NKp46 Receptor-Mediated Interferon-γ Production by Natural Killer Cells Increases Fibronectin 1 to Alter Tumor Architecture and Control Metastasis.

Natural killer (NK) cells are innate lymphoid cells, and their presence within human tumors correlates with better prognosis. However, the mechanisms by which NK cells control tumors in vivo are unclear. Here, we used reflectance confocal microscopy (RCM) imaging in humans and in mice to visualize tumor architecture in vivo. We demonstrated that signaling via the NK cell receptor NKp46 (human) and Ncr1 (mouse) induced interferon-γ (IFN-γ) secretion from intratumoral NK cells. NKp46- and Ncr1-mediated IFN-γ production led to the increased expression of the extracellular matrix protein fibronectin 1 (FN1) in the tumors, which altered primary tumor architecture and resulted in decreased metastases formation. Injection of IFN-γ into tumor-bearing mice or transgenic overexpression of Ncr1 in NK cells in mice resulted in decreased metastasis formation. Thus, we have defined a mechanism of NK cell-mediated control of metastases in vivo that may help develop NK cell-dependent cancer therapies.

BRAF and MEK inhibitor combinations induce potent molecular and immunological effects in NRAS-mutant melanoma cells: Insights into mode of action and resistance mechanisms.

About 25% of melanoma harbor activating NRAS mutations, which are associated with aggressive disease therefore requiring a rapid antitumor intervention. However, no efficient targeted therapy options are currently available for patients with NRAS-mutant melanoma. MEK inhibitors (MEKi) appear to display a moderate antitumor activity and also immunological effects in NRAS-mutant melanoma, providing an ideal backbone for combination treatments. In our study, the MEKi binimetinib, cobimetinib and trametinib combined with the BRAF inhibitors (BRAFi) encorafenib, vemurafenib and dabrafenib were investigated for their ability to inhibit proliferation, induce apoptosis and alter the expression of immune modulatory molecules in sensitive NRAS-mutant melanoma cells using two- and three-dimensional cell culture models as well as RNA sequencing analyses. Furthermore, NRAS-mutant melanoma cells resistant to the three BRAFi/MEKi combinations were established to characterize the mechanisms contributing to their resistance. All BRAFi induced a stress response in the sensitive NRAS-mutant melanoma cells thereby significantly enhancing the antiproliferative and proapoptotic activity of the MEKi analyzed. Furthermore, BRAFi/MEKi combinations upregulated immune relevant molecules, such as ICOS-L, components of antigen-presenting machinery and the "don't eat me signal" molecule CD47 in the melanoma cells. The BRAFi/MEKi-resistant, NRAS-mutant melanoma cells counteracted the molecular and immunological effects of BRAFi/MEKi by upregulating downstream mitogen-activated protein kinase pathway molecules, inhibiting apoptosis and promoting immune escape mechanisms. Together, our study reveals potent molecular and immunological effects of BRAFi/MEKi in sensitive NRAS-mutant melanoma cells that may be exploited in new combinational treatment strategies for patients with NRAS-mutant melanoma.

Tumor immunology meets oncology (TIMO) XVI, July 07-09 2022 in Halle/Saale, Germany.

During the TIMO meeting 2022, national and international scientists as well as clinicians gave novel insights as well as perspectives into basic and translational tumor immunology.

Tumor immunology meets oncology (TIMO) XVII, April 20-22 2023 in Halle/Saale, Germany.

During the TIMO meeting 2023, national and international scientists as well as clinicians gave novel insights as well as perspectives into basic and translational tumor immunology. https://dgfi.org/arbeitskreise/ak-tumorimmunologie/meeting/.

Correlation of the tumor escape phenotype with loss of PRELP expression in melanoma.

