T-cell exhaustion interrelates with immune cytolytic activity to shape the inflamed tumor microenvironment.

Direct quantification of exhausted T cells in human cancer is lacking, and its predictive value for checkpoint-based treatment remains poorly investigated. We sought to systematically characterize the pan-cancer landscape and molecular hallmarks of T-cell dysfunction for the purpose of precision immunotherapy. Here, we defined a transcriptional signature for T-cell exhaustion through analyzing differential gene expression between PD-1-high and PD-1-negative CD8+ T lymphocytes from primary non-small cell lung cancer (NSCLC), followed by positive correlation tests with PDCD1 in TCGA lung carcinomas. A 78-gene signature for exhausted CD8+ T cells (GET) was identified and validated to reflect dysfunctional immune state spanning different species and disease models. We discovered that GET estimation significantly correlated with intratumoral immune cytolytic activity (CYT) and T-cell-inflamed gene expression profile (GEP) across 30 solid tumor types. Miscellaneous tumor-intrinsic and -extrinsic properties, in particular leukocyte proportions, genomic abnormalities, specific mutational signatures, and signaling pathways, were notably associated with GET levels. Furthermore, higher GET expression predicted an increased likelihood of clinical response to immune checkpoint inhibitors. These findings highlight the interrelation between T-cell exhaustion and immune cytolytic activity at the pan-cancer scale. The resulting inflamed tumor microenvironment may further crosstalk with other molecular and clinicopathological factors, which should be properly considered during immunotherapy biomarker development. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Ferroptosis-related local immune cytolytic activity in tumor microenvironment of basal cell and squamous cell carcinoma.

BACKGROUND: Ferroptosis, a recently discovered form of cell death, whose role in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) has not been well disclosed. To improve our understanding of the differences in tumor progression and therapeutic effects between BCC and SCC, and to find potential therapeutic targets, this study systematically analyzed ferroptosis-related genes (FRGs) and their associated local immune cytolytic activity (LICA) and tumor microenvironment (TME) metabolic function differences. METHODS: Two bulk RNA-seq datasets, GSE7553 and GSE125285, from the Gene Expression Omnibus database were compared within and between groups to screen for common differentially expressed genes (DEGs) for enrichment analysis. The currently recognized FRGs in DEGs gene set were selected as the targets to analyze their correlation and difference in LICA and TME metabolic functions. And validated using immune cell populations from another single-cell RNA-seq (scRNA-seq) dataset (GSE123813) to accurately understand the difference in LICA. All of the gene sets for functional enrichment analysis comes from published results and MSigDB database. RESULTS: Ten FRGs were used to further analyze the differences in LICA and TME metabolic functions between BCC and SCC. In the SCC samples, LICA (e.g. Treg, CCR, Cytolytic activity, etc.) and TME metabolic functions (e.g. lipid and energy, etc.) were significantly related to ferroptosis genes (e.g. SLC1A5, CD44, NQO1, HMOX1 and STEAP3), and the ferroptosis potential index were also significantly higher than that in the BCC samples. Finally, based on these ten FRGs and related enrichment results, we postulated a model of NQO1 homeostasis regulated by FRGs during induction of ferroptosis in SCC. CONCLUSIONS: The results showed that three FRGs, SLC1A5, CD44 and NQO1, have significant potential in targeted therapies for SCC chemotherapy resistance. And two FRGs, STEAP3 and HMOX1, formed a synergistic effect on the occurrence of ferroptosis in tumor cells. Our findings can be used as the main research materials for metastasis and chemotherapy resistance in SCC patients.

Benchmarking HLA genotyping and clarifying HLA impact on survival in tumor immunotherapy.

Human leukocyte antigen (HLA) genotyping gains intensive attention due to its critical role in cancer immunotherapy. It is still a challenging issue to generate reliable HLA genotyping results through in silico tools. In addition, the survival impact of HLA alleles in tumor prognosis and immunotherapy remains controversial. In this study, the benchmarking of HLA genotyping on TCGA is performed and a 'Gun-Bullet' model which helps to clarify the survival impact of HLA allele is presented. The performance of HLA class I genotyping is generally better than class II. POLYSOLVER, OptiType, and xHLA perform generally better at HLA class I calling with an accuracy of 0.954, 0.949, and 0.937, respectively. HLA-HD obtained the highest accuracy of 0.904 on HLA class II alleles calling. Each HLA genotyping tool displayed specific error patterns. The ensemble HLA calling from the top-3 tools is superior to any individual one. HLA alleles show distinct survival impact among cancers. Cytolytic activity (CYT) was proposed as the underlying mechanism to interpret the survival impact of HLA alleles in the 'Gun-Bullet' model for fighting cancer. A strong HLA allele plus a high tumor mutation burden (TMB) could stimulate intensive immune CYT, leading to extended survival. We established an up to now most reliable TCGA HLA benchmark dataset, composing of concordance alleles generated from eight prevalently used HLA genotyping tools. Our findings indicate that reliable HLA genotyping should be performed based on concordance alleles integrating multiple tools and incorporating TMB background with HLA genotype, which helps to improve the survival prediction compared to HLA genotyping alone.

Immune Cytolytic Activity as an Indicator of Immune Checkpoint Inhibitors Treatment for Prostate Cancer.

