Themis suppresses the effector function of CD8+ T cells in acute viral infection.

CD8+ T cells play a central role in antiviral immune responses. Upon infection, naive CD8+ T cells differentiate into effector cells to eliminate virus-infected cells, and some of these effector cells further differentiate into memory cells to provide long-term protection after infection is resolved. Although extensively investigated, the underlying mechanisms of CD8+ T-cell differentiation remain incompletely understood. Themis is a T-cell-specific protein that plays critical roles in T-cell development. Recent studies using Themis T-cell conditional knockout mice also demonstrated that Themis is required to promote mature CD8+ T-cell homeostasis, cytokine responsiveness, and antibacterial responses. In this study, we used LCMV Armstrong infection as a probe to explore the role of Themis in viral infection. We found that preexisting CD8+ T-cell homeostasis defects and cytokine hyporesponsiveness do not impair viral clearance in Themis T-cell conditional knockout mice. Further analyses showed that in the primary immune response, Themis deficiency promoted the differentiation of CD8+ effector cells and increased their TNF and IFNγ production. Moreover, Themis deficiency impaired memory precursor cell (MPEC) differentiation but promoted short-lived effector cell (SLEC) differentiation. Themis deficiency also enhanced effector cytokine production in memory CD8+ T cells while impairing central memory CD8+ T-cell formation. Mechanistically, we found that Themis mediates PD-1 expression and its signaling in effector CD8+ T cells, which explains the elevated cytokine production in these cells when Themis is disrupted.

Themis is indispensable for IL-2 and IL-15 signaling in T cells.

To perform their antiviral and antitumor functions, T cells must integrate signals both from the T cell receptor (TCR), which instruct the cell to remain quiescent or become activated, and from cytokines that guide cellular proliferation and differentiation. In mature CD8+ T cells, Themis has been implicated in integrating TCR and cytokine signals. We investigated whether Themis plays a direct role in cytokine signaling in mature T cells. Themis was required for IL-2- and IL-15-driven CD8+ T cell proliferation both in mice and in vitro. Mechanistically, we found that Themis promoted the activation of the transcription factor Stat and mechanistic target of rapamycin signaling downstream of cytokine receptors. Metabolomics and stable isotope tracing analyses revealed that Themis deficiency reduced glycolysis and serine and nucleotide biosynthesis, demonstrating a receptor-proximal requirement for Themis in triggering the metabolic changes that enable T cell proliferation. The cellular, metabolic, and biochemical defects caused by Themis deficiency were corrected in mice lacking both Themis and the phosphatase Shp1, suggesting that Themis mediates IL-2 and IL-15 receptor-proximal signaling by restraining the activity of Shp1. Together, these results not only shed light on the mechanisms of cytokine signaling but also provide new clues on manipulating T cells for clinical applications.

m7G-related gene NUDT4 as a novel biomarker promoting cancer cell proliferation in lung adenocarcinoma.

