Endothelial dysfunction contributes to severe COVID-19 in combination with dysregulated lymphocyte responses and cytokine networks.

The systemic processes involved in the manifestation of life-threatening COVID-19 and in disease recovery are still incompletely understood, despite investigations focusing on the dysregulation of immune responses after SARS-CoV-2 infection. To define hallmarks of severe COVID-19 in acute disease (n = 58) and in disease recovery in convalescent patients (n = 28) from Hannover Medical School, we used flow cytometry and proteomics data with unsupervised clustering analyses. In our observational study, we combined analyses of immune cells and cytokine/chemokine networks with endothelial activation and injury. ICU patients displayed an altered immune signature with prolonged lymphopenia but the expansion of granulocytes and plasmablasts along with activated and terminally differentiated T and NK cells and high levels of SARS-CoV-2-specific antibodies. The core signature of seven plasma proteins revealed a highly inflammatory microenvironment in addition to endothelial injury in severe COVID-19. Changes within this signature were associated with either disease progression or recovery. In summary, our data suggest that besides a strong inflammatory response, severe COVID-19 is driven by endothelial activation and barrier disruption, whereby recovery depends on the regeneration of the endothelial integrity.

Molecular Signatures of Inflammatory Profile and B-Cell Function in Patients with Severe Fever with Thrombocytopenia Syndrome.

Dabie bandavirus (severe fever with thrombocytopenia syndrome virus [SFTSV]) induces an immunopathogenic disease with a high fatality rate; however, the mechanisms underlying its clinical manifestations are largely unknown. In this study, we applied targeted proteomics and single-cell transcriptomics to examine the differential immune landscape in SFTS patient blood. Serum immunoprofiling identified low-risk and high-risk clusters of SFTS patients based on inflammatory cytokine levels, which corresponded to disease severity. Single-cell transcriptomic analysis of SFTS patient peripheral blood mononuclear cells (PBMCs) at different infection stages showed pronounced expansion of B cells with alterations in B-cell subsets in fatal cases. Furthermore, plasma cells in which the interferon (IFN) pathway is downregulated were identified as the primary reservoir of SFTSV replication. This study identified not only the molecular signatures of serum inflammatory cytokines and B-cell lineage populations in SFTSV-induced fatalities but also plasma cells as the viral reservoir. Thus, this suggests that altered B-cell function is linked to lethality in SFTSV infections.IMPORTANCE SFTSV is an emerging virus discovered in China in 2009; it has since spread to other countries in East Asia. Although the fatality rates of SFTSV infection range from 5.3% to as high as 27%, the mechanisms underlying clinical manifestations are largely unknown. In this study, we demonstrated that SFTSV infection in fatal cases caused an excessive inflammatory response through high induction of proinflammatory cytokines and chemokines and the aberrant inactivation of adaptive immune responses. Furthermore, single-cell transcriptome sequencing (RNA-seq) analysis of SFTS patient PBMCs revealed that SFTSV targets the B-cell lineage population, especially plasma cells, as the potential viral reservoir in patients for whom the infection is fatal. Thus, SFTSV infection may inhibit high-affinity antibody maturation and secretion of plasma B cells, suppressing neutralizing antibody production and thereby allowing significant virus replication and subsequent fatality.

Virus-infected peripheral blood plasmablasts in a patient with severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne viral hemorrhagic disease with a high fatality rate. It is caused by the SFTS virus and is endemic in East Asian countries such as China, South Korea, and Japan. Previous studies have shown that plasmablasts appear transiently in peripheral blood during the acute phase of SFTS, but do not specify the characteristics of these plasmablasts. In this report, we describe the features of peripheral blood plasmablasts in a patient with SFTS. Immunohistochemical and immunofluorescence staining detected a small number of atypical lymphocytes expressing the SFTS virus antigen among peripheral leukocytes in a blood sample. The phenotype of the virus-infected cells was CD27+, CD38+, MUM1+, and CD138+, which is consistent with that of plasmablasts. This novel study demonstrates that plasmablasts in the peripheral blood of patients with SFTS are targets of the SFTS virus.

