Long COVID is a complex condition characterized by a broad spectrum of persistent, multisystemic symptoms that often impair daily activities and cause disability with significant socioeconomic impact. Although several hypotheses have been proposed to explain its pathophysiology—including immune dysregulation—the underlying mechanisms remain poorly understood. Indeed, there are no current treatments for this condition. Therefore, we aimed to characterize immune alterations induced by SARS-CoV-2 during the acute phase of infection and three months post-hospital discharge in a cohort of patients who later developed Long COVID, referred to a matched cohort who did not. We performed high-dimensional immune profiling of peripheral blood mononuclear cells using a 40-marker mass cytometry panel. Data analysis combined classical hierarchical gating strategies with unsupervised computational approaches. Our results revealed distinct immune signatures between Long COVID and non-Long COVID patients, affecting both the innate and adaptive immune compartments. Notably, individuals who subsequently developed Long COVID exhibited early abnormalities at infection in innate immunity, particularly involving dendritic cells and γδ T cells. Moreover, three months post-discharge, persistent alterations were observed in several adaptive immune cell subsets in patients who next developed Long COVID over time. In summary, our findings suggest that early immune dysregulation following viral infection may predispose individuals to persistent post-viral pathology.

BackgroundSystemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by loss of self-tolerance, causing inflammation and tissue damage in multiple organs. Although animal models have advanced our understanding of SLE’s molecular basis, recent regulatory changes and longstanding concerns regarding reproducibility and translatability have renewed the need to critically evaluate how these models mirror human disease. Understanding pathway-level similarities and differences between mouse models and human disease is essential, given the marked clinical and molecular heterogeneity of SLE.MethodsFour spontaneous SLE mouse models were studied: MRLlpr/lpr, NZB/W, BXSB.Yaa, and Tlr7.Tg6. Transcriptome sequencing from blood, spleen, and kidney; flow cytometry from the spleen; and cytokines and autoantibody measurement in plasma were performed at four time points. Similar molecular datasets from the human PRECISESADS SLE cohort were used for the integration.ResultsThe study identified specific molecular pathways driving the phenotype in each mouse model and established a framework describing the dynamics of these phenotype-associated molecular signatures, thereby facilitating the selection of time points of interest for future mouse-oriented experimental designs. In addition, by comparing these pathways with those observed in human SLE, we identified the most similar ones and their relationship with disease activity, providing crucial insight into their translational relevance. Importantly, disease severity across models was linked to both the extent and timing of molecular dysregulations. As expected, MRLlpr/lpr showed the most aggressive phenotype with early immune activation and apoptosis dysregulation, while Tlr7.Tg6 presented late-onset signatures associated with interferon and inflammation. Shared molecular features with human SLE included interferon responses, T and B cell depletion, and neutrophil activation. Integration analysis revealed distinct, yet overlapping, immune pathways between models and species, with some signatures such as age-associated B cells and double-negative memory T cells being model-specific but potentially relevant to early disease processes.ConclusionsThese findings provide a valuable framework for future SLE research and reinforce the utility of mouse models for studying specific molecular pathways related to human SLE pathogenesis and heterogeneity. The integration of longitudinal mouse and human molecular information highlights the models that best recapitulate key aspects of human disease, offering guidance for the study of specific immunopathological mechanisms or therapeutic targets.

