High-dimensional cytometric analysis of colorectal cancer reveals novel mediators of antitumour immunity

Abstract

ObjectiveA comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC.DesignThirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence.ResultsWe discovered that a previously unappreciated innate lymphocyte population (Lin–CD7+CD127–CD56+CD45RO+) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103+CD69+) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and γδ T cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated γδ T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδ T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes.ConclusionThis work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.

Pleiotropic Role and Bidirectional Immunomodulation of Innate Lymphoid Cells in Cancer

“…State-of-the-art technologies like single-cell RNA-sequencing (173) and high-dimensional mass spectrometry (CyTOF) analysis (179) have allowed us to investigate ILCPs, delineate distinct ILCs subsets and their developmental pathways, and identify novel mediators of antitumor immunity at single-cell resolution levels. These studies present new perspectives for exploring the complexity of ILCs in the diverse context of cancers and allow effective manipulation of ILC immunosurveillance to achieve optimal therapeutic opportunities.…”

Innate Lymphoid Cells in Colorectal Cancers: A Double-Edged Sword

“…These intraepithelial ILC1s can be activated by chemokines and cytokines such as IL-18 produced by intestinal epithelial cells (63) or IL-12 from dendritic cells and monocytes (62). This induces IFN-γ production in ILC1 which, in contrast to previously mentioned studies (59,60), drives the pathogenesis of anti-CD40-induced colitis in Rag1 −/− mice (62) ( Table 3 and Figure 1A). Given these divergent outcomes, further studies are required to analyze the exact role of intraepithelial ILC1, their receptor expression and cytokine production using genetically-modified conditionallydeficient mice and human samples.…”

“…High-dimensional cytometric analyses have revealed that human colorectal tumors are highly infiltrated by NK cells and intraepithelial-like ILC1 (59,60). They secrete cytotoxic molecules such as granzymes and perforin (59,60) and cytokines such as IFN-γ following stimulation (59), making them key potential players in cytotoxic anti-tumor responses ( Figure 1A).…”

“…Nevertheless, ILC2 frequency is increased in mucosal samples from IBD patients compared with healthy individuals (74). In CRC tumors, ILC2s express higher levels of ICOS and CD69 compared with ILC2 found in normal colon tissue (59,60). These phenotypically distinct ILC2s could be a potential source of IL-4, IL-5, and IL-13 as both IL-4 and IL-13 are found at elevated levels in the serum of CRC bearing mice (75).…”

“…Furthermore, local IL-23 expression drives ILC3 accumulation and activation within the colonic lamina propria (99,100). These intestinal ILC3 express higher levels of the activating receptors ICOS and CD69 (59,60), regulators of tumor responses CD244 and CD39 (59), and exhibit enhanced production of the downstream signaling cytokines IL-17 and IL-22 that contribute to the pathogenesis of CRC (96) ( Table 3 and Figure 1C).…”