Nicotinamide riboside alleviates brain dysfunction induced by chronic cerebral hypoperfusion via protecting mitochondria.

Chronic cerebral hypoperfusion (CCH) is an enduring inadequate blood flow to the brain, resulting in vascular dementia (VaD). However, the effective treatment strategies are lacking. Supplementing with nicotinamide adenine dinucleotide (NAD+) has shown neuroprotective benefits in other neurodegenerative disorders. Nicotinamide riboside (NR), as a precursor of NAD+, is believed to hold promise in improving mitochondrial health, autophagy, and cognitive function. Meanwhile, NR has unique oral bioavailability, good tolerability, and minimal side effects, and it is the most promising for clinical translation. However, the effectiveness of NR in treating CCH-related VaD is still uncertain. The present study examined the neuroprotective effects of NR supplementation and its underlying mechanisms in a CCH rat model. The rats with CCH were given NR at a daily dosage of 400 mg/kg for 3 months. NR supplementation increased blood and brain NAD+ levels and improved brain function in CCH rats, including cognitive function and oxygenation capacity. It also reduced hippocampal neuronal loss and abnormalities and mitigated the decrease in dendritic spine density. The analysis of RNA sequencing in hippocampal tissue supports these findings. Electron microscopy and protein detection results suggest that NR may maintain mitochondrial structural integrity and exert a protective role by attenuating mitochondrial fission and impaired autophagy flux caused by CCH. In conclusion, these findings offer evidence for the neuroprotective potential of NR supplementation in ameliorating cognitive impairment induced by CCH.

In-depth human immune cellular profiling from newborn to frail.

Immune functional decline and remodeling accompany aging and frailty. It is still largely unknown how changes in the immune cellular composition differentiate healthy individuals from those become frail at a relatively early age. Our aim in this exploratory study was to investigate immunological changes from newborn to frailty, and the association between health statute and various immune cell subtypes. The participants analyzed in this study covered human cord blood cells and peripheral blood cells collected from young adults, healthy and frail old individuals. A total of 30 immune cell subsets was performed by flow cytometry based on the surface markers of immune cells. Furthermore, frailty was investigated for its relations with various leukocyte subpopulations. Frail individuals exhibited a higher CD4/CD8 ratio, a higher proportion of CD4+ central memory T (TCM) cells, CD8+ effector memory T cells, CD27- switched memory B (CD27-BSM) cells, CD27+ switched memory B cells, age-associated B cells (ABCs) and CD38-CD24- B cells, and a lower proportion of naïve CD8 + T cells and progenitor B cells. The Frailty index score was found to be associated with naïve T cells, CD4/CD8 ratio, ABCs, CD27-BSM cells, and CD4+ TCM cells. Our findings conducted a relatively comprehensive and extensive atlas of age- and frailty-related changes in peripheral leukocyte subpopulations from newborn to frailty. The immune phenotypes identified in this study can contribute to a deeper understanding of immunosenescence in frailty and may provide a rationale for future interventions and diagnosis.

Global analysis of HLA-A2 restricted MAGE-A3 tumor antigen epitopes and corresponding TCRs in non-small cell lung cancer.

