Single-cell, Spatially-Resolved TCR Profiling Links T Cell Phenotype and Clonality in Human Tumors

Despite remarkable success in melanoma and other cancers, immune checkpoint blockade has shown limited efficacy in head and neck squamous cell carcinoma (HNSCC), with durable responses in only [~]10% of patients. This is paradoxical given the abundant infiltration of CD8 T cells in HNSCC, suggesting that current T cell profiling methods fail to fully capture functional anti-tumor immunity and that key immunosuppressive mechanisms remain poorly understood. To uncover mechanisms regulating T cell function in situ, we developed a multimodal profiling framework integrating high-dimensional flow cytometry, single-cell RNA- and TCR-sequencing, and spatial transcriptomics, including spatially-resolved TCR profiling at single-cell resolution. Applying this approach to paired blood and tumor samples from 27 HNSCC patients enabled single-cell resolution mapping of T cell clonality, transcriptional state, and spatial localization within intact tumors. Tumors with similar clinical features and immune profiles by flow cytometry and scRNA-seq exhibited markedly different spatial immune architectures. Within the T cell compartment, stem- and memory-like T cells localized within immune-rich niches, while exhausted cells were broadly dispersed in tumor-rich regions. Targeted spatial TCR mapping revealed that tumor-enriched TCR clones were distributed throughout the tumor yet adopted distinct, location-dependent phenotypes, indicating that both antigen specificity and spatial cues shape T cell differentiation and function in human tumors. These findings reveal fundamental principles of T cell organization in solid tumors and establish a versatile platform for spatially-resolved, antigen-specific T cell profiling.