技术资料

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

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

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

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

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

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

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

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

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

A well-balanced maternal immune system is crucial to maintain fetal tolerance in case of infections during pregnancy. Immune adaptations include an increased secretion of soluble mediators to protect the semi-allogeneic fetus from excessive pro-inflammatory response. B lymphocytes acquire a higher capacity to express CD83 and secrete soluble CD83 (sCD83) upon exposure to bacteria-derived components such as LPS. CD83 possesses immune modulatory functions and shows a promising therapeutic potential against inflammatory conditions. The administration of sCD83 to pregnant mice reduces LPS-induced abortion rates. The increased CD83 expression by endometrial B cells as compared to peripheral blood B cells suggests its modulatory role in the fetal tolerance,especially in the context of infection. We postulate that in pregnancy,CD83 expression and release is controlled by pregnancy-related hormones. The intra- and extracellular expression of CD83 in leukocytes from peripheral blood or decidua basalis and parietalis at term were analyzed by flow cytometry. After treatment with pregnancy-related hormones and LPS,ELISA and qPCR were performed to study sCD83 release and CD83 gene expression,respectively. Cleavage prediction analysis was used to find potential proteases targeting CD83. Expression of selected proteases was analyzed by ELISA. Higher levels of CD83 were found in CD11c+ dendritic cells,CD3+ T cells and CD19+ B cells from decidua basalis and decidua parietalis after LPS-stimulation in vitro. An increase of intracellular expression of CD83 was also detected in CD19+ B cells from both compartments. Stimulated B cells displayed significantly higher percentages of CD83+ cells than dendritic cells and T cells from decidua basalis and peripheral blood. Treatment of B lymphocytes with pregnancy-related molecules (E2,P4,TGF-β1 and hCG) enhanced the LPS-mediated increase of CD83 expression,while dexamethasone led to a reduction. Similarly,the release of sCD83 was increased under TGF-β1 treatment but decreased upon dexamethasone stimulation. Finally,we found that the hormonal regulation of CD83 expression is likely a result from a balance between gene transcription from CD83 and the modulation of the metalloproteinase MMP-7. Thus,data supports and complements our previous murine studies on hormonal regulation of CD83 expression,reinforcing its immunomodulatory relevance in anti-bacterial responses during pregnancy. View Publication

Background:A primary barrier to curing HIV is the HIV reservoir. The leading infectious cause of death worldwide for people living with HIV is tuberculosis (TB),but we do not know how TB impacts the HIV reservoir.Methods:Participants in identification and validation cohorts were selected from previously enrolled studies at Groupe Haïtien d'Étude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) in Port au Prince,Haiti. Intact and non-intact proviral DNA were quantified using droplet digital PCR of peripheral blood mononuclear cell (PBMC)-derived CD4+ T cells. Kruskal-Wallis tests were used to compare medians with tobit regression for censoring.Results:In the identification cohort,we found that people living with HIV with a history of active pulmonary TB (n=19) had higher levels of intact provirus than people living with HIV without a history of active TB (n=47) (median 762; IQR,183-1173 vs 117; IQR,24-279 intact provirus per million CD4,respectively; P=0.0001). This difference also was seen in the validation cohort (n=31),(median 102; IQR,0-737 vs 0; IQR,0-24.5 intact provirus per million CD4,P=0.03) for TB vs no-TB history groups,respectively. The frequencies of CD4+ T cells with any detectable proviral fragment was directly proportional to the levels of interleukin-1 beta (r=0.524,P= 0.0025) and interleukin-2 (r=0.622,P=0.0002).Conclusions:People living with HIV with a history of active pulmonary TB have more HIV pro-virus in their circulating CD4+ T cells,even years after TB cure. We need to characterize which CD4+ T cells are harboring intact provirus to consider the impact of T cell-targeting HIV cure interventions for people living in TB-endemic areas. View Publication

Single-cell RNA sequencing (scRNA-seq) can resolve transcriptional features from individual cells,but scRNA-seq techniques capable of resolving the variable regions of B cell receptors (BCRs) remain limited,especially from widely-used 3′-barcoded libraries. Here,we report a method that can recover paired,full-length variable region sequences of BCRs from 3′-barcoded scRNA-seq libraries. We first verify this method (B3E-seq) can produce accurate,full-length BCR sequences. We then apply this method to profile B cell responses elicited against the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (ST3) by glycoconjugate vaccines in five infant rhesus macaques. We identify BCR features associated with specificity for the ST3 antigen which are present in multiple vaccinated monkeys,indicating a convergent response to vaccination. These results demonstrate the utility of our method to resolve key features of the B cell repertoire and profile antigen-specific responses elicited by vaccination. A method that recovers full-length,paired heavy- and light-chain variable regions of B cell receptor transcripts from 3’barcoded scRNA-seq libraries reveals a convergent response to pneumococcus vaccination in rhesus macaques. View Publication