Intra-tumoral infiltration of GZMK(high) CD8(+) T effector memory cells is associated with poor clinical outcome in early-stage colo-rectal cancer

Background. A comprehensive understanding of the role of immune reactions in the progression of early-stage colorectal cancer (CRC) is currently lacking. Tumor infiltration by cytotoxic CD8⁺ T cells has been associated with a better prognosis in several solid tumors, including CRC, and high levels of memory CD8⁺ T cells prevent early metastatic invasion and are associated with better survival. However, the tumor immune infiltrate is highly heterogeneous and, although the influence of various myeloid cell populations on T cell activity has been reported, our understanding of the interplay between different cell compartments at the tumor site and their impact on the clinical outcome is still in its infancy. Methods. We sought to gain deeper mechanistic insights into the crosstalk between the TME and immune cells to elucidate their contribution to relapse and to implement patient stratification for immune-based therapeutics. Heterogeneity of immune cells within and across a novel prospective cohort of CRC patients (Stage I-III, treatment-naïve, n=60) was estimated by multiparametric flow cytometry, single-cell transcriptomics and multiplexed cytokine array while also obtaining functional information. Findings were validated on a larger independent cohort of CRC patients and a similar analysis extended to lung cancer (TCGA). Thus, we focused our attention on the crosstalk between CD8⁺ T cells and neutrophils, of which the contribution in inhibiting or promoting tumor progression in humans remains marginally explored and often contradictory. Experiments were conducted in a pre-clinical mouse model and the mechanistic details dissected in vitro, providing initial evidence for future therapeutic applications. Results. We defined an unique immune cell signature in which CRC tumors highly infiltrated by neutrophils contained a population of TILs with a peculiar activation and memory pattern, low levels of PD1 expression and characterized by high levels of Granzyme K (GZMK^high CD8⁺ T_EM cells). Remarkably, GZMK^high CD8⁺ T_EM cells were prospectively correlated with tumor relapse and their gene signature was found to be significantly associated with worse prognosis also in TCGA datasets. Here, we will present molecular details characterizing these interactions. Conclusions. Our study highlighted the emergence of a crosstalk between GZMK^high CD8⁺ T_EM and neutrophils as one of the most important hallmarks in the CRC immune-landscape, crucial to implement current stratification, develop new targets of intervention and guide next steps toward personalized therapeutics. Citation Format: Silvia Tiberti, Carlotta Catozzi, Caterina Scirgolea, Ottavio Croci, Danilo Cagnina, Stefano Campaner, Martin Shaefer, Nicola Fazio, Uberto Fumagalli-Romario, Guangwen Ren, Enrico Lugli, Luigi O. Nezi, Teresa Manzo. Intra-tumoral infiltration of GZMK_high CD8⁺ T effector memory cells is associated with poor clinical outcome in early-stage colo-rectal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P017.