The master antioxidant defense is activated during EBV latent infection.

IMPORTANCE: To our knowledge, this is the first report delineating the activation of the master antioxidant defense during EBV latency. We show that EBV-triggered reactive oxygen species production activates the Keap1-NRF2 pathway in EBV-transformed cells, and LMP1 plays a major role in this event, and the stress-related kinase TBK1 is required for NRF2 activation. Moreover, we show that the Keap1-NRF2 pathway is important for cell proliferation and EBV latency maintenance. Our findings disclose how EBV controls the balance between oxidative stress and antioxidant defense, which greatly improve our understanding of EBV latency and pathogenesis and may be leveraged to opportunities toward the improvement of therapeutic outcomes in EBV-associated diseases.

Tyrosine kinase signaling-independent MET-targeting with CAR-T cells.

BACKGROUND: Recent progress in cancer immunotherapy encourages the expansion of chimeric antigen receptor (CAR) T cell therapy in solid tumors including hepatocellular carcinoma (HCC). Overexpression of MET receptor tyrosine kinase is common in HCC; however, MET inhibitors are effective only when MET is in an active form, making patient stratification difficult. Specific MET-targeting CAR-T cells hold the promise of targeting HCC with MET overexpression regardless of signaling pathway activity. METHODS: MET-specific CARs with CD28ζ or 4-1BBζ as co-stimulation domains were constructed. MET-CAR-T cells derived from healthy subjects (HS) and HCC patients were evaluated for their killing activity and cytokine release against HCC cells with various MET activations in vitro, and for their tumor growth inhibition in orthotopic xenograft models in vivo. RESULTS: MET-CAR.CD28ζ and MET-CAR.4-1BBζ T cells derived from both HS and HCC patients specifically killed MET-positive HCC cells. When stimulated with MET-positive HCC cells in vitro, MET-CAR.CD28ζ T cells demonstrated a higher level of cytokine release and expression of programmed cell death protein 1 (PD-1) than MET-CAR.4-1BBζ T cells. When analyzed in vivo, MET-CAR.CD28ζ T cells more effectively inhibited HCC orthotopic tumor growth in mice when compared to MET-CAR.4-1BBζ T cells. CONCLUSION: We generated and characterized MET-specific CAR-T cells for targeting HCC with MET overexpression regardless of MET activation. Compared with MET-CAR.4-1BBζ, MET-CAR.CD28ζ T cells showed a higher anti-HCC potency but also a higher level of T cell exhaustion. While MET-CAR.CD28ζ is preferred for further development, overcoming the exhaustion of MET-CAR-T cells is necessary to improve their therapeutic efficacy in vivo.

Synthetic gRNA/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication.

The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9-mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV-infected human hepatoma cell line HepG2.2.15 with HBV-specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene-editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off-target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA/Cas9 RNP-based gene-editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.

ROS-Induced Mitochondrial Dysfunction in CD4 T Cells from ART-Controlled People Living with HIV.

We have previously demonstrated mitochondrial dysfunction in aging CD4 T cells from antiretroviral therapy (ART)-controlled people living with HIV (PLWH). However, the underlying mechanisms by which CD4 T cells develop mitochondrial dysfunction in PLWH remain unclear. In this study, we sought to elucidate the mechanism(s) of CD4 T cell mitochondrial compromise in ART-controlled PLWH. We first assessed the levels of reactive oxygen species (ROS), and we observed significantly increased cellular and mitochondrial ROS levels in CD4 T cells from PLWH compared to healthy subjects (HS). Furthermore, we observed a significant reduction in the levels of proteins responsible for antioxidant defense (superoxide dismutase 1, SOD1) and ROS-mediated DNA damage repair (apurinic/apyrimidinic endonuclease 1, APE1) in CD4 T cells from PLWH. Importantly, CRISPR/Cas9-mediated knockdown of SOD1 or APE1 in CD4 T cells from HS confirmed their roles in maintaining normal mitochondrial respiration via a p53-mediated pathway. Reconstitution of SOD1 or APE1 in CD4 T cells from PLWH successfully rescued mitochondrial function as evidenced by Seahorse analysis. These results indicate that ROS induces mitochondrial dysfunction, leading to premature T cell aging via dysregulation of SOD1 and APE1 during latent HIV infection.

