Non-classical CD45RBlo memory B-cells are the majority of circulating antigen-specific B-cells following mRNA vaccination and COVID-19 infection. (preprint)

Resting memory B-cells can be divided into classical and non-classical groups based on differential expression of markers such as CD27 and CD11c, while activated memory B-cells express a combination of markers, making their ontogeny hard to determine. Here by longitudinal analysis of COVID-19, bacterial sepsis, and BNT162b2 mRNA vaccine recipients by mass cytometry and CITE-seq we describe a three-branch structure of resting B-cell memory consisting of “classical” CD45RB+ memory and two branches of CD45RBlo memory further defined by expression of CD23 and CD11c respectively. Stable differences in CD45RB upon activation allowed tracking of activated B-cells and plasmablasts derived from CD45RB+ classical and CD45RBlo non-classical memory B-cells. In both COVID-19 patients and mRNA vaccination, CD45RBlo B-cells formed the majority of SARS-CoV2 specific memory B-cells and correlated with serum antibodies while CD45RB+ memory was most strongly activated by bacterial Sepsis. These results suggest that diverse non-classical CD45RBlo memory B-cells consisting of branches of CD11c+Tbet+ and CD23+ fractions form a critical part of responses to viral infection and vaccination.

Classification of patients with COVID-19 by blood RNA endotype: A prospective cohort study (preprint)

Background: Although the development of vaccines has considerably reduced the severity of COVID-19, its incidence is still high. Hence, a targeted approach based on RNA endotypes of a population should be developed to help design biomarker-based therapies for COVID-19. Objectives: We evaluated the major RNAs transcribed in blood cells during COVID-19 using PCR to further elucidate its pathogenesis and determine predictive phenotypes in COVID-19 patients. Study design: In a discovery cohort of 40 patients with COVID-19, 26,354 RNAs were measured on day 1 and day 7. Five RNAs associated with disease severity and prognosis were derived. In a validation cohort of 153 patients with COVID-19 treated in the intensive care unit, we focused on prolactin (PRL), and toll-like receptor 3 (TLR3) among RNAs, which have a strong association with prognosis, and evaluated the accuracy for predicting survival of PRL-to-TL3 ratios (PRL/TLR3) with the areas under the ROC curves (AUC). The validation cohort was divided into two groups based on the cut-off value in the ROC curve with the maximum AUC. The two groups were defined by high PRL/TLR3 (n=47) and low PRL/TLR3 groups (n=106) and the clinical outcomes were compared. Results: In the validation cohort, the AUC for PRL/TLR3 was 0.79, showing superior prognostic ability compared to severity scores such as APACHE II and SOFA. The high PRL/TLR3 group had a significantly higher 28-day mortality than the low PRL/TLR3 group (17.0% vs 0.9%, P<0.01). Conclusions: A new RNA endotype classified using high PRL/TLR3 was associated with mortality in COVID-19 patients.

Single-cell transcriptome landscape of circulating CD4+ T cell populations in human autoimmune diseases (preprint)

CD4+ T cells are a key mediator of various autoimmune diseases; however, how they contribute to disease development remains obscure primarily because of their cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations by decomposition-based transcriptome characterization together with canonical clustering strategies. This approach identified 12 independent transcriptional gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell data sets of over 1.8M peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloged cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.

Stimulation of dysregulated IFN-β responses by aberrant SARS-CoV-2 small viral RNAs (preprint)

Patients with severe COVID-19 exhibit a cytokine storm characterized by greatly elevated levels of cytokines during worsening disease. Despite this, the interferon (IFN) response is delayed, contributing to disease progression. Here, we report that SARS-CoV-2 generates excessive amounts of small viral RNAs (svRNAs) encoding exact 5′ ends of positive-sense genes in human cells, whereas significantly fewer similar svRNAs are produced by endemic human coronaviruses (OC43 and 229E). SARS-CoV-2 5′ end svRNAs are RIG-I agonists associated with IFN-beta expression in later stages of infection. The first 60-nt ends bearing duplex structures and 5′-triphosphates are responsible for immune-stimulation. The 5′ end svRNAs were also produced during infection ex vivo and in vivo. The delta variant retains the robust 5′ end svRNA production of the parental strain, whereas omicron (BA.1 and BA.2) produces little of these erroneous svRNAs. We propose that RIG-I activation by accumulated 5′ end svRNAs overcomes the initial IFN antagonistic ability of viral proteins and contributes to drive late over-exuberant IFN production leading to the development of severe COVID-19 and suggest that evolutionary modification of SARS-CoV-2 5′ end svRNA production may correlate with the reduced disease severity likely seen with omicron (BA.1 and BA.2).

