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Updated: Jul 9, 2025
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
PD-1 Expression on Intratumoral Regulatory T Cells Is Associated with Lack of Benefit from Anti-PD-1 Therapy in
Thomas Denize1,2, Opeyemi A Jegede3, Sayed Matar1,2
1Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
Purpose:
Programmed cell death protein 1 (PD-1) expression on CD8+TIM-3-LAG-3- tumor-infiltrating cells predicts positive response to PD-1 blockade in metastatic clear-cell renal cell carcinoma (mccRCC). Because inhibition of PD-1 signaling in regulatory T cells (Treg) augments their immunosuppressive function, we hypothesized that PD-1 expression on tumor-infiltrating Tregs would predict resistance to PD-1 inhibitors.
Experimental Design:
PD-1+ Tregs were phenotyped using multiparametric immunofluorescence in ccRCC tissues from the CheckMate-025 trial (nivolumab: n = 91; everolimus: n = 90). Expression of CD8, PD-1, TIM-3, and LAG-3 was previously determined (Ficial and colleagues, 2021). Clinical endpoints included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR).
Results:
In the nivolumab (but not everolimus) arm, high percentage of PD-1+ Tregs was associated with shorter PFS (3.19 vs. 5.78 months; P = 0.021), shorter OS (18.1 vs. 27.7 months; P = 0.013) and marginally lower ORR (12.5% vs. 31.3%; P = 0.059). An integrated biomarker (PD-1 Treg/CD8 ratio) was developed by calculating the ratio between percentage of PD-1+Tregs (marker of resistance) and percentage of CD8+PD-1+TIM-3-LAG-3- cells (marker of response). In the nivolumab (but not everolimus) arm, patients with high PD-1 Treg/CD8 ratio experienced shorter PFS (3.48 vs. 9.23 months; P < 0.001), shorter OS (18.14 vs. 38.21 months; P < 0.001), and lower ORR (15.69% vs. 40.00%; P = 0.009). Compared with the individual biomarkers, the PD-1 Treg/CD8 ratio showed improved ability to predict outcomes to nivolumab versus everolimus.
Conclusions:
PD-1 expression on Tregs is associated with resistance to PD-1 blockade in mccRCC, suggesting that targeting Tregs may synergize with PD-1 inhibition. A model that integrates PD-1 expression on Tregs and CD8+TIM-3-LAG-3- cells has higher predictive value.
Insights
Programmed cell death protein 1 (PD-1) expression on regulatory T cells (Tregs) predicts resistance to PD-1 blockade in metastatic clear-cell renal cell carcinoma (mccRCC). A novel biomarker integrating PD-1+Tregs and CD8+ cells improves outcome prediction.
Area of Science:
- Immunology
- Oncology
- Translational Medicine
Background:
- Programmed cell death protein 1 (PD-1) expression on tumor-infiltrating CD8+ cells predicts response to PD-1 blockade in metastatic clear-cell renal cell carcinoma (mccRCC).
- PD-1 signaling inhibition in regulatory T cells (Tregs) can enhance their immunosuppressive function.
Purpose of the Study:
- To investigate if PD-1 expression on tumor-infiltrating Tregs predicts resistance to PD-1 inhibitors in mccRCC.
- To develop and validate an integrated biomarker for predicting response to PD-1 blockade.
Main Methods:
- Multiparametric immunofluorescence was used to phenotype PD-1+ Tregs in ccRCC tissues from the CheckMate-025 trial (nivolumab and everolimus arms).
- Previously determined expression of CD8, PD-1, TIM-3, and LAG-3 was utilized.
- Clinical endpoints including progression-free survival (PFS), overall survival (OS), and objective response rate (ORR) were analyzed.
Main Results:
- High PD-1+ Treg percentage correlated with shorter PFS, OS, and lower ORR in the nivolumab arm, but not the everolimus arm.
- An integrated biomarker, the PD-1 Treg/CD8 ratio, demonstrated superior predictive ability for nivolumab outcomes compared to individual biomarkers.
- Patients with a high PD-1 Treg/CD8 ratio in the nivolumab arm showed significantly worse PFS, OS, and ORR.
Conclusions:
- PD-1 expression on Tregs is associated with resistance to PD-1 blockade in mccRCC.
- Targeting Tregs may offer a synergistic approach with PD-1 inhibitors.
- The integrated PD-1 Treg/CD8 biomarker model shows enhanced predictive value for PD-1 blockade outcomes.
