The immunopathogenesis of autoimmune neurological syndromes (AINS) with antibodies against the 65 kDa isoform of glutamic acid decarboxylase (anti-GAD65 AINS) remains poorly understood. To elucidate underlying disease mechanisms and identify relevant cell populations, we performed single-cell RNA and immune repertoire sequencing of cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) of eight anti-GAD65 AINS individuals compared to eight noninflammatory controls. In addition, PBMCs from 19 anti-GAD65 AINS individuals and 20 healthy controls were analyzed by multidimensional flow cytometry, and brain tissue specimens from four anti-GAD65 AINS individuals were examined histologically. We detected higher frequencies of stem cell-like memory T cells (TSCM) within the PBMCs and a marked enrichment and clonal expansion of activated CD4+TSCM in the CSF of anti-GAD65 AINS individuals. Expanded T cells exhibited increased expression of proinflammatory genes. Histological analyses confirmed intraparenchymal CD8+ TSCM in three of four anti-GAD65 AINS individuals and rare meningeal/intraparenchymal CD4+ TSCM in one person. Although CSF B cell receptors (BCRs) displayed little to no clonal expansion, recombinant expression of 40 CSF BCRs revealed that 25% were GAD65-reactive with increased somatic hypermutations compared to non-GAD65-reactive BCRs. These findings further support the concept of an antigen-specific intrathecal immune response. In summary, we characterize the immune landscape of anti-GAD65 AINS at single-cell resolution and identify clonally expanded TSCM with cytotoxic properties as a hallmark of this disease.

Bone marrow fibrosis is the most extensive matrix remodeling of the microenvironment and can include de novo formation of bone (osteosclerosis). Spatiotemporal information on the contribution of distinct bone marrow niche populations to this process is incomplete. We demonstrate that fibrosis-inducing hematopoietic cells cause profibrotic reprogramming of perivascular CXCL12-abundant reticular (CAR) progenitor cells, resulting in loss of their hematopoiesis-support and upregulation of osteogenic and pro-apoptotic programs. In turn, peritrabecular osteolineage cells (OLCs) are activated in an injury-specific, Wnt-dependent manner, comparable to skeletal repair. OLCs fuel bone marrow fibrosis through their expansion and skewed differentiation, resulting in osteosclerosis and expansion of Ly6a+ fibroblasts. NCAM1 expression marks peritrabecular OLCs and their expansion into the central marrow is specific for fibrosis in mice and patients. Peritrabecular stromal β-catenin expression is linked to fibrosis in patients, and inhibition of Wnt signaling reduces bone marrow fibrosis and osteosclerosis, possibly being a clinically relevant therapeutic target.

Complex transcriptional programs and signaling pathways control early hematopoietic lineage specification. Many key regulators have been identified; however, a substantial portion of the genome remains functionally uncharacterized. Here, we investigated six uncharacterized 'Riken' genes identified through transcriptomic profiling of Flk-1+ (also known as Kdr)/Pdgfrα- (hematoendothelial-enriched) and Flk-1+/Pdgfrα+ (cardiac mesoderm-enriched) populations at day 4 of embryoid body (EB) differentiation. We generated knockouts in mouse embryonic stem cells and performed bulk RNA-sequencing at day 4. Three of these genes (C130074G19Rik, I830077J02Rik and A530016L24Rik) were selected for further investigation by single-cell RNA-sequencing at day 7 of differentiation, which provided novel insight for two of these genes. Knockout of C130074G19Rik (hereafter G19Rik) increased the abundance of megakaryocyte progenitors and reduced endothelial populations, with differentially expressed genes enriched for hemostasis and membrane trafficking pathways. I830077J02Rik (hereafter J02Rik)-knockout cells showed subtle changes in extracellular matrix and cell adhesion genes, with a shift toward hematoendothelial lineages. Both G19Rik and J02Rik genes encode (predicted) transmembrane proteins that modulate membrane-associated processes in early hematopoietic development. This work establishes a framework for the study of uncharacterized genes with potential roles in cell fate determination.

