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The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state,and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes,such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs),which play broad regulatory roles in host gene expression. Still,it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here,we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is,at least in part,dependent on the viral Tat protein. Finally,we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART,most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together,these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation. Author summarySince the start of the HIV pandemic,it is estimated that nearly 86 million people have been infected with the virus,and about 40 million people have died. Modern antiretroviral therapies potently restrict viral replication and prevent the onset of AIDS,saving millions of lives. However,these therapies are not curative due to the persistence of the virus in a silenced or ‘latent’ state in long-lived cells of the body. One proposed strategy to clear this latent reservoir,termed “shock and kill”,is to activate these silenced viruses such that the infected cells can be cleared from the body by the immune system. While several drugs have been developed that can activate latent viruses,none have proven effective at reducing the size of the latent reservoir in patients in clinical trials. Here,we describe a new method for latency reactivation using a small molecule inhibitor of a human protein complex called the Super Elongation Complex (SEC). Inhibiting the SEC enhances viral transcription during active infection and triggers the reactivation of latent viruses,especially when in combination with other latency reversing agents. These results pave the way for developing more effective strategies to reactivate latent viruses towards a functional cure. View Publication

Immunoglobulin G (IgG) is the main isotype of antibody in human blood. IgG consists of four subclasses (IgG1 to IgG4),encoded by separate constant region genes within the Ig heavy chain locus (IGH). Here,we report a genome-wide association study on blood IgG subclass levels. Across 4334 adults and 4571 individuals under 18 years,we discover ten new and identify four known variants at five loci influencing IgG subclass levels. These variants also affect the risk of asthma,autoimmune diseases,and blood traits. Seven variants map to the IGH locus,three to the Fcγ receptor (FCGR) locus,and two to the human leukocyte antigen (HLA) region,affecting the levels of all IgG subclasses. The most significant associations are observed between the G1m (f),G2m(n) and G3m(b*) allotypes,and IgG1,IgG2 and IgG3,respectively. Additionally,we describe selective associations with IgG4 at 16p11.2 (ITGAX) and 17q21.1 (IKZF3,ZPBP2,GSDMB,ORMDL3). Interestingly,the latter coincides with a highly pleiotropic signal where the allele associated with lower IgG4 levels protects against childhood asthma but predisposes to inflammatory bowel disease. Our results provide insight into the regulation of antibody-mediated immunity that can potentially be useful in the development of antibody based therapeutics. Immunoglobulin G (IgG) is the main isotype of antibody in human blood. Here the authors describe 14 genetic variants that affect IgG levels in blood. The data provide new insight into the regulation of humoral immunity that could be useful in the development of antibody-based therapeutics. View Publication

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality worldwide. Laminar shear stress from blood flow,sensed by vascular endothelial cells,protects from ASCVD by upregulating the transcription factors KLF2 and KLF4,which induces an anti-inflammatory program that promotes vascular resilience. Here we identify clustered γ-protocadherins as therapeutically targetable,potent KLF2 and KLF4 suppressors whose upregulation contributes to ASCVD. Mechanistic studies show that γ-protocadherin cleavage results in translocation of the conserved intracellular domain to the nucleus where it physically associates with and suppresses signaling by the Notch intracellular domain. γ-Protocadherins are elevated in human ASCVD endothelium; their genetic deletion or antibody blockade protects from ASCVD in mice without detectably compromising host defense against bacterial or viral infection. These results elucidate a fundamental mechanism of vascular inflammation and reveal a method to target the endothelium rather than the immune system as a protective strategy in ASCVD. Joshi et al. show that γ-protocadherins suppress the anti-inflammatory KLF2 and KLF4 pathway and that targeting them is a viable therapeutic strategy to protect against atherosclerosis. View Publication

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

Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection,but its role has not been explored during CT infection. In the female reproductive tract (FRT),CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described,they could be at the origin of the pathology observed during CT infection. During this study,we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover,the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT,in presence or not of the pool of SP,had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation,activation and adhesion capacity,as well as the survival,ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment,in particular SP and CT infection,on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female. View Publication

Regulatory T cells (Tregs) are crucial immune cells for tissue repair and regeneration. However,their potential as a cell-based regenerative therapy is not yet fully understood. Here,we show that local delivery of exogenous Tregs into injured mouse bone,muscle,and skin greatly enhances tissue healing. Mechanistically,exogenous Tregs rapidly adopt an injury-specific phenotype in response to the damaged tissue microenvironment,upregulating genes involved in immunomodulation and tissue healing. We demonstrate that exogenous Tregs exert their regenerative effect by directly and indirectly modulating monocytes/macrophages (Mo/MΦ) in injured tissues,promoting their switch to an anti-inflammatory and pro-healing state via factors such as interleukin (IL)-10. Validating the key role of IL-10 in exogenous Treg-mediated repair and regeneration,the pro-healing capacity of these cells is lost when Il10 is knocked out. Additionally,exogenous Tregs reduce neutrophil and cytotoxic T cell accumulation and IFN-γ production in damaged tissues,further dampening the pro-inflammatory Mo/MΦ phenotype. Highlighting the potential of this approach,we demonstrate that allogeneic and human Tregs also promote tissue healing. Together,this study establishes exogenous Tregs as a possible universal cell-based therapy for regenerative medicine and provides key mechanistic insights that could be harnessed to develop immune cell-based therapies to enhance tissue healing. Regulatory T cells (Tregs) are known for suppressing inflammatory processes,but their full capacity for tissue regeneration is yet to be harnessed. Here,the authors demonstrate the efficiency of Tregs in facilitating tissue healing in mouse models of bone,muscle,and skin injury,with monocytes/macrophages and interleukin-10 playing a key mechanistic role in the process. View Publication