Identification of virus-specific B-cell epitopes by convalescent plasma from COVID-19 patients.

Identification of immunologic epitopes against SARS-CoV-2 is crucial for the discovery of diagnostic, therapeutic, and preventive targets. In this study, we used a pan-coronavirus peptide microarray to screen for potential B-cell epitopes and validated the results with peptide-based ELISA. Specifically, we identified three linear B-cell epitopes on the SARS-CoV-2 proteome, which were recognized by convalescent plasma from COVID-19 patients. Interestingly, two epitopes (S 809-823 and R1ab 909-923) strongly reacted to convalescent plasma collected at the early phase (< 90 days) of COVID-19 symptom onset, whereas one epitope (M 5-19) reacted to convalescent plasma collected > 90 days after COVID-19 symptom onset. Neutralization assays using antibody depletion with the identified spike (S) peptides revealed that three S epitopes (S 557-571, S 789-803, and S 809-823) elicited neutralizing antibodies in COVID-19 patients. However, the levels of virus-specific antibody targeting S 789-803 only positively correlated with the neutralizing rates at the early phase (<60 days) after disease onset, and the antibody titers diminished quickly with no correlation to the neutralizing activity beyond two months after recovery from COVID-19. Importantly, stimulation of peripheral blood mononuclear cells from COVID-19-recovered patients with these SARS-CoV-2 S peptides resulted in poor virus-specific B cell activation, proliferation, differentiation into memory B cells, and production of immunoglobulin G (IgG) antibodies, despite the B-cells being functionally competent as demonstrated by their response to non-specific stimulation. Taken together, these findings indicate that these newly identified SARS-CoV-2-specific B-cell epitopes can elicit neutralizing antibodies, with titers and/or neutralizing activities declining significantly within 2-3 months in the convalescent plasma of COVID-19 patients.

Mitochondrial topoisomerase 1 inhibition induces topological DNA damage and T cell dysfunction in patients with chronic viral infection.

T cells are crucial for controlling viral infections; however, the mechanisms that dampen their responses during viral infections remain incompletely understood. Here, we studied the role and mechanisms of mitochondrial topoisomerase 1 (Top1mt) inhibition in mitochondrial dysfunction and T cell dysregulation using CD4 T cells from patients infected with HCV or HIV and compared it with CD4 T cells from healthy individuals following treatment with Top1 inhibitor - camptothecin (CPT). We found that Top1mt protein levels and enzymatic activity are significantly decreased, along with Top1 cleavage complex (Top1cc) formation, in mitochondria of CD4 T cells from HCV- and HIV-infected patients. Notably, treatment of healthy CD4 T cells with CPT caused similar changes, including inhibition of Top1mt, accumulation of Top1cc in mitochondria, increase in PARP1 cleavage, and decrease in mtDNA copy numbers. These molecular changes resulted in mitochondrial dysfunction, T cell dysregulation, and programmed cell death through multiple signaling pathways, recapitulating the phenotype we detected in CD4 T cells from HCV- and HIV-infected patients. Moreover, treatment of CD4 T cells from HCV or HIV patients with CPT further increased cellular and mitochondrial reactive oxygen species (ROS) production and cell apoptosis, demonstrating a critical role for Top1 in preventing mtDNA damage and cell death. These results provide new insights into the molecular mechanisms underlying immune dysregulation during viral infection and indicate that Top1 inhibition during chronic HCV or HIV infection can induce mtDNA damage and T cell dysfunction. Thus, reconstituting Top1mt protein may restore the mtDNA topology and T cell functions in humans with chronic viral infection.

Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication.

The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.

Corrigendum: A matter of life or death: Productively infected and bystander CD4 T Cells in early HIV infection.

[This corrects the article DOI: 10.3389/fimmu.2020.626431.].

Plasma biomarkers for systemic inflammation in COVID-19 survivors.