BACKGROUND: Despite immunotherapies having revolutionized the treatment of advanced cutaneous melanoma, effective and durable responses were only reported in a few patients. A better understanding of the interaction of melanoma cells with the microenvironment, including extracellular matrix (ECM) components, might provide novel therapeutic options. Although the ECM has been linked to several hallmarks of cancer, little information is available regarding the expression and function of the ECM protein purine-arginine-rich and leucine-rich protein (PRELP) in cancer, including melanoma. METHODS: The structural integrity, expression and function of PRELP, its correlation with the expression of immune modulatory molecules, immune cell infiltration and clinical parameters were determined using standard methods and/or bioinformatics. RESULTS: Bioinformatics analysis revealed a heterogeneous, but statistically significant reduced PRELP expression in available datasets of skin cutaneous melanoma when compared to adjacent normal tissues, which was associated with reduced patients' survival, low expression levels of components of the MHC class I antigen processing machinery (APM) and interferon (IFN)-γ signal transduction pathway, but increased expression of the transforming growth factor (TGF)-ß isoform 1 (TFGB1) and TGF-ß receptor 1 (TGFBR1). In addition, a high frequency of intra-tumoral T cells directly correlated with the expression of MHC class I and PRELP as well as the T cell attractant CCL5 in melanoma lesions. Marginal to low PRELP expression levels were found in the 47/49 human melanoma cell lines analysis. Transfection of PRELP into melanoma cell lines restored MHC class I surface expression due to transcriptional upregulation of major MHC class I APM and IFN-γ pathway components. In addition, PRELP overexpression is accompanied by high CCL5 secretion levels in cell supernatant, an impaired TGF-ß signaling as well as a reduced cell proliferation, migration and invasion of melanoma cells. CONCLUSIONS: Our findings suggest that PRELP induces the expression of MHC class I and CCL5 in melanoma, which might be involved in an enhanced T cell recruitment and immunogenicity associated with an improved patients' outcome. Therefore, PRELP might serve as a marker for predicting disease progression and its recovery could revert the tumorigenic phenotype, which represents a novel therapeutic option for melanoma.

Cancer Immunology: Immune Escape of Tumors-Expression and Regulation of HLA Class I Molecules and Its Role in Immunotherapies.

The addition of "avoiding immune destruction" to the hallmarks of cancer demonstrated the importance of cancer immunology and in particular the role of immune surveillance and escape from malignancies. However, the underlying mechanisms contributing to immune impairment and immune responses are diverse. Loss or reduced expression of the HLA class I molecules are major characteristics of human cancers resulting in an impaired recognition of tumor cells by CD8 + cytotoxic T lymphocytes. This is of clinical relevance and associated with worse patients outcome and limited efficacy of T-cell-based immunotherapies. Here, we summarize the role of HLA class I antigens in cancers by focusing on the underlying molecular mechanisms responsible for HLA class I defects, which are caused by either structural alterations or deregulation at the transcriptional, posttranscriptional, and posttranslational levels. In addition, the influence of HLA class I abnormalities to adaptive and acquired immunotherapy resistances will be described. The in-depth knowledge of the different strategies of malignancies leading to HLA class I defects can be applied to design more effective cancer immunotherapies.

Induction of pulmonary HLA-G expression by SARS-CoV-2 infection.

The non-classical human leukocyte antigen (HLA)-G exerts immune-suppressive properties modulating both NK and T cell responses. While it is physiologically expressed at the maternal-fetal interface and in immune-privileged organs, HLA-G expression is found in tumors and in virus-infected cells. So far, there exists little information about the role of HLA-G and its interplay with immune cells in biopsies, surgical specimen or autopsy tissues of lung, kidney and/or heart muscle from SARS-CoV-2-infected patients compared to control tissues. Heterogeneous, but higher HLA-G protein expression levels were detected in lung alveolar epithelial cells of SARS-CoV-2-infected patients compared to lung epithelial cells from influenza-infected patients, but not in other organs or lung epithelia from non-viral-infected patients, which was not accompanied by high levels of SARS-CoV-2 nucleocapsid antigen and spike protein, but inversely correlated to the HLA-G-specific miRNA expression. High HLA-G expression levels not only in SARS-CoV-2-, but also in influenza-infected lung tissues were associated with a high frequency of tissue-infiltrating immune cells, but low numbers of CD8+ cells and an altered expression of hyperactivation and exhaustion markers in the lung epithelia combined with changes in the spatial distribution of macrophages and T cells. Thus, our data provide evidence for an involvement of HLA-G and HLA-G-specific miRNAs in immune escape and as suitable therapeutic targets for the treatment of SARS-CoV-2 infections.

Interferons and Resistance Mechanisms in Tumors and Pathogen-Driven Diseases-Focus on the Major Histocompatibility Complex (MHC) Antigen Processing Pathway.