Nodal EBV-positive polymorphic B cell lymphoproliferative disorder with plasma cell differentiation: clinicopathological analysis of five cases.

Plasma cell differentiation (PCD) is frequently observed in some entities of non-Hodgkin B cell lymphoma, including both low-grade and high-grade lymphomas. However, except for plasmablastic lymphoma and primary effusion lymphoma, EBV+ B cell lymphoproliferative disorder (LPD) with PCD has not been well addressed due to its rarity. We clinicopathologically examined five cases of nodal EBV+ polymorphic B cell LPD with PCD (PBLPD-PCD) initially diagnosed as polymorphic EBV+ diffuse large B cell lymphoma, not otherwise specified (DLBCL-NOS) with PCD (n = 3) and methotrexate-associated B cell LPD (MTX-associated B-LPD) (n = 2). One case had a concomitant brain lesion which was clinically diagnosed as EBV-related encephalitis. This patient received therapy with vidarabine, and both the brain lesion and the nodal EBV+ PBLPD-PCD lesions disappeared. Another case was characterized by Mott cell differentiation. This case was the first reported case of EBV+ B cell lymphoma or LPD with Mott cell differentiation. The two cases of MTX-associated B cell LPD which arose in patients with rheumatoid arthritis spontaneously regressed after MTX cessation. TCRγ and IGH PCR analysis was performed in four cases. Two cases had TCRγ rearrangements, but no IGH rearrangements. The other two cases had no rearrangements in these genes. We concluded that nodal EBV+ PBLPD-PCD is rare, with heterogeneous characteristics. PCR analysis revealed that nodal EBV+ PBLPD-PCD may have only TCR clonality and no IGH clonality. Considering the partial or complete loss of CD20 expression on the tumor cells, this result may be confusing for accurate diagnosis of EBV+ PBLPD-PCD, and pathologists need to be aware of this phenomenon to avoid misdiagnosis.

Analysis of KSHV B lymphocyte lineage tropism in human tonsil reveals efficient infection of CD138+ plasma cells.

Despite 25 years of research, the basic virology of Kaposi Sarcoma Herpesviruses (KSHV) in B lymphocytes remains poorly understood. This study seeks to fill critical gaps in our understanding by characterizing the B lymphocyte lineage-specific tropism of KSHV. Here, we use lymphocytes derived from 40 human tonsil specimens to determine the B lymphocyte lineages targeted by KSHV early during de novo infection in our ex vivo model system. We characterize the immunological diversity of our tonsil specimens and determine that overall susceptibility of tonsil lymphocytes to KSHV infection varies substantially between donors. We demonstrate that a variety of B lymphocyte subtypes are susceptible to KSHV infection and identify CD138+ plasma cells as a highly targeted cell type for de novo KSHV infection. We determine that infection of tonsil B cell lineages is primarily latent with few lineages contributing to lytic replication. We explore the use of CD138 and heparin sulfate proteoglycans as attachment factors for the infection of B lymphocytes and conclude that they do not play a substantial role. Finally, we determine that the host T cell microenvironment influences the course of de novo infection in B lymphocytes. These results improve our understanding of KSHV transmission and the biology of early KSHV infection in a naïve human host, and lay a foundation for further characterization of KSHV molecular virology in B lymphocyte lineages.

Epstein-Barr virus-associated enteritis with multiple ulcers: The first autopsy case.

