Novel markers for human T regulatory cells in healthy donors and cancer patients

CD4+CD25+FoxP3+ T regulatory cells (Tregs) are essential for maintaining self-tolerance and preventing autoimmune diseases. However, FoxP3+ Tregs contribute to the progression of cancer and their levels expand in cancer patients compared to healthy donors. Tregs suppress tumour-specific immune responses by accumulating in the peripheral blood and tumour microenvironment. Human Tregs secrete the latent form of transforming growth factor-beta (TGF-β), in which the mature TGF-β protein is bound to latency-associated peptide (LAP) that binds to Glycoprotein A Repetitions Predominant (GARP). Some FoxP3+ Tregs express Helios, a member of the Ikaros transcription factor family. The purpose of this study is to identify which of FoxP3+/-Helios+/- Tregs express GARP and LAP and to investigate if these receptors are vital markers for activated conventional Tregs, and also to examine the different suppressive factors and phenotypes of Tregs. This study compared the levels of Tregs in cancer patients with controls, by measuring the levels of Tregs specific and novel markers. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors (HDs), chronic pancreatitis (CP), malignant pancreatic cancer (PC) and liver metastases from colorectal cancer (LI/CRC) patients. PBMCs were then stained with anti-CD3, anti-CD4, anti-GARP, anti-LAP, anti-Helios, anti-FoxP3, anti-IFN-γ, and anti-IL-10 antibodies. The results demonstrated for the first time that GARP and LAP are mainly expressed on activated CD4+FoxP3+Helios+ Tregs and CD4+FoxP3-Helios+ Tregs for healthy donors and all patient groups. In contrast, CD4+FoxP3+Helios- and CD4+FoxP3-Helios- Tregs do not express GARP and LAP. FoxP3+Helios+ Tregs from cancer patients showed significantly higher expression of GARP and LAP, compared to healthy donors. Furthermore, there was no increase in the level of FoxP3+Helios+ Tregs in HDs and PC patients compared to LI/CRC patients. FoxP3+Helios+GARP+LAP+ Tregs secrete the IL-10 cytokine but not IFN-γ in comparison with FoxP3-Helios- Tregs. This study demonstrated that Helios, and not FoxP3, is the main marker of activated Tregs expressing GARP and LAP

A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts.

Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and βIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy

MicroRNA and mRNA profiling in the idiopathic inflammatory myopathies

Background The idiopathic inflammatory myopathies (IIMs) are heterogeneous autoimmune conditions of skeletal muscle inflammation and weakness. MicroRNAs (miRNAs) are short, non-coding RNA which regulate gene expression of target mRNAs. The aim of this study was to profile miRNA and mRNA in IIM and identify miRNA-mRNA relationships which may be relevant to disease. Methods mRNA and miRNA in whole blood samples from 7 polymyositis (PM), 7 dermatomyositis (DM), 5 inclusion body myositis and 5 non-myositis controls was profiled using next generation RNA sequencing. Gene ontology and pathway analyses were performed using GOseq and Ingenuity Pathway Analysis. Dysregulation of miRNAs and opposite dysregulation of predicted target mRNAs in IIM subgroups was validated using RTqPCR and investigated by transfecting human skeletal muscle cells with miRNA mimic. Results Analysis of differentially expressed genes showed that interferon signalling, and anti-viral response pathways were upregulated in PM and DM compared to controls. An anti-Jo1 autoantibody positive subset of PM and DM (n = 5) had more significant upregulation and predicted activation of interferon signalling and highlighted T-helper (Th1 and Th2) cell pathways. In miRNA profiling miR-96-5p was significantly upregulated in PM, DM and the anti-Jo1 positive subset. RTqPCR replicated miR-96-5p upregulation and predicted mRNA target (ADK, CD28 and SLC4A10) downregulation. Transfection of a human skeletal muscle cell line with miR-96-5p mimic resulted in significant downregulation of ADK. Conclusion MiRNA and mRNA profiling identified dysregulation of interferon signalling, anti-viral response and T-helper cell pathways, and indicates a possible role for miR-96-5p regulation of ADK in pathogenesis of IIM

Combining FoxP3 and Helios with GARP/LAP markers to identify expanded Treg subsets in cancer patients

