Search

Cell survival and apoptosis, ASM-activity, Ceramide and Sphingomyelin levels after siRNA-mediated ASM depletion in NSCLC cell lines.

Decreased NFATc1 in T cells in the tumoural region of NSCLC patients with advanced disease stages.Cytokines secreted from NFATc1Î"CD4 total lung cells did not influence lung tumour cell growth. Representative dot plots of NFATc1 levels in T cells of lung tumor-bearing B6 WT mice in vivo treated and in vitro re-challenged with αPD-1antibodies.

The lipid hydrolase enzyme acid sphingomyelinase (ASM) is required for the conversion of the lipid cell membrane component sphingomyelin into ceramide. In cancer cells, ASM-mediated ceramide production is important for apoptosis, cell proliferation, and immune modulation, highlighting ASM as a potential multimodal therapeutic target. In this study, we demonstrate elevated ASM activity in the lung tumor environment and blood serum of patients with non–small cell lung cancer (NSCLC). RNAi-mediated attenuation of SMPD1 in human NSCLC cells rendered them resistant to serum starvation–induced apoptosis. In a murine model of lung adenocarcinoma, ASM deficiency reduced tumor development in a manner associated with significant enhancement of Th1-mediated and cytotoxic T-cell–mediated antitumor immunity. Our findings indicate that targeting ASM in NSCLC can act by tumor cell–intrinsic and –extrinsic mechanisms to suppress tumor cell growth, most notably by enabling an effective antitumor immune response by the host. Cancer Res; 77(21); 5963–76. ©2017 AACR.

Regulation of T cell phenotype and serum Ceramide/Sphingomyelin levels in wild type and ASM KO naïve and lung tumour bearing mice

Supplementary Figure Legends

Supplementary Figure 3: Representative dot plots of NFATc1 levels in T cells of lung tumor-bearing B6 WT mice in vivo treated and in vitro re-challenged with αPD-1antibodies.

Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor activated by T-cell receptor (TCR) and Ca2+ signaling that affects T-cell activation and effector function. Upon tumor antigen challenge, TCR and calcium-release–activated channels are induced, promoting NFAT dephosphorylation and translocation into the nucleus. In this study, we report a progressive decrease of NFATc1 in lung tumor tissue and in tumor-infiltrating lymphocytes (TIL) of patients suffering from advanced-stage non–small cell lung cancer (NSCLC). Mice harboring conditionally inactivated NFATc1 in T cells (NFATc1ΔCD4) showed increased lung tumor growth associated with impaired T-cell activation and function. Furthermore, in the absence of NFATc1, reduced IL2 influenced the development of memory CD8+ T cells. We found a reduction of effector memory and CD103+ tissue-resident memory (TRM) T cells in the lung of tumor-bearing NFATc1ΔCD4 mice, underlining an impaired cytotoxic T-cell response and a reduced TRM tissue-homing capacity. In CD4+ICOS+ T cells, programmed cell death 1 (PD-1) was induced in the draining lymph nodes of these mice and associated with lung tumor cell growth. Targeting PD-1 resulted in NFATc1 induction in CD4+ and CD8+ T cells in tumor-bearing mice and was associated with increased antitumor cytotoxic functions. This study reveals a role of NFATc1 in the activation and cytotoxic functions of T cells, in the development of memory CD8+ T-cell subsets, and in the regulation of T-cell exhaustion. These data underline the indispensability of NFATc1 for successful antitumor immune responses in patients with NSCLC.Significance: The multifaceted role of NFATc1 in the activation and function of T cells during lung cancer development makes it a critical participant in antitumor immune responses in patients with NSCLC. Cancer Res; 78(13); 3619–33. ©2018 AACR.

