Clonal Hematopoiesis in Clinical and Experimental Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Clonal hematopoiesis (CH), which results from an array of nonmalignant driver gene mutations, can lead to altered immune cell function and chronic disease, and has been associated with worse outcomes in patients with heart failure (HF) with reduced ejection fraction. However, the role of CH in the prognosis of HF with preserved ejection fraction (HFpEF) has been understudied. This study aimed to characterize CH in patients with HFpEF and elucidate its causal role in a murine model. METHODS: Using a panel of 20 candidate CH driver genes and a variant allele fraction cutoff of 0.5%, ultradeep error-corrected sequencing identified CH in a cohort of 81 patients with HFpEF (mean age, 71±6 years; ejection fraction, 63±5%) and 36 controls without a diagnosis of HFpEF (mean age, 74±7 years; ejection fraction, 61.5±8%). CH was also evaluated in a replication cohort of 59 individuals with HFpEF. RESULTS: Compared with controls, there was an enrichment of TET2-mediated CH in the HFpEF patient cohort (12% versus 0%, respectively; P=0.02). In the HFpEF cohort, patients with CH exhibited exacerbated diastolic dysfunction in terms of E/e' (14.9 versus 11.7, respectively; P=0.0096) and E/A (1.69 versus 0.89, respectively; P=0.0206) compared with those without CH. The association of CH with exacerbated diastolic dysfunction was corroborated in a validation cohort of individuals with HFpEF. In accordance, patients with HFpEF, an age ≥70 years, and CH exhibited worse prognosis in terms of 5-year cardiovascular-related hospitalization rate (hazard ratio, 5.06; P=0.042) compared with patients with HFpEF and an age ≥70 years without CH. To investigate the causal role of CH in HFpEF, nonconditioned mice underwent adoptive transfer with Tet2-wild-type or Tet2-deficient bone marrow and were subsequently subjected to a high-fat diet/L-NAME (Nω-nitro-l-arginine methyl ester) combination treatment to induce features of HFpEF. This model of Tet2-CH exacerbated cardiac hypertrophy by heart weight/tibia length and cardiomyocyte size, diastolic dysfunction by E/e' and left ventricular end-diastolic pressure, and cardiac fibrosis compared with the Tet2-wild-type condition. CONCLUSIONS: CH is associated with worse heart function and prognosis in patients with HFpEF, and a murine experimental model of Tet2-mediated CH displays greater features of HFpEF.

CRACD loss promotes small cell lung cancer tumorigenesis via EZH2-mediated immune evasion.

The mechanisms underlying immune evasion and immunotherapy resistance in small cell lung cancer (SCLC) remain unclear. Herein, we investigate the role of CRACD tumor suppressor in SCLC. We found that CRACD is frequently inactivated in SCLC, and Cracd knockout (KO) significantly accelerates SCLC development driven by loss of Rb1, Trp53, and Rbl2. Notably, the Cracd-deficient SCLC tumors display CD8+ T cell depletion and suppression of antigen presentation pathway. Mechanistically, CRACD loss silences the MHC-I pathway through EZH2. EZH2 blockade is sufficient to restore the MHC-I pathway and inhibit CRACD loss-associated SCLC tumorigenesis. Unsupervised single-cell transcriptomic analysis identifies SCLC patient tumors with concomitant inactivation of CRACD, impairment of tumor antigen presentation, and downregulation of EZH2 target genes. Our findings define CRACD loss as a new molecular signature associated with immune evasion of SCLC cells and proposed EZH2 blockade as a viable option for CRACD-negative SCLC treatment.

Monocyte-Derived Dendritic Cells Dictate the Memory Differentiation of CD8+ T Cells During Acute Infection.

Monocyte-derived dendritic cells (moDCs) have been shown to robustly expand during infection; however, their roles in anti-infectious immunity remain unclear. Here, we found that moDCs were dramatically increased in the secondary lymphoid organs during acute LCMV infection in an interferon-γ (IFN-γ)-dependent manner. We also found that priming by moDCs enhanced the differentiation of memory CD8+ T cells compared to differentiation primed by conventional dendritic cells (cDCs) through upregulation of Eomesodermin (Eomes) and T cell factor-1 (TCF-1) expression in CD8+ T cells. Consequently, impaired memory formation of CD8+ T cells in mice that had reduced numbers of moDCs led to defective clearance of pathogens upon rechallenge. Mechanistically, attenuated interleukin-2 (IL-2) signaling in CD8+ T cells primed by moDCs was responsible for the enhanced memory programming of CD8+ T cells. Therefore, our findings unveil a specialization of the antigen-presenting cell subsets in the fate determination of CD8+ T cells during infection and pave the way for the development of a novel therapeutic intervention on infection.

