BACKGROUND: Thymocyte selection-associated HMG-BOX (TOX) belongs to a family of transcription factors containing a highly conserved region of the high mobility group box (HMG-Box). A growing body of research has shown that TOX is involved in the occurrence and development of tumors and promotes T-cell exhaustion. We assessed the role of TOX with The Cancer Genome Atlas (TCGA) Pancancer Data.
METHODS: TOX expression was examined with RNA-seq data from the TCGA and Genotype-Tissue Expression (GTEx) databases. The genetic alteration status and protein level of TOX were analyzed using databases, including the Human Protein Atlas (HPA), GeneCards, and STRING. The prognostic significance was estimated with survival data from the TCGA. Moreover, R software was used for enrichment analysis of TOX. The relationship between TOX and immune cell infiltration was assessed with the Tumor Immune Estimation Resource (TIMER) 2.0 database and the \"CIBERSORT\" method. The correlation between TOX and immune checkpoints was further explored. Immunohistochemical analysis was used to further verify the difference in TOX expression between cancerous and paracancerous tissues, and cell viability was evaluated using a CCK-8 assay.
RESULTS: In most cancer types in the TCGA cohort, differential TOX expression was observed. The genetic alteration status and protein level of TOX were examined, and the prognosis of cancers was associated with TOX expression. Moreover, TOX levels were closely related to different immune-related pathways, immune cell infiltration and immune checkpoints. Additionally, significant differences in TOX expression between several cancerous and paracancerous tissues were validated. Furthermore, TOX clearly impacted the viability of cancer cells.
CONCLUSIONS: TOX, a potential biomarker for cancer, may be involved in the regulation of the immune microenvironment and can be used for new targeted drugs.

The fundamental principle of immune checkpoint blockade (ICB) is to protect tumor-infiltrating T cells from being exhausted. Despite the remarkable success achieved by ICB treatment, only a small group of patients benefit from it. Characterized by a hypofunctional state with the expression of multiple inhibitory receptors, exhausted T (Tex) cells are a major obstacle in improving ICB. T cell exhaustion is a progressive process which adapts to persistent antigen stimulation in chronic infections and cancers. In this review, we elucidate the heterogeneity of Tex cells and offer new insights into the hierarchical transcriptional regulation of T cell exhaustion. Factors and signaling pathways that induce and promote exhaustion are also summarized. Moreover, we review the epigenetic and metabolic alterations of Tex cells and discuss how PD-1 signaling affects the balance between T cell activation and exhaustion, aiming to provide more therapeutic targets for applications of combinational immunotherapies.

Rationale: TOX is a DNA-binding factor required for the development of multiple immune cells and the formation of lymph nodes. However, the temporal regulation mode of TOX on NK cell development and function needs to be further explored. Methods: To investigate the role of TOX in NK cells at distinct developmental phases, we deleted TOX at the hematopoietic stem cell stage (Vav-Cre), NK cell precursor (CD122-Cre) stage and late NK cell developmental stage (Ncr1-Cre), respectively. Flow cytometry was used to detect the development and functional changes of NK cell when deletion of TOX. RNA-seq was used to assess the differences in transcriptional expression profile of WT and TOX-deficient NK cells. Published Chip-seq data was exploited to search for the proteins directly interact with TOX in NK cells. Results: The deficiency of TOX at the hematopoietic stem cell stage severely retarded NK cell development. To a less extent, TOX also played an essential role in the physiological process of NKp cells differentiation into mature NK cells. Furthermore, the deletion of TOX at NKp stage severely impaired the immune surveillance function of NK cells, accompanied by down-regulation of IFN-γ and CD107a expression. However, TOX is dispensable for mature NK cell development and function. Mechanistically, by combining RNA-seq data with published TOX ChIP-seq data, we found that the inactivation of TOX at NKp stage directly repressed the expression of Mst1, an important intermediate kinase in Hippo signaling pathway. Mst1 deficient at NKp stage gained the similar phenotype with Toxfl/flCD122Cre mice. Conclusion: In our study, we conclude that TOX coordinates the early mouse NK cell development at NKp stage by maintaining the expression of Mst1. Moreover, we clarify the different dependence of the transcription factor TOX in NK cells biology.

