T-cell immunity against senescence: potential role and perspectives.

The development of age-associated diseases is related to the accumulation of senescent cells in the body. These are old non-functional cells with impaired metabolism, which are unable to divide. Such cells are also resistant to programmed cell death and prone to spontaneous production of some inflammatory factors. The accumulation of senescent cells is related to the age-associated dysfunction of organs and tissues as well as chronic inflammation that enhances with age. In the young organism, senescent cells are removed with the innate immunity system. However, the efficiency of this process decreases with age. Nowadays, more and more evidences are accumulating to support the involvement of specific immunity and T-lymphocytes in the fight against senescent cells. It has great physiological importance since the efficient elimination of senescent cells requires a high diversity of antigen-recognizing receptors to cover the entire spectrum of senescent-associated antigens with high precision and specificity. Developing the approaches of T-cell immunity stimulation to generate or amplify a physiological immune response against senescent cells can provide new perspectives to extend active longevity. In this mini-review, the authors summarize the current understanding of the role of T-cell immunity in the fight against senescent cells and discuss the prospects of stimulating adaptive immunity for combating the accumulation of senescent cells that occurs with age.

Engineered AAV2.7m8 Serotype Shows Significantly Higher Transduction Efficiency of ARPE-19 and HEK293 Cell Lines Compared to AAV5, AAV8 and AAV9 Serotypes.

The level of transduction efficiency of the target retinal cells affects the choice of AAV serotype and the outcome of gene replacement therapy for inherited retinal diseases. This study focused on the tropism and transduction efficiency of AAV2.7m8-, AAV5-, AAV8-, and AAV9-GFP in ARPE-19 and HEK293 cells. Fluorescence intensity was assessed bi-hourly by means of IncuCyte S3 live imaging microscopy. Within 12 h, AAV2.7m8 demonstrated the highest transduction efficiency at four viral concentrations of 1-, 3-, 6-, and 8 × 104 VG/cell in a dose-dependent manner, followed by AAV5 in ARPE-19 and AAV9 in HEK293 cells. The transduction efficiency of AAV2.7m8 at a dose of 6 × 104 VG/cell was 21, 202, and 323 times higher in ARPE-19 cells and 324, 100, and 52 times higher in HEK293 cells compared to AAV5, AAV8, and AAV9, respectively. This trend remained for 4 days at all viral concentrations, as additionally shown by flow cytometry. At a dose of 6 × 104 VG/cell, AAV2.7m8 (97% GFP-positive cells, GFP +) was nearly two and 10 times as efficient as AAV5 (52% GFP+) and AAV9 or AAV8 (both 9%), respectively, in ARPE-19 cells. In HEK293 cells, 95% of AAV2.7m8-, 26% of AAV9-, 17% of AAV8-, and 12% of AAV5-transduced cells were GFP-positive.

Autosomal Dominant STAT6 Gain of Function Causes Severe Atopy Associated with Lymphoma.

The transcription factor STAT6 (Signal Transducer and Activator of Transcription 6) is a key regulator of Th2 (T-helper 2) mediated allergic inflammation via the IL-4 (interleukin-4) JAK (Janus kinase)/STAT signalling pathway. We identified a novel heterozygous germline mutation STAT6 c.1255G > C, p.D419H leading to overactivity of IL-4 JAK/STAT signalling pathway, in a kindred affected by early-onset atopic dermatitis, food allergy, eosinophilic asthma, anaphylaxis and follicular lymphoma. STAT6 D419H expression and functional activity were compared with wild type STAT6 in transduced HEK293T cells and to healthy control primary skin fibroblasts and peripheral blood mononuclear cells (PBMC). We observed consistently higher STAT6 levels at baseline and higher STAT6 and phosphorylated STAT6 following IL-4 stimulation in D419H cell lines and primary cells compared to wild type controls. The pSTAT6/STAT6 ratios were unchanged between D419H and control cells suggesting that elevated pSTAT6 levels resulted from higher total basal STAT6 expression. The selective JAK1/JAK2 inhibitor ruxolitinib reduced pSTAT6 levels in D419H HEK293T cells and patient PBMC. Nuclear staining demonstrated increased STAT6 in patient fibroblasts at baseline and both STAT6 and pSTAT6 after IL-4 stimulation. We also observed higher transcriptional upregulation of downstream genes (XBP1 and EPAS1) in patient PBMC. Our study confirms STAT6 gain of function (GOF) as a novel monogenetic cause of early onset atopic disease. The clinical association of lymphoma in our kindred, along with previous data linking somatic STAT6 D419H mutations to follicular lymphoma suggest that patients with STAT6 GOF disease may be at higher risk of lymphomagenesis.245 words.

