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Regulatory T cells (Treg cells) are CD4+ T cells specialized in the inhibition of immune responses. In our laboratory, we which to determine whether Treg cells contribute to the immunosuppressive environment which seems to prevail within tumors, and may limit the efficiency of cancer vaccines. To that end, a method to quantify Treg cells in human tissues is necessary. The most specific human Treg well marker is a stable epigenetic modification of a region localized in the first intron of gene FOXP3 which codes for a transcription factor essential to Treg cells. This region, named FOXP3il, is demethylated in Treg cells but completely methylated in other types of T cells. We tested the use of a methyl-specific quantitative PCR assay (MS-qPCR) to measure Treg cell frequencies in different types of human tissues.
We first verified that this assay could serve to quantify Treg cells in PBMC samples. We next applied the assay to measure Treg cell frequencies in PBMC samples from melanoma patients receiving cancer vaccines combined to potentially Treg cell-depleting therapies. No significant decrease of Treg cell frequencies was observed in six patients treated with Ontak, a diphtheria toxine-conjugated recombinant IL-2. A 50% or more decrease of Treg cell frequencies was observed in one of eleven patients treated with low dose Cyclophosphamide, and in four of thirteen patients treated with Daclizumab, a monoclonal anti-CD25 antibody. No variation of Treg cell frequencies was observed in patients treated with a cancer vaccine alone, nor in non vaccinated patients. In conclusion, by using an MS-qPCR technique that detects Treg cells but not activated T cells, we show that none of the potentially Treg cell-depleting therapies tested here can effectively decrease circulating Treg cell frequencies in a majority of patients. We also attempted to use the MS-qPCR assay to measure Treg cell frequency in tumor biopsies. However, we observed that melanoma cells may harbour demethylated FOXP3il sequences. We confirmed and extended this observation to two other regions of the FOXP3 gene using another technique named “bisulfite sequencing”. We observed different profiles of FOXP3 demethylation in melanoma cell lines. No demethylation of FOXP3il was observed in other types of tumors. In melanoma, demthylation of the FOXP3 gene was not correlated with FOXP3 mRNA expression.
In conclusion, the MS-qPCR FOXP3il assay allowed us to measure Treg cell frequencies in human PBMC samples. Its use on melanoma fragments without prior CD4+ T cell isolation will not be possible because tumor cells may also bear a demethylated FOXP3il allele Les lymphocytes T régulateurs (Treg) sont des cellules CD4+ spécialisées dans l’inhibition des réponses immunitaires. Notre laboratoire cherche à déterminer si les Treg contribuent à l’environnement immunosuppresseur qui existe au sein des tumeurs, et qui limiterait l’efficacité des vaccins anti-tumoraux. Pour ce faire, une méthode de quantification des Treg dans les tissus humains est nécessaire. La déméthylation d’une région de l’intron 1 du gène FOXP3, qui code pour un facteur de transcription essentiel aux Treg, est le marqueur le plus spécifique de l’état de méthylation de l(intron 1 de FOXP3 (Ms-qPCR FOXP3i1) pour évaluer la fréquence des Treg dans différents types de tissus humains.
Nous avons vérifié la spécificité de cette technique pour quantifier les Treg dans des échantillons de PBMC. Nous l’avons ensuite appliquée pour mesurer la fréquence des Treg dans des PBMC provenant de patients atteints de mélanome sous divers protocoles de vaccination anti-tumorale incluant l’administration d’un traitement visant à dépléter les Treg. Aucune diminution significative de la fréquence des Treg n’a été observée chez les six patients traités à l’Ontak, une protéine de fusion entre l’IL-2 et la toxine diphtérique. Une diminution de 50% ou plus de la fréquence des Treg a été observée chez un seul des onze patients traités par faible dose de cyclophosphamide, et chez quatre des treize patients traités au Daclizumab, un anticorps monoclonal anti-CD25. Aucune variation de la fréquence des Treg n’a été observée chez les patients recevant un vaccin anti-tumoral seul, ou chez des patients non vaccinés. En conclusion, en utilisant une technique de Ms-qPCR qui détecte les Treg mais pas les lymphocytes T activés, nous avons montré qu’aucun des traitements testés ici ne diminue significativement la fréquence des Treg circulants chez la majorité de patients.
Nous avons également cherché à utiliser la technique de Ms-qPCR pour mesurer la fréquence des Treg dans des biopsies de tumeurs. Cependant, nous avons observé que des cellules tumorales pouvaient porter des séquences FOXP3il déméthylées. Nous avons confirmé et étendu cette observation à deux autres régions du gène FOXP3 en utilisant une autre technique nommée « séquençage bisulfite ». Nous avons observé différents profils de déméthylation du gène FOXP3 dans les mélanomes, mais aucune déméthylation de la région FOXP3il n’a été observée dans d’autres types de tumeurs. Dans les mélanomes, la déméthylation du gène FOXP3 n’est pas corrélée avec l’expression de son ARN messager.
En conclusion, la MS-qPCR FOXP3il est une méthode qui nous a permis de mesurer la fréquence des Treg dans les échantillons de PBMC humains. Son utilisation dans des fragments de mélanome sans purification préalable des lymphocytes T CD4+ semble compromise puisque des cellules tumorales peuvent également porter un allèle FOXP3il déméthylé

