Matching cellular dimensions with molecular sizes.

This Commentary discusses the spatial perception of receptors and their nanoscale organization at the surface of the lymphocyte membrane.

Wiskott-Aldrich Syndrome Interacting Protein Deficiency Uncovers the Role of the Co-receptor CD19 as a Generic Hub for PI3 Kinase Signaling in B Cells.

Humans with Wiskott-Aldrich syndrome display a progressive immunological disorder associated with compromised Wiskott-Aldrich Syndrome Interacting Protein (WIP) function. Mice deficient in WIP recapitulate such an immunodeficiency that has been attributed to T cell dysfunction; however, any contribution of B cells is as yet undefined. Here we have shown that WIP deficiency resulted in defects in B cell homing, chemotaxis, survival, and differentiation, ultimately leading to diminished germinal center formation and antibody production. Furthermore, in the absence of WIP, several receptors, namely the BCR, BAFFR, CXCR4, CXCR5, CD40, and TLR4, were impaired in promoting CD19 co-receptor activation and subsequent PI3 kinase (PI3K) signaling. The underlying mechanism was due to a distortion in the actin and tetraspanin networks that lead to altered CD19 cell surface dynamics. In conclusion, our findings suggest that, by regulating the cortical actin cytoskeleton, WIP influences the function of CD19 as a general hub for PI3K signaling.

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.

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.

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.

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.

Metabolic stress is a barrier to Epstein-Barr virus-mediated B-cell immortalization.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that has been causally linked to the development of B-cell and epithelial malignancies. Early after infection, EBV induces a transient period of hyperproliferation that is suppressed by the activation of the DNA damage response and a G1/S-phase growth arrest. This growth arrest prevents long-term outgrowth of the majority of infected cells. We developed a method to isolate and characterize infected cells that arrest after this early burst of proliferation and integrated gene expression and metabolic profiling to gain a better understanding of the pathways that attenuate immortalization. We found that the arrested cells have a reduced level of mitochondrial respiration and a decrease in the expression of genes involved in the TCA cycle and oxidative phosphorylation. Indeed, the growth arrest in early infected cells could be rescued by supplementing the TCA cycle. Arrested cells were characterized by an increase in the expression of p53 pathway gene targets, including sestrins leading to activation of AMPK, a reduction in mTOR signaling, and, consequently, elevated autophagy that was important for cell survival. Autophagy was also critical to maintain early hyperproliferation during metabolic stress. Finally, in assessing the metabolic changes from early infection to long-term outgrowth, we found concomitant increases in glucose import and surface glucose transporter 1 (GLUT1) levels, leading to elevated glycolysis, oxidative phosphorylation, and suppression of basal autophagy. Our study demonstrates that oncogene-induced senescence triggered by a combination of metabolic and genotoxic stress acts as an intrinsic barrier to EBV-mediated transformation.

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.

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.

The B cell receptor governs the subcellular location of Toll-like receptor 9 leading to hyperresponses to DNA-containing antigens.

Synergistic engagement of the B cell receptor (BCR) and Toll-like receptor 9 (TLR9) in response to DNA-containing antigens underlies the production of many autoantibodies in systemic autoimmune diseases. However, the molecular basis of this synergistic engagement is not known. Given that these receptors are spatially segregated, with the BCR on the cell surface and TLR9 in endocytic vesicles, achieving synergy must involve unique mechanisms. We show that upon antigen binding, the BCR initiates signaling at the plasma membrane and continues to signal to activate MAP kinases as it traffics to autophagosome-like compartments. The internalized BCR signals through a phospholipase-D-dependent pathway to recruit TLR9-containing endosomes to the autophagosome via the microtubular network. The recruitment of TLR9 to the autophagosomes was necessary for hyperactivation of MAP kinases. This unique mechanism for BCR-induced TLR9 recruitment resulting in B cells hyperresponses may provide new targets for therapeutics for autoimmune diseases.

Reduced Fluorescence versus Forward Scatter Time-of-Flight and Increased Peak versus Integral Fluorescence Ratios Indicate Receptor Clustering in Flow Cytometry.