Analysis of FOXP3 DNA Methylation Patterns to Identify Functional FOXP3+ T-Cell Subpopulations.

Human regulatory CD4+CD25+FOXP3+ T cells (Tregs) are involved in the suppression of immune responses and play important roles in the maintenance of self-tolerance and immune homeostasis. Abnormal Treg function may result in disease states of varying severity. As FOXP3-expressing Treg cells are phenotypically and functionally heterogeneous, the success of Treg therapies depends on the ability to reliably distinguish subpopulations of T cells bearing a Treg-like phenotype. Methylation of cytosines within CpG dinucleotides is an important epigenetic mechanism involved in regulation (and suppression) of gene expression. On the other hand, demethylation of regulatory DNA sequences, such as promoters and enhancers, is essential for initiation of gene transcription. This protocol shows that bisulfite sequencing (BS) distinguishes methylated and unmethylated cytosines within DNA and reveals the methylation status of individual CpGs in cells within each population, identifying functionally different FOXP3+ subpopulations.

Viral Vectors in Gene Replacement Therapy.

Throughout the years, several hundred million people with rare genetic disorders have been receiving only symptom management therapy. However, research and development efforts worldwide have led to the development of long-lasting, highly efficient, and safe gene therapy for a wide range of hereditary diseases. Improved viral vectors are now able to evade the preexisting immunity and more efficiently target and transduce therapeutically relevant cells, ensuring genome maintenance and expression of transgenes at the relevant levels. Hematological, ophthalmological, neurodegenerative, and metabolic therapeutic areas have witnessed successful treatment of hemophilia and muscular dystrophy, restoration of immune system in children with immunodeficiencies, and restoration of vision. This review focuses on three leading vector platforms of the past two decades: adeno-associated viruses (AAVs), adenoviruses (AdVs), and lentiviruses (LVs). Special attention is given to successful preclinical and clinical studies that have led to the approval of gene therapies: six AAV-based (Glybera® for lipoprotein lipase deficiency, Luxturna® for retinal dystrophy, Zolgensma® for spinal muscular atrophy, Upstaza® for AADC, Roctavian® for hemophilia A, and Hemgenix® for hemophilia B) and three LV-based (Libmeldy® for infantile metachromatic leukodystrophy, Zynteglo® for ß-thalassemia, and Skysona® for ALD). The review also discusses the problems that arise in the development of gene therapy treatments, which, nevertheless, do not overshadow the successes of already developed gene therapies and the hope these treatments give to long-suffering patients and their families.

Development of Recombinant Oncolytic rVSV-mIL12-mGMCSF for Cancer Immunotherapy.

Anti-cancer therapy based on oncolytic viruses (OVs) is a targeted approach that takes advantage of OVs' ability to selectively infect and replicate in tumor cells, activate the host immune response, and destroy malignant cells over healthy ones. Vesicular stomatitis virus (VSV) is known for its wide range of advantages: a lack of pre-existing immunity, a genome that is easily amenable to manipulation, and rapid growth to high titers in a broad range of cell lines, to name a few. VSV-induced tumor immunity can be enhanced by the delivery of immunostimulatory cytokines. The targeted cytokine delivery to tumors avoids the significant toxicity associated with systemic delivery while also boosting the immune response. To demonstrate this enhanced effect on both tumor growth and survival, a novel recombinant VSV (rVSV)-mIL12-mGMCSF, co-expressing mouse IL-12 (interleukin-12) and GM-CSF (granulocyte-macrophage colony-stimulating factor), was tested alongside rVSV-dM51-GFP (rVSV-GFP) that was injected intratumorally in a syngeneic in vivo C57BL/6 mouse model infused subcutaneously with B16-F10 melanoma cells. The pilot study tested the effect of two viral injections 4 days apart and demonstrated that treatment with the two rVSVs resulted in partial inhibition of tumor growth (TGII of around 40%) and an increased survival rate in animals from the treatment groups. The effect of the two VSVs on immune cell populations will be investigated in future in vivo studies with an optimized experimental design with multiple higher viral doses, as a lack of this information presents a limitation of this study.

