Critical roles of the miR-17∼92 family in thymocyte development, leukemogenesis, and autoimmunity.

Thymocyte development requires precise control of PI3K-Akt signaling to promote proliferation and prevent leukemia and autoimmune disorders. Here, we show that ablating individual clusters of the miR-17∼92 family has a negligible effect on thymocyte development, while deleting the entire family severely impairs thymocyte proliferation and reduces thymic cellularity, phenocopying genetic deletion of Dicer. Mechanistically, miR-17∼92 expression is induced by Myc-mediated pre-T cell receptor (TCR) signaling, and miR-17∼92 promotes thymocyte proliferation by suppressing the translation of Pten. Retroviral expression of miR-17∼92 restores the proliferation and differentiation of Myc-deficient thymocytes. Conversely, partial deletion of the miR-17∼92 family significantly delays Myc-driven leukemogenesis. Intriguingly, thymocyte-specific transgenic miR-17∼92 expression does not cause leukemia or lymphoma but instead aggravates skin inflammation, while ablation of the miR-17∼92 family ameliorates skin inflammation. This study reveals intricate roles of the miR-17∼92 family in balancing thymocyte development, leukemogenesis, and autoimmunity and identifies those microRNAs (miRNAs) as potential therapeutic targets for leukemia and autoimmune diseases.

Fermented Gracilaria lemaneiformis polysaccharides alleviate food allergy by regulating Treg cells and gut microbiota.

Food allergy has a significant impact on the health and well-being of individuals, affecting both their physical and mental states. Research on natural bioactive compounds, such as polysaccharides extracted from seaweeds, holds great promise in the treatment of food allergies. In this study, fermented Gracilaria lemaneiformis polysaccharides (F-GLSP) were prepared using probiotic fermentation. Probiotic fermentation of Gracilaria lemaneiformis reduces the particle size of polysaccharides. To compare the anti-allergic activity of F-GLSP with unfermented Gracilaria lemaneiformis polysaccharides (UF-GLSP), an OVA-induced mouse food allergy model was established. F-GLSP exhibited a significant reduction in OVA-specific IgE and mMCP levels in allergic mice. Moreover, it significantly inhibited Th2 differentiation and IL-4 production and significantly promoted Treg differentiation and IL-10 production in allergic mice. In contrast, UF-GLSP only reduced OVA-specific IgE and mMCP in the serum of allergic mice. Furthermore, F-GLSP demonstrated a more pronounced regulation of intestinal flora abundance compared to UF-GLSP, significantly influencing the populations of Firmicutes, Bacteroidetes, Lactobacillus, and Clostridiales in the intestines of mice with food allergy. These findings suggest that F-GLSP may regulate food allergies in mice through multiple pathways. In summary, this study has promoted further development of functional foods with anti-allergic properties based on red algae polysaccharides.

Immunomodulatory Function of Pien Tze Huang in T Cell-Mediated Anti-tumor Activity against B16-F10, MC38 and Hep1-6 Tumor Models.

OBJECTIVE: To investigate the anti-tumor effects of Pien Tze Huang (PZH) in mouse models of B16-F10 melanoma, MC38 colorectal cancer, Hep1-6 hepatocellular carcinoma and chemically induced hepatocellular carcinoma model. METHODS: Various tumor models, including B16-F10, MC38 and Hep1-6 tumor hypodermic inoculation models, B16-F10 and Hep1-6 pulmonary metastasis models, Hep1-6 orthotopic implantation model, and chemically induced hepatocellular carcinoma model, were utilized to evaluate the anti-tumor function of PZH. Tumor growth was assessed by measuring tumor size and weight of solid tumors isolated from C57BL/6 mice. For cell proliferation and death of tumor cells in vitro, as well as T cell activation markers, cytokine production and immune checkpoints analysis, single-cell suspensions were prepared from mouse spleen, lymph nodes, and tumors after PZH treatment. RESULTS: PZH demonstrated significant therapeutic efficacy in inhibiting tumor growth (P<0.01). Treatment with PZH resulted in a reduction in tumor size in subcutaneous MC38 colon adenocarcinoma and B16-F10 melanoma models, and decreased pulmonary metastasis of B16-F10 melanoma and Hep1-6 hepatoma (P<0.01). However, in vitro experiments showed that PZH only had slight impact on the cell proliferation and survival of tumor cells (P>0.05). Nevertheless, PZH exhibited a remarkable ability to enhance T cell activation and the production of interferon gamma, tumor necrosis factor alpha, and interleukin 2 in CD4+ T cells in vitro (P<0.01 or P<0.05). Importantly, PZH substantially inhibited T cell exhaustion and boosted cytokine production by tumor-infiltrating CD8+ T cells (P<0.01 or P<0.05). CONCLUSION: This study has confirmed a novel immunomodulatory function of PZH in T cell-mediated anti-tumor immunity, indicating that PZH holds promise as a potential therapeutic agent for cancer treatment.

