技术资料

AbstractImmunodeficient mouse models are widely used for the assessment of human normal and leukemic stem cells. Despite the advancements over the years,reproducibility,as well as the differences in the engraftment of human cells in recipient mice remains to be fully resolved. Here,we used various immunodeficient mouse models to characterize the effect of donor–recipient sex on the engraftment of the human leukemic and healthy cells. Donor human cells and recipient immunodeficient mice demonstrate sex‐specific engraftment levels with significant differences observed in the lineage output of normal CD34+ hematopoietic stem and progenitor cells upon xenotransplantation. Intriguingly,human female donor cells display heightened sensitivity to the recipient mice's gender,influencing their proliferation and resulting in significantly increased engraftment in female recipient mice. Our study underscores the intricate interplay taking place between donor and recipient characteristics,shedding light on important considerations for future studies,particularly in the context of pre‐clinical research. View Publication

Toll-like receptor 9 (TLR9) recognizes bacterial,viral and self DNA and play an important role in immunity and inflammation. However,the role of TLR9 in obesity is less well-studied. Here,we generate B-cell-specific Tlr9-deficient (Tlr9fl/fl/Cd19Cre+/-,KO) B6 mice and model obesity using a high-fat diet. Compared with control mice,B-cell-specific-Tlr9-deficient mice exhibited increased fat tissue inflammation,weight gain,and impaired glucose and insulin tolerance. Furthermore,the frequencies of IL-10-producing-B cells and marginal zone B cells were reduced,and those of follicular and germinal center B cells were increased. This was associated with increased frequencies of IFNγ-producing-T cells and increased follicular helper cells. In addition,gut microbiota from the KO mice induced a pro-inflammatory state leading to immunological and metabolic dysregulation when transferred to germ-free mice. Using 16 S rRNA gene sequencing,we identify altered gut microbial communities including reduced Lachnospiraceae,which may play a role in altered metabolism in KO mice. We identify an important network involving Tlr9,Irf4 and Il-10 interconnecting metabolic homeostasis,with the function of B and T cells,and gut microbiota in obesity. Although the function of Toll-like receptor 9 (TLR9) in immunity and inflammation is well-established,its role in obesity is less well-studied. In this study,the authors demonstrate that TLR9 deficiency in B cells is associated with obesity in mice and results in altered frequencies of T and B lymphocyte subsets and gut microbiome dysbiosis. View Publication

Background:Latency reversing agents (LRAs) such as protein kinase C (PKC) modulators can reduce rebound-competent HIV reservoirs in small animal models. Furthermore,administration of natural killer (NK) cells following LRA treatment improves this reservoir reduction. It is currently unknown why the combination of a PKC modulator and NK cells is so potent and whether exposure to PKC modulators may augment NK cell function in some way.Methods:Primary human NK cells were treated with PKC modulators (bryostatin-1,prostratin,or the designed,synthetic bryostatin-1 analog SUW133),and evaluated by examining expression of activation markers by flow cytometry,analyzing transcriptomic profiles by RNA sequencing,measuring cytotoxicity by co-culturing with K562 cells,assessing cytokine production by Luminex assay,and examining the ability of cytokines and secreted factors to independently reverse HIV latency by co-culturing with Jurkat-Latency (J-Lat) cells.Results:PKC modulators increased expression of proteins involved in NK cell activation. Transcriptomic profiles from PKC-treated NK cells displayed signatures of cellular activation and enrichment of genes associated with the NFκB pathway. NK cell cytotoxicity was unaffected by prostratin but significantly decreased by bryostatin-1 and SUW133. Cytokines from PKC-stimulated NK cells did not induce latency reversal in J-Lat cell lines.Conclusions:Although PKC modulators have some significant effects on NK cells,their contribution in “kick and kill” strategies is likely due to upregulating HIV expression in CD4+ T cells,not directly enhancing the effector functions of NK cells. This suggests that PKC modulators are primarily augmenting the “kick” rather than the “kill” arm of this HIV cure approach. View Publication

