [32–34]. To investigate the cause of reduced IFN-γ production in T cells from T cell^{lncBADR−/−} mice, we performed RNA-seq on purified T cells from both the control and T cell^{lncBADR−/−} groups. Gene Set Enrichment Analysis (GSEA) revealed significant enrichment of glycolysis and oxidative phosphorylation processes in T cells from the control group (Supplementary Fig. 5 A and B), and the mTOR pathway was also notably enriched in the control group (Fig. 5A). Furthermore, research has shown that mTOR promotes the phosphorylation of Stat1 [35, 36], which provides an explanation for the regulatory role of mTOR in IFN-γ secretion. Subsequently, we conducted western blot analysis of key proteins in the mTOR-Stat1 signaling pathway, with results shown in Fig. 5B to F. Compared to the control group, phosphorylation of mTOR and its downstream effector molecule p70 S6K was inhibited in T cells from T cell^{lncBADR−/−} mice. Furthermore, phosphorylation of Stat1 was also significantly reduced in these cells. Treatment with BCAAs restored the expression levels of p-mTOR (Ser2448), p-p70 S6K (Thr389), p-Stat1 (Tyr701), and p-Stat1 (Ser727) in T cells from T cell^{lncBADR−/−} mice. These results suggest that lncBADR interacts with Mccc1 and Pcca to impede the degradation of BCAAs, leading to their accumulation within T cells. The excess BCAAs subsequently activate the mTOR-Stat1 pathway, enhancing glycolysis and oxidative phosphorylation processes, which in turn promotes the secretion of IFN-γ.

Fig. 5.

BCAAs supplementation upregulates key proteins in the mTOR-Stat1 signaling pathway. A Gene set enrichment analysis (GSEA) of T cells isolated from control and T cell^{lncBADR−/−} mice. B to F T cells isolated from control and T cell^{lncBADR−/−} were stimulated with anti-CD3 + anti-CD28 + IL-2 for 48 h, with or without the addition of BCAAs. Key proteins of the mTOR-Stat1 signaling pathway were then analyzed by Western blot. B Immunoblotting of p-mTOR (Ser2448), p-p70 S6K (Thr389), p-Stat1 (Tyr701), and p-Stat1 (Ser727). C to F The expression levels of the target proteins were normalized to the corresponding β-Actin (n = 3 or 4). Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

BCAAs supplementation partially reversed the reduction in EAE severity caused by lncBADR deletion in T cells

In vitro results demonstrate that lncBADR inhibits the degradation of BCAAs, thereby promoting their accumulation within T cells and ultimately enhancing their capacity to secrete IFN-γ. This suggests that BCAAs may play a potential pathogenic role in the EAE model of neuroinflammation. To validate this hypothesis, we employed high-BCAA feeding to induce BCAAs accumulation in T cell^{lncBADR−/−} mice, followed by EAE induction using MOG₃₅₋₅₅. We observed that compared to the normal diet group, the high-BCAA-fed T cell^{lncBADR−/−} mice exhibited increased clinical severity and greater histological damage (Fig. 6A-C). These results indicate that the deletion of lncBADR in T cells attenuates neuroinflammation, whereas high-dose BCAA feeding restores the clinical severity of the disease in mice. To further confirm whether the alleviation of EAE severity by lncBADR deletion in T cells is associated with BCAA modulation of IFN-γ secretion, we performed flow cytometry analysis on the CNS of mice at the peak of EAE. The results revealed that, compared to controls, T cell^{lncBADR−/−} mice exhibited fewer T cells infiltrating the CNS, and the expression of the inflammatory cytokine IFN-γ was significantly reduced. However, supplementation with BCAAs led to an increase in the number of infiltrating T cells and their IFN-γ expression (Fig. 6D-G). These findings suggest that in the EAE model, lncBADR contributes to encephalitis by modulating the level of IFN-γ secretion in T cells, and this regulatory effect is, in part, mediated through the modulation of BCAAs metabolism.

Fig. 6.