Epstein-Barr virus (EBV)-associated enteritis is extremely rare and has not been well characterized. Herein, we present the first autopsy case of EBV-associated enteritis with multiple ulcers in a 73-year-old Japanese male. The patient had abdominal pain and was clinically diagnosed with enteritis. An endoscopic examination revealed multiple ulcers at the terminal ileum. His condition worsened due to serosanguinous bowel discharge and the patient was then admitted to the hospital. Ileocecal and subtotal small intestinal resection was performed for repetitive hemorrhage from ulcers. However, the patient died due to uncontrolled hemorrhage. An autopsy was then performed in order to explore the cause of ulcers in the small intestine. Macroscopic findings revealed multiple ulcers with occasional cobblestone-like appearance of the ileum. Histological analysis revealed marked infiltration of lymphocytes and plasma cells around the ulcer. EBV-encoded RNA in situ hybridization (EBER-ISH) revealed positive inflammatory cells. Cytomegalovirus was immunohistochemically negative. Macroscopic and microscopic findings obtained from autopsy specimens showed no foci of inflammation and EBER-ISH-positive stromal cells in the esophagus, stomach, and colorectum. EBV-associated enteritis can cause uncontrolled repetitive hemorrhage from ulcers and result in critical condition of the patient, which can be used for differential diagnosis.

I-152, a supplier of N-acetyl-cysteine and cysteamine, inhibits immunoglobulin secretion and plasma cell maturation in LP-BM5 murine leukemia retrovirus-infected mice by affecting the unfolded protein response.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 µmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

Immunoglobulin M: An Ancient Antiviral Weapon - Rediscovered.

Recent discoveries have shed new light onto immunoglobulin M (IgM), an ancient antibody class preserved throughout evolution in all vertebrates. First, IgM - long thought to be a perfect pentamer - was shown to be asymmetric, resembling a quasi-hexamer missing one monomer and containing a gap. Second, this gap allows IgM to serve as carrier of a specific host protein, apoptosis inhibitor of macrophages (AIM), which is released to promote removal of dead-cell debris, cancer cells, or pathogens. Third, recombinant IgM delivered mucosally by passive immunization gave proof-of-concept that this antibody class can prevent mucosal simian-human immunodeficiency virus transmission in non-human primates. Finally, IgM's role in adaptive immunity goes beyond being only a first defender to respond to pathogen invasion, as long-lived IgM plasma cells have been observed predominantly residing in the spleen. In fact, IgM produced by such cells contained somatic hypermutations and was linked to protection against lethal influenza virus challenge in murine models. Importantly, such long-lived IgM plasma cells had been induced by immunization 1 year before challenge. Together, new data on IgM function raise the possibility that vaccine strategies aimed at preventing virus acquisition could include this ancient weapon.

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis.

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis.

Persistence of HIV-1 Env-Specific Plasmablast Lineages in Plasma Cells after Vaccination in Humans.

Induction of persistent HIV-1 Envelope (Env) specific antibody (Ab) is a primary goal of HIV vaccine strategies; however, it is unclear whether HIV Env immunization in humans induces bone marrow plasma cells, the presumed source of long-lived systemic Ab. To define the features of Env-specific plasma cells after vaccination, samples were obtained from HVTN 105, a phase I trial testing the same gp120 protein immunogen, AIDSVAX B/E, used in RV144, along with a DNA immunogen in various prime and boost strategies. Boosting regimens that included AIDSVAX B/E induced robust peripheral blood plasmablast responses. The Env-specific immunoglobulin repertoire of the plasmablasts is dominated by VH1 gene usage and targeting of the V3 region. Numerous plasmablast-derived immunoglobulin lineages persisted in the bone marrow >8 months after immunization, including in the CD138+ long-lived plasma cell compartment. These findings identify a cellular linkage for the development of sustained Env-specific Abs following vaccination in humans.

Murine gammaherpesvirus infection is skewed toward Igλ+ B cells expressing a specific heavy chain V-segment.