Endogenous and exogenous antigen processing and presentation through the MHC class I peptide-loading complex (PLC) are essential for initiating cytotoxic T lymphocyte responses against pathogens and tumors. Tapasin, a key component of the PLC, is produced in multiple isoforms through alternative splicing, each isoform influencing the assembly and stability of MHC class I molecules differently. While the canonical Tapasin isoform plays a critical role in stabilizing MHC class I by facilitating optimal peptide loading in the endoplasmic reticulum (ER), the other isoforms function in distinct ways that impact immune regulation. This study aimed to investigate the role of Tapasin isoforms, particularly soluble isoform 3, in modulating antigen presentation and immune responses, focusing on their effects on MHC class I peptide loading and surface expression. Our findings show that isoforms 1 and 2 stabilize TAP and facilitate efficient peptide loading onto MHC class I in the ER, promoting optimal antigen presentation. In contrast, isoform 3, which lacks both the ER retention signal and the transmembrane domain, is secreted and acts as a negative regulator. Isoform 3 inhibits the loading of exogenous peptides onto MHC class I molecules at the cell surface, thereby playing a critical role in the spatial and temporal regulation of MHC class I antigen presentation. The secreted Tapasin isoform 3 likely regulates immune responses by preventing inappropriate T cell activation and cytotoxicity, which could otherwise lead to immune-mediated tissue damage and contribute to autoimmune disorders. Understanding the distinct functions of Tapasin isoforms provides insights into immune regulation and highlights the importance of fine-tuning peptide-loading processes to ensure proper immune responses and prevent immune-related pathologies.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that often affects the kidneys, causing lupus nephritis. Diagnosis of this affection currently relies on kidney biopsy, an invasive and complex procedure. This study explores the diagnostic value of biomarkers based in the urobiome – the microbial community of the urinary tract – in patients with renal SLE. This study enrolled 585 female subjects including Healthy controls, non-renal and renal SLE patients. The taxonomic and functional differences of the urobiome in patients with SLE, as well as in the metabolites of interest, were identified by 16S rRNA profiling with PICRUSt functional inference and nuclear magnetic resonance (NMR). The accuracy of the identified biomarkers was tested by building random forest (RF) classification models. Furthermore, the results were validated in an independent cohort composed by 30 controls, 30 non-renal and 30 renal SLE patients. Bacterial gene-based biomarkers with an AUC value of 0.7 ± 0.07 and 0.67 ± 0.07 to distinguish renal from non-renal SLE cases were identified. These biomarkers were validated in a validation cohort using quantitative PCR (qPCR), demonstrating their robust diagnostic performance. Furthermore, our analysis uncovered significant urobiome dysbiosis and distinct bacterial functional profile in both groups of SLE patients, with notable differences in amino acid metabolism pathways, particularly those involving valine and leucine, which were assessed by NMR-based urinary metabolite quantification. Some bacterial genes have been identified in the urobiome of SLE patients that allow differentiation between those with renal and non-renal lupus. These findings offer valuable insight into the association between the urobiome and SLE presentation, and lay the foundation for developing novel diagnostic tools that overcome the limitations of current methods, thereby improving patient care.

Long-term consequences of SARS-CoV-2 infection are unknown since recovered individuals can experience symptoms and latent viral reactivation for months. Indeed, acute post-infection sequelae have also been observed in other respiratory viral infections, including influenza. To characterize post-COVID-19 and post-influenza induced alterations to the cellular immunome, peripheral blood mononuclear cells (PBMCs) were obtained from patients 3 months after recovery from COVID-19 (n = 93) or influenza (n = 25), and from pre-pandemic healthy controls (n = 25). PBMCs were characterized using a 40-plex mass cytometry panel. Principal component analysis (PCA), classification models, and K-means clustering were subsequently applied. PCA identified distinct immune profiles between cohorts, with both post-COVID and post-flu patients displaying an altered chemokine receptor expression compared to pre-pandemic healthy controls. These alterations were more prominent in post-COVID patients since they exhibited highly increased expression of chemokine receptors CXCR3 and CCR6 by various lymphoid populations, while post-influenza patients mainly showed a decrease in CCR4 expression by naïve T cells, monocytes, and conventional dendritic cells. Classification models using immunophenotyping data confirm the three groups, while K-means clustering revealed two subgroups among post-COVID patients, with younger patients showing more pronounced immune alterations in the chemokine receptor profile, independently of long COVID symptoms. In conclusion, post-COVID and post-influenza patients exhibit distinct and unique persistent immune alterations. Understanding these altered immune profiles can guide targeted therapies for post-COVID syndrome and highlight differences in immune recovery from various respiratory infections.