Partial human Janus kinase 1 deficiency predominantly impairs responses to interferon gamma and intracellular control of mycobacteria.

Purpose: Janus kinase-1 (JAK1) tyrosine kinase mediates signaling from multiple cytokine receptors, including interferon alpha/beta and gamma (IFN-α/ß and IFN-γ), which are important for viral and mycobacterial protection respectively. We previously reported autosomal recessive (AR) hypomorphic JAK1 mutations in a patient with recurrent atypical mycobacterial infections and relatively minor viral infections. This study tests the impact of partial JAK1 deficiency on cellular responses to IFNs and pathogen control. Methods: We investigated the role of partial JAK1 deficiency using patient cells and cell models generated with lentiviral vectors expressing shRNA. Results: Partial JAK1 deficiency impairs IFN-γ-dependent responses in multiple cell types including THP-1 macrophages, Epstein-Barr Virus (EBV)-transformed B cells and primary dermal fibroblasts. In THP-1 myeloid cells, partial JAK1 deficiency reduced phagosome acidification and apoptosis and resulted in defective control of mycobacterial infection with enhanced intracellular survival. Although both EBV-B cells and primary dermal fibroblasts with partial JAK1 deficiency demonstrate reduced IFN-α responses, control of viral infection was impaired only in patient EBV-B cells and surprisingly intact in patient primary dermal fibroblasts. Conclusion: Our data suggests that partial JAK1 deficiency predominantly affects susceptibility to mycobacterial infection through impact on the IFN-γ responsive pathway in myeloid cells. Susceptibility to viral infections as a result of reduced IFN-α responses is variable depending on cell type. Description of additional patients with inherited JAK1 deficiency will further clarify the spectrum of bacterial and viral susceptibility in this condition. Our results have broader relevance for anticipating infectious complications from the increasing use of selective JAK1 inhibitors.

Mesenchymal stromal cells induce regulatory T cells via epigenetic conversion of human conventional CD4 T cells in vitro.

Regulatory T cells (Treg) play a critical role in immune tolerance. The scarcity of Treg therapy clinical trials in humans has been largely due to the difficulty in obtaining sufficient Treg numbers. We performed a preclinical investigation on the potential of mesenchymal stromal cells (MSCs) to expand Treg in vitro to support future clinical trials. Human peripheral blood mononuclear cells from healthy donors were cocultured with allogeneic bone marrow-derived MSCs expanded under xenogeneic-free conditions. Our data show an increase in the counts and frequency of CD4+ CD25high Foxp3+ CD127low Treg cells (4- and 6-fold, respectively) after a 14-day coculture. However, natural Treg do not proliferate in coculture with MSCs. When purified conventional CD4 T cells (Tcon) are cocultured with MSCs, only cells that acquire a Treg-like phenotype proliferate. These MSC-induced Treg-like cells also resemble Treg functionally, since they suppress autologous Tcon proliferation. Importantly, the DNA methylation profile of MSC-induced Treg-like cells more closely resembles that of natural Treg than of Tcon, indicating that this population is stable. The expression of PD-1 is higher in Treg-like cells than in Tcon, whereas the frequency of PDL-1 increases in MSCs after coculture. TGF-ß levels are also significantly increased MSC cocultures. Overall, our data suggest that Treg enrichment by MSCs results from Tcon conversion into Treg-like cells, rather than to expansion of natural Treg, possibly through mechanisms involving TGF-ß and/or PD-1/PDL-1 expression. This MSC-induced Treg population closely resembles natural Treg in terms of phenotype, suppressive ability, and methylation profile.

Molecular Markers Distinguishing T Cell Subtypes With TSDR Strand-Bias Methylation.

