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.

Purpose: Clinical implications of neoadjuvant immunotherapy in patients with locally advanced but resectable head and neck squamous cell carcinoma (HNSCC) remain largely unexplored. Patients and Methods: Patients with resectable HNSCC were randomized to receive a single dose of preoperative durvalumab (D) with or without tremelimumab (T) before resection, followed by postoperative (chemo)radiotherapy based on multidisciplinary discretion and 1-year D treatment. Artificial intelligence (AI)–powered spatial distribution analysis of tumor-infiltrating lymphocytes and high-dimensional profiling of circulating immune cells tracked dynamic intratumoral and systemic immune responses. Results: Of the 48 patients enrolled (D, 24 patients; D+T, 24 patients), 45 underwent surgical resection per protocol (D, 21 patients; D+T, 24 patients). D±T had a favorable safety profile and did not delay surgery. Distant recurrence-free survival (DRFS) was significantly better in patients treated with D+T than in those treated with D monotherapy. AI-powered whole-slide image analysis demonstrated that D+T significantly reshaped the tumor microenvironment toward immune-inflamed phenotypes, in contrast with the D monotherapy or cytotoxic chemotherapy. High-dimensional profiling of circulating immune cells revealed a significant expansion of T-cell subsets characterized by proliferation and activation in response to D+T therapy, which was rare following D monotherapy. Importantly, expansion of specific clusters in CD8+ T cells and non-regulatory CD4+ T cells with activation and exhaustion programs was associated with prolonged DRFS in patients treated with D+T. Conclusions: Preoperative D±T is feasible and may benefit patients with resectable HNSCC. Distinct changes in the tumor microenvironment and circulating immune cells were induced by each treatment regimen, warranting further investigation.