Japanese Foundation for Cancer Research

TCRαβ+ CD4- CD8- double-negative T (DNT) cells are minor populations in peripheral blood, and their roles have mostly been discussed in inflammation and autoimmunity. However, the functions of DNT cells in tumor microenvironment remain to be elucidated. We investigated their characteristics, possible origins and functions in colorectal cancer tissues as well as their corresponding tumor-draining lymph nodes. We found a significant enrichment of DNT cells in tumor tissues compared with their corresponding lymph nodes, especially in tumors with lower T cell infiltration. T cell receptor (TCR) sequence analysis of CD4+ T, CD8+ T and DNT cells indicated that TCR sequences detected in DNT cells were found in CD8+ T cells, but rarely in CD4+ T cells, suggesting that a part of DNT cells was likely to be originated from CD8+ T cells. Through a single-cell transcriptomic analysis of DNT cells, we found that a DNT cell cluster, which showed similar phenotypes to central memory CD8+ T cells with low expression of effector and exhaustion markers, revealed some specific gene expression patterns, including higher GZMK expression. Moreover, in flow cytometry analysis, we found that DNT cells lost production of cytotoxic mediators. These findings imply that DNT cells might function as negative regulators of anti-tumor immune responses in tumor microenvironment.

Tumours are composed of tumour cells and the surrounding tumour microenvironment (TME), and the molecular characterisation of the various elements of the TME and their interactions is essential for elucidating the mechanisms of tumour progression and developing better therapeutic strategies. Multiplex imaging is a technique that can quantify the expression of multiple protein markers on the same tissue section while maintaining spatial positioning, and this method has been rapidly developed in cancer research in recent years. Many multiplex imaging technologies and spatial analysis methods are emerging, and the elucidation of their principles and features is essential. In this review, we provide an overview of the latest multiplex imaging techniques by type of imaging and staining method and an introduction to image analysis methods, primarily focusing on spatial cellular properties, providing deeper insight into tumour organisation and spatial molecular biology in the TME.