Background Coexpression of the immune checkpoint receptors PD-1 and TIM-3 is associated with dysfunction of tumor infiltrating lymphocytes in melanoma (MEL) and squamous cell carcinoma (SCC). To identify the mechanisms underlying this dysfunctional phenotype and to identify targets to rescue immune function, we performed transcriptional profiling of PD-1+TIM-3+CD8+ TILs sorted directly from MEL and SCC tumors in conjunction with immunologic phenotyping. We identified a number of novel dysregulated pathways and associated gene signatures in the PD-1+TIM-3+ subset that have not previously been reported in TILs, including bile acid metabolism, and demonstrated that these pathways are highly correlated not only to immune checkpoint receptor expression but also to MTOR pathway activation.
Methods Surgically excised melanoma tumors were dissociated and mononuclear cells were isolated by Ficoll gradient separation and cryopreserved. For sorting based on immune checkpoint receptor expression, a cocktail of monoclonal antibodies to the following targets, including viability dye was used: CD3, CD4, CD8, CD45RA, CD27, CCR7 PE, CD14, CD19, Live/Dead, BTLA, TIM-3, PD-1, CTLA-4, TIGIT and LAG-3. RNA was purified from 10,000 sorted cells using RNeasy Mini Kits (Qiagen), followed by RNASeq library generation using TruSeq Stranded Total RNA HT Kits (Illumina). Gene set variation analysis (GSVA) was used for pathway analysis, as well as linear regression modelling using limma (Bioconductor).
Results Transcriptomic analysis of CD8+ TILs revealed 4,228 genes (P<0.05, t-test) as differentially expressed in TILs vs. functional peripheral CD8 T cells. Using a nonlinear dimensionality-reduction technique, we found that there were several unique clusters of cell surface marker expression that were present at significantly different frequencies in the TILs. We used pathway enrichment analysis and linear regression modeling to identify gene signatures that correlate with the progressive and coordinate expression of PD-1, TIM-3, and additional checkpoint receptors thereby driving progressive dysfunction on in TILs. The peroxisome and bile acid metabolism pathways were significantly enriched in the TIL transcriptome. Moreover, higher frequencies of events in dysfunctional clusters were strongly correlated with positive enrichment of the bile acid metabolism pathway and enhanced MTOR signaling.
Conclusions Transcriptomic analysis of PD-1+TIM-3+ CD8+ TILs identified novel candidate mechanisms of immune dysfunction in CD8+ TILs from patients with metastatic melanoma who fail immunotherapy. Our study identifies the bile acid and MTOR metabolic pathways as a potential novel therapeutic targets for complementary therapy to restore immune function in melanoma and SCC patients.
Ethics Approval This study was performed under an IRB approved protocol.
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