BACKGROUND AND OBJECTIVES:Multiple sclerosis (MS) is characterized by CXCR3+ memory B cells that infiltrate the CNS to mature into antibody-secreting cells (ASCs). It remains elusive how to benefit from this disease hallmark as a prognostic tool. We aimed to uncover markers that reflect B-cell entry and maturation in the CNS and, on that basis, explore associations with response to high-efficacy MS therapies. METHODS:Ex vivo single-cell technologies were applied to blood samples and postmortem CNS suspensions from MS and control donors, either treated or untreated. In addition, in vitro assays were performed on blood samples from healthy individuals to functionally asses B-cell activation, differentiation, and transmigration. RESULTS:Single-cell RNA sequencing revealed ITGB1 (integrin β1/CD29) as a major discriminator of CXCR3+ memory B cells in blood, which was validated using spectral flow cytometry. CD29+CXCR3+ memory B cells preferentially crossed a brain microvascular endothelial layer and were highly receptive to T-cell help to become ASCs in vitro. In contrast to blood, CD29 and CXCR3 coexpression was restricted to ex vivo ASCs derived from CSF, meninges, and brain tissue of people with MS, which was supported by enhanced CD29 upregulation on in vitro-differentiated ASCs. While CD29 was downregulated on CXCR3+ memory B cells that accumulated in the blood of natalizumab-treated patients, CD29 levels were only reduced on CXCR3+ memory B cells that repopulated in cladribine-treated patients without early disease activity. DISCUSSION:These findings put forward CD29 as a putative marker on CXCR3+ precursors of CNS-residing ASCs for predicting treatment responses in MS.

OBJECTIVE:The unknown pathophysiology and the lack of specific features for systemic juvenile idiopathic arthritis and adult-onset Still disease (collectively known as Still disease; SD) delay diagnosis and appropriate treatment. The goal of this study was to identify features and mechanisms that distinguish SD from other systemic autoinflammatory diseases (SAID). METHODS:Using the SomaScan assay and RNA sequencing (RNA-Seq), we determined the plasma proteomes and immune cell microRNA (miRNA) and RNA transcriptomes of 372 patients with SAID, respectively. Proteomic findings were validated by enzyme-linked immunosorbent assays. SD (n = 72) and non-SD SAIDs (n = 300) were compared to identify distinguishing features of SD. We performed integrated and unbiased analyses of all data sets using weighted gene correlation network analysis to identify feature modules that characterize SD and stratify patients. RESULTS:Elevated plasma heme oxygenase 1 (HO-1) and interleukin-18 (IL-18) strongly correlate and characterize SD but do not associate with general inflammation. SD was characterized by ferroptosis in plasma, type I interferon (IFN) signaling in monocyte transcriptomes, and elevated natural killer cell miRNA-146a-5p, which is an IL-18 induced miRNA. Finally, we identified feature modules that distinguish SD from other SAIDs and stratified patients with SD into two distinct subgroups not attributable to disease activity or inflammation but hemophagocytosis. CONCLUSION:This unprecedented large omics data set of SAIDs revealed that complex interactions among hemophagocytosis, IL-18, and type I IFN signaling characterize SD. Furthermore, two distinct subgroups in patients with SD were distinguished by the degree of hemophagocytic activity. Finally, the large proteomics and RNA-Seq data sets generated in this study can serve as an invaluable resource for the further investigation of SD and other SAIDs.

“Cold” immunosuppressive solid tumors are hard-to-treat cancers that are non-responsive to immunotherapies. Their immunosuppressive tumor microenvironment (TME) excludes and inhibits T cells and thereby hampers the therapeutic efficacy of cancer vaccines and immune checkpoint blockade. To overcome this, we employed a “Prime-Target” (P/T) neoantigen vaccination strategy that combines subcutaneous (SQ) and intra-tumor (IT) neopeptide vaccinations to first prime potent systemic neoantigen-specific T cell immunity and then trigger intratumoral T cell recruitment. Using immunotherapy non-responsive murine tumor models with pronounced immunosuppressive TMEs, we demonstrate that P/T neopeptide vaccination resulted in extraordinary tumor control and TME remodeling. P/T vaccination elicited strong systemic anti-tumor responses as well as potent and rapid recruitment of clonal CD4+ Th1 and non-exhausted CD8+ T cells into tumors that carried novel T cell receptors (TCR) and were vaccine neopeptide-specific. Concurrently, P/T vaccination reshaped the TME by decreasing suppressive Treg and M2 macrophages, and dramatically increasing the ratio of effector T cells to Treg and M2 macrophages. Vaccination-induced tumor control was neopeptide-dependent and required concurrent IT administration of both neopeptides and adjuvants. Our study highlights P/T neoantigen vaccination as a promising strategy to overcome T cell exclusion within “cold” immunosuppressive solid tumors and thereby unleash unprecedented anti-tumor immunity. ### Competing Interest Statement The authors have declared no competing interest. Department of Immunology of Erasmus University Medical Center Dutch Cancer Society, KWF grant 12837 IMSUT International Joint Usage/Research Center Project, K22-3063, K25-3190