Thymocyte selection-associated high mobility group box protein (TOX) is expressed differently at all T lymphocytes development stages. Owing to more advanced scientific and technological means, including single-cell sequencing technology, heterogeneity of T lymphocytes and TOX has gradually been revealed. Further exploration of such heterogeneity will help us comprehend the developmental stage and functional characteristics of T lymphocytes in greater detail. Emerging evidence supports its regulation not only in exhausting, but also in activating T lymphocytes, thereby verifying TOX heterogeneity. TOX can be used not only as a latent intervention target for tumor diseases and chronic infections, and a therapeutic strategy for autoimmune diseases, but also as a critical factor predicting the drug response and overall survival of patients with malignant tumors.

Thymocyte selection-associated high-mobility group box protein (TOX) is an important molecule regulating the development and exhaustion of T lymphocytes. Our aim is to investigate the role of TOX in the immune pathogenesis of pure red cell aplasia (PRCA). TOX expression of CD8+ lymphocytes from the peripheral blood of patients with PRCA was detected by flow cytometry. Additionally, the expression of immune checkpoint molecules PD1 and LAG3 and cytotoxic molecules perforin and granzyme B of CD8+ lymphocytes was measured. The quantity of CD4+CD25+CD127low T cells was analyzed. TOX expression on CD8+ T lymphocytes in PRCA patients was significantly increased (40.73 [Formula: see text] 16.03 vs. 28.38 [Formula: see text] 12.20). The expression levels of PD1 and LAG3 on CD8+ T lymphocytes in PCRA patients were significantly higher than those in the control group (34.18 [Formula: see text] 13.26 vs. 21.76 [Formula: see text] 9.22 and 14.17 [Formula: see text] 13.74 vs. 7.24 [Formula: see text] 5.44, respectively). The levels of perforin and granzyme in CD8+ T lymphocytes of PRCA patients were 48.60 [Formula: see text] 19.02 and 46.66 [Formula: see text] 25.49, respectively, which were significantly higher than those of the control group (31.46 [Formula: see text] 7.82 and 16.17 [Formula: see text] 4.84, respectively). The number of CD4+CD25+CD127low Treg cells in PRCA patients was significantly decreased (4.30 [Formula: see text] 1.27 vs. 1.75 [Formula: see text] 1.22). In PRCA patients, CD8+ T cells were activated and exhibited overexpression of TOX, PD1, LAG3, perforin, and granzyme B, while regulatory T cells decreased. These findings suggest that T cell abnormality plays a critical role in the pathogenesis of PRCA.

BACKGROUND: Ovarian cancer is one of the most lethal gynecologic malignancies with a dismal prognosis that poses a serious threat to human health, highlighting the need for more knowledge about what is required for identifying some biomarkers for early diagnosis, prediction of prognosis and disease monitoring. TOX, a critical transcription factor related to the development of malignancies that contributing to lymphocytes not just T cells, had been proved prognostic value in some spectrum of cancers. Here, we aimed to study the prognostic role of TOX in ovarian cancer.
RESULTS: We found that TOX was not only expressed in CD8 T cells but also tumor cells. TOX expression score was higher in ovarian cancer tissues and correlated with survival status. Survival analysis revealed that ovarian cancer patients with high TOX expression score generally shorter overall survival and disease-free survival times. Univariate and Multivariate Cox demonstrated that TOX expression score could be used as an independent prognostic factor for patients with ovarian cancer.
CONCLUSIONS: TOX expression in ovarian cancer could be a promising tool for predict overall survival of ovarian cancer patients.