One of the defining characteristics of the B cell receptor (BCR) is the extensive diversity in the repertoire of immunoglobulin genes that make up the BCR, resulting in broad range of specificity. Gammaherpesviruses are B lymphotropic viruses that establish life-long infection in B cells, and although the B cell receptor plays a central role in B cell biology, very little is known about the immunoglobulin repertoire of gammaherpesvirus infected cells. To begin to characterize the Ig genes expressed by murine gammaherpesvirus 68 (MHV68) infected cells, we utilized single cell sorting to sequence and clone the Ig variable regions of infected germinal center (GC) B cells and plasma cells. We show that MHV68 infection is biased towards cells that express the Igλ light chain along with a single heavy chain variable gene, IGHV10-1*01. This population arises through clonal expansion but is not viral antigen specific. Furthermore, we show that class-switching in MHV68 infected cells differs from that of uninfected cells. Fewer infected GC B cells are class-switched compared to uninfected GC B cells, while more infected plasma cells are class-switched compared to uninfected plasma cells. Additionally, although they are germinal center derived, the majority of class switched plasma cells display no somatic hypermutation regardless of infection status. Taken together, these data indicate that selection of infected B cells with a specific BCR, as well as virus mediated manipulation of class switching and somatic hypermutation, are critical aspects in establishing life-long gammaherpesvirus infection.

Epstein-Barr virus-infected plasma cells in periodontitis lesions.

Growing evidence supports that the Epstein-Barr virus (EBV) is a putative periodontal pathogen, but little is known regarding EBV behavior in periodontitis. Here, EBV infection was monitored in saliva and periodontal pocket (PP), at baseline and 3 months after periodontal non-surgical therapy (p-NST) in 20 patients diagnosed with periodontitis. After the treatment, the patients with the improved periodontal condition (good responders) showed a significant decrease in salivary EBV load. In contrast, in poor responders, EBV load was slightly increased. Moreover, after the therapy, most patients showed clear signs of EBV infection in a deep PP (≥5 mm) selected as a study site. To investigate how EBV can persist in a PP, we further investigate cellular sites of viral replication in PP. We identified large amounts of infiltrated EBV-infected cells, mostly overlapping with CD138+ plasma cells (PC). EBV-infected PCs formed high-density clusters within the infiltrate and along the periodontal epithelium which were commonly associated with CD3+ T-cells and CD20+ B-cells to evoke diffuse ectopic lymphoid-like structures. Taking together, this study provides new insights to support a model where the periodontal condition may play a major role in oral EBV shedding. Since PC harbors the late productive phases of EBV replication, the periodontal condition may favor B-cell differentiation with possible amplification of periodontal EBV infection and viral spreading. PCs have long been recognized as pathogenic markers in inflammatory lesions. Our finding sheds new light on the role of EBV infection and PC in periodontitis.

B cell subset alteration and the expression of tissue homing molecules in dengue infected patients.

BACKGROUND: B cells play an essential role during dengue viral infection. While a major expansion of antibody secreting cells (ASCs) was observed, the importance of these increased frequencies of ASCs remains unclear. The alteration of B cell subsets may result from the expression of tissue specific homing molecules leading to their mobilization and distribution to different target organs during acute dengue viral infection. METHODS: In this study, whole blood samples were obtained from thirty pediatric dengue-infected patients and ten healthy children and then stained with fluorochrome-conjugated monoclonal antibodies against CD3, CD14, CD19, CD20, CD21, CD27, CD38, CD45, CD138 and homing molecules of interest before analyzed by polychromatic flow cytometry. B cell subsets were characterized throughout acute infection period. RESULTS: Data shows that there were no detectable differences in frequencies of resting, activated and tissue memory cells, whereas the frequency of ASCs was significantly increased and associated with the lower frequency of naïve cells. These results were found from patients with both dengue fever and dengue hemorrhagic fever, suggesting that such change or alteration of B cells was not associated with disease severity. Moreover, several homing molecules (e.g., CXCR3 and CCR2) were found in ASCs, indicating that ASCs may distribute to inflamed tissues and various organs. CONCLUSIONS: Findings from this study provide insight into B cell subset distribution. Furthermore, organ mobilization according to homing molecule expression on different B cell subsets during the course of dengue viral infection also suggests they are distributed to inflamed tissues and various organs.

Epstein-Barr Virus-Infected Plasma Cells Infiltrate Erosive Oral Lichen Planus.