Systemic anti-tumour immunity results from T cell priming in tumour-draining lymph nodes (TDLNs)1–4. Although the suppression of T cells in tumours is well characterized5–8, whether this occurs in TDLNs—and if so, through which mechanisms—remains poorly understood. Here, using imaging mass cytometry of TDLNs from patients with melanoma, we identify a spatial neighbourhood in the TDLN paracortex that is linked to the development of distant metastases. Targeted spatial transcriptomics of cells inside this neighbourhood revealed activated CD8+ T cells engaging with myeloid cells that expressed high levels of the immunosuppressive secretory phospholipase PLA2G2D. PLA2G2D+ myeloid cells were substantially more abundant in TDLNs than they were in primary tumours or metastases. Genetic loss-of-function or antibody-mediated inhibition of PLA2G2D reduced tumour growth markedly, and single-cell transcriptomics in melanoma-bearing mice revealed that expression of Pla2g2d is confined to lymph-node macrophages. Mechanistically, PLA2G2D directly suppressed the early proliferation of T cells in vitro, and inhibiting PLA2G2D resulted in an expansion of tumour-specific T cells in TDLNs, leading to an increase in these T cells in the circulation and subsequently in tumours. Notably, PLA2G2D and PD-1 act as non-redundant immune checkpoints, with combination treatment showing additive or synergistic efficacy in humanized mice treated with human-specific antibodies. Collectively, our in-depth spatial profiling identifies PLA2G2D as a TDLN-centred targetable immune checkpoint for cancer immunotherapy. A specific population of macrophages that express high levels of the enzyme PLA2G2D in tumour-draining lymph nodes is associated with poor prognosis in patients, and inhibiting PLA2G2D improves anti-tumour immunity in mouse models.

ABSTRACT:Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent infections, and an increased leukemia risk. Multiple genetic defects that underlie SCN have been identified, but a genetic diagnosis is still lacking in a significant proportion of patients. In this study, we report 4 independent pedigrees with heterozygous variants in LCP1. Variants c.740-1G>T and c.740-20_744del produced the same alternatively spliced RNA product, causing an in-frame deletion (p.A247_E254del). Variant c.509C>T in the third pedigree produced p.S170L, and variant c.806T>C in the fourth pedigree produced p.L269P. Affected individuals suffered from neutropenia, poor or complete lack of response to granulocyte colony-stimulating factor (G-CSF) treatment, and variable degrees of lymphopenia, hypogammaglobulinemia, and monocytopenia. Patients with A247_E254del and p.L269P presented with tetraploid cells in the bone marrow, indicative of disturbed cytokinesis. In one of these kindreds, 2 individuals developed acute leukemia. G-CSF nonresponsiveness and defective cell cycling were repaired upon correction of the LCP1 A247_E254del variant in patient-derived induced pluripotent stem cells, supporting the monogenic origin of the disease. Indicative of their gain-of-function effect, both the A247_E254del and S170L variants increased F-actin bundling and the formation of abnormal protrusions. Single-cell transcriptome analysis of A247_E254del bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) showed deregulation of signaling pathways that control mitosis in multilineage and lymphoid-primed HSPC subsets. We concluded that activating LCP1 variants cause a new hematopoietic disorder with autosomal dominant inheritance. Depending on the consequences of the LCP1 variants in terms of protein structure, patients may suffer from G-CSF refractory severe neutropenia, lymphopenia, hypogammaglobulinemia, monocytopenia, and defective cytokinesis.