MHC class II regulation of CD8+ T cell tolerance and implications in autoimmunity and cancer immunotherapy.

Major histocompatibility complex (MHC) class II-reactive CD8+ T cells are found in humans and animals, but little is known about their identity, development, and function. In this study, we discover a group of CD8+ T cells reactive to both MHC class I and II molecules in MHC class II-deficient mice. We clone their T cell receptors (TCRs) and analyze their development and function. In wild-type animals, thymocytes bearing those TCRs are purged by negative selection. In the absence of MHC class II, they develop into mature CD8+ T cells. When encountering MHC class II in the periphery, they undergo robust activation and proliferation, attack self-tissues, and cause lethal autoimmune diseases. In adoptive T cell therapy, those CD8+ T cells are able to efficiently control MHC class II-expressing tumors. This study opens the door to investigation of dual-reactive CD8+ T cells, their development and selection in the thymus, and the perils and promises when their normal development and selection are compromised.

The transcription factor Zeb1 controls homeostasis and function of type 1 conventional dendritic cells.

Type 1 conventional dendritic cells (cDC1) are the most efficient cross-presenting cells that induce protective cytotoxic T cell response. However, the regulation of their homeostasis and function is incompletely understood. Here we observe a selective reduction of splenic cDC1 accompanied by excessive cell death in mice with Zeb1 deficiency in dendritic cells, rendering the mice more resistant to Listeria infection. Additionally, cDC1 from other sources of Zeb1-deficient mice display impaired cross-presentation of exogenous antigens, compromising antitumor CD8+ T cell responses. Mechanistically, Zeb1 represses the expression of microRNA-96/182 that target Cybb mRNA of NADPH oxidase Nox2, and consequently facilitates reactive-oxygen-species-dependent rupture of phagosomal membrane to allow antigen export to the cytosol. Cybb re-expression in Zeb1-deficient cDC1 fully restores the defective cross-presentation while microRNA-96/182 overexpression in Zeb1-sufficient cDC1 inhibits cross-presentation. Therefore, our results identify a Zeb1-microRNA-96/182-Cybb pathway that controls cross-presentation in cDC1 and uncover an essential role of Zeb1 in cDC1 homeostasis.

The miR-17∼92 miRNAs promote plasma cell differentiation by suppressing SOCS3-mediated NIK degradation.

The miR-17∼92 family microRNAs (miRNAs) play a key role in germinal center (GC) reaction through promoting T follicular helper (TFH) cell differentiation. It remains unclear whether they also have intrinsic functions in B cell differentiation and function. Here we show that mice with B cell-specific deletion of the miR-17∼92 family exhibit impaired GC reaction, plasma cell differentiation, and antibody production in response to protein antigen immunization and chronic viral infection. Employing CRISPR-mediated functional screening, we identify Socs3 as a key functional target of miR-17∼92 in regulating plasma cell differentiation. Mechanistically, SOCS3, whose expression is elevated in miR-17∼92 family-deficient B cells, interacts with NIK and promotes its ubiquitination and degradation, thereby impairing NF-κB signaling and plasma cell differentiation. This moderate increase in SOCS3 expression has little effect on IL-21-STAT3 signaling. Our study demonstrates differential sensitivity of two key signaling pathways to alterations in the protein level of an miRNA target gene.