Background Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by a loss of self-tolerance, causing inflammation and tissue damage in multiple organs. Animal models have advanced our understanding of SLE’s molecular basis, but the FDA’s recent elimination of animal testing requirements for drug approval has raised concerns about their validity, prompting a reevaluation of their role in basic research, especially for heterogeneous diseases like SLE. Methods Four different spontaneous SLE mouse models were studied: MRL lpr/lpr , NZB/W, BXSB. Yaa , and Tlr7.Tg6. Transcriptome sequencing from blood, spleen, and kidney, flow cytometry from the spleen, and cytokines and autoantibody measurement in plasma were performed at four time points. Similar molecular data from human SLE patients was used for the integration. Results The study identified specific molecular pathways driving the phenotype in each mouse model and established optimal time points for future experimental designs. By comparing these pathways to human SLE, the most similar ones and their relationship with disease activity were identified, providing crucial insight into translational relevance. Importantly, disease severity across models was linked to the extent and timing of molecular dysregulations. As expected, MRL lpr/lpr showed the most aggressive phenotype with early immune activation and apoptosis dysregulation, while Tlr7.Tg6 presented late-onset signatures associated with interferon and inflammation. Shared molecular features with human SLE included interferon responses, T and B cell depletion, and neutrophil activation. Integration analysis revealed distinct yet overlapping immune pathways between models and species, with some signatures such as age-associated B cells and double-negative memory T cells being model-specific but potentially relevant to early disease processes. Conclusions These findings build a valuable framework for future SLE research, reinforcing the utility of mouse models in studying specific molecular pathways related to human SLE pathogenesis and heterogeneity. The integration of longitudinal mouse data with human transcriptomes highlights the models that best recapitulate key aspects of human disease, offering guidance for the study of specific immunopathological mechanisms or therapeutic targets. ### Competing Interest Statement Authors Makowska, Kageyama, Buttgereit, Lesche, and McDonald were employees of Bayer at the time of the study. * ABCs : Age-associated B cells ACR : Albumin-to-creatinine ratio ANAs : Antinuclear antibodies anti-CENP-B : Anti-centromere protein B anti-dsDNA : Anti-double-stranded DNA anti-RNP : Anti-ribonucleoprotein anti-Sm : Anti-Smith antigen anti-SSA/La : Anti-Sjögren’s-syndrome-related antigen A anti-SSB/Ro : Anti-Sjögren’s-syndrome-related antigen B CXCL-1 : Chemokine (C-X-C motif) ligand 1 DN T cells : Double-negative T cells DP T cells : Double-positive T cells EDTA : Ethylenediaminetetraacetic acid ENA-78 : Epithelial-derived neutrophil-activating peptide 78 FDA : Food and Drug Administration FDR : False discovery rate G-CSF : Granulocyte colony-stimulating factor GM-CSF : Granulocyte-macrophage colony-stimulating factor ![Graphic][1] : Growth-related oncogene-alpha GSEA : Gene Set Enrichment Analysis ![Graphic][2] : Interferon-alpha IFN-J : Interferon-gamma IgG : Immunoglobulin G IgM-IgD-B cells : Class-switched B cells IL : Interleukin IP-10 : Interferon gamma-induced protein 10 MCP-1 : Monocyte chemoattractant protein-1 MCP-3 : Monocyte chemoattractant protein-3 M-CSF : Monocyte-colony stimulating factor ![Graphic][3] : Macrophage inflammatory protein-1 alpha MIP-1ß : Macrophage inflammatory protein-1 beta MIP-2 : Macrophage inflammatory protein-2 MRL : Murphy Roths Large MZ B cells : Marginal zone B cells NZB : New Zealand Black NZW : New Zealand White PBS : Phosphate-buffered saline PCs : Plasma cells pDCs : Plasmacytoid dendritic cells RANTES : Regulated upon activation, normal T cell expressed and secreted SLE : Systemic lupus erythematosus T3 B cells : Transitional T3 B cells Tmem : Memory T cells TN : Naïve T cells [inline] : Tumor necrosis factor-alpha Innovative Medicines Initiative Joint Undertaking, 115565 Innovative Medicines Initiative 2 Joint Undertaking, 831434 MICINN, Juan de la Cierva-Incorporación, IJC2020-043364-I [1]: /embed/inline-graphic-3.gif [2]: /embed/inline-graphic-4.gif [3]: /embed/inline-graphic-5.gif

