参考文献

How the microbiome regulates responses of systemic innate immune cells is unclear. In the present study,our purpose was to document a novel mechanism by which the microbiome mediates crosstalk with the systemic innate immune system. We have identified a family of microbiome Bacteroidota-derived lipopeptides—the serine-glycine (S/G) lipids,which are TLR2 ligands,access the systemic circulation,and regulate proinflammatory responses of splenic monocytes. To document the role of these lipids in regulating systemic immunity,we used oral gavage with an antibiotic to decrease the production of these lipids and administered exogenously purified lipids to increase the systemic level of these lipids. We found that decreasing systemic S/G lipids by decreasing microbiome Bacteroidota significantly enhanced splenic monocyte proinflammatory responses. Replenishing systemic levels of S/G lipids via exogenous administration returned splenic monocyte responses to control levels. Transcriptomic analysis demonstrated that S/G lipids regulate monocyte proinflammatory responses at the level of gene expression of a small set of upstream inhibitors of TLR and NF-κB pathways that include Trem2 and Irf4. Consistent with enhancement in proinflammatory cytokine responses,decreasing S/G lipids lowered gene expression of specific pathway inhibitors. Replenishing S/G lipids normalized gene expression of these inhibitors. In conclusion,our results suggest that microbiome-derived S/G lipids normally establish a level of buffered signaling activation necessary for well-regulated innate immune responses in systemic monocytes. By regulating gene expression of inflammatory pathway inhibitors such as Trem2,S/G lipids merit broader investigation into the potential dysfunction of other innate immune cells,such as microglia,in diseases such as Alzheimer’s disease. View Publication

Streptococcus pneumoniae (Sp),a leading cause of community-acquired pneumonia,can spread from the lung into the bloodstream to cause septicemia and meningitis,with a concomitant three-fold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor,pneumolysin (PLY),triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids,including hepoxilin A3 (HXA3),a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination,we utilized bronchial stem cell-derived air-liquid interface (ALI) cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration,monolayer disruption,and concomitant Sp barrier breach. In contrast,PMN transmigration in response to the non-eicosanoid chemoattractant fMLP did not lead to epithelial disruption or bacterial translocation. Correspondingly,HXA3-ME but not fMLP increased release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus,HXA3 promotes barrier disrupting PMN transmigration and NE release,pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia. View Publication

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients,we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence,we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition,we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays,CD69,CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155,one important TIGIT-ligand,is reliably expressed on AMLs,we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally,our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype,whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively,our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-024-03766-7. 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

BackgroundThe human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body,theoretically. Therefore,hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study,we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch.MethodsFirst,we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then,a HyPSC clone was generated by introducing exogenous β-2-microglobulin (B2M),HLA-G,PD-L1,and PD-L2 genes,and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed.ResultsWe found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover,the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs),functional endothelial cells,and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further,we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo.ConclusionThe HLA class Ia/II DKO hiPSCs armed with HLA-G,PD-L1,PD-L2,and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03810-4. 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

IntroductionBispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets,bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody,which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time.MethodsThis study comprised antibodies purification,Elisa assay for antigen binding,cytotoxicity assays,T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo.ResultsWe present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin β2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially,BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo.DiscussionMoreover,PHE1 domain was further shortened with D14G and R41S mutations,named PHE-S,and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA+ tumor effect in vitro and in vivo,bringing more possibilities for the development and optimization of different bsAbs. To sum up,PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy. View Publication

BackgroundAllergen-specific immunotherapy (AIT) is able to restore immune tolerance to allergens in allergic patients. However,some patients do not or only poorly respond to current treatment protocols. Therefore,there is a need for deeper mechanistic insights and further improvement of treatment strategies. The relevance of the aryl hydrocarbon receptor (AhR),a ligand-dependent transcription factor,has been investigated in several inflammatory diseases,including allergic asthma. However,its potential role in AIT still needs to be addressed.MethodsA murine model of AIT in ovalbumin-induced allergic airway inflammation was performed in AhR-deficient (AhR-/-) and wild-type mice. Furthermore,AIT was combined with the application of the high-affinity AhR agonist 10-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (10-Cl-BBQ) as an adjuvant to investigate the effects of AhR activation on therapeutic outcome.ResultsAlthough AhR-/- mice suffer stronger allergic responses than wild-type mice,experimental AIT is comparably effective in both. Nevertheless,combining AIT with the administration of 10-Cl-BBQ improved therapeutic effects by an AhR-dependent mechanism,resulting in decreased cell counts in the bronchoalveolar fluid,decreased pulmonary Th2 and Th17 cell levels,and lower sIgE levels.ConclusionThis study demonstrates that the success of AIT is not dependent on the AhR. However,targeting the AhR during AIT can help to dampen inflammation and improve tolerogenic vaccination. Therefore,AhR ligands might represent promising candidates as immunomodulators to enhance the efficacy of AIT. View Publication

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However,it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here,Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the major histocompatibility complex-I restricted SIINFEKL epitope which is recognized by T cell receptor transgenic OT-I(OVA-TCR-I) clusters of differentiation (CD)8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of interferon-gamma (IFN-γ) that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (rhoptry protein 1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells,type 1 conventional dendritic cells were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and type 1 conventional dendritic cells is crucial for CD8+ T cell responses to Cryptosporidium. View Publication

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