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Objectives: This study, developed within the Innovative Medicines Initiative Joint Undertaking project PRECISESADS framework, aimed at functionally characterize the monocyte subsets in RA patients, and analyze their involvement in the increased CV risk associated with RA.Methods: The frequencies of monocyte subpopulations in the peripheral blood of 140 RA patients and 145 healthy donors (HDs) included in the PRECISESADS study were determined by flow cytometry. A second cohort of 50 RA patients and 30 HDs was included, of which CD14+ and CD16+ monocyte subpopulations were isolated using immuno-magnetic selection. Their transcriptomic profiles (mRNA and microRNA), proinflammatory patterns and activated pathways were evaluated and related to clinical features and CV risk. Mechanistic in vitro analyses were further performed.Results: CD14++CD16+ intermediate monocytes were extended in both cohorts of RA patients. Their increased frequency was associated with the positivity for autoantibodies, disease duration, inflammation, endothelial dysfunction and the presence of atheroma plaques, as well as with the CV risk score. CD14+ and CD16+ monocyte subsets showed distinctive and specific mRNA and microRNA profiles, along with specific intracellular signaling activation, indicating different functionalities. Moreover, that specific molecular profiles were interrelated and associated to atherosclerosis development and increased CV risk in RA patients. In vitro, RA serum promoted differentiation of CD14+CD16− to CD14++CD16+ monocytes. Co-culture with RA-isolated monocyte subsets induced differential activation of endothelial cells.Conclusions: Our overall data suggest that the generation of inflammatory monocytes is associated to the autoimmune/inflammatory response that mediates RA. These monocyte subsets, -which display specific and distinctive molecular signatures- might promote endothelial dysfunction and in turn, the progression of atherosclerosis through a finely regulated process driving CVD development in RA.

Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities., (© 2022. The Author(s).)

Objective: Clinical heterogeneity, a hallmark of systemic autoimmune diseases, impedes early diagnosis and effective treatment, issues that may be addressed if patients could be classified into groups defined by molecular pattern. This study was undertaken to identify molecular clusters for reclassifying systemic autoimmune diseases independently of clinical diagnosis., Methods: Unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data on 955 patients with 7 systemic autoimmune diseases and 267 healthy controls was undertaken. In addition, an inception cohort was prospectively followed up for 6 or 14 months to validate the results and analyze whether or not cluster assignment changed over time., Results: Four clusters were identified and validated. Three were pathologic, representing "inflammatory," "lymphoid," and "interferon" patterns. Each included all diagnoses and was defined by genetic, clinical, serologic, and cellular features. A fourth cluster with no specific molecular pattern was associated with low disease activity and included healthy controls. A longitudinal and independent inception cohort showed a relapse-remission pattern, where patients remained in their pathologic cluster, moving only to the healthy one, thus showing that the molecular clusters remained stable over time and that single pathogenic molecular signatures characterized each individual patient., Conclusion: Patients with systemic autoimmune diseases can be jointly stratified into 3 stable disease clusters with specific molecular patterns differentiating different molecular disease mechanisms. These results have important implications for future clinical trials and the study of nonresponse to therapy, marking a paradigm shift in our view of systemic autoimmune diseases., (© 2020, American College of Rheumatology.)

