Sang Jun Ha

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.

Fibroblast Growth Factor Receptor 1 Gene Amplification Is Associated With Poor Survival and Cigarette Smoking Dosage in Patients With Resected Squamous Cell Lung Cancer

PURPOSE To investigate the frequency and the prognostic role of fibroblast growth factor receptor 1 (FGFR1) amplification in patients with surgically resected squamous cell carcinoma of the lung (SCCL) and the association between smoking and FGFR1 amplification. PATIENTS AND METHODS Gene copy number of FGFR1 was investigated in microarrayed tumors from 262 patients with SCCL who had tumor tissue as well as smoking and survival data available. Gene copy number was evaluated by fluorescent in situ hybridization, and an FGFR1-amplified tumor (FGFR1 amp(+)) was prespecified as a tumor with nine or more copies of FGFR1. RESULTS Among 262 patients, the frequency of FGFR1 amp(+) was 13.0%. Patients with FGFR1 amp(+) had significantly shorter disease-free survival (DFS; 26.9 v 94.6 months; P < .001) as well as shorter overall survival (OS; 51.2 v 115.0 months; P = .002) than those without FGFR1 amp(+). Multivariate modeling confirmed that patients with FGFR1 amp(+) had a significantly greater risk of recurrence and death than those without FGFR1 amp(+) after adjusting for sex, smoking status, pathologic stage, and adjuvant chemotherapy (DFS: adjusted hazard ratio [AHR], 2.24; 95% CI, 1.45 to 3.45; P < .001; OS: AHR, 1.83; 95% CI, 1.15 to 2.89; P = .01). The frequency of FGFR1 amp(+) was significantly higher in current smokers than in former smokers and never-smokers (28.9% v 2.5% v 0%; P(trend) < .001). As the smoking dosage increased, so did the incidence of FGFR1 amp(+) (P(trend) = .002). CONCLUSION FGFR1 amplification is an independent negative prognostic factor in surgically resected SCCL and is associated with cigarette smoking in a dose-dependent manner. FGFR1 amplification is a relevant therapeutic target in Asian patients with SCCL.

Therapeutic effect of DNA vaccines combined with chemotherapy in a latent infection model after aerosol infection of mice with Mycobacterium tuberculosis

The prevention of Mycobacterium tuberculosis (M. tuberculosis) reactivation would greatly reduce the incidence of the disease, particularly among the elderly. Here, we evaluated the efficacy of DNA vaccine in combination with a conventional TB chemotherapy on the prevention of M. tuberculosis reactivation. Mice were treated with isoniazid and pyrazinamide for 3 months from 4 weeks after aerosol infection with M. tuberculosis H37Rv. During this period of chemotherapy, DNA immunization was performed three times monthly with an antigen 85A (Ag85A) DNA or an IL-12 mutant (IL-12N220L) DNA, which is known to lead to a reduction in the secretion of the p40 subunit, but not of a bioactive IL-12p70. The reactivation of M. tuberculosis was dramatically reduced in mice treated with either Ag85A DNA (P<0.01) or IL-12N220L DNA (P<0.05) in combination with chemotherapy, compared with control mice receiving only chemotherapy. Ag85A DNA vaccine showed higher IFN-γ responses to Ag85A protein, but a lower response to culture filtrate than IL-12N220L DNA vaccine. In addition, Ag85A DNA vaccine prevented the reactivation of M. tuberculosis more efficiently than IL-12N220L DNA vaccine, indicating that Ag85A-specific IFN-γ response might correlate with M. tuberculosis control. This study suggests that immunotherapy using Ag85A or IL-12N220L DNA vaccine combined with conventional chemotherapy might be effective clinically for the prevention of tuberculosis reactivation and may offer a more effective cure for humans than chemotherapy alone.

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.

