The Effect of Inducıble Costımulatory Lıgand (Icos-Lg) Expressıng Myeloıd Leukemıa Cells on Helper T Lymphocyte Actıvatıon and Exhaustıon

T cell exhaustion is characterized by progressive loss of cytokine production, decreased proliferation and resistance to reactivation especially upon continuous antigen stimulation. Despite being potent stimulators of helper T (Th) lymphocytes, AML cells employ intriguing mechanisms to limit anti-tumor responses. Thus, this study proposes an in vitro co-stimulation dependent Th cell exhaustion model that may represent a novel immune escape mechanism in AML. Firstly, ICOS-LG expression kinetics were evaluated on AML cell lines of different maturation status (HL-60, THP-1, U937, Kasumi-1, KG-1) in comparison to CD14+ monocytes by flow cytometry under steady state and pro-inflammatory conditions. Then, CD4+ T cells were co-cultured with AML cells or monocytes at different ratios and stimulated with various anti-CD3 concentrations. Media and aCD3 was regularly refreshed in extended culture periods. CD25, CD127, FoxP3, CD69, PD-L1, PD-1, CTLA-4, ICOS, TIM-3, LAG3, CD38, CD154, CCR7, HLA-DR expression, together with proliferation and viability were evaluated by flow cytometry. Assays were also performed under ICOS-LG, PD-L1/PD-L2 and CD80/CD86 blockade. IL-2, IL-10, IL-4, TGF-, IFN-γ and TNF-α levels were measured by ELISA. Exhausted (TIM-3mo/hi) and non-exhausted Th cells (TIM-3-/low) were enriched and re-stimulated for functional exhaustion assays. In the co-cultures, Th cells possessed high levels of LAG3, TIM-3, PD-1, ICOS and CTLA-4. Th cells' proliferation and activation with AML cells or monocytes were similar; however, exhaustion was significantly higher in AML co-cultures. These exhausted cells produced lower IL-2, IFN-γ and TNF-α. Exhaustion was independent of Th cells' initial proliferative activity. Co-stimulatory signals derived from AML cells were more critical than aCD3 stimulation in Th cell exhaustion. Exhaustion could be reversed by exogenous IL-2. Our findings indicate a novel immune escape mechanism employed by AML cells that can induce a co-stimulation dependent Th cell exhaustion.T hücre yorulması sürekli antijen uyarımı etkisinde kademeli olarak gelişen sitokin üretiminin kaybı, çoğalma kapasitesinde azalma ve tekrar aktivasyona direnç ile tanımlanmıştır. Yardımcı T lenfosit (Th) yanıtlarının, AML hücreleri tarafından uyarabilmesine rağmen, anti-lösemik etkisi sınırlıdır. Bu çalışmada, AML kaynaklı kostimülasyon sinyallerinin Th yorulmasına olan etkilerini incelemek amacıyla ilk kez bir in vitro model geliştirilmiştir. Öncelikle AML hücrelerinin (HL-60, THP-1, U937, Kasumi-1, KG-1) ve CD14+ monositlerin normal fizyolojik ve proinflamatuar koşullardaki ICOS-LG ifade düzeyleri ve zamana bağlı değişimleri belirlendi. Daha sonra, CD4+ T lenfositlerin AML hücre hatları veya monositler ile farklı hücre oranları ve farklı konsantrasyonlarda anti-CD3 (aCD3) uyarımı ile ko-kültürleri gerçekleştirildi. aCD3 ve kültür ortamı düzenli olarak tazelendi. Th hücrelerinin CD25, CD127, FoxP3, CD69, PD-L1, PD-1, CTLA-4, ICOS, TIM-3, LAG3, CD38, CD154, CCR7 ve HLA-DR ifade düzeyleri, çoğalma kapasiteleri ve canlılıkları akım sitometri ile saptandı. Kostimülasyon sinyallerinin T hücre yorulmasına olan etkileri ICOS-LG, PD-L1/PD-L2 ve CD80/CD86 bloklayıcı ajanlar ile test edildi. ELISA ile IL-2, IL-10, IL-4, TGF-, IFN-γ ve TNF-α düzeyleri belirlendi. Fonksiyonel yorulmanın test edilmesi için yorulmuş (TIM-3mo/hi) ve yorulmamış (TIM-3-/low) Th alt-popülasyonları zenginleştirildi ve tekrar uyarıldı. AML:Th ko-kültürlerinde Th hücreleri üzerinde LAG3, TIM-3, PD-1, CTLA-4 moleküllerin belirgin artışı saptandı. Monosit ko-kültürlerde ise Th aktivasyonu ve proliferasyonu AML hücreleri ile elde edilen sonuçlara benzer iken yorulma belirteçlerinin düzeyi anlamlı olarak düşük kaldı. Bu yorulmuş hücreler düşük düzeylerde IL-2, IFN-γ ve TNF-α üretti. Bu hücrelerin yorulmaları önceki çoğalma düzeylerinden bağımsızdı. AML-kaynaklı kostimülatör sinyallerin aCD3 uyarımına kıyasla Th yorulması için daha kritik olduğu görüldü. Yorulma dışarıdan verilen IL-2 ile geri döndürülebiliyordu. Bu bulgular, AML hücrelerinin kostimülasyon aracılığı ile Th yorulmasına neden olduğunu göstermekte ve yeni bir immün kaçış mekanizmasına işaret etmektedir

