ABSTRACT
Cyclic nucleotide phosphodiesterase (PDE) enzymes catalyze the breakdown of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which act as intracellular second messengers for signal transduction pathways and modulate various processes in the central nervous system. Recent discoveries that mutations in genes encoding different PDEs, including PDE10A, are responsible for rare forms of chorea in children led to the recognition of an emerging role of PDEs in the field of pediatric movement disorders. A comprehensive literature review of all reported cases of PDE10A mutations in PubMed and Web of Science was performed in English. We included eight studies, describing 31 patients harboring a PDE10A mutation and exhibiting a hyperkinetic movement disorder with onset in infancy or childhood. Mutations in both GAF-A, GAF-B regulatory domains and outside the GAF domains of the PDE10A gene have been reported to cause hyperkinetic movement disorders. In general, patients with homozygous mutations in either GAF-A domain of PDE10A present with a more severe phenotype and at an earlier age but without any extensive abnormalities of the striata compared with patients with dominant variants in GAF-B domain, indicating that dominant and recessive mutations have different pathogenic mechanisms. PDE10A plays a key role in regulating control of striato-cortical movement. Comprehension of the molecular mechanisms within the cAMP and cGMP signaling systems caused by PDE10A mutations may inform novel therapeutic strategies that could alleviate symptoms in young patients affected by these rare movement disorders.
Subject(s)
Hyperkinesis , Mutation , Phosphoric Diester Hydrolases , Humans , Phosphoric Diester Hydrolases/genetics , Hyperkinesis/genetics , ChildABSTRACT
Regulatory T cells (Tregs) are essential mediators of tolerance mitigating aberrant immune responses. While naturally occurring Treg (nTreg) development and function are directed by epigenetic events, induced Treg (iTreg) identity and mechanisms of action remain elusive. Mirroring the epigenetic circuits of nTregs, we and others have used hypomethylation agents (HAs) to ex vivo convert T cells into iTregs (HA-iTregs) and further showed that the suppressive properties of the HA-iTregs are predominantly confined in an emergent population, which de novo expresses the immunomodulatory molecule HLA-G, consequently providing a surface marker for isolation of the suppressive HA-iTreg compartment (G+ cells). We isolated the HA-induced G+ cells and their G- counterparts and employed high-throughput RNA-sequencing (RNA-seq) analyses to uncover the G+-specific transcriptomic changes guiding T cells toward a regulatory trajectory upon their exposure to HA. We found a distinct transcriptional upregulation of G+ cells accompanied by enrichment of immune-response-related pathways. Although single-cell RNA-seq profiling revealed regulatory G+ cells to have molecular features akin to nTregs, when assessed in conjunction with the comparative transcriptomic analysis and profiling of secreted cytokines against the non-suppressive G- cells, FOXP3 and other T-helper signatures appear to play a minor role in their suppressive phenotype. We found an ectopic expression of IDO-1 and CCL17/22 in G+ cells, denoting that in vitro exposure of T cells to HA may well unlock myeloid suppressor genes. This report provides transcriptional data shaping the molecular identity of a highly purified and potent HA-iTreg population and hints toward ectopic myeloid-specific molecular mechanisms mediating HA-iTreg function.
Subject(s)
T-Lymphocytes, Regulatory , Transcriptome , Cell Differentiation , Cytokines/metabolism , DNA MethylationABSTRACT
Microbiota and the metabolites they produce within the large intestine interact with the host epithelia under the influence of a range of host-derived metabolic, immune, and homeostatic factors. This complex host-microbe interaction affects intestinal tumorigenesis, but established microbial or metabolite profiles predicting colorectal cancer (CRC) risk are missing. Here, we aimed to identify fecal bacteria, volatile organic compounds (VOC), and their associations that distinguish healthy (non-adenoma, NA) from CRC prone (high-risk adenoma, HRA) individuals. Analyzing fecal samples obtained from 117 participants ≥15 days past routine colonoscopy, we highlight the higher abundance of Proteobacteria and Parabacteroides distasonis, and the lower abundance of Lachnospiraceae species, Roseburia faecis, Blautia luti, Fusicatenibacter saccharivorans, Eubacterium rectale, and Phascolarctobacterium faecium in the samples of HRA individuals. Volatolomic analysis of samples from 28 participants revealed a higher concentration of five compounds in the feces of HRA individuals, isobutyric acid, methyl butyrate, methyl propionate, 2-hexanone, and 2-pentanone. We used binomial logistic regression modeling, revealing 68 and 96 fecal bacteria-VOC associations at the family and genus level, respectively, that distinguish NA from HRA endpoints. For example, isobutyric acid associations with Lachnospiraceae incertae sedis and Bacteroides genera exhibit positive and negative regression lines for NA and HRA endpoints, respectively. However, the same chemical associates with Coprococcus and Colinsella genera exhibit the reverse regression line trends. Thus, fecal microbiota and VOC profiles and their associations in NA versus HRA individuals indicate the significance of multiple levels of analysis towards the identification of testable CRC risk biomarkers.