Glycogen synthase kinase 3 controls T-cell exhaustion by regulating NFAT activation.

Cellular immunity mediated by CD8+ T cells plays an indispensable role in bacterial and viral clearance and cancers. However, persistent antigen stimulation of CD8+ T cells leads to an exhausted or dysfunctional cellular state characterized by the loss of effector function and high expression of inhibitory receptors during chronic viral infection and in tumors. Numerous studies have shown that glycogen synthase kinase 3 (GSK3) controls the function and development of immune cells, but whether GSK3 affects CD8+ T cells is not clearly elucidated. Here, we demonstrate that mice with deletion of Gsk3α and Gsk3ß in activated CD8+ T cells (DKO) exhibited decreased CTL differentiation and effector function during acute and chronic viral infection. In addition, DKO mice failed to control tumor growth due to the upregulated expression of inhibitory receptors and augmented T-cell exhaustion in tumor-infiltrating CD8+ T cells. Strikingly, anti-PD-1 immunotherapy substantially restored tumor rejection in DKO mice. Mechanistically, GSK3 regulates T-cell exhaustion by suppressing TCR-induced nuclear import of NFAT, thereby in turn dampening NFAT-mediated exhaustion-related gene expression, including TOX/TOX2 and PD-1. Thus, we uncovered the molecular mechanisms underlying GSK3 regulation of CTL differentiation and T-cell exhaustion in anti-tumor immune responses.

The glycolysis/HIF-1α axis defines the inflammatory role of IL-4-primed macrophages.

T helper type 2 (Th2) cytokine-activated M2 macrophages contribute to inflammation resolution and wound healing. This study shows that IL-4-primed macrophages exhibit a stronger response to lipopolysaccharide stimulation while maintaining M2 signature gene expression. Metabolic divergence between canonical M2 and non-canonical proinflammatory-prone M2 (M2INF) macrophages occurs after the IL-4Rα/Stat6 axis. Glycolysis supports Hif-1α stabilization and proinflammatory phenotype of M2INF macrophages. Inhibiting glycolysis blunts Hif-1α accumulation and M2INF phenotype. Wdr5-dependent H3K4me3 mediates the long-lasting effect of IL-4, with Wdr5 knockdown inhibiting M2INF macrophages. Our results also show that the induction of M2INF macrophages by IL-4 intraperitoneal injection and transferring of M2INF macrophages confer a survival advantage against bacterial infection in vivo. In conclusion, our findings highlight the previously neglected non-canonical role of M2INF macrophages and broaden our understanding of IL-4-mediated physiological changes. These results have immediate implications for how Th2-skewed infections could redirect disease progression in response to pathogen infection.

Dhx33 promotes B-cell growth and proliferation by controlling activation-induced rRNA upregulation.

Upon recognition of foreign antigens, naïve B cells undergo rapid activation, growth, and proliferation. How B-cell growth and proliferation are coupled with activation remains poorly understood. Combining CRISPR/Cas9-mediated functional analysis and mouse genetics approaches, we found that Dhx33, an activation-induced RNA helicase, plays a critical role in coupling B-cell activation with growth and proliferation. Mutant mice with B-cell-specific deletion of Dhx33 exhibited impaired B-cell development, germinal center reactions, plasma cell differentiation, and antibody production. Dhx33-deficient B cells appeared normal in the steady state and early stage of activation but were retarded in growth and proliferation. Mechanistically, Dhx33 played an indispensable role in activation-induced upregulation of ribosomal DNA (rDNA) transcription. In the absence of Dhx33, activated B cells were compromised in their ability to ramp up 47S ribosomal RNA (rRNA) production and ribosome biogenesis, resulting in nucleolar stress, p53 accumulation, and cellular death. Our findings demonstrate an essential role for Dhx33 in coupling B-cell activation with growth and proliferation and suggest that Dhx33 inhibition is a potential therapy for lymphoma and antibody-mediated autoimmune diseases.

