Pattern recognition and iNKT cell regulation of B cell activation

The immune system protects individuals against infections but can cause disease if the response is unbalanced. Immune responses include both innate and adaptive response, and these systems are connected. As the first line of defense, innate immune responses are immediately induced upon infection. In contrast, adaptive immune responses respond to antigens in a delayed but more specific and effective. The adaptive immune responses are classified into T-independent and T-dependent responses, and B cells play a vital role in antigen presentation and antibody production in both types of immune responses. Moreover, B cells are crucial in maintaining immune tolerance, and unregulated B cells may enhance autoimmune disorders. This thesis focuses on B cell regulation in immune responses. Paper I identifies a novel interaction of scavenger receptor CD36 with LC3B regulating autophagosome generation in B cells in adaptive immune responses. CD36-deficient B cells exhibited a significantly reduced formation of plasma cells (PCs) and altered metabolism. These changes are accompanied by the impaired formation of autophagosomes. Autophagy induction led to colocalization of CD36 with autophagosome membrane protein LC3. Mice lacking CD36 in B cells had reduced germinal center (GC) responses and autophagosomes in GC B cells in vivo. Paper II shows that B cell response to apoptotic cells (ACs) relies on endosomal pattern recognition receptors. Syngeneic AC injections were used to break tolerance, and unc93b1 mutant mice that lack signaling from the TLR3, TLR7, and TLR9 receptors were investigated. Autoantibodies against Ro-52/60, La, cardiolipin, and DNA were all lower in unc93b1 mutant mice. We also observed significantly less formation of GC B cells and follicular help T (Tfh) cells in unc93b1 mutant mice than WT mice. Paper III reveals a balance between conventional and unconventional Tfh cells direct autoreactive B cells. Coadministration of α-GalCer with ACs initiated follicular helper iNKT (iNKTfh) cell formation, which promoted short-lived GC B cells and IgG1 autoantibody production while restricted Tfh cells. We also observed that deletion of CD1d specifically in B cells limited early B cell activation, iNKTfh cell generation, GC B cell formation, and auto-antibody production. Moreover, endosomal TLRs were required for iNKTfh cell-regulated GC response. In this thesis, we collectively evaluated the role of CD36, TLR3/7/9, and CD1d in regulating B cells in immune or autoimmune responses. It identifies critical players of CD36 in T-dependent immune response, TLR3/7/9 in autoimmunity, and iNKTfh cell-mediated help to autoreactive B cells. These studies contribute to our understanding of the connection between innate and adaptive immune responses

Live-dead assay on unlabeled cells using phase imaging with computational specificity

Existing approaches to evaluate cell viability involve cell staining with chemical reagents. However, the step of exogenous staining makes these methods undesirable for rapid, nondestructive, and long-term investigation. Here, we present an instantaneous viability assessment of unlabeled cells using phase imaging with computation specificity. This concept utilizes deep learning techniques to compute viability markers associated with the specimen measured by label-free quantitative phase imaging. Demonstrated on different live cell cultures, the proposed method reports approximately 95% accuracy in identifying live and dead cells. The evolution of the cell dry mass and nucleus area for the labeled and unlabeled populations reveal that the chemical reagents decrease viability. The nondestructive approach presented here may find a broad range of applications, from monitoring the production of biopharmaceuticals to assessing the effectiveness of cancer treatments

Artificial confocal microscopy for deep label-free imaging

Widefield microscopy methods applied to optically thick specimens are faced with reduced contrast due to spatial crosstalk, in which the signal at each point is the result of a superposition from neighboring points that are simultaneously illuminated. In 1955, Marvin Minsky proposed confocal microscopy as a solution to this problem. Today, laser scanning confocal fluorescence microscopy is broadly used due to its high depth resolution and sensitivity, which come at the price of photobleaching, chemical, and photo-toxicity. Here, we present artificial confocal microscopy (ACM) to achieve confocal-level depth sectioning, sensitivity, and chemical specificity, on unlabeled specimens, nondestructively. We augmented a laser scanning confocal instrument with a quantitative phase imaging module, which provides optical pathlength maps of the specimen on the same field of view as the fluorescence channel. Using pairs of phase and fluorescence images, we trained a convolution neural network to translate the former into the latter. The training to infer a new tag is very practical as the input and ground truth data are intrinsically registered and the data acquisition is automated. Remarkably, the ACM images present significantly stronger depth sectioning than the input images, enabling us to recover confocal-like tomographic volumes of microspheres, hippocampal neurons in culture, and 3D liver cancer spheroids. By training on nucleus-specific tags, ACM allows for segmenting individual nuclei within dense spheroids for both cell counting and volume measurements. Furthermore, taking the estimated fluorescence volumes, as annotation for the phase data, we extracted dry mass information for individual nuclei. Finally, our results indicate that the network learning can be transferred between spheroids suspended in different media.Comment: 35 pages, 6 figure