Levels of different Ceramide and Sphingomyelin molecular species measured in the blood serum of patients with ADC and SCC as well as in healthy donors

Although lung cancer is the leading cause of cancer death worldwide, the mechanisms how lung cancer cells evade the immune system remain incompletely understood. Here, we discovered IL-9-dependent signaling mechanisms that drive immune evasion in non-small cell lung cancer (NSCLC). We found increased IL-9 and IL-21 production by T cells in the tumoral region of the lung of patients with NSCLC, suggesting the presence of Th9 cells in the lung tumor microenvironment. Moreover, we noted IL-9 producing Tregs in NSCLC. IL-9 target cells in NSCLC consisted of IL-9R+ tumor cells and tumor-infiltrating lymphocytes. In two murine experimental models of NSCLC, and in vitro , IL-9 prevented cell death and controlled growth of lung adenocarcinoma cells. Targeted deletion of IL-9 resulted in successful lung tumor rejection in vivo associated with an induction of IL-21 and reduction of lung pSTAT3, Th17 and Treg cells. Finally, anti-IL-9 antibody immunotherapy resulted in suppression of tumor development even in established experimental NSCLC and was associated with reduced IL-10 production and induction of IL-21 in the lung. In conclusion, our findings indicate that IL-9 drives immune escape of lung tumor cells via effects on tumor cell survival and tumor infiltrating T cells. Thus, strategies blocking IL-9 emerge as a new approach for clinical therapy of lung cancer. Funding: This work was supported by DFG grant FI817-5-1,2 and by a DFG FI817- 5-3 granted to SF. M.T.C. work is supported by a DFG CH 1428/2-1. MHK was supported by NIH grant AI057459. Declaration of Interest: None to declare. Ethical Approval: Our human study was performed at the Friedrich-Alexander-University Erlangen-Nurnberg, Germany, after being approved by the ethics review board of the University of Erlangen (Re-No: 56_12B; DRKS-ID: DRKS00005376).

Immunotherapy is a promising treatment for lung cancer by activation of cytotoxic CD4+ and CD8+ T cells in the tumour microenvironment and suppression of Tregs cells. We previously demonstrated that the recognition and elimination of lung tumour cells by T lymphocytes requires the presence of T-box-expressed in T cells (T-bet), a transcriptional regulator that induces Th1, cytolytic CD8+ T and NK cells and inhibits Treg cells. Moreover, recent studies show that IL-2 in the tumour microenvironment supports acquisition of cytotoxic T helper cells through the transcriptional repressor B lymphocytes-induced maturation protein-1 (Blimp-1). As Blimp-1 also affects the homeostasis and function of CD4+, CD8+ T cells as well as Treg cells, we hypothesized that this transcription factor might be important in lung cancer. We thus started to analyze the Blimp-1 protein expression by Western blot analysis using proteins isolated from the lung of a cohort of patients that underwent surgery because of non-small-cell lung carcinoma (NSCLC) and subsequent histological classification as lung adenocarcinoma (ADC). We found that patients with ADC had a significant induction of Blimp-1 protein expression in the tumoural region of ADC (TU, solid tumour) as compared control lung region (CTR, tumour-free control area). We further analyzed the expression of T-bet in these patients and found that T-bet, as opposed to Blimp-1, was mainly expressed in the control region of the lung and downregulated in the tumoural region. Thus, these data suggest that Blimp-1 might represent an additional new marker to be considered in the absence of T-bet as driver of anti-tumor immune responses in lung cancer.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide health threat. Here, we report that low plasma interleukin-3 (IL-3) levels were associated with increased severity and mortality during SARS-CoV-2 infections. IL-3 promoted the recruitment of antiviral circulating plasmacytoid dendritic cells (pDCs) into the airways by stimulating CXCL12 secretion from pulmonary CD123+epithelial cells. This study identifies IL-3 as a predictive disease marker and potential therapeutic target for SARS-CoV-2 infections.

Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor activated by T-cell receptor (TCR) and Ca2+ signaling that affects T-cell activation and effector function. Upon tumor antigen challenge, TCR and calcium-release–activated channels are induced, promoting NFAT dephosphorylation and translocation into the nucleus. In this study, we report a progressive decrease of NFATc1 in lung tumor tissue and in tumor-infiltrating lymphocytes (TIL) of patients suffering from advanced-stage non–small cell lung cancer (NSCLC). Mice harboring conditionally inactivated NFATc1 in T cells (NFATc1ΔCD4) showed increased lung tumor growth associated with impaired T-cell activation and function. Furthermore, in the absence of NFATc1, reduced IL2 influenced the development of memory CD8+ T cells. We found a reduction of effector memory and CD103+ tissue-resident memory (TRM) T cells in the lung of tumor-bearing NFATc1ΔCD4 mice, underlining an impaired cytotoxic T-cell response and a reduced TRM tissue-homing capacity. In CD4+ICOS+ T cells, programmed cell death 1 (PD-1) was induced in the draining lymph nodes of these mice and associated with lung tumor cell growth. Targeting PD-1 resulted in NFATc1 induction in CD4+ and CD8+ T cells in tumor-bearing mice and was associated with increased antitumor cytotoxic functions. This study reveals a role of NFATc1 in the activation and cytotoxic functions of T cells, in the development of memory CD8+ T-cell subsets, and in the regulation of T-cell exhaustion. These data underline the indispensability of NFATc1 for successful antitumor immune responses in patients with NSCLC. Significance: The multifaceted role of NFATc1 in the activation and function of T cells during lung cancer development makes it a critical participant in antitumor immune responses in patients with NSCLC. Cancer Res; 78(13); 3619–33. ©2018 AACR.

The mechanisms of lung cancer evading the immune system are still not fully understood. As regulatory T cells (Tregs) exhibit a pro-tumoral function they are in focus of immunotherapies. Peripheral Treg differentiation is driven by TCR and its co-stimulation. A further crucial signal displays the binding of IL-2 to the high affinity receptor CD25 with downstream involvement of Signal Transducer and Activator of Transcription 5 (STAT5). STAT5 activation as well as TGF-beta stimulation in the periphery leads to expression of the Treg key factor FoxP3. In this study, we wanted to investigate the role of STAT5 and its activated status via phosphorylation (pSTAT5) within lung cancer. We thus isolated peripheral blood mononuclear cells (PBMC) from NSCLC patients and healthy donors. PBMCs were cultured under different stimulation conditions for 4 days and analyzed by FACS. Here we found CD4+ T cells with high induction of pSTAT5A and CD25 upon aCD3 and aCD28 antibody treatment. Moreover, CD4+CD25+FoxP3+ Tregs were detected under T cell activating conditions, and were found to be increased by TGFβ in smokers and NSCLC patients. The Treg population was further inducible by adding low doses of IL-2 while CD4+CD25+FoxP3− effector T cells decreased in the presence of TGFβ. Our findings point to an immunosuppressive microenvironment induced by the tumor with increased TGFβ and reduced IL-2 forcing effector T cells into a Treg state. These preliminary data suggest that current immunotherapy by aCD3/aCD28 antibody treatment could be improved by an additional blockade of TGFβ and induction of IL-2 levels. This treatment would result in an increase of CD4+CD25+pSTAT5+FoxP3− effector cells and a reduction of CD4+CD25+FoxP3+STAT5+ Tregs.

The transcription factor PU box binding-1 (PU.1), which is encoded by the SPI1 gene, is required for the development of most myeloid cell lineages as well as B- and T cells. Consequently, an alteration of the SPI1 gene and thus PU.1 can have severe impact on the immune system. However, the role of PU.1 in cancer is not yet fully understood. In this study, we were looking for a hint on the influence of PU.1 expression on various cell types during non-small cell lung cancer (NSCLC). To start the investigation, we isolated peripheral blood mononuclear cells (PBMCs) from patients with NSCLC as well as from healthy controls. The PBMCs were cultured with different cytokines for 4 days and subsequently analyzed by flow cytometry. Furthermore, RNA was isolated from the PBMCs and qPCR was performed. The qPCR results showed induced PU.1 mRNA levels in NSCLC patients in relation to GAPDH, compared to control patients. By flow cytometry, we could detect higher PU.1 expression in CD56+ Natural killer (NK) cells from lung cancer patients compared to the healthy donors. Moreover, NK-cells from lung cancer patients, in contrast to healthy controls, expressed the immunosuppressive protein Programmed Death 1 (PD-1). Furthermore, PBMCs from NSCLC patients that were cultured with aCD3/aCD28 antibodies, showed suppression of PU.1 as well as PD-1 in NK-cells, which was even more prominent after addition of IL-2. These preliminary data suggest, that conditions inducing T cell activation like IL-2 and antiCD3/antiCD28 stimulation, can downregulate both the transcription factor PU.1 and PD-1 in NK-cells in tumor patients. Thus, improvement of current immunotherapy could use additional IL-2, which targets PU-1 as well as PD-1 in NK cells in lung cancer.

