Identifying Tumor-Reactive Lymphocytes in Uveal Melanoma Liver Metastases Using Single-Cell Sequencing and Patient-Derived Xenograft Models

Core Concepts

Combining single-cell sequencing and functional analysis provides insights into tumor-reactive T cells in uveal melanoma, which can be useful for cell therapy amplification and marker selection.

Abstract

This study aimed to identify and functionally validate tumor-reactive T cells in liver metastases of patients with uveal melanoma (UM). The researchers employed single-cell RNA sequencing of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. They also created patient-derived xenograft (PDX) models of UM metastases and generated tumor sphere cultures for co-culture with autologous or MART1-specific HLA-matched allogenic TILs.
The key findings are:

Tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells.
Combining single-cell sequencing and functional analysis revealed that tumor-reactive T cells tended to have an exhausted phenotype, expressing markers like PD-1, ICOS, and HAVCR2.
Some tumor-reactive T cells recognized known melanoma antigens like MART1, PMEL, and MAGEA1, while others may have recognized unknown neoantigens.
In a PDX model where the tumor was eradicated by TILs, the remaining T cells in the liver were predominantly CD4+ T cells, with clonally expanded subsets showing signs of activation and exhaustion.
The study provides insights into the phenotypes of tumor-reactive T cells in UM, which can guide efforts to enhance immunotherapy efficacy through selective expansion or TCR transgenics.

Stats

Uveal melanoma has a 50% metastasis rate, predominantly in the liver, and patients with metastatic disease have a median survival of less than a year.
Monotherapy immune checkpoint inhibitors are less effective in uveal melanoma than in cutaneous melanoma.
Combination treatments with PD-1/CTLA-4 inhibitors or PD-1/HDAC inhibitors have demonstrated longer overall survival than historic benchmark data.
The T-cell engager tebentafusp increased median survival from 16.0 months to 21.7 months in a phase 3 trial.
Isolated hepatic perfusion tripled hepatic progression-free survival compared to the best alternative care.

Quotes

"Defining tumor-reactive T lymphocytes (TRLs) among TILs would enable the identification of candidate biomarkers for selective expansion or TCR transgenics in cell therapy experiments in mouse models and cell therapy trials."
"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."
"Part of the lack of success for ACT may be attributable to a lack of tumor-reactive lymphocytes, possibly owing to lower mutation burden in UM compared to cutaneous melanoma."

Key Insights Distilled From

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

by Karlsson,J. ... at www.biorxiv.org 05-18-2023

https://www.biorxiv.org/content/10.1101/2023.05.16.540908v2

Deeper Inquiries

What other strategies, beyond selective expansion or TCR transgenics, could be used to enhance the potency and stemness of tumor-reactive T cells for adoptive cell therapy in uveal melanoma?

In addition to selective expansion or TCR transgenics, several other strategies could be employed to enhance the potency and stemness of tumor-reactive T cells for adoptive cell therapy in uveal melanoma. One approach could involve the use of cytokines to modulate the T cell phenotype and function. For example, interleukin-15 (IL-15) has been shown to enhance the persistence and anti-tumor activity of T cells in adoptive cell therapy. By incorporating IL-15 into the culture conditions during T cell expansion, the stemness and potency of the T cells could be improved.
Another strategy is the incorporation of genetic modifications to enhance T cell function. This could involve the introduction of chimeric antigen receptors (CARs) targeting specific tumor antigens, which can redirect T cells to recognize and kill tumor cells more effectively. Additionally, genetic modifications to enhance T cell survival, proliferation, and persistence in the tumor microenvironment could be explored.
Furthermore, the use of combination therapies to target multiple pathways involved in T cell exhaustion and dysfunction could be beneficial. For example, combining immune checkpoint inhibitors with adoptive cell therapy may help overcome the inhibitory signals present in the tumor microenvironment and enhance the anti-tumor activity of T cells.

How might the tumor microenvironment in uveal melanoma liver metastases contribute to the exhausted phenotype of tumor-reactive T cells, and what interventions could be used to overcome this?

The tumor microenvironment in uveal melanoma liver metastases can contribute to the exhausted phenotype of tumor-reactive T cells through various mechanisms. The presence of inhibitory immune checkpoints, such as PD-1, CTLA-4, and LAG-3, can lead to T cell exhaustion by dampening T cell activation and effector functions. Additionally, the presence of immunosuppressive cells, such as regulatory T cells and myeloid-derived suppressor cells, can further inhibit T cell activity and promote exhaustion.
To overcome the exhausted phenotype of tumor-reactive T cells in the tumor microenvironment, interventions targeting immune checkpoints could be employed. Immune checkpoint inhibitors, such as anti-PD-1 or anti-CTLA-4 antibodies, can block inhibitory signals and restore T cell function. Combination therapies targeting multiple immune checkpoints simultaneously may provide synergistic effects and enhance T cell activity.
Furthermore, modulating the metabolic and inflammatory milieu of the tumor microenvironment could help rejuvenate exhausted T cells. Metabolic reprogramming strategies, such as targeting metabolic pathways that support T cell exhaustion, could enhance T cell function. Additionally, interventions that reduce the immunosuppressive cell populations and promote the infiltration of effector T cells into the tumor could help overcome T cell exhaustion.

Given the presence of virus-specific T cells in the tumor, how could the interplay between tumor-reactive and virus-reactive T cells be leveraged to improve immunotherapy outcomes in uveal melanoma?

The presence of virus-specific T cells in the tumor presents an opportunity to leverage the interplay between tumor-reactive and virus-reactive T cells to improve immunotherapy outcomes in uveal melanoma. One approach could involve harnessing the cytotoxic activity of virus-specific T cells to target and eliminate tumor cells. By modulating the tumor microenvironment to promote the activation and function of virus-specific T cells, their anti-tumor activity could be enhanced.
Additionally, the presence of virus-specific T cells could be exploited to enhance the overall immune response against the tumor. Strategies that promote cross-reactivity between virus-specific T cells and tumor antigens could lead to a broader and more potent anti-tumor immune response. This could involve the identification of shared epitopes between viral antigens and tumor antigens to stimulate a robust immune response against the tumor.
Furthermore, combination therapies that target both the virus and the tumor could be explored to synergistically enhance the immune response. By coordinating the activities of virus-specific and tumor-reactive T cells through combination immunotherapies, the overall efficacy of immunotherapy in uveal melanoma could be improved.