Interferons (IFNs), divided into type I, type II, and type III IFNs represent proteins that are secreted from cells in response to various stimuli and provide important information for understanding the evolution, structure, and function of the immune system, as well as the signaling pathways of other cytokines and their receptors. They exert comparable, but also distinct physiologic and pathophysiologic activities accompanied by pleiotropic effects, such as the modulation of host responses against bacterial and viral infections, tumor surveillance, innate and adaptive immune responses. IFNs were the first cytokines used for the treatment of tumor patients including hairy leukemia, renal cell carcinoma, and melanoma. However, tumor cells often develop a transient or permanent resistance to IFNs, which has been linked to the escape of tumor cells and unresponsiveness to immunotherapies. In addition, loss-of-function mutations in IFN signaling components have been associated with susceptibility to infectious diseases, such as COVID-19 and mycobacterial infections. In this review, we summarize general features of the three IFN families and their function, the expression and activity of the different IFN signal transduction pathways, and their role in tumor immune evasion and pathogen clearance, with links to alterations in the major histocompatibility complex (MHC) class I and II antigen processing machinery (APM). In addition, we discuss insights regarding the clinical applications of IFNs alone or in combination with other therapeutic options including immunotherapies as well as strategies reversing the deficient IFN signaling. Therefore, this review provides an overview on the function and clinical relevance of the different IFN family members, with a specific focus on the MHC pathways in cancers and infections and their contribution to immune escape of tumors.

Correlation between Circulating miR-16, miR-29a, miR-144 and miR-150, and the Radiotherapy Response and Survival of Non-Small-Cell Lung Cancer Patients.

Despite the success of current therapy concepts, patients with advanced non-small-cell lung cancer (NSCLC) still have a very poor prognosis. Therefore, biological markers are urgently needed, which allow the assessment of prognosis, or prediction of the success of therapy or resistance in this disease. Circulating microRNAs (miRs) have potential as biomarkers for the prognosis and prediction of response to therapy in cancer patients. Based on recent evidence that circulating miR-16, miR-29a, miR-144 and miR-150 can be regulated by ionizing radiation, the concentration of these four miRs was assessed in the plasma of NSCLC patients at different time points of radiotherapy by digital droplet PCR (ddPCR). Furthermore, their impact on patients' prognosis was evaluated. The mean plasma levels of miR-16, miR-29a, miR-144 and miR-150 significantly differed intra- and inter-individually, and during therapy in NSCLC patients, but showed a strong positive correlation. The individual plasma levels of miR-16, miR-29a and miR-144 had prognostic value in NSCLC patients during or at the end of radiotherapy in Cox's regression models. NSCLC patients with low levels of these three miRs at the end of radiotherapy had the worst prognosis. However, miR-150 plasma levels and treatment-dependent changes were not predictive. In conclusion, circulating miR-16, miR-29a and miR-144, but not miR-150, have a prognostic value in NSCLC patients undergoing radiotherapy.

Characterization of the effect of histone deacetylation inhibitors on CD8+ T cells in the context of aging.

BACKGROUND: Posttranslational protein modifications regulate essential cellular processes, including the immune cell activation. Despite known age-related alterations of the phenotype, composition and cytokine profiles of immune cells, the role of acetylation in the aging process of the immune system was not broadly investigated. Therefore, in the current study the effect of acetylation on the protein expression profiles and function of CD8+ T cells from donors of distinct age was analyzed using histone deacetylase inhibitors (HDACi). METHODS: CD8+ T cells isolated from peripheral blood mononuclear cells of 30 young (< 30 years) and 30 old (> 60 years) healthy donors were activated with anti-CD3/anti-CD28 antibodies in the presence and absence of a cocktail of HDACi. The protein expression profiles of untreated and HDACi-treated CD8+ T cells were analyzed using two-dimensional gel electrophoresis. Proteins with a differential expression level (less than 0.66-fold decrease or more than 1.5-fold increase) between CD8+ T cells of young and old donors were identified by matrix-associated laser desorption ionization-time of flight mass spectrometry. Functional enrichment analysis of proteins identified was performed using the online tool STRING. The function of CD8+ T cells was assessed by analyses of cytokine secretion, surface expression of activation markers, proliferative capacity and apoptosis rate. RESULTS: The HDACi treatment of CD8+ T cells increased in an age-independent manner the intracellular acetylation of proteins, in particular cytoskeleton components and chaperones. Despite a strong similarity between the protein expression profiles of both age groups, the functional activity of CD8+ T cells significantly differed with an age-dependent increase in cytokine secretion and expression of activation markers for CD8+ T cells from old donors, which was maintained after HDACi treatment. The proliferation and apoptosis rate of CD8+ T cells after HDACi treatment was equal between both age groups. CONCLUSIONS: Despite a comparable effect of HDACi treatment on the protein signature of CD8+ T cells from donors of different ages, an initial higher functionality of CD8+ T cells from old donors when compared to CD8+ T cells from young donors was detected, which might have clinical relevance.