AIMS:The pathogenesis of intraductal carcinoma (IDC) is still controversial. Contrary to current opinion, where IDC represents retrograde spread of invasive prostate cancer (PCa), we recently presented an alternative, unifying hypothesis named 'Repetitive Invasion, Precursor Progression' (RIPP). Little is known about genomic alterations in high-grade Prostatic Intraepithelial Neoplasia (HGPIN), IDC and adjacent invasive PCa. Our objective was to clarify the mutual clonal relationships among HGPIN, IDC, and adjacent PCa using spatial transcriptomics. METHODS AND RESULTS:Regions of interest containing HGPIN, IDC and adjacent invasive PCa were selected from six Gleason score 3 + 4 = 7 radical prostatectomy specimens. Spatial transcriptomic profiling and library preparation were executed according to the Visium workflow. Pathologist-guided manual annotations were utilized to delineate regions of interest for the integrated analysis of chromosomal copy number variants (CNV) and spatiotemporal trajectories. Adjacent HGPIN, IDC and invasive PCa shared common CNV signatures across all samples, with various subclonal events. Unsupervised clonal analysis revealed that across three samples, the adjacent invasive subclone had acquired additional genomic alterations. In two samples, HGPIN, IDC and adjacent invasive PCa had identical CNVs. Finally, in one sample, IDC had additional CNVs compared with HGPIN and invasive glands. Supervised trajectory analysis consistently placed adjacent invasive PCa as the final step in the trajectory, after HGPIN and/or IDC. CONCLUSIONS:Spatial transcriptomics revealed strong clonal relationships among adjacent HGPIN, IDC and invasive PCa. Supervised trajectory analysis did not support retrograde spread in this limited number of samples, while unsupervised analysis revealed a complex mutual relationship among HGPIN, IDC and adjacent PCa.

Pooled CRISPR screening combined with single-cell RNA sequencing (scRNA-seq) has emerged as a powerful strategy for dissecting gene function and reconstructing gene regulatory networks (GRNs) in complex biological systems. This approach enables high-throughput, parallel perturbation of multiple genes while providing transcriptome-wide readouts at single-cell resolution, overcoming many limitations of traditional arrayed screens. However, its broader application remains limited by technical challenges, including variable perturbation efficiency and difficulties in accurately identifying perturbed cells. In this study, we adapted and applied a modified CRISPR droplet sequencing (CROP-seq) protocol using CRISPR interference (CRISPRi) in K562 cells to knockdown six transcription factors (TFs): LMO2, TCF3, LDB1, MYB, GATA2, and RUNX1. Our modified approach, which allows direct capture of sgRNAs from the cDNA library without a separate enrichment step, significantly improved sgRNA assignment per cell. We successfully achieved reproducible knockdown of three TFs (MYB, GATA2, and LMO2), captured the impact of these perturbations on the TF target genes, and enabled us to reconstruct their GRNs and identify key regulons and transcriptional targets. These networks revealed both previously established (such as LMO2 GATA2 interaction) and novel regulatory interactions, which we independently validated, providing new insights into hematopoietic transcriptional control. To assess the efficiency of CRISPRi based pooled perturbation, we additionally analyzed publicly available Perturb-seq CRISPRi datasets and found that only 40–50

Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of an enteric nervous system (ENS) in the distal gut. While the ENS is critical for normal gut function, its broader role in maintaining intestinal homeostasis remains underexplored.Using single-cell RNA sequencing, we investigated the impact of ENS loss on gut composition in wildtype and ret mutant (HSCR model) zebrafish. Significant alterations were identified, including increases in immune cells and shifts in epithelial and extracellular matrix (ECM)-producing cell populations. Immune dysregulation was highlighted by impaired TNF-α signaling via NF-κB, while epithelial changes pointed to disrupted energy homeostasis with downregulated fatty acid metabolism and cell cycle pathways. The ECM producing cells showed enriched fibrotic markers. Alterations of the intestinal composition were validated in human HSCR tissues, underscoring the clinical relevance of the study. These changes can underlie the development of secondary complications and be potentially used to improve patient outcomes.