Sustained Type I Interferon Reinforces NK Cell-Mediated Cancer Immunosurveillance during Chronic Virus Infection.

The importance of natural killer (NK) cells in the early immune response to viral or bacterial infection is well known. However, the phenotype, function, and physiologic role of NK cells during the late stage of persistent viral infection have not been extensively studied. Here, we characterized NK cells in mice persistently infected with lymphocytic choriomeningitis virus clone 13 and showed that in contrast to NK cells from acutely infected or uninfected mice, NK cells from chronically infected mice expressed a terminally differentiated phenotype, stronger cytotoxicity, and reduced inhibitory receptor expression. In an in vivo tumor model, chronically infected mice exhibited significantly delayed tumor progression in an NK cell-dependent manner. NK cells from chronically infected mice also expressed high STAT1, and blocking the type I interferon (IFN) receptor revealed that type I IFN signaling directly regulated NK cell cytotoxicity. Our findings indicate that sustained type I IFN signaling during chronic viral infection potentiates the cytolytic function of NK cells and contributes to NK cell-dependent host immune surveillance.

Semi-Functional Quantitative Flow Cytometry Assay for Lymphocytic Choriomeningitis Virus Titration.

Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.

miR-150-Mediated Foxo1 Regulation Programs CD8+ T Cell Differentiation.

MicroRNA (miR)-150 is a developmental regulator of several immune-cell types, but its role in CD8+ T cells is largely unexplored. Here, we show that miR-150 regulates the generation of memory CD8+ T cells. After acute virus infection, miR-150 knockout (KO) mice exhibited an accelerated differentiation of CD8+ T cells into memory cells and improved production of effector cytokines. Additionally, miR-150 KO CD8+ T cells displayed an enhanced recall response and improved protection against infections with another virus and bacteria. We found that forkhead box O1 (Foxo1) and T cell-specific transcription factor 1 (TCF1) are upregulated during the early activation phase in miR-150 KO CD8+ T cells and that miR-150 directly targets and suppresses Foxo1. These results suggest that miR-150-mediated suppression of Foxo1 regulates the balance between effector and memory cell differentiation, which might aid in the development of improved vaccines and T cell therapeutics.

AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation.

The phosphoinositide 3-kinase-Akt signaling pathway is essential to many biological processes, including cell proliferation, survival, metabolism, and angiogenesis, under pathophysiological conditions. Although 3-phosphoinositide-dependent kinase 1 (PDK1) is a primary activator of Akt at the plasma membrane, the optimal activation mechanism remains unclear. We report that adhesion molecule with IgG-like domain 2 (AMIGO2) is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation. Loss of AMIGO2 in endothelial cells (ECs) led to apoptosis and inhibition of angiogenesis with Akt inactivation. Amino acid residues 465-474 in AMIGO2 directly bind to the PDK1 pleckstrin homology domain. A synthetic peptide containing the AMIGO2 465-474 residues abrogated the AMIGO2-PDK1 interaction and Akt activation. Moreover, it effectively suppressed pathological angiogenesis in murine tumor and oxygen-induced retinopathy models. These results demonstrate that AMIGO2 is an important regulator of the PDK1-Akt pathway in ECs and suggest that interference of the PDK1-AMIGO2 interaction might be a novel pharmaceutical target for designing an Akt pathway inhibitor.

PD-1 upregulated on regulatory T cells during chronic virus infection enhances the suppression of CD8+ T cell immune response via the interaction with PD-L1 expressed on CD8+ T cells.

Regulatory T (Treg) cells act as terminators of T cell immuniy during acute phase of viral infection; however, their role and suppressive mechanism in chronic viral infection are not completely understood. In this study, we compared the phenotype and function of Treg cells during acute or chronic infection with lymphocytic choriomeningitis virus. Chronic infection, unlike acute infection, led to a large expansion of Treg cells and their upregulation of programmed death-1 (PD-1). Treg cells from chronically infected mice (chronic Treg cells) displayed greater suppressive capacity for inhibiting both CD8(+) and CD4(+) T cell proliferation and subsequent cytokine production than those from naive or acutely infected mice. A contact between Treg and CD8(+) T cells was necessary for the potent suppression of CD8(+) T cell immune response. More importantly, the suppression required cell-specific expression and interaction of PD-1 on chronic Treg cells and PD-1 ligand on CD8(+) T cells. Our study defines PD-1 upregulated on Treg cells and its interaction with PD-1 ligand on effector T cells as one cause for the potent T cell suppression and proposes the role of PD-1 on Treg cells, in addition to that on exhausted T cells, during chronic viral infection.

Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases.

RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the mouse Prkdc gene that encodes an enzyme critical for DNA double-strand break repair resulted in immunodeficiency both in F0 and F1 mice. We propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically engineered model organisms, accelerating functional genomic research.