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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\n                    <jats:sup>+<\/jats:sup>\n                    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 more exhaustion and co-modulation when conjugated to APCs than when conjugated to tumour cells. T cells that were expanded from clusters ex vivo exerted on average ninefold increased killing activity towards autologous melanomas, which was accompanied by enhanced cytokine production. After adoptive cell transfer into mice, T cells from clusters showed improved patient-derived melanoma control, which was associated with increased T cell infiltration and activation. Together, these results demonstrate that tumour-reactive CD8\n                    <jats:sup>+<\/jats:sup>\n                    T cells are enriched in functional clusters with tumour cells and\/or APCs and that they can be isolated and expanded from clinical samples. Typically excluded by single-cell gating in flow cytometry, these distinct heterotypic T cell clusters are a valuable source to decipher functional tumour\u2013immune cell interactions and may also be\u00a0therapeutically explored.\n                  <\/jats:p>","DOI":"10.1038\/s41586-025-09754-w","type":"journal-article","created":{"date-parts":[[2025,11,19]],"date-time":"2025-11-19T16:02:08Z","timestamp":1763568128000},"page":"467-476","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":27,"title":["Tumour-reactive heterotypic CD8 T cell clusters from clinical samples"],"prefix":"10.1038","volume":"649","author":[{"given":"Sof\u00eda","family":"Ib\u00e1\u00f1ez-Molero","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Johanna","family":"Veldman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Simon Nieto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joleen J. 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J.B.A.G.H. has provided consultation, attended advisory boards and\/or provided lectures for BMS, CureVac, GSK, Imcyse, Iovance Bio, Instil Bio, Immunocore, Ipsen, Merck Serono, MSD, Molecular Partners, Novartis, Pfizer, Roche\/Genentech, Sanofi, Scenic and Third Rock Ventures; has participated in the scientific advisory board of Achilles Tx, BioNTech US, Instil Bio, PokeAcell, T-Knife, Scenic and Neogene Therapeutics; and through this the NKI has received grant support from Amgen, Asher Bio, BioNTech, BMS, MSD, Novartis, Sastra Cell Therapy, has stock options in Neogene Tx. D.S.P. is co-founder, shareholder and advisor of Flindr Therapeutics, which is unrelated to this study. J.V. is funded by a Veni-fellowship from the Dutch Research Council (NWO): 09150162210069. J.A.N. is funded by the Swedish Cancer Society, Vetenskapsr\u00e5det, the Erling-Persson and Sj\u00f6berg Foundations. D.S.P. is funded by the Oncode Institute, the Dutch Cancer Society KWF and by an ERC Advanced grant 101054465\u2014ReverT. The other authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}]}}