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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

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