In recent years, single-cell technologies have emerged as breakthrough techniques that enable the characterization of hematopoietic cell populations of normal and malignant tissue samples and will be combined in the near future with bulk technologies, currently used in clinical practice, to improve diagnosis, prognosis, and the search for novel molecular targets. These single-cell methods have the advantage of not masking cell-to-cell variation features and involve the study of genetic, epigenetic, transcriptional, and proteomic landscapes from a single-cell perspective. Latest advances in this field have enabled the development of novel strategies that significantly increase both sensitivity and high throughput. In this review, we emphasize emerging techniques aimed at assessing individual or multiomic parameters at single-cell resolution and analyze how these technologies have helped us understand hematopoietic variability and identify unknown and/or rare subpopulations. We also summarize the impact of these single-cell profiling strategies on the characterization of cell diversity within the tumor and the clonal evolution of multiple hematological malignancies in samples from untreated and treated patients, which provide valuable information for diagnosis, prognosis, and future treatments and explain why current therapies may fail. However, despite these improvements, new challenges lie ahead., Competing Interests: Conflict of interest disclosure ME is a consultant for Ferrer International and Quimatryx. The remaining authors declare that they have no conflicts of interest., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: Patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the coronavirus disease 2019 (COVID-19), exhibit a wide spectrum of disease behaviour. Since DNA methylation has been implicated in the regulation of viral infections and the immune system, we performed an epigenome-wide association study (EWAS) to identify candidate loci regulated by this epigenetic mark that could be involved in the onset of COVID-19 in patients without comorbidities., Methods: Peripheral blood samples were obtained from 407 confirmed COVID-19 patients ≤ 61 years of age and without comorbidities, 194 (47.7%) of whom had mild symptomatology that did not involve hospitalization and 213 (52.3%) had a severe clinical course that required respiratory support. The set of cases was divided into discovery (n = 207) and validation (n = 200) cohorts, balanced for age and sex of individuals. We analysed the DNA methylation status of 850,000 CpG sites in these patients., Findings: The DNA methylation status of 44 CpG sites was associated with the clinical severity of COVID-19. Of these loci, 23 (52.3%) were located in 20 annotated coding genes. These genes, such as the inflammasome component Absent in Melanoma 2 (AIM2) and the Major Histocompatibility Complex, class I C (HLA-C) candidates, were mainly involved in the response of interferon to viral infection. We used the EWAS-identified sites to establish a DNA methylation signature (EPICOVID) that is associated with the severity of the disease., Interpretation: We identified DNA methylation sites as epigenetic susceptibility loci for respiratory failure in COVID-19 patients. These candidate biomarkers, combined with other clinical, cellular and genetic factors, could be useful in the clinical stratification and management of patients infected with the SARS-CoV-2., Funding: The Unstoppable campaign of the Josep Carreras Leukaemia Foundation, the Cellex Foundation and the CERCA Programme/Generalitat de Catalunya., Competing Interests: Declaration of Competing Interest Dr. Esteller reports grants from Ferrer International, personal fees from Quimatryx, outside the submitted work. The other authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

One caveat in cancer research is the dependence of certain experimental systems that might not really reflect the properties of the primary tumours. The recent irruption of 3D cultured cells termed organoids could render a better representation of the original tumour sample. However, every laboratory has its own protocol and tissue-provider to establish these cancer models, preventing further dissemination and validation of the obtained data. To address this problem, the Human Cancer Models Initiative (HCMI) has selected the American Type Culture Collection (ATCC) to make available organoid models to the scientific community. In this regard, no epigenetic information is available for these samples and, overall, the DNA methylation profiles of human cancer organoids are largely unknown. Herein, we provide the DNA methylation landscape of 25 human cancer organoids available at the ATCC using a microarray that interrogates more than 850,000 CpG sites. We observed that the studied organoids retain the epigenetic setting of their original primary cancer type; that exhibit a DNA methylation landscape characteristic of transformed tissues excluding an overgrowth of normal-matched cells; and that are closer to the DNA methylation profiles of the corresponding primary tumours than to established 2D cell lines. Most importantly, the obtained DNA methylation results are freely available to everyone for further data mining. Thus, our findings support from the epigenetic standpoint that the ATCC human cancer organoids recapitulate many of the features of the disorder in the patient and are excellent tools to be shared among investigators for further tumour biology research.

