[SOX4 drives the differentiation of CD4⁺T cells toward an immunosuppressive phenotype]

Objective To investigate the regulatory effects of the SRY-box transcription factor 4 (SOX4) on differentiation and oxidative phosphorylation (OXPHOS) metabolism of CD4⁺T cells. Methods CD4⁺T cells were isolated from healthy human peripheral blood using magnetic bead sorting. After stimulation with CD3 and CD28 antibodies for two days, CD4⁺T cells were infected with lentivirus to establish the SOX4-overexpression (SOX4-OE) group and the empty vector (Vector) group, respectively. Flow cytometry was used to analyze the expression of phenotypic markers [CD25, programmed cell death protein 1 (PD-1), cytotoxic lymphocyte antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), tumor necrosis factor receptor superfamily member 4 (OX-40)], the transcription factor forkhead box P3 (FOXP3), intracellular cytokines [transforming growth factor β1(TGF-β1), interleukin 10(IL-10), interferon γ(IFN-γ), IL-4, IL-17A ], and OXPHOS-related indicators [adenosine triphosphate (ATP), reactive oxygen species (ROS), mitochondrial reactive oxygen species (mtROS), mitochondrial mass, mitochondrial respiratory chain complexes] in CD4⁺T cells from the SOX4-OE group and the Vector group. RNA sequencing was conducted to identify differentially expressed genes (DEGs) between the SOX4-OE and Vector groups, followed by enrichment analyses on the identified DEGs including Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and GeneSet Enrichment Analysis (GSEA). Results Compared to the Vector group, the SOX4-OE group exhibited significantly increased proportions of CD4⁺T cells expressing the activation markers CD25 and GITR, as well as the inhibitory molecules CTLA-4 and PD-1, while the proportion of cells expressing OX-40 remained unchanged between the two groups. The transcription factor FOXP3 was significantly upregulated in SOX4-OE CD4⁺T cells compared to the Vector group. Regarding intracellular cytokines, the proportions of CD4⁺T cells producing TGF-β1, IFN-γ, and IL-17A were significantly higher in the SOX4-OE group than in the Vector group. Conversely, the proportion of IL-10-producing cells was significantly lower, while no significant difference was observed for IL-4. RNA sequencing analysis of DEGs in CD4⁺T cells from the SOX4-OE and Vector groups revealed significant enrichment of the OXPHOS pathway via KEGG pathway analysis. GO enrichment analysis indicated that DEGs were significantly enriched in terms of immune response regulation, aerobic electron transport chain, and positive regulation of TGF-β production. GSEA showed that, compared with the Vector group, SOX4-OE CD4⁺T cells exhibited upregulation in the TGF-β receptor complex-mediated signaling pathway and in the transcriptional activity of Sma and Mad homolog 2 (SMAD2)/SMAD3: SMAD4 heterotrimer complex. Moreover, SOX4 overexpression enhanced ATP production and the levels of mitochondrial respiratory chain complexs while reducing ROS and mtROS levels in CD4⁺T cells, with no significant change in mitochondrial mass. Conclusion We demonstrate that SOX4 induces a plastic Treg-like state in CD4⁺T cells, modulates the expression of key molecules associated with Treg suppressive function, and enhances their oxidative phosphorylation.