BACKGROUND: While the majority of COVID-19 patients fully recover from the infection and become asymptomatic, a significant proportion of COVID-19 survivors experience a broad spectrum of symptoms lasting weeks to months post-infection, a phenomenon termed "post-acute sequelae of COVID-19 (PASC)." The aim of this study is to determine whether inflammatory proteins are dysregulated and can serve as potential biomarkers for systemic inflammation in COVID-19 survivors. METHODS: We determined the levels of inflammatory proteins in plasma from 22 coronavirus disease 2019 (COVID-19) long haulers (COV-LH), 22 COVID-19 asymptomatic survivors (COV-AS), and 22 healthy subjects (HS) using an Olink proteomics assay and assessed the results by a beads-based multiplex immunoassay. RESULTS: Compared to HS, we found that COVID-19 survivors still exhibited systemic inflammation, as evidenced by significant changes in the levels of multiple inflammatory proteins in plasma from both COV-LH and COV-AS. CXCL10 was the only protein that significantly upregulated in COV-LH compared with COV-AS and HS. CONCLUSIONS: Our results indicate that several inflammatory proteins remain aberrantly dysregulated in COVID-19 survivors and CXCL10 might serve as a potential biomarker to typify COV-LH. Further characterization of these signature inflammatory molecules might improve the understanding of the long-term impacts of COVID-19 and provide new targets for the diagnosis and treatment of COVID-19 survivors with PASC.

TRF2 inhibition rather than telomerase disruption drives CD4T cell dysfunction during chronic viral infection.

We investigated the role of telomerase and telomere repeat-binding factor 2 (TRF2 or TERF2) in T-cell dysfunction in chronic viral infection. We found that the expression and activity of telomerase in CD4+ T (CD4T) cells from patients with hepatitis C virus (HCV) infections or people living with HIV (PLWH) were intact, but TRF2 expression was significantly inhibited at the post-transcriptional level, suggesting that TRF2 inhibition is responsible for the CD4T cell dysfunction observed during chronic viral infection. Silencing TRF2 expression in CD4T cells derived from healthy subjects induced telomeric DNA damage and CD4T cell dysfunction without affecting telomerase activity or translocation - similar to what we observed in CD4T cells from HCV patients and PLWH. These findings indicate that premature T-cell aging and dysfunction during chronic HCV or HIV infection are primarily caused by chronic immune stimulation and T-cell overactivation and/or proliferation that induce telomeric DNA damage due to TRF2 inhibition, rather than telomerase disruption. This study suggests that restoring TRF2 presents a novel approach to prevent telomeric DNA damage and premature T-cell aging, thus rejuvenating T-cell functions during chronic viral infection.

Trained Immunity: An Overview and the Impact on COVID-19.

Effectively treating infectious diseases often requires a multi-step approach to target different components involved in disease pathogenesis. Similarly, the COVID-19 pandemic has become a global health crisis that requires a comprehensive understanding of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection to develop effective therapeutics. One potential strategy to instill greater immune protection against COVID-19 is boosting the innate immune system. This boosting, termed trained immunity, employs immune system modulators to train innate immune cells to produce an enhanced, non-specific immune response upon reactivation following exposure to pathogens, a process that has been studied in the context of in vitro and in vivo clinical studies prior to the COVID-19 pandemic. Evaluation of the underlying pathways that are essential to inducing protective trained immunity will provide insight into identifying potential therapeutic targets that may alleviate the COVID-19 crisis. Here we review multiple immune training agents, including Bacillus Calmette-Guérin (BCG), ß-glucan, and lipopolysaccharide (LPS), and the two most popular cell types involved in trained immunity, monocytes and natural killer (NK) cells, and compare the signaling pathways involved in innate immunity. Additionally, we discuss COVID-19 trained immunity clinical trials, emphasizing the potential of trained immunity to fight SARS-CoV-2 infection. Understanding the mechanisms by which training agents activate innate immune cells to reprogram immune responses may prove beneficial in developing preventive and therapeutic targets against COVID-19.

Inhibiting KDM6A Demethylase Represses Long Non-Coding RNA Hotairm1 Transcription in MDSC During Sepsis.

