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Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Identification of anti-TIM-3 based checkpoint inhibitor combinations with activity in immunotherapy refractory
Manali S Phadke1, Jiannong Li2, Sathya Sriramareddy1
1Department of Tumor Microenvironment and Metastasis, H Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.
Background:
A significant percentage of melanomas are refractory to immune checkpoint inhibitor (ICI) monotherapies and combinations. As there are currently no effective second-line therapies available for ICI-resistant patients, we sought to identify novel checkpoint inhibitor combinations for future clinical evaluation.
Methods:
We used responding and resistant SM1, SW1 and B16-10 melanoma models to explore the mechanism of action of anti-programmed cell death protein 1 (PD-1)+lymphocyte activation gene (LAG-3), anti-PD-1+cytotoxic lymphocyte associated protein 4 (CTLA-4), the anti-PD-1+LAG-3+CTLA-4 triplet, the anti-PD-1+T-cell immunoglobin and mucin domain 3 (TIM-3) doublet and the anti-PD-1+LAG-3+TIM-3 triplet. Flow cytometry, tetramer binding assays, T-cell depletion and analysis of the tumor-draining lymph nodes were used to determine the mechanisms of action of the ICI combinations. Single-cell RNA sequencing (scRNA-Seq) data from patients with melanoma was additionally analyzed to determine immune checkpoint expression across T-cell subsets and their association with ICI resistance.
Results:
The PD-1+CTLA-4+LAG-3 triplet combination had good efficacy in the SM1 and B16-F10 melanoma models and led to a more favorable immune microenvironment (increased CD69+CD8+ T cells and gp-100-tumor specific T cells, reduced exhausted T cells) than either doublet. The ICI-resistant SW1 model did not respond to the PD-1+CTLA-4+LAG-3 triplet and was characterized by an accumulation of TIM-3+terminally exhausted CD8+T cells. Targeting of TIM-3 using the anti-PD-1+LAG-3+TIM-3 triplet led to complete tumor regressions in the SW1 and SM1 models. The TIM-3-based triplet decreased exhausted CD8+T cells and regulatory T cells, increased numbers of gp-100 reactive CD8+T cells and increased progenitor-exhausted CD8+T cells (TCF7+Ki67+ TIM-3-) numbers in tumor-draining lymph nodes. scRNA-Seq analysis demonstrated that TIM-3 levels were higher in the CD8+T cells of patients with melanoma who were non-responsive to immunotherapy. PD-1 and TIM-3 expression was strongly correlated in the CD8+T cells from patients with melanoma, providing the rationale for co-targeting these checkpoints.
Conclusion:
Melanoma models characterized by high levels of terminally exhausted CD8+T cells (TIM-3+) can be successfully treated with the anti-PD-1+LAG-3+TIM-3 combination. The observation that high TIM-3 expression in CD8+T cells is associated with ICI resistance suggests the potential utility of anti-TIM-3-based combinations as a second-line treatment strategy in patients with advanced melanoma.
Insights
A novel anti-programmed cell death protein 1 (PD-1)+lymphocyte activation gene (LAG-3)+T-cell immunoglobulin and mucin domain 3 (TIM-3) combination effectively treats melanoma models resistant to other therapies. This suggests anti-TIM-3 strategies may offer a new second-line treatment for advanced melanoma.
Area of Science:
- Immunology
- Oncology
- Cancer Research
Background:
- Many melanomas do not respond to current immune checkpoint inhibitor (ICI) therapies.
- There is a critical need for effective second-line treatments for patients with ICI-resistant melanoma.
Purpose of the Study:
- To identify novel immune checkpoint inhibitor combinations for treating ICI-resistant melanoma.
- To investigate the mechanisms of action of various ICI combinations in melanoma models.
Main Methods:
- Utilized responsive and resistant melanoma models (SM1, SW1, B16-10) to test combinations including anti-PD-1, anti-LAG-3, anti-CTLA-4, and anti-TIM-3.
- Employed flow cytometry, tetramer binding assays, T-cell depletion, and tumor-draining lymph node analysis.
- Analyzed single-cell RNA sequencing (scRNA-Seq) data from melanoma patients to correlate immune checkpoint expression with ICI resistance.
Main Results:
- The anti-PD-1+CTLA-4+LAG-3 triplet improved the immune microenvironment in responsive models but failed in the resistant SW1 model, which showed TIM-3+ terminally exhausted CD8+ T cells.
- The anti-PD-1+LAG-3+TIM-3 triplet achieved complete tumor regressions in SW1 and SM1 models.
- scRNA-Seq revealed higher TIM-3 expression on CD8+ T cells in immunotherapy-resistant melanoma patients, correlating with PD-1 expression.
Conclusions:
- Melanoma models with high TIM-3+ terminally exhausted CD8+ T cells respond well to the anti-PD-1+LAG-3+TIM-3 combination.
- High TIM-3 expression on CD8+ T cells is linked to ICI resistance, supporting anti-TIM-3 combinations as a potential second-line therapy for advanced melanoma.
