Related Experiment Video
Updated: Dec 27, 2025
Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice
Published on: June 12, 2021
Single-cell transcriptome analysis reveals TOX as a promoting factor for T cell exhaustion and a predictor for
Kyungsoo Kim1, Seyeon Park2, Seong Yong Park3
1Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul, 03722, Korea.
Background:
T cells exhibit heterogeneous functional states in the tumor microenvironment. Immune checkpoint inhibitors (ICIs) can reinvigorate only the stem cell-like progenitor exhausted T cells, which suggests that inhibiting the exhaustion progress will improve the efficacy of immunotherapy. Thus, regulatory factors promoting T cell exhaustion could serve as potential targets for delaying the process and improving ICI efficacy.
Methods:
We analyzed the single-cell transcriptome data derived from human melanoma and non-small cell lung cancer (NSCLC) samples and classified the tumor-infiltrating (TI) CD8+ T cell population based on PDCD1 (PD-1) levels, i.e., PDCD1-high and PDCD1-low cells. Additionally, we identified differentially expressed genes as candidate factors regulating intra-tumoral T cell exhaustion. The co-expression of candidate genes with immune checkpoint (IC) molecules in the TI CD8+ T cells was confirmed by single-cell trajectory and flow cytometry analyses. The loss-of-function effect of the candidate regulator was examined by a cell-based knockdown assay. The clinical effect of the candidate regulator was evaluated based on the overall survival and anti-PD-1 responses.
Results:
We retrieved many known factors for regulating T cell exhaustion among the differentially expressed genes between PDCD1-high and PDCD1-low subsets of the TI CD8+ T cells in human melanoma and NSCLC. TOX was the only transcription factor (TF) predicted in both tumor types. TOX levels tend to increase as CD8+ T cells become more exhausted. Flow cytometry analysis revealed a correlation between TOX expression and severity of intra-tumoral T cell exhaustion. TOX knockdown in the human TI CD8+ T cells resulted in downregulation of PD-1, TIM-3, TIGIT, and CTLA-4, which suggests that TOX promotes intra-tumoral T cell exhaustion by upregulating IC proteins in cancer. Finally, the TOX level in the TI T cells was found to be highly predictive of overall survival and anti-PD-1 efficacy in melanoma and NSCLC.
Conclusions:
We predicted the regulatory factors involved in T cell exhaustion using single-cell transcriptome profiles of human TI lymphocytes. TOX promoted intra-tumoral CD8+ T cell exhaustion via upregulation of IC molecules. This suggested that TOX inhibition can potentially impede T cell exhaustion and improve ICI efficacy. Additionally, TOX expression in the TI T cells can be used for patient stratification during anti-tumor treatments, including anti-PD-1 immunotherapy.
Insights
TOX, a transcription factor, drives T cell exhaustion in tumors by increasing immune checkpoint molecules. Inhibiting TOX may enhance immunotherapy efficacy and predict patient response to anti-PD-1 treatments.
Area of Science:
- Immunology
- Cancer Biology
- Molecular Biology
Background:
- T cells in the tumor microenvironment display varied functional states.
- Immune checkpoint inhibitors (ICIs) primarily benefit T cells with stem-like progenitor exhaustion.
- Targeting T cell exhaustion regulators could enhance immunotherapy effectiveness.
Purpose of the Study:
- Identify regulatory factors of intra-tumoral T cell exhaustion.
- Investigate the role of these factors in T cell exhaustion and ICI efficacy.
- Determine the predictive value of these factors for patient outcomes.
Main Methods:
- Single-cell transcriptome analysis of human melanoma and NSCLC tumor-infiltrating CD8+ T cells.
- Classification of T cells based on PDCD1 (PD-1) expression levels.
- Identification of differentially expressed genes and validation of co-expression with immune checkpoint molecules.
- Loss-of-function assays (knockdown) and evaluation of clinical outcomes (overall survival, anti-PD-1 response).
Main Results:
- TOX identified as a key transcription factor promoting T cell exhaustion in both melanoma and NSCLC.
- TOX expression correlates with increased exhaustion and upregulation of immune checkpoints (PD-1, TIM-3, TIGIT, CTLA-4).
- TOX knockdown reduces immune checkpoint expression, suggesting TOX drives exhaustion.
- TOX levels predict overall survival and response to anti-PD-1 therapy.
Conclusions:
- TOX promotes intra-tumoral CD8+ T cell exhaustion by upregulating immune checkpoint molecules.
- TOX inhibition presents a potential strategy to enhance ICI efficacy.
- TOX expression serves as a predictive biomarker for patient stratification in anti-cancer treatments.
