TOX (thymocyte selection-associated HMG BOX) is a member of a family of transcriptional factors that contain the highly conserved high mobility group box (HMG-box) region. Increasing studies have shown that TOX is involved in maintaining tumors and promoting T cell exhaustion. In this review, we summarized the biological functions of TOX and its contribution as related to lymphocytic malignancies. We also discussed the potential role of TOX as an immune biomarker and target in immunotherapy for hematological malignancies.

Myelodysplastic syndrome (MDS) is an aggressive and genetically heterogeneous disease with poor prognosis. Cellular immune disorder is a common characteristic of this disease and is thought to be related to clinical outcome. Alterations in T cell clonal expansion and T cell dysfunction has been detected in MDS patients. Little is known about whether there are immune biomarkers to evaluate the T cell alterations with clinical outcome. Previous studies have demonstrated that B-cell leukemia/lymphoma 11B (BCL11B) plays an important role in regulating T cell development and proliferation. In this study, the prognostic value of BCL11B for MDS patients was explored by analyzing RNA-seq data from 270 patients in two datasets in the Gene Expression Omnibus (GEO) database and real-time quantitative PCR data (qRT-PCR) of 31 bone marrow (BM) samples of MDS and 6 BM samples of patients with MDS progress to secondary acute myeloid leukemia (sAML) from our clinical center. The results demonstrated that BCL11B is significantly down-regulated in MDS patients as compared with healthy individuals (HIs). Importantly, lower BCL11B expression was found in MDS patients who were of high/very high risk, older than 60 y, or male and patients with sAML. Furthermore, low BCL11B expression appeared to be associated with poor overall survival (OS) for MDS patients, though the data were not yet significant enough at this point. In addition, BCL11B low-expressing MDS patients had shorter restricted mean survival time (RMST) than those with high BCL11B expression. Interestingly, BCL11B positively correlated with naive and activated memory CD4 + T cells, CD8 + T cells, and the T cell receptor complex genes CD3E and CD3G, but it negatively correlated with regulatory T cells (Treg). Additionally, co-occurrence of low BCL11B expression and CD3E and CD3G was associated with poor OS and shorter RMST. In conclusion, lower BCL11B expression in BM samples of MDS patients was associated with adverse clinical outcome.

Thymocyte selection-associated HMG box (TOX) is a transcription factor that belongs to the high mobility group box (HMG-box) superfamily, which includes four subfamily members: TOX, TOX2, TOX3, and TOX4. TOX is related to the formation of multiple malignancies and contributes to CD8+ T cell exhaustion in solid tumors. However, little is known about the role of TOX genes in hematological malignancies. In this study, we explored the prognostic value of TOX genes from 40 patients with de novo acute myeloid leukemia (AML) by quantitative real-time PCR (qRT-PCR) in a training cohort and validated the results using transcriptome data from 167 de novo AML patients from the Cancer Genome Atlas (TCGA) database. In the training cohort, higher expression of TOX and TOX4 was detected in the AML samples, whereas lower TOX3 expression was found. Moreover, both the training and validation results indicated that higher TOX2, TOX3, and TOX4 expression of AML patients (3-year OS: 0% vs. 37%, P = 0.036; 3-year OS: 4% vs. 61%, P < 0.001; 3-year OS: 0% vs. 32%, P = 0.010) and the AML patients with highly co-expressed TOX, TOX2, TOX4 genes (3-year OS: 0% vs. 25% vs. 75%, P = 0.001) were associated with poor overall survival (OS). Interestingly, TOX2 was positively correlated with CTLA-4, PD-1, TIGIT, and PDL-2 (rs = 0.43, P = 0.006; rs = 0.43, P = 0.006; rs = 0.56, P < 0.001; rs = 0.54, P < 0.001). In conclusion, higher expression of TOX genes was associated with poor OS for AML patients, which was related to the up-regulation of immune checkpoint genes. These data might provide novel predictors for AML outcome and direction for further investigation of the possibility of using TOX genes in novel targeted therapies for AML.

Previous studies have shown increased aberrant expression of immune checkpoint (IC) proteins, such as programmed cell death receptor-1 (PD-1) and T cell immunoglobulin mucin-domain-containing-3 (Tim-3) on T cells from patients with multiple myeloma (MM), which result in T cell exhaustion and dysfunction. However, little is known about the mechanism regulating aberrant IC protein expression. In this study, we analyzed the expression of TOX (thymocyte selection-associated HMG BOX), a crucial transcription factor involved in T cell exhaustion, and its co-expression with PD-1, Tim-3, and CD244 in T cell subsets by multi-color fluorescent flow cytometry in peripheral blood (PB) and bone marrow (BM) samples from patients with MM. Significantly, the percentage of TOX + CD3 +/CD4 +/CD8 + T cells was increased, and similarly, higher numbers of TOX co-expression with PD-1, Tim-3, and CD244 on CD3 +/CD4 +/CD8 + T cells were found. Interestingly, the numbers of TOX +, TOX + PD-1 +, and TOX + Tim-3 + regulatory T (Treg) cells also significantly increased in both the PB and BM of MM patients. In summary, we for the first time observed increased TOX expression concurrent with PD-1, Tim-3, and CD244 on T cells, which may contribute to T cell exhaustion and impair their function in MM. Thus, TOX may be considered a potential target for reversing T cell exhaustion and improving T cell function in MM.

