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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

BackgroundHematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression,which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury,while if and how the niche is reshaped and regulates HSC regeneration are poorly understood.MethodsA mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number,distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry,immunofluorescence,colony assay and bone marrow transplantation,in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro,and was consolidated using megakaryocyte-specific knockout mice and transgenic mice.ResultsMegakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile,transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury,whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically,HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion,and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs,but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently,the delicate coordination between proliferation,mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly,punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury,representing a superior therapeutic approach for myelosuppression.ConclusionsOur study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01651-5. View Publication

Human brain organoids are emerging as translationally relevant models for the study of human brain health and disease. However,it remains to be shown whether human-specific protein processing is conserved in human brain organoids. Herein,we demonstrate that cell fate and composition of unguided brain organoids are dictated by culture conditions during embryoid body formation,and that culture conditions at this stage can be optimized to result in the presence of glia-associated proteins and neural network activity as early as three-months in vitro. Under these optimized conditions,unguided brain organoids generated from induced pluripotent stem cells (iPSCs) derived from male–female siblings are similar in growth rate,size,and total protein content,and exhibit minimal batch-to-batch variability in cell composition and metabolism. A comparison of neuronal,microglial,and macroglial (astrocyte and oligodendrocyte) markers reveals that profiles in these brain organoids are more similar to autopsied human cortical and cerebellar profiles than to those in mouse cortical samples,providing the first demonstration that human-specific protein processing is largely conserved in unguided brain organoids. Thus,our organoid protocol provides four major cell types that appear to process proteins in a manner very similar to the human brain,and they do so in half the time required by other protocols. This unique copy of the human brain and basic characteristics lay the foundation for future studies aiming to investigate human brain-specific protein patterning (e.g.,isoforms,splice variants) as well as modulate glial and neuronal processes in an in situ-like environment. View Publication

AbstractChronic lymphocytic leukemia (CLL) cell survival and growth is fueled by the induction of B-cell receptor (BCR) signaling within the tumor microenvironment (TME) driving activation of NFκB signaling and the unfolded protein response (UPR). Malignant cells have higher basal levels of UPR posing a unique therapeutic window to combat CLL cell growth using pharmacologic agents that induce accumulation of misfolded proteins. Frontline CLL therapeutics that directly target BCR signaling such as Bruton tyrosine kinase (BTK) inhibitors (e.g.,ibrutinib) have enhanced patient survival. However,resistance mechanisms wherein tumor cells bypass BTK inhibition through acquired BTK mutations,and/or activation of alternative survival mechanisms have rendered ibrutinib ineffective,imposing the need for novel therapeutics. We evaluated SpiD3,a novel spirocyclic dimer,in CLL cell lines,patient-derived CLL samples,ibrutinib-resistant CLL cells,and in the Eµ-TCL1 mouse model. Our integrated multi-omics and functional analyses revealed BCR signaling,NFκB signaling,and endoplasmic reticulum stress among the top pathways modulated by SpiD3. This was accompanied by marked upregulation of the UPR and inhibition of global protein synthesis in CLL cell lines and patient-derived CLL cells. In ibrutinib-resistant CLL cells,SpiD3 retained its antileukemic effects,mirrored in reduced activation of key proliferative pathways (e.g.,PRAS,ERK,MYC). Translationally,we observed reduced tumor burden in SpiD3-treated Eµ-TCL1 mice. Our findings reveal that SpiD3 exploits critical vulnerabilities in CLL cells including NFκB signaling and the UPR,culminating in profound antitumor properties independent of TME stimuli.Significance:SpiD3 demonstrates cytotoxicity in CLL partially through inhibition of NFκB signaling independent of tumor-supportive stimuli. By inducing the accumulation of unfolded proteins,SpiD3 activates the UPR and hinders protein synthesis in CLL cells. Overall,SpiD3 exploits critical CLL vulnerabilities (i.e.,the NFκB pathway and UPR) highlighting its use in drug-resistant CLL. View Publication

IntroductionImmune cells that contribute to the pathogenesis of systemic lupus erythematosus (SLE) derive from adult hematopoietic stem and progenitor cells (HSPCs) within the bone marrow (BM). For this reason,we reasoned that fundamental abnormalities in SLE can be traced to a BM-derived HSPC inflammatory signature.MethodsBM samples from four SLE patients,six healthy controls,and two umbilical cord blood (CB) samples were used. CD34+ cells were isolated from BM and CB samples,and single-cell RNA-sequencing was performed.ResultsA total of 426 cells and 24,473 genes were used in the analysis. Clustering analysis resulted in seven distinct clusters of cell types. Mutually exclusive markers,which were characteristic of each cell type,were identified. We identified three HSPC subpopulations,one of which consisted of proliferating cells (MKI67 expressing cells),one T-like,one B-like,and two myeloid-like progenitor subpopulations. Differential expression analysis revealed i) cell cycle-associated signatures,in healthy BM of HSPC clusters 3 and 4 when compared with CB,and ii) interferon (IFN) signatures in SLE BM of HSPC clusters 3 and 4 and myeloid-like progenitor cluster 5 when compared with healthy controls. The IFN signature in SLE appeared to be deregulated following TF regulatory network analysis and differential alternative splicing analysis between SLE and healthy controls in HSPC subpopulations.DiscussionThis study revealed both quantitative—as evidenced by decreased numbers of non-proliferating early progenitors—and qualitative differences—characterized by an IFN signature in SLE,which is known to drive loss of function and depletion of HSPCs. Chronic IFN exposure affects early hematopoietic progenitors in SLE,which may account for the immune aberrancies and the cytopenias in SLE. View Publication

The antitumor efficacy of adoptively transferred T cells is limited by their poor persistence,in part due to exhaustion,but the underlying mechanisms and potential interventions remain underexplored. Here,we show that targeting histone demethylase LSD1 by chemical inhibitors reshapes the epigenome of in vitro activated and expanded CD8+ T cells,and potentiates their antitumor efficacy. Upon T cell receptor activation and IL-2 signaling,a timely and transient inhibition of LSD1 suffices to improve the memory phenotype of mouse CD8+ T cells,associated with a better ability to produce multiple cytokines,resist exhaustion,and persist in both antigen-dependent and -independent manners after adoptive transfer. Consequently,OT1 cells primed with LSD1 inhibitors demonstrate an enhanced antitumor effect in OVA-expressing solid tumor models implanted in female mice,both as a standalone treatment and in combination with PD-1 blockade. Moreover,priming with LSD1 inhibitors promotes polyfunctionality of human CD8+ T cells,and increases the persistence and antitumor efficacy of human CD19-CAR T cells in both leukemia and solid tumor models. Thus,pharmacological inhibition of LSD1 could be exploited to improve adoptive T cell therapy. Phenotypic changes in exhausted T cells are linked to chromatin remodeling. Here the authors show that pharmacological inhibition of the H3K4me1/2 demethylase LSD1 promotes the persistence and enhances the therapeutic activity of adoptively transferred T cells for cancer therapy. View Publication