Intrathymic differentiation of natural antibody-producing plasma cells in human neonates.

The thymus is a central lymphoid organ primarily responsible for the development of T cells. A small proportion of B cells, however, also reside in the thymus to assist negative selection of self-reactive T cells. Here we show that the thymus of human neonates contains a consistent contingent of CD138+ plasma cells, producing all classes and subclasses of immunoglobulins with the exception of IgD. These antibody-secreting cells are part of a larger subset of B cells that share the expression of signature genes defining mouse B1 cells, yet lack the expression of complement receptors CD21 and CD35. Data from single-cell transcriptomic, clonal correspondence and in vitro differentiation assays support the notion of intrathymic CD138+ plasma cell differentiation, alongside other B cell subsets with distinctive molecular phenotypes. Lastly, neonatal thymic plasma cells also include clones reactive to commensal and pathogenic bacteria that commonly infect children born with antibody deficiency. Thus, our findings point to the thymus as a source of innate humoral immunity in human neonates.

Novel insight from the first lung transplant of a COVID-19 patient.

BACKGROUND: To reveal detailed histopathological changes, virus distributions, immunologic properties and multi-omic features caused by SARS-CoV-2 in the explanted lungs from the world's first successful lung transplantation of a COVID-19 patient. MATERIALS AND METHODS: A total of 36 samples were collected from the lungs. Histopathological features and virus distribution were observed by optical microscope and transmission electron microscope (TEM). Immune cells were detected by flow cytometry and immunohistochemistry. Transcriptome and proteome approaches were used to investigate main biological processes involved in COVID-19-associated pulmonary fibrosis. RESULTS: The histopathological changes of the lung tissues were characterized by extensive pulmonary interstitial fibrosis and haemorrhage. Viral particles were observed in the cytoplasm of macrophages. CD3+ CD4- T cells, neutrophils, NK cells, γ/δ T cells and monocytes, but not B cells, were abundant in the lungs. Higher levels of proinflammatory cytokines iNOS, IL-1ß and IL-6 were in the area of mild fibrosis. Multi-omics analyses revealed a total of 126 out of 20,356 significant different transcription and 114 out of 8,493 protein expression in lung samples with mild and severe fibrosis, most of which were related to fibrosis and inflammation. CONCLUSIONS: Our results provide novel insight that the significant neutrophil/ CD3+ CD4- T cell/ macrophage activation leads to cytokine storm and severe fibrosis in the lungs of COVID-19 patient and may contribute to a better understanding of COVID-19 pathogenesis.

Genome-wide association study of individual differences of human lymphocyte profiles using large-scale cytometry data.

Human immune systems are very complex, and the basis for individual differences in immune phenotypes is largely unclear. One reason is that the phenotype of the immune system is so complex that it is very difficult to describe its features and quantify differences between samples. To identify the genetic factors that cause individual differences in whole lymphocyte profiles and their changes after vaccination without having to rely on biological assumptions, we performed a genome-wide association study (GWAS), using cytometry data. Here, we applied computational analysis to the cytometry data of 301 people before receiving an influenza vaccine, and 1, 7, and 90 days after the vaccination to extract the feature statistics of the lymphocyte profiles in a nonparametric and data-driven manner. We analyzed two types of cytometry data: measurements of six markers for B cell classification and seven markers for T cell classification. The coordinate values calculated by this method can be treated as feature statistics of the lymphocyte profile. Next, we examined the genetic basis of individual differences in human immune phenotypes with a GWAS for the feature statistics, and we newly identified seven significant and 36 suggestive single-nucleotide polymorphisms associated with the individual differences in lymphocyte profiles and their change after vaccination. This study provides a new workflow for performing combined analyses of cytometry data and other types of genomics data.

Strong intracellular signal inactivation produces sharper and more robust signaling from cell membrane to nucleus.

For a chemical signal to propagate across a cell, it must navigate a tortuous environment involving a variety of organelle barriers. In this work we study mathematical models for a basic chemical signal, the arrival times at the nuclear membrane of proteins that are activated at the cell membrane and diffuse throughout the cytosol. Organelle surfaces within human B cells are reconstructed from soft X-ray tomographic images, and modeled as reflecting barriers to the molecules' diffusion. We show that signal inactivation sharpens signals, reducing variability in the arrival time at the nuclear membrane. Inactivation can also compensate for an observed slowdown in signal propagation induced by the presence of organelle barriers, leading to arrival times at the nuclear membrane that are comparable to models in which the cytosol is treated as an open, empty region. In the limit of strong signal inactivation this is achieved by filtering out molecules that traverse non-geodesic paths.