PD-L1 expression on immune cells, but not on tumor cells, is a favorable prognostic factor for head and neck cancer patients

To investigate the expression of programmed death-ligand 1 (PD-L1) and immune checkpoints and their prognostic value for resected head and neck squamous cell cancer (HNSCC). PD-L1 expression on tumor cells (TC) and tumor-infiltrating immune cells (IC), abundance of tumor-infiltrating lymphocytes (TILs), and expression of the immune checkpoints were investigated in 402 HNSCC patients. PD-L1 expression on TC and IC was categorized into four groups according to the percentage of PD-L1-positive cells. PD-L1 positivity was defined as ≥5% of cells based on immunohistochemistry. High PD-L1 expression on IC, but not TC, was an independent favorable prognostic factor for RFS and OS adjusted for age, gender, smoking, stage, and HPV. High frequencies of CD3+ or CD8+ TILs, Foxp3+ Tregs, and PD-1+ TILs were strongly associated with favorable prognosis. PD-L1 was exclusively expressed on either TC or IC. Transcriptome analysis demonstrated that IC3 expressed higher levels of the effector T cell markers than TC3, suggesting that PD-L1 expression is regulated via an adaptive IFNγ-mediated mechanism. High PD-L1 expression on IC, but not TC, and high abundance of PD-1+ T cells and Foxp3+ Tregs are favorable prognostic factors for resected HNSCC. This study highlights the importance of comprehensive assessment of both TC and IC.

Enhanced immunogenicity and protective efficacy with the use of interleukin-12-encapsulated microspheres plus AS01B in tuberculosis subunit vaccination.

ABSTRACT Tuberculosis subunit vaccines codelivered with interleukin-12 (IL-12)-encapsulated microspheres (IL-12EM) are designed for a sustained release of IL-12 and could induce strong Th1 immune responses specific to Ag85A and ESAT-6. The adjuvant combination of IL-12EM plus AS01B was a more efficient way to induce a sustained Th1 immunity and protection against Mycobacterium tuberculosis.

Discrimination between Active and Latent Tuberculosis Based on Ratio of Antigen-Specific to Mitogen-Induced IP-10 Production

ABSTRACT Mycobacterium tuberculosis is the major causative agent of tuberculosis (TB). The gamma interferon (IFN-γ) release assay (IGRA) has been widely used to diagnose TB by testing cell-mediated immune responses but has no capacity for distinguishing between active TB and latent TB infection (LTBI). This study aims to identify a parameter that will help to discriminate active TB and LTBI. Whole-blood samples from 33 active TB patients, 20 individuals with LTBI, and 26 non-TB controls were applied to the commercial IFN-γ release assay, QuantiFERON-TB Gold In-Tube, and plasma samples were analyzed for interleukin-2 (IL-2), IL-6, IL-8, IL-10, IL-13, tumor necrosis factor-alpha (TNF-α), IFN-γ, monokine induced by IFN-γ (MIG), interferon gamma inducible protein 10 (IP-10), interferon-inducible T cell alpha chemoattractant (I-TAC), and monocyte chemoattractant protein 1 (MCP-1) by using a commercial cytometric bead array. The Mycobacterium tuberculosis antigen-specific production of most of the assayed cytokines and chemokines was higher in the active TB than in the LTBI group. The mitogen-induced responses were lower in the active TB than in the LTBI group. When the ratio of TB-specific to mitogen-induced responses was calculated, IL-2, IL-6, IL-10, IL-13, TNF-α, IFN-γ, MIG, and IP-10 were more useful in discriminating active TB from LTBI. In particular, most patients showed higher IP-10 production to Mycobacterium tuberculosis antigens than to mitogen at the individual level, and the ratio for IP-10 was the strongest indicator of active infection versus LTBI with 93.9% sensitivity and 90% specificity. In conclusion, the ratio of the TB-specific to the mitogen-induced IP-10 responses showed the most promising accuracy for discriminating active TB versus LTBI and should be further studied to determine whether it can serve as a biomarker that might help clinicians administer appropriate treatments.

