技术资料

Midnolin is an essential gene with previously unknown effects in vivo. This paper shows that midnolin stimulates proteasome activity necessary for lymphopoiesis and B cell cancer growth in mice. In a genetic screen,we identified two viable missense alleles of the essential gene Midnolin (Midn) that were associated with reductions in peripheral B cells. Causation was confirmed in mice with targeted deletion of four of six MIDN protein isoforms. MIDN was expressed predominantly in lymphocytes where it augmented proteasome activity. We showed that purified MIDN directly stimulated 26S proteasome activity in vitro in a manner dependent on the ubiquitin-like domain and a C-terminal region. MIDN-deficient B cells displayed aberrant activation of the IRE-1/XBP-1 pathway of the unfolded protein response. Partial or complete MIDN deficiency strongly suppressed Eμ-Myc–driven B cell leukemia and the antiapoptotic effects of Eμ-BCL2 on B cells in vivo and induced death of Sp2/0 hybridoma cells in vitro,but only partially impaired normal lymphocyte development. Thus,MIDN is required for proteasome activity in support of normal lymphopoiesis and is essential for malignant B cell proliferation over a broad range of differentiation states. View Publication

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by anti-nuclear autoantibodies whose production is promoted by autoreactive T follicular helper (TFH) cells. During SLE pathogenesis,basophils accumulate in secondary lymphoid organs (SLO),amplify autoantibody production and disease progression through mechanisms that remain to be defined. Here,we provide evidence for a direct functional relationship between TFH cells and basophils during lupus pathogenesis,both in humans and mice. PD-L1 upregulation on basophils and IL-4 production are associated with TFH and TFH2 cell expansions and with disease activity. Pathogenic TFH cell accumulation,maintenance,and function in SLO were dependent on PD-L1 and IL-4 in basophils,which induced a transcriptional program allowing TFH2 cell differentiation and function. Our study establishes a direct mechanistic link between basophils and TFH cells in SLE that promotes autoantibody production and lupus nephritis. Basophils have been implicated in systemic lupus erythematosus (SLE),as evidenced by the fact that basophil-deficient mice do not develop the disease. Here,the authors demonstrate that PD-L1 and IL-4 expression in basophils promotes the pathogenic accumulation of follicular helper T cells in patients with SLE and murine models. 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

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

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

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

BackgroundNeutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation,emergency granulopoiesis leads to release of immature neutrophils from the bone marrow,serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood,partly due to a lack of genetically amenable models of neutrophil development.MethodsWe optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry,we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis,and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors.ResultsWe obtain high yields of ex vivo cultured neutrophils,which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation,cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins,as demonstrated by proteomic analysis.ConclusionEx vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-024-05337-x. View Publication

Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells,which binds to CD47,a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells,therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer,several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However,the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited,suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end,DS-1103a,a newly developed anti-human SIRPα antibody (Ab),was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd),DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2,respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model,vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab,implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore,in syngeneic mouse models,both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together,this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs. View Publication

Over 4% of the global population is estimated to live with autoimmune disease,necessitating immunosuppressive treatment that is often chronic,not curative,and carries associated risks. B cells have emerged as key players in disease pathogenesis,as evidenced by partial responsiveness to B cell depletion by antibody-based therapies. However,these treatments often have transient effects due to incomplete depletion of tissue-resident B cells. Chimeric antigen receptor (CAR) T cells targeting B cells have demonstrated efficacy in refractory systemic lupus erythematosus. To this end,we developed an anti-CD19 CAR T cell product candidate,CABA-201,containing a clinically evaluated fully human CD19 binder (IC78) with a 4-1BB costimulatory domain and CD3 zeta stimulation domain for treatment refractory autoimmune disease. Here,we demonstrate specific cytotoxic activity of CABA-201 against CD19+ Nalm6 cells with no off-target effects on primary human cells. Novel examination of CABA-201 generated from primary T cells from multiple patients with autoimmune disease displayed robust CAR surface expression and effective elimination of the intended target autologous CD19+ B cells in vitro. Together,these findings support the tolerability and activity of CABA-201 for clinical development in patients with autoimmune disease. Graphical abstract Basu and colleagues show CABA-201,a B cell-targeting CAR T cell product with a fully human CD19 binder and 4-1BB costimulatory domain,can precisely eliminate autoimmune patient B cells without off-target deleterious effects,demonstrating its ability as a robust therapeutic for B cell-driven autoimmune disorders. View Publication

BackgroundAllergen-specific immunotherapy (AIT) is able to restore immune tolerance to allergens in allergic patients. However,some patients do not or only poorly respond to current treatment protocols. Therefore,there is a need for deeper mechanistic insights and further improvement of treatment strategies. The relevance of the aryl hydrocarbon receptor (AhR),a ligand-dependent transcription factor,has been investigated in several inflammatory diseases,including allergic asthma. However,its potential role in AIT still needs to be addressed.MethodsA murine model of AIT in ovalbumin-induced allergic airway inflammation was performed in AhR-deficient (AhR-/-) and wild-type mice. Furthermore,AIT was combined with the application of the high-affinity AhR agonist 10-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (10-Cl-BBQ) as an adjuvant to investigate the effects of AhR activation on therapeutic outcome.ResultsAlthough AhR-/- mice suffer stronger allergic responses than wild-type mice,experimental AIT is comparably effective in both. Nevertheless,combining AIT with the administration of 10-Cl-BBQ improved therapeutic effects by an AhR-dependent mechanism,resulting in decreased cell counts in the bronchoalveolar fluid,decreased pulmonary Th2 and Th17 cell levels,and lower sIgE levels.ConclusionThis study demonstrates that the success of AIT is not dependent on the AhR. However,targeting the AhR during AIT can help to dampen inflammation and improve tolerogenic vaccination. Therefore,AhR ligands might represent promising candidates as immunomodulators to enhance the efficacy of AIT. View Publication

BackgroundThe human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body,theoretically. Therefore,hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study,we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch.MethodsFirst,we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then,a HyPSC clone was generated by introducing exogenous β-2-microglobulin (B2M),HLA-G,PD-L1,and PD-L2 genes,and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed.ResultsWe found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover,the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs),functional endothelial cells,and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further,we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo.ConclusionThe HLA class Ia/II DKO hiPSCs armed with HLA-G,PD-L1,PD-L2,and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03810-4. View Publication

Mitochondria react to infection with sub-lethal signals in the apoptosis pathway. Mitochondrial signals can be inflammatory but mechanisms are only partially understood. We show that activation of the caspase-activated DNase (CAD) mediates mitochondrial pro-inflammatory functions and substantially contributes to host defense against viral infection. In cells lacking CAD,the pro-inflammatory activity of sub-lethal signals was reduced. Experimental activation of CAD caused transient DNA-damage and a pronounced DNA damage response,involving major kinase signaling pathways,NF-κB and cGAS/STING,driving the production of interferon,cytokines/chemokines and attracting neutrophils. The transcriptional response to CAD-activation was reminiscent of the reaction to microbial infection. CAD-deficient cells had a diminished response to viral infection. Influenza virus infected CAD-deficient mice displayed reduced inflammation in lung tissue,higher viral titers and increased weight loss. Thus,CAD links the mitochondrial apoptosis system and cell death caspases to host defense. CAD-driven DNA damage is a physiological element of the inflammatory response to infection. View Publication