Themis orchestrates T-cell development through coordinated regulation of mitochondrial metabolism and Jun family transcription factors

To the Editor: T cells are key components of the immune system, playing a critical role in defending against infections and tumors. Dysregulated T-cell immunity can trigger and exacerbate autoimmune diseases. Themis, independently identified by five research groups in 2009,[1–5] is crucial for T-cell development and function. In peripheral T cells, Themis significantly influences CD8+ T cell homeostasis and enhances the responsiveness of TH1 cells to foreign antigen stimulation, playing a pivotal role in immune responses during infection and inflammation.[6–8] We performed single-cell RNA sequencing on three genotypes of Themis mice to further investigate the role of Themis in T-cell development at the single-cell level. The results show that Themis influences mitochondrial biogenesis and function, altering cellular metabolism. Furthermore, Jun family transcription factors were upregulated in Themis–/– mice, ThemisY34F/Y34F mice, and primary T cells from knockout (KO) mice expressing the Themis Delta NLS mutant, highly suggesting that Themis exerts its function in T cell development via nuclear regulation of Jun family members. All animal experiments undertaken in this study were approved by the Institutional Animal Care and Use Committee of ShanghaiTech University (P2021-0174) and conducted in accordance with governmental regulations of China for the care and use of animals. In our prior study, we generated ThemisY34F/Y34F and Themis–/– mice, establishing the essential role of Y34 phosphorylation in T cell development.[9] To investigate thymic T cell developmental dynamics, we performed single-cell RNA sequencing (scRNA-seq) on thymocytes from three genetically distinct mouse models: Themis+/+, Themis–/–, and ThemisY34F/Y34F. Unsupervised clustering analysis identified nine distinct cell populations, DN3, DN4, DN_DP, DP, DP_SP, CD4, CD8, type 1 innate lymphoid cells (ILC1), and a non-T cell population [Figure 1A, Supplementary Figure 1A, https://links.lww.com/CM9/C792].Figure 1: Themis deficiency impairs T-cell development through coordinated mitochondrial dysfunction and Jun family dysregulation. (A) Distribution of UMAP of single-cell RNA-seq data colored by cell type. (B) The same UMAP colored by mouse genotype (WT, Y34F, and KO; n = 1 mouse per genotype). (C) Boxplot of Themis expression in different cell types. (D) Proportion of apoptotic T cells (MT transcripts ratio ≥10%) across three genotypes. (E) Proportion of MT transcripts per cell in viable T cells (MT transcripts ratio <10%) across three genotypes. (F–G) Flow cytometry analysis of MitoTracker Green staining (F) and quantification of mitochondrial staining intensity (G) in total thymocytes from WT and Themis –/– mice (n = 3, 3). (H) Total thymocytes were isolated from Themis WT and KO mice, and mitochondrial parameters were quantified. OCR of indicated cells at baseline and in response to oligomycin (Oligo), FCCP, and rotenone plus antimycin A (Rot/AA). (I) Western blotting of total thymocytes from three genotypes was performed to detect the expression levels of the indicated proteins. (J–O) Themis WT and the Delta NLS mutant were expressed in thymocytes (J–L) and splenocytes (M–O) from Themis –/– mice, and the mRNA expression levels of Jun, JunB, and JunD were analyzed by RT-qPCR (n = 4, 3 in J–L, n = 4 in M–O, biologically independent samples over 2 independent experiments). Statistical significance was assessed using Wilcoxon rank sum test in E and two-tailed unpaired Student’s t-test in G and J–O. ns: P >0.05; *: P ≤0.05; †: P ≤0.01; ‡: P ≤0.001; §: P ≤0.0001. FCCP: Carbonyl cyanide 4-(trifluoromethoxy phenylhydrazone); KO: Knockout; MT: Mitochondrial; NLS: Nuclear localization signal; OCR: Oxygen consumption rate; RNA-seq: RNA sequencing; UMAP: Uniform manifold approximation and projection; WT: Wild type.Compared to wild-type, ThemisY34F/Y34F mice showed increased DN3 and DN_DP proportions, whereas Themis–/– mice had similar levels. At the DN4 stage, both mutants showed reduced cell populations [Figure 1B]. Themis–/– mice exhibited elevated proportions of DP_SP thymocytes, while both mutants displayed increased DP cells but decreased mature CD4SP, CD8SP, ILCs, and other subsets [Supplementary Figure 1B–D, https://links.lww.com/CM9/C792]. Themis expression peaks during the DP stage and declines sharply during the DP_SP transition [Figure 1C], consistent with its role in T cell maturation. The single-cell RNA sequencing (scRNA-seq) data corroborate these findings and match protein-level profiles from prior flow cytometry studies.