Objective: We have previously shown, in a cohort of untreated rheumatoid arthritis (RA) patients, that the suppressive function of Treg cells is defective. However, other studies in cohorts of patients with established RA have shown that Treg cell function is normal. We hypothesized that treatment may restore Treg cell function and lead to reduced disease activity. The aim of this study was to investigate whether treatment with methotrexate (MTX) can result in epigenetic changes that lead to restoration of the Treg cell suppressive function in RA.Methods: Peripheral blood samples from RA patients were assessed using ³H‐thymidine incorporation to measure Treg cell suppression of T cell proliferation, and by enzyme‐linked immunosorbent assay to determine Treg cell suppression of interferon‐γ production. CTLA‐4 and FoxP3 expression was measured by flow cytometry and quantitative polymerase chain reaction (qPCR) in Treg cells from healthy individuals and RA patients. CD4+ T cells isolated from healthy individuals were cultured with interleukin‐2 (IL‐2), IL‐6, and tumor necrosis factor α in the presence or absence of MTX, and FoxP3 expression was determined using qPCR and flow cytometry. Methylation of the FOXP3 upstream enhancer was analyzed by bisulfite sequencing PCR.Results: Defective Treg cell function was observed only in RA patients who had not been treated with MTX, whereas Treg cells from MTX‐exposed RA patients had restored suppressive function. This restored suppression was associated with increased expression of FoxP3 and CTLA‐4 in Treg cells. Bisulfite sequencing PCR of Treg cells cultured in MTX revealed a significant reduction in methylation of the FOXP3 upstream enhancer.Conclusion: This study identifies a novel mechanism of action of MTX, in which treatment of RA patients with MTX restores defective Treg cell function through demethylation of the FOXP3 locus, leading to a subsequent increase in FoxP3 and CTLA‐4 expression.

Objective: Functionally impaired Treg cells expressing abnormally low levels of CTLA‐4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA‐4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function.Methods: CTLA‐4 expression in Treg cells from RA patients and healthy controls was measured by quantitative polymerase chain reaction (PCR) and flow cytometry. Methylation of the CTLA‐4 gene promoter was analyzed by bisulfite‐specific PCR, followed by sequencing. Methylation‐dependent transcriptional activity of the CTLA‐4 gene promoter was measured by luciferase assay, and NF‐AT binding to the CTLA‐4 gene promoter was determined by chromatin immunoprecipitation. The role of CTLA‐4 expression in controlling Teff cells was analyzed using an autologous mixed lymphocyte reaction.Results: Down‐regulation of CTLA‐4 expression in Treg cells from RA patients was caused by methylation of a previously unidentified NF‐AT binding site within the CTLA‐4 gene promoter. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan‐degrading enzyme indoleamine 2,3‐dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway.Conclusion: We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn²⁺ in modulating B cell receptor signal strength and positive selection.

Here we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.

Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming.

Significance: Inflammation is an important component of the pathogenesis of cardiovascular disease, the world’s biggest killer. No antiinflammatory treatments have yet been developed to treat cardiovascular disease. Indoleamine 2,3-dioxygenase (IDO) is a critical enzyme in the metabolism of tryptophan that has been shown to be immune-regulatory in many diseases. ApoE^−/−mice deficient in IDO (ApoE^−/−Indo^−/−) developed larger atherosclerotic lesions and an unfavorable lesion phenotype that may predispose to cardiovascular complications. Furthermore, administration of an orally active synthetic tryptophan metabolite (3,4-DAA) reduced disease development in mice and cytokine production in human atheroma. Our data demonstrate that endogenous production of tryptophan metabolites via IDO is an essential feedback loop that controls atherogenesis and athero-inflammation, defining a path toward the development of new therapeutics.