T-Lymphocytes, Regulatory --- FOXP3 protein, human --- Methylation --- Introns --- Polymerase Chain Reaction

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This eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contact

Medicine --- Immunology --- TNF --- TNFR2 --- CD4+Foxp3+ regulatory T cells --- myeloid-derived suppressive cells --- mesenchymal stem cells --- inflammation --- cancer --- autoimmune diseases

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Oligonucleotides (ON) constitute a new group of molecular agents, the object of significant interest due to their potential value as drugs for diagnostic and therapeutic applications. Their special interest derives from the intrinsic characteristics of ONs: a) ONs are informative agents, a property that derives from the order in which the nucleotides of each particular ON are arranged; b) ONs can act as ligands (ASO, TFO, aptamers, G-quadruplex, etc.) of complementary nucleic acid sequences (DNA or RNA) due to their high capacity to hybridize (by means of Watson and Crick or Hoogsteen links) with other nucleotide sequences, resulting in specific gene modulatory effects. However, nonspecific sequences may also be of interest, as is the case with repetitive nucleotide sequences (CpG) with adjuvant effects of vaccines; c) ONs can also rapidly evolve to achieve specific advantages of utility (targeting, stability, efficacy, toxicity, etc.) or high-sensitivity diagnostic technology (markers, analyzes, biosensors, FISH, microarrays, etc.), by chemical modification of nucleotides in any of their atoms. These properties show that ONs are first-order molecules due to their potential usefulness in practice.In this collection of research articles and review papers, we aim to highlight their therapeutic, but also diagnostic and technological utility as drugs.

Medicine --- quantum dots (QDs) --- DNAzyme --- ROS --- Amplex Red --- light-induced activity --- DNA methylation --- histone code --- microRNA --- nanoparticles --- noncoding RNA --- pulmonary arterial hypertension --- aptamer --- aptasensor --- influenza --- SERS --- virus detection --- α-synuclein --- antisense oligonucleotide --- dopamine neurotransmission --- double mutant A30P*A53T* --- motor deficits --- Parkinson’s disease --- transgenic mouse model --- G-quadruplexes --- covalent dimer construct --- anti-proliferative activity --- primary cell culture of human glioma --- antisensense oligonucleotide --- Foxp3 --- regulatory T cells --- vaccine immunogenicity --- Sporothrix schenckii --- Marfan syndrome --- fibrillin-1 --- antisense oligonucleotides --- exon skipping --- splice-switching --- quantum dots (QDs) --- DNAzyme --- ROS --- Amplex Red --- light-induced activity --- DNA methylation --- histone code --- microRNA --- nanoparticles --- noncoding RNA --- pulmonary arterial hypertension --- aptamer --- aptasensor --- influenza --- SERS --- virus detection --- α-synuclein --- antisense oligonucleotide --- dopamine neurotransmission --- double mutant A30P*A53T* --- motor deficits --- Parkinson’s disease --- transgenic mouse model --- G-quadruplexes --- covalent dimer construct --- anti-proliferative activity --- primary cell culture of human glioma --- antisensense oligonucleotide --- Foxp3 --- regulatory T cells --- vaccine immunogenicity --- Sporothrix schenckii --- Marfan syndrome --- fibrillin-1 --- antisense oligonucleotides --- exon skipping --- splice-switching

