参考文献

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

SummarySevere COVID-19 often leads to secondary infections and sepsis that contribute to long hospital stays and mortality. However,our understanding of the precise immune mechanisms driving severe complications after SARS-CoV-2 infection remains incompletely understood. Here,we provide evidence that the SARS-CoV-2 envelope (E) protein initiates innate immune inflammation,via toll-like receptor 2 signaling,and establishes a sustained state of innate immune tolerance following initial activation. Monocytes in this tolerant state exhibit reduced responsiveness to secondary stimuli,releasing lower levels of cytokines and chemokines. Mice exposed to E protein before secondary lipopolysaccharide challenge show diminished pro-inflammatory cytokine expression in the lung,indicating that E protein drives this tolerant state in vivo. These findings highlight the potential of the SARS-CoV-2 E protein to induce innate immune tolerance,contributing to long-term immune dysfunction that could lead to susceptibility to subsequent infections,and uncovers therapeutic targets aimed at restoring immune function following SARS-CoV-2 infection. Graphical abstract Highlights•SARS-CoV-2 envelope (E) protein activated innate immune cells through TLR2•E protein promoted a long-term tolerant immune state after initial activation•Monocytes in this tolerant state had reduced responsiveness to secondary stimuli•E protein priming reduced lung inflammation markers to LPS in neonatal mice Molecular biology; Immunity; Components of the immune system; Virology; Transcriptomics. View Publication

Alternative splicing events are a major causal mechanism for complex traits,but they have been understudied due to the limitation of short-read sequencing. Here,we generate a full-length isoform annotation of human immune cells from an individual by long-read sequencing for 29 cell subsets. This contains a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE in the Alzheimer’s disease locus. We profile characteristics of isoforms and show that repetitive elements significantly explain the diversity of unannotated isoforms,providing insight into the human genome evolution. In addition,some of the isoforms are expressed in a cell-type specific manner,whose alternative 3’-UTRs usage contributes to their specificity. Further,we identify disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the mechanism of complex diseases via alternative splicing. This paper unveils the complexity of human immune cell splicing,highlighting cell-specific isoforms and establishing connections between alternative splicing and complex traits. These findings have implications for understanding diseases and the evolution of the genome. View Publication