Small extracellular vesicles (sEVs) are important mediators of intercellular communication between tumor cells and their surrounding environment. Furthermore,the mechanisms by which miRNAs carried in tumor sEVs regulate macrophage polarization remain largely unknown. To concentrate sEVs,we used the traditional ultracentrifugation method. Western blot,NanoSight,and transmission electron microscopy were used to identify sEVs. To determine the function of sEVs-miR-487a,we conducted in vivo and in vitro investigations. The intercellular communication mechanism between osteosarcoma cells and M2 macrophages,mediated by sEVs carrying miR-487a,was validated using luciferase reporter assays,transwell assays,and Western blot analysis. In vitro,sEVs enriched in miR-487a and delivered miR-487a to macrophages,promoting macrophage polarization toward an M2-like type,which promotes proliferation,migration,invasion,and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. In vivo,sEVs enriched in miR-487a facilitate lung metastasis of osteosarcoma. Moreover,plasma miR-487a in sEVs was shown to be a potential biomarker applicable for osteosarcoma diagnosis. In summary,miR-487a derived from osteosarcoma cells can be transferred to macrophages via sEVs,then promote macrophage polarization towards an M2-like type by targeting Notch2 and activating the GATA3 pathway. In a feedback loop,the activation of macrophages accelerates epithelial-mesenchymal transition (EMT),which in turn promotes the migration,invasion,and lung metastasis of osteosarcoma cells. This reciprocal interaction between activated macrophages and osteosarcoma cells contributes to the progression of the disease. Our data demonstrate a new mechanism that osteosarcoma tumor cells derived exosomal-miR-487a which is involved in osteosarcoma development by regulating macrophage polarization in tumor microenvironment (TME).Supplementary InformationThe online version contains supplementary material available at 10.1186/s12935-024-03488-x. 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

CD19-targeted chimeric antigen receptor (CAR) T cell therapies have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However,>50% of CD19-CAR-T-treated patients experience progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore,it remains a challenge to efficiently,cost-effectively,and consistently manufacture clinically relevant numbers of virally engineered CAR-T cells. Using a highly efficient piggyBac transposon-based vector,Quantum pBac™ (qPB),we developed a virus-free cell-engineering system for development and production of multiplex CAR-T therapies. Here,we demonstrate in vitro and in vivo that consistent,robust and functional CD20/CD19 dual-targeted CAR-T stem cell memory (CAR-TSCM) cells can be efficiently produced for clinical application using qPB™. In particular,we showed that qPB™-manufactured CAR-T cells from cancer patients expanded efficiently,rapidly eradicated tumors,and can be safely controlled via an iCasp9 suicide gene-inducing drug. Therefore,the simplicity of manufacturing multiplex CAR-T cells using the qPB™ system has the potential to improve efficacy and broaden the accessibility of CAR-T therapies. View Publication

AbstractIntroductionNovel strategies are needed to address the rising burden of osteoporosis and fragility fractures. High-intensity resistance and impact (HiRIT) exercise has shown benefit in improving bone density in postmenopausal women with osteoporosis/osteopenia. Whether HiRIT can enhance the therapeutic effects of osteoporosis pharmacotherapy has not been established. ROLEX-DUO is a randomised controlled trial designed to assess the efficacy of romosozumab on various bone and muscle outcomes in combination with different exercise interventions in women with postmenopausal osteoporosis/osteopenia.Methods and analysisROLEX-DUO is an 8-month randomised placebo-controlled trial conducted at two tertiary referral centres for patients with osteoporosis/osteopenia in Sydney,New South Wales,Australia. The study is implementing the combination of romosozumab or placebo with different forms of exercise in postmenopausal women with osteoporosis/osteopenia without recent fragility fracture (n=102). Eligible women will be randomised 1:1:1 into one of three groups: (1) romosozumab with supervised HiRIT,(2) romosozumab with unsupervised low-intensity exercise or (3) placebo with unsupervised low-intensity exercise. Co-primary outcomes are the mean percentage change in lumbar spine bone mineral density (BMD),and mean change in five times sit-to-stand test performance (seconds) at 8 months. Secondary/exploratory outcomes include BMD changes at the femoral neck,total hip and distal radius,three-dimensional dual-energy X-ray absorptiometry (DXA) hip outcomes,DXA-derived lean and fat mass,serum markers of bone turnover (procollagen type 1 peptide,C-telopeptide of type 1 collagen) and bone biomarkers (dickkopf-1),serum extracellular vesicle analyses,36-Item Short Form Survey (SF-36) quality-of-life scores,Menopause-Specific Quality Of Life (MENQOL) Questionnaire menopause symptom burden scores,number of falls and fractures. Mixed-effects models will be performed to compare longitudinal outcome results between groups using intention-to-treat analysis.Ethics and disseminationThe trial was approved by the Northern Sydney Local Health District Human Research Ethics Committee (2022/ETH01794,protocol V.8,dated 03 July 2024). Participants will provide written informed consent prior to inclusion. Findings will be disseminated via peer-reviewed journals,scientific conferences and summary reports to funding bodies.Trial registration numberACTRN12623000867695. 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

mRNA applications have undergone unprecedented applications—from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation,involving high costs,risks,and toxicity,emphasizing the need for alternative way as mRNA technology. We successfully developed,screened,and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells,KHYG-1 cells,and primary NK cells with high efficiency without cytotoxicity,while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover,the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably,the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether,our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies. Graphical abstract Pichon and colleagues developed imidazole lipids-based mRNA platforms very efficient to transfect both NK-92 cells,KHYG-1 cells and primary NK cells without cytotoxicity. They succeeded to replace IL-2 protein by IL-2 mRNA transfection and obtained NK cells with extended viability with preserved biomarkers and full functionalities to kill target cells. View Publication