Human regulatory CD4+CD25+FOXP3+ T cells (Treg) play important roles in the maintenance of self-tolerance and immune homeostasis in various disease settings and are also involved in the suppression of effective immune responses. These cells are heterogeneous in phenotype and function, and the ability to reliably distinguish between various FOXP3-expressing subpopulations can affect the development of successful therapies. This study demonstrates that hypomethylated CpG sites, present in four regions of the FOXP3 locus, CAMTA1 and FUT7 gene regions, can be used to distinguish several subsets of Treg from conventional CD4+ T lymphocytes (Tcon) in donors of both genders. We describe a previously unreported strand-bias hemimethylation pattern in FOXP3 promoter and TSDR in donors of both genders, with the coding strand being demethylated within promoter and methylated within TSDR in all CD4+ lymphocyte subtypes, whereas the template strand follows the previously described pattern of methylation with both regions being more demethylated in Treg subtypes and mostly methylated in Tcon. This strand-specific approach within the TSDR may prove to be instrumental in correctly defining Treg subsets in health and in disease.

Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications.

BACKGROUND: Many biomedical applications require the expression or production of therapeutic hetero-multimeric proteins/protein complexes: in most cases only accomplished by co-ordinated co-expression within the same cell. Foot-and-mouth disease virus 2A (F2A) and '2A-like' sequences are now widely used for this purpose. Since 2A mediates a co-translational 'cleavage' at its own C-terminus, sequences encoding multiple proteins (linked via 2As) can be concatenated into a single ORF: a single transgene. It has been shown that in some cases, however, the cleavage efficiency of shorter versions of F2A may be inhibited by the C-terminus of certain gene sequences immediately upstream of F2A. This paper describes further work to optimise F2A for co-expression strategies. RESULTS: We have inserted F2A of various lengths in between GFP and CherryFP 'reporter' proteins (in reciprocal or tandem arrangements). The co-expression of these proteins and cleavage efficiencies of F2As of various lengths were studied by in vitro coupled transcription and translation in rabbit reticulocyte lysates, western blotting of HeLa cell lysates and fluorescence microscopy. CONCLUSIONS: Optimal and suboptimal lengths of F2A sequences were identified as a result of detailed 'fine-tuning' of the F2A sequence. Based on our data and the model according to which 2A activity is a product of its interaction with the exit tunnel of the ribosome, we suggest the length of the F2A sequence which is not 'sensitive' to the C-terminus of the upstream protein that can be successfully used for co-expression of two proteins for biomedical applications.

Protein coexpression using FMDV 2A: effect of "linker" residues.

Many biomedical applications absolutely require, or are substantially enhanced by, coexpression of multiple proteins from a single vector. Foot-and-mouth disease virus 2A (F2A) and "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A) are used widely for this purpose since multiple proteins can be coexpressed by linking open reading frames (ORFs) to form a single cistron. The activity of F2A "cleavage" may, however, be compromised by both the use of shorter versions of F2A and the sequences (derived from multiple-purpose cloning sites) used to link F2A to the upstream protein. To characterise these effects, different lengths of F2A and T2A were inserted between green and cherry fluorescent proteins. Mutations were introduced in the linker region immediately upstream of both F2A- and T2A-based constructs and activities determined using both cell-free translation systems and transfected cells. In shorter versions of F2A, activity may be affected by both the C-terminal sequence of the protein upstream and, equally strikingly, the residues immediately upstream introduced during cloning. Mutations significantly improved activity for shorter versions of F2A but could decrease activity in the case of T2A. These data will aid the design of cloning strategies for the co-expression of multiple proteins in biomedical/biotechnological applications.

Human coronavirus 229E papain-like proteases have overlapping specificities but distinct functions in viral replication.

Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PLpros). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/pp1ab region by two paralogous PLpro activities. Reverse-genetics data revealed that replacement of the PL2pro active-site cysteine was lethal. By contrast, the PL1pro activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1pro active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1pro activity. Further experiments showed that both PL1pro and PL2pro were able to cleave the nsp1-nsp2 cleavage site, with PL2pro cleaving the site less efficiently. The PL1pro-negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1pro mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PLpro paralogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2pro plays a universal and essential proteolytic role that appears to be assisted by the PL1pro paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed.

Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis.

Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'-->5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of approximately 8-12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.