The innovative medicine initiative project called PRECISESADS will study 2.500 individuals affected by systemic autoimmune diseases (SADs) and controls. Among extensive OMICS approaches, multi-parameter flow cytometry analyses will be performed in eleven different centers. Therefore, the integration of all data in common bioinformatical and biostatistical investigations requires a fine mirroring of all instruments. We describe here the procedure elaborated to achieve this prerequisite. One flow cytometer chosen as reference instrument fixed the mean fluorescence intensities (MFIs) of 8 different fluorochrome-conjugated antibodies (Abs) using VersaComp Ab capture beads. The ten other centers adjusted their own PMT voltages to reach the same MFIs. Subsequently, all centers acquired Rainbow 8-peak beads data on a daily basis to follow the stability of their instrument overtime. One blood sample has been dispatched and concomitantly stained in all centers. Comparison of leukocytes frequencies and cell surface marker MFIs demonstrated the close sensitivity of all flow cytometers, allowing a multicenter analysis. The effective multi-center harmonization enables the constitution of a workable wide flow cytometry database for the identification of specific molecular signatures in individuals with SADs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Adaptor molecules are essential in organizing signaling molecules and in coordinating and compartmentalizing their activity. SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein mainly expressed by hematopoietic cells that has been shown to act as a positive regulator in T, B, and NK cell signal transduction. 3BP2 is an important regulator of cytotoxic granule release in NK cells. Mast cells (MCs) similarly degranulate following Ag-dependent aggregation of the FcεRI on the cell surface. Activation of these cells induces the release of preformed inflammatory mediators and the de novo synthesis and secretion of cytokines and chemokines. Thus, MCs participate in both innate and acquired responses. We observed that 3BP2 is expressed in human MCs (huMCs) from diverse origins. Moreover, 3BP2 coimmunoprecipitates with essential MC signaling mediators such as Lyn, Syk, and phospholipase C γ; thus, a role for this adaptor in MC function was postulated. In the present work, we used the short hairpin RNA lentiviral targeting approach to silence 3BP2 expression in huMCs. Our findings point to a requirement for 3BP2 in optimal immediate and late MCs responses such as degranulation and IL-8 or GM-CSF secretion. 3BP2 was determined to be necessary for optimal phosphorylation of Syk, linker for activation of T cells, and phospholipase C γ(1), critical signals for calcium release from intracellular stores. Taken together, our results show that by participating in FcεRI- mediated signal transduction 3BP2 is an important regulator of huMC activation. Thus, 3BP2 could be a potential therapeutic target for IgE-dependent MC-mediated inflammatory disease.

It is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). We have analyzed the function of cluster of differentiation (CD)300f immunoreceptor in a model of excitotoxic rat brain damage. First, to explore the presence of endogenous ligand(s) for this receptor we used a human CD300f-Ig soluble protein and confocal microscopy, showing specific staining mainly in CNS white matter and on the surface of oligodendrocytes and certain astrocytes. Next, we demonstrated in a model of in vivo rat brain excitotoxic damage that the overexpression of human CD300f induced a significant reduction in the lesion volume. To validate these results, we cloned the rat ortholog of CD300f protein (rCD300f). The overexpression of rCD300f receptor had a comparable neuroprotective effect after the acute brain injury and a similar CNS staining pattern when stained with the rCD300f-Ig soluble protein. Interestingly, when we analyzed the expression pattern of rCD300f in brain cells by quantitative polymerase chain reaction and immunohistochemistry, we detected the expression of CD300f as expected in microglial cells, but also in oligodendrocytes and neurons. These data suggest that the neuroprotective role of CD300f would be the result of a complex network of cell interactions., (© 2011 The Authors; Brain Pathology © 2011 International Society of Neuropathology.)

The development of chronic allergic dermatitis in early life has been associated with increased onset and severity of allergic asthma later in life. However, the mechanisms linking these two diseases are poorly understood. In this study, we report that the development of oxazolone-induced chronic allergic dermatitis, in a mouse model, caused enhanced OVA-induced allergic asthma after the resolution of the former disease. Our findings show that oxazolone-induced dermatitis caused a marked increase in tissue mast cells, which persisted long after the resolution of this disease. Subsequent OVA sensitization and airway challenge of mice that had recovered from dermatitis resulted in increased allergic airway hyperreactivity. The findings demonstrate that the accumulation of mast cells during dermatitis has the detrimental effect of increasing allergic airway hypersensitivity. Importantly, our findings also show that exposure to a given allergen can modify the immune response to an unrelated allergen.

