biostudies-literatureUnknown649(8096)Ibanez-Molero SEmerging evidence suggests a correlation between CD8⁺ T cell-tumour cell proximity and anti-tumour immune response^1,2. However, it remains unclear whether these cells exist as functional clusters that can be isolated from clinical samples. Here, using conventional and imaging flow cytometry, we show that from 21 out of 21 human melanoma metastases, we could isolate heterotypic clusters, comprising CD8⁺ T cells interacting with one or more tumour cells and/or antigen-presenting cells (APCs). Single-cell RNA-sequencing analysis revealed that T cells from clusters were enriched for gene signatures associated with tumour reactivity and exhaustion. Clustered T cells exhibited increased TCR clonality indicative of expansion, whereas TCR-matched T cells showed morNature467-476https://www.ebi.ac.uk/biostudies/studies/S-EPMC12779571biostudies-literatureTumour-reactive heterotypic CD8 T cell clusters from clinical samples.PMC12779571Tissier RGeorge Avan den Broek BIbanez-Molero SNilsson JASeignette IMPeeper DSNilsson LMHoefakker KVeldman JAlkemade MHarkes Rvan Hal-van Veen SEStenqvist Svan Baalen MHaanen JBAGPozniak JSimon Nieto JAlondiga-Merida PNieuwland MTol SVisser NLTraets JJHMul EKaromi APack SMvan Houdt WJTas LfalseTumour-reactive heterotypic CD8 T cell clusters from clinical samples.Emerging evidence suggests a correlation between CD8⁺ T cell-tumour cell proximity and anti-tumour immune response^1,2. However, it remains unclear whether these cells exist as functional clusters that can be isolated from clinical samples. Here, using conventional and imaging flow cytometry, we show that from 21 out of 21 human melanoma metastases, we could isolate heterotypic clusters, comprising CD8⁺ T cells interacting with one or more tumour cells and/or antigen-presenting cells (APCs). Single-cell RNA-sequencing analysis revealed that T cells from clusters were enriched for gene signatures associated with tumour reactivity and exhaustion. Clustered T cells exhibited increased TCR clonality indicative of expansion, whereas TCR-matched T cells showed mor2026-01-01T00:00:00Z2026 Jan2026-07-15T14:32:56.063Z2026-07-06T03:09:04.332ZS-EPMC127795714126113510.1038/s41586-025-09754-w