A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation.

Plasma cells (PC) are antibody-secreting cells and terminal effectors in humoral responses. PCs differentiate directly from activated B cells in response to T cell-independent (TI) antigens or from germinal center B (GCB) cells in T cell-dependent (TD) antigen-induced humoral responses, both of which pathways are essentially regulated by the transcription factor BLIMP1. The p38 mitogen-activated protein kinase isoforms have already been implicated in B cell development, but the precise role of p38α in B cell differentiation is still largely unknown. Here we show that PC differentiation and antibody responses are severely impaired in mice with B cell-specific deletion of p38α, while B cell development and the GCB cell response are spared. By utilizing a Blimp1 reporter mouse model, we show that p38α-deficiency results in decreased BLIMP1 expression. p38α-driven BLIMP1 up-regulation is required for both TI and TD PCs differentiation. By combining CRISPR/Cas9 screening and other approaches, we identify TCF3, TCF4 and IRF4 as downstream effectors of p38α to control PC differentiation via Blimp1 transcription. This study thus identifies an important signalling pathway underpinning PC differentiation upstream of BLIMP1, and points to a highly specialized and non-redundant role for p38α among p38 isoforms.

Myc inhibition tips the immune balance to promote antitumor immunity.

Aberrant expression of Myc is one of the most common oncogenic events in human cancers. Scores of Myc inhibitors are currently under development for treating Myc-driven cancers. In addition to directly targeting tumor cells, Myc inhibition has been shown to modulate the tumor microenvironment to promote tumor regression. However, the effect of Myc inhibition on immune cells in the tumor microenvironment remains poorly understood. Here, we show that the adaptive immune system plays a vital role in the antitumor effect of pharmacologic inhibition of Myc. Combining genetic and pharmacologic approaches, we found that Myc inhibition enhanced CD8 T cell function by suppressing the homeostasis of regulatory T (Treg) cells and the differentiation of resting Treg (rTreg) cells to activated Treg (aTreg) cells in tumors. Importantly, we demonstrated that different Myc expression levels confer differential sensitivity of T cell subsets to pharmacologic inhibition of Myc. Although ablation of the Myc gene has been shown to suppress CD8 T cell function, Treg cells, which express much less Myc protein than CD8 T cells, are more sensitive to Myc inhibitors. The differential sensitivity of CD8 T and Treg cells to Myc inhibitors resulted in enhanced CD8 T cell function upon Myc inhibition. Our findings revealed that Myc inhibitors can induce an antitumor immune response during tumor progression.

Sulfate oligosaccharide of Gracilaria lemaneiformis modulates type 1 immunity by restraining T cell activation.

Seaweeds are important "blue food" and some seaweeds have been demonstrated that have the immunoregulation activities and benefit for human health. However, which immune cell subsets are targeted and how immune processes are regulated by those seaweeds remain poorly understood. Here we identified sulfated oligosaccharide of Gracilaria lemaneiformis (GLSO) inhibits IFNγ production by T cells in ovalbumin (OVA) immunized mice and in vitro activation system of OVA-specific CD4+ T cells. Furthermore, GLSO predominantly targets T cells but not dendritic cells, and regulates the late stage of expression of T cell activation and differentiation markers. Mechanistically, results of transcriptomic and proteome analysis indicate that GLSO inhibits mTOR activity, glycolysis, cell cycle and DNA replication. Thus, GLSO has an immunomodulatory function in Th1 immune responses by restraining T cell activation. Our study unravels the anti-inflammatory mechanism of GLSO and provides a new insight for industrial development of seaweeds as health food.

Mitochondrial C1qbp promotes differentiation of effector CD8+ T cells via metabolic-epigenetic reprogramming.