Hyperoxia Alters Ultrastructure and Induces Apoptosis in Leukemia Cell Lines.

Oxygenation conditions are crucial for growth and tumor progression. Recent data suggests a decrease in cancer cell proliferation occurring after exposure to normobaric hyperoxia. Those changes are associated with fractal dimension. The purpose of this research was to study the impact of hyperoxia on apoptosis and morphology of leukemia cell lines. Two hematopoietic lymphoid cancer cell lines (a T-lymphoblastoid line, JURKAT and a B lymphoid line, CCRF-SB) were tested under conditions of normobaric hyperoxia (FiO2 > 60%, ± 18h) and compared to a standard group (FiO2 = 21%). We tested for apoptosis using a caspase-3 assay. Cell morphology was evaluated by cytospin, microphotography after coloration, and analysis by a fractal dimension calculation software. Our results showed that exposure of cell cultures to transient normobaric hyperoxia induced apoptosis (elevated caspase-3) as well as significant and precocious modifications in cell complexity, as highlighted by increased fractal dimensions in both cell lines. These features are associated with changes in structure (pycnotic nucleus and apoptosis) recorded by microscopic analysis. Such morphological alterations could be due to several molecular mechanisms and rearrangements in the cancer cell, leading to cell cycle inhibition and apoptosis as shown by caspase-3 activity. T cells seem less resistant to hyperoxia than B cells.

Tetraspanin 33 (TSPAN33) regulates endocytosis and migration of human B lymphocytes by affecting the tension of the plasma membrane.

B lymphocytes are a leukocyte subset capable of developing several functions apart from differentiating into antibody-secreting cells. These processes are triggered by external activation signals that induce changes in the plasma membrane properties, regulated by the formation of different lipid-bilayer subdomains that are associated with the underlying cytoskeleton through different linker molecules, thus allowing the functional specialization of regions within the membrane. Among these, there are tetraspanin-enriched domains. Tetraspanins constitute a superfamily of transmembrane proteins that establish lateral associations with other molecules, determining its activity and localization. In this study, we identified TSPAN33 as an active player during B-lymphocyte cytoskeleton and plasma membrane-related phenomena, including protrusion formation, adhesion, phagocytosis, and cell motility. By using an overexpression model of TSPAN33 in human Raji cells, we detected a specific distribution of this protein that includes membrane microvilli, the Golgi apparatus, and extracellular vesicles. Additionally, we identified diminished phagocytic ability and altered cell adhesion properties due to the aberrant expression of integrins. Accordingly, these cells presented an enhanced migratory phenotype, as shown by its augmented chemotaxis and invasion rates. When we evaluated the mechanic response of cells during fibronectin-induced spreading, we found that TSPAN33 expression inhibited changes in roughness and membrane tension. Contrariwise, TSPAN33 knockdown cells displayed opposite phenotypes to those observed in the overexpression model. Altogether, our data indicate that TSPAN33 represents a regulatory element of the adhesion and migration of B lymphocytes, suggesting a novel implication of this tetraspanin in the control of the mechanical properties of their plasma membrane.

Expression of mucosal addressin cell adhesion molecule-1 on the reticular framework between white pulp and the marginal zone in the human spleen.

The antigenic heterogeneity of the reticular framework of the white pulp and marginal zone is well documented in the human adult spleen. Immunostaining of α-smooth muscle actin characterizes the heterogeneity of the reticular framework of the white pulp and marginal zone. In the human spleen, the blood cells flow in an open circulation. T and B lymphocytes flow out from the arterial terminal, and migrate in the reticular framework. Homing of lymphocytes to lymphoid tissues is regulated by selective interactions between cell surface homing receptors and tissue vascular addressins at sites of lymphocyte recruitment from the blood. In the present study, mucosal addressin cell adhesion molecule-1 was selectively expressed on α-smooth muscle actin-positive reticular framework. The reticular framework may function in lymphocyte homing and segregation into the periarteriolar lymphoid sheath, lymph follicle and marginal zone.

"Hairiness" is a Facsimile of Reorganized Cytoskeletons: A Cytopathic Effect of Coxiella burnetii.

