Natural killer (NK) cells are a promising tool for the use in adoptive immunotherapy, since they efficiently recognize and kill tumor cells. In this context,
cultivation is an attractive option to ...increase NK cells in numbers and to improve their antitumor potential prior to clinical applications. Consequently, various strategies to generate NK cells for adoptive immunotherapy have been developed. Here, we give an overview of different NK cell cultivation approaches and their impact on shaping the NK cell antitumor activity. So far, the cytokines interleukin (IL)-2, IL-12, IL-15, IL-18, and IL-21 are used to culture and expand NK cells. The selection of the respective cytokine combination is an important factor that directly affects NK cell maturation, proliferation, survival, distribution of NK cell subpopulations, activation, and function in terms of cytokine production and cytotoxic potential. Importantly, cytokines can upregulate the expression of certain activating receptors on NK cells, thereby increasing their responsiveness against tumor cells that express the corresponding ligands. Apart from using cytokines, cocultivation with autologous accessory non-NK cells or addition of growth-inactivated feeder cells are approaches for NK cell cultivation with pronounced effects on NK cell activation and expansion. Furthermore,
cultivation was reported to prime NK cells for the killing of tumor cells that were previously resistant to NK cell attack. In general, NK cells become frequently dysfunctional in cancer patients, for instance, by downregulation of NK cell activating receptors, disabling them in their antitumor response. In such scenario,
cultivation can be helpful to arm NK cells with enhanced antitumor properties to overcome immunosuppression. In this review, we summarize the current knowledge on NK cell modulation by different
cultivation strategies focused on increasing NK cytotoxicity for clinical application in malignant diseases. Moreover, we critically discuss the technical and regulatory aspects and challenges underlying NK cell based therapeutic approaches in the clinics.
Cancer immunotherapies utilize the capabilities of the immune system to efficiently target malignant cells. In recent years, chimeric antigen receptor (CAR) equipped T cells showed promising results ...against B cell lymphomas. Autologous CAR-T cells require patient-specific manufacturing and thus extensive production facilities, resulting in high priced therapies. Along with potentially severe side effects, these are the major drawbacks of CAR-T cells therapies. Natural Killer (NK) cells pose an alternative for CAR equipped immune cells. Since NK cells can be safely transferred from healthy donors to cancer patients, they present a suitable platform for an allogeneic "off-the-shelf" immunotherapy. However, administration of activated NK cells in cancer therapy has until now shown poor anti-cancer responses, especially in solid tumors. Genetic modifications such as CARs promise to enhance recognition of tumor cells, thereby increasing anti-tumor effects and improving clinical efficacy. Although the cell biology of T and NK cells deviates in many aspects, the development of CAR-NK cells frequently follows within the footsteps of CAR-T cells, meaning that T cell technologies are simply adopted to NK cells. In this review, we underline the unique properties of NK cells and their potential in CAR therapies. First, we summarize the characteristics of NK cell biology with a focus on signaling, a fine-tuned interaction of activating and inhibitory receptors. We then discuss why tailored NK cell-specific CAR designs promise superior efficacy compared to designs developed for T cells. We summarize current findings and developments in the CAR-NK landscape: different CAR formats and modifications to optimize signaling, to target a broader pool of antigens or to increase
persistence. Finally, we address challenges beyond NK cell engineering, including expansion and manufacturing, that need to be addressed to pave the way for CAR-NK therapies from the bench to the clinics.
Immunological therapy principles are increasingly determining modern medicine. They are used to treat diseases of the immune system, for tumors, but also for infections, neurological diseases, and ...many others. Most of these therapies base on antibodies, but small molecules, soluble receptors or cells and modified cells are also used. The development of immune checkpoint inhibitors is amazingly fast. T-cell directed antibody therapies against PD-1 or CTLA-4 are already firmly established in the clinic. Further targets are constantly being added and it is becoming increasingly clear that their expression is not only relevant on T cells. Furthermore, we do not yet have any experience with the long-term systemic effects of the treatment. Flow cytometry can be used for diagnosis, monitoring, and detection of side effects. In this review, we focus on checkpoint molecules as target molecules and functional markers of cells of the innate and acquired immune system. However, for most of the interesting and potentially relevant parameters, there are still no test kits suitable for routine use. Here we give an overview of the detection of checkpoint molecules on immune cells in the peripheral blood and show examples of a possible design of antibody panels.