There is increasing knowledge in the recognition of individuals at risk for progression to rheumatoid arthritis (RA) before the clinical manifestation of the disease. This prodromal phase preceding the manifestation of RA may represent a “window of opportunity” for preventive interventions that may transform the clinical approach to this disease. However, limited evidence exists in support of effective interventions to delay the onset or even halt the manifestation of RA. Given the multifactorial nature of RA development and disease progression, the latest guidelines for established RA stress the use of integrative interventions and multidisciplinary care strategies, combining pharmacologic treatment with non-pharmacological approaches. Accordingly, individuals at risk of RA could be offered an integrative, multifactorial intervention approach. Current data point toward pharmacological intervention reverting the subclinical inflammation and delay in the disease onset. In addition, targeting life style modifiable factors (smoking cessation, dental health, physical activity, and diet) may presumably improve RA prognosis in individuals at risk, mainly by changes in epigenetics, autoantibodies, cytokines profiles, and microbiome. Nonetheless, the benefits of multidisciplinary interventions to halt the manifestation of RA in at-risk individuals remain unknown. As there is a growing knowledge of possible pharmacological intervention in the preclinical phase, this narrative review aims to provide a comprehensive overview of non-pharmacological treatments in individuals at risk of RA. Considering the mechanisms preceding the clinical manifestation of RA we explored all aspects that would be worth modifying and that would represent an integrative non-pharmacological care for individuals at risk of RA.

1 Abstract Background Systemic autoimmune diseases (SADs) are characterized by internal heterogeneity, overlapping clinical symptoms, and shared molecular pathways. Therefore, they are difficult to diagnose and new tools allowing precise diagnosis are needed. Molecular-based reclassification studies enable to find patterns in a diagnosis-independent way. Objective To evaluate the possibility of using high-content immunophenotyping for detecting patient subgroups in the context of precise treatment. Methods Whole blood high-content immunophenotyping of 101 patients with 7 systemic autoimmune diseases and 22 controls was performed using 36-plex mass cytometry panel. Patients were compared across diagnostic entities and re-classified using Monte Carlo reference-based consensus clustering. Levels of 45-plex multiplexed cytokine were measured and used for cluster characterization. Results Differential analysis by diagnosis did not reveal any disease-specific pattern in the cellular compositions and phenotypes but rather their relative similarities. Accordingly, patients were classified into phenotypically distinct groups composed of different diagnostic entities sharing common immunophenotypes and cytokine signatures. These features were mainly based on granulocyte activation and CD38 expression in discrete lymphocyte populations and were related to Th17 or IFN-dependent cytokines. Conclusions Our data indicate that specific individuals could potentially benefit from the same line of treatment independently of their diagnosis and emphasize the possibility of using immunophenotyping as a stratification tool in precision rheumatology. 2 Graphical abstract Key messages Whole blood immmunophenotyping could be used to stratify systemic autoimmune patients, thus it is a useful tool in precision medicine. Patients’ groups could benefit from the same line of treatment.

Lupus nephritis (LN) represents one of the most severe complications of systemic lupus erythematosus, leading to end-stage kidney disease in worst cases. Current first-line therapies for LN, including mycophenolate mofetil (MMF) and azathioprine (AZA), fail to induce long-term remission in 60-70% of the patients, evidencing the urgent need to delve into the molecular knowledge-gap behind the non-response to these therapies. A longitudinal cohort of treated LN patients including clinical, cellular and transcriptomic data, was analyzed. Gene-expression signatures behind non-response to different drugs were revealed by differential expression analysis. Drug-specific non-response mechanisms and cell proportion differences were identified. Blood cell subsets mediating non-response were described using single-cell RNASeq data. We show that AZA and MMF non-response implicates different cells and regulatory functions. Mechanistic models were used to suggest add-on therapies to improve their current performance. Our results provide new insights into the molecular mechanisms associated with treatment failures in LN.

The heterogeneity of systemic lupus erythematosus (SLE) can be explained by epigenetic alterations that disrupt transcriptional programs mediating environmental and genetic risk. This study evaluated the epigenetic contribution to SLE heterogeneity considering molecular and serological subtypes, genetics and transcriptional status, followed by drug target discovery. We performed a stratified epigenome-wide association studies of whole blood DNA methylation from 213 SLE patients and 221 controls. Methylation quantitative trait loci analyses, cytokine and transcription factor activity - epigenetic associations and methylation-expression correlations were conducted. New drug targets were searched for based on differentially methylated genes. In a stratified approach, a total of 974 differential methylation CpG sites with dependency on molecular subtypes and autoantibody profiles were found. Mediation analyses suggested that SLE-associated SNPs in the HLA region exert their risk through DNA methylation changes. Novel genetic variants regulating DNAm in disease or in specific molecular contexts were identified. The epigenetic landscapes showed strong association with transcription factor activity and cytokine levels, conditioned by the molecular context. Epigenetic signals were enriched in known and novel drug targets for SLE. This study reveals possible genetic drivers and consequences of epigenetic variability on SLE heterogeneity and disentangles the DNAm mediation role on SLE genetic risk and novel disease-specific meQTLs. Finally, novel targets for drug development were discovered.