Immune checkpoint inhibitors (ICIs) treatment is becoming a new hope for cancer treatment. However, most prostate cancer (PCa) patients do not benefit from it. In order to achieve the accuracy of ICIs treatment in PCa and reduce unnecessary costs for patients, we have analyzed the data from TCGA database to find a indicator that can assist the choice of treatment. By analyzing the data of PCa patients with TMB analysis and immune infiltration analysis, we found the expression of immune cells in different immune infiltration groups. Commonly used markers of ICIs, expressed on CD8+ T cell, were highly expressed in the high immune group. Then we used the forimmune cytolytic activity (CYT) to determine its relationship with the target of ICIs treatment. Through the analysis of CYT score and the ligands of immune checkpoints, we found that there was a significant correlation between them. With the increase of CYT score, the expression of CD80/86, PD-L1/L2, TNFSF14, and LGALS9 also increased gradually. Similarly, CD8+ T cells were significantly increased in the CYT high group compared with the CYT low group in PRAD. The present research provides novel insights into the immune microenvironment of PRAD and potential immunotherapies. The proposed CYT score is a clinically promising indicator that can serve as a marker to assist anti-PD-L1 or other ICIs treatment. At the same time, it also provides a basis for the selection of other immune checkpoint drugs.

Immune Cytolytic Activity Is Associated With Genetic and Clinical Properties of Glioma.

Background: Immunotherapy provided unprecedented advances in the treatment of several previously untreated cancers. However, these immunomodulatory maneuvers showed limited response to patients with glioma in clinical trials. Our aim was to depict the immune characteristics of glioma with immune cytolytic activity at genetic and transcriptome levels. Methods: In total, 325 gliomas from CGGA dataset as training cohort and 699 gliomas from TCGA dataset as validation cohort were enrolled in our analysis. We calculated the immune cytolytic activity for 1,000 of gliomas. The characteristics of immune cytolytic activity in gliomas were interpreted by the corresponding clinical, molecular genetics and radiological information. Results: We found that immune cytolytic activity was highly associated with molecular, clinical, and edema extent. High cytolytic activity gliomas were more likely to be diagnosed as glioblastoma and might be a potential marker of mesenchymal subtype. Moreover, those gliomas exhibited significantly increased copy number alterations including recurrent focal amplifications of PDGFA and EGFR, as well as recurrent deletions of CDKN2A/B. Subsequent biological function analysis revealed that the immune response and immune checkpoints expression were significantly correlated with the cytolytic activity of gliomas. Immune cytolytic activity was significantly positively associated with the extent of peri-tumor edema and was independently correlated with reduced survival time. Conclusion: Our results highlighted the immunoregulatory mechanism heterogeneity of gliomas. Cytolytic activity, indirectly reflected by the extent of peri-tumor edema, may provide a potential index to evaluate the status of immune microenvironment and immune checkpoints in glioma, which should be fully valued for precision classification and immunotherapy.

The Expression and Prognostic Impact of Immune Cytolytic Activity-Related Markers in Human Malignancies: A Comprehensive Meta-analysis.

BACKGROUND: Recently, immune-checkpoint blockade has shown striking clinical results in different cancer patients. However, a significant inter-individual and inter-tumor variability exists among different cancers. The expression of the toxins granzyme A (GZMA) and perforin 1 (PRF1), secreted by effector cytotoxic T cells and natural killer (NK) cells, were recently used as a denominator of the intratumoral immune cytolytic activity (CYT). These levels are significantly elevated upon CD8+ T-cell activation as well as during a productive clinical response against immune-checkpoint blockade therapies. Still, it is not completely understood how different tumors induce and adapt to immune responses. METHODS: Here, we calculated the CYT across different cancer types and focused on differences between primary and metastatic tumors. Using data from 10,355, primary tumor resection samples and 2,787 normal samples that we extracted from The Cancer Genome Atlas and Genotype-Tissue Expression project databases, we screened the variation of CYT across 32 different cancer types and 28 different normal tissue types. We correlated the cytolytic levels in each cancer type with the corresponding patient group's overall survival, the expression of several immune-checkpoint molecules, as well as with the load of tumor-infiltrating lymphocytes (TILs), and tumor-associated neutrophils (TANs) in these tumors. RESULTS: We found diverse levels of CYT across different cancer types, with highest levels in kidney, lung, and cervical cancers, and lowest levels in glioma, adrenocortical carcinoma (ACC), and uveal melanoma. GZMA protein was either lowly expressed or absent in at least half of these tumors; whereas PRF1 protein was not detected in almost any of the different tumor types, analyzing tissue microarrays from 20 different tumor types. CYT was significantly higher in metastatic skin melanoma and correlated significantly to the TIL load. In TCGA-ACC, skin melanoma, and bladder cancer, CYT was associated with an improved patient outcome and high levels of both GZMA and PRF1 synergistically affected patient survival in these cancers. In bladder, breast, colon, esophageal, kidney, ovarian, pancreatic, testicular, and thyroid cancers, high CYT was accompanied by upregulation of at least one immune-checkpoint molecule, indicating that similar to melanoma and prostate cancer, immune responses in cytolytic-high tumors elicit immune suppression in the tumor microenvironment. CONCLUSION: Overall, our data highlight the existence of diverse levels of CYT across different cancer types and suggest that along with the existence of complicated associations among various tumor-infiltrated immune cells, it is capable to promote or inhibit the establishment of a permissive tumor microenvironment, depending on the cancer type. High levels of immunosuppression seem to exist in several tumor types.