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Partial absence of PD?1 expression by tumor?infiltrating EBV?specific CD8+ T cells in EBV?driven lymphoepithelioma?like carcinoma

ABSTRACT: Abstract Objectives Lymphoepithelioma?like carcinoma (LELC) is an uncommon lung cancer, typically observed in young, non?smoking Asian populations. LELC is associated with Epstein–Barr virus (EBV) infection of lung tumor cells of epithelial origin, suggesting a carcinogenic role of EBV as observed in nasopharyngeal carcinoma (NPC). Here, we studied the antigen specificity and phenotype of EBV?specific CD8+ T cells in blood and tumor of one LELC patient positive for EBV infection in lung tumor cells. Methods Using multiplex MHC class I tetramers, mass cytometry and mRNA sequencing, we studied EBV?specific CD8+ T cells at the transcriptomic and phenotypic levels in blood and tumor tissues of the LELC patient. Results Lymphoepithelioma?like carcinoma lung tumor cells were positive for EBV infection. In both blood and tumor tissues, we detected two populations of EBV?specific CD8+ T cells targeting the EBV lytic cycle proteins: BRLF1 and BMLF1. Transcriptomic analyses of these two populations in the tumor, which can be considered as tumor?specific, revealed their distinct exhausted profile and polyclonal TCR repertoire. High?dimensional phenotypical analysis revealed the distinct phenotype of these cells between blood and tumor tissues. In tumor tissue, EBV?specific CD8+ TILs were phenotypically heterogeneous, but consistently expressed CD39. Unexpectedly, although the LELC tumor cells expressed abundant PD?L1, these tumor?specific CD8+ tumor?infiltrating lymphocytes (TILs) mostly did not express PD?1. Conclusion Epstein–Barr virus?specific CD8+ TILs in EBV?driven tumor are heterogeneous and partially lack PD?1 expression, suggesting that anti?PD1/PD?L1 immunotherapy may not be an appropriate strategy for disinhibiting EBV?specific cells in the treatment of LELC patients. We studied the antigen specificity and phenotype of CD8+ tumor?infiltrating lymphocytes (TILs) in one LELC patient positive for EBV infection in lung tumor cells. Transcriptomic analyses of EBV?specific CD8+ TILs reveal their distinct exhausted profiles and polyclonal TCR repertoire. Although the LELC tumor cells expressed abundant PD?L1, EBV?specific CD8+ TILs mostly did not express PD?1, but express CD39.

SUBMITTER: Simoni Y

PROVIDER: S-EPMC7503213 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundStratification of patients who could benefit from immune checkpoint inhibitor (ICI) therapy is of much importance. PD-1hiCD8+ T cells represent a newly identified and effective biomarker for ICI therapy response biomarker in lung cancer. Accurately quantifying these T cells using commonly available RNA sequencing (RNA-seq) data may extend their applications to more cancer types.MethodWe built a transcriptome signature of PD-1hiCD8+ T cells from bulk RNA-seq and single-cell RNA-seq (scRNA-seq) data of tumor-infiltrating immune cells. The signature was validated by flow cytometry and in independent datasets. The clinical applications of the signature were explored in non-small-cell lung cancer, melanoma, gastric cancer, urothelial cancer, and a mouse model of breast cancer samples treated with ICI, and systematically evaluated across 21 cancer types in The Cancer Genome Atlas (TCGA). Its associations with other biomarkers were also determined.ResultsSignature scores could be used to identify the PD-1hiCD8+ T subset and were correlated with the fraction of PD-1hiCD8+ T cells in tumor tissue (Pearson correlation, R=0.76, p=0.0004). Furthermore, in the scRNA-seq dataset, we confirmed the capability of PD-1hiCD8+ T cells to secrete CXCL13, as well as their interactions with other immune cells. In 581 clinical samples and 204 mouse models treated with ICIs, high signature scores were associated with increased survival, and the signature achieved area under the receiver operating characteristic curve scores of 0.755 (ranging from 0.61 to 0.91) in predicting therapy response. In TCGA pan-cancer datasets, our signature scores were consistently correlated with therapy response (R=0.78, p<0.0001) and partially explained the diverse response rates among different cancer types. Finally, our signature generally outperformed other mRNA-based predictors and showed improved predictive performance when used in combination with tumor mutational burden (TMB). The signature score is available in the R package "PD1highCD8Tscore" (https://github.com/Liulab/PD1highCD8Tscore).ConclusionThrough estimating the fraction of the PD-1hiCD8+ T cell, our signature could predict response to ICI therapy across multiple cancers and could serve as a complementary biomarker to TMB.

Project description:BackgroundThe functions of infiltrating CD8+ T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment. Thus, the mechanisms of CD8+ T cell dysfunction have become a hot research topic, and there is increased interest on how changes in metabolomics correlate with CD8+ T cell dysfunction.AimTo investigate whether and how glutamine metabolism affects the function of infiltrating CD8+ T cells in hepatocellular carcinoma.MethodsImmunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients. Differentially expressed genes in infiltrating CD8+ T cells in hepatocellular carcinoma were detected using RNA sequencing. Activated CD8+ T cells were co-cultured with Huh-7 cells for 3 d. The function and mitochondrial status of CD8+ T cells were analyzed by flow cytometry, quantitative real-time polymerase chain reaction, and transmission electron microscopy. Next, CD8+ T cells were treated with the mitochondrial protective and damaging agents. Functional alterations in CD8+ T cells were detected by flow cytometry. Then, complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.ResultsThere were a large number of infiltrating PD-1+CD8+ T cells in liver cancer tissues. Next, we co-cultured CD8+ T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8+ T cells. Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin (PRF1) and granzyme B (GZMB) by CD8+ T cells in the co-culture group. Meanwhile, JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8+ T cells of the co-culture group; additionally, the mitochondria of these cells were swollen. When CD8+ T cells were treated with the mitochondrial protective and damaging agents, their function was restored and inhibited, respectively, through the mitochondrial damage and apoptotic pathways. Subsequently, complete medium without glutamine was used to culture cells. As expected, CD8+ T cells showed functional downregulation, mitochondrial damage, and apoptosis.ConclusionGlutamine deprivation impairs the function of infiltrating CD8+ T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.

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Partial absence of PD?1 expression by tumor?infiltrating EBV?specific CD8+ T cells in EBV?driven lymphoepithelioma?like carcinoma

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