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Oligonucleotides (ON) constitute a new group of molecular agents, the object of significant interest due to their potential value as drugs for diagnostic and therapeutic applications. Their special interest derives from the intrinsic characteristics of ONs: a) ONs are informative agents, a property that derives from the order in which the nucleotides of each particular ON are arranged; b) ONs can act as ligands (ASO, TFO, aptamers, G-quadruplex, etc.) of complementary nucleic acid sequences (DNA or RNA) due to their high capacity to hybridize (by means of Watson and Crick or Hoogsteen links) with other nucleotide sequences, resulting in specific gene modulatory effects. However, nonspecific sequences may also be of interest, as is the case with repetitive nucleotide sequences (CpG) with adjuvant effects of vaccines; c) ONs can also rapidly evolve to achieve specific advantages of utility (targeting, stability, efficacy, toxicity, etc.) or high-sensitivity diagnostic technology (markers, analyzes, biosensors, FISH, microarrays, etc.), by chemical modification of nucleotides in any of their atoms. These properties show that ONs are first-order molecules due to their potential usefulness in practice.In this collection of research articles and review papers, we aim to highlight their therapeutic, but also diagnostic and technological utility as drugs.

Medicine --- quantum dots (QDs) --- DNAzyme --- ROS --- Amplex Red --- light-induced activity --- DNA methylation --- histone code --- microRNA --- nanoparticles --- noncoding RNA --- pulmonary arterial hypertension --- aptamer --- aptasensor --- influenza --- SERS --- virus detection --- α-synuclein --- antisense oligonucleotide --- dopamine neurotransmission --- double mutant A30P*A53T* --- motor deficits --- Parkinson’s disease --- transgenic mouse model --- G-quadruplexes --- covalent dimer construct --- anti-proliferative activity --- primary cell culture of human glioma --- antisensense oligonucleotide --- Foxp3 --- regulatory T cells --- vaccine immunogenicity --- Sporothrix schenckii --- Marfan syndrome --- fibrillin-1 --- antisense oligonucleotides --- exon skipping --- splice-switching

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The mechanistic/mammalian target of rapamycin (mTOR), a serine/threonine kinase, is a central regulator for human physiological activity. Deregulated mTOR signaling is implicated in a variety of disorders, such as cancer, obesity, diabetes, and neurodegenerative diseases. The papers published in this Special Issue summarize the current understanding of the mTOR pathway and its role in the regulation of tissue regeneration, regulatory T cell differentiation and function, and different types of cancer including hematologic malignancies, skin, prostate, breast, and head and neck cancer. The findings highlight that targeting mTOR pathway is a promising strategy to fight against certain human diseases.

mTOR --- histone deacetylase --- prostate cancer --- integrins --- adhesion --- invasion --- cell metabolism --- T cells --- Foxp3 --- Acute Lymphoblastic leukemia --- targeted therapy --- metabolism --- cell signalling --- mTOR signalling --- head and neck cancer --- mutant genes --- biomarkers --- targeted therapies --- clinical trials --- cancers --- inhibitors --- photodynamic therapy --- PI3K --- Akt --- skin cancers --- phytochemicals --- melanoma --- basal cell carcinoma --- squamous cell carcinoma --- Merkel cell carcinoma --- TNBC --- eribulin --- PI3K/AKT/mTOR --- everolimus --- combination --- synergy --- mTOR signaling --- tissue regeneration --- neuron --- muscle --- liver --- intestine --- hematologic malignancies --- regulatory T cells --- tumor