In 1993, I reported that Coxiella burnetii transforms human B cells into hairy cells (cbHCs), the first hairy cell reported outside of hairy cell leukemia (HCL). Over last few decades, advances in molecular biology have provided evidence supporting that C. burnetii induces hairiness and inhibits the apoptosis of host cells. The present review summarizes new information in support of cbHC. C. burnetii was shown to induce reorganization of the cytoskeleton and to inhibit apoptosis in host cells. Peritoneal B1a cells were found to be permissive for virulent C. burnetii Nine Mile phase I (NMI) strains in mice. C. burnetii severely impaired E-cad expression in circulating cells of Q fever patients. B-cell non-Hodgkin lymphoma was linked to C. burnetii. Mutation of BRAF V600E was pronounced in HCL, but "hairiness" was not linked to the mutation. Risk factors shared among coxiellosis and HCL in humans and animals were reported in patients with Q-fever. Accordingly, I propose that C. burnetii induces reorganization of the cytoskeleton and inhibits apoptosis as cytopathic effects that are not target cell specific. The observed hairiness in cbHC appears to be a fixed image of dynamic nature, and hairy cells in HCL are distinct among lymphoid cells in circulation. As the cytoskeleton plays key roles in maintaining cell structural integrity in health and disease, the pathophysiology of similar cytopathic effects should be addressed in other diseases, such as myopathies, B-cell dyscrasias, and autoimmune syndromes.

Elevated internalization and cytotoxicity of polydispersed single-walled carbon nanotubes in activated B cells can be basis for preferential depletion of activated B cells in vivo.

Uptake of polydispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) in resting and LPS-activated B cells was studied using fluorescence-tagged AF-SWCNTs (FAF-SWCNTs). Activated B cells internalized substantially higher amounts of FAF-SWCNTs [76.5% AF-SWCNT+ B cells, mean fluorescence intensity (MFI) 720.6] as compared to the resting B cells [39.5% AF-SWCNT+ B cells, MFI 198.5]. B cells in S and G2/M phases were found to have significantly higher uptake of FAF-SWCNTs as compared to cells in G0/G1 phase. Confocal microscopy indicated that AF-SWCNTs were essentially localized on cell membrane in resting B cells, whereas in activated B cells, AF-SWCNTs were distributed throughout the cytoplasm. Targeting of AF-SWCNTs specifically to activated B cells in vivo was examined by first administering intravenously LPS-activated B cells tagged with fluorescence tracer (CFSE) in mice, followed by FAF-SWCNTs through the same route. It was found that FAF-SWCNTs were specifically taken up by CFSE+CD19+-activated B cells (95% FAF-SWCNT+ B cells, MFI 3725) as compared to CFSE- CD19+ resting B cells (31.1% FAF-SWCNT+ B cells, MFI 428). Administration (i.v.) of LPS resulted in a significant increase in the proportion of B cell in mouse spleen that was reduced by 68% by administering AF-SWCNTs. In control mice, the corresponding decrease in B cell proportion was 49%, which was significantly lower (p < 0.005) than the decline in LPS-treated mice. These results indicate that AF-SWCNTs may have the potential as an agent for depleting activated B cells in vivo.

LAMTOR2 (p14) Controls B Cell Differentiation by Orchestrating Endosomal BCR Trafficking.

B-cell development and function depend on stage-specific signaling through the B-cell antigen receptor (BCR). Signaling and intracellular trafficking of the BCR are connected, but the molecular mechanisms of this link are incompletely understood. Here, we investigated the role of the endosomal adaptor protein and member of the LAMTOR/Ragulator complex LAMTOR2 (p14) in B-cell development. Efficient conditional deletion of LAMTOR2 at the pre-B1 stage using mb1-Cre mice resulted in complete developmental arrest. Deletion of LAMTOR2 using Cd19-Cre mice permitted analysis of residual B cells at later developmental stages, revealing that LAMTOR2 was critical for the generation and activation of mature B lymphocytes. Loss of LAMTOR2 resulted in aberrant BCR signaling due to delayed receptor internalization and endosomal trafficking. In conclusion, we identify LAMTOR2 as critical regulator of BCR trafficking and signaling that is essential for early B-cell development in mice.

Activation-induced deaminase (AID) localizes to the nucleus in brief pulses.