Immunotherapy improves the life expectancy of patients with lung cancer by targeting the immune inhibitory T cell surface markers resulting in the activation of anti-tumor T cell survival and suppression of tumor cell growth. The transcription factor repressor Positive Regulatory Domain Containing Protein 1 (encoded in humans by the Prdm1 gene) also known as B lymphocytes-induced maturation protein-1 (Blimp-1) affects the homeostasis and function of CD4+ and CD8+ T cells as well as T regulatory cells (T regs). We recently reported that Blimp-1 is induced in the lung tumoral region of patients with lung ADC. To further investigate whether Blimp-1 could be a candidate gene involved in the regulation of the anti-tumor immune-response in lung cancer, we started to investigate peripheral blood mononuclear cells (PBMCs) isolated from healthy control subjects and NSCLC patients for Blimp-1 expression in immunosuppressive CD4+CD25+Foxp3+ T regs. The PBMCs were cultured with and without antiCD3/CD28 antibodies, without and with TGF-beta, to mimic the tumor microenvironment, for 4 days and subsequently analyzed by flow cytometry using antibodies against Blimp-1, Foxp3, CD4 and CD25(IL-2R alpha chain). Here we found, by trend, induced CD4+Blimp1+CD25+Foxp3+ Tregs in unstimulated PBMCs from patients with lung cancer. Moreover, TGF-beta further induced these Blimp1+CD4+CD25+Foxp3+ Tregs in PBMCs obtained both from healthy controls and from NSCLC patients. Taken together these data indicate that the tumor microenvironment induces Blimp-1 in immunosuppressive CD4+CD25+Foxp3+ Tregs via TGF-beta. Thus, current immunotherapy should be combined to TGF-beta inhibitors to target CD4+CD25+Blimp1+Foxp-3+ T regulatory cells.

Lung cancer is one of the most common cancer types in the western world, with a high lethality and increasing incidence. One major factor, supporting tumor growth is a high sugar diet. Thus, reducing the sugar intake by different diets might be a way to improve current cancer treatment. These metabolic changes are accompanied by regulation of systemic levels of Insulin and insulin like growth factors. As oppose to normal cells, tumor cells metabolism mainly depends on glycolysis. Thus, reducing diet carbohydrate intake might be a way to improve current immunotherapy by inducing immunological changes caused by the extracellular glucose concentration in tumor microenvironment. Therefore, we cultured the human lung adenocarcinoma (ADC) cell line A549 with different extracellular glucose concentrations to analyze effects by flow cytometry and qPCR. Moreover, lung tissue samples from patients with non small cell lung cancer (NSCLC), were analyzed by qPCR. In a murine model of lung ADC, tumor cells were injected intravenously and mice were fed with different diets. Lung cells were isolated and analyzed by flow cytometry. Here we show that, IGF1R expression is upregulated in A549 cells line, depending on increasing extracellular glucose concentration. Moreover, in studies on human cohorts of patients with NSCLC, we found a direct correlation between BMI and IGF1R expression and its downstream signaling. Finally, in a murine model of disease, diet glucose restriction decreased significantly tumor load associated with increased lung infiltrating CD8+ T-cells and tissue-resident memory T-cells. Thus, this study supports the need to introduce glucose restrictions in the diet of patients with NSCLC in association with current immunotherapy.

