Tracking antigen-specific TCR clonotypes in SARS-CoV-2 infection reveals distinct severity trajectories.

Despite the importance of antigen-specific T cells in infectious disease, characterizing and tracking clonally amplified T cells during the progression of a patient's symptoms remain unclear. Here, we performed a longitudinal, in-depth single-cell multiomics analysis of samples from asymptomatic, mild, usual severe, and delayed severe patients of SARS-CoV-2 infection. Our in-depth analysis revealed that hyperactive or improper T-cell responses were more aggressive in delayed severe patients. Interestingly, tracking of antigen-specific T-cell receptor (TCR) clonotypes along the developmental trajectory indicated an attenuation in functional T cells upon severity. In addition, increased glycolysis and interleukin-6 signaling in the cytotoxic T cells were markedly distinct in delayed severe patients compared to usual severe patients, particularly in the middle and late stages of infection. Tracking B-cell receptor clonotypes also revealed distinct transitions and somatic hypermutations within B cells across different levels of disease severity. Our results suggest that single-cell TCR clonotype tracking can distinguish the severity of patients through immunological hallmarks, leading to a better understanding of the severity differences in and improving the management of infectious diseases by analyzing the dynamics of immune responses over time.

Longitudinal Analysis of Human Memory T-Cell Response According to the Severity of Illness up to 8 Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: Understanding the memory T-cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for assessing the longevity of protective immunity after SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) vaccination. However, the longitudinal memory T-cell response up to 8 months post-symptom onset (PSO) according to the severity of illness is unknown. METHODS: We analyzed peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with COVID-19 who experienced asymptomatic, mild, or severe illness at 2, 5, and 8 months PSO. SARS-CoV-2 spike, nucleocapsid, and membrane protein-stimulated PBMCs were subjected to flow cytometry analysis. RESULTS: A total of 24 patients (7 asymptomatic, 9 with mild disease, and 8 with severe disease) and 6 healthy volunteers were analyzed. SARS-CoV-2-specific OX40+CD137+CD4+ T cells and CD69+CD137+CD8+ T cells persisted at 8 months PSO. Also, antigen-specific cytokine-producing or polyfunctional CD4+ T cells were maintained for up to 8 months PSO. Memory CD4+ T-cell responses tended to be greater in patients who had severe illness than in those with mild or asymptomatic disease. CONCLUSIONS: Memory response to SARS-CoV-2, based on the frequency and functionality, persists for 8 months PSO. Further investigations involving its longevity and protective effect from reinfection are warranted.

Kidney residency of VISTA-positive macrophages accelerates repair from ischemic injury.

Tissue-resident macrophages have unique tissue-specific functions in maintaining homeostasis and resolving inflammation. However, the repair role and relevant molecules of kidney-resident macrophages after ischemic injury remain unresolved. To this end, mice without kidney-resident R1 macrophages but containing infiltrating monocyte-derived R2 macrophages were generated using differential cellular kinetics following clodronate liposome treatment. When ischemia-reperfusion injury was induced in these mice, late phase repair was reduced. Transcriptomic and flow cytometric analyses identified that V-domain Ig suppressor of T cell activation (VISTA), an inhibitory immune checkpoint molecule, was constitutively expressed in kidney-resident R1 macrophages, but not in other tissue-resident macrophages. Here, VISTA functioned as a scavenger of apoptotic cells and served as a checkpoint to control kidney-infiltrating T cells upon T cell receptor-mediated stimulation. Together these functions improved the repair process after ischemia-reperfusion injury. CD14+ CD33+ mononuclear phagocytes of human kidney also expressed VISTA, which has similar functions to the mouse counterpart. Thus, VISTA is upregulated in kidney macrophages in a tissue-dependent manner and plays a repair role during ischemic injury.

Transient expression of ZBTB32 in anti-viral CD8+ T cells limits the magnitude of the effector response and the generation of memory.

