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Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
PD-1 and LAG-3 Dominate Checkpoint Receptor-Mediated T-cell Inhibition in Renal Cell Carcinoma
Henning Zelba1, Jens Bedke2, Jörg Hennenlotter2
1Department of Immunology, University of Tübingen, Tübingen, Germany. henning_zelba@gmx.de cecile.gouttefangeas@uni-tuebingen.de.
Abstract:
Drugs targeting the programmed cell death protein 1 (PD-1) pathway are approved as therapies for an increasing number of cancer entities, including renal cell carcinoma. Despite a significant increase in overall survival, most treated patients do not show durable clinical responses. A combination of checkpoint inhibitors could provide a promising improvement. The aim of the study was to determine the most promising checkpoint blockade combination for renal cell carcinoma patients. Tumor-infiltrating lymphocytes (TIL) and autologous peripheral blood mononuclear cells (PBMC) were isolated from patients undergoing surgery for primary tumors. Cells were stained for multicolor flow cytometry to determine the (co)expression of five inhibitory receptors (iR), PD-1, LAG-3, Tim-3, BTLA, and CTLA-4, on T-cell populations. The function of these TILs was assessed by intracellular cytokine staining after in vitro stimulation in the presence or absence of PD-1 ± LAG-3 or Tim-3-specific antibodies. Although the percentage of iR+ T cells was low in PBMCs, both CD4+ and CD8+ T cells showed increased frequencies of PD-1+, LAG-3+, and Tim-3+ cells on TILs. The most frequent iR combination was PD-1 and LAG-3 on both CD4+ and CD8+ TILs. Blockade of PD-1 resulted in significant LAG-3, but not Tim-3, upregulation. The dual blockade of PD-1 and LAG-3, but not PD-1 and Tim-3, led to increased IFNγ release upon in vitro stimulation. Together, these data suggest that dual blockade of PD-1 and LAG-3 is a promising checkpoint blockade combination for renal cell carcinoma.
Insights
Combining PD-1 and LAG-3 checkpoint inhibitors shows promise for renal cell carcinoma treatment. This dual blockade enhances T-cell responses, potentially improving patient outcomes where single therapies fall short.
Area of Science:
- Immunology
- Oncology
- Cancer Therapy
Background:
- Programmed cell death protein 1 (PD-1) pathway inhibitors are approved for various cancers, including renal cell carcinoma (RCC).
- While improving overall survival, current PD-1 therapies often lack durable responses in most RCC patients.
- Combination immunotherapy strategies are being explored to enhance clinical efficacy.
Purpose of the Study:
- To identify the most effective checkpoint blockade combination for renal cell carcinoma (RCC) patients.
- To investigate the co-expression of inhibitory receptors (iRs) on tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs).
- To assess the functional impact of dual PD-1 and other iR blockade on T-cell responses.
Main Methods:
- Isolation of TILs and PBMCs from RCC patients undergoing primary tumor surgery.
- Multicolor flow cytometry to analyze the co-expression of PD-1, LAG-3, Tim-3, BTLA, and CTLA-4 on T-cell populations.
- In vitro stimulation of TILs with PD-1 ± LAG-3 or Tim-3 specific antibodies, followed by intracellular cytokine staining to assess T-cell function.
Main Results:
- Increased frequencies of PD-1+, LAG-3+, and Tim-3+ cells were observed on CD4+ and CD8+ TILs compared to PBMCs.
- The most common inhibitory receptor combination on TILs was PD-1 and LAG-3.
- Dual blockade of PD-1 and LAG-3 significantly increased IFNγ release, unlike PD-1 and Tim-3 blockade.
Conclusions:
- Dual blockade of PD-1 and LAG-3 represents a promising combination immunotherapy strategy for renal cell carcinoma.
- This combination enhances T-cell effector function, potentially overcoming resistance to single-agent PD-1 therapy.
- Further clinical investigation of PD-1 and LAG-3 blockade in RCC is warranted.
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