In human tumors of distinct origin including renal cell carcinoma (RCC), the non-classical human leukocyte antigen G (HLA-G) is frequently expressed, thereby inhibiting the cytotoxic activity of T and natural killer (NK) cells. Recent studies demonstrated a strong post-transcriptional gene regulation of the HLA-G by miR-152, -148A, -148B and -133A. Standard methods were applied to characterize the expression and function of HLA-G, HLA-G-regulatory microRNAs (miRs) and the immune cell infiltration in 453 RCC lesions using a tissue microarray and five RCC cell lines linking these results to clinical parameters. Direct interactions with HLA-G regulatory miRs and the HLA-G 3' untranslated region (UTR) were detected and the affinities of these different miRs to the HLA-G 3'-UTR compared. qPCR analyses and immunohistochemical staining revealed an inverse expression of miR-148A and -133A with the HLA-G protein and. Stable miR overexpression caused a downregulation of HLA-G protein enhancing the NK and LAK cell-mediated cytotoxicity in CD107a activation assays revealing a HLA-G-dependent cytotoxic activity of immune effector cells. A significant higher frequency of CD3/CD8 T cell lymphocytes, but no differences in the activation markers CD69, CD25 or in the presence of CD56, FoxP3 and CD4 immune cells were detected in HLA-G compared to HLA-G RCC lesions. This could be associated with higher WHO grade, but not with a disease-specific survival. These data suggest a miR-mediated control of HLA-G expression in RCC, which is associated with a distinct pattern of immune cell infiltration.

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Ligands for receptors of natural killer (NK) cells and CD8(+) cytotoxic T lymphocytes (CTL), such as the inhibitory nonclassical HLA-G, the activating stress-induced major histocompatibility complex class I-related antigens MICA and MICB, and/or the UL16-binding proteins (ULBPs), are often aberrantly expressed upon viral infection and neoplastic transformation, thereby preventing virus-infected or malignant-transformed cells from elimination by immune effector cells. Recently, it has been shown that ligands of both NK and CD8(+) T cells are regulated by a number of cellular and/or viral microRNAs (miRs). These miRs are involved in shaping the antiviral and/or antitumoral immune responses as well as neoplastic growth properties. This review summarizes the expression pattern and function of miRs directed against selected NK and T cell receptor ligands, their putative role in shaping immune surveillance and tumorigenicity, and their clinical relevance. In addition, the potential role of RNA-binding proteins in the post-transcriptional gene regulation of these ligands will be discussed.

The detailed mechanisms of Epstein–Barr virus (EBV) infection in the initiation and progression of EBV-associated malignancies are not yet completely understood. During the last years, new insights into the mechanisms of malignant transformation of EBV-infected cells including somatic mutations and epigenetic modifications, their impact on the microenvironment and resulting unique immune signatures related to immune system functional status and immune escape strategies have been reported. In this context, there exists increasing evidence that EBV-infected tumor cells can influence the tumor microenvironment to their own benefit by establishing an immune-suppressive surrounding. The identified mechanisms include EBV gene integration and latent expression of EBV-infection-triggered cytokines by tumor and/or bystander cells, e.g., cancer-associated fibroblasts with effects on the composition and spatial distribution of the immune cell subpopulations next to the infected cells, stroma constituents and extracellular vesicles. This review summarizes (i) the typical stages of the viral life cycle and EBV-associated transformation, (ii) strategies to detect EBV genome and activity and to differentiate various latency types, (iii) the role of the tumor microenvironment in EBV-associated malignancies, (iv) the different immune escape mechanisms and (v) their clinical relevance. This gained information will enhance the development of therapies against EBV-mediated diseases to improve patient outcome.

BackgroundThe importance of B7-H molecules for the T cell/tumor communication and its impact on renal cell carcinoma (RCC) progression and prognosis has been recently described. Cytokine treatment of RCC has earlier been shown to be beneficial in preclinical settings, but its clinical implementation has not proven to be as effective. This might be partially explained by the yet incomplete picture of cellular alterations in tumor cells upon cytokine treatment investigated in detail in this study.MethodsRCC tumor cell lines were treated with different cytokines alone or in combination. The constitutive and/or cytokine-induced expression of cytokine receptors signaling components and B7-H molecules in RCC cells were analysed by qPCR and flow cytometry. A mcherry reporter gene construct containing B7-H1 promoter was cloned and its activity was determined upon transfection in cytokine-stimulated cells. Cytokine pretreated tumor cells were co-cultured with allogeneic CD8+ T cells from healthy donors and T cell proliferation as well as cytokine secretion was determined.ResultsA heterogeneous, but constitutive B7-H1,-H2,-H3 and H4 expression was found on human RCC cell lines. IL-4 and TNFα treatment led to strong synergistic induction of B7-H1 in RCC cells, whereas B7-H2 was only increased by TNFα. In contrast, B7-H3 and B7-H4 expression were not altered by these cytokines. Treatment of RCC cells with TNFα and IL-4 was accompanied by an activation of signaling molecules like NF-κB, IκB and STAT6. The cytokine-mediated up-regulation of B7-H1 was due to transcriptional control as determined by an increased B7-H1 promoter activity in the presence of IL-4 and TNFα. Despite HLA class I and LFA-1 were also increased, the cytokine-mediated up-regulation of B7-H1 was more pronounced and caused an inhibition of allospecifc CD8+ T cell proliferation.ConclusionThus, IL-4 and TNFα, which could be released by immune cells of the tumor microenvironment, are able to control the B7-H1 expression in RCC thereby altering T cell responses. These data are of importance for understanding the complex interplay of tumor cells with immune cells orchestrated by a number of different soluble and membrane bound mediators and for the implementation of check point antibodies directed against B7-H1.

