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BackgroundPlasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins are expressed on the surface of infected erythrocytes,mediating parasite sequestration in the vasculature. PfEMP1 is a major target of protective antibodies,but the features of the antibody response are poorly defined.MethodsIn Malawian children with cerebral or uncomplicated malaria,we characterized the antibody response to 39 recombinant PfEMP1 Duffy binding like (DBL) domains or cysteine-rich interdomain regions (CIDRs) in detail,including measures of antibody classes,subclasses,and engagement with Fcγ receptors and complement. Using elastic net regularized logistic regression,we identified a combination of seven antibody targets and Fc features that best distinguished between children with cerebral and uncomplicated malaria. To confirm the role of the selected targets and Fc features,we measured antibody-dependent neutrophil and THP-1 cell phagocytosis of intercellular adhesion molecule-1 (ICAM-1) and endothelial protein C (EPCR) co-binding infected erythrocytes.ResultsThe selected features distinguished between children with cerebral and uncomplicated malaria with 87% accuracy (median,80–96% interquartile range) and included antibody to well-characterized DBLβ3 domains and a less well-characterized CIDRγ12 domain. The abilities of antibodies to engage C1q and FcγRIIIb,rather than levels of IgG,correlated with protection. In line with a role of FcγRIIIb binding antibodies to DBLβ3 domains,antibody-dependent neutrophil phagocytosis of ICAM-1 and EPCR co-binding IE was higher in uncomplicated malaria (15% median,8–38% interquartile range) compared to cerebral malaria (7%,30–15%,p < 0.001).ConclusionsAntibodies associated with protection from cerebral malaria target a subset of PfEMP1 domains. The Fc features of protective antibody response include engagement of FcγRIIIb and C1q,and ability to induce antibody-dependent neutrophil phagocytosis of infected erythrocytes. Identifying the targets and Fc features of protective immunity could facilitate the development of PfEMP1-based therapeutics for cerebral malaria.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12916-024-03604-8. View Publication

Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. Here,we investigate the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using an in vitro model for exhaustion,RNA and ATAC sequencing on baseline and exhausted CART cells,and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identify interleukin (IL)-4 as a regulator of CART cell dysfunction. Further,IL-4-treated CD8+ CART cells develop signs of exhaustion independently of the presence of CD4+ CART cells. Conversely,IL-4 pathway editing or the combination of CART cells with an IL-4 monoclonal antibody improves antitumor efficacy and reduces signs of CART cell exhaustion in mantle cell lymphoma xenograft mouse models. Therefore,we identify both a role for IL-4 in inducing CART exhaustion and translatable approaches to improve CART cell therapy. The application and therapeutic success of CAR-T cell approaches are limited by the development of T cell exhaustion. Here,Stewart et al discover a role for IL-4 in driving CD8+ CAR-T cell exhaustion and demonstrate the improvement of CAR-T cell effectivity with interruption of IL-4 signalling. View Publication

A diverse antibody repertoire is essential for humoral immunity. Antibody diversification requires the introduction of deoxyuridine (dU) mutations within immunoglobulin genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR). dUs are normally recognized and excised by the base excision repair (BER) protein uracil-DNA glycosylase 2 (UNG2). However,FAM72A downregulates UNG2 permitting dUs to persist and trigger SHM and CSR. How FAM72A promotes UNG2 degradation is unknown. Here,we show that FAM72A recruits a C-terminal to LisH (CTLH) E3 ligase complex to target UNG2 for proteasomal degradation. Deficiency in CTLH complex components result in elevated UNG2 and reduced SHM and CSR. Cryo-EM structural analysis reveals FAM72A directly binds to MKLN1 within the CTLH complex to recruit and ubiquitinate UNG2. Our study further suggests that FAM72A hijacks the CTLH complex to promote mutagenesis in cancer. These findings show that FAM72A is an E3 ligase substrate adaptor critical for humoral immunity and cancer development. Antibody diversification relies on the intentional mutagenesis of immunoglobulin genes for adaptive immune responses. Here,the authors identified a CTLH E3 ubiquitin ligase complex that co-opts FAM72A to recruit and degrade the UNG2 base excision repair factor to permit mutagenesis. View Publication

