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CD4+ T helper (TH)-17 cells play a pivotal role in mucosal immune defense and are implicated in autoimmune diseases and cancer. Although Th17 cell plasticity is well-studied in mice,the factors driving their transition between pro-inflammatory and immunomodulatory states in humans remain less understood. Our study explored the transcriptional and epigenetic landscapes of single-cell cultures of human memory TH17 cells,focusing on clones that produce either immunomodulatory IL-10 or pro-inflammatory IFNγ and IL-22. We found that IL-10+ TH17 cells exhibit a T cell exhaustion-like profile with increased CTLA-4 expression and reduced IL-2 levels,while Ikaros zinc finger (IkZF) transcription factors,Aiolos and Eos,are differentially expressed in IL-10+ and IL-22+ TH17 cells,respectively. While exogenous IL-2 promotes IL-10 production in TH17 cells,lenalidomide induces IL-2 and promotes inflammatory TH17 cells,shifting TH17 cells towards a pro-inflammatory phenotype by reducing IL-10 and increasing IL-22 and IFNγ levels. Conversely,upregulation of Eos enhanced pro-inflammatory cytokine production. These findings highlight the crucial role of IkZF transcription factors in regulating human TH17 cell functions. Moreover,single-cell RNA sequencing of PBMCs from lenalidomide-treated patients confirmed an enrichment of inflammatory signatures,including interferon and IL-2/STAT5 pathways in TH17 cells. The ability to modulate this axis through targeted interventions,such as lenalidomide-induced Aiolos degradation or enforced Eos expression,presents new therapeutic opportunities for managing TH17 cell states in cancer and autoimmune diseases. View Publication

SummaryT cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues,including bone marrow (BM),liver,heart,and kidneys. We describe steps for tissue harvesting,preparation of single-cell suspensions,and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses. Graphical abstract Highlights•Isolation of leukocytes from murine BM,liver,heart and kidneys•Non-enzymatic dissociation of kidney and heart tissue•Protocol for immunomagnetic enrichment of T cells•Flow cytometry analysis of T cell purity and viability Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. T cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues,including bone marrow (BM),liver,heart,and kidneys. We describe steps for tissue harvesting,preparation of single-cell suspensions,and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses. View Publication

Objective and designChronic rhinosinusitis (CRS) is a heterogeneous inflammatory disease of the paranasal sinuses,which is divided into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRSwNP is typically caused by type 2 inflammation,which is characterized by elevated IL-4 and IL-13 levels,impairment of the epithelial barrier,and tissue remodeling. While the involvement of immune cells is well known,it remains unclear to what extent structural cells intrinsically maintain disease-specific functional programs. The aim of this study was to determine whether epithelial cells and fibroblasts derived from CRSwNP and CRSsNP differ in their barrier properties,inflammatory reactivity,and type 2-associated functional characteristics.MethodsAir–liquid interface (ALI) epithelial cultures and primary fibroblast cultures were generated from CRSwNP and CRSsNP tissue. Epithelial barrier integrity was assessed by transepithelial electrical resistance (TEER),and inflammatory responses to TLR stimulation were analyzed by qRT-PCR. Fibroblast migration was evaluated using scratch assays. Cellular responses to IL-4/IL-13 with or without Dupilumab were quantified by qRT-PCR.ResultsCRSwNP-derived epithelial cells exhibited delayed tight junction formation and impaired differentiation compared to CRSsNP cells. Poly(I:C) stimulation induced stronger expression of Th2-associated cytokines in CRSwNP cultures. CRSwNP fibroblasts showed reduced migratory capacity and a heightened induction of Th2 cytokines and extracellular matrix genes following IL-4/IL-13 stimulation relative to CRSsNP fibroblasts.ConclusionEpithelial cells and fibroblasts derived from CRSwNP retain disease-associated type 2 characteristics in vitro,indicating persistent disease-aligned programmed functional alterations of the polyp microenvironment. In contrast,CRSsNP-derived cells lacked comparable enhanced type 2 responsiveness. These findings support CRSwNP as a distinct,self-sustaining inflammatory endotype and underscore the value of patient-derived models for investigating disease mechanisms and targeted therapies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12950-026-00497-7. View Publication

Classical intracellular delivery methods such as transfection and transduction are inefficient,particularly with confluent cells and organoids,and lack cell type‐specific programmability. We demonstrate that an innovative methodology called calcium shock (CaSh) dramatically improves particle delivery into single cells,colonies,and organoids,and enables programmable delivery (CaSh‐Pro) into specific cell types within heterocellular populations. Calcium shock works by increasing endocytotic uptake while simultaneously disarming cell‐cell junctions. CaSh‐Pro further incorporates specific molecular targeting agents and amphiphilic peptides for preferential editing of different cell types. Calcium shock improves expression of plasmid,ribonucleoprotein,or adeno‐associated viral vectors with minimal toxicity in intact organoids representing diverse lineages. CaSh and CaSh‐Pro provide simple,versatile protocols for genome editing in complex systems,to enable biological discovery and therapeutic development. Classical transfection and transduction are inefficient,particularly with confluent cells and organoids,and lack cell type‐specific programmability. This study presents calcium shock (CaSh),a method that dramatically improves particle delivery into single cells,colonies,and organoids. CaSh is further utilized to enable programmable delivery (CaSh‐Pro) into specific cell types within heterocellular populations. View Publication

Background: Immunotherapy efficacy in esophageal squamous cell carcinoma (ESCC) is often limited by an immunosuppressive tumor microenvironment (TME). NLRC5,a key regulator of MHC-I antigen presentation,exhibits context-dependent roles in tumor immunity; however,its function in ESCC remains unclear. This study aimed to systematically investigate the expression pattern,prognostic value,and immunological role of NLRC5 in ESCC. Methods: An integrated analysis of bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) data was performed using multiple cohorts,including The Cancer Genome Atlas,Gene Expression Omnibus,and an in-house ESCC cohort. Differential expression,survival analysis,immune infiltration estimation,and functional enrichment analyses were conducted to elucidate the role of NLRC5 in the tumor microenvironment. Results: NLRC5 was significantly upregulated in ESCC and its high expression independently predicted poor patient survival. Although NLRC5 expression was associated with increased CD8+ T cell infiltration,it was paradoxically accompanied by features of T-cell exhaustion and elevated expression of multiple immune checkpoints. Moreover,NLRC5-high tumors were enriched in transcriptional programs related to PANoptosis,indicating an additional immunosuppressive mechanism within the TME. Conclusions: NLRC5 is not only a prognostic biomarker but also a key modulator of an immune-active yet functionally suppressed tumor microenvironment in ESCC. These findings highlight NLRC5 as a potential therapeutic target for restoring effective antitumor immunity. View Publication