技术资料

Siglec-6 is a lectin receptor with restricted expression in the placenta,mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL),its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8,a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand,sTn,results in Cdc42 activation,WASP protein recruitment and F-actin polymerization,which are all associated with cell migration. Therapeutically,a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL,which can be therapeutically targeted using a Siglec-6 specific T-biAb. Siglec-6 is often overexpressed in chronic lymphocytic leukaemia (CLL),but its role is unclear. Here,the author report that Siglec-6 regulates the migration and adhesion of CLL B cells via interaction with sialyl Tn on bone marrow stromal cells driving invasion which could be therapeutically targeted using a Siglec-6/CD3-bispecfiic antibody. View Publication

Long interspersed element 1 (LINE-1; L1) are a family of transposons that occupy ~17% of the human genome. Though a small number of L1 copies remain capable of autonomous transposition,the overwhelming majority of copies are degenerate and immobile. Nevertheless,both mobile and immobile L1s can exert pleiotropic effects (promoting genome instability,inflammation,or cellular senescence) on their hosts,and L1’s contributions to aging and aging diseases is an area of active research. However,because of the cell type-specific nature of transposon control,the catalogue of L1 regulators remains incomplete. Here,we employ an eQTL approach leveraging transcriptomic and genomic data from the GEUVADIS and 1000Genomes projects to computationally identify new candidate regulators of L1 RNA levels in lymphoblastoid cell lines. To cement the role of candidate genes in L1 regulation,we experimentally modulate the levels of top candidates in vitro,including IL16,STARD5,HSD17B12,and RNF5,and assess changes in TE family expression by Gene Set Enrichment Analysis (GSEA). Remarkably,we observe subtle but widespread upregulation of TE family expression following IL16 and STARD5 overexpression. Moreover,a short-term 24-hour exposure to recombinant human IL16 was sufficient to transiently induce subtle,but widespread,upregulation of L1 subfamilies. Finally,we find that many L1 expression-associated genetic variants are co-associated with aging traits across genome-wide association study databases. Our results expand the catalogue of genes implicated in L1 RNA control and further suggest that L1-derived RNA contributes to aging processes. Given the ever-increasing availability of paired genomic and transcriptomic data,we anticipate this new approach to be a starting point for more comprehensive computational scans for regulators of transposon RNA levels. Author summaryTransposable elements,or jumping genes,are fragments of DNA that have or once had the ability to mobilize to a new location within our genome. In humans,the most abundant transposable element is LINE-1 (L1),accounting for ~17% of our total DNA. Though L1 is generally repressed in healthy human cells,derepression of transposable elements (including L1) has been observed in aging and in aging-associated diseases. Additionally,there is increasing evidence that L1 transcriptional levels may promote features of aging,highlighting the importance of understanding the mechanisms that regulate L1 RNA levels. Here,we computationally identify new candidate regulators of L1 RNA levels,provide experimental evidence that candidate regulators influence L1 RNA levels,and demonstrate that genetic variants associated with differences in L1 RNA levels are co-associated with aging phenotypes. Our approach expands the toolkit that can be used to characterize transposable element regulation and highlights specific genes for further study. Importantly,our results reiterate the notion that L1 levels are linked with aging phenotypes and represent a potential therapeutic target for age-related decline. View Publication

Hypoimmune gene edited human pluripotent stem cells (hPSCs) are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive (e.g.,T cell) immune responses,but have largely not addressed the innate immune cells (e.g.,monocytes,neutrophils) that mediate inflammation and rejection processes occurring early after graft transplantation. We identified the adhesion molecule ICAM-1 as a novel hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In a series of studies,we found that ICAM-1 blocking or knock-out (KO) in hPSC-derived cardiovascular therapies imparted significantly diminished binding of multiple immune cell types. ICAM-1 KO resulted in diminished T cell proliferation responses in vitro and in longer in vivo retention/protection of KO grafts following immune cell encounter in NeoThy humanized mice. The ICAM-1 KO edit was also introduced into existing first-generation hypoimmune hPSCs and prevented immune cell binding,thereby enhancing the overall hypoimmune capacity of the cells. This novel hypoimmune editing strategy has the potential to improve the long-term efficacy and safety profiles of regenerative therapies for cardiovascular pathologies and a number of other diseases. Graphical Abstract ICAM-1 Knock-out in Transendothelial Migration and at the Immune Synapse. Abbreviations: PSC-EC - pluripotent stem cell-derived endothelial cells; KO – knock-out; dSMAC – distal supramolecular activation complex; pSMAC – peripheral supramolecular activation complex; cSMAC – central supramolecular activation complex. 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

Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression,but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28,CTLA4 and ICOS,encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells,both conventional and regulatory subsets,uncovered gene-,cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets,this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex,multi-gene locus critical to immune homeostasis. Functional characterization of the regulatory landscape of the adjacent costimulatory genes CD28,CTLA4 and ICOS in primary human T cell subsets identifies context-dependent programs controlling this locus critical for immune homeostasis. View Publication

Given the marginal penetration of most drugs across the blood-brain barrier,the efficacy of various agents remains limited for glioblastoma (GBM). Here we employ low-intensity pulsed ultrasound (LIPU) and intravenously administered microbubbles (MB) to open the blood-brain barrier and increase the concentration of liposomal doxorubicin and PD-1 blocking antibodies (aPD-1). We report results on a cohort of 4 GBM patients and preclinical models treated with this approach. LIPU/MB increases the concentration of doxorubicin by 2-fold and 3.9-fold in the human and murine brains two days after sonication,respectively. Similarly,LIPU/MB-mediated blood-brain barrier disruption leads to a 6-fold and a 2-fold increase in aPD-1 concentrations in murine brains and peritumoral brain regions from GBM patients treated with pembrolizumab,respectively. Doxorubicin and aPD-1 delivered with LIPU/MB upregulate major histocompatibility complex (MHC) class I and II in tumor cells. Increased brain concentrations of doxorubicin achieved by LIPU/MB elicit IFN-γ and MHC class I expression in microglia and macrophages. Doxorubicin and aPD-1 delivered with LIPU/MB results in the long-term survival of most glioma-bearing mice,which rely on myeloid cells and lymphocytes for their efficacy. Overall,this translational study supports the utility of LIPU/MB to potentiate the antitumoral activities of doxorubicin and aPD-1 for GBM. Ultrasound-mediated blood-brain barrier opening has been exploited to improve drug delivery in the brain. Here the authors show that low-intensity pulsed ultrasound in combination with intravenous injection of microbubbles enhances the delivery of doxorubicin and anti-PD1 in gliomas,improving anti-tumor immune responses. 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

AbstractEncouraged by the observations of significant B7-H3 protein overexpression in many human solid tumors compared to healthy tissues,we directed our focus towards targeting B7-H3 using chimeric antigen receptor (CAR) T cells. We utilized a nanobody as the B7-H3–targeting domain in our CAR construct to circumvent the stability issues associated with single-chain variable fragment–based domains. In efforts to expand patient access to CAR T-cell therapy,we engineered our nanobody-based CAR into human Epstein-Barr virus–specific T cells (EBVST),offering a readily available off-the-shelf treatment. B7H3.CAR-armored EBVSTs demonstrated potent in vitro and in vivo activities against multiple B7-H3–positive human tumor cell lines and patient-derived xenograft models. Murine T cells expressing a murine equivalent of our B7H3.CAR exhibited no life-threatening toxicities in immunocompetent mice bearing syngeneic tumors. Further in vitro evaluation revealed that while human T,B,and natural killer cells were unaffected by B7H3.CAR EBVSTs,monocytes were targeted because of upregulation of B7-H3. Such targeting of myeloid cells,which are key mediators of cytokine release syndrome (CRS),contributed to a low incidence of CRS in humanized mice after B7H3.CAR EBVST treatment. Notably,we showed that B7H3.CAR EBVSTs can target B7-H3–expressing myeloid-derived suppressor cells (MDSC),thereby mitigating MDSC-driven immune suppression. In summary,our data demonstrate that our nanobody-based B7H3.CAR EBVSTs are effective as an off-the-shelf therapy for B7-H3–positive solid tumors. These cells also offer an avenue to modulate the immunosuppressive tumor microenvironment,highlighting their promising clinical potential in targeting solid tumors.Significance:Clinical application of EBVSTs armored with B7-H3–targeting CARs offer an attractive solution to translate off-the-shelf CAR T cells as therapy for solid tumors. View Publication