The transcription factor MECOM, located at 3q26, is essential for hematopoietic stem cells in healthy individuals. Enhancer translocations, due to 3q26 rearrangements, drive out-of-context MECOM expression in one of the most aggressive subtypes of acute myeloid leukemia (AML). Aberrantly expressed MECOM is essential for the survival and immature phenotype of these leukemia cells. Direct depletion of MECOM using an endogenous auxin-inducible degron immediately upregulates expression of CEBPA, which encodes a transcription factor required for neutrophil development and is frequently mutated in other AML subtypes. MECOM depletion is accompanied by a severe loss of CD34 and gain of mature myeloid cell surface marker CD15. MECOM exerts its inhibitory effect on differentiation by binding to the +42-kilobase CEBPA enhancer. This is partially dependent on the interaction between MECOM and its corepressor CTBP2. We demonstrate that CEBPA overexpression can bypass the MECOM-mediated block of differentiation. In addition, patients with AML with MECOM overexpression through enhancer hijacking show significantly reduced CEBPA levels. Our study directly connects 2 major players in normal and malignant hematopoiesis, MECOM and CEBPA, and unveils how MECOM maintains self-renewal by repressing CEBPA-induced differentiation.

Background Glutamic acid decarboxylase (GAD) antibody-associated autoimmune neurological syndromes (AINS) are a spectrum of autoimmune-mediated CNS disorders. While antibodies targeting the 65 kDa isoform of GAD are of high diagnostic value, T cell mediated cytotoxicity has been identified as a key component of disease pathogenesis. The precise pathophysiological mechanisms by which the disease is triggered and maintained, however, remain incompletely understood. Methods We performed single-cell transcriptome and immune repertoire sequencing (sc-seq) in CSF and blood of 8 anti-GAD65 AINS patients compared to 8 non-inflammatory controls. Monoclonal antibodies (mAbs) were synthesized from B cell receptor (BCR) data to evaluate the B cellular immune response. Findings We identified an increase and expansion of activated CD4+ stem cell-like memory T cells (TSCM) in the CSF of anti-GAD65 AINS patients. Expanded T cells showed increased expression of proinflammatory genes. The mAb analysis revealed a high frequency of GAD65-reactive BCRs in the CSF of anti-GAD65 AINS patients with increased somatic hypermutations compared to non-GAD-reactive BCRs and BCRs from controls. Conclusions Sc-seq identified clonally expanded CD4+ TSCM in the CSF of anti-GAD65 AINS patients harboring cytotoxic properties likely contributing to disease pathogenesis. GAD-reactive B cells circulate in the CSF of anti-GAD65 AINS patients further supporting the concept of an antigen-specific intrathecal immune response. Future studies need to clarify the actual pathogenicity of these immune cells and the link between T and B cellular immune mechanisms in the pathogenesis of anti-GAD65 AINS. Funding German Research Foundation (ERARE18-202 UltraAIE), German Federal Ministry of Education and Research (CONNECT GENERATE (2.0); 01GM1908A and 01GM2208A). ### Competing Interest Statement SB is affiliated with Novartis, Basel, Switzerland; SR received travel grants from Merck Healthcare Germany GmbH, Alexion Pharmaceuticals, Bristol Myers Squibb, the German Neurological Society, and the American Academy of Neurology. She served on a scientific advisory board from Merck Healthcare Germany GmbH and received honoraria for lecturing from Roche and Merck Healthcare Germany GmbH. Her research was supported by Novartis, "Stiftung zur Foerderung junger Neurowissenschaftler", and "Else Kroener-Fresenius-Stiftung"; KE was supported by the "Friedrich-Baur-Stiftung"; EK has received personal honoraria for lectures or advice from UCB, UNEEG, Medtronic, Desitin and Precisis and has participated in trials sponsored by Medtronic, UCB, Ergomed, and Precisis. Her research is supported by the Munich Clinican Scientist Program (MCSP); MR received lecture fees and/or served on advisory boards for AstraZeneca, Echosens, Eli Lilly, Madrigal, Merck-MSD, Novo Nordisk, Synlab and Target RWE and performed