Regulatory T-cells are central hubs for age-, sex- and severity-associated cellular networks during COVID-19 (preprint)

Using single-cell proteomics by mass cytometry, we investigate changes to a broad selection of over 10,000,000 immune cells in a cohort of moderate, severe, and critical Japanese COVID-19 patients and healthy controls with a particular focus on regulatory T-cells (Tregs). We find significant disruption within all compartments of the immune system and the emergence of atypical CTLA-4high CD4 T-cells and proliferating HLA-DRlowCD38high Tregs associated with critical patients. We also observed disrupted regulation of humoral immunity in COVID-19, with a loss of circulating T follicular regulatory T cells (Tfr) and altered T follicular helper (Tfh)/Tfr and plasma cell/Tfr ratios, all of which are significantly lower in male patients. Shifting ratios of CXCR4 and CXCR5 expression in B-cells provides further evidence of an autoimmune phenotype and dysregulated humoral immunity. These results suggest that Tregs are central to the changing cellular networks of a wide range of cells in COVID-19 and that sex specific differences to the balance of Tfr, Tfh and plasma cells may have important implications for the specificity of the humoral immune response to SARS-CoV-2.

Resistin Forms A Network With Inflammatory Cytokines And Endothelial Damage Markers And Provides Prognostic And Therapeutic Information For COVID-19 (preprint)

Background: Resistin increases in septic subjects and is associated with severity and prognosis. Its role in Coronavirus disease 2019 (COVID-19) is unknown. We investigated relationships between resistin and the severity, prognosis and time to wean off mechanical ventilation (MV) in two cohorts. Methods: : Plasma resistin was available for 306 mild-to-critical COVID-19 patients on days 1, 4 and 8 from the Massachusetts General Hospital Emergency Department COVID-19 (MGH) cohort public proteomics data. The relationship between resistin and severity (World Health Organization COVID-19 outcomes) and the prognosis were evaluated. A cohort of 62 critical COVID-19 patients (Osaka cohort) was used to evaluate the relationship between resistin on days 1 (day of ICU admission), 2–3, 6–8 and 11–15 and the prognosis and time to wean off MV. Correlations among resistin, inflammatory cytokines and endothelial damage markers were evaluated. Results: : In the MGH cohort, day 1 resistin was associated with severity and predicted the prognosis in an ROC analysis (AUC, 0.739; 95% CI, 0.659–0.819). Twenty-eight-day non-survivors showed significantly greater resistin levels than 28-day survivors on days 1, 4 and 8. In the Osaka cohort, a Cox proportional hazards model (time dependent) showed a significant relationship between resistin and time to wean off MV (crude hazard ratio, 0.702 [95% CI, 0.508–0.969]). Resistin formed a network with inflammatory cytokines and endothelial damage markers. Conclusions: : Resistin was associated with severity, prognosis and time to wean off MV in COVID-19 patients. Resistin formed a network with inflammatory cytokines and endothelial damage markers, suggesting its contribution to the pathogenesis of COVID-19.

Generation of human bronchial organoids for SARS-CoV-2 research (preprint)

Coronavirus disease 2019 (COVID-19) is a disease that causes fatal disorders including severe pneumonia. To develop a therapeutic drug for COVID-19, a model that can reproduce the viral life cycle and evaluate the drug efficacy of anti-viral drugs is essential. In this study, we established a method to generate human bronchial organoids (hBO) from commercially available cryopreserved human bronchial epithelial cells and examined whether they could be used as a model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research. Our hBO contain basal, club, ciliated, and goblet cells. Angiotensin-converting enzyme 2 (ACE2), which is a receptor for SARS-CoV-2, and transmembrane serine proteinase 2 (TMPRSS2), which is an essential serine protease for priming spike (S) protein of SARS-CoV-2, were highly expressed. After SARS-CoV-2 infection, not only the intracellular viral genome, but also progeny virus, cytotoxicity, pyknotic cells, and moderate increases of the type I interferon signal could be observed. Treatment with camostat, an inhibitor of TMPRSS2, reduced the viral copy number to 2% of the control group. Furthermore, the gene expression profile in SARS-CoV-2-infected hBO was obtained by performing RNA-seq analysis. In conclusion, we succeeded in generating hBO that can be used for SARS-CoV-2 research and COVID-19 drug discovery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/115600v2_ufig1.gif" ALT="Figure 1"> View larger version (99K): org.highwire.dtl.DTLVardef@13a6908org.highwire.dtl.DTLVardef@1c59300org.highwire.dtl.DTLVardef@362167org.highwire.dtl.DTLVardef@1cb31ed_HPS_FORMAT_FIGEXP M_FIG C_FIG