技术资料

Tyrosine kinase inhibitors (TKIs) are the standard treatment for advanced hepatocellular carcinoma (HCC). However,their therapeutic efficacy is often limited by drug resistance,primarily driven by tumoral intrinsic mechanisms. In this study,we demonstrate that IFNγ in the tumor microenvironment can potentiate TKI response,and that ablation of IFNγ receptor on HCC cells leads to TKI resistance in vivo. Mechanistically,IFNγ synergizes with TKI to induce GSDME-mediated pyroptosis of HCC cells. The PERK-mediated unfolded protein response (UPR) protects HCC cells from TKI-induced pyroptosis. IFNγ attenuates PERK activation by inducing the expression of PDIA1,which alleviates the stress of protein unfolding. In vivo,PERK inhibition augments TKI therapy,and elevated PERK expression correlates with poor overall survival of patients with HCC. Moreover,IFNγ-producing CD8+ T cells can potentiate TKI efficacy. Combining PD-1 blockade to activate T-cell response with TKI therapy synergistically suppresses the growth of GSDME-expressing HCC tumors,which is further enhanced by the PERK inhibitor. Our findings reveal how IFNγ signaling modulates TKI response and demonstrate the potential of a sequential combination of ICB-mediated immunotherapy and TKI therapy for patients with GSDME+ HCC. T cell-derived IFNγ enhances TKI-induced pyroptosis in HCC. Mechanistic illustration of IFNγ secreted from CD8+ T cells enhancing TKI-induced GSDME-mediated pyroptosis in hepatocellular carcinoma via suppression of the PERK pathway. Created with BioRender.com. View Publication

FOXP3+ regulatory T cells (Tregs) play a central role in the regulation of the immune system. Human Tregs preferentially express a FOXP3 isoform known as delta 2,which lacks exon 2. In addition to FOXP3,Tregs also express isoforms of other Treg-related molecules,such as CTLA-4 and IKZF-2. It is hypothesized that Tregs possess a unique isoform repertoire based on their unique gene and isoform expression profiles,which include FOXP3. Here,we identified a Treg-specific unique isoform repertoire confirmed by long-read high-throughput isoform sequencing called Iso-seq,which is uniquely capable of providing data on genome-wide isoform usage. Notably,while conventional T cells (Tconvs) do not exhibit this pattern,Tregs preferentially express the full-length FOXP3 isoform. Interestingly,the preferential expression of ICOS and PD-L1 upon T-cell receptor (TCR) stimulation was noted in activated Tregs but not in Tconvs or non-activated Tregs. Moreover,using a PD-L1 antibody blockade on Tregs did not diminish FOXP3 expression; however,it significantly reduced the suppressive function. Therefore,Tregs may have a unique isoform repertoire,which becomes pronounced upon polyclonal TCR stimulation. View Publication

AbstractNonsense mutations arise from single nucleotide substitutions that result in premature termination codons (PTCs). PTCs result in little to no full-length protein production and decreased mRNA stability due to the nonsense-mediated mRNA decay (NMD) pathway. We provide evidence that anticodon-edited (ACE-) tRNAs efficiently suppress the most prevalent cystic fibrosis (CF)-causing PTCs,promoting significant rescue of endogenous cystic fibrosis transmembrane conductance regulator (CFTR) transcript abundance and channel function in different model systems. We show that our best-performing ACE-tRNA,which decodes all UGA PTCs to a leucine amino acid,markedly rescues CFTR function from the most prevalent CF-causing PTCs,all of which arose from nonleucine encoding codons. Using this single ACE-tRNA variant,we demonstrate significant rescue of CFTR function in an immortalized airway cell line and two different primary CF patient-derived intestinal cell models with CFTR nonsense mutations. Further,we demonstrate that leucine substitution CFTR variants are highly functional. Thus,ACE-tRNAs have promise as a platform therapeutic for CF and other nonsense-associated diseases. Graphical Abstract Graphical Abstract View Publication