Activation-induced deaminase (AID) converts C to U and 5-methyl-C to T. These mutagenic activities are critical to immunoglobulin (Ig) gene diversification and epigenetic reprogramming, but they must be tightly controlled to prevent compromising cell fitness. AID acts in the nucleus but localizes predominately to the cytoplasm. To address this apparent paradox, we have carried out time-lapse imaging of AID in single living B cells and fibroblasts. We demonstrate that AID enters the nucleus in brief (30 min) pulses, evident in about 10% of cells in the course of a single cell cycle (24 hr imaging). Pulses do not depend on AID catalytic activity, but they are coordinated with nuclear accumulation of P53. Pulsing may protect cells from pathologic consequences of excess exposure to AID, or enable AID to synchronize its activity with transcription of genes that are AID targets or with nuclear entry of factors that act at sites of AID-catalyzed DNA deamination to promote Ig gene diversification or epigenetic reprogramming.

Detailed endometrial immune assessment of both normal and adverse reproductive outcome populations.

PURPOSE: Using a comprehensive flow cytometric panel, do endometrial immune profiles in adverse reproductive outcomes such as repeat implantation failure (RIF) and repeat pregnancy loss (RPL) differ from each other and male-factor controls? METHODS: Six-hundred and twelve patients had an endometrial biopsy to assess the immunophenotype. History on presentation was used to subdivide the population into recurrent implantation failure (RIF) [n = 178], recurrent pregnancy loss (RPL) [n = 155], primary infertility [n = 130] and secondary infertility [n = 114]. A control group was utilised for comparative purposes [n = 35] and lymphocyte subpopulations were described. RESULTS: Distinct lymphocyte percentage differences were noted across the populations. Relative to controls and RPL, patients with a history of RIF had significantly raised uterine NKs (53.2 vs 45.2 & 42.9%, p < 0.0001). All sub-fertile populations had increased percentage peripheral type NKs (p = 0.001), and exhibited increased CD69+ activation (p = 0.005), higher levels of B cells (p < 0.001), elevated CD4:CD8 ratio (p < 0.0001), lower T-regs (p = 0.034) and a higher proportion of Th1+ CD4s (p = 0.001). Patient aetiology confers some distinct findings, RPL; pNK, Bcells and CD4 elevated; RIF; uNK and CD56 raised while CD-8 and NK-T lowered. CONCLUSIONS: Flow cytometric endometrial evaluation has the ability to provide a rapid and objective analysis of lymphocyte subpopulations. The findings show significant variations in cellular proportions of immune cells across the patient categories relative to control tissue. The cell types involved suggest that a potential differential pro-inflammatory bias may exist in patients with a history of adverse reproductive outcomes. Immunological assessment in appropriate populations may provide insight into the underlying aetiology of some cases of reproductive failure.

Tracking EBV-encoded RNAs (EBERs) from the nucleus to the excreted exosomes of B-lymphocytes.

Epstein-Barr virus-encoded RNAs (EBER1 and EBER2) are two highly abundant, non-protein coding RNAs consistently expressed in all EBV infected cells, but their function remains poorly understood. Conventional in situ hybridization studies have indicated that these RNAs are present exclusively in the nucleus. We have recently demonstrated that EBERs can be excreted from infected cells via exosomes. However, the details of the steps involved in their excretion remain unknown. In this study, we aimed to directly track the journey of EBERs from the nucleus to the excretory exosomes of EBV immortalized B-lymphocytes. Using a combination of molecular and novel immuno-gold labelled electron microscopy (EM) based techniques, we demonstrate the presence of EBERs, not only in the nucleus, but also in the cytoplasm of EBV infected B cell lines. EBERs were also seen in exosomes shed from infected cells along with the EBER binding protein La. Our results show, for the first time, that at least a proportion of EBERs are transported from the nucleus to the cytoplasm where they appear to be loaded into multi-vesicular bodies for eventual excretion via exosomes.

Decreased Expression of MicroRNA-107 in B Lymphocytes of Patients with Antibody-Mediated Renal Allograft Rejection.