Virus infections induce CD8+ T cell responses comprised of a large population of terminal effector cells and a smaller subset of long-lived memory cells. The transcription factors regulating the relative expansion versus the long-term survival potential of anti-viral CD8+ T cells are not completely understood. We identified ZBTB32 as a transcription factor that is transiently expressed in effector CD8+ T cells. After acute virus infection, CD8+ T cells deficient in ZBTB32 showed enhanced virus-specific CD8+ T cell responses, and generated increased numbers of virus-specific memory cells; in contrast, persistent expression of ZBTB32 suppressed memory cell formation. The dysregulation of CD8+ T cell responses in the absence of ZBTB32 was catastrophic, as Zbtb32-/- mice succumbed to a systemic viral infection and showed evidence of severe lung pathology. We found that ZBTB32 and Blimp-1 were co-expressed following CD8+ T cell activation, bound to each other, and cooperatively regulated Blimp-1 target genes Eomes and Cd27. These findings demonstrate that ZBTB32 is a key transcription factor in CD8+ effector T cells that is required for the balanced regulation of effector versus memory responses to infection.

Dual oxidase 1 and NADPH oxidase 2 exert favorable effects in cervical cancer patients by activating immune response.

BACKGROUND: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) not only can promote cancer progression, but also they have recently emerged as mediators of the mucosal immune system. However, the roles and clinical relevance of the collective or individual NADPH oxidase (NOX) family genes in cervical cancer have not been studied. METHODS: We investigated the clinical significance of the NOX family genes using data from 307 patients with cervical cancer obtained from The Cancer Genome Atlas. Bioinformatics and experimental analyses were performed to examine NOX family genes in cervical cancer patients. RESULTS: Dual Oxidase1 (DUOX1) and Dual Oxidase 2 (DUOX2) mRNA levels were upregulated 57.9- and 67.5-fold, respectively, in cervical cancer patients. The protein expression of DUOX1, DUOX2, and NOX2 also identified in cervical squamous cell carcinoma tissues. Especially, DUOX1 and DUOX2 mRNA levels were significantly increased in patients infected with human papillomavirus (HPV) 16. Moreover, high DUOX1 mRNA levels were significantly associated with both favorable overall survival and disease-free survival in cervical cancer patients. High NOX2 mRNA levels was significantly associated with favorable overall survival. Gene set enrichment analyses revealed that high DUOX1 and NOX2 expression was significantly correlated with the enrichment of immune pathways related to interferon (IFN)-alpha, IFN-gamma, and natural killer (NK) cell signaling. Cell-type identification by estimating relative subsets of known RNA transcript analyses indicated that the fraction of innate immune cells, including NK cells, monocytes, dendritic cells, and mast cells, was elevated in patients with high DUOX1 expression. CONCLUSIONS: DUOX1 and NOX2 expression are associated with mucosal immunity activated in cervical squamous cell carcinoma and predicts a favorable prognosis in cervical cancer patients.

CRIF1 deficiency induces FOXP3LOW inflammatory non-suppressive regulatory T cells, thereby promoting antitumor immunity.

Recently identified human FOXP3lowCD45RA- inflammatory non-suppressive (INS) cells produce proinflammatory cytokines, exhibit reduced suppressiveness, and promote antitumor immunity unlike conventional regulatory T cells (Tregs). In spite of their implication in tumors, the mechanism for generation of FOXP3lowCD45RA- INS cells in vivo is unclear. We showed that the FOXP3lowCD45RA- cells in human tumors demonstrate attenuated expression of CRIF1, a vital mitochondrial regulator. Mice with CRIF1 deficiency in Tregs bore Foxp3lowINS-Tregs with mitochondrial dysfunction and metabolic reprograming. The enhanced glutaminolysis activated α-ketoglutarate-mTORC1 axis, which promoted proinflammatory cytokine expression by inducing EOMES and SATB1 expression. Moreover, chromatin openness of the regulatory regions of the Ifng and Il4 genes was increased, which facilitated EOMES/SATB1 binding. The increased α-ketoglutarate-derived 2-hydroxyglutarate down-regulated Foxp3 expression by methylating the Foxp3 gene regulatory regions. Furthermore, CRIF1 deficiency-induced Foxp3lowINS-Tregs suppressed tumor growth in an IFN-γ-dependent manner. Thus, CRIF1 deficiency-mediated mitochondrial dysfunction results in the induction of Foxp3lowINS-Tregs including FOXP3lowCD45RA- cells that promote antitumor immunity.

