LAG-3 sustains TOX expression and regulates the CD94/NKG2-Qa-1b axis to govern exhausted CD8 T cell NK receptor expression and cytotoxicity.

Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.

Multi-omics analysis reveals a feedback loop amplifying immune responses in acute graft-versus-host disease due to imbalanced gut microbiota and bile acid metabolism.

Acute graft-versus-host disease (aGVHD) is primarily driven by allogeneic donor T cells associated with an altered composition of the host gut microbiome and its metabolites. The severity of aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not solely determined by the host and donor characteristics; however, the underlying mechanisms remain unclear. Using single-cell RNA sequencing, we decoded the immune cell atlas of 12 patients who underwent allo-HSCT: six with aGVHD and six with non-aGVHD. We performed a fecal microbiota (16SrRNA sequencing) analysis to investigate the fecal bacterial composition of 82 patients: 30 with aGVHD and 52 with non-aGVHD. Fecal samples from these patients were analyzed for bile acid metabolism. Through multi-omic analysis, we identified a feedback loop involving "immune cell-gut microbes-bile acid metabolites" contributing to heightened immune responses in patients with aGVHD. The dysbiosis of the gut microbiota and disruption of bile acid metabolism contributed to an exaggerated interleukin-1 mediated immune response. Our findings suggest that resistin and defensins are crucial in mitigating against aGVHD. Therefore, a comprehensive multi-omic atlas incorporating immune cells, gut microbes, and bile acid metabolites was developed in this study and used to propose novel, non-immunosuppressive approaches to prevent aGVHD.

Leukemia inhibitory factor is a therapeutic target for renal interstitial fibrosis.

BACKGROUND: The role of the IL6 family members in organ fibrosis, including renal interstitial fibrosis (TIF), has been widely explored. However, few studies have ever simultaneously examined them in the same cohort of patients. Besides, the role of leukemia inhibitory factor (LIF) in TIF remains unclear. METHODS: RNA-seq data of kidney biopsies from chronic kidney disease (CKD) patients, in both public databases and our assays, were used to analyze transcript levels of IL6 family members. Two TIF mouse models, the unilateral ureteral obstruction (UUO) and the ischemia reperfusion injury (IRI), were employed to validate the finding. To assess the role of LIF in vivo, short hairpin RNA, lenti-GFP-LIF was used to knockdown LIF receptor (LIFR), overexpress LIF, respectively. LIF-neutralizing antibody was used in therapeutic studies. Whether urinary LIF could be used as a promising predictor for CKD progression was investigated in a prospective observation patient cohort. FINDINGS: Among IL6 family members, LIF is the most upregulated one in both human and mouse renal fibrotic lesions. The mRNA level of LIF negatively correlated with eGFR with the strongest correlation and the smallest P value. Baseline urinary concentrations of LIF in CKD patients predict the risk of CKD progression to end-stage kidney disease by Kaplan-Meier analysis. In mouse TIF models, knockdown of LIFR alleviated TIF; conversely, overexpressing LIF exacerbated TIF. Most encouragingly, visible efficacy against TIF was observed by administering LIF-neutralizing antibodies to mice. Mechanistically, LIF-LIFR-EGR1 axis and Sonic Hedgehog signaling formed a vicious cycle between fibroblasts and proximal tubular cells to augment LIF expression and promote the pro-fibrotic response via ERK and STAT3 activation. INTERPRETATION: This study discovered that LIF is a noninvasive biomarker for the progression of CKD and a potential therapeutic target of TIF. FUNDINGS: Stated in the Acknowledgements section of the manuscript.

Dissection of the MEF2D-IRF8 transcriptional circuit dependency in acute myeloid leukemia.

Transcriptional dysregulation is a prominent feature in leukemia. Here, we systematically surveyed transcription factor (TF) vulnerabilities in leukemia and uncovered TF clusters that exhibit context-specific vulnerabilities within and between different subtypes of leukemia. Among these TF clusters, we demonstrated that acute myeloid leukemia (AML) with high IRF8 expression was addicted to MEF2D. MEF2D and IRF8 form an autoregulatory loop via direct binding to mutual enhancer elements. One important function of this circuit in AML is to sustain PU.1/MEIS1 co-regulated transcriptional outputs via stabilizing PU.1's chromatin occupancy. We illustrated that AML could acquire dependency on this circuit through various oncogenic mechanisms that results in the activation of their enhancers. In addition to forming a circuit, MEF2D and IRF8 can also separately regulate gene expression, and dual perturbation of these two TFs leads to a more robust inhibition of AML proliferation. Collectively, our results revealed a TF circuit essential for AML survival.