[9] Interestingly, Themis–/– mice showed a higher proportion of apoptotic cells than WT and ThemisY34F/Y34F mice [Figure 1D]. In viable cells, Themis–/– mice had elevated mitochondrial (MT) transcript levels across all T cell stages, suggesting mitochondrial dysfunction [Figures 1E and Supplementary Figure 2A, B, https://links.lww.com/CM9/C792].[7] Flow cytometry with Mitotracker Green staining revealed increased mitochondrial mass in DN, DP, CD8SP, and total thymocytes from Themis–/– mice, but not in CD4SP subsets [Figures 1F–G and Supplementary Figure 2C, D, https://links.lww.com/CM9/C792]. To assess mitochondrial function, we performed a mitochondrial stress test. Basal respiration and adenosine triphosphate (ATP) production—measured after oligomycin inhibition of complex V—were comparable between Themis-KO and Themis-WT thymocytes [Figures 1H and Supplementary Figure 2E–F, https://links.lww.com/CM9/C792]. These findings indicate that Themis does not significantly affect overall energy utilization or ATP-linked respiration. Maximal respiration, induced by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) to dissipate the proton gradient, was significantly reduced in KO thymocytes. Spare respiratory capacity was also reduced by ~30%. Rotenone and antimycin A abolished mitochondrial respiration, with no significant difference in non-mitochondrial oxygen consumption [Figures 1H and Supplementary Figure 2G–I, https://links.lww.com/CM9/C792]. These results show Themis deficiency impairs maximal respiration and spare capacity, indicating mitochondrial dysfunction. Analysis of differentially expressed genes revealed consistent alterations in Jun family transcription factors (Jun, JunB, JunD) across T cell subsets. The Jun family, part of the AP-1 complex, regulates gene expression in response to stimuli such as stress, growth factors, and cytokines. Notably, compared to WT mice, Jun family expression was significantly increased in DN3, DN4, DN–DP, DP, DP_SP, and CD4 cell types in both mutants, except for unchanged Jun levels in DN3 of Themis–/– mice and stable JunD in DN_DP across genotypes. In CD8 and ILC1 thymocytes, JunB was markedly upregulated in both mutants, whereas JunD and Jun were upregulated in Themis–/– mice and ThemisY34F/Y34F mice, respectively [Supplementary Figures 3A–H, https://links.lww.com/CM9/C792]. To confirm these findings, we isolated total thymocytes and assessed the protein levels of key regulators: PGC1β, Jun, JunB, and JunD [Figure 1I]. PGC1β (PPARGC1B), a transcriptional coactivator involved in mitochondrial biogenesis and metabolic regulation[10], was significantly elevated in Themis–/– mice but not in ThemisY34F/Y34F mice compared to wild-type controls [Figure 1I and Supplementary Figure 3I, https://links.lww.com/CM9/C792]. This is consistent with the aforementioned data showing increased mitochondrial mass [Figure 1D–G and Supplementary Figure 2A–D, https://links.lww.com/CM9/C792]. Jun, JunB, and JunD expression was increased in both mutants [Figure 1I and Supplementary Figure 3J–L, https://links.lww.com/CM9/C792], aligning with scRNA-seq results. Given the observed nuclear translocation of Themis, we hypothesized that nuclear import is required for suppressing Jun gene expression. To test this, we generated a Themis Delta NLS mutant lacking the nuclear localization signal (deletion of residues 343–347: KRRPR), resulting in cytoplasmic retention of the protein.[11] Thymocytes [Figure 1J–L] and splenocytes [Figure 1M–O] from Themis–/– mice were transfected with either wild-type or the Delta NLS mutant construct. Notably, Jun, JunB, and JunD mRNA levels were significantly higher in cells expressing the Delta NLS mutant, indicating that Themis nuclear localization is essential for repressing Jun family gene transcription. In conclusion, scRNA sequencing and biochemical validation experiments demonstrate that Themis deficiency leads to an increase in mitochondrial content within thymocytes, thereby altering their metabolic profiles. The nuclear translocation of Themis suppresses the expression of Jun family genes, thereby affecting T cell activation, proliferation, and apoptosis during T cell development. Funding This work was supported by grants from the National Natural Science Foundation of China (Nos. 3240050774, 32370764 and 32070776, 31871332) and the China Postdoctoral Science Foundation (No. 2024M753227). Conflicts of interest None.