Proliferative T cell infiltration is associated with colorectal cancer molecular subtypes and cancer specific survival

The immune microenvironment is a critical regulator of colorectal cancer (CRC) biology and represents a clinically targetable tumor feature in a subset of patients. T cells are one of the most common cell types in the CRC immune microenvironment and exist in a broad variety of subtypes and activation states. Proliferation status represents a general measure of T cell activity that is applicable to many subtypes. Evidence from other tumor types suggests that proliferating cytotoxic T cells may mediate adaptive immunity in cancer. However, T cell proliferation in CRC is incompletely characterized. To investigate the role of proliferating T cells in CRC, we developed a multiplexed immunofluorescence assay assessing CD3, CD4, CD8, FOXP3, PTPRC (CD45RA and CD45RO), MKI67 (Ki67), and KRT (cytokeratin) expression, and assessed 1,610 primary resected CRC specimens from four US/Canadian cohort studies. Digital imaging and pathologist-supervised machine learning were used to decompose image data into single-cell level information. The Kaplan-Meier method was used to describe CRC-specific survival (CSS) and multivariable Cox proportional hazards models were used to examine associations of proliferating and non-proliferating T cell densities with CSS. Multivariable ordinal logistic regression was used to adjust for confounding factors (age, cancer site, sex, and study batch) in analyses of T cell densities and tumor features. Across one million T cells, 4.5 % were classified as proliferating, as defined by positive MKI67 expression. While higher densities of both proliferating and non-proliferating T cells were positively associated with microsatellite instability (MSI), a CpG island methylator high phenotype, BRAF mutant status and KRAS wildtype status, these associations were stronger for proliferating T cells (e.g., adjusted odds ratio (OR) 2.09, CI 1.62-2.70, p<0.0001 for MSI and higher proliferative T cell density versus adjusted OR 1.40, CI 1.08-1.81, p=0.01 for MSI and higher non-proliferative T cell density). Higher proliferative and non-proliferative T cell densities were both positively associated with CSS (adjusted hazard ratio (HR) 0.75, CI 0.55-1.03, comparing highest versus lowest quartile of proliferative T cell densities (Ptrend=0.018) and adjusted HR 0.42, CI 0.31-0.57, comparing highest versus lowest quartile of non-proliferative T cell densities (Ptrend<0.001). Despite comprising a minor subset of total T cell infiltration in the CRC microenvironment, proliferative T cells are more strongly associated with specific molecular subtypes and are also associated with CSS. These results, across a large series of primary human tumors, suggest that proliferation marks a subset of functionally distinct subset of T cells in the CRC microenvironment.