ABSTRACT
Cancer recurrence and metastasis, following successful treatment, constitutes a critical threat in clinical oncology and are the leading causes of death amongst cancer patients. This phenomenon is largely attributed to metastatic tumor dormancy, a rate-limiting stage during cancer progression, in which disseminated cancer cells remain in a viable, yet not proliferating state for a prolonged period. Dormant cancer cells are characterized by their entry into cell cycle arrest and survival in a quiescence state to adapt to their new microenvironment through the acquisition of mutations and epigenetic modifications, rendering them resistant to anti-cancer treatment and immune surveillance. Under favorable conditions, disseminated dormant tumor cells 're-awake', resume their proliferation and thus colonize distant sites. Due to their rarity, detection of dormant cells using current diagnostic tools is challenging and, thus, therapeutic targets are hard to be identified. Therefore, unraveling the underlying mechanisms required for keeping disseminating tumor cells dormant, along with signals that stimulate their "re-awakening" are crucial for the discovery of novel pharmacological treatments. In this review, we shed light into the main mechanisms that control dormancy induction and escape as well as emerging therapeutic strategies for the eradication of metastatic dormant cells, including dormancy maintenance, direct targeting of dormant cells and re-awakening dormant cells. Studies on the ability of the metastatic cancer cells to cease proliferation and survive in a quiescent state before re-initiating proliferation and colonization years after successful treatment, will pave the way toward developing innovative therapeutic strategies against dormancy-mediated metastatic outgrowth.
Subject(s)
Neoplasm Recurrence, Local , Neoplasms, Second Primary , Humans , Neoplasm Recurrence, Local/pathology , Cell Cycle Checkpoints , Cell Division , Epigenesis, Genetic , Tumor Microenvironment/physiologyABSTRACT
Nano-immunotherapy improves breast cancer outcomes but not all patients respond and none are cured. To improve efficacy, research focuses on drugs that reprogram cancer-associated fibroblasts (CAFs) to improve therapeutic delivery and immunostimulation. These drugs, however, have a narrow therapeutic window and cause adverse effects. Developing strategies that increase CAF-reprogramming while limiting adverse effects is urgent. Here, taking advantage of the CAF-reprogramming capabilities of tranilast, we developed tranilast-loaded micelles. Strikingly, a 100-fold reduced dose of tranilast-micelles induces superior reprogramming compared to free drug owing to enhanced intratumoral accumulation and cancer-associated fibroblast uptake. Combination of tranilast-micelles and epirubicin-micelles or Doxil with immunotherapy increases T-cell infiltration, resulting in cures and immunological memory in mice bearing immunotherapy-resistant breast cancer. Furthermore, shear wave elastography (SWE) is able to monitor reduced tumor stiffness caused by tranilast-micelles and predict response to nano-immunotherapy. Micellar encapsulation is a promising strategy for TME-reprogramming and SWE is a potential biomarker of response.
Subject(s)
Drug-Related Side Effects and Adverse Reactions , Neoplasms , Mice , Animals , Micelles , Tumor Microenvironment , Immunotherapy , ortho-Aminobenzoates/pharmacology , ortho-Aminobenzoates/therapeutic use , Immunologic Factors , PolymersABSTRACT
Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS) with an unknown etiology, although genetic, epigenetic, and environmental factors are thought to play a role. Recently, coagulation components have been shown to provide immunomodulatory and pro-inflammatory effects in the CNS, leading to neuroinflammation and neurodegeneration. The current study aimed to determine whether patients with MS exhibited an overrepresentation of polymorphisms implicated in the coagulation and whether such polymorphisms are associated with advanced disability and disease progression. The cardiovascular disease (CVD) strip assay was applied to 48 MS patients and 25 controls to analyze 11 genetic polymorphisms associated with thrombosis and CVD. According to our results, FXIIIVal34Leu heterozygosity was less frequent (OR: 0.35 (95% CI: 0.12-0.99); p = 0.04), whereas PAI-1 5G/5G homozygosity was more frequent in MS (OR: 6.33 (95% CI: 1.32-30.24); p = 0.016). In addition, carriers of the HPA-1a/1b were likely to have advanced disability (OR: 1.47 (95% CI: 1.03-2.18); p = 0.03) and disease worsening (OR: 1.42 (95% CI: 1.05-2.01); p = 0.02). The results of a sex-based analysis revealed that male HPA-1a/1b carriers were associated with advanced disability (OR: 3.04 (95% CI: 1.22-19.54); p = 0.01), whereas female carriers had an increased likelihood of disease worsening (OR: 1.56 (95% CI: 1.04-2.61); p = 0.03). Our findings suggest that MS may be linked to thrombophilia-related polymorphisms, which warrants further investigation.
