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Objective: Neonatal lupus erythematosus (NLE) may develop after transplacental transfer of maternal autoantibodies with cardiac manifestations (congenital heart block, CHB) including atrioventricular block, atrial and ventricular arrhythmias, and cardiomyopathies. The association with anti-Ro/SSA antibodies is well established, but a recurrence rate of only 12%-16% despite persisting maternal autoantibodies suggests that additional factors are required for CHB development. Here, we identify fetal genetic variants conferring risk of CHB and elucidate their effects on cardiac function.Methods: A genome-wide association study was performed in families with at least one case of CHB. Gene expression was analysed by microarrays, RNA sequencing and PCR and protein expression by western blot, immunohistochemistry, immunofluorescence and flow cytometry. Calcium regulation and connectivity were analysed in primary cardiomyocytes and cells induced from pleuripotent stem cells. Fetal heart performance was analysed by Doppler/echocardiography.Results: We identified DNAJC6 as a novel fetal susceptibility gene, with decreased cardiac expression of DNAJC6 associated with the disease risk genotype. We further demonstrate that fetal cardiomyocytes deficient in auxilin, the protein encoded by DNAJC6, have abnormal connectivity and Ca2+ homoeostasis in culture, as well as decreased cell surface expression of the Cav1.3 calcium channel. Doppler echocardiography of auxilin-deficient fetal mice revealed cardiac NLE abnormalities in utero, including abnormal heart rhythm with atrial and ventricular ectopias, as well as a prolonged atrioventricular time intervals.Conclusions: Our study identifies auxilin as the first genetic susceptibility factor in NLE modulating cardiac function, opening new avenues for the development of screening and therapeutic strategies in CHB. [ABSTRACT FROM AUTHOR]

Summary: TRIM21 is an interferon‐stimulated E3 ligase that controls the activity of pattern‐recognition signaling via ubiquitination of interferon regulatory factors and DDX41. Previous studies on the role of TRIM21 in innate immune responses have yielded contradictory results, suggesting that the role of TRIM21 is cell specific. Here, we report that bone‐marrow‐derived macrophages (BMDMs) generated from Trim21−/− mice have reduced expression of mature macrophage markers. Reflecting their reduced differentiation in response to macrophage colony‐stimulating factor (M‐CSF), Trim21−/− BMDMs had decreased expression of M‐CSF signature genes. Although Trim21−/− BMDMs responded normally to Toll‐like receptor 9 (TLR9) activation, they produced lower levels of pro‐inflammatory cytokines in response to the TLR2 agonist PAM3CSK4. In line with this, the response to infection with the Bacillus Calmette–Guérin strain of Mycobacterium bovis was also diminished in Trim21−/− BMDMs. Our results indicate that TRIM21 controls responses to TLR2 agonists. [ABSTRACT FROM AUTHOR]

Systemic autoimmune diseases are characterized by the overexpression of type I IFN stimulated genes, and accumulating evidence indicate a role for type I IFNs in these diseases. However, the underlying mechanisms for this are still poorly understood. To explore the role of type I IFN regulated miRNAs in systemic autoimmune disease, we characterized cellular expression of miRNAs during both acute and chronic type I IFN responses. We identified a T cell‐specific reduction of miR‐31‐5p levels, both after intramuscular injection of IFNβ and in patients with Sjögren's syndrome (SjS). To interrogate the role of miR‐31‐51p in T cells we transfected human CD4+ T cells with a miR‐31‐5p inhibitor and performed metabolic measurements. This identified an increase in basal levels of glucose metabolism after inhibition of miR‐31‐5p. Furthermore, treatment with IFN‐α also increased the basal levels of human CD4+ T‐cell metabolism. In all, our results suggest that reduced levels of miR‐31‐5p in T cells of SjS patients support autoimmune T‐cell responses during chronic type I IFN exposure. Quiescent and naive T cells depend on oxidative phosphorylation but switch to glycolysis during their effector stage. We identified miR‐31‐5p as a type I IFN‐suppressed miRNA that negatively regulates glycolysis in human CD4+ T cells. This suggests that miR‐31‐5p regulates T‐cell metabolism in autoimmune diseases with type I IFN signatures. [ABSTRACT FROM AUTHOR]

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 × 10 8 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes., Competing Interests: B.J.S., S.B., F.F., L.H.W., and R.S.P. are or were employed by and have an equity interest in Indee Labs. L.M.R.F., J.B., L.L., F.L.P. and J.E. are or were consultants to Indee Labs. R.S.P. is an investor in and a venture partner at both Pioneer Fund and Axial, which have a financial interest in Indee Labs. The Ferreira Lab received support from Indee Labs as a subaward from the National Institute of Diabetes and Digestive and Kidney Diseases (Grant No. 1R43DK133029-01). Indee Labs has a commercial interest in developing patents related to Hydropore™ (WO2016109864A1 & WO2019084624A1). M.F. and R.B. are or were employed by and have an equity interest in CellChorus. CellChorus received support from the National Center for Translational Sciences (R44TR005137) and the National Institute of General Medical Sciences (R44GM149106) of the National Institutes of Health and the National Science Foundation (NSF2229323). CellChorus has a commercial interest in developing the TIMING assay. J.E. is a compensated co-founder at Mnemo Therapeutics; owns stocks in Mnemo Therapeutics and Cytovia Therapeutics; is a compensated scientific advisor for Enterome, Treefrog Therapeutics and Resolution Therapeutics; and is a holder of patents pertaining to but not resulting from this work. The Eyquem Lab received research support from Cytovia Therapeutic, Mnemo Therapeutics, Takeda and Indee Labs as a subaward from the National Cancer Institute (Contract No. 7591022C00053).

Discovering the role of fibroblasts residing in the tumor microenvironment (TME) requires controlled, localized perturbations because fibroblasts play critical roles in regulating immunity and tumor biology at multiple sites. Systemic perturbations can lead to unintended, confounding secondary effects, and methods to locally genetically engineer fibroblasts are lacking. To specifically investigate murine stromal cell perturbations restricted to the TME, we developed an adeno-associated virus (AAV)-based method to target any gene-of-interest in fibroblasts at high efficiency (>80%). As proof of concept, we generated single (sKO) and double gene KOs (dKO) of Osmr , Tgfbr2 , and Il1r1 in cancer-associated fibroblasts (CAFs) and investigated how their cell states and those of other cells of the TME subsequently change in mouse models of melanoma and pancreatic ductal adenocarcinoma (PDAC). Furthermore, we developed an in vivo knockin-knockout (KIKO) strategy to achieve long-term tracking of CAFs with target gene KO via knocked-in reporter gene expression. This validated in vivo gene editing toolbox is fast, affordable, and modular, and thus holds great potential for further exploration of gene function in stromal cells residing in tumors and beyond., Competing Interests: M.F.K. is a founder and shareholder of FOUNDERY innovations. J.E. is a compensated co-founder at Mnemo Therapeutics and a compensated scientific advisor to Cytovia Therapeutics. J.E. owns stocks in Mnemo Therapeutics and Cytovia Therapeutics. J.E. has received a consulting fee from Casdin Capital, Resolution Therapeutics and Treefrog Therapeutics. The J.E. lab has received research support from Cytovia Therapeutics, Mnemo Therapeutics, and Takeda Pharmaceutical Company.