Myeloid-derived suppressor cells (MDSCs) prolong sepsis by promoting immunosuppression. We reported that sepsis MDSC development requires long non-coding RNA Hotairm1 interactions with S100A9. Using a mouse model that simulates the immunobiology of sepsis, we find that histone demethylase KDM6A promotes Hotairm1 transcription by demethylating transcription repression H3K27me3 histone mark. We show that chemical targeting of KDM6A by GSK-J4 represses Hotairm1 transcription, which coincides with decreases in transcription activation H3K4me3 histone mark and transcription factor PU.1 binding to the Hotairm1 promoter. We further show that immunosuppressive IL-10 cytokine promotes KDM6A binding at the Hotairm1 promoter. IL-10 knockdown repletes H3K27me3 and reduces Hotairm1 transcription. GSK-J4 treatment also relocalizes nuclear S100A9 protein to the cytosol. To support translation to human sepsis, we demonstrate that inhibiting H3K27me3 demethylation by KDM6A ex vivo in MDSCs from patients with protracted sepsis decreases Hotairm1 transcription. These findings suggest that epigenetic targeting of MDSCs in human sepsis might resolve post-sepsis immunosuppression and improve sepsis survival.

KDM6A Lysine Demethylase Directs Epigenetic Polarity of MDSCs during Murine Sepsis.

Sepsis-induced myeloid-derived suppressor cells (MDSCs) increase mortality risk. We previously identified that long non-coding RNA Hotairm1 supports myeloid precursor shifts to Gr1+CD11b+ MDSCs during mouse sepsis. A major unanswered question is what molecular processes control Hotairm1 expression. In this study, we found by a genetic deletion that a specific PU.1-binding site is indispensable in controlling Hotairm1 transcription. We then identified H3K4me3 and H3K27me3 at the PU.1 site on the Hotairm1 promoter. Controlling an epigenetic switch of Hotairm1 transcription by PU.1 was histone KDM6A demethylase for H3K27me3 that derepressed its transcription with possible contributions from Ezh2 methyltransferase for H3K27me3. KDM6A knockdown in MDSCs increased H3K27me3, decreased H3K4me3, and inhibited Hotairm1 transcription activation by PU.1. These results enlighten clinical translation research of PU.1 epigenetic regulation as a potential sepsis immune-checkpoint treatment site.

The US Department of Veterans Affairs Science and Health Initiative to Combat Infectious and Emerging Life-Threatening Diseases (VA SHIELD): A Biorepository Addressing National Health Threats.

Background: The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has demonstrated the need to share data and biospecimens broadly to optimize clinical outcomes for US military Veterans. Methods: In response, the Veterans Health Administration established VA SHIELD (Science and Health Initiative to Combat Infectious and Emerging Life-threatening Diseases), a comprehensive biorepository of specimens and clinical data from affected Veterans to advance research and public health surveillance and to improve diagnostic and therapeutic capabilities. Results: VA SHIELD now comprises 12 sites collecting de-identified biospecimens from US Veterans affected by SARS-CoV-2. In addition, 2 biorepository sites, a data processing center, and a coordinating center have been established under the direction of the Veterans Affairs Office of Research and Development. Phase 1 of VA SHIELD comprises 34 157 samples. Of these, 83.8% had positive tests for SARS-CoV-2, with the remainder serving as contemporaneous controls. The samples include nasopharyngeal swabs (57.9%), plasma (27.9%), and sera (12.5%). The associated clinical and demographic information available permits the evaluation of biological data in the context of patient demographics, clinical experience and management, vaccinations, and comorbidities. Conclusions: VA SHIELD is representative of US national diversity with a significant potential to impact national healthcare. VA SHIELD will support future projects designed to better understand SARS-CoV-2 and other emergent healthcare crises. To the extent possible, VA SHIELD will facilitate the discovery of diagnostics and therapeutics intended to diminish COVID-19 morbidity and mortality and to reduce the impact of new emerging threats to the health of US Veterans and populations worldwide.