What turns CREB on? And off? And why does it matter?

Altered expression and function of the transcription factor cyclic AMP response-binding protein (CREB) has been identified to play an important role in cancer and is associated with the overall survival and therapy response of tumor patients. This review focuses on the expression and activation of CREB under physiologic conditions and in tumors of distinct origin as well as the underlying mechanisms of CREB regulation by diverse stimuli and inhibitors. In addition, the clinical relevance of CREB is summarized, including its use as a prognostic and/or predictive marker as well as a therapeutic target.

A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells.

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.

"Tumor immunology meets oncology (TIMO) XV", April 25th-27th 2019, Halle/Saale, Germany.

Novel insights into basic and translational tumor immunology including immunotherapies were presented by national and international scientists and clinicians at the TIMO XV meeting in Halle.

Characterization of the expression and immunological impact of the transcriptional activator CREB in renal cell carcinoma.

BACKGROUND: The non-classical human leukocyte antigen (HLA)-G is a strong immunomodulatory molecule. Under physiological conditions, HLA-G induces immunological tolerance in immune privileged tissues, while under pathophysiological situations it contributes to immune escape mechanisms. Therefore, HLA-G could act as a potential immune checkpoint for future anti-cancer immunotherapies. Recent data suggest an aberrant expression of the cAMP response element binding protein (CREB) in clear cell renal cell carcinoma (ccRCC), which is correlated with tumor grade and stage. Furthermore, preliminary reports demonstrated a connection of CREB as a control variable of HLA-G transcription due to CREB binding sites in the HLA-G promoter region. This study investigates the interaction between CREB and HLA-G in different renal cell carcinoma (RCC) subtypes and its correlation to clinical parameters. METHODS: The direct interaction of CREB with the HLA-G promoter was investigated by chromatin immunoprecipitation in RCC cell systems. Furthermore, the expression of CREB and HLA-G was determined by immunohistochemistry using a tissue microarray (TMA) consisting of 453 RCC samples of distinct subtypes. Staining results were assessed for correlations to clinical parameters as well as to the composition of the immune cell infiltrate. RESULTS: There exists a distinct expression pattern of HLA-G and CREB in the three main RCC subtypes. HLA-G and CREB expression were the lowest in chromophobe RCC lesions. However, the clinical relevance of CREB and HLA-G expression differed. Unlike HLA-G, high levels of CREB expression were positively associated to the overall survival of RCC patients. A slightly, but significantly elevated number of tumor infiltrating regulatory T cells was observed in tumors of high CREB expression. Whether this small increase is of clinical relevance has to be further investigated. CONCLUSIONS: An interaction of CREB with the HLA-G promoter could be validated in RCC cell lines. Thus, for the first time the expression of CREB and its interaction with the HLA-G in human RCCs has been shown, which might be of clinical relevance.

Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated.

BACKGROUND: Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. METHODS: DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation. RESULTS: Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines. CONCLUSION: Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

The Role of the RNA-Binding Protein Family MEX-3 in Tumorigenesis.

The muscle excess 3 (MEX-3) protein was first identified in Caenorhabditis elegans (C. elegans), and its respective homologues were also observed in vertebrates, including humans. It is a RNA-binding protein (RBP) with an additional ubiquitin E3 ligase function, which further acts as a post-transcriptional repressor through unknown mechanisms. In humans, MEX-3 proteins post-transcriptionally regulate a number of biological processes, including tumor immunological relevant ones. These have been shown to be involved in various diseases, including tumor diseases of distinct origins. This review provides information on the expression and function of the human MEX-3 family in healthy tissues, as well after malignant transformation. Indeed, the MEX-3 expression was shown to be deregulated in several cancers and to affect tumor biological functions, including apoptosis regulation, antigen processing, and presentation, thereby, contributing to the immune evasion of tumor cells. Furthermore, current research suggests MEX-3 proteins as putative markers for prognosis and as novel targets for the anti-cancer treatment.