Effects of host and pathogenicity on mutation rates in avian influenza A viruses.

Mutation is the primary determinant of genetic diversity in influenza viruses. The rate of mutation, measured in an absolute time-scale, is likely to be dependent on the rate of errors in copying RNA sequences per replication and the number of replications per unit time. Conditions for viral replication are probably different among host taxa, potentially generating the host specificity of the viral mutation rate, and possibly between highly and low pathogenic (HP and LP) viruses. This study investigated whether mutation rates per year in avian influenza A viruses depend on host taxa and pathogenicity. We inferred mutation rates from the rates of synonymous substitutions, which are assumed to be neutral and thus equal to mutation rates, at four segments that code internal viral proteins (PB2, PB1, PA, NP). On the phylogeny of all avian viral sequences for each segment, multiple distinct subtrees (clades) were identified that represent viral subpopulations, which are likely to have evolved within particular host taxa. Using simple regression analysis, we found that mutation rates were significantly higher in viruses infecting chickens than domestic ducks and in those infecting wild shorebirds than wild ducks. Host dependency of the substitution rate was also confirmed by Bayesian phylogenetic analysis. However, we did not find evidence that the mutation rate is higher in HP than in LP viruses. We discuss these results considering viral replication rate as the major determinant of mutation rate per unit time.

Corrigendum: Distinct Immune Response at 1 Year Post-COVID-19 According to Disease Severity.

[This corrects the article DOI: 10.3389/fimmu.2022.830433.].

Distinct Immune Response at 1 Year Post-COVID-19 According to Disease Severity.

Background: Despite the fact of ongoing worldwide vaccination programs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), understanding longevity, breadth, and type of immune response to coronavirus disease-19 (COVID-19) is still important to optimize the vaccination strategy and estimate the risk of reinfection. Therefore, we performed thorough immunological assessments 1 year post-COVID-19 with different severity. Methods: We analyzed peripheral blood mononuclear cells and plasma samples at 1 year post-COVID-19 in patients who experienced asymptomatic, mild, and severe illness to assess titers of various isotypes of antibodies (Abs) against SARS-CoV-2 antigens, phagocytic capability, and memory B- and T-cell responses. Findings: A total of 24 patients (7, 9, and 8 asymptomatic, mild, and severe patients, respectively) and eight healthy volunteers were included in this study. We firstly showed that disease severity is correlated with parameters of immune responses at 1 year post-COVID-19 that play an important role in protecting against reinfection with SARS-CoV-2, namely, the phagocytic capacity of Abs and memory B-cell responses. Interpretation: Various immune responses at 1 year post-COVID-19, particularly the phagocytic capacity and memory B-cell responses, were dependent on the severity of the prior COVID-19. Our data could provide a clue for a tailored vaccination strategy after natural infection according to the severity of COVID-19.

Chromatin accessibility of circulating CD8+ T cells predicts treatment response to PD-1 blockade in patients with gastric cancer.

Although tumor genomic profiling has identified small subsets of gastric cancer (GC) patients with clinical benefit from anti-PD-1 treatment, not all responses can be explained by tumor sequencing alone. We investigate epigenetic elements responsible for the differential response to anti-PD-1 therapy by quantitatively assessing the genome-wide chromatin accessibility of circulating CD8+ T cells in patients' peripheral blood. Using an assay for transposase-accessible chromatin using sequencing (ATAC-seq), we identify unique open regions of chromatin that significantly distinguish anti-PD-1 therapy responders from non-responders. GC patients with high chromatin openness of circulating CD8+ T cells are significantly enriched in the responder group. Concordantly, patients with high chromatin openness at specific genomic positions of their circulating CD8+ T cells demonstrate significantly better survival than those with closed chromatin. Here we reveal that epigenetic characteristics of baseline CD8+ T cells can be used to identify metastatic GC patients who may benefit from anti-PD-1 therapy.

Aberrant hyperactivation of cytotoxic T-cell as a potential determinant of COVID-19 severity.