Impact of high-BCAAs diet on EAE induction with MOG₃₅₋₅₅. A Clinical scores of EAE (n = 4 or 5 per group). B and C MBP staining of spinal cord on day 17 after EAE induction (with white arrows indicating areas of myelin loss, n = 3 or 4 per group). D to G CNS-infiltrating cells were isolated on day 17 after EAE induction and analyzed by flow cytometry. D and E Dot plots and corresponding percentages of CD45⁺CD3⁺ T cells (n = 4). F and G Dot plots and corresponding percentages showing IFN-γ expression in T cells (n = 4). Statistical analysis was performed using two-way ANOVA or one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Discussion

MS is a chronic autoimmune disease characterized primarily by inflammation and demyelination within the CNS. In recent years, the incidence of MS has continued to rise. While environmental and genetic factors are considered to increase the risk of developing the disease, its fundamental pathogenesis remains incompletely understood [37–39]. Although current therapeutic options are effective, they are associated with significant side effects and limited efficacy [40–42]. The pathological progression of MS is predominantly mediated by activated T cells, with CD4⁺ T cells differentiating into Th1 and Th17 subtypes that secrete pro-inflammatory cytokines such as IFN-γ and IL-17, initiating CNS inflammation and driving disease progression [7, 43–45]. CD8⁺ T cells exert cytotoxic effects by secreting IFN-γ and granzyme B, thereby activating other immune cells and amplifying the inflammatory response in the CNS [46, 47].

The role of lncRNAs in immune regulation has garnered significant attention. Although lncRNAs do not encode proteins, studies have shown that they play a critical role in maintaining immune homeostasis, cellular development, differentiation, proliferation, and the balance between pro-inflammatory and anti-inflammatory factors [16–18]. Based on the potential of lncRNAs in immune regulation, we selected lncRNA-NONMMUT031096, which exhibits the most significant differential expression in naïve and EAE T cells, as the subject of our study, naming it lncBADR. We generated T cell-specific lncBADR knockout mice (T cell^{lncBADR−/−}) to investigate the immune regulatory role of lncBADR in T cells. First, we confirmed that the absence of lncBADR in T cells did not significantly affect the development of immune organs or immune cells (Supplementary Fig. 1). Next, in the EAE induction model, we observed that T cell^{lncBADR−/−} mice displayed resistance to EAE, with delayed onset of disease, faster clinical recovery, and histological analysis revealing reduced inflammatory infiltration (Fig. 1E-I). These findings suggest that lncBADR may play a role in the progression of EAE by modulating T cell effector functions. Further experiments indicated that the absence of lncBADR led to a significant reduction in IFN-γ secretion in T cells, and a concomitant suppression of Granzyme B secretion, although IL-17 production remained unaffected. Specifically, both CD4⁺ T and CD8⁺ T cells secreted substantial amounts of IFN-γ, while Granzyme B was primarily produced by CD8⁺ T cells (Fig. 2 and Supplementary Fig. 2). This phenomenon was also confirmed in in vitro experiments (Fig. 3). Therefore, we hypothesize that the absence of lncBADR in T cells primarily reduces neuroinflammation through diminished IFN-γ secretion, a process likely mediated by direct effects on T cell intracellular signaling pathways and metabolic processes.

We next investigated the mechanism through which lncBADR regulates IFN-γ secretion in T cells. RNA pull-down assays identified Mccc1 and Pcca as direct lncBADR-interacting proteins. Both enzymes function within the BCAAs catabolic pathway: Mccc1 serves as the α-catalytic subunit of MCC (mitochondrial 3-methylcrotonyl-CoA carboxylase), partnering with the β-subunit Mccc2 to catalyze leucine degradation and facilitate entry of leucine-derived carbon skeletons into the TCA cycle [20]. Pcca functions as the α-subunit of PCC (propionyl-CoA carboxylase), collaborating with the β-subunit Pccb to mediate valine and isoleucine catabolism, ultimately generating succinyl-CoA [21]. These enzymes are indispensable for BCAAs degradation. We demonstrated that lncBADR binds Mccc1 and Pcca, thereby modulating intracellular BCAAs concentrations in T cells. Significantly, culturing cells in BCAA-free medium reversed the lncBADR-mediated enhancement of IFN-γ secretion, indicating that Mccc1 and Pcca participate in BCAAs metabolism and regulate cytokine production (specifically IFN-γ and IL-17) in T cells. Clinical evidence links Mccc1 and Pcca mutations to metabolic disorders: Mccc1 variants cause MCC deficiency characterized by acute metabolic crises [48], while Pcca mutations precipitate propionic acidemia featuring metabolic acidosis and multi-organ damage [49, 50]. Current research predominantly focuses on their roles in metabolic diseases, whereas investigation into their immunological functions remains limited—particularly their impact on T cell effector mechanisms remains unreported. To address this knowledge gap, we knocked down Mccc1 and Pcca expression in vitro, confirming their essential roles in BCAAs degradation and T cell-mediated immune responses (Fig. 4K-R). Considering that BCAAs can effectively regulate immune responses by activating the mTORC1 signaling pathway [32–34], promote IFN-γ secretion, and enhance glycolysis and oxidative phosphorylation [51, 52], and that mTOR facilitates the phosphorylation of Stat1 [35, 36], a key transcription factor for IFN-γ, we hypothesize that lncBADR regulates BCAAs accumulation in T cells, thereby activating the mTOR-Stat1 signaling pathway to promote IFN-γ secretion. This mechanism is supported by multiple experiments (Figs. 4 and 5).