Current Understanding of the HIF-1-Dependent Metabolism in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the 10th most frequent human malignancy and is thus a global burden. Despite some progress in diagnosis and therapy, patients' overall survival rate, between 40 and 55%, has stagnated over the last four decades. Since the tumor node metastasis (TNM) system is not precise enough to predict the disease outcome, additive factors for diagnosis, prognosis, prediction and therapy resistance are urgently needed for OSCC. One promising candidate is the hypoxia inducible factor-1 (HIF-1), which functions as an early regulator of tumor aggressiveness and is a key promoter of energy adaptation. Other parameters comprise the composition of the tumor microenvironment, which determines the availability of nutrients and oxygen. In our opinion, these general processes are linked in the pathogenesis of OSCC. Based on this assumption, the review will summarize the major features of the HIF system-induced activities, its target proteins and related pathways of nutrient utilization and metabolism that are essential for the initiation, progression and therapeutic stratification of OSCC.

Immune Escape Mechanisms and Their Clinical Relevance in Head and Neck Squamous Cell Carcinoma.

Immunotherapy has been recently approved for the treatment of relapsed and metastatic human papilloma virus (HPV) positive and negative head and neck squamous cell carcinoma (HNSCC). However, the response of patients is limited and the overall survival remains short with a low rate of long-term survivors. There exists growing evidence that complex and partially redundant immune escape mechanisms play an important role for the low efficacy of immunotherapies in this disease. These are caused by diverse complex processes characterized by (i) changes in the expression of immune modulatory molecules in tumor cells, (ii) alterations in the frequency, composition and clonal expansion of immune cell subpopulations in the tumor microenvironment and peripheral blood leading to reduced innate and adaptive immune responses, (iii) impaired homing of immune cells to the tumor site as well as (iv) the presence of immune suppressive soluble and physical factors in the tumor microenvironment. We here summarize the major immune escape strategies of HNSCC lesions, highlight pathways, and molecular targets that help to attenuate HNSCC-induced immune tolerance, affect the selection and success of immunotherapeutic approaches to overcome resistance to immunotherapy by targeting immune escape mechanisms and thus improve the HNSCC patients' outcome.

Expression, Regulation and Function of microRNA as Important Players in the Transition of MDS to Secondary AML and Their Cross Talk to RNA-Binding Proteins.

Myelodysplastic syndromes (MDS), heterogeneous diseases of hematopoietic stem cells, exhibit a significant risk of progression to secondary acute myeloid leukemia (sAML) that are typically accompanied by MDS-related changes and therefore significantly differ to de novo acute myeloid leukemia (AML). Within these disorders, the spectrum of cytogenetic alterations and oncogenic mutations, the extent of a predisposing defective osteohematopoietic niche, and the irregularity of the tumor microenvironment is highly diverse. However, the exact underlying pathophysiological mechanisms resulting in hematopoietic failure in patients with MDS and sAML remain elusive. There is recent evidence that the post-transcriptional control of gene expression mediated by microRNAs (miRNAs), long noncoding RNAs, and/or RNA-binding proteins (RBPs) are key components in the pathogenic events of both diseases. In addition, an interplay between RBPs and miRNAs has been postulated in MDS and sAML. Although a plethora of miRNAs is aberrantly expressed in MDS and sAML, their expression pattern significantly depends on the cell type and on the molecular make-up of the sample, including chromosomal alterations and single nucleotide polymorphisms, which also reflects their role in disease progression and prediction. Decreased expression levels of miRNAs or RBPs preventing the maturation or inhibiting translation of genes involved in pathogenesis of both diseases were found. Therefore, this review will summarize the current knowledge regarding the heterogeneity of expression, function, and clinical relevance of miRNAs, its link to molecular abnormalities in MDS and sAML with specific focus on the interplay with RBPs, and the current treatment options. This information might improve the use of miRNAs and/or RBPs as prognostic markers and therapeutic targets for both malignancies.