技术资料

Trained immunity may play a role in vaccine-induced protection against infections. We showed that the highly efficacious recombinant VZV-gE zoster vaccine (RZV) generated trained immunity in monocytes,natural killer (NK) cells,and dendritic cells (DCs) and that the less efficacious live zoster vaccine did not. RZV stimulated ex vivo gE-specific monocyte,DC and NK cell responses that did not correlate with CD4 + T-cell responses. These responses were also elicited in purified monocyte and NK cell cocultures stimulated with VZV-gE and persisted above prevaccination levels for ≥ 4 years post-RZV administration. RZV administration also increased ex vivo heterologous monocyte and NK cell responses to herpes simplex and cytomegalovirus antigens. ATAC-seq analysis and ex vivo TGFβ1 supplementation and inhibition experiments demonstrated that decreased tgfβ1 transcription resulting from RZV-induced chromatin modifications may explain the development of monocyte trained immunity. The role of RZV-trained immunity in protection against herpes zoster and other infections should be further studied. 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

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases,including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs,most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets,may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes,a human β cell line and human islet β cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet β cells. In exploring subsets and mechanisms that may explain this pattern,we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39− CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly,β cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased β cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet β cells in the setting of CAR immunotherapy. In summary,introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction. View Publication

AbstractObjectiveUnique metabolic requirements accompany the development and functional fates of immune cells. How cellular metabolism is important in natural killer (NK) cells and their memory‐like differentiation in bacterial infections remains elusive.MethodsHere,we utilise our established NK cell memory assay to investigate the metabolic requirement for memory‐like NK cell formation and function in response to the Gram‐negative intracellular bacteria Burkholderia pseudomallei (BP),the causative agent of melioidosis.ResultsWe demonstrate that CD160+ memory‐like NK cells upon BP stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients which is maintained at least 3‐month post‐hospital admission. Using an in vitro assay,human BP‐specific CD160+ memory‐like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake,increased mTOR activation and mitochondrial membrane potential upon BP re‐stimulation. Antigen‐specific and cytokine‐induced IFN‐γ production of this memory‐like NK cell subset are highly dependent on oxidative phosphorylation (OXPHOS) with some dependency on glycolysis,whereas the formation of CD160+ memory‐like NK cells in vitro is dependent on fatty acid oxidation and OXPHOS and further increased by metformin.ConclusionThis study reveals the link between metabolism and cellular function of memory‐like NK cells,which can be exploited for vaccine design and for monitoring protection against Gram‐negative bacterial infection. This study reveals the link between metabolism and cellular function of memory‐like NK cells in melioidosis. We demonstrate that CD160+ memory‐like NK cells upon Burkholderia pseudomallei (BP) stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients. Using an in vitro assay,human BP‐specific CD160+ memory‐like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake,increased mTOR activation and mitochondrial membrane potential upon BP re‐stimulation. View Publication

AbstractBackgroundCancer‐targeted T‐cell receptor T (TCR‐T) cells hold promise in treating cancers such as hematological malignancies and breast cancers. However,approaches to obtain cancer‐reactive TCR‐T cells have been unsuccessful.MethodsHere,we developed a novel strategy to screen for cancer‐targeted TCR‐T cells using a special humanized mouse model with person‐specific immune fingerprints. Rare steady‐state circulating hematopoietic stem and progenitor cells were expanded via three‐dimensional culture of steady‐state peripheral blood mononuclear cells,and then the expanded cells were applied to establish humanized mice. The human immune system was evaluated according to the kinetics of dendritic cells,monocytes,T‐cell subsets,and cytokines. To fully stimulate the immune response and to obtain B‐cell precursor NAML‐6‐ and triple‐negative breast cancer MDA‐MB‐231‐targeted TCR‐T cells,we used the inactivated cells above to treat humanized mice twice a day every 7 days. Then,human T cells were processed for TCR β‐chain (TRB) sequencing analysis. After the repertoires had been constructed,features such as the fraction,diversity,and immune signature were investigated.ResultsThe results demonstrated an increase in diversity and clonality of T cells after treatment. The preferential usage and features of TRBV,TRBJ,and the V–J combination were also changed. The stress also induced highly clonal expansion. Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice.ConclusionsWe constructed a personalized humanized mouse model to screen cancer‐targeted TCR‐T pools. Our platform provides an effective source of cancer‐targeted TCR‐T cells and allows for the design of patient‐specific engineered T cells. It therefore has the potential to greatly benefit cancer treatment. Cancer‐targeted T‐cell receptor T (TCR‐T) cells hold promise in treating malignancies but with limited source. We applied steady‐state peripheral blood mononuclear cells via three‐dimensional culture to construct humanized mouse model for cancer‐targeted TCR‐T repertoire screening. 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

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

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

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

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

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