RESUMO
The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.
Assuntos
Retrovirus Endógenos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Superantígenos/genética , Superantígenos/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Células A549 , Animais , Células COS , Linhagem Celular , Sistema Livre de Células , Chlorocebus aethiops , Retrovirus Endógenos/química , Retrovirus Endógenos/genética , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Proteínas de Membrana/química , Conformação Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/química , Superantígenos/química , Transcrição Gênica , Proteínas do Envelope Viral/químicaRESUMO
INTRODUCTION: The immunosuppressive tumor microenvironment promotes progression of pancreatic ductal adenocarcinoma (PDAC). γδ T cells infiltrate the pancreatic tumor stroma and support tumorigenesis through αß T cell inhibition. Pancreatic stellate cell (PSC) activation contributes to pancreatic fibrosis in PDAC, limiting the delivery and efficacy of therapeutic agents. Whether γδ T cells have direct effects on PSC activation is unknown. METHODS: In this study, we analyzed tumor tissue from 68 patients with PDAC and determined the frequency and location of γδ T cells using immunohistochemistry and immunofluorescence. PDAC samples from the TCGA database with low and high TRGC2 expression were correlated with the expression of extracellular matrix genes. Further, PSCs were isolated from pancreatic tumor tissue and co-cultured with γδ T cells for 48 hours and cytokine production was measured using a cytometric bead array. RESULTS: γδ T cells infiltrated the pancreatic tumor stroma and were located in proximity to PSCs. A high infiltration of γδ T cells was associated with increased expression of several extracellular matrix genes in human PDAC. In vitro, γδ T cells stimulated IL-6 production by PDAC-derived PSCs. CONCLUSION: γδ T cells activated PSCs and modulation of this interaction may enhance the efficacy of combinational therapies in human PDAC.
Assuntos
Adenocarcinoma/imunologia , Carcinoma Ductal Pancreático/imunologia , Interleucina-6/genética , Linfócitos Intraepiteliais/imunologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Carcinogênese/genética , Carcinogênese/imunologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Células Estreladas do Pâncreas/imunologia , Células Estreladas do Pâncreas/metabolismo , Microambiente Tumoral/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) responds poorly to checkpoint blockade, such as anti-CTLA-4 and anti-PD-1. Galectin-9, a ß-galactoside-binding lectin, promotes immune suppression through T-cell inhibition, and programming of tolerogenic macrophages. Of all cancers tested, PDAC showed the highest expression of LGALS9 (galectin-9) mRNA. We analyzed formalin-fixed and paraffin-embedded specimens from 83 patients with PDAC stained for galectin-9. Using flow cytometry, we determined galectin-9 expression on immune cells from tumor and matched blood samples from 12 patients with resectable PDAC. Furthermore, we analyzed galectin-9 serum levels by enzyme-linked immunosorbent assay using serum samples from 70 patients with PDAC, from 36 individuals with benign pancreatic disease, and from 28 healthy controls. Galectin-9 was highly expressed in human PDAC compared with normal pancreas and present on both tumor and immune cells. Tumor-infiltrating immune cells, especially CD3+ T cells, showed upregulation of galectin-9 compared with immune cells from matched blood. Blood γδ T cells from PDAC patients had higher galectin-9 expression than γδ T cells from healthy individuals. Galectin-9 polarized macrophages toward a protumoral M2 phenotype leading to suppressed T-cell cytokine secretion. Furthermore, serum concentration of galectin-9 was able to discriminate PDAC from benign pancreatic disease and healthy individuals, and was prognostic for stage IV patients. Galectin-9 is a new biomarker for the detection of PDAC.