Efficient immune responses rely on the capacity of leukocytes to traverse diverse and complex tissues. To meet such changing environmental conditions,leukocytes usually adopt an ameboid configuration,using their forward-positioned nucleus as a probe to identify and follow the path of least resistance among pre-existing pores. We show that,in dense environments where even the largest pores preclude free passage,leukocytes position their nucleus behind the centrosome and organelles. The local compression imposed on the cell body by its surroundings triggers assembly of a central F-actin pool,located between cell front and nucleus. Central actin pushes outward to transiently dilate a path for organelles and nucleus. Pools of central and front actin are tightly coupled and experimental depletion of the central pool enhances actin accumulation and protrusion formation at the cell front. Although this shifted balance speeds up cells in permissive environments,migration in restrictive environments is impaired,as the unleashed leading edge dissociates from the trapped cell body. Our findings establish an actin regulatory loop that balances path dilation with advancement of the leading edge to maintain cellular coherence. Sixt and colleagues show that,in environments where even the largest pores preclude free passage,leukocytes position their nucleus behind the centrosome and assemble a central F-actin pool that pushes outward to transiently dilate a path for the nucleus. View Publication

AbstractBackgroundPublic neoantigens,including KRAS,TP53,and PIK3CA mutations,which are shared across various tumor types,have demonstrated significant immunogenicity and offer great promise for cancer immunotherapy. Clinical trials targeting these public neoantigens have yielded encouraging results,including tumor regression and prolonged relapse-free survival. This study evaluates the human leukocyte antigen (HLA) binding properties of T-cell epitopes derived from these public neoantigens to identify optimal T-cell target and further develops T-cell receptor (TCR)-based therapeutics.MethodsThe binding properties of public neoantigens to HLA-I molecules were evaluated using peptide-HLA binding affinity and stability assays. Naive T-cell repertoires were used to expand and detect neoantigen-specific TCRs. TCR clones were characterized for functionality using TCR-Jurkat cells and TCR-T cells. Peptide specificity was assessed using an HLA transgenic cell panel and the X-scan assay. In vivo antitumor efficacy of TCR-T cells was tested in xenograft mouse models of solid tumors.ResultsThe analysis of HLA binding properties for public neoantigens revealed that HLA-A*11:01-presented KRASG12V epitopes exhibited the strongest HLA binding stability. Four TCR clones specific to the 9-mer KRASG12V peptide (KRASG12V[9]) were identified. All KRASG12V[9]-specific TCRs,both newly identified by us and previously reported,exhibited varying degrees of cross-recognition of the exogenous self-antigen RAB7B. Among the four TCR clones,one TCR (KT18) exhibited superior functional avidity,effectively recognizing and eliminating KRASG12V mutant tumor cells without off-target activity against endogenous RAB7B or similar peptides. Significantly,KT18 TCR-T cells efficiently mediated tumor regression in multiple xenograft models of solid tumors.ConclusionsThese findings highlight significant differences in peptide-HLA binding affinity and stability across public neoantigen-HLA pairings. The cross-recognition of RAB7B13-21 represents a critical safety consideration when developing HLA-A*11:01-restricted KRASG12V[9]-specific TCRs. KT18 TCR-T cells are highly cytotoxic,exhibiting no off-target recognition and significant potential for clinical applications against KRASG12V-driven solid tumors. View Publication

AbstractDendritic cells (DCs) and natural killer (NK) cells engage in reciprocal interactions to trigger an efficient innate immune response while governing downstream adaptive immunity. Here,we used an ex vivo autologous human primary immune cell coculture system of DCs and NK cells to examine their impact on naïve CD4+ and CD8+ T cell (CD3+CD45RA+CD197+) activation in response to influenza A viral (IAV) infection. Using multiparameter flow cytometry,we observed that culturing T cells with both DCs and NK cells enhanced CD69 expression on CD4+ and CD8+ T cells,increased CD25 on CD4+ T cells,and promoted CD8+ T cell proliferation,compared with cultures with only NK cells or DCs. When DCs were exposed to the pandemic A/California/07/2009 (H1N1) strain or the A/Victoria/361/2011 (H3N2) strain,subsequent coculture with NK cells reduced the frequency of CD4+CD69+ and CD8+CD69+ naïve T cells. Notably,H3N2,but not H1N1,exposure also reduced CD4+CD25+ T cell frequencies. The IAV-mediated curtailment of T cell activation was dependent on viral replication because exposure to DCs with irradiated the H1N1 strain followed increased the frequency of CD4+CD69+,CD8+CD69+,CD4+CD25+,and CD8+CD25+ T cells,while irradiation of H3N2 increased the frequency of CD4+CD69+,CD8+CD69+ and proliferation of CD4+ and CD8+ T cells. These findings demonstrate that IAV can partially subvert DC-NK cell crosstalk to impair naïve T cell activation in a strain-dependent manner. This knowledge may guide the design of next-generation influenza vaccines to elicit robust cellular immune responses. View Publication

A major knowledge gap exists in understanding immune effects of adjuvants in early life. As environmental stimuli shape the infant immune system,adjuvants may also influence this process. Using a neonatal mouse model,we investigated the differential effects of adjuvants in neonates vs. adults. Mice were immunized with an adjuvanted hepatitis B vaccine followed by exposure to ovalbumin to determine whether prior immunization alters subsequent heterologous immune responses. Neonatal immunization with a Th2-biased alum-adjuvanted vaccine predisposed mice to develop Th2-biased immunity to subsequent ovalbumin exposures. Conversely,neonatal immunization with a Th1-polarizing CpG-adjuvanted vaccine resulted in preferential priming of Th1-biased heterologous responses. Immunization in adulthood did not alter heterologous immune responses. Early-life immunization modified the ability of bone marrow DCs to prime Th1/Th2 immune responses,suggesting a role for immune training in these antigen agnostic effects. These data suggest that rational adjuvant selection for early-life vaccines may beneficially shape immune development. View Publication

SummaryHemozoin (HZ),the malaria pigment,is associated with the disease when released during the pro-inflammatory blood stage and co-infections with bacteria lead to a more severe disease progression. The underlying mechanisms are poorly understood and,here,we show that the impact of co-exposure to HZ and lipopolysaccharide (LPS) on monocyte-derived dendritic cells (moDC) alters the early transcriptional response to a subset of IFNγ controlled genes,HLA-DR,and PD-L1. HZ-exposure had no effect on inflammatory genes,which were substantially induced by LPS. The reduced expression of HLA-DR and PD-L1 by HZ was associated with the chromatin remodeling complex NuRD and a decreased binding of the NF-κB transcription factor RELA compared to cells stimulated with LPS alone. NuRD replaced the SWI/SNF complex variant PBAF at the specific promoters,without chromatin accessibility changes. The immune modulatory effect of HZ may lead to changed immune responses to bacterial co-infections. Graphical abstract Highlights•Hemozoin dampens the early acute response of LPS by impeding a subset of ISG promoters•LPS activates ISG through the SWI/SNF complex PBAF,HZ represses the activation through NuRD•Inflammatory genes are activated through the SWI/SNF complex ncBAF and not affected by HZ•HZ activates NF-κB and IRF3,keeping DC in an immature state by maintaining DC SIGN and CD206 Immune response; Molecular mechanism of gene regulation; Parasitology View Publication

Probiotics offer a non-pharmacological approach to support immune function,yet clinical evidence for strain-specific benefits remains limited. We conducted an 8-week,randomized,double-blind,placebo-controlled trial of Lacticaseibacillus rhamnosus LM1019 in 121 generally healthy adults. Both the active and placebo arms produced comparable within-group increases in natural killer (NK) cell cytotoxicity and modest,non-differential declines in circulating cytokines; safety and tolerability were excellent,with mild adverse events evenly distributed. In a post-hoc subgroup defined by age ≥ 40 years,baseline white blood cell count ≥ 5.0 × 103/µL,and LDL cholesterol < 130 mg/dL,the probiotic arm demonstrated statistically significant enhancements in NK activity (p = 0.021–0.008 across all effector-to-target ratios),whereas no change was observed in the placebo group. These findings suggest that this intervention may selectively boost NK-mediated immunity in individuals with preserved baseline immune and lipid profiles. Future larger trials using phenotype-driven enrollment and controlled dietary intake are warranted to confirm and extend these results. View Publication

Eccrine sweat glands play a vital role in human thermoregulation; however,their self-repair function is minimal. Therefore,developing methods to regenerate and improve sweat gland function that use cultured sweat gland cells presents an urgent issue. The tissue microenvironment,especially hypoxic niches,essentially maintain cell stemness,highlighting the importance of oxygen concentration in the culture environment. Therefore,we evaluated the effects of different oxygen environments on human sweat glands and their regulatory mechanisms. Human eccrine sweat glands express HIF-1α and HIF-2α,suggesting that they respond to hypoxia in vivo. Primary human-derived eccrine sweat gland cells were cultured for two weeks using the spheroid culture method at 0.5%,2%,10%,and 21% O2 concentration. HIF-1,Wnt/β-Catenin,and TGFβ1 signaling increased in sweat gland cells cultured in 0.5% O2 conditions,along with increased undifferentiated cell marker expression. The results of this study will contribute to in vitro research models of sweat glands and treatment development for damage to sweat glands,including burns. View Publication

Glioblastoma multiforme (GBM) is an aggressive and molecularly heterogeneous brain cancer with a poor prognosis. Despite advancements in standard-of-care therapies,including surgery,radiotherapy,and temozolomide (TMZ),the median survival remains approximately 15 months,with a 5-year survival rate of less than 10%. We and others have demonstrated that FOXM1 is a critical oncogenic driver of GBM cell proliferation. However,the role of FOXM1 and its interaction with other oncogenic signaling pathways in GBM remains incompletely understood. In this study,we identified FOXM1,AXL,and eEF2K as highly upregulated oncogenes in GBM patient tumors. We demonstrated,for the first time,that FOXM1 directly interacts with AXL and eEF2K,regulating their expression and promoting GBM cell proliferation,migration,and invasion. Knockdown of these genes disrupted cell proliferation,spheroid formation,migration,and invasion,and induced apoptosis and ferroptosis. Additionally,inhibiting the FOXM1-AXL/eEF2K signaling axis sensitized GBM cells to TMZ,further enhancing apoptotic and ferroptotic responses. These findings highlight the critical role of the FOXM1-AXL/eEF2K signaling pathway in GBM progression and suggest that targeting this axis may offer a novel multitargeted therapeutic strategy in GBM. View Publication

The lymphocyte-specific transcription factor interferon regulatory factor 4 (IRF4) is a key player in immune evasion in cancers,with the complex mechanism(s) being barely understood. In this study,we have focused on the role of IRF4 in regulating T cell functions through its transcriptional regulation of programmed death 1 (PD1) and its ligand PD1 ligand 1 (PD-L1),which were identified as IRF4 transcriptional targets in multi-omics analysis. We have shown that IRF4 transcriptionally regulates both PD1 and PD-L1,promoting immune suppression in the context of Epstein–Barr virus (EBV) infection. Co-culturing EBV+ JiJoye lymphoma cells with CD4+ T cells or with peripheral blood mononuclear cells (PBMCs) downregulates CD4+ T cell functions,but the depletion of IRF4 in EBV+ JiJoye lymphoma cells reduces PD1 and PD-L1 expression,and partially restores CD4+ T cell functions. Moreover,CD4+ T cell depletion from PBMCs enhances EBV transformation,and EBV has a greater efficiency in transforming PBMCs from HIV patients with impaired CD4+ T cell functions. These findings support the role of IRF4 in immune evasion by upregulating PD1/PD-L1 during EBV transformation,and that functional CD4+ T cells are essential for limiting EBV transformation. View Publication