Background: Advanced non-small cell lung cancer (NSCLC) is the most common type of lung cancer with poor prognosis. Adoptive cell therapy using engineered T-cell receptors (TCRs) targeting cancer-testis antigens, such as Melanoma-associated antigen 3 (MAGE-A3), is a potential approach for the treatment of NSCLC. However, systematic analysis of T cell immune responses to MAGE-A3 antigen and corresponding antigen-specific TCR is still lacking. Methods: In this study, we comprehensively screened HLA-A2 restricted MAGE-A3 tumor epitopes and characterized the corresponding TCRs using in vitro artificial antigen presentation cells (APC) system, single-cell transcriptome and TCR V(D)J sequencing, and machine-learning. Furthermore, the tumor-reactive TCRs with killing potency was screened and verified. Results: We identified the HLA-A2 restricted T cell epitopes from MAGE-A3 that could effectively induce the activation and cytotoxicity of CD8+ T cells using artificial APC in vitro. A cohort of HLA-A2+ NSCLC donors demonstrated that the number of epitope specific CD8+ T cells increased in NSCLC than healthy controls when measured with tetramer derived from the candidate MAGE-A3 epitopes, especially epitope Mp4 (MAGE-A3: 160-169, LVFGIELMEV). Statistical and machine-learning based analyses demonstrated that the MAGE-A3-Mp4 epitope-specific CD8+ T cell clones were mostly in effector and proliferating state. Importantly, T cells artificially expressing the MAGE-A3-Mp4 specific TCRs exhibited strong MAGE-A3+ tumor cell recognition and killing effect. Cross-reactivity risk analysis of the candidates TCRs showed high binding stability to MAGE-A3-Mp4 epitope and low risk of cross-reaction. Conclusions: This work identified candidate TCRs potentially suitable for TCR-T design targeting HLA-A2 restricted MAGE-A3 tumor antigen.

Immunological risk factors for sepsis-associated delirium and mortality in ICU patients.

Background: A major challenge in intervention of critical patients, especially sepsis-associated delirium (SAD) intervention, is the lack of predictive risk factors. As sepsis and SAD are heavily entangled with inflammatory and immunological processes, to identify the risk factors of SAD and mortality in the intensive care unit (ICU) and determine the underlying molecular mechanisms, the peripheral immune profiles of patients in the ICU were characterized. Methods: This study contains a cohort of 52 critical patients who were admitted to the ICU of the First Affiliated Hospital of Jinan University. Comorbidity, including sepsis and SAD, of this cohort was diagnosed and recorded. Furthermore, peripheral blood samples were collected on days 1, 3, and 5 of admission for peripheral immune profiling with blood routine examination, flow cytometry, ELISA, RNA-seq, and qPCR. Results: The patients with SAD had higher mortality during ICU admission and within 28 days of discharge. Compared with survivors, nonsurvivors had higher neutrophilic granulocyte percentage, higher CRP concentration, lower monocyte count, lower monocyte percentage, lower C3 complement level, higher CD14loCD16+ monocytes percentage, and higher levels of IL-6 and TNFα. The CD14hiCD16- monocyte percentage manifested favorable prediction values for the occurrence of SAD. Differentially expressed genes between the nonsurvival and survival groups were mainly associated with immune response and metabolism process. The longitudinal expression pattern of SLC2A1 and STIMATE were different between nonsurvivors and survivors, which were validated by qPCR. Conclusions: Nonsurvival critical patients have a distinct immune profile when compared with survival patients. CD14hiCD16- monocyte prevalence and expression levels of SLC2A1 and STIMATE may be predictors of SAD and 28-day mortality in ICU patients.

Optimization of antigen-specific CD8+ T cell activation conditions for infectious diseases including COVID-19.

Here, we describe the use of the artificial antigen-presenting cell (aAPC) system for the verification of T-cell epitopes. We purify and activate CD8+ T cells from blood samples from HLA-A2 that are negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). CD8+ T cells are combined with peptide-loaded T2-A2 cells, which are then stained with a SARS-CoV-2-specific MHC-1 tetramer to identify specific HLA-A2-restricted T-cell epitopes. The use of aAPC and healthy donors means that only BSL2 lab conditions are needed. For details of the use and implementation of this protocol, please refer to Deng et al. (2021).

Identification of HLA-A2 restricted CD8+ T cell epitopes in SARS-CoV-2 structural proteins.