The immune cellular landscape from the gastric mucosa remains largely unknown despite its relevance in several inflammatory conditions. Human gastric biopsies were obtained from the antrum, body and incisura from 10 individuals to obtain lamina propria mononuclear cells that were further characterized by spectral cytometry. Phenotypic hierarchical analyses identified a total of 52 different immune cell subsets within the human gastric mucosa revealing that T-cells (> 60%) and NK cells (> 20%) were the main populations. Within T-cells, CD4+ and CD8+ were equally represented with both subsets displaying mainly a memory and effector phenotype. NK cells, on the contrary, were largely of the early phenotype. No regional differences were observed for any subsets among the 3 locations. Following unsupervised analysis, a total of 82 clusters were found. Again, no differences were observed amongst locations although a great degree of inter-individual variability was found, largely influenced by the presence of H. pylori infection and dyspepsia. We have unraveled the human gastric immune cellular subset composition and a unique interindividual immune fingerprint with no inter-regional variations.

Clinical studies are conducted to better understand the pathological mechanism of diseases and to find biomarkers associated with disease activity, drug response, or outcome prediction. Mass cytometry (MC) is a high-throughput single-cell technology that measures hundreds of cells per second with more than 40 markers per cell. Thus, it is a suitable tool for immune monitoring and biomarker discovery studies. Working in translational and clinical settings requires a careful experimental design to minimize, monitor, and correct the variations introduced during sample collection, preparation, acquisition, and analysis. In this review, we will focus on these important aspects of MC-related experiments and data curation in the context of translational clinical research projects.

ABSTRACT Objective The heterogeneity of systemic lupus erythematosus (SLE) can be explained by epigenetic alterations that disrupt transcriptional programs mediating environmental and genetic risk. This study evaluated the epigenetic contribution to SLE heterogeneity considering molecular and serological subtypes, genetics and transcriptional status, followed by drug target discovery. Methods We performed a stratified epigenome-wide association studies of whole blood DNA methylation from 213 SLE patients and 221 controls. Methylation quantitative trait loci analyses, cytokine and transcription factor activity - epigenetic associations and methylation-expression correlations were conducted. New drug targets were searched for based on differentially methylated genes. Results In a stratified approach, a total of 974 differential methylation CpG sites with dependency on molecular subtypes and autoantibody profiles were found. Mediation analyses suggested that SLE-associated SNPs in the HLA region exert their risk through DNA methylation changes. Novel genetic variants regulating DNAm in disease or in specific molecular contexts were identified. The epigenetic landscapes showed strong association with transcription factor activity and cytokine levels, conditioned by the molecular context. Epigenetic signals were enriched in known and novel potential drug targets for SLE. Conclusion This study expands the number of genes associated with SLE and reveals novel pathways of disease. The findings reveal possible genetic drivers and consequences of epigenetic variability on SLE heterogeneity and disentangles the DNAm mediation role on SLE genetic risk and the genetic architecture of DNAm in different molecular contexts. Finally, novel targets for drug development were discovered.

