Increased TOX expression concurrent with PD-1, Tim-3, and CD244 expression in T cells from patients with acute myeloid leukemia.

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.

Increased TOX expression concurrent with PD-1, Tim-3, and CD244 in T cells from patients with non-Hodgkin lymphoma.

AIM: To characterize immune suppression in lymphoma, thymocyte selection-associated high mobility group box protein (TOX) expression and co-expression with programmed cell death receptor-1 (PD-1), T cell immunoglobulin mucin-domain-containing-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. RESULTS: An increased percentage of TOX+ CD3+, CD4+, and CD8+ T cells was found in PB from patients with B cell non-Hodgkin's lymphoma (B-NHL) in comparison with healthy controls. Moreover, TOX+PD-1+ and TOX+Tim-3+ double-positive T cells increased among the CD3+, CD4+, and CD8+T cell populations in the B-NHL group. There was apparent heterogeneity in TOX expression and co-expression with PD-1, Tim-3, and CD244 in CD3+, CD4+, and CD8+ T cells in different lymphoma patients. In addition, the percentage of CD4+CD25+FoxP3+ T cells (Treg) among the CD3+ and CD4+ T cells significantly increased, and the number of TOX+ and TOX+PD-1+ Treg cells also significantly increased in the B-NHL group. CONCLUSIONS: Higher expression of TOX concurrent with PD-1, Tim-3, and CD244 in T cells from patients with B-NHL may contribute to T cell exhaustion and impair their special anti-tumor T cell activity. TOX may be considered a potential target for reversing T cell exhaustion and improving T cell function in hematological malignancies.

Increased TOX expression associates with exhausted T cells in patients with multiple myeloma.

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.

Higher TOX Genes Expression Is Associated With Poor Overall Survival for Patients With Acute Myeloid Leukemia.

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.