Background The immunosuppressive role of the cytokine IL-35 in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, we analysed the localisation and regulation of IL-35 in the lung of patients with non-small cell lung cancer (NSCLC) to further elucidate the immune-escape of cancer cells in perioperative course of disease. Methods Interleukin 35 (IL-35) was measured by ELISA in postoperative serum from 7 patients with NSCLC as well as 8 samples from healthy controls. Immunohistochemistry, FACS analysis, real-time PCR, as well as western blot from samples of the control (CTR), peri-tumoural (PT) and the tumoural (TU) region of the lung derived from patients with NSCLC and 10 controls were performed. Results Here we found elevated levels of IL-35 in the TU region as well as postoperative serum from patients with lung adenocarcinoma. Consistently, we found an increased expression of IL-35+Foxp-3+ cells, which associated with ARG1 mRNA expression and decreased TNFA in the TU region of the lung of patients with NSCLC as compared to their CTR region. Furthermore, in the CTR region of the lung of patients with NSCLC, CD68+ macrophages were induced and correlated with IL-35+ cells. Finally, IL-35 positively correlated with TTF-1+PD-L1+ cells in the TU region of NSCLC patients. Conclusions Induced IL-35+Foxp3+ cell numbers in the TU region of the lung of patients with NSCLC associated with ARG1 mRNA expression and with TTF-1+PD-L1+ cells. In the tumour-free CTR area, IL-35 correlated with CD68+ macrophages. Thus inhibitors to IL-35 would probably succeed in combination with antibodies against immune checkpoints like PD-L1 and PD-1 currently used against NSCLC because they would inhibit immunosuppressive macrophages and T regulatory cells while promoting T cell-mediated anti-tumoural immune responses in the microenvironment as well as the TU region of NSCLC patients.

Despite the opposite roles of Tbet and Foxp3 in the immune system as well as in tumour biology, recent studies have demonstrated the presence of of CD4(+) T cells, expressing both, Tbet and Foxp3. Although Tbet(+)Foxp3(+) T cells are currently a subject of intense research, less is known about their biological function especially in cancer. Here we found a considerable accumulation of Tbet(+)Foxp3(+)CD4(+) T cells, mediated by the immunosuppressive cytokine TGFβ in the lungs of tumour bearing mice. This is in line with previous studies, demonstrating the important role of TGFβ for the immunopathogenesis of cancer. By gathering results both in murine model and in human disease, we demonstrate that, the conversion of IFNγ producing anti-tumoral T-bet+Th1 CD4+ T cells into immunosuppressive Tbet and Foxp3-PD1 co-expressing regulatory cells could represent an additional important mechanism of TGFβ-mediated blockade of anti-tumour immunity.

Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor activated by T-cell receptor (TCR) and Ca

The lipid hydrolase enzyme acid sphingomyelinase (ASM) is required for the conversion of the lipid cell membrane component sphingomyelin into ceramide. In cancer cells, ASM-mediated ceramide production is important for apoptosis, cell proliferation, and immune modulation, highlighting ASM as a potential multimodal therapeutic target. In this study, we demonstrate elevated ASM activity in the lung tumor environment and blood serum of patients with non–small cell lung cancer (NSCLC). RNAi-mediated attenuation of SMPD1 in human NSCLC cells rendered them resistant to serum starvation–induced apoptosis. In a murine model of lung adenocarcinoma, ASM deficiency reduced tumor development in a manner associated with significant enhancement of Th1-mediated and cytotoxic T-cell–mediated antitumor immunity. Our findings indicate that targeting ASM in NSCLC can act by tumor cell–intrinsic and –extrinsic mechanisms to suppress tumor cell growth, most notably by enabling an effective antitumor immune response by the host. Cancer Res; 77(21); 5963–76. ©2017 AACR.

Genome-wide association studies (GWAS) associated Family with sequence similarity 13, member A (FAM13A) with non-small cell lung cancer (NSCLC) occurrence. Here, we found increased numbers of FAM13A protein expressing cells in the tumoral region of lung tissues from a cohort of patients with NSCLC. Moreover, FAM13A inversely correlated with CTLA4 but directly correlated with HIF1α levels in the control region of these patients. Consistently, FAM13A RhoGAP was found to be associated with T cell effector molecules like HIF1α and Tbet and was downregulated in immunosuppressive CD4+CD25+Foxp3+CTLA4+ T cells. TGFβ, a tumor suppressor factor, as well as siRNA to FAM13A, suppressed both isoforms of FAM13A and inhibited tumor cell proliferation. RNA-Seq analysis confirmed this finding. Moreover, siRNA to FAM13A induced TGFβ levels. Finally, in experimental tumor cell migration, FAM13A was induced and TGFβ accelerated this process by inducing cell migration, HIF1α, and the FAM13A RhoGAP isoform. Furthermore, siRNA to FAM13A inhibited tumor cell proliferation and induced cell migration without affecting HIF1α. In conclusion, FAM13A is involved in tumor cell proliferation and downstream of TGFβ and HIF1α, FAM13A RhoGAP is associated with Th1 gene expression and lung tumor cell migration. These findings identify FAM13A as key regulator of NSCLC growth and progression.