Dendritic cells (DCs) are pivotal in regulating allergic asthma. Our research has shown that the absence of Sema3E worsens asthma symptoms in acute and chronic asthma models. However,the specific role of PlexinD1 in these processes,particularly in DCs,remains unclear. This study investigates the role of PlexinD1 in CD11c+ DCs using a house dust mite (HDM) model of asthma. We generated CD11c+ DC-specific PlexinD1 knockout (CD11cPLXND1 KO) mice and subjected them,alongside wild-type controls (PLXND1fl/fl),to an HDM allergen protocol. Airway hyperresponsiveness (AHR) was measured using FlexiVent,and immune cell populations were analyzed via flow cytometry. Cytokine levels and immunoglobulin concentrations were assessed using mesoscale and ELISA,while collagen deposition and mucus production were examined through Sirius-red and periodic acid Schiff (PAS) staining respectively. Our results indicate that CD11cPLXND1 KO mice exhibit significantly exacerbated AHR,characterized by increased airway resistance and tissue elastance. Enhanced mucus production and collagen gene expression were observed in these mice compared to wild-type counterparts. Flow cytometry revealed higher CD11c+ MHCIIhigh CD11b+ cell recruitment into the lungs,and elevated total and HDM-specific serum IgE levels in CD11cPLXND1 KO mice. Mechanistically,co-cultures of B cells with DCs from CD11cPLXND1 KO mice showed significantly increased IgE production compared to wild-type mice.These findings highlight the critical regulatory role of the plexinD1 signaling pathway in CD11c+ DCs in modulating asthma features. View Publication

A portion of the genetic basis for many common autoimmune disorders has been uncovered by genome-wide association studies (GWAS),but GWAS do not reveal causal variants,effector genes,or the cell types impacted by disease-associated variation. We have generated 3D genomic datasets consisting of promoter-focused Capture-C,Hi-C,ATAC-seq,and RNA-seq and integrated these data with GWAS of 16 autoimmune traits to physically map disease-associated variants to the effector genes they likely regulate in 57 human cell types. These 3D maps of gene cis-regulatory architecture are highly powered to identify the cell types most likely impacted by disease-associated genetic variation compared to 1D genomic features,and tend to implicate different effector genes than eQTL approaches in the same cell types. Most of the variants implicated by these cis-regulatory architectures are highly trait-specific,but nearly half of the target genes connected to these variants are shared across multiple autoimmune disorders in multiple cell types,suggesting a high level of genetic diversity and complexity among autoimmune diseases that nonetheless converge at the level of target gene and cell type. Substantial effector gene sharing led to the common enrichment of similar biological networks across disease and cell types. However,trait-specific pathways representing potential areas for disease-specific intervention were identified. To test this,we pharmacologically validated squalene synthase,a cholesterol biosynthetic enzyme encoded by the FDFT1 gene implicated by our approach in MS and SLE,as a novel immunomodulatory drug target controlling inflammatory cytokine production by human T cells. These data represent a comprehensive resource for basic discovery of gene cis-regulatory mechanisms,and the analyses reported reveal mechanisms by which autoimmune-associated variants act to regulate gene expression,function,and pathology across multiple,distinct tissues and cell types. View Publication

IntroductionThe early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins,including the E3/49K protein,which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45,thereby modulating activation of T and NK cells.MethodsConsidering that E3/49K represents the most polymorphic viral protein among species D HAdVs,we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus,this feature is conserved regardless of the pathological associations of the respective HAdV types.ResultsIt appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover,E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45,suggesting E3/49K as a potential tool to investigate CD45 specific functions.ConclusionWe identified B cells as new direct target of E3/49K-mediated immune modulation,representing a novel viral immunosubversive mechanism. View Publication

AbstractChromobox 2 (CBX2),an epigenetic reader and component of polycomb repressor complex 1,is highly expressed in >75% of high-grade serous carcinoma. Increased CBX2 expression is associated with poorer survival,whereas CBX2 knockdown leads to improved chemotherapy sensitivity. In a high-grade serous carcinoma immune-competent murine model,knockdown of CBX2 decreased tumor progression. We sought to explore the impact of modulation of CBX2 on the tumor immune microenvironment (TIME),understanding that the TIME plays a critical role in disease progression and development of therapy resistance. Exploration of existing datasets demonstrated that elevated CBX2 expression significantly correlated with specific immune cell types in the TIME. RNA sequencing and pathway analysis of differentially expressed genes demonstrated immune signature enrichment. Confocal microscopy and co-culture experiments found that modulation of CBX2 leads to increased recruitment and infiltration of macrophages. Flow cytometry of macrophages cultured with CBX2-overexpressing cells showed increased M2-like macrophages and decreased phagocytosis activity. Cbx2 knockdown in the Trp53-null,Brca2-null ID8 syngeneic murine model (ID8 Trp53−/−Brca2−/−) led to decreased tumor progression compared with the control. NanoString immuno-oncology panel analysis suggested that knockdown in Cbx2 shifts immune cell composition,with an increase in macrophages. Multispectral immunohistochemistry (mIHC) further confirmed an increase in macrophage infiltration. Increased CBX2 expression leads to recruitment and polarization of protumor macrophages,and targeting CBX2 may serve to modulate the TIME to enhance the efficacy of immune therapies.Significance:CBX2 expression correlates with the TIME. CBX2 modulation shifts the macrophage population,potentially leading to an immunosuppressive microenvironment,highlighting CBX2 as a target to improve efficacy of immunotherapy. View Publication

