PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rβ–IL2Rγc membrane dimers. This results in impaired IL-2–mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.


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Life sciences
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Life sciences study design
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Sample size
No statistical methods were used to predetermine sample size.Sample sizes for in vitro and in vivo assays were determined empirically based on previous work and minimum of 3 biological replicates were used in most of the studies to allow for statistical comparisons.
Data exclusions No data were excluded from our analysis Replication Data was collected using biological replicates to ensure reproducibility.The number of independent replicates for each experiment is noted in all figure legends.All experimental findings were reproduced succesfully at least 2 times.
Randomization For in vitro experiments, no randomization was performed.For murine Winn assay, mice were randomly allocated to the different treatment groups based on weight of the mice while for the murine adoptive cell therapy tumor control experiment, mice were randomized based on tumor size.

Blinding
Blinding was not conducted for most in vitro research because the same people performed the experiments, collected the data, and analysed it.Imaging flow cytometry analysis, Cytoff analysis and electron microscopy acquistion and analysis were performed blindly.
Murine experiments were performed blind to experimental conditions.
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Data collection
Tumour samples were collected between October 2016 and August 2023 at the Centre Hospitalier Universitaire Vaudoise (CHUV), Lausanne, Switzerland was under a specific protocol TIL-ME study with the number 247/13.After that, samples were collected by using the Pre-IT protocol (2016-02094).
Informed consent was obtained from any patients undergoing surgery at the CHUV.Informed consent was obtained from any patients undergoing surgery at the CHUV.Patients were approached and requested to consent to donating their samples for translational research if the samples were not required for clinical pathological evaluation.There is no selection based on previous history, age, previous treatments and thus no potential selection bias exists.The population characteristics were blinded to researchers.
For the analysis of the melanoma cohort, we re-analyzed results already published from a phase 1 trial of ACT with TILs in melanoma patients (ClinicalTrials.govNCT03475134).For correlation of PGE2 in the supernatant and TIL expansion, we used collected supernatant of TIL cultures from patients enrolled in a phase 1 trial of ACT with TILs in solid tumours (CHUV-DO-0018-NeoTIL-2019, NCT04643574).

Outcomes
This study did not evaluate any clinical outcomes.Clinical samples from prior phase I clinical trials were only utilised in this project for translational purposes and described in Barras et al. (DOI: 10.1126/sciimmunol.adg7995).

Flow Cytometry Plots
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Methodology Sample preparation
Cells analyzed by flowcytometry were derived from human dissociated tumour, expanded TILs and peripheral blood lymphocytes isolated from PBMC or murine T cells extracted from the spleen of OTI mice or from the tumours of tumoursbearing mice.Methods for cell isolation and staining are described in the material and method section.

Instrument
Cells were analyzed on Fortessa flow cytometer (BD Biosciences) and on IntelliCyt iQue® Screener PLUS (Bucher Biotec)

Software
BD FACSDiva was used for data acquisition.Flowjo v10.5.3 was used for data analysis.ForeCyt® was used for data acquisition on IntelliCyt iQue® Screener PLUS.

Cell population abundance
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