Background Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease that leads to significant worsening of quality of life and mortality. The enormous molecular heterogeneity of SLE is reflected in different clinical manifestations, disease progression and also in a different drug efficacy across patients[1]. Lupus nephritis (LN) is the most severe SLE manifestation with the potential of rapidly evolving into irreversible chronic kidney disease and kidney failure if not adequately followed and treated. Mycophenolate mofetil (MMF) is the most widely used first-line treatment for LN, being ineffective or partially effective in 15-30 percent of the patients[2]. Reasons for non-response are still unknown and low or moderate drug efficacy may lead to aggravation of the disease. Treat-to-target approaches where personalized molecular patterns guide therapeutic decisions, are rapidly growing in medical fields such as oncology, but remain unmet within clinical rheumatology[3]. In addition, pathological molecular dysregulation behind SLE fluctuates within a non-linear clinical course and unpredictable patterns of flares and remissions, hindering the development of effective and robust predictive biomarkers for both diagnosis and drug responsiveness[4]. Objectives The development of new, more effective therapies to treat LN is an urgent unmet need. The objective of this work is to define the cellular and molecular immune landscape behind non-response to MMF to finally apply this knowledge within routine clinical practice. Methods A longitudinal cohort comprising gene-expression and clinical data of 97 MMF responder and 28 non-responder blood samples was retrospectively analyzed. Differential gene expression and functional analysis were performed. Response rate was measured based on blood cell proportions. Single-cell RNA sequencing data was analyzed to identify the cell subtypes influencing non-response and their contributing genes and regulation mechanisms. Results A robust signature comprising 41 differentially expressed genes defined non-response to MMF and cellular profiles that favor the response to the drug in the patients were revealed. The response rate to MMF increases the lower the B cell/T cell ratio. Single-cell RNA sequencing showed that overexpression of the IFITM-family of genes in age-associated B cells, plasma cells, myeloid dendritic cells and macrophages was behind the non-response signature. Conclusion Blood cell subtypes and genes mediating non-response to MMF were revealed, opening a new scenario for the development of new therapeutic strategies for LN. References [1]Toro-Domínguez D, Martorell-Marugán J, Martinez-Bueno M, López-Domínguez R, Carnero-Montoro E, Barturen G, et al. Scoring personalized molecular portraits identify Systemic Lupus Erythematosus subtypes and predict individualized drug responses, symptomatology and disease progression. Brief Bioinform. 2022 Sep 20;23(5):bbac332. [2]Chan TM, Li FK, Tang CSO, Wong RWS, Fang GX, Ji YL, et al. Efficacy of Mycophenolate Mofetil in Patients with Diffuse Proliferative Lupus Nephritis. N Engl J Med. 2000 Oct 19;343(16):1156–62. [3]Pitzalis C, Choy EHS, Buch MH. Transforming clinical trials in rheumatology: towards patient-centric precision medicine. Nat Rev Rheumatol. 2020 Oct;16(10):590–9. [4]Guthridge JM, Wagner CA, James JA. The promise of precision medicine in rheumatology. Nat Med. 2022 Jul;28(7):1363–71. Acknowledgements: NIL. Disclosure of Interests None Declared.

A recommendation of: Moana Peylhard, David Berthier, Guiguigbaza-Kossigan Dayo, Isabelle Chantal, Souleymane Sylla, Sabine Nidelet, Emeric Dubois, Guillaume Martin, Guilhem Sempéré, Laurence Flori, Sophie Thévenon Whole blood transcriptome profiles of trypanotolerant and trypanosusceptible cattle highlight a differential modulation of metabolism and immune response during infection by Trypanosoma congolense https://doi.org/10.1101/2022.06.10.495622

With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.

Summary Background Type I IFN (IFN-I) is a family of cytokines involved in the pathogenesis of autoimmune and autoinflammatory diseases such as psoriasis. SIDT1 is an ER-resident protein expressed in the lymphoid lineage, and involved in anti-viral IFN-I responses in vivo, through an unclear mechanism. Herein we have dissected the role of SIDT1 in the natural IFN-producing cells, the plasmacytoid dendritic cells (pDC). Methods The function of SIDT1 in pDC was determined by silencing its expression in human primary pDC and GEN2.2 cell line. SIDT1 role in vivo was assessed using the imiquimod-induced psoriasis model in the SIDT1-deficient mice (sidt1-/-). Findings Silencing of SIDT1 in GEN2.2 led to a blockade of the IFN-I response after stimulation of TLR7 and TLR9, without affecting the pro-inflammatory responses or upregulation of maturation markers. We found that SIDT1 migrates from the ER to the endosomal and lysosomal compartments together with TLR9 after CpG stimulation, participating in the access of the TLR9-CpG complex to lysosome-related vesicles, and therefore mediating the activation of TBK1 and the nuclear migration of IRF7, but not of NF -KB. sidt1-/- mice showed a significant decrease in severity parameters of the imiquimod-induced acute psoriasis-like model, associated with a decrease in the production of IFN-I and IFN-dependent chemokines. Interpretation Our findings indicate that SIDT1 is at the cross-road between the IFN-I and the proinflammatory pathways and constitutes a promising drug target for psoriasis and other diseases mediated by IFN-I responses. Funding This work was supported by the Consejer?a de Salud y Familias de la Junta de Andaluc?a (PIER_S1149 and C2_S0050) and Instituto de Salud Carlos III (PI18/00082 and PI21/01151), partly supported by European FEDER funds, and prior funding to MEAR from the Alliance for Lupus Research and the Swedish Research Council. Copyright (c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) eBioMedicine 103808 Published uary https://doi.org/10.1016/j. ebiom.2021.103808 Superscript/Subscript Available