Aim: To characterize immune suppression in lymphoma, thymocyte selectionassociated high mobility group box protein (TOX) expression and co-expression with programmed cell death receptor-1 (PD-1), T cell immunoglobulin mucin-domaincontaining-3 (Tim-3), and CD244 in CD3+, CD4+, CD8+, and regulatory T (Treg) cells from patients with lymphomas were analyzed.Methods: TOX expression and co-expression with PD-1, Tim-3, and CD244 in CD3+, CD4+, Treg, and CD8+ T cells were analyzed by multi-color fluorescent flow cytometry using peripheral blood (PB) from 13 newly diagnosed, untreated lymphoma patients, and 11 healthy individuals.

Background T cell dysregulation is a common event in leukemia. Recent findings have indicated that aberrant expression of immune checkpoint proteins may be associated with disease relapse and progression in acute myeloid leukemia (AML). TOX, a transcription factor in the HMG‐box protein superfamily, was found to be a potential target for immunotherapy not only in solid tumors but also in hematological malignancies. However, little is known about TOX expression and co‐expression with immune checkpoint proteins or the exhausted phenotype in the T cell subsets in AML. Thus, in this study, we analyzed TOX expression and co‐expression with PD‐1, Tim‐3, and CD244 in T cells. Methods TOX expression and co‐expression with PD‐1, Tim‐3, and CD244 in CD3+, CD4+, regulatory T (Treg), and CD8+ T cells were analyzed by multi‐color fluorescent flow cytometry in peripheral blood (PB) and bone marrow (BM) samples from patients with de novo AML and AML in complete remission (CR) and healthy individuals (HIs). Results A significantly increased percentage of TOX+CD3+, CD4+, and CD8+ T cells was found in PB from patients with de novo AML in comparison with HIs. Double‐positive TOX+CD244+, TOX+PD‐1+, and TOX+Tim‐3+ T cells markedly increased in the CD3+, CD4+, and CD8+ T cell populations in de novo AML patients compared with HIs, and similar trends were demonstrated for TOX+Tim‐3+CD3+/CD4+/CD8+ T cells in de novo AML compared with AML‐CR patients. In addition, the number of TOX+, TOX+PD‐1+, and TOX+Tim‐3+Treg cells significantly increased in de novo AML patients compared with HIs, and TOX+PD‐1+Treg cells were higher in de novo AML compared with AML‐CR patients. Moreover, TOX positively correlated with Tim‐3 expression in CD8+ and Treg cells, and a positive correlation between the expression of TOX+ CD4+ and CD244+CD4+ T cells was found. Furthermore, an increased percentage of TOX+Tim‐3+ T cells in BM was also found in de novo AML patients compared with HIs. Conclusions Increased TOX concurrent with PD‐1, Tim‐3, and CD244 in T cells may contribute to T cell exhaustion and impair their function in AML. Such exhausted T cells may be partially revised when AML patients achieve CR after chemotherapy. TOX may be considered a potential target for reversing T cell exhaustion and improving T cell function in AML.

V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) is considered as an immunosuppressive factor and potential therapeutic target for anticancer therapy. However, little is known about VISTA expression and its role in immunosuppression in multiple myeloma (MM). In this study, VISTA expression and co-expression with programmed cell death receptor-1 (PD-1), T cell immunoglobulin mucin-domain-containing-3 (Tim-3), and T cell immunoglobulin and ITIM domain (TIGIT) in CD3+, CD4+, CD8+, and regulatory T (Treg) cells were analyzed in patients with MM by multi-color fluorescent flow cytometry of peripheral blood (PB) and bone marrow (BM) samples from 36 patients with MM and compared to 36 PB samples and 10 BM samples from healthy individuals (HIs), which served as controls. The results demonstrated a significant increased percentage of VISTA co-expression with PD-1, Tim-3, and TIGIT in CD3+, CD4+, CD8+, and Treg cells in PB from MM patients compared with HIs. A similar trend for VISTA+CD8+ T cells was found in BM. Moreover, a trend of a high percentage on VISTA expression and co-expression in PB rather than BM was found. Furthermore, significant positive correlations existed for VISTA expression concurrent with PD-1, Tim-3, and TIGIT in T cell subsets and clinical indicators, including Revised International Staging System (R-ISS) staging of multiple myeloma, Eastern Cooperative Oncology Group (ECOG) score, and beta-2-microglobulin (β2-MG). In conclusion, higher VISTA expression concurrent with PD-1, Tim-3, and TIGIT on T cells, particularly in the PB of patients with MM, may result in T cell exhaustion and dysfunction and be closely associated with disease progression and clinical indicators. Thus, VISTA may be considered a potential target for reversing T cell exhaustion and improving T cell function in MM.

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