IntroductionHeme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation,and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors,and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation,that is,NOD-like receptor protein 3 (NLRP3),glutathione (GSH),GSH disulfide (GSSG),and interleukin 6 (IL-6).MethodsThe study population comprised three age groups: young adults (age range,19–23 years),older adults aged under 85 years (age range,73–84 years),and older adults aged over 85 years (age range,85–92 years). NLRP3,GSH,and GSSG concentrations were measured in serum,whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48 h and stimulated with IL-2,lipopolysaccharide (LPS),or phorbol 12-myristate 13-acetate (PMA) with ionomycin.ResultsThe analysis of serum NLRP3,GSH,and GSSG concentrations revealed no statistically significant differences among the studied age groups. However,some typical trends of aging were observed,such as a decrease in GSH concentration and an increase in both GSSG level,and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover,statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells.ConclusionsThese results showed that NK cells can express HO-1 at a basal level,which was significantly increased in activated cells,even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters. View Publication

IntroductionHumanization is typically adopted to reduce the immunogenicity of murine antibodies generated by hybridoma technology when used in humans.MethodsTwo different strategies of antibody humanization are popularly employed,including “complementarity determining region (CDR) grafting” and “framework (FR) shuffling” to humanize a murine antibody against human programmed death-1 (PD-1),XM PD1. In CDR-grafting humanization,the CDRs of XM PD-1,were grafted into the human FR regions with high homology to the murine FR counterparts,and back mutations of key residues were performed to retain the antigen-binding affinities. While in FR-shuffling humanization,a combinatorial library of the six murine CDRs in-frame of XM PD-1 was constructed to a pool of human germline FRs for high-throughput screening for the most favorable variants. We evaluated many aspects which were important during antibody development of the molecules obtained by the two methods,including antibody purity,thermal stability,binding efficacy,predicted humanness,and immunogenicity,along with T cell epitope prediction for the humanized antibodies.ResultsWhile the ideal molecule was not achieved through CDR grafting in this particular instance,FR-shuffling proved successful in identifying a suitable candidate. The study highlights FR-shuffling as an effective complementary approach that potentially increases the success rate of antibody humanization. It is particularly noted for its accessibility to those with a biological rather than a computational background. DiscussionThe insights from this comparison are intended to assist other researchers in selecting appropriate humanization strategies for drug development,contributing to broader application and understanding in the field. View Publication

Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However,it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics,particularly those with Latin American ancestry. Using dPCR,we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both,suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification,early detection of cancers,and reduction of associated cancer health disparities. Rates of Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) continue to rise in Hispanics and Latinos. Authors developed a digital PCR assay to quantify activation-induced cytidine deaminase activity at risk loci involved in cancer etiology that may contribute to this health disparity. View Publication

MIF is a ubiquitous protein involved in proinflammatory processes,which undergoes an oxidation-driven conformational change to oxidized (ox)MIF. We demonstrate that hypochlorous acid,produced by neutrophil-released myeloperoxidase (MPO) under inflammatory conditions,effectively oxidizes MIF into the oxMIF isoform,which is specifically recognized by the anti-oxMIF therapeutic antibody,ON104. NMR investigation of MIF oxidized by the MPO system revealed increased flexibility throughout the MIF structure,including at several catalytic and allosteric sites. Mass spectrometry of MPO-oxMIF revealed methionines as the primary site of oxidation,whereas Pro2 and Tyr99/100 remained almost unmodified. ELISA,SPR and cell-based assays demonstrated that structural changes caused by MPO-driven oxidation promoted binding of oxMIF to its receptor,CD74,which does not occur with native MIF. These data reveal the environment and modifications that facilitate interactions between MIF and its pro-inflammatory receptor,and a route for therapeutic intervention targeting the oxMIF isoform. View Publication

Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways. However,targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here,we have developed an mRNA therapeutic for the lower lung using one-component Ionizable Amphiphilic Janus Dendrimers (IAJDs) as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA,transforming growth factor-beta (TGF-β),to produce transient protein expression in the lower regions of the lung. This study highlights IAJD’s potential for precise,effective,and safe delivery of TGF-β mRNA to the lung. This delivery system offers a promising approach for targeting therapeutics to the specific tissues,a strategy necessary to fill the current clinical gap in treating parenchymal lung injury and disease. View Publication