Background: Lung cancer is the leading cause of mortality in cancer patients. N7-methylguanosine (m7G) modification as a translational regulation pattern has been reported to participate in multiple types of cancer progression, but little is known in lung cancer. This study attempts to explore the role of m7G-related proteins in genetic and epigenetic variations in lung adenocarcinoma, and its relationship with clinical prognosis, immune infiltration, and immunotherapy. Methods: Sequencing data were obtained from the Genomic Data Commons (GDC) Data Portal and Gene Expression Omnibus (GEO) databases. Consensus clustering was utilized to distinguish m7G clusters, and responses to immunotherapy were also evaluated. Moreover, univariate and multivariate Cox and Least absolute shrinkage and selection operator LASSO Cox regression analyses were used to screen independent prognostic factors and generated risk scores for constructing a survival prediction model. Multiple cell types such as epithelial cells and immune cells were identified to verify the bulk RNA results. Short hairpin RNA (shRNA) Tet-on plasmids, Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 for knockout plasmids, and nucleoside diphosphate linked to moiety X-type motif 4 (NUDT4) overexpression plasmids were constructed to inhibit or promote tumor cell NUDT4 expression, then RT-qPCR, Cell Counting Kit-8 CCK8 proliferation assay, and Transwell assay were used to observe tumor cell biological functions. Results: Fifteen m7G-related genes were highly expressed in tumor samples, and 12 genes were associated with poor prognosis. m7G cluster-B had lower immune infiltration level, worse survival, and samples that predicted poor responses to immunotherapy. The multivariate Cox model showed that NUDT4 and WDR4 (WD repeat domain 4) were independent risk factors. Single-cell m7G gene set variation analysis (GSVA) scores also had a negative correlation tendency with immune infiltration level and T-cell Programmed Death-1 PD-1 expression, but the statistics were not significant. Knocking down and knocking out the NUDT4 expression significantly inhibited cell proliferation capability in A549 and H1299 cells. In contrast, overexpressing NUDT4 promoted tumor cell proliferation. However, there was no difference in migration capability in the knockdown, knockout, or overexpression groups. Conclusions: Our study revealed that m7G modification-related proteins are closely related to the tumor microenvironment, immune cell infiltration, responses to immunotherapy, and patients' prognosis in lung adenocarcinoma and could be useful biomarkers for the identification of patients who could benefit from immunotherapy. The m7G modification protein NUDT4 may be a novel biomarker in promoting the progression of lung cancer.

In Situ biomimetic Nanoformulation for metastatic cancer immunotherapy.

Metastasis is the leading cause of death in cancer patients. Eliciting anti-tumor immune responses against lung metastasis is hindered by the immunosuppressive microenvironment. This study explored a biomimetic nanoformulation, comprising a nanovaccine (OP) that delivers tumor antigens and adjuvants spatially and temporally in a virus-like manner, and a pulmonary surfactant-biomimetic liposome with an immunomodulator, JQ1 (PS-JQ1). The findings of this study showed that intratracheal administration of OP+PS-JQ1 activated lung immune cells without concomitant excess inflammation, enhanced tumor antigen cross-presentation, generated a significantly high antigen-specific CD8+ T cell response, and reshaped the immunocellular composition in B16 melanoma tumor-bearing lung. OP+PS-JQ1 nanoformulation exhibited a striking immunotherapeutic efficacy, induced local and systemic tumor suppression, improved survival of mice, initiated immune memory that prevents recurrence of secondary tumors. This stable and nontoxic nanoformulation provides a simple, flexible, and robust strategy for augmenting anti-tumor immunity for metastatic cancer. STATEMENT OF SIGNIFICANCE: Egg glue proteins are produced by female insects, which can make the eggs firmly attached to the oviposition sites, not affected by wind and rain. However, genes encoding insect egg glue proteins have not yet been reported, and the molecular mechanism underpinning their adhesion is still unknown. Our study makes a significant contribution to the literature as it identifies the sequence, structure, adhesive property, and mechanism of silkworm egg glue protein. Furthermore, it outlines key insights into the structure-function relationships associated with egg glue proteins. We believe that this paper will be of interest to the readership of your journal as it identifies the first complete sequence of insect egg glue proteins, thereby highlighting their potentials future applications in both the biomedical and technical fields.

Tubule-specific protein nanocages potentiate targeted renal fibrosis therapy.