Oxidative Stress Induces Mitochondrial Compromise in CD4 T Cells From Chronically HCV-Infected Individuals.

We have previously shown that chronic Hepatitis C virus (HCV) infection can induce DNA damage and immune dysfunctions with excessive oxidative stress in T cells. Furthermore, evidence suggests that HCV contributes to increased susceptibility to metabolic disorders. However, the underlying mechanisms by which HCV infection impairs cellular metabolism in CD4 T cells remain unclear. In this study, we evaluated mitochondrial mass and intracellular and mitochondrial reactive oxygen species (ROS) production by flow cytometry, mitochondrial DNA (mtDNA) content by real-time qPCR, cellular respiration by seahorse analyzer, and dysregulated mitochondrial-localized proteins by Liquid Chromatography-Mass Spectrometry (LC-MS) in CD4 T cells from chronic HCV-infected individuals and health subjects. Mitochondrial mass was decreased while intracellular and mitochondrial ROS were increased, expressions of master mitochondrial regulators peroxisome proliferator-activated receptor 1 alpha (PGC-1α) and mitochondrial transcription factor A (mtTFA) were down-regulated, and oxidative stress was increased while mitochondrial DNA copy numbers were reduced. Importantly, CRISPR/Cas9-mediated knockdown of mtTFA impaired cellular respiration and reduced mtDNA copy number. Furthermore, proteins responsible for mediating oxidative stress, apoptosis, and mtDNA maintenance were significantly altered in HCV-CD4 T cells. These results indicate that mitochondrial functions are compromised in HCV-CD4 T cells, likely via the deregulation of several mitochondrial regulatory proteins.

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells.

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress-mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells-the major effectors of host adaptive immunity against infection and malignancy-is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (1 O2 ) only at telomeres or mitochondria in Jurkat T cells. We found that targeted 1 O2 production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted 1 O2 formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X-ray repair cross-complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet 1 O2 formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging-associated diseases.

The Ubiquitin Sensor and Adaptor Protein p62 Mediates Signal Transduction of a Viral Oncogenic Pathway.

The Epstein-Barr virus (EBV) protein LMP1 serves as a paradigm that engages complicated ubiquitination-mediated mechanisms to activate multiple transcription factors. p62 is a ubiquitin sensor and a signal-transducing adaptor that has multiple functions in diverse contexts. However, the interaction between p62 and oncogenic viruses is poorly understood. We recently reported a crucial role for p62 in oncovirus-mediated oxidative stress by acting as a selective autophagy receptor. In this following pursuit, we further discovered that p62 is upregulated in EBV type 3 compared to type 1 latency, with a significant contribution from NF-κB and AP1 activities downstream of LMP1 signaling. In turn, p62 participates in LMP1 signal transduction through its interaction with TRAF6, promoting TRAF6 ubiquitination and activation. As expected, short hairpin RNA (shRNA)-mediated knockdown (KD) of p62 transcripts reduces LMP1-TRAF6 interaction and TRAF6 ubiquitination, as well as p65 nuclear translocation, which was assessed by Amnis imaging flow cytometry. Strikingly, LMP1-stimulated NF-κB, AP1, and Akt activities are all markedly reduced in p62-/- mouse embryo fibroblasts (MEFs) and in EBV-negative Burkitt's lymphoma (BL) cell lines with CRISPR-mediated knockout (KO) of the p62-encoding gene. However, EBV-positive BL cell lines (type 3 latency) with CRISPR-mediated KO of the p62-encoding gene failed to survive. In consequence, shRNA-mediated p62 KD impairs the ability of LMP1 to regulate its target gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of lymphoblastic cell lines (LCLs). These important findings have revealed a previously unrecognized novel role for p62 in EBV latency and oncogenesis, which advances our understanding of the mechanism underlying virus-mediated oncogenesis. IMPORTANCE As a ubiquitin sensor and a signal-transducing adaptor, p62 is crucial for NF-κB activation, which involves the ubiquitin machinery, in diverse contexts. However, whether p62 is required for EBV LMP1 activation of NF-κB is an open question. In this study, we provide evidence that p62 is upregulated in EBV type 3 latency and, in turn, p62 mediates LMP1 signal transduction to NF-κB, AP1, and Akt by promoting TRAF6 ubiquitination and activation. In consequence, p62 deficiency negatively regulates LMP1-mediated gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of LCLs. These important findings identified p62 as a novel signaling component of the key viral oncogenic signaling pathway.