ABSTRACT
Alzheimer's disease (AD) is a progressive neurodegenerative disease of the brain causing either familial or sporadic dementia. We have previously administered the modified C5a receptor agonist (EP67) for a short period to a transgenic mouse model of AD (5XFAD) and have observed not only reduction in ß-amyloid deposition and gliosis but also improvement in cognitive impairment. Inquiring, however, on the effects of EP67 in an already heavily burdened animal, thus representing a more realistic scenario, we treated 6-month-old 5XFAD mice for a period of 14 weeks. We recorded a significant decrease in both fibrillar and pre-fibrillar ß-amyloid as well as remarkable amelioration of cognitive impairment. Following proteomic analysis and pathway association, we postulate that these events are triggered through the upregulation of ß-adrenergic and GABAergic signaling. In summary, our results reveal how inflammatory responses can be employed in inducing tangible phenotype improvements even in advanced stages of AD.
Subject(s)
Alzheimer Disease , Cognitive Dysfunction , Oligopeptides , Receptor, Anaphylatoxin C5a , Receptors, Adrenergic, beta , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Animals , Cognitive Dysfunction/drug therapy , Cognitive Dysfunction/genetics , Cognitive Dysfunction/metabolism , Disease Models, Animal , Mice , Mice, Transgenic , Oligopeptides/pharmacology , Oligopeptides/therapeutic use , Proteomics , Receptor, Anaphylatoxin C5a/agonists , Receptor, Anaphylatoxin C5a/genetics , Receptor, Anaphylatoxin C5a/metabolism , Receptors, Adrenergic, beta/genetics , Receptors, Adrenergic, beta/metabolismABSTRACT
Poland syndrome is a rare developmental disorder characterized by unilateral, complete or partial, absence of the pectoralis major (and often minor) muscle, accompanied with ipsilateral hand malformations. To date, no clear genetic cause has been associated with Poland syndrome, although familial cases have been reported. We report the employment of trio exome investigation and the identification of a heterozygous de novo pathogenic variant in the SFMBT1 gene, a transcription factor associated with transcriptional repression during development, in a 14-yr-old boy with Poland syndrome. We further demonstrate by means of cDNA sequencing and western blot analysis that this variant results in SFMBT1 exon 10 skipping and a lower concentration of the SFMBT1 wild-type protein. To our knowledge, the heterozygous pathogenic SFMBT1 variant identified in association with this condition is novel as it has not been elsewhere described in the literature and it can be incorporated to the limited reported cases published.
Subject(s)
Poland Syndrome , Adolescent , Exome , Heterozygote , Humans , Male , Poland Syndrome/genetics , Repressor Proteins/genetics , Transcription Factors/genetics , Exome SequencingABSTRACT
Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.
Subject(s)
Antigens, Neoplasm/immunology , Dendritic Cells/immunology , Fetal Blood/cytology , Immunotherapy, Adoptive/methods , Leukemia/therapy , T-Cell Antigen Receptor Specificity , T-Lymphocyte Subsets/immunology , WT1 Proteins/immunology , Antigens, CD/analysis , Blood Banks/economics , Cell Differentiation , Cells, Cultured , Cord Blood Stem Cell Transplantation/standards , Cytotoxicity, Immunologic , Dendritic Cells/cytology , Dendritic Cells/transplantation , Humans , Immunomagnetic Separation , Immunophenotyping , Immunotherapy, Adoptive/economics , Leukemia/economics , Memory T Cells/immunology , Memory T Cells/transplantation , T-Lymphocyte Subsets/transplantation , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantationABSTRACT
Nano-immunotherapy regimens have high potential to improve patient outcomes, as already demonstrated in advanced triple negative breast cancer with nanoparticle albumin-bound paclitaxel and the immune checkpoint blocker (ICB) atezolizumab. This regimen, however, does not lead to cures with median survival lasting less than two years. Thus, understanding the mechanisms of resistance to and development of strategies to enhance nano-immunotherapy in breast cancer are urgently needed. Here, in human tissue it is shown that blood vessels in breast cancer lung metastases are compressed leading to hypoxia. This pathophysiology exists in murine spontaneous models of triple negative breast cancer lung metastases, along with low levels of perfusion. Because this pathophysiology is consistent with elevated levels of solid stress, the mechanotherapeutic tranilast, which decompressed lung metastasis vessels, is administered to mice bearing metastases, thereby restoring perfusion and alleviating hypoxia. As a result, the nanomedicine Doxil causes cytotoxic effects into metastases more efficiently, stimulating anti-tumor immunity. Indeed, when combining tranilast with Doxil and ICBs, synergistic effects on efficacy, with all mice cured in one of the two ICB-insensitive tumor models investigated is resulted. These results suggest that strategies to treat breast cancer with nano-immunotherapy should also include a mechanotherapeutic to decompress vessels.
