Quantification of Immune Checkpoint Interactions in RFA-Treated Lung Metastases
What This Study Shows
This study was the first to quantify CTLA-4/CD80 checkpoint interactions at molecular resolution (1-10nm) in human patient samples. It examined lung metastases treated with radiofrequency ablation (RFA), a technique that heats and destroys tumors.
A key finding: checkpoint interaction levels did NOT correlate with ligand expression—more evidence that you can't predict function from expression alone.
Key Findings
First CTLA-4/CD80 Quantification at Molecular Scale
iFRET enabled the first measurement of CTLA-4/CD80 interaction at distances only achievable by direct molecular contact.
Interaction ≠ Expression
Checkpoint interaction (iFRET signal) did not correlate with CD80 or PD-L1 ligand expression levels measured by IHC.
PD-1/PD-L1 vs. T-Cell Density
Higher PD-1/PD-L1 interaction was associated with LOWER CD3+ and CD8+ T-cell infiltration—suggesting active immune suppression.
RFA and Immunotherapy
Radiofrequency ablation (RFA) destroys tumors with heat, but it also releases tumor antigens that can stimulate immune responses. Understanding the checkpoint landscape in RFA-treated tumors helps inform combination therapy strategies.
The finding that high PD-1/PD-L1 interaction correlates with low T-cell infiltration suggests these checkpoints are actively suppressing immune responses—making them therapeutic targets.
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Abstract
Radiofrequency ablation (RFA) induces immunogenic cell death and may synergize with checkpoint inhibitors. However, the checkpoint interaction landscape in RFA-treated lesions is unknown. We applied iFRET to quantify PD-1/PD-L1 and CTLA-4/CD80 interactions at the tumor-immune interface in colorectal cancer lung metastases treated with RFA, correlating with T-cell infiltration and ligand expression.
Methods
Samples: FFPE sections from 24 colorectal cancer lung metastases treated with RFA.
iFRET: Two-site amplified FRET using donor-labeled anti-receptor (PD-1 or CTLA-4) and acceptor-labeled anti-ligand (PD-L1 or CD80) antibodies. FLIM acquisition with TCSPC.
IHC: Serial sections stained for CD3, CD8, PD-L1, and CD80 expression. Quantified by digital image analysis.
Correlations: Spearman correlation between iFRET signals, expression markers, and T-cell densities.
Key Results
PD-1/PD-L1 iFRET vs. CD8+ Density
Negative correlation between PD-1/PD-L1 interaction and CD8+ T-cell infiltration, suggesting checkpoint-mediated immune exclusion.
PD-1/PD-L1 iFRET vs. PD-L1 Expression
No significant correlation between functional interaction and ligand expression—high PD-L1 does not guarantee high engagement.
"CTLA-4/CD80 interaction was successfully quantified at 1-10nm resolution for the first time in patient samples, demonstrating the feasibility of multiplexed checkpoint profiling."
Implications for RFA-ICI Combinations
- 🔥 Abscopal potential: RFA-induced immunogenic cell death releases tumor antigens; understanding checkpoint engagement helps predict ICI synergy.
- 🎯 Patient selection: iFRET could identify RFA-treated patients most likely to benefit from subsequent checkpoint inhibition.
- 📊 Multiplexed profiling: Simultaneous CTLA-4/CD80 and PD-1/PD-L1 measurement enables comprehensive checkpoint assessment.