The incidence of chronic allergic dermatitis is rapidly increasing. Regulatory control of this disease has not been adequately explored. Here we report that mast cell-derived interleukin-2 (IL-2) contributes to the suppression of chronic allergic dermatitis. Mice deficient in IL-2 production, or deficient in mast cells (Kit(W-sh/W-sh)), showed exacerbated dermatitis upon repeated oxazolone challenge when compared to their wild-type counterparts. Adoptive transfer of wild-type, but not Il2(-/-), mast cells into Kit(W-sh/W-sh) mice dampened the inflammatory response. During the course of disease, mast cell expansion occurred at the site of inflammation and also in the spleen, where production of IL-2 by mast cells was markedly enhanced. In the absence of mast cell IL-2 production, the ratio of activated to regulatory T cells at the site of inflammation was increased. Thus, MC-derived IL-2 contributes to the maintenance of suppression in chronic allergic skin inflammation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Engagement of FcepsilonRI causes its phosphorylation by Lyn kinase. Two alternatively spliced variants, Lyn A and B, are expressed in mast cells, and both isoforms interact with FcepsilonRI. Unlike Lyn A, Lyn B lacks a 21-aa region in the N-terminal unique domain. In this study, we investigated the role of Lyn A and B isoforms in mast cell signaling and responses. Lyn B was found to be a poor inducer of mast cell degranulation and was less potent in both inositol 1,4,5-triphosphate production and calcium responses. Expression of Lyn B alone showed reduced phosphorylation of both phospholipase Cgamma-1 and -2 and decreased interaction of phospholipase Cgamma-1 with the phosphorylated linker for activation of T cells. Lyn B also showed increased binding of tyrosine-phosphorylated proteins, which included the negative regulatory lipid phosphatase SHIP-1. In contrast, both Lyn A and B caused similar total cellular tyrosine phosphorylation and FcepsilonRI phosphorylation and neither Lyn A nor Lyn B alone could completely restore mast cell degranulation or dampen the excessive cytokine production seen in the absence of Lyn. However, expression of both isoforms showed complementation and normalized responses. These findings demonstrate that Lyn B differs from Lyn A in its association with SHIP-1 and in the regulation of calcium responses. However, complementation of both isoforms is required in mast cell activation.

Mast cells are pivotal in innate immunity and play an important role in amplifying adaptive immunity. Nonetheless, they have long been known to be central to the initiation of allergic disorders. This results from the dysregulation of the immune response whereby normally innocuous substances are recognized as non-self, resulting in the production of IgE antibodies to these 'allergens'. Preformed and newly synthesized inflammatory (allergic) mediators are released from the mast cell following allergen-mediated aggregation of allergen-specific IgE bound to the high-affinity receptors for IgE (FcepsilonRI). Thus, the process by which the mast cell is able to interpret the engagement of FcepsilonRI into the molecular events necessary for release of their allergic mediators is of considerable therapeutic interest. Unraveling these molecular events has led to the discovery of a functional class of proteins that are essential in organizing activated signaling molecules and in coordinating and compartmentalizing their activity. These so-called 'adapters' bind multiple signaling proteins and localize them to specific cellular compartments, such as the plasma membrane. This organization is essential for normal mast cell responses. Here, we summarize the role of adapter proteins in mast cells focusing on the most recent advances toward understanding how these molecules work upon FcepsilonRI engagement.

The immune receptor expressed by myeloid cell 1 (IREM-1) (CD300f) inhibitory receptor displays five cytoplasmic tyrosine residues, two of them (Y205 and Y249) fit with ITIMs, whereas Y236 and Y263 constitute putative binding sites for PI3K. In the present study, immunoprecipitation analysis revealed that both the p85alpha subunit of PI3K and Src homology region 2 domain-containing phosphatase-1 could be recruited by IREM-1 in transfected cells as well as in the U937 monocytic leukemia cells, which constitutively express the receptor. By assaying the ability of different IREM-1 mutants to regulate the secretion of beta-hexosaminidase induced via FcRepsilonI in rat basophilic leukemia cells, both Y205 and Y249 appeared crucial for IREM-1-mediated inhibition. Remarkably, engagement of an IREM-1 mutant (Y(205,249,284)F), which did not recruit Src homology region 2 domain-containing phosphatase-1 and lost its inhibitory function, induced rat basophilic leukemia cell degranulation. This effect was dependent on the recruitment of PI3K, requiring the integrity of Y236 and Y263, and was blocked by PI3K inhibitors (i.e., wortmannin and LY-294002). Altogether, these data reveal a putative functional duality of the IREM-1 myeloid cell receptor.

