✍🏼 Joakim Karlsson, Vasu R Sah, Roger Olofsson Bagge, Irina Kuznetsova, Munir Iqbal, Samuel Alsén, Sofia Stenqvist, Alka Saxena, Lars Ny, Lisa M Nilsson, Jonas A Nilsson

🏠 Harry Perkins Institute of Medical Research and University of Western Australia, Nedlands, WA, Australia

📑 eLife (2024)

Abstract
Uveal melanoma (UM) is a rare melanoma originating in the eye’s uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients succumb to recurrent disease after two years. This study aimed to provide a path towards enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA sequencing of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR sequencing, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

How the WOLF was used in this study
In this study, the WOLF cell sorter was employed to isolate activated tumor-reactive T cells from co-cultures of tumor spheroids and tumor-infiltrating lymphocytes (TILs). Specifically, after co-culturing PDX-derived tumor spheroids with TILs, cells expressing both CD3 and the activation marker CD137 were identified and sorted using the WOLF. The purpose of this sorting step was to enrich for antigen-specific, activated T cells for downstream analyses, including RNA extraction and T-cell receptor (TCR) profiling. This approach enabled precise characterization of the TCR repertoire of tumor-reactive T cells, facilitating subsequent single-cell RNA sequencing and functional studies of immune responses in uveal melanoma models.