Epstein-Barr virus (EBV), in addition to its transforming properties, contributes to the pathogenesis of several inflammatory diseases. Here, we investigated its involvement in oral lichen planus (OLP), a common autoimmune-like disease of unknown etiopathogenesis that can display a malignant potential. EBV-infected cells (EBV+ cells) were sought in a large series of clinically representative OLPs ( n = 99) through in situ hybridization to detect small noncoding EBV-encoded RNAs. Overall, our results demonstrated that EBV was commonly found in OLP (74%), with significantly higher frequency (83%) in the erosive form than in the reticular/keratinized type mild form (58%). Strikingly, many erosive OLPs were massively infiltrated by large numbers of EBV+ cells, which could represent a large part of the inflammatory infiltrate. Moreover, the number of EBV+ cells in each OLP section significantly correlated with local inflammatory parameters (OLP activity, infiltrate depth, infiltrate density), suggesting a direct relationship between EBV infection and inflammatory status. Finally, we characterized the nature of the infiltrated EBV+ cells by performing detailed immunohistochemistry profiles ( n = 21). Surprisingly, nearly all EBV+ cells detected in OLP lesions were CD138+ plasma cells (PCs) and more rarely CD20+ B cells. The presence of EBV+ PCs in erosive OLP was associated with profound changes in cytokine expression profile; notably, the expression of key inflammatory factors, such as IL1-ß and IL8, were specifically increased in OLP heavily infiltrated with EBV+ PCs. Moreover, electron microscopy-based experiments showed that EBV+ PCs actively produced EBV viral particles, suggesting possible amplification of EBV infection within the lesion. Our study thus brings conclusive evidence showing that OLP is commonly infiltrated with EBV+ PCs, adding a further puzzling element to OLP pathogenesis, given that PCs are now considered to be major regulatory immune cells involved in several autoimmune diseases (ClinicalTrials.gov NCT02276573).

sIgM-FcµR Interactions Regulate Early B Cell Activation and Plasma Cell Development after Influenza Virus Infection.

Previous studies with mice lacking secreted IgM (sIgM) due to a deletion of the µs splice region (µs-/- ) had shown sIgM involvement in normal B cell development and in support of maximal Ag-specific IgG responses. Because of the changes to B cell development, it remains unclear to which extent and how sIgM directly affects B cell responses. In this study, we aimed to explore the underlying mechanisms of sIgM-mediated IgG response regulation during influenza virus infection. Generating mice with normally developed µs-deficient B cells, we demonstrate that sIgM supports IgG responses by enhancing early Ag-specific B cell expansion, not by altering B cell development. Lack of FcµR expression on B cells, but not lack of Fcα/µR expression or complement activation, reduced antiviral IgG responses to the same extent as observed in µs-/- mice. B cell-specific Fcmr-/- mice lacked robust clonal expansion of influenza hemagglutinin-specific B cells early after infection and developed fewer spleen and bone marrow IgG plasma cells and memory B cells, compared with controls. However, germinal center responses appeared unaffected. Provision of sIgM rescued plasma cell development from µs-/- but not Fcmr-/- B cells, as demonstrated with mixed bone marrow chimeric mice. Taken together, the data suggest that sIgM interacts with FcµR on B cells to support early B cell activation and the development of long-lived humoral immunity.

Massive intracerebral Epstein-Barr virus reactivation in lethal multiple sclerosis relapse after natalizumab withdrawal.

Rebound of disease activity in multiple sclerosis patients after natalizumab withdrawal is a potentially life-threatening event. To verify whether highly destructive inflammation after natalizumab withdrawal is associated with Epstein-Barr virus (EBV) reactivation in central nervous system infiltrating B-lineage cells and cytotoxic immunity, we analyzed post-mortem brain tissue from a patient who died during a fulminating MS relapse following natalizumab withdrawal. Numerous EBV infected B cells/plasma cells and CD8+ T cells infiltrated all white matter lesions; the highest frequency of EBV lytically infected cells and granzyme B+ CD8+ T cells was observed in actively demyelinating lesions. These results may encourage switching to B-cell depleting therapy after natalizumab discontinuation.