Therapeutic usage of cytokines in patients is limited by toxicity. The authors report that blocking a cytokine binding protein,IL18BP,to enhance the cytokine’s natural activity yields antitumor activity in preclinical models and shows promise for clinical translation. AbstractRecombinant cytokines have limited anticancer efficacy mostly due to a narrow therapeutic window and systemic adverse effects. IL18 is an inflammasome-induced proinflammatory cytokine,which enhances T- and NK-cell activity and stimulates IFNγ production. The activity of IL18 is naturally blocked by a high-affinity endogenous binding protein (IL18BP). IL18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. In this study,we found that IL18 is upregulated in the TME compared with the periphery across multiple human tumors and most of it is bound to IL18BP. Bound IL18 levels were largely above the amount required for T-cell activation in vitro,implying that releasing IL18 in the TME could lead to potent T-cell activation. To restore the activity of endogenous IL18,we generated COM503,a high-affinity anti-IL18BP that blocks the IL18BP:IL18 interaction and displaces precomplexed IL18,thereby enhancing T- and NK-cell activation. In vivo,administration of a surrogate anti-IL18BP,either alone or in combination with anti-PD-L1,resulted in significant tumor growth inhibition and increased survival across multiple mouse tumor models. Moreover,the anti-IL18BP induced pronounced TME-localized immune modulation including an increase in polyfunctional nonexhausted T- and NK-cell numbers and activation. In contrast,no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together,blocking IL18BP using an Ab is a promising approach to harness cytokine biology for the treatment of cancer. View Publication

IntroductionThe effector function of T cells is regulated via immune checkpoints,activating or inhibiting the immune response. The BTLA-HVEM complex,the inhibitory immune checkpoint,may act as one of the tumor immune escape mechanisms. Therefore,interfering with the binding of these proteins can prove beneficial in cancer treatment. Our study focused on peptides interacting with HVEM at the same place as BTLA,thus disrupting the BTLA-HVEM interaction. These peptides’ structure and amino acid sequences are based on the gD protein,the ligand of HVEM. Here,we investigated their immunomodulatory potential in melanoma patients.MethodsFlow cytometry analyses of activation,proliferation,and apoptosis of T cells from patients were performed. Additionally,we evaluated changes within the T cell memory compartment.ResultsThe most promising compound – Pep(2),increased the percentages of activated T cells and promoted their proliferation. Additionally,this peptide affected the proliferation rate and apoptosis of melanoma cell line in co-culture with T cells.DiscussionWe conclude that the examined peptide may act as a booster for the immune system. Moreover,the adjuvant and activating properties of the gD-derived peptide could be used in a combinatory therapy with currently used ICI-based treatment. Our studies also demonstrate that even slight differences in the amino acid sequence of peptides and any changes in the position of the disulfide bond can strongly affect the immunomodulatory properties of compounds. View Publication

Thrombopoietin (Tpo),which binds to its specific receptor,the Mpl protein,is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor,as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565,Mpl-Y599 and Mpl-Y604. Here,we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599,mutation of which resulted in thrombocytopenia,deficient megakaryopoiesis,low hematopoietic stem cell (HSC) number and function,and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN),where Mpl is required for pathogenesis,thrombocytosis was dependent on intact Mpl-Y599. In contrast,Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression,and exacerbated thrombocytosis associated with MPN. 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