MicroRNAs (miRNAs) are small noncoding RNA molecules that participate in normal B cell lineage development through posttranscriptional gene regulation. Antibody-mediated renal allograft rejection (ABMR) is emerging as one of the most common serious threats to renal transplant patients. In this study, we explored the role of miRNAs in the pathogenesis of ABMR. The differentially expressed miRNAs were identified by Affymetrix miRNA microarray analysis using B lymphocytes from 5 recipients and 5 volunteers. Based on quantitative RT-PCR, the expression levels of miR-107 were lower in the B lymphocytes from recipients than in those from volunteers. Computational analysis predicted that 3'-untranslated region of the autophagy-related protein 12 (ATG12) mRNA was targeted by miR-107, and we identified ATG12 as a target of miR-107 by Luciferase assay. Importantly, the expression levels of ATG12 in B lymphocytes of recipients were higher than those in the volunteer group, and miR-107 mimic significantly decreased ATG12 expression and formation of autolysosomes in B lymphocytes of recipients. Furthermore, we observed that levels of autophagy in B lymphocytes of transplant recipients were higher than those in B cells from volunteers. These findings suggest that miR-107 may contribute to the regulation of autophagy via targeting ATG12. Lastly, treatment with an miR-107 mimic caused the decrease in the secretion of IgG and IgM antibodies from B lymphocytes of transplant recipients, indicating that deregulated miR-107 could be involved in the pathogenesis of ABMR. Taken together, we propose that decreased miR-107 expression is associated with autophagy activation in B lymphocytes from patients with ABMR.

Osmotic modulation of chromatin impacts on efficiency and kinetics of cell fate modulation.

Chromatin structure is a major regulator of transcription and gene expression. Herein we explore the use of osmotic modulation to modify the chromatin structure and reprogram gene expression. In this study we use the extracellular osmotic pressure as a chromatin structure and transcriptional modulator. Hyposmotic modulation promotes chromatin loosening and induces changes in RNA polymerase II (Pol II) activity. The chromatin decondensation opens space for higher amounts of DNA engaged RNA Pol II. Hyposmotic modulation constitutes an alternative route to manipulate cell fate decisions. This technology was tested in model protocols of induced pluripotency and transdifferentiation in cells growing in suspension and adherent to substrates, CD34+ umbilical-cord-blood (UCB), fibroblasts and B-cells. The efficiency and kinetics of these cell fate modulation processes were improved by transient hyposmotic modulation of the cell environment.

Low level Hg2+ exposure modulates the B-cell cytoskeletal phosphoproteome.

Exposure of Wehi-231 B-cells to Hg2+ for 5min resulted in concentration dependent changes in protein phosphorylations. Phosphorylation was quantified using mass spectrometry to analyze TiO2 and anti-pTyr antibody selected phosphopeptides from Wehi-231 digests. The most frequent and largest amplitude responses to Hg2+ exposure were increased phosphorylation although a decrease was observed for 1% of phosphoproteins detected in the untreated cells. A subset of proteins responded with an increase in phosphorylation to Hg2+ exposure at low micromolar concentrations. The majority of proteins required Hg2+ over 20µM in order to increase phosphorylation. Ser/Thr phosphorylations are prominent in the cytoskeletal organization and the GTPase signaling systems and these systems are notable as the primary ones responding to the lowest concentrations of Hg2+. Systems that required higher concentrations of Hg2+ to increase phosphorylation included immune receptor signaling. The proteins for which an increase in phosphorylation occurred at Hg2+ above 20µM have a higher proportion of pTyr sites. Anti Ig stimulation of Wehi-231 cells confirmed that cytoskeletal protein phosphorylation and GTPase signaling are modulated in physiologically relevant B-cell receptor activation. Candidate kinases that respond to Hg2+ exposure at the low µM concentrations include MAP Kinase 1, CaM Kinase II delta and PAK2. SIGNIFICANCE: Mercury (Hg) is a wide spread environmental toxicant. Epidemiological and laboratory studies suggest that exposure to environmental Hg at current levels, which have been perceived to be non-toxic, may contribute to immune system dysfunction and autoimmune disease in humans and animals respectively. While we have previously shown that exposure of B lymphocytes to low levels of mercury interferes with B-cell receptor signaling mediated by post transcriptional phosphorylation events, overall the mechanism that is responsible for increased autoimmunity in mercury exposed human or animal populations is not well understood. The current study evaluated the dose dependent actions of mercury to change phosphorylation in the Wehi-231 cell line, an immature B-cell model in which actions of mercury on development of cell function can be evaluated. The study identified the cytoskeletal proteins as the most sensitive to modulation by mercury with changes in Ser/Thr phosphorylation being observed at the lowest concentrations of mercury. These findings indicate that the actions of mercury on B-cell immune function and development are at least in part likely mediated through changes in cytoskeletal protein phosphorylation.