Early-activated CD8+ T cells increase both aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). However, whether and how the augmentation of OXPHOS regulates differentiation of effector CD8+ T cell remains unclear. Here, we found that C1qbp was intrinsically required for such differentiation in antiviral and antitumor immune responses. Activated C1qbp-deficient CD8+ T cells failed to increase mitochondrial respiratory capacities, resulting in diminished acetyl­coenzyme A as well as elevated fumarate and 2-hydroxyglutarate. Consequently, hypoacetylation of H3K27 and hypermethylation of H3K27 and CpG sites were associated with transcriptional down-regulation of effector signature genes. The effector differentiation of C1qbp-sufficient or C1qbp-deficient CD8+ T cells was reversed by fumarate or a combination of histone deacetylase inhibitor and acetate. Therefore, these findings identify C1qbp as a pivotal positive regulator in the differentiation of effector CD8+ T cells and highlight a metabolic-epigenetic axis in this process.

Glycogen synthase kinase 3 drives thymocyte egress by suppressing ß-catenin activation of Akt.

Molecular pathways controlling emigration of mature thymocytes from thymus to the periphery remain incompletely understood. Here, we show that T cell­specific ablation of glycogen synthase kinase 3 (GSK3) led to severely impaired thymic egress. In the absence of GSK3, ß-catenin accumulated in the cytoplasm, where it associated with and activated Akt, leading to phosphorylation and degradation of Foxo1 and downregulation of Klf2 and S1P1 expression, thereby preventing emigration of thymocytes. A cytoplasmic membrane-localized ß-catenin excluded from the nucleus promoted Akt activation, suggesting a new function of ß-catenin independent of its role as a transcriptional activator. Furthermore, genetic ablation of ß-catenin, retroviral expression of a dominant negative Akt mutant, and transgenic expression of a constitutively active Foxo1 restored emigration of GSK3-deficient thymocytes. Our findings establish an essential role for GSK3 in thymocyte egress and reveal a previously unidentified signaling function of ß-catenin in the cytoplasm.

Ddb1 Is Essential for the Expansion of CD4+ Helper T Cells by Regulating Cell Cycle Progression and Cell Death.

Follicular helper T (TFH) cells are specialized CD4+ helper T cells that provide help to B cells in humoral immunity. However, the molecular mechanism underlying generation of TFH cells is incompletely understood. Here, we reported that Damage-specific DNA binding protein 1 (Ddb1) was required for expansion of CD4+ helper T cells including TFH and Th1 cells, germinal center response, and antibody response to acute viral infection. Ddb1 deficiency in activated CD4+ T cells resulted in cell cycle arrest at G2-M phase and increased cell death, due to accumulation of DNA damage and hyperactivation of ATM/ATR-Chk1 signaling. Moreover, mice with deletion of both Cul4a and Cul4b in activated CD4+ T cells phenocopied Ddb1-deficient mice, suggesting that E3 ligase-dependent function of Ddb1 was crucial for genome maintenance and helper T-cell generation. Therefore, our results indicate that Ddb1 is an essential positive regulator in the expansion of CD4+ helper T cells.

Diverse immunoglobulin gene usage and convergent epitope targeting in neutralizing antibody responses to SARS-CoV-2.

It is unclear whether individuals with enormous diversity in B cell receptor repertoires are consistently able to mount effective antibody responses against SARS-CoV-2. We analyzed antibody responses in a cohort of 55 convalescent patients and isolated 54 potent neutralizing monoclonal antibodies (mAbs). While most of the mAbs target the angiotensin-converting enzyme 2 (ACE2) binding surface on the receptor binding domain (RBD) of SARS-CoV-2 spike protein, mAb 47D1 binds only to one side of the receptor binding surface on the RBD. Neutralization by 47D1 is achieved independent of interfering RBD-ACE2 binding. A crystal structure of the mAb-RBD complex shows that the IF motif at the tip of 47D1 CDR H2 interacts with a hydrophobic pocket in the RBD. Diverse immunoglobulin gene usage and convergent epitope targeting characterize neutralizing antibody responses to SARS-CoV-2, suggesting that vaccines that effectively present the receptor binding site on the RBD will likely elicit neutralizing antibody responses in a large fraction of the population.

Endothelial Scaffolding Protein ENH (Enigma Homolog Protein) Promotes PHLPP2 (Pleckstrin Homology Domain and Leucine-Rich Repeat Protein Phosphatase 2)-Mediated Dephosphorylation of AKT1 and eNOS (Endothelial NO Synthase) Promoting Vascular Remodeling.

