Functional γδT-omics pipeline reveals compartmentalization of Vδ1⁺ T cell migration, tumor-reactivity, and clonality in human colorectal cancer

Gatti, L. C. D. E.; Nicolasen, M. J. T.; Keramati, F.; Brazda, P.; Meringa, A. D.; De Bont, D. A.; Aarts-Riemens, T.; Brandwijk, W. J. C.; van der Wijst, M. I. M.; Stuut, A. H. G.; Gasull Celades, L.; Zawal, D.; Vazaios, K.; Daudeij, A.; Huismans, M. A.; Parigiani, M. A.; Straetemans, T.; Beringer, D. X.; Kranenburg, O.; Berlin, C.; Minguet, S.; Kesselring, R.; Stunnenberg, H. G.; Roodhart, J. M. L.; Sebestyen, Z.; Kuball, J.

doi:10.1101/2025.08.19.671055

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bioRxiv · 10.1101/2025.08.19.671055

Functional γδT-omics pipeline reveals compartmentalization of Vδ1⁺ T cell migration, tumor-reactivity, and clonality in human colorectal cancer

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Abstract

{gamma}{delta}T cells play a pivotal role in cancer immune surveillance, yet the current knowledge of their function across the compartments in solid tumors is meager. To address this gap, we developed a comprehensive {gamma}{delta}T-omics platform that integrates functional screening, biomimetic migration assays, {gamma}{delta}TCR repertoire analysis, and transcriptomic profiling. Using matched samples from 31 patients with microsatellite-stable colorectal cancer (CRC), we analyzed {gamma}{delta}T cells from peripheral blood (PBLs{gamma}{delta}), adjacent colon (LPLs{gamma}{delta}), primary tumors (pTILs{gamma}{delta}), and liver metastases (mTILs{gamma}{delta}). This approach uncovered striking compartmentalization of {gamma}{delta}T cell phenotypes, clonality, and function. Tumor-reactive, clonally expanded V{delta}1 {gamma}{delta}T cells were enriched in primary tumors and shared transcriptional and functional features with lamina propria lymphocytes (LPLs). In contrast, V{delta}1 {gamma}{delta}T cells from liver metastases lacked tumor reactivity, exhibited distinct {gamma}{delta}TCR repertoires, and expressed transcriptional signatures associated with TGF-{beta}-mediated suppression and cellular quiescence, suggesting they are shaped by tissue-specific environmental cues. CXCL16 secretion by tumor cells initiated V{delta}1 LPLs{gamma}{delta} migration, which was further amplified by {gamma}{delta}TCR-mediated CCL5 induction from pTILs{gamma}{delta}, leading to CCR5 downregulation and subsequent entrapment of pTILs{gamma}{delta} within the tumor microenvironment. Accordingly, our clinical data from an independent second cohort of 69 patients showed that infiltration by pTILs{gamma}{delta}, but not mTILs{gamma}{delta}, is associated with a protective effect against CRC progression. In summary, our study offers a compartment-resolved perspective on {gamma}{delta}T cell behavior in CRC, revealing key trafficking and functional mechanisms, and enabling the identification of novel tumor-reactive {gamma}{delta}TCRs and migratory cues to inform immunotherapeutic strategies for both primary and metastatic CRC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/671055v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@ff3bborg.highwire.dtl.DTLVardef@14a7e1forg.highwire.dtl.DTLVardef@19e4de4org.highwire.dtl.DTLVardef@bc600_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Gatti, L. C. D. E., Nicolasen, M. J. T., Keramati, F., Brazda, P., Meringa, A. D., De Bont, D. A., Aarts-Riemens, T., Brandwijk, W. J. C., van der Wijst, M. I. M., Stuut, A. H. G., Gasull Celades, L., Zawal, D., Vazaios, K., Daudeij, A., Huismans, M. A., Parigiani, M. A., Straetemans, T., Beringer, D. X., Kranenburg, O., Berlin, C., Minguet, S., Kesselring, R., Stunnenberg, H. G., Roodhart, J. M. L., Sebestyen, Z., Kuball, J.. 2025-08-23. Functional γδT-omics pipeline reveals compartmentalization of Vδ1⁺ T cell migration, tumor-reactivity, and clonality in human colorectal cancer. https://doi.org/10.1101/2025.08.19.671055

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