The interferon-stimulated gene RIPK1 regulates cancer cell intrinsic and extrinsic resistance to immune checkpoint blockade.

Interferon-gamma (IFN-γ) has pleiotropic effects on cancer immune checkpoint blockade (ICB), including roles in ICB resistance. We analyzed gene expression in ICB-sensitive versus ICB-resistant tumor cells and identified a strong association between interferon-mediated resistance and expression of Ripk1, a regulator of tumor necrosis factor (TNF) superfamily receptors. Genetic interaction screening revealed that in cancer cells, RIPK1 diverted TNF signaling through NF-κB and away from its role in cell death. This promoted an immunosuppressive chemokine program by cancer cells, enhanced cancer cell survival, and decreased infiltration of T and NK cells expressing TNF superfamily ligands. Deletion of RIPK1 in cancer cells compromised chemokine secretion, decreased ARG1+ suppressive myeloid cells linked to ICB failure in mice and humans, and improved ICB response driven by CASP8-killing and dependent on T and NK cells. RIPK1-mediated resistance required its ubiquitin scaffolding but not kinase function. Thus, cancer cells co-opt RIPK1 to promote cell-intrinsic and cell-extrinsic resistance to immunotherapy.

ZMYND8-regulated IRF8 transcription axis is an acute myeloid leukemia dependency.

The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML.

In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer.

Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8+ T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8+ T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.

HTNV Sensitizes Host Toward TRAIL-Mediated Apoptosis-A Pivotal Anti-hantaviral Role of TRAIL.

Hantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and have led to public health threat in China. The pathogenesis of HFRS is complex and involves capillary leakage due to the infection of vascular endothelial cells. Accumulating evidence has demonstrated that hantavirus can induce apoptosis in many cells, but the mechanism remains unclear. Our studies showed that Hantaan virus (HTNV) infection could induce TNF-related apoptosis-inducing ligand (TRAIL) expression in primary human umbilical vein endothelial cells (HUVECs) and sensitize host cells toward TRAIL-mediated apoptosis. Furthermore, TRAIL interference could inhibit apoptosis and enhance the production of HTNV as well as reduce IFN-ß production, while exogenous TRAIL treatment showed reverse outcome: enhanced apoptosis and IFN-ß production as well as a lower level of viral replication. We also observed that nucleocapsid protein (NP) and glycoprotein (GP) of HTNV could promote the transcriptions of TRAIL and its receptors. Thus, TRAIL was upregulated by HTNV infection and then exhibited significant antiviral activities in vitro, and it was further confirmed in the HTNV-infected suckling mice model that TRAIL treatment significantly reduced viral load, alleviated virus-induced tissue lesions, increased apoptotic cells, and decreased the mortality. In conclusion, these results demonstrate that TRAIL-dependent apoptosis and IFN-ß production could suppress HTNV replication and TRAIL treatment might be a novel therapeutic target for HTNV infection.

High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening.

CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.

lncRNA PCAT18 inhibits proliferation, migration and invasion of gastric cancer cells through miR-135b suppression to promote CLDN11 expression.

BACKGROUND: Gastric cancer is a severe disease with a high occurrence rate worldwide. And lncRNAs are demonstrated to be responsible for cancer growth and metastasis. So, it is of great importance to explore the lncRNAs involved mechanism of gastric cancer occurrence and development deeply. METHODS: Transfection was conducted to build over-expression and down-expression models. Moreover, RT-qPCR and western blot were used to detect the transcriptional and translational levels. The biological functions such as proliferation, migration and invasion of AGS cells were evaluated by MTT analysis, colony formation assay, scarification detection and transwell assay, respectively. The potential binding of miR-135b and its downstream and upstream molecules was validated by dual luciferase reporter gene assay or RIP. Also, the in-vivo mice model was further used to demonstrate the role of lncRNA PCAT18 in gastric cancer. RESULTS: PCAT18 down-expression promoted proliferation, migration and invasion of gastric cancer cells. Furtherly, over-expression of miR-135b also promoted these biological characteristics of AGS cells. Importantly, we found that PCAT18 could bind miR-135b which also was bound with CLDN11. We found that miR-135b is negatively correlated with CLDN11; PCAT18 and CLDN11 are positively correlated. Moreover, miR-135b mimics could down-regulate protein level of CLDN11, whereas CLDN11 could reverse this effect. In in-vivo experiment, PCAT18 over-expression restrained tumor growth and metastasis. CONCLUSIONS: Over-expressed lncRNA PCAT18 inhibits proliferation, migration and invasion of gastric cancer cells through regulation of miR-135b/CLDN11.