investigator-initiated research with support from Boehringer Ingelheim and Novo Nordisk to the German Diabetes Center (DDZ); HW received speaker honoraria from Alexion, Biogen, Bristol Myers Squibb, Genzyme, Merck, Neurodiem, Novartis, Ology, Roche, TEVA, and WebMD Global. He received honoraria for consulting services from Abbvie, Actelion, Argenx, BD, Bristol Myers Squibb, EMD Serono, Fondazione Cariplo, Gossamer Bio, Idorsia, Immunic, Immunovant, INmune Bio_Syneos Health, Janssen, Merck, NexGen, Novartis, Roche, Sanofi, Swiss MS Society, UCB and Worldwide Clinical Trials. His research is supported by the German Myasthenia Gravis Society; NHS declares financial ties to Menarini Silicon Biosystems (CellSearch Assay) in the form of third-party funding for research support, as well as to Illumina (NGS products) in the form of lecture fees; SGM receives honoraria for lecturing, and travel expenses for attending meetings from Academy 2, Argenx, Alexion, Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, BioNtech, BMS, Celgene, Datamed, Demecan, Desitin, Diamed, Diaplan, DIU Dresden, DPmed, Gen Medicine and Healthcare products, Genzyme, Hexal AG, IGES, Impulze GmbH, Janssen Cilag, KW Medipoint, MedDay Pharmaceuticals, Merck Serono, MICE, Mylan, Neuraxpharm, Neuropoint, Novartis, Novo Nordisk, ONO Pharma, Oxford PharmaGenesis, QuintilesIMS, Roche, Sanofi-Aventis, Springer Medizin Verlag, STADA, Chugai Pharma, Teva, UCB, Viatris, Wings for Life international and Xcenda, his research is funded by the German Ministry for Education and Research (BMBF), Bundesinstitut fuer Risikobewertung (BfR), Deutsche Forschungsgemeinschaft (DFG), Else Kroener Fresenius Foundation, Gemeinsamer Bundesausschuss (G-BA), German Academic Exchange Service, Hertie Foundation, Interdisciplinary Center for Clinical Studies (IZKF) Muenster, German Foundation Neurology and Alexion, Almirall, Amicus Therapeutics Germany, Biogen, Diamed, DGM e.v., Fresenius Medical Care, Genzyme, Gesellschaft von Freunden und Foerderern der Heinrich-Heine-Universitaet Duesseldorf e.V., HERZ Burgdorf, Merck Serono, Novartis, ONO Pharma, Roche, and Teva; MJT M.J. Titulaer has received funds for serving on a scientific advisory board of AmGen, ArgenX, Arialys and UCB, received funds from Dioraphte (2001 0403) EpilepsieNL (19-18), filed a patent for methods for typing neurologic disorders and cancer, and devices for use therein, received research funds for consultation at Guidepoint Global LLC, an unrestricted research grant from CSL Behring, and an unrestricted research grant from Euroimmun, was supported by an Interlaken Leadership Award and an E-RARE3 grant (Netherlands Medical Research Foundation [ZonMW], ERARE-JTC2018 202: UltraAIE).; FL is also supported by E-Rare Joint Transnational research support (ERA-Net, LE3064/2-1), Stiftung Pathobiochemie of the German Society for Laboratory Medicine and HORIZON MSCA 2022 Doctoral Network 101119457 - IgG4-TREAT and discloses speaker honoraria from Grifols, Teva, Biogen, Bayer, Roche, Novartis, Fresenius, travel funding from Merck, Grifols and Bayer and serving on advisory boards for Roche, Biogen and Alexion; GMzH received research support from Biogen and Merck Germany; he received honoraria from Alexion and LFB Pharma and participated in Data Safety Monitoring and/or Advisory Boards of LFB Pharma, Roche and Immunovant; FT received grant support from Novartis Pharma GmbH and speaker honoraria from Alexion Pharmaceuticals; NM received honoraria for lecturing and travel expenses for attending meetings from Biogen Idec, GlaxoSmithKline, Teva, Novartis Pharma, Bayer Healthcare, Genzyme, Alexion Pharmaceuticals, Fresenius Medical Care, Diamed, UCB Pharma, AngeliniPharma, BIAL and Sanofi-Aventis, received royalties for consulting from UCB Pharma, Alexion Pharmaceuticals and Sanofi, and received financial research support from Euroimmun, Fresenius Medical Care, Diamed, Alexion Pharmaceuticals, Novartis Pharma, and Sanofi. MS, RM, and LK are employees of EUROIMMUN. The remaining authors declare no conflict of interest. German Research Foundation, ERARE18-202 UltraAIE German Federal Ministry of Education and Research, CONNECT GENERATE (2.0); 01GM1908A and 01GM2208A