Malnutriția este o constatare frecventă și importantă în secțiile chirurgicale. Deşi consecințele sale includ complicații postoperatorii și costuri mai mari, evaluarea nutrițională nu face parte din protocoalele preoperatorii de rutină. Evaluarea nutriţională include parametri clinici și biologici și este vitală pentru a începe tratamentul și a îmbunătăți rezultatele. Prealbumina este în prezent recunoscută ca un marker fidel al malnutriției şi este inclusă în ghidurile de practică. Unul dintre cele mai importante aspecte legate de prealbumină este faptul că variațiile sale în timp sunt mai valoroase decât valorile absolute. Scopul acestui studiu a fost de a evalua și compara evoluția nutrițională perioperatorie a pacienţilor care necesită intervenţii de chirurgie toracică, cu şi fără cancer, folosind prealbumina - preoperator și postoperator - ca marker principal. Treizeci şi şase de pacienţi de la Clinica de Chirurgie Toracică au fost evaluaţi înainte de intervenţia chirurgicală, prin indicele de masă corporală, scorul de risc nutriţional Subjective Global Assessment (SGA) și parametri biochimici de rutină. Prealbumina a fost evaluată înainte de intervenţia chirurgicală şi la 3 zile după operaţie.Vârstă, durata spitalizării postoperatorii și prezența complicaţiilor au fost notate. Pacienții cu cancer (n = 19) au fost semnificativ mai în vârstă decât pacienții fără cancer (p = 0,007) și au fost mai frecvent, dar nu în mod semnificativ, evaluaţi prin SGA ca fiind malnutriţi (42,1% față de 11,6%). Prealbumina preoperator și alți parametri nu diferă semnificativ între grupuri. Cu toate acestea, a existat o scădere semnificativă a prealbuminei postoperator doar la pacienții cu cancer. Prin urmare, prealbumina s-a dovedit a fi valoroasă în evaluarea malnutriţiei acute la pacienţii cu cancer, în special prin monitorizarea variaţiilor în timp, ceea ce ar putea fi util în planificarea tratamentului nutriţional.

Here we describe increased expression of IL6R in the tumoural region of lung tissue from patients affected by lung adenocarcinoma as compared to squamous cell lung carcinoma. Moreover, here we found increased IL6R in the tumour free part of the lung. By using a murine model of lung adenocarcinoma, we discovered that few lung tumour cells expressed IL-6R and CD4+CD25+Foxp-3+ T regulatory cells down-regulated IL-6R in the tumour bearing lungs. Downstream of IL-6R, the Th17 lineage-specification factors: Signal transducer and activator of transcription 3 (STAT3), Basic leucine zipper transcription factor, BATF and a protein encoded by the RORC in human (RAR-related orphan receptor C) (RORγT), were also found induced in the tumoural region of lung tissue from patients affected by lung adenocarcinoma as compared to those carrying squamous cell carcinoma. Moreover, pSTAT3 protein was found phosphorylated and auto-phosphorylated in the tumoural region of patients with adeno cell carcinoma of the lung as compared to the tumoural region of patients with squamous cell carcinoma of the lung. Intranasal application of anti-IL-6R antibodies in a murine model of lung adenocarcinoma, induced T regulatory cell markers such as Foxp3, Ctla4, Icos, Il10, Il21, Folr4 and Lag3 and inhibited Rorc in lung adenocarcinoma.

Tyrosine kinase 2 (TYK2) is a member of the Janus family of non-receptor tyrosine kinases involved in cytokine signaling. TYK2 deficiency is associated with increased susceptibility to mycobacterial and viral infections, hyper IgE syndrome as well as with allergic asthma. However the precise role of TYK2 in oncogenesis and tumor progression is not clear yet. Tyk2-deficient mice are prone to develop tumors because they lack efficient cytotoxic CD8+ T-cell antitumor responses as a result of deficient Type I interferon signaling. However, as TYK2 functions downstream of growth factor receptors that are often hyperactivated in cancer, inhibiting TYK2 might also have beneficial effects for cancer treatment.