Background: Regulatory T cells (Tregs) comprise numerous heterogeneous subsets with distinct phenotypic and functional features. Identifying Treg markers is critical to investigate the role and clinical impact of various Treg subsets in pathological settings, and also for developing more effective immunotherapies. Methods: We investigated different Treg subsets as defined by expression of FoxP3 and Helios transcription factors and GARP and LAP immunosuppressive markers in peripheral blood samples isolated from patients with pancreatic cancer (PC) and liver metastases from colorectal cancer (LICRC), and compared their levels to control groups. Results: We have recently shown that non-activated FoxP3–Helios+ and activated FoxP3+/–Helios+ CD4+ T cells express GARP/LAP immunosuppressive markers in healthy donors. In this study, we report similar observations in the peripheral blood of patients with pancreatic cancer (PC) and liver metastases from colorectal cancer (LICRC). Comparing levels of different Treg subpopulations in cancer patients and controls, we report that in PC patients, and unlike LICRC patients, there was no increase in Treg levels as defined by FoxP3 and Helios. However, defining Tregs based on GARP/LAP expression showed that FoxP3–LAP+Tregs in non-activated and activated settings, and FoxP3+Helios+GARP+LAP+activated Tregs were significantly increased in both groups of patients, compared with controls. Additionally, GARP–/+LAP+ CD4+ T cells made IL-10, and not IFN-g, and levels of IL-10-secreting CD4+ T cells were elevated in LICRC patients, especially with higher tumor staging. Conclusions: This work implies that a combination of Treg-specific markers could be used to more accurately determine expanded Treg subsets and to understand their contribution in cancer settings, and investigations of Treg levels in different cancers should consider diverse Treg-related markers such as GARP, LAP, Helios, and others and not only FoxP3 as a sole Treg-specific marker

A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts

Marwah Abd Al Samid,1 Jamie S McPhee,2 Jasdeep Saini,1 Tristan R McKay,1 Lorna M Fitzpatrick,1 Kamel Mamchaoui,3 Anne Bigot,3 Vincent Mouly,3 Gillian Butler-Browne,3 Nasser Al-Shanti1 1Healthcare Science Research Institute, School of Healthcare Science, Manchester Metropolitan University, Manchester, UK; 2Department of Sport and Exercise Science, Manchester Metropolitan University, Manchester, UK; 3Center for Research in Myology, Sorbonne Université-INSERM, Paris, France Background: Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. Objective: We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. Methods: Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. Results: Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and βIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. Conclusion: We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy. Keywords: motor unit, neuromuscular junctions, human embryonic stem cells, neuronal progenitor cells, human myoblast

Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells

Jasdeep Saini,1 Alessandro Faroni,2,3 Marwah Abd Al Samid,1 Adam J Reid,2,3 Adam P Lightfoot,1 Kamel Mamchaoui,4 Vincent Mouly,4 Gillian Butler-Browne,4 Jamie S McPhee,5 Hans Degens,1,6,7 Nasser Al-Shanti1 1Musculoskeletal Science & Sports Medicine Research Centre, School of Healthcare Science, Manchester Metropolitan University, Manchester, UK; 2Blond McIndoe Laboratories, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK; 3Department of Plastic Surgery & Burns, University Hospitals of South Manchester, Manchester Academic Health Science Centre, Manchester, UK; 4Center for Research in Myology, Sorbonne Université–INSERM, Paris, France; 5Department of Sport and Exercise Science, Manchester Metropolitan University, Manchester, UK; 6Institute of Sport Science and Innovations, Lithuanian Sports University, Kaunas, Lithuania; 7University of Medicine and Pharmacy of Targu Mures, Targu Mures, Romania Background: Neuromuscular junctions (NMJs) consist of the presynaptic cholinergic motoneuron terminals and the corresponding postsynaptic motor endplates on skeletal muscle fibers. At the NMJ the action potential of the neuron leads, via release of acetylcholine, to muscle membrane depolarization that in turn is translated into muscle contraction and physical movement. Despite the fact that substantial NMJ research has been performed, the potential of in vivo NMJ investigations is inadequate and difficult to employ. A simple and reproducible in vitro NMJ model may provide a robust means to study the impact of neurotrophic factors, growth factors, and hormones on NMJ formation, structure, and function. Methods: This report characterizes a novel in vitro NMJ model utilizing immortalized human skeletal muscle stem cells seeded on 35 mm glass-bottom dishes, cocultured and innervated with spinal cord explants from rat embryos at ED 13.5. The cocultures were fixed and stained on day 14 for analysis and assessment of NMJ formation and development. Results: This unique serum- and trophic factor-free system permits the growth of cholinergic motoneurons, the formation of mature NMJs, and the development of highly differentiated contractile myotubes, which exhibit appropriate configuration of transversal triads, representative of in vivo conditions. Conclusion: This coculture system provides a tool to study vital features of NMJ formation, regulation, maintenance, and repair, as well as a model platform to explore neuromuscular diseases and disorders affecting NMJs. Keywords: neuromuscular junction, NMJ, coculture, myoblast, myotube, motor neuron, motoneuro