Abstract
Glioblastoma multiforme (GBM) is an extremely aggressive brain tumor, characterized by its high genetic heterogeneity. In search of novel putative therapeutic RNA targets we investigated the ...role of the oncogenic long noncoding RNA LINC00152 (CYTOR, and STAiR18) in A172 glioblastoma cells. Here, we are the first to describe, that LINC00152 unexpectedly acts in a tumor suppressive manner in this cell line. SiRNA-based knockdown of LINC00152 enhanced malignant tumor behaviors including proliferation, cell cycle entry, migration, and invasion, contradicting previous studies using U87-MG and LN229 glioblastoma cells. Furthermore, LINC00152 knockdown had no influence on survival of A172 glioblastoma cells. In a genome wide transcription analysis of A172 and U87-MG glioblastoma cells, we identified 70 LINC00152 target genes involved in locomotion, cell migration, and motility in A172 cells, whereas in U87-MG cells only 40 target genes were detected. The LINC00152-regulated genes found in A172 differed from those identified in U87-MG glioblastoma cells, none of them being regulated in both cell lines. These findings underline the strong genetic heterogeneity of glioblastoma and point to a potential, yet unknown risk addressing LINC00152 lncRNA as a prospective therapeutic target in GBM.
The coordinated transcriptional regulation of activated T-cells is based on a complex dynamic behavior of signaling networks. Given an external stimulus, T-cell gene expression is characterized by ...impulse and sustained patterns over the course. Here, we analyze the temporal pattern of activation across different T-cell populations to develop consensus gene signatures for T-cell activation.
Here, we identify and verify general biomarker signatures robustly evaluating T-cell activation in a time-resolved manner. We identify time-resolved gene expression profiles comprising 521 genes of up to 10 disjunct time points during activation and different polarization conditions. The gene signatures include central transcriptional regulators of T-cell activation, representing successive waves as well as sustained patterns of induction. They cover sustained repressed, intermediate, and late response expression rates across multiple T-cell populations, thus defining consensus biomarker signatures for T-cell activation. In addition, intermediate and late response activation signatures in CAR T-cell infusion products are correlated to immune effector cell-associated neurotoxicity syndrome.
This study is the first to describe temporally resolved gene expression patterns across T-cell populations. These biomarker signatures are a valuable source, e.g., monitoring transcriptional changes during T-cell activation with a reasonable number of genes, annotating T-cell states in single-cell transcriptome studies, or assessing dysregulated functions of human T-cell immunity.
Prosthetic valve endocarditis (PVE) remains a serious condition with a high mortality rate. Precise identification of the PVE-associated pathogen/s and their virulence is essential for successful ...therapy and patient survival. The commonly described PVE-associated pathogens are staphylococci, streptococci, and enterococci, with Staphylococcus aureus being the most frequently diagnosed species. Furthermore, multi-drug resistance pathogens are increasing in prevalence and continue to pose new challenges mandating a personalized approach. Blood cultures in combination with echocardiography are the most common methods to diagnose PVE, often being the only indication, it exists. In many cases, the diagnostic strategy recommended in the clinical guidelines does not identify the precise microbial agent, and frequently, false-negative blood cultures are reported. Despite the fact that blood culture findings are not always a good indicator of the actual PVE agent in the valve tissue, only a minority of re-operated prostheses are subjected to microbiological diagnostic evaluation. In this review, we focus on the diversity and the complete spectrum of PVE-associated bacterial, fungal, and viral pathogens in blood and prosthetic heart valve, their possible virulence potential, and their challenges in making a microbial diagnosis. We are curious to understand if the unacceptable high mortality of PVE is associated with the high number of negative microbial findings in connection with a possible PVE. Herein, we discuss the possibilities and limits of the diagnostic methods conventionally used and make recommendations for enhanced pathogen identification. We also show possible virulence factors of the most common PVE-associated pathogens and their clinical effects. Based on blood culture, molecular biological diagnostics, and specific valve examination, better derivations for the antibiotic therapy as well as possible preventive intervention can be established in the future.