Development of a novel in vitro screening method using genetically modified NK-92 cells against various tumor cells

Natural killer (NK) cells of the innate immune system are recognized for their ability to potently kill tumor cells. NK cell-mediated lysis is maintained by an intricate balance between several activating and inhibitory receptors that either trigger or dampen effector functions upon ligand engagement. In this study, we aim to dissect this complex balance by developing a cell-based screening tool to identify receptor specific anti-tumor responses. As the character of the heterogeneous tumor cell populations differs among patients, such a tool may be instrumental in developing patient-tailored cancer immunotherapies. Genes encoding 20 NK cell surface receptors were cloned into lentiviral vectors for genetic modification of the NK-92 cell line. Genetically modified (GM) NK-92 cells were enriched and overexpression of receptors was confirmed by flow cytometry. We analyzed the effector functions of all GM NK-92 cells against human cancer cell lines as well as against primary human sarcoma explants. Overall, genetic modifications did not hamper cytotoxic capacity of GM NK-92 cells; rather induced enhanced tumor cell targeting by receptors such as DNAM-1 and NKG2D. We further confirmed that this response was indeed DNAM-1 or NKG2D-dependent by using blocking antibodies. We also evaluated the synergistic response of prominent receptors in triggering degranulation and cytotoxicity by co-expressing DNAM-1 and NKG2D. Our results show the feasibility of an in vitro genetic screening approach to identify response-triggering receptors in genetically modified NK cells expressing different activating receptors. This tool has the potential to rapidly identify patient-specific targets for adoptive immunotherapy of cance

Th1 cells in cancer-associated inflammation

The immune system is not only evolved to protect the body from pathogens, but it also recognizes and eliminates cancer cells. CD4+ helper T (Th) lymphocytes are central intercessors differentiated according to the character of physiological or pathological status. Generation of type 1 Th (Th1) cells is primarily associated with a pathological insult that must be removed through immune elimination. Upon interacting with other immune and transformed cells, Th1 cells can hamper cancer progression. Therefore, it is a major obstacle for tumor cells to become insensitive or resistant to Th1-oriented actions. The organism employs various mechanisms to return to a steady state and ensure tissue repair following a destructive inflammatory response. Th1 cells are also tightly regulated during the termination of immune responses. They can reduce the production of inflammatory cytokines, both generate and be prone to inhibitory signals, and undergo activation-induced cell death for inflammation resolution. Additionally, Th1 cells may become hyporesponsive, exhausted, and decorated with many inhibitory receptors and eventually lose functionality. There is growing evidence about tumor cells taking advantage of the strategies used for the resolution of Th1-oriented inflammation. Here, the current insights on Th1 cells during cancer-associated inflammatory responses are reviewed.The immune system is not only evolved to protect the body from pathogens, but it also recognizes and eliminates cancer cells. CD4+ helper T (Th) lymphocytes are central intercessors differentiated according to the character of physiological or pathological status. Generation of type 1 Th (Th1) cells is primarily associated with a pathological insult that must be removed through immune elimination. Upon interacting with other immune and transformed cells, Th1 cells can hamper cancer progression. Therefore, it is a major obstacle for tumor cells to become insensitive or resistant to Th1-oriented actions. The organism employs various mechanisms to return to a steady state and ensure tissue repair following a destructive inflammatory response. Th1 cells are also tightly regulated during the termination of immune responses. They can reduce the production of inflammatory cytokines, both generate and be prone to inhibitory signals, and undergo activation-induced cell death for inflammation resolution. Additionally, Th1 cells may become hyporesponsive, exhausted, and decorated with many inhibitory receptors and eventually lose functionality. There is growing evidence about tumor cells taking advantage of the strategies used for the resolution of Th1-oriented inflammation. Here, the current insights on Th1 cells during cancer-associated inflammatory responses are reviewed