Peptidylarginine deiminase inhibition impairs Toll-like receptor agonist-induced functional maturation of dendritic cells, resulting in the loss of T cell–proliferative capacity: a partial mechanism with therapeutic potential in inflammatory settings

Cl‐amidine, which is a small‐molecule inhibitor of PAD, has therapeutic potential for inflammation‐mediated diseases. However, little is known regarding the manner by which PAD inhibition by Cl‐amidine regulates inflammatory conditions. Here, we investigated the effects of PAD inhibition by Cl‐amidine on the functioning of DCs, which are pivotal immune cells that mediate inflammatory diseases. When DC maturation was induced by TLR agonists, reduced cytokine levels (IL‐6, IL‐1β, and IL‐12p70) were observed in Cl‐amidine‐treated DCs. Cl‐amidine‐treated, LPS‐activated DCs exhibited alterations in their mature and functional statuses with up‐regulated antigen uptake, down‐regulated CD80, and MHC molecules. In addition, Cl‐amidine‐treated DCs dysregulated peptide‐MHC class formations. Interestingly, the decreased cytokines were independent of MAPK/NF‐κB signaling pathways and transcription levels, indicating that PAD inhibition by Cl‐amidine may be involved in post‐transcriptional steps of cytokine production. Transmission electron microscopy revealed morphotypical changes with reduced dendrites in the Cl‐amidine‐treated DCs, along with altered cellular compartments, including fragmented ERs and the formation of foamy vesicles. Furthermore, in vitro and in vivo Cl‐amidine treatments impaired the proliferation of nai¨ve CD4+ and CD8+ T cells. Overall, our findings suggest that Cl‐amidine has therapeutic potential for treating inflammation‐mediated diseases.

Generation of Tolerogenic Dendritic Cells and Their Therapeutic Applications.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that bridge innate and adaptive immune responses, thereby leading to immune activation. DCs have been known to recognize pathogen-associated molecular patterns such as lipopolysaccharides (LPS) and nucleic acids via their pattern recognition receptors, which trigger signaling of their maturation and effector functions. Furthermore, DCs take up and process antigens as a form of peptide loaded on the major histocompatibility complex (MHC) and present them to T cells, which are responsible for the adaptive immune response. Conversely, DCs can also play a role in inducing immune suppression under specific circumstances. From this perspective, the role of DCs is related to tolerance rather than immunity. Immunologists refer to these special DCs as tolerogenic DCs (tolDCs). However, the definition of tolDCs is controversial, and there is limited information on their development and characteristics. In this review, we discuss the current concept of tolDCs, cutting-edge methods for generating tolDCs in vitro, and future applications of tolDCs, including clinical use.

Differentiation of Antigen-Specific T Cells with Limited Functional Capacity during Mycobacterium tuberculosis Infection

ABSTRACT Despite the generation of Mycobacterium tuberculosis-specific T cell immune responses during the course of infection, only 5 to 10% of exposed individuals develop active disease, while others develop a latent infection. This phenomenon suggests defective M. tuberculosis-specific immunity, which necessitates more careful characterization of M. tuberculosis-specific T cell responses. Here, we longitudinally analyzed the phenotypes and functions of M. tuberculosis-specific T cells. In contrast to the functional exhaustion of T cells observed after chronic infection, M. tuberculosis-specific CD8+ T cells differentiated into either effector (CD127lo CD62Llo) or effector memory (CD127hi CD62Llo) cells, but not central memory cells (CD127hi CD62Lhi), with low programmed death 1 (PD-1) expression, even in the presence of high levels of bacteria. Additionally, M. tuberculosis-specific CD8+ and CD4+ T cells produced substantial levels of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), but not interleukin 2 (IL-2), upon in vitro restimulation. Among M. tuberculosis-specific CD8+ T cells, CD127hi effector memory cells displayed slower ongoing turnover but greater survival potential. In addition, these cells produced more IFN-γ and TNF-α and displayed lytic activity upon antigen stimulation. However, the effector function of M. tuberculosis-specific CD8+ CD127hi effector memory T cells was inferior to that of canonical CD8+ CD127hi memory T cells generated after acute lymphocytic choriomeningitis virus infection. Collectively, our data demonstrate that M. tuberculosis-specific T cells can differentiate into memory T cells during the course of M. tuberculosis infection independent of the bacterial burden but with limited functionality. These results provide a framework for further understanding the mechanisms of M. tuberculosis infection that can be used to develop more effective vaccines.