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) remains the standard of care for chemotherapy-refractory leukemia patients,but cure rates are still dismal. To prevent leukemia relapse following HSCT,we aim to improve the early graft-versus-leukemia effect mediated by natural killer (NK) cells. Our approach is based on the adoptive transfer of Therapeutic Inducers of Natural Killer cell Killing (ThINKK). ThINKK are expanded and differentiated from HSC,and exhibit blood plasmacytoid dendritic cell (pDC) features. We previously demonstrated that ThINKK stimulate NK cells and control acute lymphoblastic leukemia (ALL) development in a preclinical mouse model of HSCT for ALL. Here,we assessed the cellular identity of ThINKK and investigated their potential to activate allogeneic T cells. We finally evaluated the effect of immunosuppressive drugs on ThINKK-NK cell interaction.MethodsThINKK cellular identity was explored using single-cell RNA sequencing and flow cytometry. Their T-cell activating potential was investigated by coculture of allogeneic T cells and antigen-presenting cells in the presence or the absence of ThINKK. A preclinical human-to-mouse xenograft model was used to evaluate the impact of ThINKK injections on graft-versus-host disease (GvHD). Finally,the effect of immunosuppressive drugs on ThINKK-induced NK cell cytotoxicity against ALL cells was tested.ResultsThe large majority of ThINKK shared the key characteristics of canonical blood pDC,including potent type-I interferon (IFN) production following Toll-like receptor stimulation. A minor subset expressed some,although not all,markers of other dendritic cell populations. Importantly,while ThINKK were not killed by allogeneic T or NK cells,they did not increase T cell proliferation induced by antigen-presenting cells nor worsened GvHD in vivo. Finally,tacrolimus,sirolimus or mycophenolate did not decrease ThINKK-induced NK cell activation and cytotoxicity.ConclusionOur results indicate that ThINKK are type I IFN producing cells with low T cell activation capacity. Therefore,ThINKK adoptive immunotherapy is not expected to increase the risk of GvHD after allogeneic HSCT. Furthermore,our data predict that the use of tacrolimus,sirolimus or mycophenolate as anti-GvHD prophylaxis regimen will not decrease ThINKK therapeutic efficacy. Collectively,these preclinical data support the testing of ThINKK immunotherapy in a phase I clinical trial. View Publication

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case of Trypanosoma cruzi infection,specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein,we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acute T. cruzi infection. Using the DEREG mouse model,we found that Treg cells play a role during the initial stages after T. cruzi infection,restraining the magnitude of CD8+ T cell responses and parasite control. Early Treg cell depletion increased the frequencies of polyfunctional short-lived,effector T cell subsets,without affecting memory precursor cell formation or the expression of activation,exhaustion and functional markers. In addition,Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell immunity. Crucially,the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses,preventing increased parasite replication in T. cruzi infected mice adoptively transferred with Treg cells. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model. Author summaryChagas disease,caused by Trypanosoma cruzi,can result in severe health complications. While the exact mechanisms underlying the disease’s pathogenesis remain incompletely understood,the host’s inflammatory immune response is believed to play a critical role. To shed light on disease mechanisms and potential treatments,we investigated the impact of regulatory T (Treg) cells on the development of effector immune responses against T. cruzi. Our findings reveal that Treg cells dampen parasite-specific CD8+ T cells,a crucial arm of the immune response in counteracting the parasite. Notably,this regulatory influence occurs primarily during the early stages of T. cruzi infection. Furthermore,we observed that while Treg cells have minimal effects on antigen-presenting cells,they modulate the magnitude and phenotype of conventional CD4+ T cells. Importantly,we identified CD39,a molecule involved in the purinergic pathway,as essential for the suppressive functions of Treg cells during T. cruzi infection. Our findings enhance the understanding of the regulatory response during the acute phase of T. cruzi infection and may have implications for the development of novel therapeutic strategies. View Publication