Furthermore, given the pivotal role of BCAAs metabolism in macrophage-mediated inflammation [53–55], we quantified lncBADR expression in macrophages isolated from EAE mice and observed a 5-fold upregulation relative to controls (Supplementary Fig. 6 A), suggesting potential functional significance of lncBADR in macrophage biology. Key unresolved questions include whether lncBADR regulates macrophage effector functions through BCAAs metabolic reprogramming—analogous to its established mechanism in T cells—and how altered lncBADR expression influences EAE pathogenesis. Addressing these questions—through both in vivo and in vitro approaches—will necessitate substantial experimental investment. Essential methodologies include: (1) generating myeloid-specific lncBADR-conditional knockout murine models; and (2) establishing lentiviral-based RNA interference systems for targeted lncBADR knock down in primary macrophages. These investigations constitute immediate priorities for subsequent research to delineate lncBADR-dependent mechanisms governing macrophage effector functionality and disease pathogenesis.

In addition to the most significant changes in IFN-γ, we also observed a decrease in the expression of CD25 on CD4⁺ T cells in T cell^{lncBADR−/−} mice (Fig. 3L), and a significant reduction in the expression of activation markers CD69, CD25, and CD44 on CD8⁺ T cells (Fig. 3U-W). Furthermore, Granzyme B expression was also notably diminished (Fig. 3AD). The widespread nature of these changes reflects the crucial role of BCAAs in T cell metabolic reprogramming [31, 51, 52]. Clearly, the knockout of lncBADR inhibits T cell activation and effector molecule secretion, particularly in CD8⁺ T cells, which is likely due to the relatively lower expression of amino acid transporters in CD4⁺ T cells [56], resulting in a decreased BCAA uptake efficiency and a smaller intracellular response to the absence of lncBADR. Notably, IL-17 production remained largely unaffected by lncBADR knockout (Figs. 2C-D, I-J and O-P and 3I and S, AC). Through comprehensive literature review, we identified that IFN-γ-mediated regulation of Th17 cells predominantly occurs within the CNS pathological microenvironment, as evidenced by studies demonstrating that CNS-infiltrating Th1 cells facilitate Th17 cells recruitment [57]. Concurrently, established literature indicates T-bet critically governs Th17 pathogenicity during EAE pathogenesis [58, 59]. In our experimental system utilizing T cell^lncBADR-/- mice—where lncBADR deficiency commences from embryonic development—we observed that this genetic deletion does not compromise naïve T cell development or population size (Supplementary Fig. 1D-H). However, within antigen-stimulated microenvironments of peripheral lymphoid organs (in vivo: antigen-specific; in vitro: non-specific stimulation), it significantly impedes naïve T cell differentiation toward Th1 lineage (Fig. 2G, H; Supplementary Fig. 2G, H; Fig. 3R) while exerting no discernible effect on Th17 differentiation (Fig. 2I, J; Supplementary Fig. 2I, J; Fig. 3S). Crucially, unlike the CNS pathological context involving secondary activation of antigen-experienced T cells, peripheral lymphoid organs constitute sites of primary antigen encounter where naïve T cells undergo initial activation and differentiation. This fundamental microenvironmental divergence likely underlies altered T cell activation states, signaling transduction pathways, and metabolic reprogramming requirements. Although the precise mechanistic basis for microenvironment-specific regulation—particularly why IFN-γ/T-bet modulates Th17 cells within the CNS but not peripheral compartments—remains incompletely characterized, this phenomenon potentially reflects sophisticated, context-dependent adaptations in T cell response patterns. We acknowledge this constitutes an intriguing biological question warranting rigorous future investigation.