ABSTRACT
Myeloid cells include various cellular subtypes that are distinguished into mononuclear and polymorphonuclear cells, derived from either common myeloid progenitor cells (CMPs) or myeloid stem cells. They play pivotal roles in innate immunity since, following invasion by pathogens, myeloid cells are recruited and initiate phagocytosis and secretion of inflammatory cytokines into local tissues. Moreover, mounting evidence suggests that myeloid cells may also regulate cancer development by infiltrating the tumor to directly interact with cancer cells or by affecting the tumor microenvironment. Importantly, mononuclear phagocytes, including macrophages and dendritic cells (DCs), can have either a positive or negative impact on the efficacy of chemotherapy, radiotherapy as well as targeted anti-cancer therapies. Tumor-associated macrophages (TAMs), profusely found in the tumor stroma, can promote resistance to chemotherapeutic drugs, such as Taxol and Paclitaxel, whereas the suppression of TAMs can lead to an improved radiotherapy outcome. On the contrary, the presence of TAMs may be beneficial for targeted therapies as they can facilitate the accumulation of large quantities of nanoparticles carrying therapeutic compounds. Tumor infiltrating DCs, however, are generally thought to enhance cytotoxic therapies, including those using anthracyclines. This review focuses on the role of tumor-infiltrating and stroma myeloid cells in modulating tumor responses to various treatments. We herein report the impact of myeloid cells in a number of therapeutic approaches across a wide range of malignancies, as well as the efforts toward the elimination of myeloid cells or the exploitation of their presence for the enhancement of therapeutic efficacy against cancer.
ABSTRACT
Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored. Methods: Here, we re-purposed the TGF-ß inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors. Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4. Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.
Subject(s)
Immunotherapy/methods , Transforming Growth Factor beta/drug effects , Triple Negative Breast Neoplasms , Tumor Microenvironment/drug effects , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/pharmacology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , CTLA-4 Antigen/drug effects , Chemotherapy, Cancer, Regional Perfusion/methods , Disease Models, Animal , Doxorubicin/administration & dosage , Doxorubicin/analogs & derivatives , Doxorubicin/pharmacology , Drug Combinations , Extracellular Matrix/drug effects , Female , Immunization/methods , Mice , Nanomedicine/methods , Nanoparticles/therapeutic use , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/pharmacology , Programmed Cell Death 1 Receptor/drug effects , T-Lymphocytes/drug effects , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/immunology , ortho-Aminobenzoates/administration & dosage , ortho-Aminobenzoates/pharmacologyABSTRACT
According to the amyloid hypothesis of Alzheimer's disease (AD) the deposition of prefibrillar and fibrillar Aß peptide sets off the pathogenic cascades of neuroinflammation and neurodegeneration that lead to synaptic and neuronal loss resulting in cognitive decline. Various approaches to reduce amyloid load by reducing production of the Aß peptide or enhancing amyloid clearance by primary or secondary immunization have not proven successful in clinical trials. Interfering with the normal function of secretases and suboptimal timing of Aß peptide removal have been put forward as possible explanations. Complement, an innate component of the immune system, has been found to modulate disease pathology and in particular neuronal loss in the AD mouse model but its mechanism of action is complex. C1Q has been shown to facilitate phagocytosis of Aß peptide but its Ablation attenuates neuroinflammation. Experiments in AD mouse models show that inhibition of complement component C5a reduces amyloid deposition and alleviates neuroinflammation. Phagocytes including microglia, monocytes and neutrophils carry C5a receptors. Here, a widely used mouse model of AD, 5XFAD, was intermittently treated with the oral C5a receptor agonist EP67 and several neuronal and neuroinflammatory markers as well as memory function were assessed. EP67 treatment enhanced phagocytosis, resulting in a significant reduction of both fibrillar and non-fibrillar Aß, reduced astrocytosis and preserved synaptic and neuronal markers as well as memory function. Timely and phasic recruitment of the innate immune system offers a new therapeutic avenue of treating pre-symptomatic Alzheimer disease.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid/drug effects , Memory/drug effects , Oligopeptides/pharmacology , Phagocytosis/drug effects , Alzheimer Disease/genetics , Amyloid/metabolism , Animals , Brain/drug effects , Brain/metabolism , Disease Models, Animal , Mice , Mice, Transgenic , Neurons/drug effects , Neurons/metabolismABSTRACT