Using a three-hybrid strategy, we have identified a novel cell surface molecule which interacts with the Src homology 2 (SH2) domains of SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1), termed "immune receptor expressed on myeloid cells 1" (IREM-1). The full-length cDNA coding for a polypeptide of 290 amino acids presents an extracellular single V-type Ig domain, a transmembrane region and a cytoplasmic tail with five tyrosine residues, two of which are in the context of an immunoreceptor tyrosine-based inhibitory motif. Moreover, cDNA encoding for three other splicing forms of IREM-1, named IREM-1 splice variant (Sv)1, Sv2 and Sv3 were cloned by reverse transcription (RT)-PCR. The gene encoding for IREM-1 contains nine exons, is located on human chromosome 17 (17q25.1) and is homologous to previously identified molecules termed CMRF-35 and IRp60. RT-PCR, northern blot and FACS analysis with specific monoclonal antibodies indicated that IREM-1 is expressed on monocytes, granulocytes, and myeloid leukemia cell lines. Western blot analysis confirmed the recruitment of SHP-1 to IREM-1 and demonstrated that phosphotyrosine residue 205 is the main docking site for this interaction. Finally, cross-linking of IREM-1 results in the inhibition of FcRepsilon-induced activation. Our results indicate that IREM-1 is a novel inhibitory receptor of the Ig superfamily in myeloid cells.

Homology basic local alignment search tool search was conducted using a sequence encoding for a novel inhibitory receptor (IREM-1) cloned in our laboratory and a previously described homologous sequence termed CMRF-35. On the basis of this information, we cloned a full length cDNA corresponding to a novel member of this family, termed immune receptor expressed by myeloid cells 2 (IREM-2). The gene, located in chromosome 17q25.1, encodes for a protein of 205 aa that contains an extracellular region comprising an Ig-like domain and a transmembrane region with a positively charged amino acid residue (lysine), that predicted its putative association with an adapter molecule. Indeed, the interaction between IREM-2 and DAP-12 was confirmed in transfected COS-7 cells. By generating specific Abs and using bone marrow and PBMCs, we observed that IREM-2 expression appeared to be restricted to mature hemopoietic cells of the monocytic and myeloid dendritic cell lineages. In vitro differentiation to macrophages or immature dendritic cells down-regulated IREM-2 expression. Upon engagement with the specific mAbs, IREM-2 expressed in rat basophilic leukemia cells together with DAP-12, induced NFAT transcriptional activity; moreover, IREM-2 engagement on monocytes induced TNF-alpha production. Taken together, our results indicate that IREM-2 is a novel activating receptor of the Ig-superfamily in the monocytic lineage.

In the present work we demonstrate that the cancer-associated O-glycosylated Tn antigen (GalNAc-O-Ser/Thr) is expressed by the cestode Echinococcus granulosus. This antigen was detected in both larval and adult worm extracts, with the highest specific activity observed in the adult excretion/secretion preparation. Histochemical analysis showed that Tn is preferentially expressed in the parenchyma in both parasite stages and the external part of tegument in adult worms. A similar pattern was observed for sialyl-Tn, a related O-linked antigen. Tn glycoproteins from protoscoleces were resolved by SDS-PAGE in two main components of 43 and 49 kDa. After purification, this material was reactive with lectins which bind GlcNAc/sialic acid, GalNAc, and T antigen. In a preliminary evaluation, high levels of Tn antigen were detected in serum samples from patients with hydatid cyst, suggesting that the measure of Tn in serum could be a biomarker of this disease, although extensive work is necessary in order to determine the clinical usefulness of this assay. The results reported here, the first evidence of O-glycosylation pathways in E. granulosus and the presence of Tn antigen in cestodes, suggest that the evaluation of O-glycosylated antigens might give new insights in the host-parasite relationship., (Copyright 2001 Academic Press.)