Dot shape indicates similar tumour samples. (LinCCD7+CD127CCD56+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 T cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated T cells were almost exclusively found in MMR-deficient cancers. Non-activated Bambuterol counterparts of tumour-resident cytotoxic and T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes. Conclusion This 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. (figure 2D). Open in a separate window Figure 2 Activated CD8+ and T cells are tumour tissue-specific and enriched in mismatch repair-deficient colorectal cancers. (A) HSNE embedding of 1 1.6105 landmarks representing the memory CD8+/ T cell compartment (1.1106 cells) from the discovery cohort of patients with CRC coloured by tissue type (first plot) and relative expression of indicated markers. (B) A heatmap showing median marker expression values (left) and frequencies of selected memory CD8+/ T cell clusters (right). Hierarchical clustering was performed on cluster frequencies using Spearmans rank correlation. Colour bars indicate tissue type. (C) Frequencies of selected memory CD8+/ T cell clusters among?CRC tissues (n=35, MMR-deficient (n=13) and MMR-proficient (n=22)), colorectal healthy mucosa (n=17), tumour-associated lymph nodes (n=26) and peripheral blood (n=19) as percentage of total CD45+ cells (upper panel) and memory CD8+ or T cells (lower panel). Cluster IDs correspond to the ones in (B). Bars indicate medianIQR. Each dot represents an individual sample. Data from 22 independent Bambuterol experiments with mass cytometry. *Pon CD4+ T cells was confirmed by single-cell RNA-sequencing, which also revealed the expression of (((figure 5A). We performed additional single-cell RNA-sequencing on CD45+ cells Bambuterol from one MMR-deficient tumour with high numbers of LinCCD7+CD127CCD56+CD45RO+ ILCs (70% of the ILC cluster), as revealed by mass cytometry data. Here, we also observed high expression levels of cytotoxic molecules (eg, and in the ILC cluster (online supplementary figure S7). Cell surface expression of KIRs was confirmed by flow cytometry in LinCCD7+CD127CCD56+CD45RO+ ILCs from this tumour (online supplementary figure S7). Open in a separate window Figure 5 Tumour-resident ILCs are involved in the antitumour immune response. (A) Violin Bambuterol plot showing log-transformed expression levels of the top 20 differentially expressed genes within ILCs (n=74) analysed by single-cell RNA-sequencing on CD45+ cells from CRC tissues (n=7)?(figure 1D). Each dot represents a single cell. (B) Representative plots of a MMR-deficient tumour analysed by flow cytometry without stimulation showing the distinction between CD45RO+ ILCs and CD45RA+ NK cells within LinCCD7+CD127CCD56+ cells (first plot) and their expression of Bambuterol cytotoxic molecules. (C) Granzyme B/perforin expression in different immune cell populations of CRC tissues (n=6, of which 4 MMR-deficient and 2 MMR-proficient). Dot shape indicates similar tumour samples. Data from three independent experiments with flow cytometry. CRC,?colorectal cancer; ILC, innate lymphoid cell; MMR-d, mismatch repair-deficient; MMR-p, mismatch repair-proficient; NK, natural killer. To further investigate functional properties of tumour-resident lymphocytes, we designed a flow cytometry antibody panel to analyse the cytotoxic potential of LinCCD7+CD127CCD56+CD45RO+ ILCs, LinCCD7+CD127CCD56+CD45RA+ NK cells and memory CD8+ T cells in CRC tissues. Strikingly, up to 82.3% of unstimulated CD127CCD56+CD45RO+ ILCs displayed granzyme B/perforin expression TSPAN5 in the tumour tissues (figure 5B). Granzyme B/perforin expression by the ILCs was most abundant in MMR-deficient cancers as compared with MMR-proficient cancers (figure 5C). Interestingly, the cytotoxic capacity of CD127CCD56+CD45RO+ ILCs was accompanied by similar profiles in CD127CCD56+CD45RA+ NK cells and memory CD8+ T cells across samples (figure 5C), suggesting a coordinated cytotoxic innate and adaptive immune response in CRC tissues. To investigate the spatial localisation of the ILCs in.