OBJECTIVE: A decrease in nitric oxide, leading to vascular smooth muscle cell proliferation, is a common pathological feature of vascular proliferative diseases. Nitric oxide synthesis by eNOS (endothelial nitric oxide synthase) is precisely regulated by protein kinases including AKT1. ENH (enigma homolog protein) is a scaffolding protein for multiple protein kinases, but whether it regulates eNOS activation and vascular remodeling remains unknown. Approach and Results: ENH was upregulated in injured mouse arteries and human atherosclerotic plaques and was associated with coronary artery disease. Neointima formation in carotid arteries, induced by ligation or wire injury, was greatly decreased in endothelium-specific ENH-knockout mice. Vascular ligation reduced AKT and eNOS phosphorylation and nitric oxide production in the endothelium of control but not ENH-knockout mice. ENH was found to interact with AKT1 and its phosphatase PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2). AKT and eNOS activation were prolonged in VEGF (vascular endothelial growth factor)-induced ENH- or PHLPP2-deficient endothelial cells. Inhibitors of either AKT or eNOS effectively restored ligation-induced neointima formation in ENH-knockout mice. Moreover, endothelium-specific PHLPP2-knockout mice displayed reduced ligation-induced neointima formation. Finally, PHLPP2 was increased in the endothelia of human atherosclerotic plaques and blood cells from patients with coronary artery disease. CONCLUSIONS: ENH forms a complex with AKT1 and its phosphatase PHLPP2 to negatively regulate AKT1 activation in the artery endothelium. AKT1 deactivation, a decrease in nitric oxide generation, and subsequent neointima formation induced by vascular injury are mediated by ENH and PHLPP2. ENH and PHLPP2 are thus new proatherosclerotic factors that could be therapeutically targeted.

Noninvasive photoacoustic and fluorescent tracking of optical dye labeled T cellular activities of diseased sites at new depth.

The migration of immune cells is crucial to the immune response. Visualization of these processes has previously been limited because of the imaging depth. We developed a deep-penetrating, sensitive and high-resolution method to use fast photoacoustic tomography (PAT) to image the dynamic changes of T cells in lymph node and diseases at new depth (up to 9.5 mm). T cells labeled with NIR-797-isothiocyanate, an excellent near-infrared photoacoustic and fluorescent agent, were intravenously injected to the mice. We used fluorescence imaging to determine the location of T cells roughly and photoacoustic imaging is used to observe T-cell responses in diseased sites deeply and carefully. The dynamic changes of T cells in lymph node, acute disease (bacterial infection) and chronic disease (tumor) were observed noninvasively by photoacoustic and fluorescence imaging at different time points. T cells accumulated gradually and reached a maximum at 4 hours and declined afterwards in lymph node and bacterial infection site. At tumor model, T cells immigrated to the tumor with a maximum at 12 hours. Our study can not only provide a new observing method for immune activities tracking, but also enable continuous monitoring for therapeutic interventions.

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper cells.

MicroRNA (miRNA) deficiency impairs the generation of T follicular helper (Tfh) cells, but the contribution of individual miRNAs to this phenotype remains poorly understood. In this study, we performed deep sequencing analysis of miRNAs expressed in Tfh cells and identified a five-miRNA signature. Analyses of mutant mice deficient of these miRNAs revealed that miR-22 and miR-183/96/182 are dispensable, but miR-155 is essential for the generation and function of Tfh cells. miR-155 deficiency led to decreased proliferation specifically at the late stage of Tfh cell differentiation and reduced CD40 ligand (CD40L) expression on antigen-specific CD4(+) T cells. Mechanistically, miR-155 repressed the expression of Peli1, a ubiquitin ligase that promotes the degradation of the NF-κB family transcription factor c-Rel, which controls cellular proliferation and CD40L expression. Therefore, our study identifies a novel miR-155-Peli1-c-Rel pathway that specifically regulates Tfh cell generation and function.