Through in vitro experiments, we uncovered the critical role of BCAAs in the immune regulation induced by lncBADR deficiency. As essential amino acids, BCAAs must be obtained through dietary supplementation [30]. To validate the role of BCAAs in lncBADR regulation, we fed T cell^{lncBADR−/−} mice a high-BCAA diet to restore their BCAAs levels and then induced the EAE model. The results demonstrated that high-BCAA feeding partially reversed the alleviation of EAE symptoms and histopathological damage caused by lncBADR deficiency (Fig. 6A-C), and significantly increased T cell infiltration and IFN-γ secretion in the CNS (Fig. 6D-G). This suggests that the absence of lncBADR alleviates the severity of EAE to some extent by modulating BCAAs metabolism, and the close connection between BCAAs metabolism and T cell effector functions further confirms the pivotal role of lncBADR in this process.

Conclusions

This study unveils a critical role for lncBADR in T cell-mediated autoimmunity, demonstrating that its deletion alleviates EAE symptoms by reducing IFN-γ production through modulation of BCAAs metabolism. Mechanistically, lncBADR binds to Mccc1 and Pcca, inhibiting BCAAs degradation and activating the mTOR-Stat1 pathway(see Graphical abstract). In vivo experiments further confirmed that high-BCAAs feeding partially reverses the protective effects of lncBADR deletion, underscoring the importance of BCAAs metabolism in T cell effector functions. These findings not only deepen our understanding of lncRNA-mediated immune regulation but also highlight lncBADR and BCAAs metabolism as potential therapeutic targets for autoimmune diseases, including MS.

Abbreviations

AUC

Area under the curve

BCAAs

Branched-chain amino acids

BCAT

Branched-Chain Amino Acid Transaminase

BCKAs

Branched-Chain α-Keto Acids

BCKDC

Branched-Chain α-Keto Acid Dehydrogenase Complex

CFA

Complete Freund's adjuvant

CKO

Conditional knockout

CNS

Central nervous system

DN

Double-negative

DP

Double-positive

DPI

Days after immunization

EAE

Experimental autoimmune encephalomyelitis

GSEA

Gene Set Enrichment Analysis

H&E

Hematoxylin and eosin (H&E) staining

IFN-γ

Interferon gamma

IL

Interleukin

lncBADR

LncRNA Branched-chain Amino acids Degradation Regulator

LncRNAs

Long non-coding RNAs

MBP

Myelin Basic Protein

MOG_35-55

Myelin Oligodendrocyte Glycoprotein peptide 35-55

MS

Multiple sclerosis

RT-PCR

Real-time PCR

SDS-PAGE

SDS-polyacrylamide gel electrophoresis

SP

Single-negative

SPF

Specific pathogen-free

TCA

Tricarboxylic Acid Cycle

T cell^lncBADR-/-

T cell-specific lncBADR knockout mice

Th

T helper cells

Treg

Regulatory T cells

Authors’ contributions

YTL: wrote the main manuscript and performed the research. JY, XRH, ZYL: contributed to the experimental procedures. JW, YXJ and WKJ: conducted data analysis. YY and YH: created the graphical abstract, performed RNA-seq data analysis. JTT, DK, YML, XJL and XJL: proofread articles. HLL and BS: designed the research and acquired funding. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the National Natural Science Foundation of China (81870955, 82371821, 82271845), the Natural Science Foundation of Heilongjiang Province (LH2022H004), “XingLian” Outstanding Youth Foundation of Harbin Medical University (2024-202026), and “Pandeng Program” of Harbin Medical University Cancer Hospital (PDYS2024-08).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study was conducted in compliance with the principles outlined in Harbin Medical University’s Guide to the Care and Use of Laboratory Animals, as published by the China National Institute of Health.

Consent for publication

All authors contributed to the article and approved the submitted version.

Competing interests

The authors declare no competing interests.