SARS-CoV-2 specific memory T cell epitopes identified in COVID-19-recovered subjects.

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to public health. An explicit investigation of COVID-19 immune responses, particularly the host immunity in recovered subjects, will lay a foundation for the rational design of therapeutics and/or vaccines against future coronaviral outbreaks. Here, we examined virus-specific T cell responses and identified T cell epitopes using peptides spanning SARS-CoV-2 structural proteins. These peptides were used to stimulate peripheral blood mononuclear cells (PBMCs) derived from COVID-19-recovered subjects, followed by an analysis of IFN-γ-secreting T cells by enzyme-linked immunosorbent spot (ELISpot). We also evaluated virus-specific CD4 or CD8 T cell activation by flow cytometry assay. By screening 52 matrix pools (comprised of 315 peptides) of the spike (S) glycoprotein and 21 matrix pools (comprised of 102 peptides) spanning the nucleocapsid (N) protein, we identified 28 peptides from S protein and 5 peptides from N protein as immunodominant epitopes. The immunogenicity of these epitopes was confirmed by a second ELISpot using single peptide stimulation in memory T cells, and they were mapped by HLA restrictions. Notably, SARS-CoV-2 specific T cell responses positively correlated with B cell IgG and neutralizing antibody responses to the receptor-binding domain (RBD) of the S protein. Our results demonstrate that defined levels of SARS-CoV-2 specific T cell responses are generated in some, but not all, COVID-19-recovered subjects, fostering hope for the protection of a proportion of COVID-19-exposed individuals against reinfection. These results also suggest that these virus-specific T cell responses may induce protective immunity in unexposed individuals upon vaccination, using vaccines generated based on the immune epitopes identified in this study. However, SARS-CoV-2 S and N peptides are not potently immunogenic, and none of the single peptides could universally induce robust T cell responses, suggesting the necessity of using a multi-epitope strategy for COVID-19 vaccine design.

Immune Activation Induces Telomeric DNA Damage and Promotes Short-Lived Effector T Cell Differentiation in Chronic HCV Infection.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) leads to a high rate of chronic infection and T cell dysfunction. Although it is well known that chronic antigenic stimulation is a driving force for impaired T cell functions, the precise mechanisms underlying immune activation-induced T cell dysfunctions during HCV infection remain elusive. APPROACH AND RESULTS: Here, we demonstrated that circulating CD4+ T cells from patients who are chronically HCV-infected exhibit an immune activation status, as evidenced by the overexpression of cell activation markers human leukocyte antigen-antigen D-related, glucose transporter 1, granzyme B, and the short-lived effector marker CD127- killer cell lectin-like receptor G1+ . In contrast, the expression of stem cell-like transcription factor T cell factor 1 and telomeric repeat-binding factor 2 (TRF2) are significantly reduced in CD4+ T cells from patients who are chronically HCV-infected compared with healthy participants (HP). Mechanistic studies revealed that CD4+ T cells from participants with HCV exhibit phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling hyperactivation on T cell receptor stimulation, promoting proinflammatory effector cell differentiation, telomeric DNA damage, and cellular apoptosis. Inhibition of Akt signaling during T cell activation preserved the precursor memory cell population and prevented inflammatory effector cell expansion, DNA damage, and apoptotic death. Moreover, knockdown of TRF2 reduced HP T cell stemness and triggered telomeric DNA damage and cellular apoptosis, whereas overexpression of TRF2 in CD4 T cells prevented telomeric DNA damage. CONCLUSIONS: These results suggest that modulation of immune activation through inhibiting Akt signaling and protecting telomeres through enhancing TRF2 expression may open therapeutic strategies to fine tune the adaptive immune responses in the setting of persistent immune activation and inflammation during chronic HCV infection.