BackgroundCancer is the leading cause of death among older adults. Although the integration of immunotherapy has revolutionized the therapeutic landscape of cancer,the complex interactions between age and immunotherapy efficacy remain incompletely defined. Here,we aimed to elucidate the relationship between aging and immunotherapy resistance.MethodsFlow cytometry was performed to evaluate the infiltration of immune cells in the tumor microenvironment (TME). In vivo T cell proliferation,cytotoxicity and migration assays were performed to evaluate the antitumor capacity of tumor antigen-specific CD8+ T cells in mice. Real-time quantitative PCR (qPCR) was used to investigate the expression of IFN-γ-associated gene and natural killer (NK)-associated chemokine. Adoptive NK cell transfer was adopted to evaluate the effects of NK cells from young mice in overcoming the immunotherapy resistance of aged mice.ResultsWe found that elderly patients with advanced non-small cell lung cancer (aNSCLC) aged ≥ 75 years exhibited poorer progression-free survival (PFS),overall survival (OS) and a lower clinical response rate after immunotherapy. Mechanistically,we showed that the infiltration of NK cells was significantly reduced in aged mice compared to younger mice. Furthermore,the aged NK cells could also suppress the activation of tumor antigen-specific CD8+ T cells by inhibiting the recruitment and activation of CD103+ dendritic cells (DCs). Adoptive transfer of NK cells from young mice to aged mice promoted TME remodeling,and reversed immunotherapy resistance.ConclusionOur findings revealed the decreased sensitivity of elderly patients to immunotherapy,as well as in aged mice. This may be attributed to the reduction of NK cells in aged mice,which inhibits CD103+ DCs recruitment and its CD86 expression and ultimately leads to immunotherapy resistance.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40164-024-00511-9. View Publication

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration,thereby converting tumours to an immunologically cold phenotype. Moreover,synchronous analysis of multiple checkpoint molecules,including PD-1,CD80 and PD-L1,on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether,our study shows that sEVs carry multiple inhibitory immune checkpoints proteins,which form a potentially targetable adaptive loop to suppress antitumour immunity. Immune checkpoint inhibition is a successful form of immune therapy; however response rates vary widely among individual patients. Here authors show that circulating small extracellular vesicles might contribute to poor response to anti-PD-1 treatment by carrying PD-1 and CD80 which results in higher level of vesicular PD-L1 expression in the circulation at the expense of expression on tumour cell membranes,causing immunosuppression. View Publication

IntroductionSepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness.MethodsCellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression,RNA velocities,and biologic process clustering.ResultsWe identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally,we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome.DiscussionThe origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however,these cells exhibit a dynamic phenotype with considerable plasticity. View Publication

Innate antiviral factors are essential for effective defense against viral pathogens. However,the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here,we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN,CIITA,EHMT2,CEACAM3,CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene,we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry,transcription,release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle. Innate immune mechanisms are critical for antiviral defense. Here,the authors developed a CRISPR/Cas9-based HIV-driven approach to identify cellular factors compromising viral transcription,assembly,release or infectivity in human T cells. They identify targets of the Nef protein as antiviral factors. View Publication

Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here,we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore,gene repression in ZF-R-expressing T cells was highly efficient in vitro and in vivo during the entire monitoring period (up to 10 weeks),and it was accompanied by epigenetic remodeling events. Finally,we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion,we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing. Graphical abstract David Fenard and colleagues developed a lentiviral backbone for the multiplexing of up to three ZF-R sequences,allowing an efficient,stable,and specific epigenetic control of multiple genes in T cells or Tregs after a single lentiviral transduction event. View Publication

Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex,with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T cell population,a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP),where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. T cell products were evaluated using spectral flow cytometry,metabolic assays,cytokine production,cytotoxicity assays in vitro,and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared with standard TRAC-CAR T cells,MP TRAC-CAR T cells showed less glycolysis,higher CCR7/CD62L expression,more bound NAD(P)H activity,and reduced IFN-γ,IL-2,IP-10,IL-1β,IL-17,and TGF-β production at the end of manufacturing ex vivo,with increased central memory CAR T cells and better persistence observed in vivo. MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo,which could lead to better responses against solid tumors in vivo. Graphical abstract Cappabianca and colleagues manufactured virus-free CAR T cells at scale with CRISPR-Cas9 and “metabolically primed” them by attenuating activation in low-glucose/glutamine medium with expansion in high-glucose/glutamine medium. Priming made CAR T cells with increased stem cell memory properties,including enriched central memory phenotypes in vivo while lysing solid tumors. View Publication

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2–6,suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes,promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function,memory potential and metabolic fitness in settings of chronic stimulation,and exhibited enhanced persistence and tumour control in vivo. By contrast,overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably,FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes,underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells,and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states. The transcription factor FOXO1 has a key role in human T cell memory,and manipulating FOXO1 expression could provide a way to enhance CAR T cell therapies by increasing CAR T cell persistence and antitumour activity. View Publication