The non-classical human leukocyte antigen G (HLA-G) is expressed at a high frequency in renal cell carcinoma (RCC) and is associated with a higher tumor grade and a poor clinical outcome. This might be caused by the HLA-G-mediated inhibition of the cytotoxicity of T and NK cells. Therefore a selective targeting of HLA-G might represent a powerful strategy to enhance the immunogenicity of RCC lesions. Recent studies identified a number of HLA-G-regulating microRNAs (miRs) and demonstrated an inverse expression of some of these miRs with HLA-G in RCC in vitro and in vivo. However, it was postulated that further miRs might exist contributing to the tightly controlled selective HLA-G expression.By application of a miR enrichment assay (miTRAP) in combination with in silico profiling two novel HLA-G-regulatory miRs, miR-548q and miR-628-5p, were identified. Direct interactions of both miRs with the 3′ untranslated region of HLA-G were confirmed with luciferase reporter gene assays. In addition, qPCR analyses and immunohistochemical staining revealed an inverse, expression of miR-628-5p, but not of miR-548q to the HLA-G protein in primary RCC lesions and cell lines. Stable overexpression of miR-548q and miR-628-5p caused a downregulation of HLA-G mRNA and protein. This leads in case of miR-548q to an enhanced NK cell-mediated HLA-G-dependent cytotoxicity, which could be reverted by ILT2 blockade suggesting a control of the immune effector cell activity at least by this miR. The identification of two novel HLA-G-regulatory miRs extends the number of HLA-G-relevant miRs tuning the HLA-G expression and might serve as future therapeutic targets.

The non-classical human leukocyte antigen E (HLA-E) expression is frequently overexpressed in tumor diseases, transplants and virus-infected cells and represents an immunomodulatory molecule by binding to the receptors CD94/NKG2A, -B and –C on NK and T cells. Due to its immune suppressive features HLA-E expression might represent an important mechanism of tumors to escape immune surveillance.While an aberrant expression of the non-classical HLA-G antigen in human renal cell carcinoma (RCC) has been demonstrated to be associated with a worse outcome of patients and reduced sensitivity to immune effector cell-mediated cytotoxicity, the expression and function of HLA-E has not yet been analyzed in this tumor entity.Higher levels of HLA-E transcripts were detected in all RCC cell lines and tumor lesions, which were tested in comparison to normal kidney epithelium. Immunohistochemical staining of a tissue microarray (TMA) using the HLA-E-specific monoclonal antibody TFL-033 recognizing the cytoplasmic HLA-E α-chain as monomer revealed a heterogeneous HLA-E expression in RCC lesions with the highest frequency in chromophobe RCC when compared to other RCC subtypes. HLA-E expression did not correlate with the frequency of CD3+, CD4+, CD8+ and FoxP3+ immune cell infiltrations, but showed an inverse correlation with infiltrating CD56+ cells. In contrast to HLA-G, HLA-E expression in RCCs was not statistically significant associated with a decreased disease specific survival. These data suggest that HLA-E overexpression frequently occurs in RCC and correlates with reduced immunogenicity.

Tumor escape is often associated with abnormalities in the surface expression of the human leukocyte antigen class I (HLA-I) antigens thereby limiting CD8 + cytotoxic T cell responses. This impaired HLA-I surface expression can be mediated by deficient expression of components of the antigen processing and presentation machinery (APM) due to epigenetic, transcriptional and/or post-transcriptional processes. Since a discordant mRNA and protein expression pattern of APM components including the peptide transporter associated with antigen processing 1 (TAP1) has been frequently described in tumors of distinct origin, a post-transcriptional control of APM components caused by microRNAs (miR) was suggested. Using an in silico approach, miR-200a-5p has been identified as a candidate miR binding to the 3ʹ untranslated region (UTR) of TAP1. Luciferase reporter assays demonstrated a specific binding of miR-200a-5p to the TAP1 3ʹ-UTR. Furthermore, the miR-200a-5p expression is inversely correlated with the TAP1 protein expression in HEK293T cells and in a panel of melanoma cell lines as well as in primary melanoma lesions. High levels of miR-200a-5p expression were associated with a shorter overall survival of melanoma patients. Overexpression of miR-200a-5p reduced TAP1 levels, which was accompanied by a decreased HLA-I surface expression and an enhanced NK cell sensitivity of melanoma cells. These data show for the first time a miRmediated control of the peptide transporter subunit TAP1 in melanoma thereby leading to a reduced HLA-I surface expression accompanied by an altered immune recognition and reduced patients' survival.