BACKGROUND:Retinoic acid (RA) is pivotal for the development of the enteric nervous system (ENS), guiding the migration and differentiation of vagal neural crest cells in the gastrointestinal tract. Despite its recognized influence on other intestinal cell types, the specific roles of RA on these cells are less understood. This study investigates the extensive impact of RA inhibition on the intestinal composition using zebrafish, while also considering its potential use as a model for Hirschsprung disease (HSCR). METHODS:Zebrafish larvae were treated with diethylaminobenzaldehyde (DEAB), an inhibitor of RA synthesis, to induce phenotypes resembling total colonic aganglionosis. Single-cell RNA sequencing was performed to compare the intestinal cell composition of DEAB-treated zebrafish with wildtype and a ret mutant HSCR model. RESULTS:Inhibition of RA reduced ENS cell numbers but also induced significant changes within non-neuronal cell populations. Notably, there was an increase in epithelial cells, a decrease in immune cells, and suppression of inflammatory pathways. Additionally, we observed increased myofibroblast numbers and decreased fibroblasts, which might suggest fibrosis. Comparative analysis of the ret mutant and DEAB-treated models showed profound effects on non-neuronal cells, yet most of these changes differed between the two models, underscoring the distinct roles of RA and Ret signaling on intestinal development. CONCLUSION:These findings highlight RA's pivotal role in maintaining intestinal structure and immune homeostasis, while further supporting the importance of considering both neuronal and non-neuronal cell populations in HSCR research.

Hematopoiesis is the process of producing blood cells. In mammalian embryos, hematopoiesis occurs in three consecutive overlapping waves (Neo et al. 2021; Dzierzak and Bigas 2018) that are regulated by transcription factors (TFs) and signaling proteins. We investigated the functions of three relatively poorly studied TFs in early embryonic hematopoietic development at single-cell resolution: Activating transcription factor 3 (Atf3), Zinc finger protein 711 (Zfp711), and B cell CLL/lymphoma 6, member B (Bcl6b). These TFs are upregulated early in development when hematopoietic and endothelial lineages separate from cardiac and other mesodermal lineages. We combined multiplexed single-cell RNA sequencing (scRNA-seq) and cell identity analysis using Flow Cytometric Analysis (FCA) with TF knockouts (KO) in in-vitro differentiating mouse embryonic stem cells (mESCs) to dissect the function of these TFs in lineage induction, specification, and separation. The Atf3-KO showed an increase of distinct mesodermal subpopulations, but a decrease of endothelial and erythro-myeloid progenitors (EMPs) by downregulation of important genes ( i.e. Runx1, Mafb, Egr1, Jun, Jund, Fos, Batf3, and Zf608) that likely explains the effects on EMPs and the increased expression of interferon-related genes. In Zfp711-KO cells, the number of blood progenitor cells and erythroid cells increased, while the number of endothelial cells decreased. Furthermore, Hoxa expressing mesoderm decreased, while Hoxb expressing mesoderm increased. In contrast, the Bcl6b-KO had no observable effects on early hematopoiesis. In conclusion, we report the function of these three TFs function at different stages of hemato-endothelial lineage specification. ### Competing Interest Statement The authors have declared no competing interest.

Hematopoiesis occurs in three consecutive overlapping waves in mammals, regulated by transcription factors. We investigated the role of three relatively poorly studied transcription factors in early embryonic hematopoietic development at single-cell resolution: Atf3, Zfp711 and Bcl6b. These transcription factors are upregulated early in development, when hematopoietic and endothelial lineages separate from cardiac and other mesodermal lineages. We combined multiplexed single-cell RNA sequencing and flow cytometric analysis with knockouts in in vitro differentiating mouse embryonic stem cells to dissect the function of these transcription factors in lineage specification. ΔAtf3 cells showed increased mesodermal differentiation but decreased endothelial cells and erythro-myeloid progenitors, accompanied by aberrant interferon signaling. Mechanistically, loss of Atf3 disrupted key hematopoietic regulatory genes (Runx1, Egr1, Jun, Fos, Mafb and Batf3) required for the formation of erythro-myeloid progenitors. ΔZfp711 cells exhibited increased blood progenitors and erythroid cells, but decreased endothelial cells, with a striking shift from Hoxa+ mesoderm (allantois and limb mesoderm) to Hoxb+ mesoderm (mesenchyme and epicardium). Notably, Zfp711 binds the Atf3 promoter, suggesting a hierarchical regulation. In contrast, ΔBcl6b had no observable effects on early hematopoiesis, despite specific expression in hemato-endothelial progenitors.