The detection of early coronary atherosclerosis (ECA) is still a challenge and the mechanism of endothelial dysfunction remains unclear. In the present study, we aimed to identify differentially expressed genes (DEGs) and the regulatory network of miRNAs as well as TFs in dysfunctional endothelium to elucidate the possible pathogenesis of ECA and find new potential markers. The GSE132651 data set of the GEO database was used for the bioinformatic analysis. Principal component analysis (PCA), the identification of DEGs, correlation analysis between significant DEGs, the prediction of regulatory networks of miRNA and transcription factors (TFs), the validation of the selected significant DEGs, and the receiver operating characteristic (ROC) curve analysis as well as area under the curve (AUC) values were performed. We identified ten genes with significantly upregulated signatures and thirteen genes with significantly downregulated signals. Following this, we found twenty-two miRNAs regulating two or more DEGs based on the miRNA-target gene regulatory network. TFs with targets ≥ 10 were E2F1, RBPJ, SSX3, MMS19, POU3F3, HOXB5, and KLF4. Finally, three significant DEGs (TOX, RasGRP3, TSPAN13) were selected to perform validation experiments. Our study identified TOX, RasGRP3, and TSPAN13 in dysfunctional endothelium and provided potential biomarkers as well as new insights into the possible molecular mechanisms of ECA.

The specification of the αβ/γδ lineage and the maturation of medullary thymic epithelial cells (mTECs) coordinate central tolerance to self-antigens. However, the mechanisms underlying this biological process remain poorly clarified. Here, we report that dual-stage loss of TOX in thymocytes hierarchically impaired mTEC maturation, promoted thymic IL-17A-producing γδ T-cell (Tγδ17) lineage commitment, and led to the development of fatal autoimmune hepatitis (AIH) via different mechanisms. Transfer of γδ T cells from TOX-deficient mice reproduced AIH. TOX interacted with and stabilized the TCF1 protein to maintain the balance of γδ T-cell development in thymic progenitors, and overexpression of TCF1 normalized αβ/γδ lineage specification and activation. In addition, TOX expression was downregulated in γδ T cells from AIH patients and was inversely correlated with the AIH diagnostic score. Our findings suggest multifaceted roles of TOX in autoimmune control involving mTEC and Tγδ17 development and provide a potential diagnostic marker for AIH.

T-cell exhaustion is one of the key reasons for attenuated T-cell cytotoxicity against tumours. At both the expression and epigenetic levels, a number of genes, including the transcription factor TOX, are believed to be implicated in regulating T-cell exhaustion. In the present study, we found that in NB patients, the ratio of exhausted T cells, featuring upregulated PD-1 and Tim-3, was increased. Meanwhile, the expression of inhibitory surface receptors, including Lag-3, CD160, VISTA and KLRG1, was also increased, but this was accompanied by a reduced ability to release the effector molecules IL-2, IFN-γ, TNF-α and Granzyme B in CD3+ T cells from NB patients. It is noteworthy that NB-derived memory T cells (Tm) showed more obvious exhausted characteristics than other T cells. Moreover, the T cells from NB patients possessed a higher potential for exhaustion conversion upon in vitro TCR stimulation in our time-course culture experiment. In NB patients, T-cell exhaustion was demonstrated to correlate with the elevated expression of TOX in freshly sorted CD3+ T cells as well as in anti-CD3 stimulated PBMCs. Most importantly, our data supported the idea that the hypomethylation of the TOX promoter may be one of the initiators that regulates TOX expression and enables TOX to play a crucial role in T-cell exhaustion reprogramming in NB patients.

The thymocyte selection-related HMG box protein (TOX) subfamily comprises evolutionarily conserved DNA-binding proteins, and is expressed in certain immune cell subsets and plays key roles in the development of CD4+ T cells, innate lymphoid cells (ILCs), T follicular helper (Tfh) cells, and in CD8+ T-cell exhaustion. Although its roles in CD4+ T and natural killer (NK) cells have been extensively studied, recent findings have demonstrated previously unknown roles for TOX in the development of ILCs, Tfh cells, as well as CD8+ T-cell exhaustion; however, the molecular mechanism underlying TOX regulation of these immune cells remains to be elucidated. In this review, we discuss recent studies on the influence of TOX on the development of various immune cells and CD8+ T-cell exhaustion and the roles of specific TOX family members in the immune system. Moreover, this review suggests candidate regulatory targets for cell therapy and immunotherapies.