Quantitative investigation into the influence of intravenous fluids on human immune and cancer cell lines

The effect of intravenous fluids (IVF) has been investigated clinically through the assessment of post-treatment reactions. However, the responses to IVF vary from patient-to-patient. It is important to understand the response of IVF treatment to be able to provide optimal IVF care. Herein, we investigated the impact of commonly used IVFs, Dextrose, NaCl and Ringer on different human cancer (HepG2 (liver hepatocellular carcinoma) and MCF7 (breast adenocarcinoma)) and immune cell lines (U937 (lymphoma) monocyte and macrophages). The effect of IVF exposure on single cells was characterized using hemocytometer, fluorescence microscopy and flow cytometry. Quantitative data on the viability and morphology of the cells were obtained. Our results emphasize that different IVFs demonstrate important differences in how they influence distinct cell lines. Particularly, we observed that the lactated ringer and dextrose solutions altered the viability and nuclear size of cancer and immune cells differently. Our findings present valuable information to the knowledge of cellular-level IVF effects for further investigations in IVF usage on diverse patient populations and support the importance and necessity of developing optimal diluents not only for drug stability but also for patient benefits

Dielectrophoretic characterization and separation of monocytes and macrophages using 3D carbon-electrodes

Monocyte heterogeneity and its prevalence are revealed as indicator of several human diseases ranking from cardiovascular diseases to rheumatoid arthritis, chronic kidney diseases, autoimmune multiple sclerosis, and stroke injuries. When monocytes and macrophages are characterized and isolated with preserved genetic, phenotypic and functional properties, they can be used as label‐free biomarkers for precise diagnostics and treatment of various diseases. Here, the dielectrophoretic responses of the monocytes and macrophages were examined. We present 3D carbon‐electrode dielectrophoresis (carbon‐DEP) as a separation tool for U937 monocytes and U937 monocyte‐differentiated macrophages. The carbon‐electrodes advanced the usability and throughput of DEP separation, presented wider electrochemical stability. Using the 3D carbon‐DEP chip, we first identified the selective positive and negative DEP responses and specific crossover frequencies of monocytes and macrophages as their signatures for separation. The crossover frequency of monocytes and macrophages was 17 and 30 kHz, respectively. Next, we separated monocyte and macrophage subpopulations using their specific dielectrophoretic responses. Afterward, we used a fluorescence‐activated cell sorter to confirm our results. Finally, we enriched 70% of monocyte cells from the mixed cell population, in other words, concentration of monocyte cells to macrophage cells was five times increased, using the 30‐kHz, 10‐Vpp electric field and 1 μL/min flow rate

Engineering antigen‐specific NK cell lines against the melanoma‐associated antigen tyrosinase via TCR gene transfer

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T‐lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL‐TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off‐target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen‐specific TCR complex on NK cell lines NK‐92 and YTS, demonstrated by using a TCR against the HLA‐A2‐restricted tyrosinase‐derived melanoma epitope, Tyr368‐377. Most importantly, the introduction of a TCR complex to NK cell lines enables MHC‐restricted, antigen‐specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of “mispairing” in TCR gene therapy