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Ab-targeted vaccination involves targeting a receptor of choice expressed by dendritic cells (DCs) with Ag-coupled Abs. Currently,there is little consensus as to which criteria determine receptor selection to ensure superior Ag presentation and immunity. In this study,we investigated parameters of DC receptor internalization and determined how they impact Ag presentation outcomes. First,using mixed bone marrow chimeras,we established that Ag-targeted,but not nontargeted,DCs are responsible for Ag presentation in settings of Ab-targeted vaccination in vivo. Next,we analyzed parameters of DEC205 (CD205),Clec9A,CD11c,CD11b,and CD40 endocytosis and obtained quantitative measurements of internalization speed,surface turnover,and delivered Ag load. Exploiting these parameters in MHC class I (MHC I) and MHC class II (MHC II) Ag presentation assays,we showed that receptor expression level,proportion of surface turnover,or speed of receptor internalization did not impact MHC I or MHC II Ag presentation efficiency. Furthermore,the Ag load delivered to DCs did not correlate with the efficiency of MHC I or MHC II Ag presentation. In contrast,targeting Ag to CD8(+) or CD8(-) DCs enhanced MHC I or MHC II Ag presentation,respectively. Therefore,receptor expression levels,speed of internalization,and/or the amount of Ag delivered can be excluded as major determinants that dictate Ag presentation efficiency in setting of Ab-targeted vaccination. View Publication

The Sox6 transcription factor plays critical roles in various cell types,including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells,we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar,evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo,and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied,in erythroid cells,by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation,mediated by the double Sox6 binding site within its own promoter,may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation. View Publication

Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression,reproducibility,length,and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation,embryoid body (EB) differentiation,and direct neuronal differentiation. Here,we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage. View Publication

$\$-Thalassemia ($\$-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in $\$-globin gene (HBB). It is estimated that 4.5% of the population in the world carry $\$-Thal mutants (1),posing a persistent threat to public health. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However,homologous recombination-based gene correction in human iPSCs remains largely inefficient. Here,we describe a robust process combining efficient generation of integration-free $\$-Thal iPSCs from the cells of patients and transcription activator-like effector nuclease (TALEN)-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN-induced off targeting mutations by sequencing. More importantly,the gene-corrected $\$-Thal iPS cell lines from each patient can be induced to differentiate into hematopoietic progenitor cells and then further to erythroblasts expressing normal $\$-globin. Our studies provide an efficient and universal strategy to correct different types of $\$-globin mutations in $\$-Thal iPSCs for disease modeling and applications. View Publication

Microglia positively affect neural progenitor cell physiology through the release of inflammatory mediators or trophic factors. We demonstrated previously that reactive microglia foster K(ATP) -channel expression and that blocking this channel using glibenclamide administration enhances striatal neurogenesis after stroke. In this study,we investigated whether the microglial K(ATP) -channel directly influences the activation of neural precursor cells (NPCs) from the subventricular zone using transgenic Csf1r-GFP mice. In vitro exposure of NPCs to lipopolysaccharide and interferon-gamma resulted in a significant decrease in precursor cell number. The complete removal of microglia from the culture or exposure to enriched microglia culture also decreased the precursor cell number. The addition of glibenclamide rescued the negative effects of enriched microglia on neurosphere formation and promoted a 20% improvement in precursor cell number. Similar results were found using microglial-conditioned media from isolated microglia. Using primary mixed glial and pure microglial cultures,glibenclamide specifically targeted reactive microglia to restore neurogenesis and increased the microglial production of the chemokine monocyte chemoattractant protein-1 (MCP-1). These findings provide the first direct evidence that the microglial K(ATP) -channel is a regulator of the proliferation of NPCs under inflammatory conditions. View Publication

Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotidebackslashnrepeat (CAG) expansion in the coding region of the ATAXN2 gene onbackslashnchromosome 12,which produces an elongated,toxic polyglutamine tract,backslashnleading to Purkinje cell loss. There is currently no effective therapy.backslashnOne of the main obstacles that hampers therapeutic development is lackbackslashnof an ideal disease model. In this study,we have generated andbackslashncharacterized SCA2-induced pluripotent stem (iPS) cell lines as an inbackslashnvitro cell model. Dermal fibroblasts (FBs) were harvested from primarybackslashncultures of skin explants obtained from a SCA2 subject and a healthybackslashnsubject. For reprogramming,hOct4,hSox2,hKlf4,and hc-Myc werebackslashntransduced to passage-3 FBs by retroviral infection. Both SCA2 iPS andbackslashncontrol iPS cells were successfully generated and showed typical stembackslashncell growth patterns with normal karyotype. All iPS cell lines expressedbackslashnstem cell markers and differentiated in vitro into cells from threebackslashnembryonic germ layers. Upon in vitro neural differentiation,SCA2 iPSbackslashncells showed abnormality in neural rosette formation but successfullybackslashndifferentiated into neural stem cells (NSCs) and subsequent neuralbackslashncells. SCA2 and normal FBs showed a comparable level of ataxin-2backslashnexpression; whereas SCA2 NSCs showed less ataxin-2 expression thanbackslashnnormal NSCs and SCA2 FBs. Within the neural lineage,neurons had thebackslashnmost abundant expression of ataxin-2. Time-lapsed neural growth assaybackslashnindicated terminally differentiated SCA2 neural cells were short-livedbackslashncompared with control neural cells. The expanded CAG repeats of SCA2backslashnwere stable throughout reprogramming and neural differentiation. Inbackslashnconclusion,we have established the first disease-specific human SCA2backslashniPS cell line. These mutant iPS cells have the potential for neuralbackslashndifferentiation. These differentiated neural cells harboring mutationsbackslashnare invaluable for the study of SCA2 pathogenesis and therapeutic drugbackslashndevelopment. View Publication

Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus,HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here,we show that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro,most probably by binding to the catalytic center of HDACs. Most importantly,valproic acid induces differentiation of carcinoma cells,transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over,tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore,valproic acid might serve as an effective drug for cancer therapy. View Publication

BackgroundLevels of the human cationic antimicrobial host defence peptide LL-37 are enhanced in the lungs during neutrophilic airway inflammation. LL-37 drives Th17 differentiation,and Th17 cells produce IL-17A and IL-17F which form the biologically active heterodimer IL-17A/F. While IL-17 is a critical mediator of neutrophilic airway inflammation,LL-37 exhibits contradictory functions; LL-37 can both promote and mitigate neutrophil recruitment depending on the inflammatory milieu. The impact of LL-37 on IL-17-induced responses in the context of airway inflammation remains largely unknown. Therefore,we examined signaling intermediates and downstream responses mediated by the interplay of IL-17A/F and LL-37 in human bronchial epithelial cells (HBEC). As LL-37 can become citrullinated during airway inflammation,we also examined LL-37-mediated downstream responses compared to that with citrullinated LL-37 (citLL-37) in HBEC.ResultsUsing an aptamer-based proteomics approach,we identified proteins that are altered in response to IL-17A/F in HBEC. Proteins enhanced in response to IL-17A/F were primarily neutrophil chemoattractants,including chemokines and proteins associated with neutrophil migration such as lipocalin-2 (LCN-2). We showed that selective depletion of LCN-2 mitigates neutrophil migration,functionally demonstrating LCN-2 as a critical neutrophil chemoattractant. We further demonstrated that LL-37 and citLL-37 selectively suppress IL-17A/F-induced LCN-2 abundance in HBEC. Mechanistic studies revealed that LL-37 and citLL-37 suppresses IL-17 A/F-mediated enhancement of C/EBPβ,a transcription factor required for LCN-2 production. In contrast,LL-37 and citLL-37 enhance the abundance of ribonuclease Regnase-1,which is a negative regulator of IL-17 and LCN-2 in HBEC. In an animal model of allergen-challenged airway inflammation with elevated IL-17A/F and neutrophil elastase in the lungs,we demonstrated that CRAMP (mouse orthologue of LL-37) negatively correlates with LCN-2.ConclusionsOverall,our findings showed that LL-37 and citLL-37 can selectively suppress the abundance of IL-17A/F-mediated LCN-2,a protein that is critical for neutrophil migration in HBEC. These results suggest that LL-37,and its modified citrullinated form,have the potential to negatively regulate IL-17-mediated neutrophil migration during airway inflammation. To our knowledge,this is the first study to report that the immunomodulatory function of LL-37 enhances the RNA binding protein Regnase-1,suggesting that a post-transcriptional mechanism of action is mediated by the peptide.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12950-025-00446-w. View Publication

Glioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor and is associated with poor survival. Recently,stem-like cell populations have been identified in numerous malignancies including GBM. To identify genes whose expression is changed with differentiation,we compared transcript profiles from a GBM oncosphere line before and after differentiation. Bioinformatic analysis of the gene expression profiles identified podocalyxin-like protein (PODXL),a protein highly expressed in human embryonic stem cells,as a potential marker of undifferentiated GBM stem-like cells. The loss of PODXL expression upon differentiation of GBM stem-like cell lines was confirmed by quantitative real-time PCR and flow cytometry. Analytical flow cytometry of numerous GBM oncosphere lines demonstrated PODXL expression in all lines examined. Knockdown studies and flow cytometric cell sorting experiments demonstrated that PODXL is involved in GBM stem-like cell proliferation and oncosphere formation. Compared to PODXL-negative cells,PODXL-positive cells had increased expression of the progenitor/stem cell markers Musashi1,SOX2,and BMI1. Finally,PODXL expression directly correlated with increasing glioma grade and was a marker for poor outcome in patients with GBM. In summary,we have demonstrated that PODXL is expressed in GBM stem-like cells and is involved in cell proliferation and oncosphere formation. Moreover,high PODXL expression correlates with increasing glioma grade and decreased overall survival in patients with GBM. View Publication

Severe cutaneous adverse drug reactions (SCARs) are life-threatening diseases,which are associated with human leukocyte antigen ( HLA ) risk variants. However,the low positive predictive values of HLA variants suggest additional factors influence disease susceptibility. Using dapsone hypersensitivity syndrome (DHS) as a paradigm for SCARs,we show that the DHS patients harbor a sex-related global reduction in blood NK cells,contributing to the higher incidence of reactions in females. Single-cell RNA sequencing revealed a decrease in the immunoregulatory CD56 low XCL1/2 low NK cell subset and an expansion of CD56 high XCL1/2 high NK cell subsets with an effector phenotype in DHS patients compared to dapsone-tolerant individuals. Functionally,interleukin-15 superagonist-induced activation of NK cells exacerbated SCARs-like symptoms in a murine model. Mechanistically,TSC22 domain family member 3 (TSC22D3) deficiency enhanced NK cell effector function,shifting the immune response from CD4+ T cell to CD8+ T cell function. These results demonstrate that TSC22D3-regulated NK cells play a critical role in predisposing to drug hypersensitivity reactions,bridging innate and adaptive immune dysregulation in SCARs pathogenesis. Our study highlights the importance of NK cell heterogeneity and TSC22D3 in immune-mediated hypersensitivity disorders,offering potential therapeutic targets for SCARs and related conditions. Subject terms: Innate immunity,Innate immunity View Publication