Purpose
Increased numbers of OX40+ T cells in the tumor microenvironment of colorectal cancer (CRC) patients have been associated with improved outcome. However, the OX40+ T cell population is heterogeneous and includes, among others, CD4+Foxp3+ regulatory T cells (Treg) as well as CD4+Foxp3- effector T cells (Teff). In this study, we used a novel triple immunofluorescence assay for CD4, FoxP3 and OX40 to determine the relationship between tumor-associated CD4+ T cell subsets and patient disease stage as well as outcome in CRC.