OBJECTIVES: We hypothesized that immune response may contribute to progression of coronavirus disease-19 (COVID-19) at the second week of illness. Therefore, we compared cell-mediated immune (CMI) responses between severe and mild COVID-19 cases. METHODS: We examined peripheral blood mononuclear cells of laboratory-confirmed COVID-19 patients from their first and third weeks of illness. Severe pneumonia was defined as an oxygen saturation ≤93% at room air. Expressions of molecules related to T-cell activation and functions were analyzed by flow cytometry. RESULTS: The population dynamics of T cells at the first week were not different between the two groups. However, total numbers of CD4+ and CD8+ T cells tended to be lower in the severe group at the third week of illness. Expressions of Ki-67, PD-1, perforin, and granzyme B in CD4+ or CD8+ T cells were significantly higher in the severe group than in the mild group at the third week. In contrast to the mild group, the levels of their expression did not decrease in the severe group. CONCLUSIONS: Severe COVID-19 had a higher degree of proliferation, activation, and cytotoxicity of T-cells at the late phase of illness without cytotoxic T-cell contraction, which might contribute to the development of severe COVID-19.

Integrative analysis of KIF4A, 9, 18A, and 23 and their clinical significance in low-grade glioma and glioblastoma.

To determine the prognostic significance of kinesin superfamily gene (KIF) expression in patients with brain cancer, including low-grade glioma (LGG) and glioblastoma (GBM), we comprehensively analyzed KIFs in 515 LGG and 595 GBM patients. Among KIFs, KIF4A, 9, 18A, and 23 showed significant clinical implications in both LGG and GBM. The mRNA and protein expression levels of KIF4A, 9, 18A, and 23 were significantly increased in LGG and GBM compared with those in the normal control groups. The mRNA expression levels of KIF4A, 9, 18A, and 23 in LGG were significantly increased in the high-histologic-grade group compared with those with a low histologic grade. Genomic analysis showed that the percent of mRNA upregulation of KIF4A, 9, 18A, and 23 was higher than that of other gene alterations, including gene amplification, deep deletion, and missense mutation. In addition, LGG patients with KIF4A, 18A, and 23 gene alterations were significantly associated with a poor prognosis. In survival analysis, the group with high expression of KIF4A, 9, 18A, and 23 mRNA was significantly associated with a poor prognosis in both LGG and GBM patients. Gene Set Enrichment Analysis (GSEA) revealed that high mRNA expression of KIF4A, 18A, and 23 in LGG and GBM patients showed significant positive correlations with the cell cycle, E2F targets, G2M checkpoint, Myc target, and mitotic spindle. By contrast, high mRNA expression of KIF9 in both LGG and GBM patients was significantly negatively correlated with the cell cycle, G2M checkpoint, and mitotic spindle pathway. However, it was significantly positively correlated with EMT and angiogenesis. This study has extended our knowledge of KIF4A, 9, 18A, and 23 in LGG and GBM and shed light on their clinical relevance, which should help to improve the treatment and prognosis of LGG and GBM.

Smart Hybrid Nanocomposite for Photodynamic Inactivation of Cancer Cells with Selectivity.

Photodynamic therapy has been efficiently applied for cancer therapy. Here, we have fabricated the folic acid (FA)- and pheophorbide A (PA)-conjugated FA/PA@Fe3O4 nanoparticle (smart hybrid nanocomposite, SHN) to enhance the photodynamic inactivation (PDI) of specific cancer cells. SHN coated with the PDI agent is designed to have selectivity for the folate receptor (FR) expressed on cancer cells. Structural characteristics and morphology of the fabricated MNPs were studied with X-ray diffraction and scanning electron microscopy. The photophysical properties of SHN were investigated with absorption, emission spectroscopies, and Fourier transform infrared spectroscopy. In addition, the magnetic property of Fe3O4 nanoparticle (MNP) can be utilized for the collection of SHNs by an external magnetic field. The photofunctionality was given by the photosensitizer, PA, which generates reactive oxygen species by irradiation of visible light. Generation of singlet oxygen was directly evaluated with time-resolved phosphorescence spectroscopy. Biocompatibility and cellular interaction of SHN were also analyzed by using various cancer cells, such as KB, HeLa, and MCF-7 cells which express different levels of FR on the surface. Cellular adsorption and the PDI effect of SHN on the various cancer cells in vitro were correlated well with the surface expression levels of FR, suggesting potential applicability of SHN on specific targeting and PDI of FR-positive cancers.