The contribution of specific HLA Class II alleles in type 1 diabetes is determined by polymorphic amino acid epitopes that direct antigen binding therefore, along with conventional allele frequency analysis, epitope analysis can provide important insights into disease susceptibility. We analyzed the highly heterogeneous Cypriot population for the HLA class II loci of T1DM patients and controls and we report for the first time their allele frequencies. Within our patient cohort we identified a subgroup that did not carry the DRB1*03:01-DQA1*05:01-DQB1*02:01 and DRB1*04:xx-DQA1*03:01-DQB1*03:02 risk haplotypes but a novel recombinant one, DRB1*04:XX-DQA1*03:01-DQB1*02:01 designated DR4-DQ2.3. Through epitope analysis we identified established susceptibility (DQB1 A57, DRB1 H13) and resistance (DQB1 D57) residues as well as other novel susceptibility residues DRB1 Q70, DQB1 L26 and resistance residues DRB1 D70, R70 and DQB1 Y47. Prevalence of susceptibility epitopes was higher in patients and was not exclusively a result of linkage disequilibrium. Residues DRB1 Q70, DQB1 L26 and A57 and a 10 amino acid epitope of DQA1 were the most significant in discriminating risk alleles. An extended haplotype containing these epitopes was carried by 92% of our patient cohort. Sharing of susceptibility epitopes could also explain the absence of risk haplotypes in patients. Finally, many significantly associated epitopes were non-pocket residues suggesting that critical immune functions may exist spanning further from the binding pockets.
Subject(s)
Alanine/genetics , Diabetes Mellitus, Type 1/genetics , Epitope Mapping/methods , Glutamine/genetics , HLA-DQ beta-Chains/genetics , HLA-DRB1 Chains/genetics , Age of Onset , Binding Sites , Cohort Studies , Cyprus , Diabetes Mellitus, Type 1/immunology , Female , Gene Frequency , Genetic Predisposition to Disease , HLA-DQ beta-Chains/chemistry , HLA-DRB1 Chains/chemistry , Haplotypes , Humans , Linkage Disequilibrium , MaleABSTRACT
BACKGROUND: Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the "semi-allogeneic" fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs. METHODS: Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties. RESULTS: HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4+HLA-Gpos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-Gpos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD). DISCUSSION: We propose, in vitro generation of HLA-G-expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy.
Subject(s)
Cell Engineering/methods , Graft vs Host Disease/therapy , HLA-G Antigens/metabolism , Immunotherapy, Adoptive/methods , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/transplantation , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cell Culture Techniques , Cells, Cultured , DNA Methylation/drug effects , Decitabine , Gene Expression Regulation/drug effects , Graft vs Host Disease/immunology , HLA-G Antigens/genetics , Humans , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunologyABSTRACT
The HLA-G molecule presents immunomodulatory properties that might inhibit immune responses when interacting with specific Natural Killer and T cell receptors, such as KIR2DL4, ILT2 and ILT4. Thus, HLA-G might influence the outcome of situations in which fine immune system modulation is required, such as autoimmune diseases, transplants, cancer and pregnancy. The majority of the studies regarding the HLA-G gene variability so far was restricted to a specific gene segment (i.e., promoter, coding or 3' untranslated region), and was performed by using Sanger sequencing and probabilistic models to infer haplotypes. Here we propose a massively parallel sequencing (NGS) with a bioinformatics strategy to evaluate the entire HLA-G regulatory and coding segments, with haplotypes inferred relying more on the straightforward haplotyping capabilities of NGS, and less on probabilistic models. Then, HLA-G variability was surveyed in two admixed population samples of distinct geographical regions and demographic backgrounds, Cyprus and Brazil. Most haplotypes (promoters, coding, 3'UTR and extended ones) were detected both in Brazil and Cyprus and were identical to the ones already described by probabilistic models, indicating that these haplotypes are quite old and may be present worldwide.
Subject(s)
HLA-G Antigens/genetics , Haplotypes/genetics , Adult , Base Sequence , Brazil , Computational Biology , Cyprus , Female , Genetic Variation , High-Throughput Nucleotide Sequencing , Humans , Male , Polymerase Chain ReactionABSTRACT
The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers.