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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.

Gallbladder cancer (GBC) is the most common malignancy of the biliary tract, with high metastasis incidence and extremely low survival rate. The tumor endothelial cells (TECs) are fundamental components in the tumor microenvironment and significantly contribute to various tumor progression; however, the roles of TECs in GBC are poorly understood. Here, using single-cell RNA sequencing, we identify a GBC-enriched endothelial population-CD34+CD90+ ECs (SAEndo2). The CD34+CD90+ endothelial subset correlates with patients’ poor prognosis and liver metastasis. In vitro and in vivo experiments suggest that CD34+CD90+ ECs promote the GBC cell migration and metastasis, showing EndoMT properties. Moreover, CD34+CD90+ ECs display enhanced activation of TGF-β signaling, and TGF-β inhibition abolishes the CD34+CD90+ ECs’ promotion effect on GBC cell migration. Collectively, our study provides a detailed profiling of endothelial cells in GBC and identifies an essential endothelial population that regulates GBC metastasis, laying new theoretical insight and offering a potential therapeutic target for GBC metastasis.

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.

BACKGROUND: Gastric cancer (GC) is a leading cause of cancer-related mortality worldwide. Oxaliplatin (OXA) based therapy plus/minus targeted therapy and immune check point inhibitors is the standard first-line treatment for advanced GC; however, its clinical efficacy is often hindered by the development of drug resistance. Cyclin-dependent kinase 12 (CDK12), a transcriptional regulator linked to DNA repair, plays a crucial role in transcription, cancer progression as well as drug resistance, where its exact role is unclear. In this study, we will investigate the role of CDK12 in GC progression and its potential as a therapeutic target specifically to enhance the efficacy of OXA. METHODS: CDK12 expression in GC tissues was analyzed by quantitative polymerase chain reaction (qPCR) and tissue microarrays (TMAs). A Kaplan-Meier survival analysis was conducted to assess the relationship between CDK12 levels and clinical outcomes. The effect of the CDK12 inhibitor combined with OXA was evaluated through in vivo and in vitro models. RNA-sequencing and western blots were used to investigate the molecular mechanisms of CDK12 inhibitor sensitizing OXA. RESULTS: CDK12 exhibited significant amplification frequency in GC. The Mendelian-randomization analysis revealed a positive causal association between elevated CDK12 expression and an increased risk of GC. Additionally, CDK12 was significantly overexpressed in GC tissues compared with adjacent normal tissues, and its high expression was significantly associated with a worse prognosis. The functional assays revealed that combining the CDK12 inhibitor THZ531 with OXA synergistically suppressed GC cell proliferation, induced apoptosis, and reduced colony formation in vitro, while substantially inhibiting tumor growth in xenograft models. Mechanistically, CDK12 inhibition disrupted MAPK signaling, leading to enhanced OXA-induced DNA damage and potentiated anti-tumor effects. CONCLUSIONS: Our findings suggest that CDK12 inhibition may represent a promising strategy for overcoming OXA resistance and improving GC treatment outcomes.

Cryptosporidium parvum is a major pathogen responsible for neonatal calf diarrhoea, but research has been hampered due to the lack of in vitro models that can complete the life cycle. In this scenario, human and murine small intestinal organoids (enteroids) are emerging as new in vitro tools. However, models employing bovine cells, relevant for the pathogenesis in the target species, are lacking. Thus, a panel of bovine enteroids was isolated in this study. Enteroids have an enclosed apical lumen, and the parasite must be delivered to the apical side of the cells to facilitate infection. Two different methods of reversing cell polarity were used to generate bovine apical-out enteroids: dissociation in ethylenediaminetetraacetic acid (EDTA), and dissociation in trypsin. Infection of these enteroids with C. parvum was attempted by incubation of the enteroids with viable, bleach-treated oocysts and subsequent cultivation of the two different enteroid set-ups. Apical-out enteroids dissociated in trypsin supported C. parvum infection and asexual replication, whilst dissociation in EDTA did not. However, only when a high dose of oocysts was administered, were all enteroids included able to support C. parvum replication consistently. When the apical-out enteroids were inoculated with a low dose of oocysts, only one isolate supported C. parvum replication, suggesting enteroid-specific variability. This study reports on infection and asexual replication of C. parvum in bovine apical-out ileal organoids. Graphical abstractImage 1 Highlights•Bovine apical-out enteroids can be established by dissociation in EDTA or trypsin.•Apical-out enteroids dissociated in EDTA did not support C. parvum infection.•Trypsinized apical-out enteroids supported C. parvum infection and replication.•Different isolates of enteroids supported C. parvum replication to different extents.

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.

Monoallelic mutations, p.A116V and p.R126P, in MMD2 disturb fMLP-induced activation of Ras/ERK signaling and underlie the impairment of neutrophil chemotaxis, which leads to the development of the autosomal dominant form of aggressive periodontitis. Aggressive periodontitis causes rapid destruction of periodontal tissue. It occurs at a young age with familial clustering. We report on the first time on molecular and cellular basis of a Mendelian form of autosomal dominant aggressive periodontitis. Monoallelic mutations in the monocyte to macrophage differentiation-associated 2 (MMD2) gene, encoding MMD2, in two Japanese families with autosomal dominant aggressive periodontitis are identified. Mutations, c.347 C>T (p.A116V) and c.377 G>C (p.R126P) in MMD2, disturbed fMLP-induced activation of Ras/ERK signaling. Additionally, abnormalities in the proteins of Golgi apparatus, a crucial contributor to innate immune signaling pathways, were identified in patients’ neutrophils. The knock-in and knockout mice exhibited alveolar bone loss by ligature-induced periodontitis, along with impaired fMLP-induced chemotaxis, as found in the patients with MMD2 mutation. Our studies revealed that monoallelic mutations in MMD2 underlie the impairment of neutrophil chemotaxis, which leads to the development of autosomal dominant aggressive periodontitis.

Zika virus (ZIKV) is a neurotropic Orthoflavivirus transmitted by mosquito vectors, which has evolved into two lineages, namely African and Asian. ZIKV from the Asian lineage has been responsible for epidemics in the Pacific and the Americas, the largest of which occurred in Brazil in 2015 and was associated with severe neurological disorders, including cases of microcephaly and other congenital fetal malformations. Although never implicated in human epidemics, African strains exhibit faster replication, higher virus production, and greater virulence in animal models compared to their Asian counterparts. A key feature that may account for the better fitness of African ZIKV strains compared to Asian ones is the fact that they are more resistant to interferon (IFN). IFN response is a major host defense mechanism against viral infections, which culminates in the induction of hundreds of IFN-induced genes (ISGs) whose products inhibit viral replication. By screening an array of ISGs known for their antiviral activity, we show that African ZIKV strains are globally more resistant than their Asian counterparts to ISG-mediated restriction. In particular, SHFL, RTP4 and IFI6, which were the three most active ISGs against Asian viruses, had little or no effect on the replication of African ZIKV strains. These observations therefore suggest that if African strains are more resistant to the antiviral effect of IFN than Asian strains, this is not because they have greater capacity to inhibit IFN signaling, but rather because they are able to escape ISG-mediated restriction. Our results provide an explanation as to why viruses of African origin spread more rapidly and efficiently in vitro than their Asian counterparts as repeatedly demonstrated. However, it remains unclear why, despite their greater virulence and resistance to cellular antiviral defenses, ZIKV strains of the African lineage have never been identified in large-scale epidemics. Author summaryInterferons (IFNs) are secreted antiviral cytokines, which commonly exert their activity by inducing an antiviral state, conferred by antiviral factors encoded by IFN-stimulated genes (ISGs). Collectively, ISGs can inhibit the replication of all known viruses, although some have developed resistance mechanisms. In this study, we compared the sensitivity to ISGs of different strains of Zika virus (ZIKV), a neurotropic arbovirus that exists as two distinct lineages, African and Asian. The Asian lineage of ZIKV has been responsible for major epidemics associated with severe neurological and congenital disorders, while the African lineage of ZIKV has not been reported to be responsible for epidemics or severe disease so far. In this study, we demonstrate that African ZIKV strains are more resistant than their Asian counterparts to the antiviral activity of IFN, and that ISGs that inhibit replication of Asian viruses have little or no effect on strains of African origin. While our results explain why African ZIKV strains propagate better than Asian ones, as demonstrated in various cellular and animal models, they do not provide an explanation for the higher pathogenicity and epidemic propensity of the Asian lineage.

AbstractT‐cell immunoglobulin and mucin‐domain containing‐3 (TIM3), generally known as an immune checkpoint receptor, is expressed on leukemic stem and progenitor cells (LSPCs) in acute myeloid leukemia (AML), and has an active role in LSC self‐renewal. Therefore, TIM3 has been suggested as a potential target for AML treatment. Hence, we explored the feasibility of targeting TIM3 with chimeric antigen receptor (CAR) T‐cells. Despite the expression of TIM3 on activated T‐cells, TIM3 CAR T‐cells were successfully generated from different healthy individuals with excellent in vitro expansion without signs of fratricide and sustained central‐memory phenotype with minimal expression of exhaustion‐related markers, including complete loss of TIM3 expression. TIM3 loss also did not affect effector functions since TIM3 CAR T‐cells efficiently lysed TIM3+ leukemic cell lines, produced Th1‐predominant cytokines, successfully inhibited the colony‐forming of TIM3+ AML‐derived LSPCs, and showed excellent AML tumor control in xenogeneic mouse models. Notably, TIM3 CAR T‐cells did not affect healthy hematopoietic progenitor cells and healthy mature hematopoietic cells that express TIM3 at moderate levels, suggesting an optimal therapeutic window for the treatment of AML.

INTRODUCTION: Necrotizing enterocolitis (NEC) is a leading cause of neonatal death and long-term morbidity, involving complex pathophysiology including prematurity, abnormal bacterial colonization, and ischemia-reperfusion injury, partially mediated by neutrophils. However, the limitations of current animal models hinder the development of targeted therapies for NEC. Thus, this study aimed to develop a human intestinal organoid model for NEC to investigate its pathophysiology, understand neutrophil involvement, and bridge animal and human research. METHODS: Organoid cultures were established from human neonatal intestinal samples with NEC (n=7) and without gut inflammation (controls, n=7), treated with lipopolysaccharides (LPS), and/or cocultured with neutrophils. Flow cytometry quantified neutrophil survival (propidium iodide/Annexin-V), activation (CD11b/CD66b), and TLR-4 expression, as well as organoid TLR-4 expression and apoptosis markers. NEC status and neutrophil recruitment were analyzed using immunofluorescence. RESULTS: After LPS administration, NEC organoids showed significantly increased TLR-4 expression, intestinal apoptosis markers, and NEC scores compared to controls, with more pronounced differences after neutrophil addition. Neutrophil activation markers were elevated when cocultured with both NEC and control organoids, but TLR-4 expression increased only with NEC organoids. DISCUSSION: The findings suggest that epithelial cells from NEC patients have a heightened innate TLR-4 expression upon LPS stimulation, potentially contributing to NEC development. LPS stimulation resulted in more pronounced NEC-like lesions in NEC organoids, which were exacerbated by neutrophils. This model demonstrates that neutrophils might contribute to NEC manifestation and maintenance and that NEC organoids can reflect disease aspects, potentially aiding in the development of targeted therapies.

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

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.