Immunization with a recombinant antigen composed of conserved blocks from TSA56 provides broad genotype protection against scrub typhus.

Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Despite the wide range of approaches explored during the last seventy years, an effective prophylactic vaccine is not yet available. Here, we developed a novel recombinant antigen derived from conserved regions of 56 kDa type-specific antigen (TSA56), a major outer membrane protein responsible for genetic heterogeneity and antigenicity, and evaluated it as a protective vaccine antigen. Our findings demonstrate that immunization with conserved blocks of TSA56 (cTSA56) not only provides protective immunity against lethal challenges with the homologous genotype, but also confers significantly better protection against heterologous genotypes than TSA56. Adoptive transfer of CD4+ or CD8+ T cells from immunized mice provided significantly enhanced protection against lethal challenge, whereas immune B cells failed to do so, indicating that cellular immunity against the conserved epitopes plays a protective role. Moreover, immunization with a 10-mer peptide mixture, screened from CD8+ T cell epitopes within the conserved region of TSA56, provided enhanced protection against lethal challenge with O. tsutsugamushi. Therefore, this novel recombinant antigen is a promising candidate for scrub typhus vaccine against a wide range of O. tsutsugamushi genotypes.

Interleukin-7 Contributes to the Invasiveness of Prostate Cancer Cells by Promoting Epithelial-Mesenchymal Transition.

Precise mechanisms underlying interleukin-7 (IL-7)-mediated tumor invasion remain unclear. Thus, we investigated the role of IL-7 in tumor invasiveness using metastatic prostate cancer PC-3 cell line derivatives, and assessed the potential of IL-7 as a clinical target using a Janus kinase (JAK) inhibitor and an IL-7-blocking antibody. We found that IL-7 stimulated wound-healing migration and invasion of PC-3 cells, increased phosphorylation of signal transducer and activator of transcription 5, Akt, and extracellular signal-regulated kinase. On the other hand, a JAK inhibitor and an IL-7-blocking antibody decreased the invasiveness of PC-3 cells. IL-7 increased tumor sphere formation and expression of epithelial-mesenchymal transition (EMT) markers. Importantly, lentiviral delivery of IL-7Rα to PC-3 cells significantly increased bone metastasis in an experimental murine metastasis model compared to controls. The gene expression profile of human prostate cancer cells from The Cancer Genome Atlas revealed that EMT pathways are strongly associated with prostate cancers that highly express both IL-7 and IL-7Rα. Collectively, these data suggest that IL-7 and/or IL-7Rα are promising targets of inhibiting tumor metastasis.

Expression of Hippo pathway genes and their clinical significance in colon adenocarcinoma.

Yes-associated protein 1 (YAP1) is a transcriptional regulator of the Hippo pathway, which regulates the development and progression of a number of types of cancer, including that of the colon. In the present study, the expression levels of Hippo pathway genes and their clinical significance were investigated in 458 patients with colon adenocarcinoma (COAD), the most frequently diagnosed neoplastic disease globally, using data obtained from The Cancer Genome Atlas database. Notably, mRNA expression of YAP1 was higher in COAD than in other types of gastrointestinal tract cancer. Expression of YAP1 mRNA was higher in COAD than in normal colon samples and was significantly higher in Tumor-Node-Metastasis (TNM) stages III-IV than in stages I-II. YAP1 protein levels, a protein primarily localized in the nucleus, was greater in TNM stages III-IV than in stages I-II. The level of pYAP1, which is inactive and localized in the cytoplasm, was significantly higher in TNM stages III-IV than in stages I-II. However, the YAP1/pYAP1 ratio, which is representative of activity, was higher in TNM stages III-IV than in stages I-II. High mRNA expression of YAP1, TAZ and TEAD4 was associated with a poor prognosis in patients with COAD. Bioinformatics analysis revealed that YAP1 was associated with DNA duplication, cell proliferation and development. Wnt signaling and transforming growth factor-ß signaling were significantly higher in the high-YAP1 group, according to data from Gene Set Enrichment Analysis. Taken together, the results indicate that the subcellular distribution of YAP1 and high mRNA expression of YAP1, TAZ and TEAD4 may be associated with poorer overall survival rates in patients with COAD.

