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Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article,we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE,combined with single-molecule,real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV),D (IGHD),and J (IGHJ) gene usage,complementarity-determining region 3 length,and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together,these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs,purified B cells,and whole blood,which recapitulated results generated by commonly used methods,while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide,for the first time,to our knowledge,simultaneous single-molecule characterization of IGHV,IGHD,IGHJ,and IGHC region genes and alleles,allele-resolved subisotype definition,and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes,FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles,28 (87%) of which were previously uncharacterized. Together,these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV,IGHD,IGHJ,and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date. View Publication

UNLABELLED As part of the Multiple Myeloma Research Foundation (MMRF) immune atlas pilot project,we compared immune cells of multiple myeloma bone marrow samples from 18 patients assessed by single-cell RNA sequencing (scRNA-seq),mass cytometry (CyTOF),and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to understand the concordance of measurements among single-cell techniques. Cell type abundances are relatively consistent across the three approaches,while variations are observed in T cells,macrophages,and monocytes. Concordance and correlation analysis of cell type marker gene expression across different modalities highlighted the importance of choosing cell type marker genes best suited to particular modalities. By integrating data from these three assays,we found International Staging System stage 3 patients exhibited decreased CD4+ T/CD8+ T cells ratio. Moreover,we observed upregulation of RAC2 and PSMB9,in natural killer cells of fast progressors compared with those of nonprogressors,as revealed by both scRNA-seq and CITE-seq RNA measurement. This detailed examination of the immune microenvironment in multiple myeloma using multiple single-cell technologies revealed markers associated with multiple myeloma rapid progression which will be further characterized by the full-scale immune atlas project. SIGNIFICANCE scRNA-seq,CyTOF,and CITE-seq are increasingly used for evaluating cellular heterogeneity. Understanding their concordances is of great interest. To date,this study is the most comprehensive examination of the measurement of the immune microenvironment in multiple myeloma using the three techniques. Moreover,we identified markers predicted to be significantly associated with multiple myeloma rapid progression. View Publication

Pre-existent cardiovascular disease is a risk factor for weak anti-viral immunity,but underlying mechanisms remain undefined. Here,we report that patients with coronary artery disease (CAD) have macrophages (M??) that actively suppress the induction of helper T cells reactive to two viral antigens: the SARS-CoV2 Spike protein and the Epstein-Barr virus (EBV) glycoprotein 350. CAD M?? overexpressed the methyltransferase METTL3,promoting the accumulation of N�?-methyladenosine (m6A) in Poliovirus receptor (CD155) mRNA. m6A modifications of positions 1635 and 3103 in the 3'UTR of CD155 mRNA stabilized the transcript and enhanced CD155 surface expression. As a result,the patients' M?? abundantly expressed the immunoinhibitory ligand CD155 and delivered negative signals to CD4+ T cells expressing CD96 and/or TIGIT receptors. Compromised antigen-presenting function of METTL3hi CD155hi M?? diminished anti-viral T cell responses in vitro and in vivo. LDL and its oxidized form induced the immunosuppressive M?? phenotype. Undifferentiated CAD monocytes had hypermethylated CD155 mRNA,implicating post-transcriptional RNA modifications in the bone-marrow in shaping anti-viral immunity in CAD. View Publication

Exosomes derived from mesenchymal stem cells (MSCs) could protect against myocardial infarction (MI). TLR4 is reported to play an important role in MI,while microRNA-182-5p (miR-182-5p) negatively regulates TLR4 expression. Therefore,we hypothesize that MSCs-derived exosomes overexpressing miR-182-5p may have beneficial effects on MI. We generated bone marrow mesenchymal stem cells (BM-MSCs) and overexpressed miR-182-5p in these cells for exosome isolation. H2O2-stimulated neonatal mouse ventricle myocytes (NMVMs) and MI mouse model were employed,which were subjected to exosome treatment. The expression of inflammatory factors,heart function,and TLR4 signaling pathway activation were monitored. It was found that miR-182-5p decreased TLR4 expression in BM-MSCs and NMVMs. Administration of exosomes overexpressing miR-182-5p to H2O2-stimulated NMVMs enhanced cell viability and suppressed the expression of inflammatory cytokines. In addition,they promoted heart function,suppressed inflammatory responses,and de-activated TLR4/NF-$\kappa$B signaling pathway in MI mice. In conclusion,miR-182-5p transferred by the exosomes derived from BM-MSCs protected against MI-induced impairments by targeting TLR4. View Publication