To identify potential early biomarkers of treatment response and immune-related adverse events (irAE), a pilot immune monitoring study was performed in stage IV melanoma patients by flow cytometric ...analysis of peripheral blood mononuclear cells (PBMC). Overall, 17 patients were treated with either nivolumab or pembrolizumab alone, or with a combination of nivolumab and ipilimumab every three weeks. Of 15 patients for which complete response assessment was available, treatment responders (n = 10) as compared to non-responders (n = 5) were characterized by enhanced PD-1 expression on CD8+ T cells immediately before treatment (median ± median absolute deviation/MAD 26.7 ± 10.4% vs. 17.2 ± 5.3%). Responders showed a higher T cell responsiveness after T cell receptor ex vivo stimulation as determined by measurement of programmed cell death 1 (PD-1) expression on CD3+ T cells before the second cycle of treatment. The percentage of CD8+ effector memory (CD8+CD45RA−CD45RO+CCR7−) T cells was higher in responders compared to non-responders before and immediately after the first cycle of treatment (median ± MAD 39.2 ± 7.3% vs. 30.5 ± 4.1% and 37.7 ± 4.6 vs. 24.0 ± 6.4). Immune-related adverse events (irAE) were accompanied by a higher percentage of activated CD4+ (CD4+CD38+HLADR+) T cells before the second treatment cycle (median ± MAD 14.9 ± 3.9% vs. 5.3 ± 0.4%). In summary, PBMC immune monitoring of immune-checkpoint inhibition (ICI) treatment in melanoma appears to be a promising approach to identify early markers of treatment response and irAEs.
Here we present a simple and sensitive flow cytometric-based assay to assess T cell proliferation. Given the critical role STAT5A phosphorylation in T cell proliferation, we decided to evaluate ...phosphorylation of STAT5A as an indicator of T cell proliferation. We determined pSTAT5A in T cell treated with either CD3/CD28 or PHA. After stimulation, T cells from adult healthy donors displayed a strong long-lasting phosphorylation of STAT5A, reaching a peak value after 24 h. The median fluorescence intensity (MFI) of pSTAT5A increased from 112 ± 17 to 512 ± 278 (CD3/CD28) (24 h) and to 413 ± 123 (PHA) (24 h), the IL-2 receptor-α (CD25) expression was greatly enhanced and after 72 h T cell proliferation amounted to 52.3 ± 10.3% (CD3/CD28) and to 48.4 ± 9.7% (PHA). Treatment with specific JAK3 and STAT5 inhibitors resulted in a complete blockage of phosphorylation of STAT5A, CD25 expression, and suppression of T cell proliferation. Compared with currently available methods, STAT5A phosphorylation is well-suited to predict T cell proliferation. Moreover, the method presented here is not very time consuming (several hours) and delivers functional information from which conclusions about T cell proliferation can be drawn.
Extracellular vesicles (EVs) derived from the secretome of human mesenchymal stromal cells (MSC) contain numerous factors that are known to exert anti‐inflammatory effects. MSC‐EVs may serve as ...promising cell‐based therapeutics for the inner ear to attenuate inflammation‐based side effects from cochlear implantation which represents an unmet clinical need. In an individual treatment performed on a ‘named patient basis’, we intraoperatively applied allogeneic umbilical cord‐derived MSC‐EVs (UC‐MSC‐EVs) produced according to good manufacturing practice. A 55‐year‐old patient suffering from Menière's disease was treated with intracochlear delivery of EVs prior to the insertion of a cochlear implant. This first‐in‐human use of UC‐MSC‐EVs demonstrates the feasibility of this novel adjuvant therapeutic approach. The safety and efficacy of intracochlear EV‐application to attenuate side effects of cochlea implants have to be determined in controlled clinical trials.
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