Clustering of surface receptors is often required to initiate signal transduction, receptor internalization, and cellular activation. To study the kinetics of clustering, we developed an economic high-throughput method using flow cytometry. The quantification of receptor clustering by flow cytometry is based on the following two observations: first, the fluorescence signal length (FL time-of-flight [ToF]) decreases relative to the forward scatter signal length (FSc-ToF), and second, the peak FL (FL-peak) increases relative to the integral FL (FL-integral) upon clustering of FL-labeled surface receptors. Receptor macroclustering can therefore be quantified using the ratios FL-ToF/FSc-ToF (method ToF) or FL-peak/FL-integral (method Peak). We have used these methods to analyze clustering of two immune receptors known to undergo different conformational and oligomeric states: the BCR and the complement receptor 3 (CR3), on murine splenocytes, purified B cells, and human neutrophils. Engagement of both the BCR and CR3, on immortalized as well as primary murine B cells and human neutrophil, respectively, resulted in decreased FL-ToF/FSc-ToF and increased FL-peak/FL-integral ratios. Manipulation of the actin-myosin cytoskeleton altered BCR clustering which could be measured using the established parameters. To confirm clustering of CR3 on neutrophils, we applied imaging flow cytometry. Because receptor engagement is as a biological process dependent on cell viability, energy metabolism, and temperature, receptor clustering can only be quantified by gating on viable cells under physiological conditions. In summary, with this novel method, receptor clustering on nonadherent cells can easily be monitored by high-throughput conventional flow cytometry.

Vesicular transport of progeny parvovirus particles through ER and Golgi regulates maturation and cytolysis.

Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway.

Auer Rod-Like Inclusions in Prolymphocytic Leukemia.

BACKGROUND: Prolymphocytic leukemia (PLL) is a generalized malignancy of the lymphoid tissue, usually of B cell type. Auer rod-like inclusions in prolymphocytic leukemia cells are an extremely rare event; the inclusions are very similar to the Auer rods morphologically. METHODS: We describe a case of B-cell PLL presenting with Auer rod-like inclusions. The diagnosis was eventually proven by the morphology, cytochemical staining, immunophenotypes, and electron microscopy. RESULTS: Auer rod-like inclusions are pathological changes of mitochondria with increasing density of matrix and disappearing internal instructure seen through a scanning electronic microscope. CONCLUSIONS: Auer rod-like inclusions can present in pathologically changed prolymphocytic leukemia cells.

Telomeres in T and B cells.

Telomeres are the structures at the ends of linear chromosomes. In mammalian cells, they consist of hexanucleotide (TTAGGG) repeats, together with many associated proteins. In the absence of a compensatory mechanism, dividing cells undergo gradual telomere erosion until a critical degree of shortening results in chromosomal abnormalities and cell death or senescence. For T and B cells, the ability to undergo extensive cell division and clonal expansion is crucial for effective immune function. This article describes our current understanding of telomere-length regulation in lymphocytes and its implications for immune function.

[Frontiers in Live Bone Imaging Researches. In vivo imaging of immune tissues].

In vivo imaging analysis of the immune tissues, especially secondary lymphoid tissues such as lymph nodes, has greatly increased our understanding of how immune responses are promoted and regulated by immune cell trafficking. Recently, in vivo tracking of follicular helper T (Tfh) cells, a vital T cell subset for B cell responses to produce antibodies, by imaging analysis and light-induced cell labeling not only revealed their migration dynamics, but also provided new insights into how Tfh cells may be involved in the generation of immunological memory.

Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells.

Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14.

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3' untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

CD4+ T-cell synapses involve multiple distinct stages.

One very striking feature of T-cell recognition is the formation of an immunological synapse between a T cell and a cell that it is recognizing. Formation of this complex structure correlates with cytotoxicity in the case of killer (largely CD8(+)) T-cell activity, or robust cytokine release and proliferation in the case of the much longer lived synapses formed by helper (CD4(+)) T cells. Here we have used electron microscopy and 3D tomography to characterize the synapses of antigen-specific CD4(+) T cells recognizing B cells and dendritic cells at different time points. We show that there are at least four distinct stages in synapse formation, proceeding over several hours, including an initial stage involving invasive T-cell pseudopodia that penetrate deeply into the antigen-presenting cell, almost to the nuclear envelope. This must involve considerable force and may serve to widen the search for potential ligands on the surface of the cell being recognized. We also show that centrioles and the Golgi complex are always located immediately beneath the synapse and that centrioles are significantly shifted toward the late contact zone with either B lymphocytes or bone marrow-derived dendritic cells such as antigen-presenting cells, and that there are dynamic, stage-dependent changes in the organization of microtubules beneath the synapse. These data reinforce and extend previous data on cytotoxic T cells that one of the principal functions of the immunological synapse is to facilitate cytokine secretion into the synaptic cleft, as well as provide important insights into the overall dynamics of this phenomenon.