BACKGROUND Specific immune response is a hallmark of cancer immunotherapy and shared tumor-associated antigens (TAAs) are important targets. Recent advances using combined cellular therapy against multiple TAAs renewed the interest in this class of antigens. Our study aims to determine the role of TAAs in esophago-gastric adenocarcinoma (EGA). METHODS RNA expression was assessed by NanoString in tumor samples of 41 treatment-na{\{i}}ve EGA patients. Endogenous T cell and antibody responses against the 10 most relevant TAAs were determined by FluoroSpot and protein-bound bead assays. Digital image analysis was used to evaluate the correlation of TAAs and T-cell abundance. T-cell receptor sequencing in vitro expansion with autologous CD40-activated B cells (CD40Bs) and in vitro cytotoxicity assays were applied to determine specific expansion clonality and cytotoxic activity of expanded T cells. RESULTS 68.3% of patients expressed ??5 TAAs simultaneously with coregulated clusters which were similar to data from The Cancer Genome Atlas (n=505). Endogenous cellular or humoral responses against ??1??TAA were detectable in 75.0% and 53.7% of patients respectively. We found a correlation of T-cell abundance and the expression of TAAs and genes related to antigen presentation. TAA-specific T-cell responses were polyclonal could be induced or enhanced using autologous CD40Bs and were cytotoxic in vitro. Despite the frequent expression of TAAs co-occurrence with immune responses was rare. CONCLUSIONS We identified the most relevant TAAs in EGA for monitoring of clinical trials and as therapeutic targets. Antigen-escape rather than missing immune response should be considered as mechanism underlying immunotherapy resistance of EGA." View Publication

The single antigen bead (SAB) assay is the most used test for the identification of HLA specific antibodies pre- and post-transplant. Nevertheless,detection of spurious reactivities remains a recognized assay limitation. In addition,the presence of weak reactivity patterns can complicate unacceptable antigen assignment. This work presents the evaluation of the adsorption with crossmatch cells and elution (AXE) technique,which was designed to help differentiate weak HLA specific antibodies targeting native antigens from spurious and background SAB assay reactivity. The AXE protocol uses selected donor cells to adsorb HLA specific antibodies from sera of interest. Bound antibodies are then eluted off washed cells and identified using the SAB assay. Only antibodies targeting native HLA are adsorbed. Assay evaluation was performed using five cell donors and pooled positive control serum. AXE efficiency was determined by comparing SAB reactivity of adsorbed/eluted antibody to that of the antibodies in unadsorbed sera. A robust efficiency was seen across a wide range of original MFI for donor specific antibodies (DSA). A higher absorption/elution recovery was observed for HLA class I antigens vs. class II. Locus-specific variation was also observed,with high-expression HLA loci (HLA-A/B/DR) providing the best recovery. Importantly,negligible reactivity was detected in the last wash control,confirming that AXE eluates were not contaminated with HLA antibody carry-over. Donor cells incubated with autologous and DSA-containing allogeneic sera showed that AXE selectively adsorbed HLA antibodies in a donor antigen-specific manner. Importantly,antibodies targeting denatured epitopes or other non-HLA antigens were not detected by AXE. AXE was particularly effective at distinguishing weak HLA antibodies from background reactivity. When combined with epitope analysis,AXE enhanced precise identification of antibody-targeted eplets and even facilitated the characterization of a potential novel eplet. Comparison of AXE to flow cytometric crossmatching further revealed that AXE was a more sensitive technique in the detection of weak DSA. Spurious reactivities on the current SAB assay have a deleterious impact on the assignment of clinically relevant HLA specificities. The AXE protocol is a novel test that enables users to interrogate reactive patterns of interest and discriminate HLA specific antibodies from spurious reactivity. View Publication