SOCS3 Expression by Thymic Stromal Cells Is Required for Normal T Cell Development

The suppressor of cytokine signaling 3 (SOCS3) is a major regulator of immune responses and inflammation as it negatively regulates cytokine signaling. Here, the role of SOCS3 in thymic T cell formation was studied in Socs3fl/fl Actin-creER mice (Δsocs3) with a tamoxifen inducible and ubiquitous Socs3 deficiency. Δsocs3 thymi showed a 90% loss of cellularity and altered cortico-medullary organization. Thymocyte differentiation and proliferation was impaired at the early double negative (CD4-CD8-) cell stage and apoptosis was increased during the double positive (CD4+CD8+) cell stage, resulting in the reduction of recent thymic emigrants in peripheral organs. Using bone marrow chimeras, transplanting thymic organoids and using mice deficient of SOCS3 in thymocytes we found that expression in thymic stromal cells rather than in thymocytes was critical for T cell development. We found that SOCS3 in thymic epithelial cells (TECs) binds to the E3 ubiquitin ligase TRIM 21 and that Trim21−/− mice showed increased thymic cellularity. Δsocs3 TECs showed alterations in the expression of genes involved in positive and negative selection and lympho-stromal interactions. SOCS3-dependent signal inhibition of the common gp130 subunit of the IL-6 receptor family was redundant for T cell formation. Together, SOCS3 expression in thymic stroma cells is critical for T cell development and for maintenance of thymus architecture.publishedVersio

Severity of enterovirus A71 infection in a human SCARB2 knock-in mouse model is dependent on infectious strain and route

Enterovirus A71 (EV-A71) is a major etiological agent of human hand, foot and mouth disease, and it can cause severe neurological complications. Although several genotypes of EV-A71 strains are prevalent in different regions of the world, the genotype C4 has circulated in mainland China for more than 20 years. The pathogenicity of different EV-A71 clinical isolates varies and needs to be explored. In this study, hSCARB2 knock-in mice (N = 181) with a wide range of ages were tested for their susceptibility to two EV-A71 strains with the subgenotypes C4 and C2, and two infection routes (intracranial and venous) were compared. The clinical manifestations and pathology and their relationship to the measured viral loads in different tissues were monitored. We observed that 3 weeks is a crucial age, as mice younger than 3-week-old that were infected became extremely ill. However, mice older than 3 weeks displayed diverse clinical symptoms. Significant differences were observed in the pathogenicity of the two strains with respect to clinical signs, disease incidence, survival rate, and body weight change. We concluded that hSCARB2 knock-in mice are a sensitive model for investigating the clinical outcomes resulting from infection by different EV-A71 strains. The intracranial infection model appears to be suitable for evaluating EV-A71 neurovirulence, whereas the venous infection model is appropriate for studying the pathogenicity of EV-A71

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

A physical based model to predict performance degradation of FinFET accounting for interface state distribution effect due to hot carrier injection

A physical based model for predicting the performance degradation of the FinFET is developed accounting for the interface state distribution effect due to hot carrier injection (HCI). The non-uniform distribution of interface state along the FinFET channel is first extracted by a forward gated-diode method and then reproduced by an empirical model. From this, a physical-based device model, which accounts for the interface state distribution effect, is developed to predict the performance degradation of FinFET. The result shows that the developed model not only matches well with the experimental data of FinFET in all operation regions, but also predicts the asymmetric degradation of saturation drain current in forward and reverse operation mode. Finally, the impact of HCI to a 6-T SRAM cell is simulated using HSPICE. (C) 2010 Elsevier Ltd. All rights reserved

Impact of Random Dopant Fluctuation Effect on Surrounding Gate MOSFETs: From Atomic Level Simulation to Circuit Performance Evaluation

This paper investigates the impact of random dopant fluctuation effect on surrounding gate MOSFET, from atomic statistical simulation of device to circuit performance evaluation. The doping profile is generated by an analysis of each lattice atom and then the threshold voltage variation is obtained by device Drift-Diffusion simulation. Then the circuit performance evaluation is performed by feeding the result into a surrounding-gate MOSFET model. It is shown that a significant fluctuation in threshold voltage is due to the decreasing volume. The circuit simulation results also reveal that a surrounding gate MOSFET based 6-T SRAM presents a promising resistibility to noise disturbance