Functional heterogeneity of lymphocytic patterns in primary melanoma dissected through single-cell multiplexing
- KU Leuven and UZ Leuven, Belgium
- ProtATonce Ltd, Greece
- Università degli studi di Milano-Bicocca, Italy
- KU Leuven, Belgium
Abstract
In melanoma, the lymphocytic infiltrate is a prognostic parameter classified morphologically into 'brisk', 'non-brisk' and 'absent' entailing a functional association that has never been proved. Recently, it has been shown that lymphocytic populations can be very heterogeneous, and that anti-PD-1 immunotherapy supports activated T cells. Here, we characterize the immune landscape in primary melanoma by high-dimensional single cell multiplex analysis in tissue sections (MILAN technique) followed by image analysis, RT-PCR and shotgun proteomics. We observed that the brisk and non-brisk patterns are heterogeneous functional categories that can be further sub-classified into active, transitional or exhausted. The classification of primary melanomas based on the functional paradigm also shows correlation with spontaneous regression, and an improved prognostic value when compared to that of the brisk classification. Finally, the main inflammatory cell subpopulations that are present in the microenvironment associated with activation and exhaustion and their spatial relationships are described using neighbourhood analysis.
Data availability
All data generated or analysed during this study are included in the submission as source data files. We also included the codes to ease the review in process.
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TCGA-SKCMTCGA, TCGSKCM phs000178.
Article and author information
Author details
Francesca Maria Bosisio
Giorgio Cattoretti
Funding
Horizon 2020 Framework Programme (642295)
- Francesca Maria Bosisio
Horizon 2020 Framework Programme (675585)
- Asier Antoranz
BEL114054 (HGS1006-C1121)
- Maddalena Maria Bolognesi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Human subjects: Ethical approval was obtained from the Ethical Committee/IRB OG032 of the University Hospital of Leuven. After the approval, the study was identified with the number S57266. According to the Clinical Trial regalement no informed consent was needed due to the use of post-diagnostic left-over material.
Copyright
© 2020, Bosisio et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Functional heterogeneity of lymphocytic patterns in primary melanoma dissected through single-cell multiplexing
eLife 9:e53008.
https://doi.org/10.7554/eLife.53008
Further reading
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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 with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq 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-seq, 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.