The outbreak of coronavirus disease 2019 (COVID-19) has now become a pandemic, and the etiologic agent is the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). T cell mediated immune responses play an important role in virus controlling; however, the understanding of the viral protein immunogenicity and the mechanisms of the induced responses are still limited. So, identification of specific epitopes and exploring their immunogenic properties would provide valuable information. In our study, we utilized the Immune Epitope Database and Analysis Resource and NetMHCpan to predict HLA-A2 restricted CD8+ T cell epitopes in structural proteins of SARS-CoV-2, and screened out 23 potential epitopes. Among them, 18 peptides showed strong or moderate binding with HLA-A2 with a T2A2 cell binding model. Next, the mixed peptides induced the increased expression of CD69 and highly expressed levels of IFN-γ and granzyme B in CD8+ T cells, indicating effective activation of specific CD8+ T cells. In addition, the peptide-activated CD8+ T cells showed significantly increased killing to the target cells. Furthermore, tetramer staining revealed that the activated CD8+ T cells mainly recognized seven epitopes. All together, we identified specific CD8+ T cell epitopes in SARS-CoV-2 structural proteins, which could induce the production of specific immune competent CD8+ T cells. Our work contributes to the understanding of specific immune responses and vaccine development for SARS-CoV-2.

CD8+ T-Cell Epitope Variations Suggest a Potential Antigen HLA-A2 Binding Deficiency for Spike Protein of SARS-CoV-2.

We identified SARS-CoV-2 specific antigen epitopes by HLA-A2 binding affinity analysis and characterized their ability to activate T cells. As the pandemic continues, variations in SARS-CoV-2 virus strains have been found in many countries. In this study, we directly assess the immune response to SARS-CoV-2 epitope variants. We first predicted potential HLA-A*02:01-restricted CD8+ T-cell epitopes of SARS-CoV-2. Using the T2 cell model, HLA-A*02:01-restricted T-cell epitopes were screened for their binding affinity and ability to activate T cells. Subsequently, we examined the identified epitope variations and analyzed their impact on immune response. Here, we identified specific HLA-A2-restricted T-cell epitopes in the spike protein of SARS-CoV-2. Seven epitope peptides were confirmed to bind with HLA-A*02:01 and potentially be presented by antigen-presenting cells to induce host immune responses. Tetramers containing these peptides could interact with specific CD8+ T cells from convalescent COVID-19 patients, and one dominant epitope (n-Sp1) was defined. These epitopes could activate and generate epitope-specific T cells in vitro, and those activated T cells showed cytolytic activity toward target cells. Meanwhile, n-Sp1 epitope variant 5L>F significantly decreased the proportion of specific T-cell activation; n-Sp1 epitope 8L>V variant showed significantly reduced binding to HLA-A*02:01 and decreased proportion of n-Sp1-specific CD8+ T cell, which potentially contributes to the immune escape of SARS-CoV-2. Our data indicate that the variation of a dominant epitope will cause the deficiency of HLA-A*02:01 binding and T-cell activation, which subsequently requires the formation of a new CD8+ T-cell immune response in COVID-19 patients.

Zika Virus Induced More Severe Inflammatory Response Than Dengue Virus in Chicken Embryonic Livers.

Dengue (DENV) and Zika virus (ZIKV) are important flaviviruses in tropical and subtropical regions, causing severe Dengue Hemorrhagic Fever (DHF)/Dengue Shock Syndrome (DSS) and microcephaly, respectively. The infection of both viruses during pregnancy were reported with adverse fetal outcomes. To investigate the effects of ZIKV and DENV infections on fetal development, we established an infection model in chicken embryos. Compared with DENV-2, the infection of ZIKV significantly retarded the development of chicken embryos. High viral loads of both DENV-2 and ZIKV was detected in brain, eye and heart 7 and 11 days post-infection, respectively. Interestingly, only ZIKV but not DENV-2 was detected in the liver. Even both of them induced apparent liver inflammation, ZIKV infection showed a more severe inflammatory response than DENV-2 infection based on the inflammation scores and the gene expression levels of IL-1ß, TNF, IL-6, and TGFß-2 in liver. Our results demonstrated that ZIKV induced more severe inflammatory response in chicken embryo liver compared to DENV-2, which might partially attribute to viral replication in liver cells. Clinicians should be aware of the potential liver injury associated with ZIKV infection in patients, especially in perinatal fetuses.