Background: Chronic pulmonary inflammation strongly contributes to respiratory failure and mortality in patients with cystic fibrosis (pwCF). Effective anti-microbial immunity and maintaining lung homeostasis require continuous structural-immune cell communication. Whether and how this crosstalk is altered in CF remains poorly understood, obscuring potential new angles for therapy development to restore airway homeostasis in pwCF. Methods: We performed droplet-based single cell RNA-sequencing on bronchial biopsies from pwCF to investigate structural-immune cell crosstalk. Computational analyses were used to compare these data to samples obtained from healthy controls. Results: CF airway wall biopsies showed lower proportions and altered transcriptomes of basal cells, submucosal gland cells and endothelial cells, and a higher abundance of ciliated cells, monocytes, macrophages and T cells. Both B and T lymphocytes displayed aberrantly activated phenotypes with transcriptional changes linked to hypoxia and vascular endothelial growth factor signaling, indicative of crosstalk with endothelial cells. The CF lung displayed unique changes in intercellular communication potential involving ionocytes, macrophages, endothelial cells and lymphocytes. This included interactions between HLA-E on structural cells and the druggable CD94/NKG2A immune checkpoint on CD8+ T cells. Conclusions: We report the first single cell transcriptome atlas of the CF lung containing the full spectrum of structural and immune cells, providing a valuable resource for investigating changes to cellular composition, phenotypes and crosstalk linked to CF. Our analyses highlight dysregulated basal cell function and adaptive immunity in pwCF - despite favorable responses to CFTR modulator therapy. We identify novel aspects of CF pathophysiology and potential entry points for therapeutic strategies.

Anti-leucine-rich glioma inactivated-1 (LGI1) and anti-contactin-associated-protein-2 (CASPR2) autoimmune encephalitis (AIE) are common and characterized by pathogenic antibodies targeting neuronal autoantigens. However, the drivers of the antibody-secreting cells and involvement of T cells remain unresolved. We performed single-cell RNA sequencing of fresh CSF and parallel blood samples of 15 patients with LGI1-AIE (n = 9) and CASPR2-AIE (n = 6) compared with control patients [multiple sclerosis (n = 15) and idiopathic intracranial hypertension (n = 18)]. We validated our observations in independent cohorts using flow cytometry of CSF and blood. We confirmed autoantibody specificity using recombinant human monoclonal antibodies. In comparison to idiopathic intracranial hypertension and multiple sclerosis controls, we observed clonal CSF-specific antibody-secreting cell expansion in LGI1/CASPR2-AIE despite mostly normal CSF findings. Antibody-secreting cells were dominantly plasmablasts and transcribed IgG4 and IgG1/2 heavy chains. Expanded clones showed signs of affinity maturation and bound the respective neuronal autoantigen. Within CD4 and CD8 T-cell clusters, CD4 and CD8 central memory T cells were activated, clonally restricted and expanded. T-cell clones were often shared between CSF and blood. We also observed a shift of natural killer cells and loss of mucosa-associated invariant T (MAIT) cells in the CSF of LGI1-AIE and the blood of LGI1- and CASPR2-AIE compared with idiopathic intracranial hypertension and multiple sclerosis controls. MAIT-like T cells were detected in autopsied brains of LGI1- and CASPR2-AIE patients, and mice lacking MAIT cells displayed an increased antibody seroconversion and higher titres following active LGI1/CASPR2 immunization. Our data: (i) confirm the intrathecal antigen-specific plasma cell expansion in LGI1- and CASPR2-AIE in a large cohort of untreated AIE patients; (ii) suggest that activated and expanded central memory CD4 and CD8 T cells in the CSF participate in disease pathogenesis; and (iii) implicate invariant T-cell receptor-expressing lymphocytes in the brain, CSF and blood in disease pathogenesis.