Diversification of Orientia tsutsugamushi genotypes by intragenic recombination and their potential expansion in endemic areas.

BACKGROUND: Scrub typhus is a mite-borne febrile disease caused by O. tsutsugamushi infection. Recently, emergence of scrub typhus has attracted considerable attention in several endemic countries in Asia and the western Pacific. In addition, the antigenic diversity of the intracellular pathogen has been a serious obstacle for developing effective diagnostics and vaccine. METHODOLOGY/PRINCIPAL FINDINGS: To understand the evolutionary pathway of genotypic diversification of O. tsutsugamushi and the environmental factors associated with the epidemiological features of scrub typhus, we analyzed sequence data, including spatiotemporal information, of the tsa56 gene encoding a major outer membrane protein responsible for antigenic variation. A total of 324 tsa56 sequences covering more than 85% of its open reading frame were analyzed and classified into 17 genotypes based on phylogenetic relationship. Extensive sequence analysis of tsa56 genes using diverse informatics tools revealed multiple intragenic recombination events, as well as a substantially higher mutation rate than other house-keeping genes. This suggests that genetic diversification occurred via frequent point mutations and subsequent genetic recombination. Interestingly, more diverse bacterial genotypes and dominant vector species prevail in Taiwan compared to other endemic regions. Furthermore, the co-presence of identical and sub-identical clones of tsa56 gene in geographically distant areas implies potential spread of O. tsutsugamushi genotypes. CONCLUSIONS/SIGNIFICANCE: Fluctuation and diversification of vector species harboring O. tsutsugamushi in local endemic areas may facilitate genetic recombination among diverse genotypes. Therefore, careful monitoring of dominant vector species, as well as the prevalence of O. tsutsugamushi genotypes may be advisable to enable proper anticipation of epidemiological changes of scrub typhus.

Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8+ T Cells.

The voltage-gated potassium channel, Kv1.3, and the Ca2+-activated potassium channel, KCa3.1, regulate membrane potentials in T cells, thereby controlling T cell activation and cytokine production. However, little is known about the expression and function of potassium channels in human effector memory (EM) CD8+ T cells that can be further divided into functionally distinct subsets based on the expression of the interleukin (IL)-7 receptor alpha (IL-7Rα) chain. Herein, we investigated the functional expression and roles of Kv1.3 and KCa3.1 in EM CD8+ T cells that express high or low levels of the IL-7 receptor alpha chain (IL-7Rαhigh and IL-7Rαlow, respectively). In contrast to the significant activity of Kv1.3 and KCa3.1 in IL-7Rαhigh EM CD8+ T cells, IL-7Rαlow EM CD8+ T cells showed lower expression of Kv1.3 and insignificant expression of KCa3.1. Kv1.3 was involved in the modulation of cell proliferation and IL-2 production, whereas KCa3.1 affected the motility of EM CD8+ T cells. The lower motility of IL-7Rαlow EM CD8+ T cells was demonstrated using transendothelial migration and motility assays with intercellular adhesion molecule 1- and/or chemokine stromal cell-derived factor-1α-coated surfaces. Consistent with the lower migration property, IL-7Rαlow EM CD8+ T cells were found less frequently in human skin. Stimulating IL-7Rαlow EM CD8+ T cells with IL-2 or IL-15 increased their motility and recovery of KCa3.1 activity. Our findings demonstrate that Kv1.3 and KCa3.1 are differentially involved in the functions of EM CD8+ T cells. The weak expression of potassium channels in IL-7Rαlow EM CD8+ T cells can be revived by stimulation with IL-2 or IL-15, which restores the associated functions. This study suggests that IL-7Rαhigh EM CD8+ T cells with functional potassium channels may serve as a reservoir for effector CD8+ T cells during peripheral inflammation.