Systemic lupus erythematosus (SLE) patients display an increased risk of cardiovascular disease (CVD). With the improved clinical management of other classical severe manifestation of the disease, CVD is becoming one of the most relevant complications of SLE, and it is an important factor causing morbidity and mortality. Several immune constituents have been shown to be involved in the pathogenesis of atherosclerosis and endothelial damage in SLE patients, including specific circulating cell populations, autoantibodies, and inflammatory mediators. In this review, we summarize the presentation of CVD in SLE and the role of the autoimmune responses present in SLE patients in the induction of atherogenesis, endothelial impairment and cardiac disease. Additionally, we discuss the utility of these immune mediators as early CVD biomarkers and targets for clinical intervention in SLE patients.

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.

BackgroundSystemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSC), Sjögren’s syndrome (SJS), mixed connective tissue disease (MCTD), primary antiphospholipid syndrome (PAPS) and undifferentiated connective tissue disease (UCTD) are classified as systemic autoimmune diseases (SADs). They are diagnosed based on different clinical and laboratory criteria. Due to their high internal heterogeneity and overlapping symptoms, SADs are difficult to diagnose. Therefore, molecular and cellular-based studies need to be undertaken to precisely classify the patients. Mass cytometry is a single-cell proteomics technology that measures approximately 50 markers per cell, thus it is a suitable tool to perform deep-phenotyping studies in SADs.ObjectivesExplore differences and similarities between SADs and build reclassification framework using high-dimensional cytometry data.MethodsThe whole blood samples collected from 129 individuals, including patients and controls were stained with a 39-plex antibody panel and acquired in 9 batches on a CyTOF (HELIOS) instrument. Data were cleaned, and normalized for batch effects using semi-automated cytof analysis pipeline. Cell frequencies and median signal intensities (MSI) for each population were extracted using FlowSOM for mononuclear cells (PBMC) and Phenograph for granulocytes. Secretion of 44 cytokines and chemokines were analyzed using a multiplexed luminex assay. Diseases were compared by Kruskal-Wallis analysis and hierarchical clustering and reclassification was done using unsupervised k-means clustering. Cytokine analysis across clusters was performed using Kruskal-Wallis test.ResultsDifferently expressed features were observed between patient groups, regarding frequency of classical monocytes, B and T cells subpopulations, mature and immature granulocytes and intensities of CD38, HLA-DR and CD95 across various populations. However, none of them were disease specific. K-means clustering identified four patient clusters, which were composed by a mixture of different diagnosis. Cluster C1 was characterized by increased levels of circulating cells from PBMC compartment, and lower activation of different populations of the T cell compartment. It presented lower frequency in multiple granulocyte populations and the highest expression of CD95 and CD38. This cluster was also associated with antimalarial and steroid treatment. Clusters C1 and C2 were exactly opposite to each other, cluster C3 was characterized by intermediate features between C1 and C2 and cluster C4 could be considered as undifferentiated, mixed group. Higher production of TNFα, IL-10 and IP-10 were found in patients from C1 compared to C2, suggesting more active phenotype in C1 and physiological one in C2. The cytokine levels were independent of the treatment.ConclusionWe constructed a patient reclassification framework using cell frequencies and expression levels of functional markers. To our knowledge this is the first time when 7 different SADs were compared using mass cytometry. In agreement with other reports we did not detect any disease-specific cellular markers. Distribution of diagnosis across different clusters confirms diseases heterogeneity. Patients can be classified into phenotypically similar groups, that could potentially benefit from the same line of treatment.AcknowledgementsThis project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831434 (3TR) and The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. Also from No 115565 PRECISESADS.Disclosure of InterestsNone declared