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Updated: May 6, 2026
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets
Luisa Chocarro1, Ester Blanco2,3, Leticia Fernandez-Rubio2
1OncoImmunology Unit, Navarrabiomed - Fundación Miguel Servet, Universidad Pública de Navarra (UPNA), Hospital Universitario de Navarra (HUN), Instituto de Investigación Sanitaria de Navarra (IdiSNA), 31008, Pamplona, Spain. lchocard@navarra.es.
Abstract:
Many cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. To identify shared features associated to PD-1/LAG-3 dysfunctionality in human cancers and T-cells, multiomic expression profiles were obtained for all TCGA cancers immune infiltrates. A PD-1/LAG-3 dysfunctional signature was found which regulated immune, metabolic, genetic, and epigenetic pathways, but especially a reinforced negative regulation of the TCR signalosome. These results were validated in T-cell lines with constitutively active PD-1, LAG-3 pathways and their combination. A differential analysis of the proteome of PD-1/LAG-3 T-cells showed a specific enrichment in ubiquitin ligases participating in E3 ubiquitination pathways. PD-1/LAG-3 co-blockade inhibited CBL-B expression, while the use of a bispecific drug in clinical development also repressed C-CBL expression, which reverted T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD-1 blockade. The combination of CBL-B-specific small molecule inhibitors with anti-PD-1/anti-LAG-3 immunotherapies demonstrated notable therapeutic efficacy in models of lung cancer refractory to immunotherapies, overcoming PD-1/LAG-3 mediated resistance.
Insights
Many cancer patients resist PD-L1/PD-1 immunotherapy due to T-cell dysfunction. Targeting PD-1/LAG-3 pathways and CBL-B/C-CBL ubiquitination can overcome this resistance, improving outcomes in refractory lung cancers.
Area of Science:
- Immunology
- Oncology
- Molecular Biology
Background:
- Many cancer patients do not respond to PD-L1/PD-1 blockade immunotherapies.
- PD-1 and LAG-3 co-upregulation in T-cells drives resistance by inducing T-cell dysfunction.
- Understanding shared features of PD-1/LAG-3 dysfunction is crucial for improving cancer treatment.
Purpose of the Study:
- To identify shared molecular features of PD-1/LAG-3 dysfunction in human cancers and T-cells.
- To investigate the role of ubiquitin ligases in PD-1/LAG-3 mediated T-cell exhaustion.
- To evaluate therapeutic strategies combining PD-1/LAG-3 blockade with E3 ubiquitination pathway modulation.
Main Methods:
- Multiomic expression profiling of TCGA cancer immune infiltrates.
- Validation in T-cell lines with constitutively active PD-1 and LAG-3 pathways.
- Proteomic analysis of PD-1/LAG-3 T-cells and assessment of ubiquitin ligase enrichment.
Main Results:
- A PD-1/LAG-3 dysfunctional signature was identified, negatively regulating immune, metabolic, genetic, epigenetic, and TCR signaling pathways.
- PD-1/LAG-3 T-cells showed enrichment in ubiquitin ligases involved in E3 ubiquitination.
- Co-blockade of PD-1/LAG-3 and inhibition of CBL-B/C-CBL reversed T-cell dysfunctionality in resistant lung cancer models.
Conclusions:
- PD-1/LAG-3 co-upregulation establishes T-cell dysfunction through specific molecular pathways, including E3 ubiquitination.
- Targeting CBL-B and C-CBL in combination with PD-1/LAG-3 blockade offers a promising strategy to overcome immunotherapy resistance.
- This approach demonstrated therapeutic efficacy in preclinical models of refractory lung cancer.