BACKGROUND: Despite the dramatic advances in modern medicine, efficient therapeutic measures for renal fibrosis remain limited. Celastrol (CLT) is effective in treating renal fibrosis in rat models, while causing severe systemic toxicity. Thus, we designed a tubule-specific nanocage (K3-HBc NCs) that effectively deliver CLT to tubular epithelial cell in a virus-like manner. The targeting ligand (K3) to tubular epithelial cells was displayed on the surface of Hepatitis B core protein (HBc) NCs by genetic fusion to the major immunodominant loop region. Ultra-small CLT nanodots were subtly encapsulated into the cavity through electrostatic interaction with the disassembly and reassembly of K3-HBc NCs, to yield K3-HBc/CLT complex. The efficacy of K3-HBc/CLT NCs were demonstrated in Unilateral ureteral obstruction (UUO)-induced renal fibrosis. RESULTS: The self-assembled K3-HBc/CLT could specifically target tubular epithelial cells via affinity with K3 ligand binding to the megalin receptor, significantly attenuating renal fibrosis. Remarkably, K3-HBc/CLT NCs significantly increased therapeutic efficacy and reduced the systemic toxicity in comparison with free CLT in UUO-induced mouse renal fibrosis model. Importantly, analysis of RNA sequencing data suggested that the anti-fibrotic effect of K3-HBc/CLT could be attributed to suppression of premature senescence in tubular epithelial cells via p21Cip1 and p16Ink4a pathway. CONCLUSION: The tubule-specific K3-HBc/CLT represented a promising option to realize precise treatment for renal fibrosis.

The T Cell Receptor Immune Repertoire Protects the Liver From Reconsitution.

Aberrant immune cell infiltrates and microcircumstances represent characteristic features of liver fibrosis. In this study, we profiled the transcriptomes of intrahepatic CD45+ immune cells, from mice, using single-cell RNA sequencing (scRNA-seq) technology to understand the landscape of intrahepatic immune cells during the pathogenesis of fibrosis. Analysis of approximately 10,000 single-cell transcriptomes revealed an increase in dendritic cells (DCs), macrophages, and neutrophils and a decrease in T and natural killer T (NKT) cells. In addition, we report changes in the transcriptomes of diverse immune cell types, implying a deteriorating intrahepatic immune microcircumstance. Furthermore, we uncovered a novel fibrosis-associated CD8 T (Ccl5+, Ccl4+) and CD4 T (mt-Co1+) cell subpopulation, which infiltrates fibrotic liver and is characterized by abnormal activation or inactivation as well as a TCR decline. The results from scRNA-seq and bulk immune repertoire sequencing (IR-seq) revealed an obvious decline in T cell receptor (TCR) clonotypes combined with shrinking VJ and VDJ segment usage, as well as lower complementarity-determining region 3 (CDR3) amino acid (AA) diversity from fibrotic liver. Interestingly, a deficiency of TCR IR (TcrbKO mice) led to a deterioration of liver fibrosis, coupled with activation of hepatic stellate cells (HSCs) induced by the upregulation of macrophage and γδ T cell distribution in fibrotic TcrbKO livers. Our findings reveal the landscape and dynamics of single immune cells in liver fibrosis, and clarify the protective role of TCR IR in response to chronic liver injury.

Synthetic lethality of combined AT-101 with idarubicin in acute myeloid leukemia via blockade of DNA repair and activation of intrinsic apoptotic pathway.

Leukemia stem cells (LSCs) are deemed to the mainspring for treatment failure in acute myeloid leukemia (AML). Conventional chemotherapeutic drugs fail to eradicate leukemia stem cells, which becomes the root of drug resistance and disease recurrence. Hence, new therapeutic strategies targeting LSCs are supposed to be critical for patients with AML. Here we report that combination of Bcl-2 inhibitor AT-101 and chemotherapeutic drug idarubicin (IDA) results in synergistic lethality in CD34+CD38- leukemia stem-like cells sorted from KG-1α and Kasumi-1 AML cell lines and primary CD34+ AML cells in vitro while sparing the normal counterparts. In addition, combinatorial treatment also significantly inhibits the growth of patient-derived xenograft (PDX) mouse models generated from FLT3-ITDmut AML patient in vivo. Mechanistically, the synergistic effects of AT-101 with IDA to induce cell death are closely associated with blockage of DNA damage repair and thus activates the intrinsic apoptotic pathway. In summary, these findings suggest that combinatorial therapy with AT-101 and IDA selectively eliminates leukemia stem-like cells both in vitro and in vivo, representing a potent and alternative salvage therapy for the treatment of relapsed and refractory patients with AML.