IFN-λs inhibit Hantaan virus infection through the JAK-STAT pathway and expression of Mx2 protein.

Hantaan virus (HTNV), member of the newly defined Hantaviridae family, within the order Bunyavirales, can cause a hemorrhagic fever with renal syndrome with high fatality rates in humans. However, no specific antiviral agents are currently available for HTNV infection approved by the US Food and Drug Administration. Although interferon lambdas (IFN-λs) have been shown to induce an antiviral state against HTNV, the molecular mechanisms remain to be determined. In this study, we found that IFN-λs exerted its anti-HTNV effect by activating Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway-mediated antiviral immunity in A549 cells. Simultaneously, IFN-λs downregulated suppressor of cytokine signaling proteins, which are the known negative feedback regulators of the JAK-STAT signaling pathway. Additionally, we demonstrated the role of IFN-λs-induced myxovirus resistance 2 (Mx2, also known as MxB) protein as a potential inhibitor for HTNV infection. These findings indicate that IFN-λs play an important role in cellular defenses against HTNV infection at an early stage and that human Mx2 may represent a potential therapeutic target for HTNV infection.

DNA damage triggers tubular endoplasmic reticulum extension to promote apoptosis by facilitating ER-mitochondria signaling.

The endoplasmic reticulum (ER) is composed of the nuclear envelope, perinuclear sheets and a peripheral tubular network. The peripheral ER and mitochondria form tight contacts at specific subdomains, which coordinate the functions of the two organelles and are required for multiple cellular processes such as Ca2+ transfer and apoptosis. However, it is largely unknown how ER morphology and ER-mitochondria signaling are dynamically regulated under different physiological or pathological conditions such as DNA damage. Here we show that the peripheral, tubular ER undergoes significant extension in response to DNA damage, and that this process is dependent on p53-mediated transcriptional activation of the ER-shaping proteins REEP1, REEP2 and EI24 (alias PIG8). This promotes the formation of ER-mitochondria contacts through EI24 and the mitochondrial outer membrane protein VDAC2, facilitates Ca2+ transfer from ER to mitochondria and promotes DNA damage-induced apoptosis. Thus, we identify a unique DNA damage response pathway involving alterations in ER morphology, ER-mitochondria signaling, and apoptosis.

The dynamic changes of CD3e-CD11c+ dendritic cells in spleens and bone marrow of mice infected with Schistosoma japonicum.

Schistosoma japonicum as a pathogeny requires dendritic cells to activate immune response. So, the research is to study the dynamic changes of CD3e-CD11c+ dendritic cells in mice infected with S. japonicum. Zero, 7, 28, 35, and 63 days were selected to study the variation of dendritic cells, and the proportions of CD3e-CD11c+ dendritic cells and CD86+ mature dendritic cells in spleens and bone marrow were tested by flow cytometry. As a result, the variation trends of dendritic cells in spleen and bone marrow are similar as follows: the proportions of CD3e-CD11c+ dendritic cells increased first and then decreased from day 35, but the percentages of CD86+ mature dendritic cells decreased from day 28 and increased in day 63. In vitro, cultured dendritic cells treated with SEA and SAWA were tested by flow cytometry, the variation trends of CD86 on dendritic cells are consistent with the results in days 28 and 63. Besides CD86, the expression of MHC-II also hints immune regulation. In conclusion, it is speculated that dendritic cells play a role of immune regulation through MHC-II and CD86 in S. japonicum infection. Immune regulation of dendritic cells is not only in favor of the survival of host and parasite but also can be used in the therapy for immune diseases.