BACKGROUND There is considerable evidence that epigenetic mechanisms and DNA methylation are critical drivers of immune cell lineage differentiation and activation. However,there has been limited coordinated investigation of common epigenetic pathways among cell lineages. Further,it remains unclear if long-lived memory cell subtypes differentiate distinctly by cell lineages. RESULTS We used the Illumina EPIC array to investigate the consistency of DNA methylation in B cell,CD4 T,and CD8 T na{\{i}}ve and memory cells states. In the process of na{\"{i}}ve to memory activation across the three lineages we identify considerable shared epigenetic regulation at the DNA level for immune memory generation. Further in central to effector memory differentiation our analyses revealed specific CpG dinucleotides and genes in CD4 T and CD8 T cells with DNA methylation changes. Finally we identified unique DNA methylation patterns in terminally differentiated effector memory (TEMRA) CD8 T cells compared to other CD8 T memory cell subtypes. CONCLUSIONS Our data suggest that epigenetic alterations are widespread and essential in generating human lymphocyte memory. Unique profiles are involved in methylation changes that accompany memory genesis in the three subtypes of lymphocytes." View Publication

Asthmatic women tend to develop severe airway disease in their reproductive years,and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells,or ILC2,are readily detected in allergic conditions,such as rhinosinusitis,in individuals that develop nasal polyps do to allergen exposures,and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33,an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in na{\{i}}ve and ovalbumin (OVA) challenged female BALB/c mice in comparison to male mice following stimulation with IL-33. Here we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-$\kappa$B signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated and had reduced NF-$\kappa$B activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2 and Nfkb1 was found among top-ranked regulatory interactions. Together these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases." View Publication

Neutrophils are central players in the innate immune system. To protect against invading pathogens,neutrophils can externalize chromatin to create neutrophil extracellular traps (NETs). While NETs are critical to host defense,they also have deleterious effects,and dysregulation of NETs formation has been implicated in autoimmune diseases,atherosclerosis and thrombotic conditions,cancer progression and dissemination,and acute respiratory distress syndrome. Here,we report that selinexor,a first-in-class selective inhibitor of nuclear export approved for the treatment of multiple myeloma and diffuse large B-cell lymphoma,markedly suppressed the release of NETs in vitro. Furthermore,we demonstrate a significant inhibitory effect of selinexor on NETs formation,but not on oxidative burst or enzymatic activities central to NETs release such as neutrophil elastase,myeloperoxidase or peptidyl arginine deiminase type IV. The inhibitory effect of selinexor was demonstrated in neutrophils activated by a variety of NETs-inducers,including PMA,TGF-$\beta$,TNF-$\alpha$ and IL-8. Maximal inhibition of NETs formation was observed using TGF-$\beta$,for which selinexor inhibited NETs release by 61.6%. These findings pave the way to the potential use of selinexor in an effort to reduce disease burden by inhibition of NETs. View Publication

Tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF) and their precursor mesenchymal stromal cells (MSC) are often detected together in tumors,but how they cooperate is not well understood. Here,we show that TAM and CAF are the most abundant nonmalignant cells and are present together in untreated human neuroblastoma (NB) tumors that are also poorly infiltrated with T and natural killer (NK) cells. We then show that MSC and CAF-MSC harvested from NB tumors protected human monocytes (MN) from spontaneous apoptosis in an interleukin (IL)-6 dependent mechanism. The interactions of MN and MSC with NB cells resulted in a significant induction or increase in the expression of several pro-tumorigenic cytokines/chemokines (TGF-$\beta$1,MCP-1,IL-6,IL-8,and IL-4) but not of anti-tumorigenic cytokines (TNF-$\alpha$,IL-12) by MN or MSC,while also inducing cytokine expression in quiescent NB cells. We then identified a TGF-$\beta$1/IL-6 pathway where TGF-$\beta$1 stimulated the expression of IL-6 in NB cells and MSC,promoting TAM survival. Evidence for the contribution of TAM and MSC to the activation of this pathway was then provided in xenotransplanted NB tumors and patients with primary tumors by demonstrating a direct correlation between the presence of CAF and p-SMAD2 and p-STAT3. The data highlight a new mechanism of interaction between TAM and CAF supporting their pro-tumorigenic function in cancer. View Publication