B cell generation of autoantibodies is a crucial step in the pathogenesis of systemic lupus erythematosus (SLE). After their differentiation in the bone marrow, B cells populate the secondary ...lymphatic organs, where they undergo further maturation leading to the development of memory B cells as well as antibody‐producing plasmablasts and plasma cells. Targeting B cells is an important strategy to treat autoimmune diseases such as SLE, in which B cell tolerance is disturbed and autoimmune B cells and autoantibodies emerge. This review discusses the functional aspects of antibody‐ and cell‐based B cell–depleting therapy in SLE. It thereby particularly focuses on lessons learned from chimeric antigen receptor (CAR) T cell treatment on the role of B cells in SLE for understanding B cell pathology in SLE. CAR T cells model a deep B cell depletion and thereby allow understanding the role of aberrant B cell activation in the pathogenesis of SLE. Furthermore, the effects of B cell depletion on autoantibody production can be better described, ie, explaining the concept of different cellular sources of (auto‐) antibodies in the form of short‐lived plasmablasts and long‐lived plasma cells, which differ in their susceptibility to B cell depletion and require different targeted therapeutic approaches. Finally, the safety of deep B cell depletion in autoimmune disease is discussed.
Objective
Findings from recent transcriptome analyses of the synovium of patients with rheumatoid arthritis (RA) have revealed that 15‐fold expanded HLA–DR+CD90+ synovial fibroblasts potentially act ...as key mediators of inflammation. The reasons for the expansion of HLA–DR+CD90+ synovial fibroblasts are unclear, but genetic signatures indicate that interferon‐γ (IFNγ) plays a central role in the generation of this fibroblast subset. The present study was undertaken to investigate the generation, function and therapeutically intended blockage of HLA–DR+CD90+ synovial fibroblasts.
Methods
We combined functional assays using primary human materials and focused bioinformatic analyses of mass cytometry and transcriptomics patient data sets.
Results
We detected enriched and activated Fcγ receptor type IIIa–positive (CD16+) NK cells in the synovial tissue from patients with active RA. Soluble immune complexes were recognized by CD16 in a newly described reporter cell model, a mechanism that could be contributing to the activation of natural killer (NK) cells in RA. In vitro, NK cell–derived IFNγ induced HLA–DR on CD90+ synovial fibroblasts, leading to an inflammatory, cytokine‐secreting HLA–DR+CD90+ phenotype. HLA–DR+CD90+ synovial fibroblasts consecutively activated CD4+ T cells upon receptor crosslinking via superantigens. HLA–DR+CD90+ synovial fibroblasts also activated CD4+ T cells in the absence of superantigens, an effect that was initiated by NK cell–derived IFNγ and that was 4 times stronger in patients with RA compared to patients with osteoarthritis. Finally, JAK inhibition in synovial fibroblasts prevented HLA–DR induction and blocked proinflammatory signals to T cells.
Conclusion
The HLA–DR+CD90+ phenotype represents an activation state of synovial fibroblasts during the process of inflammation in RA that can be induced by IFNγ, likely generated from infiltrating leukocytes such as activated NK cells. The induction of these proinflammatory, interleukin‐6–producing, and likely antigen‐presenting synovial fibroblasts can be targeted by JAK inhibition.
Neutrophils are versatile innate effector cells essential for immune defense but also responsible for pathologic inflammation. This dual role complicates therapeutic targeting. However, neither ...neutrophils themselves nor the mechanisms they employ in different forms of immune responses are homogeneous, offering possibilities for selective intervention. Here we review heterogeneity within the neutrophil population as well as in the pathways mediating neutrophil recruitment to inflamed tissues with a view to outlining opportunities for therapeutic manipulation in inflammatory disease.
Neutrophils are implicated in multiple homeostatic and pathological processes, but whether functional diversity requires discrete neutrophil subsets is not known. Here, we apply single-cell RNA ...sequencing to neutrophils from normal and inflamed mouse tissues. Whereas conventional clustering yields multiple alternative organizational structures, diffusion mapping plus RNA velocity discloses a single developmental spectrum, ordered chronologically. Termed here neutrotime, this spectrum extends from immature pre-neutrophils, largely in bone marrow, to mature neutrophils predominantly in blood and spleen. The sharpest increments in neutrotime occur during the transitions from pre-neutrophils to immature neutrophils and from mature marrow neutrophils to those in blood. Human neutrophils exhibit a similar transcriptomic pattern. Neutrophils migrating into inflamed mouse lung, peritoneum and joint maintain the core mature neutrotime signature together with new transcriptional activity that varies with site and stimulus. Together, these data identify a single developmental spectrum as the dominant organizational theme of neutrophil heterogeneity.
CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody ...antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177pos and CD177neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration.
•Ligation of CD177, a GPI-linked surface protein expressed selectively on neutrophils, blocks neutrophil migration independently of PECAM-1.•Blockade reflects activation through β2 integrins, immobilizing cells via stronger integrin attachments and impaired chemokine signaling.
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Neutrophils are frequently studied in mouse models, but the extent to which findings translate to humans remains poorly defined. In an integrative analysis of 11 mouse and 13 human datasets, we find ...a strong correlation of neutrophil gene expression across species. In inflammation, neutrophils display substantial transcriptional diversity but share a core inflammation program. This program includes genes encoding IL-1 family members, CD14, IL-4R, CD69, and PD-L1. Chromatin accessibility of core inflammation genes increases in blood compared to bone marrow and further in tissue. Transcription factor enrichment analysis implicates members of the NF-κB family and AP-1 complex as important drivers, and HoxB8 neutrophils with JunB knockout show a reduced expression of core inflammation genes in resting and activated cells. In independent single-cell validation data, neutrophil activation by type I or type II interferon, G-CSF, and E. coli leads to upregulation in core inflammation genes. In COVID-19 patients, higher expression of core inflammation genes in neutrophils is associated with more severe disease. In vitro treatment with GM-CSF, LPS, and type II interferon induces surface protein upregulation of core inflammation members. Together, we demonstrate transcriptional conservation in neutrophils in homeostasis and identify a core inflammation program shared across heterogeneous inflammatory conditions.
To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for ...transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising <75 miRNAs; however, each cell type had a unique miRNA signature. Integration of miRNA expression with chromatin accessibility revealed putative regulatory elements for differentially expressed miRNAs, including miR-21a, miR-146a and miR-223. The integrated maps suggest that many miRNAs utilize multiple promoters to reach high abundance and identified dominant and divergent miRNA regulatory elements between lineages and during development that may be used by clustered miRNAs, such as miR-99a/let-7c/miR-125b, to achieve distinct expression. These studies, with web-accessible data, help delineate the cis-regulatory elements controlling miRNA signatures of the immune system.
BackgroundSystemic Lupus Erythematosus (SLE) is a prototypic, systemic autoimmune disease that can affect many organs. Current treatment of SLE is largely empirical, while the existing ...immunosuppressive treatments fail to induce remission in over 40% of patients.MethodsWhole blood transcriptome samples were obtained from 95 patients with moderate to severe SLE at baseline, 1 month and 6 months after initiation of treatment with cytotoxic agents (cyclophosphamide, mycophenolate mofetil), mycophenolate mofetil/anti-CD40 antibody, rituximab or belimumab. Disease activity was assessed using the SLEDAI-2K. Response to treatment was defined as achievement of Low Disease Activity State (LLDAS) or remission at 6 months. Differentially expressed genes (DEGs) were identified using the DEseq2. Weighted correlation network analysis (WGCNA) was applied to detect modules of co-expressed transcripts. Abundances of cell types were assessed by CIBERSORTx.Results95 patients were enrolled in our study. Most of the patients were women (93.7%) with a mean SD age at SLE diagnosis of 42.9 13.6 years and a mean disease duration SD at sampling of 5.1 7.2 years. Cyclophosphamide was the most frequently used immunosuppressive agent (n=46), followed by belimumab (n=24) and rituximab (n=21). 43 patients responded to treatment. Transcriptional disturbances related to type I interferon signaling (p=0.04, r= 0.15) and leukocyte chemotaxis (p=0.005, r=0.21) positively correlated with response to treatment at 6 months. Enrichment of processes linked to complement activation and PI3KK/Akt pathway distinguishes active Lupus Nephritis (LN) responders from LN non-responders at baseline. Gene expression signatures indicative of cell cycle checkpoint regulation and humoral immunity emerged as potential determinants of resistant disease 6 months after treatment initiation. Marked reduction in the naïve B cell compartment uniquely characterized successful response induction, while the neutrophilic fraction exhibited a statistically significant reduction upon treatment, irrespective of the 6-month outcome.ConclusionsBaseline transcriptional signatures related to innate immunity correlated with 6-month response to treatment in SLE. Disturbances linked to cell cycle regulation decisively shaped the transcriptional landscape of ‘resistant’ disease.AcknowledgementsThis work was supported by grants from EU (SYSCID grant agreement number 733100), ERC (LUPUSCARE grant agreement number 742390), FOREUM all to DTB.
Treatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant ...autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission.
We evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology-European League against Rheumatism (ACR-EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded.
The median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR-EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell-associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization.
In this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.).
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